Atlas of Avian Hematology

ALFRED M. LUCAS • CASIMIR JAMROZ

Agriculture Monograph 25

UNITED STATES DEPARTMENT OF AGRICULTURE Marine Biological Laboratory Library Woods Hole. Massachusetts

Gift of the Author - 1976 7. t/u JZt^^-w-y /7 -^^

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Atlas of Avian Hematology

Alfred M. Lucas, A.B., Ph.D., Cytopathologist

Casimir Jamroz, B.S., A.B., Medical Illustrator

Regional Poultry Research Laboratory, East Lansing, Mich.

Animal Husbandry Research Division

Agricultural Research Service

Agricuhiire Monograph 25

UNITED STATES DEPARTMENT OF AGRICULTURE

WASHINGTON : 1961

For sale by the Superintendent of Documents, U.S. Government Prmting Office - Washington 25, D.C.

Preface

An atlas of hematology is a picture Itook that functions as a dictionary. Minot, in his foreword to Atlas of the Blood in Children, hy Blackfan, Dia- mond, and Leister (1944), aptly expressed the need for illustrations in

hematology when he stated, "Illustration is essential in hematology. The in- adequacy of language to convey the appearance of disease to the mind renders

an appeal to the senses desirable whenever it can be employed, and when

the objects themselves cannot be presented the best substitute for them is to be found in pictures."

It is hoped that this Atlas will enable investigators to forge ahead without the necessity for long delay in determining how the normal cell types and developmental stages should appear. The identification of the early and intermediate stages of development for most of the cell types has been worked out for the first time. The results of this research have been inte- grated with previous knowledge. All of the illustrations are original. With the population expanding at an ever-increasing rate, the demand already is upon us to have available a stockpile of sound biological information on many subjects in order quickly and accurately to solve future problems of agriculture that will grow out of the necessity for making farm production more efficient. The control of disease is an obvious method of increasing our efficiency and is the objective of the long-range program on normal avian hematology that was undertaken at this Laboratory.

The subject matter covers not only tlie circulating blood of the adult bird, ]jut also that of the embryo during its incubation from 2 days to hatching, and it includes the developmental stages found in blood-forming organs of both tlie adult and the embryo. The illustrations and text are concerned not only with the appearance of typical blood cells but also with the recognition of the atypical, the unusual, the abnormal, and the false. It is believed that future investigators of lalood diseases of poultry will rarely find a cell in their preparations that has not been illustrated here, except cells invaded by organisms such as protozoan and other parasites.

A biopsy that involves Jjlood is simpler and easier to procure than is a biopsy of any other tissue of the body, and even from a drop of blood nuich useful information concerning the health of the organism can be obtained.

It is intended that this publication shall serve the needs of the poultry- man, the veterinarian, and the research worker in zoology, embryology, endocrinology, physiology, virology, and nutrition when these persons are confronted with the problem of identifying Ijlood cells in birds. It will

lU worker in bird wikliile, in that rep- sei-ve the ornitliologist and the research than the domestic chicken; resentative studies have been made on species other of chickens, they have and where cells of wild species differed from those been illustrated and described. cells and Innumerable names have been applied to the different blood synonyms, many carry the their developmental stages. Many of these are hematopoiesis, and many implication of adherence to a particular theory of anatomical labora- are the products of the clinical laboratory and of the suitability of the names that, in tory. Whether all workers agree on the are summarized in table this book, have l)een attached to the drawings and mental image of a particular 2 is not important but it is important that the discuss it. and to this cell should be the same in the minds of all who end an atlas functions as liaison among many persons. theory of hematopoiesis; the total No attempt is made to champion any putting the cells in serial effort has gone into recording what was seen and impossible to carry order whenever possijjle. In some cases it has been force a point. Ijlanks have a series back to a stem cell and, rather than are just l)eginning a study l)een left in the record. In the avian field, we the stage attained in of normal blood morphology and thus are now at investigations on histology the human field 50 to 75 years ago. Wlien were being vigorously and anatomy in the mammalian and human fields counterpart of such studies pursued at about the turn of the century, the it was not realized that in the avian field was neglected, perhaps because to form the foundation the same kind of prerequisite information was needed pathology, physiology, for later exact investigations in pouUry diseases, embryology, and nutrition. have been Critical studies in the field of lilood diseases of poultry and almor- retarded because a reference work on the morphology of normal Avian Hematology is mal cells has not previously existed. The Atlas of program on the basic the first publication within the framework of a broad program can be histology and anatomy of the fowl. It is hoped that the carried to completion. manuscript, new informa- In the interim following the completion of tlie hematology has Ijcen published. tion on the subject of avian and mammalian Two addenda, Therefore, a few references have been added on page 242. end of one on page 93 and another on page 140, have l)een inserted at the chapters so that pagination would not be disturbed.

Alfred M. Lucas Casimir Jamroz

IV Contents

Chapter Page Chapter Poge

Preface Ill 2 Circulating blood of the hatched CHICKEN—Continued 1 General rkm\i!Ks and definitions 1 granular leukocytes—Continued Methods of study 1 Eosinophi Is Con t inued Terminology 8 — Deyelopniental stages found in circulating Magnification 14 blood 91 Arrangement of subject matter 15 Abnormal cells 91 2 Circulating blood of the hatched Technic artifacts 91 CHICK EN 17 Basophils 91 Erythrocytes 17 Normal mature basophils 91 Normal mature ervthrocvtes 17 Developmental stages found in circulating Develoj»riiciital stages found in circidating blood 92 blood 24 Technic artifacts 92 Atypical and al)norinal erythrocytes 30 hemokoma and serum granules 93 Technic artifacts 33 Thrombocytes 41 3 Circulating blood of the embryo 104

Normal mature thrombocytes 42 Erythrocyte changes during incubation. . . . 104 Deselopmental stages found in circulating Description of the cells 115 blood 44 Primary erythrocytes 115

te generations . 128 Abnormal cells 45 Later embryonic ery throcy Technic artifacts 46 Embryo thrombocytes 130 Cells occasionalK found in circulating 47 nongranulak leukocytes blood of the embryo 133 50 Lymphocytes 4 Blood cells from hematopoietic organs Normal mature lymphocytes 50 of the embryo 141 Deyelopniental stages found in circulating Embryo bone marrow 141 53 blood Embryo spleen 155 54 Vbnormal hmphocytes Blood changes at hatching 157 65 Monocytes Lymphocytogenesis in the thymus 167 mature monocytes 65 Normal Eeather sheath cell 169 De\ciopmental stages found in circidating 5 Blood cells from bone MARHo^^ of the blood 70 hatched chicken 181 Abnormal monocytes 72 Erythrocytes and thrombocytes 181 Technic artifacts 72 Granulocytes 193 granular leukocytes 73 Plasma cells 197 Osteoclasts 198 Heterophils 73 Differential counts on bone marrow 198 Normal mature heterophils 73 Developmental stages found in circidating 6 Blood cells of other avian species 202 blood 86 Description of cells 202 Abnormal heterophils 87 Size of cells 211 Technic artifacts 87 Cell counts, hemoglobin levels, and hemato- Eosinophils 89 crits 214 Normal mature eosinophils 89 Arneth counts 220 V Page Chapter ^"g«" Chapter Continued 7 Technics for avian blood 222 7 Technics for avian blood— The microscope and light 222 Staining—Continued 230 Procuring blood 225 Wright -Giemsa 230 Preparation of cannulas for taking blood Petrunkevitch No. 2 and M. G. G 230 from early embryos 225 Metln 1 alcohol and thionin 230 Method for taking blood from the dorsal Reticulocyte stain Ralph's modification of the benzidine tech- aorta of the 48-to-72-hour embryo. . . . 226 231 Method for taking blood from the heart of nic for hemoglobin in cells 231 embryos of 96 hours and older 227 Miscellaneous technics

determination. . . 231 The infrared lamp and its use in drying Method for hemoglobin

. . 232 and warming slides 227 Method for hematocrit determination. Method for collecting and carrying blood Method for making thrombocyte coimts. 232 samples 227 Method for white-cell coimts 232 Staining 228 Literature cited 235 Selection, preparation, and use of Wright's stain 228 Acknowledgments 243 Bulk staining with Wright's stain 229 Mav-Griinwald Giemsa 230 Index 245

VI CHAPTER 1

General Remarks and Definitions

METHODS OF STUDY this Atlas is the air-dried smear. It was chosen

for the reasons given in tahle 1 and hecause it is Blood cells may he studied in a variety of the method most commonly used for routine blood ways. A perusal of the literature sometimes examination. gives the impression that one technical method is Wright's stain was employed for the circu- far superior to another; hut actually each method lating blood because it is the stain that is most

has its particular merit, and it is often found that familiar to a large number of veterinarians and the advantages of a different approach compen- research workers who are not specialists in the sate for the shortcoming of the method that has field of blood. Other stains would undoubtedly

heen selected as the one generally to he followed. have made it possible to carry out certain phases Some of the principal methods for studying of the study with greater precision, but Wiight's

iilood and some of the advantages and disad- stain, in solution, keeps well, is easy to apply, vantages that have heen claimed for each method and may be procured from any medical or bio- are listed in table 1. The method chosen for logical supply house.

Table 1.—Advantages and disadvantages of various methods that have been used to study hlood cells

Metliod of study Advantages Disadvantages KILLED CELLS 1. This meltiod permits a great variety of L Considerable alteration occurs in the tran- technics to bring various components and sition from life to death; thus it becomes reactions of cells into view. Many technics difficult to

1. Tissue, fixed, sectioned, and 1. Cells are fixed in a]>proxinialelv their 1. Cells are not killed as quickly in the center stained. normal shape; that is, they are not flattened of a mass of tissue as they are in a smear, so as in dry smears. that alterations in shape and organization An example would he to drop a piece can occur. of tissue such as embryo spleen in 2. Maintains topographicrelationshipof cells Zenker ffjrmol, wash, rim through so that daughter cells and clusters of cells 2. The method is time consimiing, especially alcohols, embed, section, and stain having a connuon ancestor can be identified it a celloidin technic is employed. This im- in hematoxvlin and azure It eosin. by their proximity. poses a greater limitation on a survey type of study than does the smear metliod. 3. Shows fixed cells of tissue as well as blood cells. 3. Most investigators consider that minute differences in nuclei and cytoplasm are not as 4. Some regard the cytological appearance clearly differentiated in tissue sections as in of fixed and sectioned blood cells as more dry smears. reliable than smears for distinguishing differences.

2. Wet fixed smears. 1. Cells by this method duplicate the ap- 1. Topographic relations with other cells pearance tlie\ ha\'e in fixed preparations so and tissues are lost. .\ lliiu smear of blood or other tissue that cells studied by either method can be ci'lU on a slide that is dropped into readily compared. 2. Cells do not show the delicate structural the fixative before it tlries. Usual and tinctorial gradations seen in dry smears. lerhniques from here on. 2. A fixing agent can be chosen that will in the serve a particular purpose— i. e., the use of 3. Has some practical disadvantages methyl alcohol for preservation of granules field, since it involves carrying a nundier of in blood and tissue basophils. solutions.

3. Requires less time and equipment than the fixed-tissue method.

4. Cells usually not as severely flattened as in dry fixed smears. Table 1. —Advantages and disadvantages of various methods that have been used to study blood cells—Continued

Method of study Advantaiies Disadvantages 3. Dry fixed smears. 1. Reveal maxinnim slriietnral and tine- 1. Lose topcgrajihic relations with other torial gradation ihat permit differentia- cells and tissues. A thin smear of blood or other tissue tion hetween closely similar cells cells on a slide, dried in the air, 2. Because cells in dry smears appear struc- stained, and dried again after stain- 2. Easy to prepare. A technic useful for turally different from cells in fixed-tissue ing. work in field or laboratory research. Mini- preparations, they cannot be readily com- mum equipment and time required. pared with cells in fixed tissues.

4. Electron microscopy. 1. Offers higher magnification and resolving 1. It is necessary, not only to kill the cells, power than any previous method. Its but also to dehydrate them cftmpletely in Spread cells on a gelatin screen and greatest usefulness is to make visible minute vacuum, thus removing all volatile mole- place in a vacuum chamber of the structural details within the cell. cules. apparatus. Killed cells sometimes jireviously treated >vith osmic acid 2. This treatment of tissue cells may pro- or other metals. duce distortions even greater than those that occur in the usual fixed and stained preparations.

3. Staining technics thus far are limiled to the use of metallic stains.

4. In order to see most cells with the electron microscope, the material needs to be cut into sections thinner than one micron.

LIVING CELLS L Living cells are generally tlioiighl lo give 1. The perception ol the cell and its internal a better stantlarfl for measuring reality of structure is dependent ii|)on differences in structures than do killed and stained cells. refractive indices. Therefore, the fact that an object catinol be seen \\ilhin a living cell 2. Measurements of size are more accu- is not pniof that the object's j>resence in rately made on live than on dead cells. killed and stained cell is an artifact.

A. In vim methods. L Study of cells in their nalnral location L Technic difficulties are often insurmount- within the body bathed by normal body able. Low-power studies have their useful- .\ll technics where cells are studied fluids gives the most reliable informalion ness, but individual cells are scarcely visible. within the body, such as the blood of any method. That w bicli can be clearly L'se of water or oil immersion lenses means studies made on the living tadpole seen may" he regarded with considerable as- a short working distance with all its limita- tail, the transparent chamber built surance as a true picture. tions. in the rabbit ear, and the use of the quartz tube to concentrate light for 2. The same cell can be followed over a con- 2. It is often difficidt lo transmit enough the study of vascidar problems in si

4. Often an operation is required to see the cells and this introdiues a disturbing factor.

B. In vitro methods. 1. Cells taken outside the body can be sub- 1. Cells removed from the body are no jected to a witler variety of technical longer boimd by the same physical and Tissue cidture chiefly', but it in- methods, and the cells can be followed more chemical environmental forces that existed cludes, also, all technics where li\ ing closely than within the body. inside the body, and new equilibriums are set cells are studied outside the IxkIv. up that may lie atvjiical.

2. In temporary moinits expected to last for merely a mailer of hours, the cells begin their degenerative jirocess as soon as they leave the body, and it becomes difficult to determine between the extremes of normal variability and the early stages of irreversible degeneration. Table 1. —Advantages and disadvantages of various methods that have been used to study blood cells—Continued

Method of study Advantages Disadtantages

1. \ ital staining. 1. Motility and reactivity can be correlated 1. Vital dyes used in studies of this sort are with cell cytology. usually slightly toxic. Afellivlene blue (vital), neutral red, Janus green H. and brilliant cresyl 2. Gives a sharper separation of lympho- 2. Clean slides are very important in supra- bine are examples of vital dves that cytes and monocytes tlian with other tech- vital technics and may refpiire 3 to 4 weeks have been used extensively. nics. to prepare |>roperly. Thus this teclmic cannot be set up on short notice. 3. Some degenerative reactions may be use- fnl, such as the occurrence of vacuolization 3. Fails to show variations in cytoplasmic in granidocytes that appear 30 to W niiimles basophiUa and in nuclear details as \\ell as after the preparation is made. they are shown in drv smears.

1. Degenerative processes inherent in vital

preparations are a limiting factor in I he number of slides that may be studied at one lime; thus, weekK c-(kuUs on 23 birds wouhl be more of a task by this method than by using drv smears.

2. Darkfield illumination. 1. Often reveals extremely delicate jiroto- 1. Not adaptable to routine study of cells plasmic ]>rocesses such as filanicnls of or to making differential C4)unts. A microscopic technic (usually erythrocytes and the undulating membrane with a special condenser) whereby of monocytes. 2. The reflected light gives a dislorleil im- light enters the field at such a wide pression of the real size of lines and dots. angle that the field appears black 2. t seful for study of formed structures except where the light strikes a within the cell and movements of cells particle and bends upward towartl without the introduction of toxic agents. the observer.

3. Phase microscopy. 1. Has advantages of vital staining in ]. Does not get the full range of tinctorial showing movements and the existence of variations obtained in stained slides. A microscopic technic that utilizes [ireformed structures such as granules and some principles of darkfield. inter- mitochondria without the disadvantage of ference phenomena, and differences introducing toxic dves. in refractive indices of various materials within the cell. 2. Reveals almost as wide a variety of structures as may be shown in fixed and stained preparations.

3. Excellent for exposing technic artifacts induced by killing and staining cells.

Wright's stain is often capricious when ap- study has been applied to all embryonic blood plied to circulating Llood of embryos, to bone and to impression smears of embryonic and adult marrow, to spleen, and to pathological blood that hematopoietic tissues. contains blast cells. The chief objection is its The great variability of approaches to frequent failure to penetrate adequately and the study of blood-cell morphology has been stain the nuclei; the nuclei remain pale blue and brought out in table 1, and it is quite evident seemingly structureless, but close examination that no one method has all the advantages with under oil immersion reveals that the structures no disadvantages. The numerous theories of are actually present, and duplicate slides stained blood-cell genesis and developmental potentiali- with May-Griinwald Giemsa demonstrate that ties often have been associated witli a particular the lack of staining is not due to degeneration technic; for example, the Maximow school de- of the cell. Additional conunents on Wright's veloped and used celloidin on fixed and cut sec- stain are given in chapter 7, page 228. May- tions and arrived at the unitarian theory of Griinwald Giemsa has been used routinely in this hemocytogenesis, and many proponents of this Laboratorv for a mmiljer of vears and in this theorv continue with the same technics. The be a network upon sur- clinical hematologists use the smear method ex- dots in cross section may of a prophase figure tensively and, in general, hold to the polyphy- face view came from study cell containing a plasmosome nucleolus letic theory. The statement that the theories are of a liver determined by the technic used would, of course, (Lucas and Riser, 194.5, fig. 29). been fixed and sectioned has be too broad, but technic has certainly exerted A cell that has size and shape, where- an influence that cannot be ignored. approximately its normal method is spread out Much of the early basic concept of general as a cell fixed by the smear into a very thin, flat disk. The cell cytology was procured from sectioned mate- and distorted might seem to he ample reason rial, and the hundreds of textbook figures de- rough treatment method as reliable pro- picting the nucleus of the typical cell show its for discarding the latter in this way show many internal structure only, the surface structure be- cedure, but cells flattened never seen in a sec- ing practically ignored. Studying only the in- cytological details that are three diagrams (fig. 1, ternal structure of a cell, or only the surface tioned cell. A series of illustrates what an observer sees structure, is comparable to gaining an impres- A, B, and C) sectioned nucleus. A, as com- sion of a house by noting the arrangement of when viewing a a surface view of a flattened cell, C, and, the rooms or by looking at it from the outside; pared to already pointed out, this is a change from the thus it is no wonder that hematologists seem so as interior the nucleus to the surface far apart in their basic concepts when often a study of the of merely dia- particular school sees only one aspect of the of the nucleus. These figures are are compared nucleus. grams but if A and C of figure 1 seen in section and The realization that the internal structure and with the nuclear structures detailed draw- external surface of a nucleus can present en- surface views, as illustrated in the and Downey tirely different pictures came with the study of ings presented by Kirschbaum the cytology of intranuclear inclusions produced (1937), the similarity is striking. happens to a cell by viruses. In these cells the chromatin that In order to determine what was marginated did not disappear (Lucas and Herr- when it is spread on a slide, an experiment first coated with mann, 1935), and the appearance of a thin line performed in which a slide was dry, a blood against the nuclear membrane was not a line at a thin layer of celloidin and, when in the normal manner on the all but part of a reticulum just beneath tlie nu- smear was made surface. The celloidin with the flattened cells clear membrane (Lucas, 1940) ; that this was ac- in fluid celloidin, tually a reticulum could be seen only by focus- was peeled off and embedded the blood ing on the membrane surface. Then, during de- hardened, and sectioned transversely to cells were finally generation, the marginated chromatin aggre- cells. Wlien these flattened they were found to gated into larger and larger clumps. In cross- located under the microscope, more than a section these appeared to be a string of closely be exceedingly thin, certainly no region of the nucleus set beads, but in surface view they proved to be micron in thickness. The cytoplasm rather widely spaced, irregular clumps, ar- was scarcely thicker than that of the the whole content of the ranged at the interstices of a network. Still an- alone. This means that the other example illustrating that what appears as cell lies in the same level and, being thin,

Figure 1.

measuring cells. A, B, C: Three stages in the shift of viewpoint from the D, E: Scales for interior of the nucleus to the surface. D Scale for measuring cells drawn at low magnification, A Cut section of a nucleus, showing nuclear membrane, 1,.370X. measuring cells drawn at high magnification, chromatin chimps, and a plasmosome nucleohis E Scale for 2,470X. (stippled disk). B A transitional step showing both interior and surface G: Appearance hlood smears from chickens. views of the nucleus. F, of

C A flattened nucleus as it appears in a blood smear in F Smear from young birds, males and nonlaying females. droplets of which the nuclear plattern is determined by the chro- G Smear from laying hens. Clear areas due to is made. matin reticulum at the nuclear membrane. fat that flatten and spread when the smear 10 ZO 30 40// X 1370 D

I I I I I I I I I I I I I I I I I I I I I

10 20 30 4r0 yU X 2470 -g

I I I I I I I I I I I I I I I I I M M I I I I I I I I I I

r '-#-*-« V ^^^- whole cell falls wilhiii the depth of focus of even focused on the structure of the screen itself; the the oil immersion lens. This is readily con- latter offers verv little obstruction to a clear view firmed hy looking at any dried hlood cell of of the objects beyond: in fact, attention must be average size. shifted in order to see the wire net. Examples The cytosome is flattened in the same way as will bring out these differences as they occur in the nucleus, and the disk-shaped nucleus is cov- avian blood cells. ered by a thin layer of cytoplasm above and Erythroblasts and thromboblasts are charac- another below. The stainable liodies of the cyto- terized by the presence of nucleoli; whereas, plasm may thus be superimposed on the nuclear granuloblasts, lymphoblasts, and the most imma- structure; likewise stainable particles in the fluid ture monocytes that we have been able to find, around the cell may come to lie upon the flat- do not appear to have nucleoli, owing in part at tened cell, and it is impossible, because of the least to the difference in screen effect as deter- shallow depth of focus, to tell whether they are mined by the coarseness of the chromatin reticu- inside or outside the cell. lum. Whether nucleoli actually exist in all blast There are numerous illustrations of the points cells is not the point for consideration at this that have been presented thus far. Figures 70, time. Granuloblasts (figs. 366 and 367) have 7], and 72 show examples of substances outside a close screenlike pattern of chromatin reticulum the cell that appear to be inside; in figures 70 and. if a nucleolus exists, it cannot be seen. The and 71, parts of snuidged nuclei from other cells same is true for the lymphoblast (fig. 334) . So fell upon the cytosome and produced stained far, all efforts to demonstrate a luicleolus in the bodies. In figure 72, stained serum granules granuloblast and lymphoblast of chicken blood fell upon the cell, and had not other granules have failed. Yet. in a few cells, a vague bluish around them been stained also, thev might have image seemed to be present below this network l)een considered as lying within the cell instead (fig. 382). For practical purposes of cell-type of on it. In other instances, granules having an identificalion, it makes no dift'erence whether a identical appearance mav lie inside the cell. nucleolus is actually present or not, but it is im- Considei'able judgment may be required to de- portant and would have a Ijearing on the accept- termine what is artifact and what is real. ance of one hematopoietic theory over another. Figures 103, 105-107, and 110 show cyto- In the box-turtle the inmiature heterophil pos- somal inclusions that appear to l)e in the luiclei; sesses a blue-staining plasmosome nucleolus, ac- thev stain intensely, and the nuclei in some cases cording to Ryerson (1943). In sectioned chick are lighter than normal, so that the particles stand material studied by Dantschakoff (19081)) a out in contrast to the Ijackground. Cytoplasmic nucleolus was present inside the nucleus of the inclusions such as pale straining azurophilic granuloblast. bodies of the monoc}1e usually are not visible The blast stages of both erythrocytes and

when they overlie a darkly stained nucleus (figs. thrombocytes show nucleoli ( figs. 345, 346, 357, more clearlv than : is shown 132-134) but they may appear to be located and 358 ) the former latter, which is to be expected, since the latter inside the nucleus if they stain intensely I fig. the 135). has a denser chromatin layer at the nuclear sur- nucleoli are There is need at this point to explain more face. Yet. at best, these plasmosome fully what has been said regarding the desira- never sufficiently sharply defined to show clearly to be bility of viewing both the outside and the inside the boundary of this body. There seem the masking effect of of the nucleus. It was stated that in the flattened, two reasons for this— (1) has dried cell one sees primarily the surface of the overlying surface chromatin particles which the penetration nucleus rather than its interior. It is the coarse- already been discussed, and (2) ness of this network at the surface that in part of the stain. rup- determines the extent to which oljjects within Normally, when the cell dries it does not membranes become visible. It is the same experience that ture; instead, the cell and nuclear like balloons one has in attempting to view an object through remain intact and merely flatten out resist- a very fine screen, as against a coarse wire net; partlv filled with water, and membrane a dried the former almost completely blocks the view of ance to penetration is far more effective in to a thick- things bevond and attention is automatically cell, even though the cell is compressed ness of less than one niitron, tlian it is in sectioned may play a part, yet believe that the pattern is tissue of 5 to 7 microns in thickness. Drying, due chiefly to the arrangement of chromatin at the like tanning, seems to toughen the meml)ranes. nuclear meml)rane. kack of stain penetration is well illustrated in The question can justifialily l^e raised. How tlie use of Wright's stain on heterophils (figs. should the study of normal hematology be ap- 154—167) where tlie micleus does not stain in jnoached? In one book on human hematology lliose portions that lie within the central part of approximately the first hundred pages are de- the cell. When the chai^acter of the membrane voted to the cytology of blast-cell types and their is changed by a strong fixative, the stain pene- derivatives. These figures form the basis of com- trates readily and colors the chromatin brilliantly parison for the subject matter of the body of the (figs. 203—214). Another example is the baso- book on blood diseases, yet approximately 85 ])hil nucleus, whieli often appears to be pale and jicrcent of the cells selected for illustration and ghostlike with no evidence of chromatin or other description in the section illustrating stages in structure. This effect is in addition to the mask- development of each blood-cell type came from ing of the micleus by the basophilic granules patients suffering from different types of leuke- (figs. 190 and 191). In these illustrations, the mias or infectious diseases. Only a few were stained bodies simulating chromatin clumps taken from normal, healthy individuals, and usu- williin the l)oundary of each nucleus are actually ally these were the mature stages of cell lines. cytoplasmic basophilic granules; yet when the It is recognized that pathologic conditions often membrane resistance is Ijroken down, the cell reveal what cannot lie deciphered readily from proves to have a normal nucleus capable of in- llie normal, where all processes of formation and ternal staining (fig. 221). destruction of blood cell lines are in balance; yet Dried immature cells appear to offer greater from our limited experience with pathologic resistance to penetration of stain than do mature avian blood, undoubtedly we would have gone cells, and thus Wright's stain, which is able to astray had the picture of the normal been built penetrate the memlnanes of normal circulating upon abnormal blood conditions. It was ob- cells, does not readily do so in the case of served for example, that heterophil myelocytes precursor cells found in bone marrow, spleen, found in circulating blood after irradiation were tliymus, and other hematopoietic organs. On slightly different in general appearance and cyto- the other hand, May-Griinwald Giemsa can pene- logic detail from those found in normal bone trate inuuature cells quite well, with an occa- marrow of the chicken. In leukemias as well as sional exception (figs. 259, 281, and 334). Neo- in this example from irradiation, inunature cells plastic blood cells are usually at various stages have been pushed into a new environment, and of inunaturitv, and it is for this reason that fol- thus two variables are operating, either of which lowing Wright's stain they often appear as baso- may be responsible for their slightly different ap- philic rings with empty nuclei. A May-Griin- pearance— (1) the chemical constitution of the wald Giemsa stain on these cells will generally new environment and (2) the abnormal condi- bring out the details of nuclear structure. Al- tions that produced them. though Emmel ( 1936) did not give the stain used Isaacs ( 1928) was dealing with essentially the in his studies on hemocytolilastosis, many of the same problem when he noted that bone-marrow figures he has depicted as degenerated cells are cells and tumor cells stained more brilliantly identical in appearance with cells that have been when mixed with blood senim than when im- inadef|uately colored with Wright's stain. His prints from these tissues were made directly on figure .3B is a good example. In this study such the slide.

conditions are regarded as defective technic, not As a side effect, the fact must not lje overlooked as degenerated cells. that often disease conditions weaken and change

Jones (1948) discussed the problem of the the permeability of the cell membrane; thus it appearance of nuclei in cut sections versus dried may well be that some of the differences we see smears for embryo rat jjlood. and on most points are merely a matter of degree derived from the we are in agreement, except that he considers the more effective penetration of the blood stains nuclear pattern as due to overlying mitochon- used. Determination of the correct answer must dria: whereas, we recognize that cell organelles await further collection of data. The same complaint was made hy Dantscha- layer in the embryo. But thereafter the theories diverge or less radically, koff (1908b) against the use of Mood from cases and hypotheses more that the dif- of leukemia and other blood diseases as source though I would venture to assert material for estaljlishing the normal. She says ferences arise more in the interpretation than in B. opposing objective observations, where experi- (p. 477),". . . NachnieinerMeinungistesz. methods." heute bei der verwirrenden Menge von einzelnen, ments have paralleled in materials and coiuparative hematology by Jordan nicht systematischen Beobachtungen geradezu A sui-vey of brings out the point that regardless of the aussichtslos, aus Beobachtungen an krankliaft ( 1938) verandertem Blut des erwachsenen Menschen z. wide differences in form and habitat from fish to tlie general blood patterns are remarkably B. bei den verschiedenen Leukamien etc.. auf die man, normale Entstehung und die Verwandtschaft der constant and similar. verschiedenen Blutzellenformen zu schliessen, Kindred (1940), using the rat, illustrated the cell type as seen by the wie es jetzt von vielen tatsachlich gemacht appearance of the same ' wi rd." section and by the smear metliod. This work brought out clearly the difference in appearance of the nuclei of cells by these two different tech- TERMINOLOGY nics. A still more striking comparison was made by Kirschbaum and Downey (1937) when they One of the big problems in morphologic hema- placed some of Maximow's drawings (1909) de- tology is the choice of an acceptable terminology. rived from celloidin sectioned material beside Theories of hematology liave influenced the ter- drawings of corresponding cells made from air- minology and for the early stages, at least, each dried smears. The material for both came from school has its own set of names. This fact makes 14-day-old rabbit embryos. The wide differ- a selection of working terminology, independent ences in cell identification and even terminology of any particular theory, often impossible to find. that can be attributable largely to the different The reader should bear in mind, therefore, that appearance of similar cells in two different tech- when a particular term is applied to a cell, the nics is provocative. Our own studies on cell authors have selected it without any implication identification agree closely with those of Kirsch- that they favor the theory of hematopoiesis com- bauiu and Downey, except that we have avoided monly associated with the term. No single in- the use of the term "megaloblast" because of the vestigator has been able to encompass the whole controversy associated with the identity of the field of hematology from his own researches, and cell and the correct usage of the term (Jones, the terms he uses in his own studies are influ- 1943). enced by the appearance of the cells as revealed Kracke and Gai-ver (1937) and Osgood and by the particular technics he has used. Were Ashworth (1937) emphasize in their atlases the one unbiased person able to thoroughly review need for staiulardized terminology and in the first the wliole hematologic picture of the normal and of the books mentioned, the origins of the words abnormal of the embryo and of the adult for coiumonly and uncommonly used are discussed just one species of reptile, bird, or manunal, by rather fully. Exact terminology often moves sectioned tissue, by smear method, and by vital contrary to siiuplified terminology. Proposals technic. supplemented Ijy tissue culture, prolj- made by a conmiittee for standardized terminol- ably all conflicting theories could be merged into ogy are highly commendable (Anonymous, a uniform concept of hematopoiesis in health and 1949), but the names proposed are specifically disease. A similar thought was expressed by adapted to luan and are not broad enough to ". Doan (1932) when he said, . . It is agreed fulfill entirely the needs for terminology in other tliat all [l)lood cells] take their first beginnings classes of vertebrates. Although a serious ef- the mesenchymal cells of tlie mesodermal from fort has been made by the authors to fit the terminology of the bird into the exact framework 'Translation: In my opinion, it is tlo\vnrif;lit hopeless, for designed for clinical medicine of the human, instance to draw conclusions about the normal origin and the relationships of the various blood cell forms from today's the effort did not succeed aiul one cannot avoid bewildering mass of separate unsystematic observations of the conclusion that any satisfactory universal pathologically altered blood of adults in various leukemias, for terminologv should be broad enough to include example: but this is actually done today by many students.

8 all vertebrates and, if possible, all theories of view of blast cells in mammals cannot be applied hematopoiesis. as readily to avian blood because, as seen by Table 2 shows the lineages of cell lines as seen the smear method, cytologic differences do exist in the birds. For eacli line, there is listed at the among the blast cells. top of the column a primordial stem cell or blast All schools of hematology recognize the fact cell. In human hematology, blast cells are pic- that if the genealogy of cell types is carried far tured and described as containing one or more enough back into the embryology of the organism, nucleoli that appear as pale blue homogeneous there will be a common cell for all blood-cell bodies following the commonly used blood stains. types. The point of controversy is not on this In avian blood, no more than one nucleolus has matter but on whether a blast cell of a particular been seen in any cell line except in the primary cell line is a fixed type, incapable of differentia- generation of embryonic erythrocytes, and nu- tion under stimulus into other cell types. An- cleoli are absent from granuloblasts, lympho- swers to such questions must come from experi-

blasts, and probably monoblasts, at least by the mentation and, as far as the present study is same technics that have revealed them in human concerned, there is no evidence that blood cells blood. have or have not any potentiality beyond the par- Erythroblasts, thromboblasts, lymphoblasts, ticular line that they represent. granuloblasts, and primordial osteogenic cells of On the basis of what has been observed in birds are somewhat more easily distinguished the chicken, its embryology, and its hematopoietic than in the human species. Each represents the organs, a blast cell may be defined as the earliest earliest recognizable member of its respective recognizable cell belonging to a particular cell line, and although some do not show nucleoli, type, and all following stages observed consist of each is believed to be equivalent functionally to progressive steps toward the mature cell. To this

the same blast cell by the same name in the hu- should be added the observation that if, antece- man series. The important point is that in many dent to the blast cell, there does exist a totipotent cases they can be separated on the basis of their cell type other than mesenchyme, reticular, and structure, even when isolated from the other cells possibly connective and endothelial tissues, it has on the slide. This does not alway hold true in not been found in this study. It should be added, the human field, and in the atlas by Osgood and also, that the terminology presented in table 2 is Ashworth (1937) it is stated (p. 36), "In the based on the assumption that the usual medium authors opinion [Osgood], the individual and small lymphocyte found in circulating blood granuloblast (myeloblast) is morphologically in- is a mature cell, on a par with all other fully distinguishaljle from the most immature lympho- differentiated mature cells found in the circulat- blast, monoblast, plasmoblast, or karyoblast ing blood, and that it is not a totipotent primor- (megaloblast). The differentiation of the type dial cell capable, at least in the course of normal of cell has to be made by identification of the hematopoiesis, of producing all other blood-cell cells found associated with the stem cell under types. In normal blood only two types of mature consideration. A stem cell found in association lymphocytes exist, medium and small; the large with a progranulocyte (promyelocyte) is classed lymphocyte is not a mature cell but is an imma- as a granuloblast." (References to figures have ture cell usually standing early in the lineage of been omitted in the quotation just given.) It is the cell line to which it belongs, which may not this type of dependence upon the presence of necessarily be the lymphocyte line.^ more highly differentiated cells in the same field More than one erythrocyte series exists in the for exact placement of the different blast cells life of the embryo up to hatching. There is a pri- that has led the unitarian hematologist to sug- mary series representing the first generation of gest that if blast cells all look alike and are all red cells in the embryo; then follow several characterized by a narrow rim of ]:)lue-staining generations, each less precocious in its hemo- cytoplasm around the nucleus, and the nucleus globin formation than the preceding one and, contains a nucleolus, it is just as logical to say "For further clarification tliat there is a single common stem cell capable of viewpoint see: Lucas, A. M. 1959. A discussion of synonymy in avian and of differentiation into any blood cell type. As mammalian hematologic nomenclature. Amer. Jour. Vet. Res. already mentioned, the criticism leveled at this 78:887-897. V S finally, the definitive cell line arises. Thrombo- THROMBOCYTE SERIES cyte stages of development in the embryo differ Thromboblast.—A cell with basophilic cytoplasm morphologically also from those found in the de- that is amoeboid in the early embryonic generations. finitive stages. Therefore, to cover all these vari- The cytosome forms a narrow rim around the nucleus. ations, definitions must be broad. Spaces are present in the cytosome. It has a round nucleus with a plasmosome nucleolus that may be masked by the density of chromatin granules. The ERYTHROCYTE SERIES chromatin tends to he punctate in contrast to that of the erythroblast. where it is more angular, merging Erythroblast.—A large cell with more cytoplasm in into the linin network. relation to the nucleus than in most blast cells. The Thrombocytes appear to be functional in the embryo cytoplasm of the primary generation is strongly baso- from an early stage and hence probably should not be philic, but in the definitive cell line it stains less in- called immature; thus, in table 2 two columns of terms tensely. The cytosonie shows mitochondrial spaces are given for the thrombocytes—one for the embryo and may have an amoeboid shape. The nucleus is an and one for the bone marrow of the adult and circu- open, coarse network with chromatin that is clumped lating blood. more than usually found in other blast cells. The plasmosome nucleolus is large and more conspicuous than Embryo Thrombocytes in the thromboblast. Large embryo thrombocyte.—This is a large cell with Early polychromatic erythrocyte.—A smaller cell a moderate amount of cytoplasm around the nucleus. than the blast stage, and the cytosome is rounded. The cytosome shows partial to nearlv complete vacu- Mitochondrial spaces are largely replaced by a more olization and stains more lightly than in the preceding homogeneous cytojilasm. The cytosome has a strong stage. The nucleus has large irregular chromatin basophilic color. The chromatin of the nucleus is defi- cluni|)s and no visible nucleolus. nitely clumped. The nucleolus is smaller than in the blast stage but is often visible. Medium embryo thrombocyte.—A cell of medium size with about the same nucleocell ratio as before. Mid-polychromatic erythrocyte.—This cell is usually The cytosome is fully vacuolated and S|)ecific granules smaller than in the preceding stage, of rounded shape, sometimes are present. Early degeneration is marked and has a gray cytoplasmic color that ranges from by pinkish coloration and by crumpling of the borders nearly basophilic to slightly eosinophilic. No mito- of the c)tosome. The nucleus is pachychroniatic. chondrial spaces are visible but the cytosome often shows an artifact of fractured spaces usually concentric Small embryo thrombocyte.—A small cell of which to the cell perimeter. The nucleus is small relative to the cytosonie is highly vacuolated and pale staining. the cytoplasm. It is round and has a clumped chro- The nucleus is dense and individual chromatin clumps matin pattern. No nucleolus is seen in the definitive are fairly distinct. Nearly all cells of this type, as cells. seen in the smear of embryo blood, have lost most of the cytosome and are clumjied. Late polychromatic erythrocyte.—This cell is round to slightly oval. Staining of the cytoplasm varies from Definitive Thrombocytes an eosinophilic gray color to a pale eosin and. in the Early immature thrombocyte. large cell, often older cells, to a moderate eosin color. Fracture arti- —A not round, and with the nucleocell ratio less than in facts tend to appear in tlie younger forms of this stage. the preceding stage. The cytoplasm is basophilic but The luicleus is round to slightly oval and there is ir- is presence regular clumping of the chromatin. the overall color lightened by the of vacuoles. The nucleolus may still be visible and the chromatin is aggregated into clumps of nonuniform Reticulocyte.—Without special stain, this cell ap- size. pears as a late polychromatic erythrocyte with almost full development of hemoglobin or even as a mature Mid-immature thrombocyte. cell of medium size erythrocyte. A reticulocyte stain reveals the presence —A that is often elongated slightly or has an irregular of granules in the cytosonie. concentric to the nucleus. shape. The cytoplasm is vacuolated and stains a light The basophilic granules characteristic of a ty])ical re- blue color. Specific granules may be present. The ticulocyte may be found at earlier stages of develop- chromatin is clumjied extensively but linin network ment, after the appearance of hemoglobin in the cell. is still visible.

Mature erythrocyte.—This cell has an oval shape and a cytosome of uniform texture, colored a reddish Late immature thrombocyte.—This cell is elongated orange. The nucleus is elongated, oval, and sometimes but the nucleus fills up a larger proportion of it than rodlike with rounded ends. The nucleus is lepto- at the mature stage. Clumps of chromatin are still chromatic to pachychroniatic. Aged cells have dense clearly visible. Specific cytoplasmic granules are gen- homogeneous or nearlv structureless nuclei. erally present.

11 SERIES Mature thrombocyte.—The mature definitive throm- HETEROPHIL shape and is slightly smaller than bocyte has an ovoid Granuloblast.—A large round cell with a narrow rim cytosome takes on a pale the mature erythrocyte. The of cytoplasm around the nucleus. The cytosome stains blood stains. The intensity of blue color with most an intense blue color and is only slighdy interrupted presence of vaguely defined rare- color is varied by the by light-staining areas. The chromatin forms a reticu- fied areas. lum more delicate than for any other blast cell. The rather than elongated The nucleus is usually round smear method would indicate that a nucleolus is absent. The chromatin is as in the mature erythrocytes. The morphology of the granuloblast is the same for are closely packed, which clum])ed and the clumps heterophils, eosinophils, and basophils. equal to or gives to the nucleus an effect of density lymphocyte. greater than that of the small Metagranuloblast.—The cytoplasm on the side op- granules, having a weak affin- One or more specific posite the eccentrically placed nucleus is vacuolated visible in most mature ity for acidophilic dyes, are with spaces of approximately equal size. The nuclear at one end thrombocytes. These granules usually lie chromatin may be aggregated slightly, but more often of the cell. the chromatin remains in the form of a delicate reticulum, and the boundary between the nucleus and cytoplasm becomes indistinct. At this stage no granules LYMPHOCYTE SERIES characteristic of this cell line have appeared.

Lymphoblast.—This is a large round cell with a Promyelocyte.—This stage also precedes the appear- narrow rim of cytoplasm that stains dark blue but may ance of specific granules. The boundary of the nucleus contain colorless spaces. The chromatin forms a deli- often becomes indistinct and in the cytosome there ap- reticular pattern. No nucleolus is visible. cate pear dark-stained magenta granules and rings. The magenta bodies are highly characteristic for the hetero- Immature lymphocyte.—General appearance the phil but are not specific for it. Orange-stained spheres as in the blast stage, except that the chromatin same present in the vacuolated cytoplasm and these are smaller. are of the nucleus shows clumping and the cell is the precursors of the definitive rods.

lymphocvte.—KucleoceW ratio is similar to Mature From the precursor orange spheres nucleus Mesomyelocyte.— the' blast cell. The chromatin of the that of come the definitive rods. Cells with less than half the clumped but not always in massive blocks is definitely number of definitive rods are included in this stage. the small mature lymphocyte. The separation even in Magenta rings and granules may still be present. The arbitrary. into "medium" and "small" is purely of size nuclear boundary often is still vague. In some cells there is evidence of nuclear condensation.

MONOCYTE SERIES Metamyelocyte.—This cell usually is smaller than the preceding one. The cytosome contains more than half Monoblast.—Thus far the monoblast stage has not the normal complement of rods. The chromatin of definitely identified. been the nucleus is condensed and in older stages the nucleus may have a bean shape. The nucleus begins to show monocyte. The cytoplasm of this Early immature — staining refractiveness characteristic of adult cells. basophilic cell stains a clear blue color with or without The granules. Azurophilic granules may be present. Mature heterophil.—The cell contains a full comple- nucleus has a granularity on a reticulum quite similar ment of rods and the nuclear lobes may vary from 1 to that of the mature cell. The cytosome is large rela- to 5 or more. The chromatin of the nucleus is aggre- tive to the nucleus. gated into clumps.

Late immature monocyte.—The cytoplasm often shows a basophilic granulation, or reticulum, and EOSINOPHIL SERIES azurophilic bodies may be present. The nucleus is Granuloblast.—Morphologically this cell is the same round and has an eccentric position in the cell and as described for the heterophil series. a Hof may be present. Metagranuloblast.—The nucleus is eccentric and this cytosome is large relative Mature monocyte.—The position produces a crescent of cytoplasm at one side. to the nucleus. The cytoplasm has a reticular struc- The vacuoles are more uniform and more sharply de- ture and contains azurophilic substances, either on the fined than in the heterophil at this stage. The nuclear reticulum or as discrete granules. The nucleus usually structure is more definite than in the heterophil, and

is indented and adjacent to it is a Hof area with spheres chromatin clumps are larger. faintly stained an orange color present in the vacuoles. defined as the one in The chromatin is usually a delicate reticulum but may Promyelocyte.—This stage is produces the de- be composed of coarse blocks. which only precursor substance that

12 finitive specific granule is present, but in immature oles. There are relatively few mitochondrial spaces. eosinophils, all degrees of development from the pre- The cytosome is large relative to the nucleus. The cursor substance to the definitive granule may be nuclear chromatin is finely punctate and a nucleolus found. Therefore, this stage overlaps the next, and is present that stains light blue. the term "mesomyelocyte" has been used to cover both stages. Osteocyte Series

Mesomyelocyte.—Definitive granules arise at this Immature osteoblast.—The nucleus is placed eccen- stage, and all cells are included under this term that trically. Large and definite mitochondrial spaces are have less than half the usual number found in the adult present. Usually there is a clear area on the side cell. The nucleus is more definite and chromatin more of the nucleus toward the center of the cell. Clear condensed than in the heterophil. spherical vacuoles are present in the cytosome. The nucleus stains darkly with uniform granulation, and Metamyelocyte.—This is an immature myelocyte with usually one blue-stained nucleolus is present. more than half the number of specific granules. The nucleus may be round, indented, or constricted, and its Mature osteoblast.—A darkly stained cell with well chromatin clumped irregularly. developed, clear areas adjacent to the nucleus. All parts of the cytosome are filled with mitochondrial Mature eosinophil.—Contains a full complement of spaces. The nucleus has a pattern of granular chrom- specific granules, and the number of lobes in the nu- atin, and a nucleolus is present. cleus may vary from one to five. The chromatin clumps are close together. Osteocyte.—This cell has not been seen in smear preparations of bone marrow since it is embedded BASOPHIL SERIES within the substance of the bone.

Granuloblast.—Morphologically this cell is the same Osteoclast Series as described for the heterophil series. Mononuclear osteoclast.—This cell closely resembles Metagranuloblast.—This stage is present but. concur- the primordial osteogenic cell. The nucleus may be in rently with the characteristic vacuolization of the cyto- the center of a round cell or at one end of an elongated some, the magenta granules appear also, and thus the cell. It contains a mixture of delicate vacuoles com- term "promyelocyte" has been used for the two stages posed of mitochondrial spaces and eosinophilic accum- combined. ulations. Also present are some clear vacuoles with a definite spherical shape. The nucleus is composed of Promyelocyte.—Scattered magenta bodies are pres- particulate granules on a linin network and beneath ent. They are closely similar to those of the heterophil, this a nucleolus is visible. except that there is less tendency to form rings. If rings are present, they are usually small. Vacuoles are Muhinuclear osteoclast.—This is the only giant cell present in the cytoplasm but they are less uniform in of the avian bone marrow. The cytosome contains size than for the other two preceding granulocytes. basophilic and sometimes eosino])hilic substances, and The nucleus is eccentric and has a coarse chromatin the latter usually are concentrated in the central part pattern. of the cell. Many vacuoles and granules and sometimes irregularly shaped bodies are present in the Mesomyelocyte.—This cell is smaller than the pre- cytosome. The borders of the cell are poorly defined. ceding one. The nucleus usually is not in the center The nucleus is large and round; it contains relatively but is not completely eccentric. The cytosome confine, punctate granules; a single nucleolus is present. tains less than half the number of granules found in the adult cell. The water solubility of the granules PLASMACYTE SERIES is a confusing factor in estimating number. Plasmablast.—This cell may be a primordial osteo- Metamyelocyte.—This cell contains more than half genic cell or a reticular type cell. It has not been identified the total number of basophil granules. The nucleus is thus far. near the center and is composed of a dense chromatin network. Early immature plasmacyte.—The ratio of cytosomal to nuclear size is about the same as in the monocyte. Mature basophil.—This cell contains a full comple- The cytosome contains vacuoles and mitochondrial ment of granules. The nucleus is a single body in spaces, and the ground substance stains a clear blue most cases, but occasionally it is divided into two lobes. that is more transparent than the basophilic cytoplasm of most other young cells. OSTEOGENIC CELLS Late immature plasmacyte.—The cytoplasm in this Primordial osteogenic cell.—A large amoebid cell cell is colored the same shade of blue as in the pre- with lightly stained cytoplasm containing clear vacu- ceding stage, but there are fewer mitochondrial spaces

13 helpfid adjunct to cellular morphol- and more vacuoles. The nucleus is small in relation Size is a stained. There is a cells into types and to the cell area, and is darkly ogy for the categorizing of side of the nucleus. clear area in the cytosome at the into stages of development, but if one takes away color, form, and internal structure, then size plasmacyte.—The cell may vary in size from Mature tool for cell contains numerous alone becomes a rather inadequate large to small, and the cytosome nucleus is especially true of cells granules, ranging from large to small. The identification. This is located which contains condensed blocks of chromatin and prepared for study by the smear method, clear area adjacent to the against the cell wall. A flattens them as broadly as their membranes will present in most mature plasmacytes. nucleus is permit. Smears from bone marrow and other hematopoietic organs showed this quite clearly: The definitions that have heen presented are the smear was thick, the cells cell type, in an area where in reality brief descriptions of each were smaller, but at the edges of a group of cells and the use of these, combined with the illustra- in many cases, were together they were larger and, tions, should make it possible to put stretched to the breaking point in drying. In the quickly the necessary facts for identification of wet-fixed smear the cells are luuch smaller than any mature or immature cell without extensive in the dried smear and they often appear reading. shrunken. Compare, for example, the size of Brief mention of some terms used in general basophils that have been fixed by drying in air cytology may be helpful. The cell is composed (fiijs. 385-387) with similar cells fixed in methyl of two main parts—nucleus and cytosome; the ak^ohol (figs. 388-390). former contains nucleoplasm and the latter, cyto- Most often the visual impression of size as plasm. If a large dense mass of basichromatin seen microscopically forms the basis for the lies within the nucleus, it is called a karyosome statement that a cell is large, or medium, or small or karyosome nucleolus, and if the mass or sphere and, only rarely, is the filar micrometer taken takes acidophilic dyes, it is called a plasmosome out of the box and actual measurements of size or plasmosome nucleolus, and if both karyosome made. True, actual measurements should be plasruosome are present, the two form an amphi- taken more often than they are, but the fact re- nucleolus. mains that we depend to a great extent on mental Wherever color is mentioned, it is understood impressions for a comparative estimate of the size the cir- that Wright's stain was used on smears of of objects. Therefore, in order to avoid con- culating blood of the hatched chicken, and May- fusion in making these impressions from the il- Giemsa was used for immature cells Griinwald only two magnifications have been organs of lustrations, found in embryos and in hematopoietic high ^igeJ—one called low power and the other, both embryo and hatched chick. Any exceptions power. Two scales have been constructed (fig. noted in the legends or text. to this have been to the 1, D and E). One, D, is equivalent These two stains give closely similar colors on measurements in microns at a magnification of the same cell, but the latter produces a somewhat 1370 X and the other, E, at 2470 X- With these more intense coloration. scales the size of any cell or its part can be estimated fairly closely, since all cells, both at low and high magnification, were drawn carefully MAGNIFICATION with a camera lucida. The low-power drawings were made at an op- measureiuent of lilood cells is an im- magnification The tical magnification of 400 X and a on eryth- portant field of study, and especially so when projected on drawing paper of 91 3.3 X. and are rocytes, which have a definite shape The high-power drawings were made at an opti- of held by a firm stroma. Some measurements cal magnification of 1125X and a projected mag- length and of width of elongated cells and of nification of 2470 X. By optical magnification ir- obtained when the diameters of circular cells and of areas of is meant the theoretical value multiplied by regularly shaped cells have been undertaken in magnification of the objective is magnification of the eyepiece. The low- this study. Not as much emphasis has been the drawings were made by using a 20X ob- put on cell size as in some atlases on human power jective and a ocular, and the high-power blood. 20X

14 drawings were made with a objective and which 90X diere is a relatively even distribution of 12.5 ocular. X During the engraving process, cells is obtained from all hatched chickens, ex- all the low-power drawings thai originally were cept laying hens, as shown in figure 1 F. The approximately 3 x 3% inches were increased in scratches and abraded spots that often come when size 50 percent, so that, as presented here, they the slides are blotted or handled roughly are are 4^/2 x SWie inches or slightly more. There- nicluded. The appearance of a smear made from fore, in the low-power drawings, the cells are the blood of a laying hen is shown shown in figure 1 G; at about 1370 times their natural size. the fat globules in the senmi spread when the The purpose of the low-power drawings is to smear was made and pushed the cells of the un- give the overall impression that one has when dried layer aside. This, however, looking disturbed through the microscope and ojjsei-ving only slightly the uniformity of distribution of different kinds of cells in the same field. Under cells and did not cause certain cell types such to conditions one can distinguish minute dif- segregate. ferences in color, tone, or texture that often van- ish when single cells are removed from the environment of other cells, even when they have ARRANGEMENT OF SUBJECT been drawn at a much higher magnification. The MATTER low-power drawings serve another purpose in that It is often helpful to the reader they of a scientific contain about four times as many cells as liook if the writer reviews briefly the general plan are represented in the high-power drawings; of organization and what was in his mind when therefore, the same cell type shown at high mag- seemingly unrelated things sometimes nification were can be presented sufficiently often so placed beside each other. In this study, the that deviations list from the typical are fully illus- of chapter headings reflects the scope and se- trated. If all these variations were presented as cpience of the fields covered. high-power drawings the Atlas would be unduly More emphasis has been placed on the large. subject matter of the second chapter than on any other. An outline of the individual cells of each low- It contains almost as many power drawings as do all drawing is given on the facing page along other chapters combined. The purpose of this witli the legend. Identification of the cells in the emphasis is to give as nmch help as possible to field is made by numbers placed on or near the the field worker in poultry diseases, whose first cells of the outline drawing. consideration when confronted with an Blood unknown from mammals often spreads unevenly condition is to arrive as quickly as possible at a over the slide when the smear is made and in par- preliminary diagnosis. ticular shows a clumping of platelets and an The four categories into which the study of aggregation of leukocytes along the margins of each cell type is grouped is best exemplified in the preparation. In order to reduce this tend- the erythrocyte series from the ency circulating blood as much as possible, the mammalian hema- of the hatched chicken. These series fall into tologist has often used coverglass smears. This the following classifications: (1) Normal mature has not been necessary for preparations of avian cells, (2) normal immature forms found blood in cir- because the cells in the average well-made culating blood, (3) abnoi-mal cells, and arti- smear do not (4) segregate, there is no clumping of facts. Sometimes the variety of immature cells thrombocytes, and the erythrocytes do not rear- found in circulating blood is so great that these range themselves in rouleaux formation. The cells appear to represent the complete develop- simplest and easiest method is to place a small mental series, but they were included under the drop of blood on the end of a "pusher" slide and circulating blood to indicate the range of cell to touch this to one end of another slide where it types that might be picked up in a general ex- is held for a moment—long enough for the drop amination. In the study of embryo circulating to spread to each edge of the pusher slide. The blood and blood from hematopoietic organs, latter slide is then steadily and rather quickly these cells are shown in their proper setting in slid to the opposite end of the "smear" slide at the form of a complete series. This may appear an angle of about 45 degrees. to be a duplication of effort, but it has proved The appearance of the typical blood smear in to be of great help in the exact characterization

15 are because, amples showing abnormalities and artifacts of a cell type or stage of development they much fewer in the chapters on embryo blood and as already mentioned, immature cells when have on bone marrow cells than in chapter 2. are carried by the circulating blood often Chapters 3, 4, and 5 have much in common appearance that is different from that pre- an deal the in regard to their subject matter; they all sented when they are in the environment of with cells during development. This is true, organ from which they came. whether the cells were collected from functional A distinction has been made between abnor- or from various is a third circulating blood of the embryo mal cells and artifacts; actually there hematopoietic organs of embryo or adult. category, namely, variations from the typically descrip- Chapter 6 is devoted primarily to a normal, and to place each atypical cell in the tion of blood cells of avian species other than correct one of these three categories has taken the chicken. It was soon discovered that sim- far more study and experimentation than find- ilarities in the morphology of blood cells of ing, illustrating, and describing the "typical" greater than error is different species were much cells. Likewise, the possibility of differences. Included also in this chapter is tab- greater, so that future research may well discover ular material on cell sizes and cell counts. that what has been called abnormal is, in reality, A chapter on technic was included at the end an artifact, or merely a variation of the normal. that seem- that of the volume. Sometimes it happens Cells are called abnormal on the assumption ing differences in cell morphology can be traced the abnormality in them was present within the the use of different technics by different inves- body of the bird, and artifacts are considered to to tigators. It was hoped that this difficulty might be deviations from the normal that presumably be avoided if the technics used in this study were caused in the process of taking blood, or in addition to so much were set forth. Many methods drying it, or applying the stain. Since blood those discussed here have been applied to avian less is known of the cytology of immature blood studies. cells than of the mature types, the series of ex-

16 CHAPTER 2

Circiilatino Blood of the Hatched Chicken

ERYTHROCYTES them, or they may be eccentrically placed (figs. 2,

9 and 3, 9) ; the nuclei may not be in the center Normal mature erythrocytes (figs. 4—8) of the cells and may be blunt at one end and pointed at the other. The "typical" erythrocyte of birds has often Most conspicuous of all are the indentations l)een described as an oval cell with an oval nu- (figs. 2, 11, 12 and 3, 11, 12), constrictions cleus (Goodall, 1909; Foot, 1913; Magath and (fig. 2, 13), and protrusions (figs. 2, 14 and Higgins, 1934; and many others). Forkner 3, 14). Even duplications of the nuclei (figs. 7, 8, and may (1929) has described in detail its appearance 29) be found. As far as can be determined, these are all in vital stained preparations. The nucleus is normal cells and in spite of their multiplicity not quite concentric with the contour of the cell; of shape they are all instantly recognizable there is a wider margin at the poles of the cell as mature erythrocytes, because the than at the sides. The cytoplasm takes an orange hemoglobin gives to the cytosome a strong pink color with Wright's stain and with May- affinity for acid dyes and a nearly homogeneous texture. In Griinwald Giemsa gives a distinctly more red- some cases a narrow rim of cytosome around tlie nucleus stains dish color. The nucleus stains intensely but re- lighter than the more peripheral part, but veals a pattern of chromatin clumps more or less this is probably an artifact that developed uniforndy distributed. If tlie nucleus has an when the cell was flattened oval shape, there are no massive chromatin in the process of making the smear. This perimiclear space is in clumps. If the nucleus is contracted to an elon- shown figure 2 but not ni figure 3. The clear space, gated rod-shaped structure, dense clumps of as suggested, may arise as an artifact but its occurrence in chromatin are usually present. A nucleolus is one absent. smear and not in another may be worthy of further study. A perinuclear space appears Low-power views are presented in figures 2 in all types of Idood cells, except the and 3. The slide from which figure 2 was made heterophil, when the smear has lieen fixed in Petrunkevitch came from the flock of Single Comb Wliite Leg- No. 2, and stained in May-Griinwald liorn chickens maintained at this Laboratory, Giemsa (figs. 198-202, 215, and 221). Following this tech- and figure 3 was drawn from a set of 25 slides nic the nucleus of the erythrocyte obtained from the same breed at another location, appears to be almost a solid chromatin mass. which for convenience has been designated as Suggestions concerning the origin Laboratory No. 2. Thus, even in these two of multipolar and giant erythrocytes and leukocytes samples, difl:erences can be observed and prob- in man have been given by Schwarz ably could be extended if a careful study were (1946). He be- lieves these conditions can made of blood from many sources. be traced back to multinuclear conditions in the immature stages. A typical cell is shown in figure 4. It was Certain types of variability have necessary to do considerable searching to find significance. Among the 25 blood this "typical" cell. Examples are shown also smears received from Lab- oratory No. in figures 2, 7 and 3, 7. All the other cells de- 2 there were several in which the viate from it in shape of nucleus or cytosome or nuclei of the erythrocytes were longer and nar- both. The cells may be too round or too elon- rower (figs. 3, 8, and 5) than any found in smears gate or irregular (fig. 3, 5) . The nuclei may be from our flock. Also, the chromatin was more too large or too small for the cytosome (figs. 2, condensed and more heavily stained. The sig- 10 and the 3, 10) ; long axis of the nuclei may nificance is not known but the same type of eryth- not coincide with those of the cells that contain rocyte has been observed in some of the smears

17 blood of thebasilic (eubUal) vein of a Singk Comb 2.-Appcai-ance of a typical smear made from the FiGUHK smeai ^^as isolation, ^^•as 2,095 days ot age when the ^Yhite Lejiorn chicken. This bird, raised in taken. 1,370X- placed nuclei. 9 Erythrocytes with eccentrically 1 Heterophil. 10 Round undersized nucleus. 2, 3 Lymphocytes. end. 11 Erythrocyte nuclei indented at one 4, 5 Thrombocytes. 12 Erythrocyte nuclei indented on one side. G Squashed erythrocyte nucleus. 13 Erythrocyte with constricted nucleus. 7 Typical erythrocytes. 14 Erythrocyte nuclei with protrusions. 8 Erythrocytes smaller than average.

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Leghorn. The cause of the Figure 3.—Appearance of an atypical smear from a Single Comb White is not known. chromophobic bands in the nuclei of heterophils and of erythrocytes 1,370 X.

Erythrocyte in which the nucleus is undersized Heterophil showing chromophobic bands across 10 almost round. the nuclear lobes. and with nucleus indented at one end. Erythroplastid. 11 Erythrocyte with nucleus indented on one side. Late polychromatic erythrocytes. 12 Erythrocyte Erythrocyte with a pyknotic nucleus. Erythrocytes in which the distorted shapes may 13 nucleus with a protrusion. have been acquired during the making of the smear. 14 Erythrocyte Erythrocytes showing chromophobic bands across A "typical" erythrocyte. 15-22 Erythrocyte with an elongated, dense nucleus. the nuclei. Erythrocyte with nucleus eccentrically placed.

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22 from wild birds. In starling blood, for example, relative merit of each type in relation to the the condensed chromatin beneath the nuclear general vigor of the bird. This is a point of memlnane produces a bumpy contour like that more than academic interest. One hypothesis of a mulberry. A narrow condensed nucleus is might be that a smaller, more condensed nu- found also in the common mallard duck. In cleus permits the existence of a proportionally fact, in this species cells showing the two nuclear larger cytosome and hence a greater content of types are found in the same smears. For con- hemoglobin. Another hypothesis stems from venience, one (fig. 4) may be designated as the the fact that an oval leptochromatic nucleus is ovoid, leptochromatic type, and the other as characteristic of immature cells and a conden- the elongated, pachychromatic type (fig. 5). sation of chromatin is associated with older cells. Breusch (1928) makes the statement 224), (p. The condition in tlie common mallard duck of a ". . . Allgemein kann man weiterhin feststel- mixture of these two types of erythrocytes is len, dasz je junger die orthochromatische Zelle some evidence for this hypothesis. Therefore, is, um so mehr bezitzt der Kern Blaschenform. it is possible that in our Laboratory stock the ." ' . . However, no data to support this opin- erythrocytes show a type of ion are presented. nucleus not fully developed because the cells are Studies are needed on blood physiology and destroyed before they reach full maturity. chemistry that would aid in the evaluation of the The existence of amitosis in mature erythro- 'Translation: In general one may state that the younger the cytes has been suggested in amphibians (Charip- orthochromatic cell is, the more its nucleus has the vesicular form. per and Dawson, 1928). Up to a certain point

Figures 4-28.—Normal erythrocytes—immature, mature, and aged—found in the circulating blood of the hatched chicken. 2,470 X.

Figures 4-8: Typical mature erythrocytes and variations. 17 An atypical late polychromatic erythrocyte. 18 Late polychromatic erythrocyte. This and figure 4 Mature erythrocyte, typical for otir Laboratory 16 are typical of cells at this stage. stock. Niicleu.s—oval leptochromatic type. 19 Mature erythrocyte. Nucleus and stroma differen- 5 Mature erythrocyte, typical for some other stocks tiated ahead of changes in cell shape, and thus the and breeds of chickens, for other domesticated round shape is fi.xed. A similar reaction is often birds, and for wild birds. Nucleus—elongated, found in first generation of erythrocytes in the em- pachychromatic type. bryo. 6 Normal erythrocyte with nucleus indented at the end; similar to figure 2, 11. Figures 20-23: Reticulocytes from the circulating blood of 7, 8 Mature erythrocytes with two nuclei. The cell in 1-day-old chicks. figure 8 shows that the two nuclei may be completely separated. 20 Early stage of reticuloycte; granules are abundant. 21 Same stage as preceding cell. The color obtained with Wright's counterstain indicates that reticulo- Figures 9-23: Immature cells. cytes have full complement of hemoglobin. fl An erythroblast, equivalent to that in figure 25.5, 22 Partial loss of reticulocyte granules.

which is from the embryo. 23 Granular material is minimal in amount but is suffi- 10 A late erythroblast, equivalent to, but smaller than, cient to establish the cell as a reticulocyte. Smaller that in figure 347, which is from the bone marrow. amounts are confused with precipitated stain. 11 Early poh'chromatic erythrocyte, equivalent to those in figures 264 and 348, which are from the Figures 24, 25: Mature erythrocytes. embryo and the bone marrow, respectively. 24 The type characteristic of Laboratory stock. 12 Early polychromatic erythrocyte. A few lightly 25 A cell approaching senility in Laboratory stock but stained spaces characteristic of the erythroblast are typical for most other stocks of chickens and for still visible in the cytoplasm. most other birds. Compare with figures 2 and 3. 13 Mid-polychromatic erythrocyte, equivalent to those in figures 350 and 351, which are from the bone Figures 26-28: Aged erythrocytes. marrow. 14 Mid-polychromatic erythrocyte. 26 Aged erythrocyte, undergoing p3'knosis. 15 Transition between mid- and late-polychromatic 27 Aged erythrocyte with vacuolization of the nucleus. erythrocyte, but included with the latter type. 28 Aged erythrocyte showing continuity between intra- 16 Late polychromatic erythrocyte. nuclear vacuole and cytosome.

23 necessary, but birds, such as cytes, considerable searching is there is some evidence for it in even erytliroblasts so immature that they might figures 2, 12 and 13; 3, 12; and 6, 7, shown in found rather be classed as large lymphocytes have been 8, 17, and 29. Some indentations extend (figs. 9 and 10). far into the nucleus and sometimes there are two Since blast cells of various sorts and poten- completely separate nuclei in the cell. Figure have close morphologic similarity among 29 demonstrates fairly clearly that a cell with tialities themselves and in turn resemble what has been two nuclei such as shown in figures 7 and 8 can called a large lymphocyte, it seemed best as far be derived by constriction of a single nucleus as circulating blood is concerned to discuss them and not by mitosis in which the cytosome failed and four ex- in the under the subject of lymphocytes, to divide. Figure 17 is an early stage amples have been illustrated (figs. 121-124). process of nuclear constriction. Cells A and B Some differential counts given in the literature of figure 29 were from chickens used in an irradi- that large lymphocytes were pres- experiment. The history of the birds that would indicate ation This ent to the extent of 1 percent and over. furnished these cells is given in the legend. be a different cell from the one described Neither of these two cells can be considered nor- must here since the "large lymphocytes" observed in mal, but in B the cytosome is partly divided and these studies occurred so infrequently that they even though abor-tive, as it obviously is in this would not be included in a differential count. cell, fulfills some of the criteria for amitosis. Further amplification of the point will be made Charipper and Dawson (1928) believed that later 50), but figure 121 has the character- erythrocytes of amphibians, which showed (p. the thus istics that identify it as an erythroblast and the same range of morpliological variations found of develop- occur- could be included among the series here in chickens, offered evidence for the mental stages shown in figures 9-23. A dis- rence of amitosis. As already discussed, cells cussion of other structural features that distin- with two nuclei or cells with constricted cyto- blast cells is in guish an erythroblast from other somes (figs. 34, 35, and 36) may be found given on page 9. avian blood, but the same two processes have When development has reached the stage of not yet been found in the same cell except in an early polychromatic erythrocyte, there is no figure 29 B and in a primary erythrocyte (fig. confusion with other cells, and typical ex- The idea of amitosis would be more longer 246). Mito- in amples are shown in figures 11 and 12. convincing if one could find series of stages chondrial spaces may or may not be present in which the nucleus was first involved and divided develop- the cytosome, and cells at this stage of into halves and each half moved to opposite poles ment have not as yet acquired the homogeneity when the cytosome divided. Tlie point will be cytoplasm that comes later. Sometimes the discussed again when artifacts are considered. of eryth- cytoplasm is vacuolar as shown in basophil rocytes from bone marrow (figs. 348 and 349). a In figure 11 there is a faint suggestion of in circulating Developmental stages found nucleolus near the lower nuclear margin. The blood (figs. 9-28) term "erytlnoblast" has been reserved for the where a nucleolus so early stages of development Cells more immature than reticulocytes are red-cell line the nucleolus normal, is present, yet in the rare in the circulating blood of the be visible at the early polychromatic that the presence of even an may still healthy mammal of the em- stage and in the primary generation occasional one in a smear is suggestive of a the mid- and late stages. The avian blood the pres- bryo, even to padiological condition. In been early polychromatic erythrocyte often has ence of immature erythrocytes is common and basophil erythroblast, but actually some know now, an occasional immature called a as far as we acquired at this Im- hemoglobin has already been cell does not indicate a blood dysfunction. stage mature stages are found in birds of all ages, and The polychromatic phases of erythrocyte de- three birds that were over 5 years of age con- velopment are represented by cells in which the tributed examples of polychromatic erythrocytes possesses an affinity for both baso- figures presented here. If one cytoplasm in the series of propor- philic and acidophilic dyes in various seeks stages earlier than polychromatic erythro-

24 tions. Thus, there be may found a complete The classification of stages in the development range of color from a basophilic cytoplasm with of erythrocytes on the basis of hemoglobin con- a trace of hemoglobin (fig. 11) to a cell that tent has not found general acceptance by hemo- has a high hemoglobin content and only a trace tologists working on human blood, and the point of basophilia (figs. 16-18). The early poly- is illustrated by Osgood's (1938) statement (p. chromatic erythrocyte is characterized by a blue 67), "However, it seems to the author unjusti- cytosome, tlie mid-polychromatic erythrocyte by fiable to use die amount of hemoglobin in the a gray coloration, and the late stage by various cytoplasm as the criterion of the age of the indi- tints of orange. A mixture of blue and orange vidual cell since many polychromatophilic akary- produces gray, and in some cases the yellow por- ocytes (nonnucleated red cells) are seen which tion of eosin mixed with blue adds a slightly contain practically no hemoglobin and these must greenish tinge. With a shift from a predomi- certainly be more mature than nucleated red nantly basophilic to predominantly acidophilic cells which contain much hemoglobin. If one cytoplasm there is an accompanying progres- uses the nucleus alone, however, as the criterion sion of changes involving cytoplasmic texture, of the maturity of the cell, one can arrange a nuclear structure, nucleocytoplasmic ratio, and continuous series, each one differing from the cell shape. Each cell passes through an infinite neighboring cell by an almost imperceptible de- number of steps, but for purposes of communi- gree, from the most immature karyoblast (mega- cation we arbitrarily break up a continuous series loblast) to the most mature metakaryocyte (nor- into segments ; three seems to be the most work- moblast) which is just losing its nucleus." This able number, and these, as already indicated, are point of view is probably entirely justified for called, early, mid-, and late. erythrogenesis of mammalian blood, but in avian The color of the cytoplasm serves as the pri- blood where the red cells do not lose their nuclei, mary criterion in identifying each of these the three color changes withhi the cytosome seem to phases. Figures 11 and 12 are examples of be a much more reliable criterion of progressive early polychromatic erythrocytes, as has already cellular differentiation than the alternate ones been mentioned. An important nuclear suggested change by Osgood for man. It is agreed that is an increase in the amount of chromatin clump- hemoglobin uptake and structural differentiation ing beyond that obsei-ved in the erythroblast. are not always synchronized. The cytoplasm has taken on some of the homo- Dantschakoff ( 1908b) faced the same problem geneous textural characteristics found in the main her use of the term "polychromatic." Her ture erythrocyte. This is variable, as shown in comments (p. 519) are interesting. these two figures, and is not veiy closely synchro- "Da die jungen, eben erst aus den farblosen nized with the degree of basophilia. The size of Elementen entstandenen primitiven Erythro- nucleus in comparison with the size of cytoplasm blasten noch sehr wenig Hamoglobin enthalten, usually shows a definite decrease in the shift erscheint ihr Protoplasma nach D-, EA- und G- from erythroblast to early polychromatic erythro- Farbung in einem Mischton von blau und rosa cyte, but this, also, is not constant. In general, tingiert, weil es eben seine urspriingliche Baso- this cell is somewhat smaller than the erythro- philic nur noch zum kleinsten Teil eingebiisst blast but size in itself is not a reliable criterion hat. Hamoglobinfiihrende Zellen mit ahnlich for separating the two stages of development. reagierendem Protoplasma werden bekanntlich These points are examples of the lack of close bei verschiedenen Tieren und auch beim Men- synchronism between different parts of the cell schen im erwachsenen Korper bei verschiedenen during development. Actually the cell in figure Kranklieitszustanden im Blute gefunden and sie 11 is relatively rare, even in bone marrow. Its erhielten in der Pathologic den Namen 'poly- homogeneous cytosome combined with strong chromatophile Erythrocyten resp. Erythro- basophilia is not typical. Figure 12 is more typ- blasten'. Das Wesen dieser sogen. Polychroma- ical in that there is some evidence of mitochon- tophilie wurde von verschiedenen Autoren sehr drial spaces and of irregularities in cytoplasmic verschieden aufgefasst. Ehrlich (15 u. 16) structure. These may persist even up through betrachtet sie als Folge anamischer Degenera- the mid-polychromatic erythrocyte stage of de- tion, wobei die Erythrocyten den Blutfarbstoff velopment (figs. 13 and 14). ins Plasma diffundieren lassen; einen ahnlichen

25 audi often remain. The characteristic by which this Standpunkt nehnien ferner f iir manche Fiille wobei stage is identified is the presence of a gray- Aschheim (2) iind Pappenheim (37) ein, Polychromato- stained cytoplasm that may vary from bluish sie jedoch in anderen Fallen die nocli niclit vollen- gray to a slightly orange gray. philie fiir den Ausdiuck einer Gegensatz The late polychromatic erythrocyte is the third deten Reife der Zelle erklaren. Im stage of the polychromatic series. The cyto- dazu halten Heinz (23) und Troje (49) die Poly- plasm shows a definite orange tinge. In figures chromasie als Folge der Auflosung des Chroma- 15-18, which are examples of this stage, the tins im ZelUeib. cytosome, both from circulating blood (fig. 3, 3 "In meinem Falle treten ausgesprochen poly- and and from bone marrow (figs. 352-354), chromatophile Hamoglobinzellen, die primitiven 4) is about as homogeneous as in the mature erythro- Erythroblasten in friihen Stadien normaler Em- pro- Fall ist also cyte. Clumping of nuclear chromatin has bryonalentwickelung auf ; in diesem der gressed almost to that of the mature erythrocyte diese Erscheinung sicherlich das Symptom but the nucleus itself is not as compressed later- Jugendlichkeit der Zelle. Fiir das Blut kann ally as it will be later. Even at this stage there man vielleicht iiberhaupt den Satz aufstellen, junger, is evidence of the variability in nuclear form dass die Basophilie das typische Merkmal ' found in the mature cell; the nucleus in figure noch nicht differenzierter Zellformen ist." 17 is deeply indented on one side. It may be Cells of the mid-polychromatic erythrocyte are usu- a stage leading to a binucleated cell but, as al- stage of development (figs. 13 and 14) that stage. ready stated, this is insufficient evidence ally smaller than they are in the preceding amitosis is a common method of multiplication nucleus may be large or small in relation The which clear- chromatin for these cells. Figure 29, A and B. the total cell size, and the pattern of to separation of the nucleus into two parts, two ly shows condensation is intermediate between the was made from late polychromatic erythrocytes, represented by the erythroblast and the extremes also. erythrocyte, in that there is considerable mature The cell shape in the late polychromatic eryth- condensation, yet there still remain numerous rocyte is approaching that of the mature in the linin network through which open spaces like the nucleus, is still less com- The erythrocyte but, the nonstaining nucleoplasm is exposed. pressed than it will be later. The slight angu- in many cells is not entirely homoge- cytosome larity of cells found in dried smears has no and vague traces of mitochondrial spaces neous, biological significance; it is part of the tendency hexagonal form due to crowding on erythroblasts that toward a = Translation: Since the young primitive elements still contam mentioned, shape alone have just appeared from the colorless the slide. As already protoplasm^ appears tmged a very little hemoglobin, their cells assume and G-colonng, is a poor criterion of cell age—some mixed tone of blue and pink, after D-, Ea-, the least bit of its original baso- the process of since it has so far lost just an oval shape quite early during with protoplasm of a philia. Hemoglobin-containing cells accumulate the blood m ditter- hemoglobin acquisition while others similar reaction are known to be found in mature body, under various ent animals and also in men in the their full complement of hemoglobin and still these have the name pathological conditions. In pathology i he na- cell and nuclear shape usu- -polychromatophilic erythrocytes or erythroblasts. retain the spherical has beeii conceived ture of these so-called polychromatophilia associated with a relatively undifferentiated Ehrlich (15 and 161 ally of very differently by different authors. degeneration, in which the as figure 19 cannot be cata- viewed it as the result of anemic cell. Such a cell diffuse into the pla^ma; erythrocytes let the blood pigment erythrocyte. It might cases by Ascheim 2U) loged properly as a mature a similar viewpoint is taken for many the polychromatophilia or with and Pappenheim (37), who explain be labeled an orthochromatic erythrocyte, of a still uncompleted even in other cases as the expression Heinz and justification a cell showing anisocytosis." maturity of the cells. On the other hand, (23) equal of the dissolu- Troje (49) consider polychromasia as the result

in the cell body. , . ., tion of the chromatin .,. , , observa- , = might be made from the polychromatophihc hemoglobin cells Another interpretation In my case, pronounced the changes in shape development: tions of Shattuck (1928), who followed in the early stages of normal embryonic appear of lysins. He noted that chicken this case this phenomenon of red cells under the action the primitive erythroblasts; thus in similar oval shape and became round. A of the younthfulness of the cell. cells lost their is certainly the symptom Nesterow an oval to a round shape was noted by can establish the principle generally for the change from Perhaps one degeneration reaction when chicken typical sign of young, still un- (1935) as an initial blood, that basophilia is the dogs and rabbits. erythrocytes were injected intravenously into differentiated cell forms. possible that the round shape of an Dantsclia- Therefore, it is at least in parentheses refer to references in cases, indicate the The numbers in the chicken might, in some refer to Dominici, erythrocyte koff's bibliography. "D-," "Ea-," and "G-" first stage in degeneration. Eosin-azure, and Giemsa stains.

26 — )

developmental form found in bone marrow. Their type 3 might be a thrombocyte, since thrombocytes are not othenvise mentioned in their paper. Reticulocytes have been mentioned occasionally in avian literature. Magath and Higgins (1934) found that the percentages of reticulocytes for adult mallard ducks varied from 16.6 to 27.7 percent. This is considerably higher than the normal value of 1.47 percent given for children of various ages (Osgood, Baker, and Wil- helm, 1934). Wills (1932) demonstrated that ni Figure 29. pigeons, reticulocyte counts do not remain at a steady and low level. Individual birds showed A A double nucleus in an erythrocyte from a 6-week- old Single high counts Comb White Leghorn that, 3 days earlier, during the holding period, for no had received 900r total body irradiation. accountable reason. Peabody and Neale ( 1933 B An atypical erythrocyte found in a nonirradiated bird had a somewhat similar experience. During 25 from the same stock and the same age. days confinement, the counts usually fell from 15-22 percent to 8-10 percent, but in some individuals the counts went up again to 11-13 This again points to the fact that the shape percent. To our knowledge no one has as yet of the cell is not closely synchronized with color carried out a study on the changes in reticulocyte change, and not infrequently a cell may show counts in birds from the day of hatching to ma- its full complement of hemoglobin and yet re- turity, such as was done by Orten and Smith main as round as an early polychromatic erythro- (1934) on rats. According to Magath and Hig- cyte. Thus, in any attempt to pigeonhole cells, gins, the reticulations in the avian erythrocyte one must select one "chief criterion and let all are composed of little dots along the strands of others become subsidiary to it. Color, in the a basket network. As the cells grow older, the erythrocyte series, has been selected as the chief network disappears, leaving a few strands and criterion, whereas nuclear development, vacuoli- dots. zation of the cytosome, and cell shape are re- Their description agrees closely with the cytol- garded as subsidiary. This does not minimize ogy of this stage as observed in the day-old the importance or significance of these subsidi- chick, where at first (fig. 20) there was an abun- ary criteria. They are indispensible for iden- dance of granules arranged on a reticulum. tification of cells and for an understanding of These form a band of uniform width around the progressive change, but terminology based on nucleus, and when cells of this type are given a nuiltiple criteria leads to ambiguity and counterstain it is evident that the cell, if the re- contradictions. ticular granules had not been revealed, would It was probably some such cell as that repre- be classed as a mature cell (fig. 21), but others sented by figure 19 that led Bizzozero and Torre with reticulations appear to he late polychro- (1881) to divide erythrocytes into three types matic erythrocytes. During the final step in the (1) the typical erythrocyte, oval in shape; (2) maturation of the cell die reticulum breaks apart the type that is spherical but has an oval nucleus and the amount grows less, but it still retains the and is intensely stained; and a (3) rare type perinuclear arrangement (fig. 22) and later be- distinguished from the typical erythrocyte by its comes dissipated throughout the cytosome (fig. more delicate contour, weaker staining, and 23). It was hoped that the last stages in the somewhat shorter length, as well as by its larger disappearance of the reticular granules could be and sometimes almost spherical nucleus. That traced, but with the technics used (ch. 7, p. 230), type 2 includes immature cells is suggested by there always persisted a certain amount of pre- the further statement that these spherical forms cipitate over the slide, which, as it fell on the appear somewhat more abundantly in the blood cells, resembled in size and color those granula- of anemic animals and correspond to an earlier tions. These illustrations for reticulocytes are

27 that in the past have in the pigeon for the types of study similar to those given by Hewitt (1940) been applied to pigeons. frontispiece. points that can be settled by counted only those cells These are all Richardson (1937) present completely further study, but more significant at as reticulocytes in which the granules observations of Seyfarth (1927) that re- nucleus. In his material he are the surrounded the blood are ticulate granulations in mammalian found the normal count to be 7.8 percent with to die phases of erythrocyte matura- of ±2.39. Robertson et al. not limited a standard deviation normoblast that follow the extrusion of the that chicks that had been de- tion (1947) showed staining nucleus. By the use of reticulocyte folic acid for 4 weeks held a relatively prived of that these on cells from bone marrow, he found low percentage level of reticulocytes. One in- were present at very early stages gave a sudden increase in granulations jection of folic acid only a of development, when the cytoplasm was proportion of reticulocytes. Tlie peak of the the erythroblast nucleus. As sixth day. The per- narrow rim around increase was reached on the normo- the nucleus became pycnotic at the late subsided to a normal level liy the centage curve perinuclear blast stage, the granules retained a thirteenth day. of the position, forming a ring around the last An excellent review of the occurrence of retic- chromatin. After the chromatin had been en- various classes of vertebrates ulocytes among (he calls tirely discharged, the reticular granules and of the significance of the basophilic granu- tliem substantia granulo-filamentosa) again scat- lations in these cells has been presented by Orten tered throughout" the cytosome of the erythrocyte (1934). His statement that sometimes nearly and then gradually disappeared. all of the erythrocytes in pigeons, chickens, rep- series of stages was reticulocyte An exactly comparable tiles, frogs, and fishes may be in the presented by Seyfarth from studies on the bone stage, is based on observations made by Seyfarth and blood of the chicken, except that Graam (1934) found a similar result marrow (1927). the cell. tlie nucleus was not eliminated from in pigeons when she stained for 10 to 30 minutes, In birds the reticular granulations almost entire- Seyfarth was aware of the fact that staining but in the late ly fill the narrow rim of cytoplasm for a long time damaged the cells. He found erythroblast or the early polychromatic erythro- that reticulocyte granules took up the stain in cyte and as the nucleus condenses, the granules a few seconds. Yet, in spite of the precaution its peripheral margin, thus leaving staining time short, sometimes he accompany to keep the granules. the periphery of the cytosome free from obtained preparations from lower vertebrates at which is no normoblast stage in birds proportion of the erythrocytes with There with a large behavior stage the nucleus is eliminated, but the reticular granulations. From his colored illus- of the reticular granules gives an indication when of reticulocytes in the bone marrow and trations reached in stage of development has been blood of the fowl, he has included in the last this reticulocyte. the maturation process of the avian one or two steps of his developmental series, cells indicated by the change from a condensed contained only a few granulations. Had This is which the nu- of reticular granulation around such cells in our studies as jjenig band we included granules a subsequent scattering of the then we also would have been cleus to reticulocytes, Disappearance of the all throughout the cytosome. forced to the point of view that practically same time. granules is taking place at the in the circulating blood of either erythrocytes appear that From these observations, it would chicks or adult birds were reticulocytes because, the bird is homologous pre- the mature erythrocyte of as mentioned (last paragraph, p. 27), a mature erythrocyte of the mammal, and present over these slides and stain- to the cipitate was suggested by not to the erythrolilast of man, as able granules existed both between the cells and (1912). Those on the cells resembled retic- Burckhardt on the cells. the The reticular granulations first appear m ular cell granulations. following the development of blood from normal adult cell, immediately From our study of This the cell (Seyfarth, 1927) . are extremely rare and hemoglobin in birds, the reticulocytes of with Dawson's data on the occurrence at least exists that the chicken agrees the possibility erythrocytes vftally stained granules in primary would be a better experimental animal than the

28 : :

over a wide range of developmental stages (Daw- not reach the normal level, probably owing to the son, 1936a). presence of polychromatophils A normal in the cir- mature erythrocyte (fig. 24) has culation." been included in order to make the developmental Some physiological differences between ma- series complete. This forms the point of departure and immature erythrocytes of birds, the ture for the discussion of over-aged cells that rate of maturation and the differences in these follows. In summary, it is quite evident that all respects between ijirds and mammals have been stages in erythropoiesis from the erythroblast to brought out in studies made by Wright ( 1930a the mature erythrocyte may be found in the cir- and b) and Wright and VanAlstyne (1931), and culating blood. It would appear that birds in reviewed by Orten (1934). Wright made general, including the chicken, have a more labile use of the weU-established fact that immature hematopoietic system than mammals, and the erythrocytes have a lower specific gravity than presence of an occasional immature red cell can- mature erythrocytes. By centrifugation he sepa- not, at present, be regarded as abnormal or in- rated the reticulocytes and other more immature dicative of a pathological or diseased condition erythrocytes from mature cells of chicken blood. in birds. Wirth (1950) also obsei-ved greater He obtained the immature cells by repeated reactivity in chickens than in mammals and stated bleeding of adult birds and by injection of phen- that regeneration in the chicken was very vigor- ylhydrazine hydrochloride. He estaljlished the ous; that it ends in about a week, and that in mam- fact that the oxygen consumption of all types of mals a]:)out 3 weeks are necessary for the same immature cells was greater than for mature cells. result. Polychromatic erythrocytes and ery- This was true for mammals also, and it has throblasts occurred in very large numbers (up to been suggested that perhaps most of the respira- a half million per cubic millimeter) and the re- tion which occurs in mammalian erythrocytes is ticulocytes became so numerous that they rose due to the reticulocytes present. In summary, from to 33 percent. Wright (1930b) says (p. 213): Splenectomy in pigeons (Toiyii, 1930) raises "A comparison is made of the respiration of the number of polychromatic erythrocytes from the reticulated nucleated red cells present in the a control level of none to a quarter of a million blood of anemic fowls and the nonnucleated retic- and more. This increase comes the first day ulated red cells of rabbits. On the basis of after splenectomy and continues for about 2 equal volumes of cells, the respiration of the weeks and even after .50 days the level of im- former is about twice that of the latter, while mature erythrocytes does not return to the nor- this in turn is aljout six times as great as the mal. Jordan and Robeson (1942) observed nucleated but nonreticulated normal red cells that splenectomy in pigeons increased the number of the fowl." of plugged vessels and lymphoid foci in the bone Wright and VanAlstyne (1931) has brought marrow. These authors interpret this as a com- out some significant points concerning the rate pensatory reaction but the possibility of a differ- of maturation of avian erythrocytes that may ent interpretation is discussed on page 181. help to account for the fact that recovery from Toryu (1931) also performed splenectomy on injury apparently is more rapid in ]>irds than pigeons and an abstract of his article (1933) in mammals. They found in vitro that young states: red cells could differentiate into mature erythro- "After complete splenectomy the marrow of cytes within 36 hours, with a full complement of the femur and tijjia becomes fatty and inactive hemoglobin. In fact, they state (p. 36) for erythrocyte formation, but active for ". lympho- . . the conclusions are drawn that the baso- cyte formation; new haemopoietic tissue appears philic staining characteristic of the more primi- m the lobules of the liver and various stages of tive cells is no indication of any lack of hemo- erythrocytes are seen in the central veins and the globin. Indeed the most primitive cells exam- capillaries of the acini. Splenectomy in adult ined seem to have possessed almost, if not quite, carrier pigeons brings about a general circula- as much of this substance as the ordinary red tion of polychromatophil cells, which amount to corpuscles." 3-8 percent of the red corpuscles in the blood. On the subject of rate of maturation they ob- The hemoglobin content after the operation does served (p. 32)

29 contraction there is usually extensive of the fowl's blood takes a the body, "This maturation considerable development of the nucleus that brings about shorter time than the equivalent Some change in the nucleocytoplasmic ratio. cells. Erythropoiesis can be mo- of mammalian figure 26 but than evidence of this shift is shown in bilized much more rapidly in the fowl m reaction is equally common, from the regeneration a different type of the rabbit, as can be seen nucleus 1930b), namely, vacuole formation within the curves for the two (Wright, 1930a, and 27 and 28). This would appear to be a on the basis of relative Ijlood vol- (figs. even though nucleus durmg fowl. compensatory reaction. The umes the loss of blood is greater in the pyknosis should shrink but apparently the at- Possibly the delay in the mammal is related to will not permit an disposal of tachment of nucleus to stroma the additional time necessary for the The overall retraction of the nuclear membrane. the nucleus." figure 28, with its stem find goblet-shaped vacuole in Since immature stages are not hard to nuclear surface, suggests that that over-age extending to the in circulating blood, it was thought fluids have been sucked in to form find. A few were cytosomal cells might also be easy to condensa- these vacuoles, permitting increased after much searching. They discovered but only of nuclear immature tion of the chromatin without loss are not nearly so common as are the volume. However, this statement is applicable stages. in the spleeii ex- and degenera- Degenerating erythroc^-tes only to stages showing pyknosis without hibit predominantly nuclear contraction indications of aging are fairly tion; the early show karyorrhexis. The one slide there vacuolization; some ( fig.' 25) . Within any abimdant observed in circulating the nr- latter has never been exists considerable varialnlity ni usually erythocytes from normal birds. variability is blood in mature tensity of nuclear staining. This degradation changes in the cell and its particularly in figure These illustrated in figure 2 and describing nucleus are included under the section in some nuclei has the form of 3; the chromatin because aging and the color, normal circulating blood particles and gives to the nuclei a light fine as well as death itself, and the processes leading to death, but in others the chromatin is condensed normal phases of life's progression. But these latter that are all nuclei are darkly stained. It is the whether degenerating cells normally occur in presumably are the older cells, and were they blood of a healthy bird is another the spleen, circulating not removed from the circulation Ijy present. for which there is no answer at pyknotic condition (fig. question they would go on to a in study on the occurrence of over-aged cells pyknotic stages are rare. A 26 ) . The circulating blood under various experimental of chro- the In pyknosis the first reactions consist re- conditions should produce some interesting condensation and nuclear contraction, and matin bird lends itself to this type of study are no sults. The the spaces between the chromatin clumps indicator Ijecause the nucleus is a more delicate the same staining reaction longer clear but take hemoglobin-bearing cytosome. In shade. than is the the chromatin, although in a lighter as there is no means for recogniz- whether mammalian blood sectioned material it is hard to decide On erythrocyte because the nucleus is basichroma- ing the old this reaction is due to dissolution of enters the ejected or disintegrated before the cell effect of tin into the nucleoplasm or to the filter circulation. underlying chromatin clumps that are out of focus because the nucleus has greater thickness focus. (figs. than the oil inmierision lens has depth of Atypical and abnormal erythrocytes smear, The flattened nucleus of a cell in a blood 30-49) range of the however, lies within sharp-focus which, included in this group are those in conclusion may be justified that Cells lens ; thus, the condition observed ex- basichromatni presumablv. the atvpical in the process of pyknosis some bird before the blood was drawn, nucleoplasm. This conclu- isted in the is dissolved in the that a particu- there is alwavs the possibility agreement with observations made on but sion is in produced by the technic the lar result observed was a previous study (Lucas, 1940) in which on the nu- employed. Feulgen test gave a positive reaction blood cells (figs. 30-32) are rare in cells. Spindle cleoplasm of degenerating tissue the from normal birds. It is conceivable that As pyknosis proceeds in most tissue cells of

30 occasional cell of this type in normal blood is a some process such as indicated in figures 35 and technic artifact, produced when the smear is made 38, and the anisocytosis sometimes observed by the mechanical stretching of the cell. On the in cells could be accounted for by a diminu- other hand, poikilocytosis does occur in chick- tion in size following the production of an eiyth- ens; it is indicative of a disturbance in the blood, roplastid. It is conceivable that a small one and among the various shapes are many that have (fig. 41) could be derived from a cell like figure a spindle form. Poikilocytosis obviously de- and 37, a medium-sized erythroplastid (fig. 42) velops within the bird and is not a technic artifact, from figure 40, and large ones (figs. 3, 2, and but this does not exclude the possibility that tech- 43) from such a cell as figure 39. Primary nic can play a role. Spindle cells have been seen erythrocytes of embryonic blood break off anu- in the counting chamber of the hemocytometer cleated portions of cytosome more frequently and, of course, under these conditions there than do the definitive erythrocytes. would be no stretching effect on the cells. Amitosis has already been mentioned in con- Such spindle cells have been observed by nection widi the study made by Charipper and others, even in the embryo. Sugiyama (1926) Dawson ( 1928) on the blood of Necturus. They in his study says (p. 134), "It is noteworthy that include the elongations of cells and constrictions there are a few spindle-shaped red cells in the of either nucleus or cytosome under their evi- blood of chicks, not only in embryonic life but dence for amitosis, but, as already pointed out, also after hatching. These red cells ordinarily only rarely has there been any evidence of di- vary from medium size to exceedingly small, vision of nucleus followed by division of cyto- sometimes with one end pointed and the other some. The opinion is held that they are ex- rounded, sometimes with both ends pointed. tending tlie definition of amitosis too far when Such red cells have appeared by the time the em- they include the formation of erythroplastids. bryo has 22 to 29 somites, ahat is to say, from the They state, "Their fomiation may be considered stage of early erythroblasts; at this stage they are to be by an amitotic division of the erythrocytes usually pointed at one end and rounded at the involving only the cytosome." Wilson (1925) other (figs. 18)." 17, His figure 17 is similar in his glossary defines amitosis as "mass-division to our figure 32 and his figure 18 is equivalent of the nucleus without the formation of spireme, to our figure 30. He goes on to say, "As an evi- chromosomes or spindle-figure." Erythroplas- dence that such spindle-shaped tid red cells are by formation is equivalent to the throwing off no means to be considered as artificial products, of blebs of cytoplasm, which occurs so frequently one finds them in the circulating blood widiin the in lymphocytes, or to the pinching off of pseu- vessels of the area pellucida." dopodia. After discussing the evidence for and Distortions of cells often produce rarefied against amitosis as a normal process of cell areas in the cytosome ; an example of this is shown nuiltiplication involving genetic continuity of to a slight extent cells, in figure 30 and more clearly Wilson (1925) states (p. 221), "It is clear, in figure 31. A variation in the production of therefore, that evidence of amitosis, unless based a spindle cell is shown in figure 32. One end on direct study of the living cell, must be re- is round and the other drawn ceived with the ." out into a long greatest caution; . . This tapering point. The fact that intermediate point desei-ves reemphasis. The existence of stages between figures 31 and 32 can be found amitosis in avian blood can only be established leads to the suggestion that all these various dis- after careful in vivo studies of the type Speidel tortions of cell shape have a common under- (1932) has carried out on the tadpole tail and lying cause. Knisely, et al. (1947) on mammals. Distortion of cells may not necessarily pro- In birds, erythroplastids are relatively com- duce pointed ends. There may be a slight ineak mon and somewhere during the course of evolu- in the side of the cell (fig. 34), a splitting apart tion from reptiles to the ancestor of the mammals of the nucleus (fig. 33), a constriction of one the process of erythroplastid formation became end of the cell (fig. 35), an elongation of the fully estajjlished. It would be interesting to cell (fig. 36), and the production of erythroplas- know whether the survival value of such cell frag- tids of various sizes (figs. 41-43). ments depended upon the more economical utili- The erythroplastid is probably produced by zation of space without the nucleus present or

31 indentations because there came about because the nucleus aged and died matic bands are not enougli of the nuclear boundary still before the cytosome had reached its senility and is usually see that it is not curved inward. When by the elimination of the nucleus, a longer life visible to examined closely there is no span was obtained. individual cells are that the nucleus has been fractured and The life span of erythrocytes among mammals evidence portions pulled apart by pressure in varies greatly, from 8 or 9 days for rabbits and the two making the slide. rats to about 100 days for monkeys ( Harne, Lutz. extend chickens it Sometimes tlie chromophobic streaks Zimmerman, and Davis, 1945) ; for lengthwise in the nucleus (fig. 47) , leaving a cen- is said to be about 28 days (Hevesy and Ottesen, chromatin granules that stains nor- 1945). In the pigeon after hemorrhage there tral axis of washed-out band shows no trace of followed recurrent reticulocyte peaks at about 11- mally. The granules; it shows only a faintly day intervals (Graam, 1935). chromatin 47-49). These Another type of abnormal cell involves only stained linin network (figs. figures illustrate a transition leading to a the nucleus and is illustrated in figure 3, 15 to three chromophobic nucleus. The late 22, and figures 44-49. This defect was found completely (completely empty nucleus) might l^e con- only in slides from Laboratory No. 2 and has stage of emptiness sometimes never been observed in any of the hundreds of fused Avith the illusion after Wright's stain on immature cells. slides made at this Laboratoiy or in slides made found however, different; the former is prac- from farm stock. These cells are included here They are, tically structureless and colorless but the latter because it is not known whether these abnormal pale blue color over the nucleus althougli nuclei develop within the bird or appear on the shows a details are hardly visible. slide as a result of faulty technic. The pre- structural reaction might be a type of ponderance of evidence points to an abnormal The chromophobic chromatolysis but, if it is, it dift'ers from the com- cell. If this is true the cytopadiology deserves obsei-ved liquefaction process in that there thorough study since it is a very conspicuous han- monly is a sharp boundary between the staining and non- dle, or label, that the veterinarian could easily staining parts of the nucleus, whereas usually use in the identification of a disease condition. chromatolysis is a progressive process affecting It is the type of abnormality tliat could be recog- nucleus equally. nized readily from field cases and requires only all parts of the are not limited to er^-thro- low-power magnification to locate the cells. Nuclear fractures found pictures cells show- cytes. In the same set of slides they were Wirth ( 1950) in his figure 43 heterophils (fig. 3. 1) and in lymphocytes ing the same type of cleft nuclei. He labels also in heterophil illustrated, them pathological erythrocytes of birds but gives (figs. 117-120). In the areas extend lengthwise down the mid- no further information about them or the species the clear two of the nuclear lobes. The nuclear of bird in which they were found. It is possible dle of degeneration seen in lymphocytes will be de- to go one step further than this and say that No. scribed later. It is usually more vacuolar and it is not a breed difference since Laboratory irregular than in the erythrocytes, and rarely are 2 and this Laboratory are using the same breed, width, the fact that nuclei namely. Single Comb White Leghorn. the clefts of uniform different kinds of cells are affected The defect appears as an achromatic or chro- of several as evidence that this is a mophobic band across the nucleus; sometimes might be considered technic artifact. If these chromophobic hands it is narrow (figs. 3, 16. and 44) and sometimes expected Sometimes are due to faulty technic, it would be broad (figs. 3, 19 and 20, and 45) . nucleus that some slides prepared at this Laboratory it does not cut all the way through the would also show them, because certainly every (fig. 3, 21). Sometimes it cleaves straight one of the manv thousands made here has not across the middle (fig. 44) but frequently it is of top quality. Moreover. Laboratory No. diagonal (figs. 3, 17 and 18, and 45). Some- been several years later to prepare an- times there are two clefts (fig. 46) and some- 2 was asked other set from the same flock and stain them, and times the ])reak is subterminal (figs. 3, 15 and in the second set showed this particular 19, and 45) witli nuclear substance visible at none defect. tip, or it may appear as if the tip of the the had achro- Numerous visitors to this Laboratory who nucleus had been lost (fig. 3, 22) . These

32 worked with poultry diseases or in the field of the smear and are absent from other intervening hematology have been asked if they had ever ob- areas, it is concluded that this is a technic defect. served this type of reaction in any of their studies The defect shown in figure 50 is commonly and thus far the answer has always been in the found in polychromatic erythrocytes and, there- negative. fore, is quite characteristic for embryonic blood Tate and Vincent (1932) have reported the at certain ages (figs. 227 and 273-275) and for occurrence of sharply delimited spherical bodies leukemic blood. It is readily recognizable by in the cytoplasm of erythrocytes of canaries and the irregular pale spaces scattered through the mice treated with R59 and P25 two compounds — cytosome and by a loss of homogeneity in the used in antimalarial tests. The bodies stained remaining chromophilic masses of cytoplasm. blue in dried smears following Leishman's stain The spaces are not vacuoles in the cytoplasm or but were not visible when other types of fixatives breaks in the continuity of the cell membrane, were used, and could not be seen in dark field. smce sharp boundaries or refractile margins are The peculiar bodies were found, not only in ery- never associated with this type of artifact. It throcytes, but also in eosinophils, leukocytes and occurs predominately in the mid-polychromatic reticuloendothelial cells. Their significance is and the early part of the late polychromatic ery- not blown but nothing similar to these bodies has throcyte development. This fact aids in under- been seen in our studies. Nor were the small standing what causes this atypical reaction. The spherical bodies called stigmata described by mid-polychromatic erythrocyte is in a delicate Nittis (1930) after vital staining with brilliant- transitional condition. It has lost approxi- cresyl-blue observed in our preparations. He mately half of its basophilic substance and has found these bodies associated with nucleated replaced it with about half of its final content erythrocytes of various classes of vertebrates. of hemoglobin. The transition from basophilic They were not visible after Wright's stain. Al- to acidophilic cytoplasm in immature blood cells, though Nittis did not believe that the stigmata like the molting of insects, is a vulnerable period. were the same as the refractile granule found in The cytoplasm is distorted when the smear is nearly mature mammalian erythrocytes by Isaacs dried. The distortion occurs most readily where (1925) yet the granules resemble each other in the serum layer is thick and the slide dries appearance as illustrated by the two authors. slowly; in thin portions of the smear the cells dry quickly and here the normal homogeneous appearance of the cytoplasm is retained. Cells pulled into two pieces have already been Technic artifacts (figs. 50-72) shown (figs. 35, 36, and 38) but those illustrated in figures 51 and 52 differ from them in All smears of avian blood will show some de- that the cytoplasm was already fixed before the fective cells. It often becomes a difficult prob- pulling began or in that the stretching took place lem to separate those that are atypical jjecause when the smear was made. It is obvious in the they are truly abnormal from those that have been latter two figures that the cytoplasm had some made to appear abnormal by the technics used rigidity before it was forced apart. Cells do to make the smear and stain the blood. Under not divide normally by the kind of process in- the previous heading were listed those abnor- dicated in figures 51 and 52. In both of these malities about which there might be some ques- cells the nuclei lie at one pole and it is quite tion of whether they occurred in situ or were re- probable that these cells were caught in the proc- lated to the technics used, but the group of cells ess of producing erythroplastids. The cyto- now to be considered are all quite probably tech- plasm was weakened and the pressure from sur- nic defects. Since it is difficult to tell whether face tension when the smear was made or when a peculiar appearance found in cells should be it was blotted was sufficient to pull the halves referred back to the animal or to technic, a rule of the cells apart along the planes already set of thumb has been adopted and found helpful. up for the separation of the cell into nucleated It is based on the distribution of the abnormal and anucleated portions. cells on the slide; if a number of cells showing Price-Jones (1910) studied the differentia- the same defect are grouped in the same region on tion of the erythrocyte in the early chick embryo.

33 . .

clumped into a dense mass and it seemed to pass, The technic used was equal parts of glycerine His phantomlike, through the nuclear membrane and distilled water, followed by drying. without rupturing it or even denting it. Often colored drawings illustrate examples of lack of basichromatin mass lay beyond the limits of homogeneity in the cytoplasm of the partially the cell, and again apparently without rupture of developed erythrocyte, formation of erythro- the membranes. It must be assumed that the dense plastids, and elongation and distortion of the cell basichromatin leaves by the top of the nucleus body. He regarded these atypical cells as evi- instead of laterally, so that, as viewed dence of degeneration, but some of them ap- and cell breaks in the membranes were peared atypical probably because of the technic from above, the not visible. Sometimes the chromatin masses used. their original shapes but were Too nuich pressure exerted during blotting did not retain out into elongate bodies with bizarre of the slide will damage the cells in other ways, drawn forms. The iiuclear hull remaining behind was as shown in figures 53 and 54. These frac- anchored to the stroma of the cytosome tures of the cytosome and the cell membrane are firmly showed no evidence of displacement, and it of the kind that come after the cytoplasm has and nucleoplasm that was tinged with dis- become rigid; the clefts have sharp borders and retained extend no farther than the nucleus. In figure solved basichromatin. displaced basichromatin masses are so 54 the damage is greater dian in figure 53 in The peas popped out of a pod that slides that the nucleus, as well as the cytosome, has been suggestive of normal birds and the un- partly squashed. The former does not show were made later from pressed against anotlier fractures but the chromatin is spread out into stained cells vigorously top of the smear. There were no a thinner layer than normal and stains more slide laid on nuclei. The whole phenomenon is lightly. effects on the needs to be studied further. The peculiar nuclear reaction shown in a se- an intriguing one and Sometimes other slides are found in which the ries of three cells, figures 55 to 57, has been have been drawn out into long observed only once. It occurred in a routine nuclear contents Usually they are roughly parallel slide made from a moribund young chick that streamers. or cui-ved. Flies and had previously l)een inoculated with neoplastic and they may be straight should be suspected when this type lymphoid tumor cells. It is listed under the cockroaches observed. Flies tend to heading of technic artifacts because additional of nuclear dissolution is touched by their proboscis l)et- smears made from the same bird, only an hour clean up the spot than do cockroaches, which have a different or two later, failed to produce these odd-looking ter type of mouth structure. The salivary secre- cells. The basichromatin of the nucleus was

considered to be normal. FiGURES 30-49.—Atypical and abnormal cells found in smears from chickens 2,470X.

erythroplastid. Figures 30-43; Poikilocytes (P), amsoctjtes (A), and 41 Small 42 Medium erythroplastid. erythroplastkls. 43 Large erythroplastid. 30 Bipolar spindle cell (P) cell, with light staining 31 Large elongated bipolar spindle in Figures 44-49: Cells showing chromophobic reactions areas at the ends (P). 15-22. the nuclei. Compare with figure 3, 1, and 32 Unipolar spindle cell (P). small 33 Cell with nucleus constricted longitudinally. 44 Chromophobic band across the nucleus and a infolding of 34 Transverse constriction of nucleus with area at one pole. across the cytoplasm on one side (P). 45 A single chromophobic band diagonally at one pole (P) 35 Constriction of the cytoplasm nucleus at its lower end. cell and nucleus (P). 36 Elongated 46 Two transverse chromophobic bands. with eccentric nucleus (A). Probably a portion down 37 Cell 47 Chromophilic area restricted to a narrow band the cytoplasm has been lost. of the center of the nucleus. protrusion of cytosome. 38 Cell with nucleus earned into of the 48 Chromophilic area limited to the center Probably a stage in the formation of erythroplastid. nucleus. 39 Microcyte (A). 49 Nucleus entirely chromophobic. 40 Microcyte (A).

34 % 1^

:i(i 34 33

32 31

^ 39 35 ^ 38 37

i I f

40 41 42 43 44

< *

45 46 47 48 49

35 Figures 50-72.—Technic artifacts in erythrocytes. 2,470X.

50 Late polychromatic erythrocyte with internal frac- 60 Partially ruptured cell in which the nucleus was turing of the cytosome. Probably occurs as the almost completely chromophobic. slide dries. Figures 61-63: Cells showing varying degrees of non- Figures 51-54: Mechanical rupturing of the cell; occurred refractile vacuolization of the cytosome. when smear was made or when it ivas blotted. 61 A few vacuoles of varying size lateral to the nucleus. of a cell that already had its nucleus at 51 Pulling apart 62 Half the cytosome filled with large vacuoles. to pinching off an erythroplastid. one pole prior 63 Small vacuoles filling the entire cell. 52 Partial rupture of a cell in which nucleus lies near one pole. Figures 64-69: Artifacts due to overheating the slide. 53 Fracture of the cytosome. 54 Partial squashing of the nucleus and fracture of the 64 Heating has produced a few scattered refractile cytosome. — vacuoles in the cytosome. 65 Large and small refractile vacuoles in the cytosome. Figures 55-57: Extrusion of nuclei, found in a bird pre- 66 Coalescence of refractile vacuoles. viously inoculated with lymphoid tumor cells. Manifes- 67 A single large refractile vacuole at one pole of the tation of the technic artifact probably enhanced by the nucleus. disease condition. 68 Effect of excessive heat. Substance of refractile vacuoles driven off leaving empty spaces. 55 Condensation of basichromatin. 69 Staining of serum granules in an overheated slide. 56 Shifting of the basichromatin outside the nucleus. 57 Complete displacement of basichromatin outside the nuclear membrane. Figures 70, 71: Artifacts due to parts of smudged cells falling on top of normal cells. Figures 58-60: Smudged cells. Fragile cells broken at the time the smear loas made. 70 Vacuoles and chain of three bodies beside the nucleus due to overlying smudged nucleus. of 58 Partially ruptured cell with squeezing out of liquid 71 Two cells with a smudged nucleus overlying both basichromatin. Later stage shown in figure 2, 6. them. 59 Partially ruptured cell with early chromophobic 72 Serum granules which have taken the stain. Compare reaction of the nucleus. with figure 322.

36 9 >i/ 50

53 55 52 54 51

% 0*

56 57 60 58 59

' ( • ^

(^^ €^ 66 67

68 %\

70 71 T • ^

74 75 73 76 77

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78 79 80 81 82

#' #3 •

87 85 86 84 83

FiGUiiES 73-87.— irombocytes. 2,470 X. Figures 80-84: Reactive ihrombocytcs. Figures 73, 74: Normal throinbocyies. 80 Multiplication of specific thrombocyte granules to 73 A typical thrombocyte. form a sphere. the reactive 74 A variation from the tyjiical toward granules to 81 Multiplication of specific thrombocyte type. tlirombocyte form spheres. 82 Vacuolization of cytosome and presence of two Figures 75-79: Devdopmcnlal stages fowid in thecirculaiing irregular spheres. Aggregation of specific thrombocyte granules around blood. 83 vacuoles of various sizes. 75 Stage about midway in the differentiation process. 84 Numerous faint spheres and large, dark granules. same 76 Early elongation stage. Cytoplasm about the Specific granule present. as in preceding cell. Figures 85-87: Stages in disintegration of the thrombocyte elongation, beginning rarefaction. Two 77 Early when exposed to air. specific granules present. stage of disintegration of the cell membrane. 78 Thrombocyte character definitely established. 85 Early of disintegration. Lightly stained cytoplasm. Granulation still very 86 Mid-stage Late stage of disintegration. Specific granule still faint. 87 visible. 79 Nearly mature thrombocyte.

38 tions put the proteins from the nucleus into sohi- when blood was diluted with the animals' own tion and in the wet condition they are spread serum. around tlie slide. These dry and later, when Smudged cells in chickens are so numerous stained, appear as long strands, irregular masses, that it is easy to find all stages. The initial and fine filaments that take the typical nuclear reaction is liquefaction of chromatin which gives stain. If the slides are on edge, there will be the nucleus a light staining homogeneous appear- long streamers flowing downward. ance, and the cjiiosome is flattened beyond its It has been found from experience that if blood usual limits. In anytliing beyond this initial slides must be kept over until the next day before stage, the liquefied chromatin flows beyond the staining, the rack should be put inside a glass dish nuclear membrane (fig. 58) and the cytoplasm with a good cover and the edges sealed with ad- shows various stages of rapid disintegration. hesive tape. No drawings have been made show- In most smudged cells the reaction goes all the ing these effects but there should be no difficulty way and the nucleus is recognizable only as an in recognizing them and in reproducing them. irregular, magenta-colored mass (fig. 2, 6). The If a slide held up to the light shows minute clear cytoplasm is less durable than the nucleus and areas punched out here and there over the smear, even when slightly flattened it loses its affinity for those areas are fairly the a good indication that flies stain (fig. 60) . With only slightly more pres- have had access to the slide. sure, the cytoplasm becomes almost completely (figs. Squashed cells are exceedingly common in lysed 58 and 59) . When cells are severely avian blood (fig. 2, 6) . The part of the cell that squashed, the nucleus also disappears and die

stains is the nucleus, and in the case of the cell only indication that a cell previously existed is mentioned the cytoplasm has disappeared en- found when some of the squashed material hap- tirely. When it has been squashed to this degree pens to overlie the cytoplasm of another cell (figs.

it often becomes and ) impossible to determine whether 70 71 . Mere gossamer traces of squashed it had been an erythrocyte, lymphocyte, mono- chromatin can be seen between the cells but that cyte, or thrombocyte. If granulocyte cells be- which falls on top of odier cells takes an intense come broken they can be identified by their spe- stain. In figure 70 the remains of the squashed cific inclusions (figs. 175, 176, 187, 195, and nucleus have taken the form of a row of bead- 196). Broken monocytes can sometimes be like structures, and have interfered widi the identified (figs. 148 and 149). proper staining of the cytoplasm of the under- Smudged cells are much more abundant in lying cell. In figure 71 the nuclear substance bird than in mannnalian Ijlood, has iDcen stretched because, in birds between two cells ; the cell to all blood cells are nucleated and there is much the left received a bow-shaped strand extending more opportunity to find a smudged cell, than half way around the nucleus. The other por- where only the leukocytes nuclei, as in cany tion of the same squashed nucleus is only faintly mammals. visible on the cell to the right, and a small por- It was noted by Furth, Seibold, and Rathbone tion extends beyond the lower side. The vacu- (1933) that in mice, smudged cells were rare ole at the upper edge of the same cell is caused in normal animals but very numerous in cases by the presence of the squashed nucleus, which with lymphomatosis. They suggested that the interfered with the straining of the cytoplasm. incidence of smudged cells might represent a These small fragments of darkly stained material measure of the cells that were fragile, but they tliat fall on the cytoplasm of normal cells are knew of no way to put this idea to the test. They confusing in that they may resemble foreign bod- were not found in the counting chamber. Al- ies, parasites, bacteria, or even Cabot's tliough no adequately controlled studies have rings. Careful study may sometimes been made on chickens, the indications are the be necessary to distinguish same as for mice, that there are fewer squashed between such artifacts and some types cells from smears of normal individuals than of pathogenic organisms. from those inoculated with tumor transplants. Two drawings of partially squashed cells from Kyes (1929) found a certain proportion of Laboratory No. 2 (figs. 59 and 60) have been in- erythrocytes in fowl and pigeon so susceptible to cluded because they contribute some additional hemolysis that nucleated stromata appeared even information on several subjects—squashed cells, 39 .

either air or moisture and the bul:)bles chromophobic nuclear bands, and bird differ- contain shown in cells were often break through the cell wall as ences. In the set of slides, squashed the type figure 65. An extreme condition is shown in rare but tliose that occurred did not show the cell in- figure 68 where all the bubbles have left of nuclear reaction illustrated in figure 58; discrete. except one, which is in the process of extrusion, stead, the chromatin clumps remained and with the extrusion of the bubbles the cyto- There was no indication of basicromatin lique- refractile. figure 59, plasm is still vacuolated but not faction and spread. The nucleus in the bubbles coalesce and form bizarre squashing, was probably similar to the Often before one l^and cut shapes (figs. 66 and 67). They may form cell in figure 45, where a chromophobic large vacuole with its margin intensely colored one end of the nucleus, and the cell in fig- across mass, shown (fig. 67), or a refractile, sausage-shaped ure 60 was probably something like that last named is chromo- or irregular bodies ( fig. 66) . The in figure 48. As the flattened nuclei with a common type and often appears in slides in phobic bands expanded, their structural details which there was no indication that too much heat became increasingly clear. Wlien one looks at of the cause of mind, it had been applied. The problem figures 59 and 60 with that thought in artifacts still exists, and causes for their becomes questionable whether "chromophobic" these are not excluded. designating the clear production, other than heat, is the best term to use in Dawson (1931) has photographed erythrocytes nuclear areas. The basichromatiu failed to re- similar to figure 67 affinity for of Nectiuus that appear very veal itself, not because it had lost its and they occurred in fresh, unstained blood. In nuclear stains but because it had actually disap- conclusion he stated, "The vacuoles have been peared. A study of chromophobic reactions in interpreted as degenerative in nature, but no lymphocytes (figs. 117-120) raises the same specific cause for such changes in the erythro- question. difi;erences cytes has been discovered." It is pertinent to ask whether such artifacts produce appearances simulat- respect to nuclear reactions are Some between birds in The case, ing cell abnormalities (figs. 69-72). genetic or pathologic in origin. In any use cause of the magenta bodies found in the erythro- these differences emphasize the fact that full cyte cytoplasm (figs. 70 and 71) and their pos- has not yet been made of cytologic details that sible confusion with Cabot's rings or intracellu- could be applied as labels in various kinds of parasites have already been discussed. A experimental studies. lar red cell falling on top of another cell produces common artifact is the presence of vacuoles A under- types are a refractile curved line across the cell in the cytoplasm (figs. 61-68). Two neath and a concentric clear band of cytoplasm. illustrated in this series of drawings—non- line is re- It is so obvious that the clear cytoplasmic refractile vacuoles (figs. 61 and 63) and caused by pressure from the overlying cell that fractile vacuoles (figs. 64^68). The former no questions are raised, Init die same phenome- type was found in slides from Laboratory No. 2 non originating from a small granule or dust and the latter in slides from this Laboratory. falling on the surface of the cell before The cause of the nonrefractile vacuole is not particle erroneous interpreta- fault since it dries will often lead to known, but it is undoubtedly a technic it is heavy enough to of the slides tion. The tiny particle, if it was found on localized regions depress the cell surface as it dries, will thin out with large expanses of normal erythrocytes in- the underlying cytoplasmic layer and this will tervening. The vacuoles are fairly uniform in vacuole when the slide is stained. perhaps widi some coalescence. They vary look like a size, of Wliat one sees is a granule lying in the center from a few to many and may fill up the entire vacuole that appears to be located inside the cytosome. The nucleus was not affected by a cytosome. Focusing does not help to determine vacuole formation in the cell body even when the cell is whether it is on top or inside, because the condition became extreme (fig. 63) to such an extent that its whole thick- In an attempt to produce similar structures, flattened of the lens, and result obtained, ness lies within the depdi of focus slides were overheated ; the only even if focal levels could be separated it would however, was a vacuole that was refractile. The look as if it were inside the cell, because the spheres may vary from very minute bodies (fig. still surface. (figs. 65-67) They particle has depressed the 64) to relatively large ones .

40 Avian blood serum contains more particles sites that are frequently found and misinter- than mammalian serum. Usually the serum preted. Their studies covered several classes does not give trouble in stained smears from of vertebrates, including birds. Some addi- young birds or males, but it may be quite annoy- tional artifacts and parasites found in avian ing in laying birds, in circulating blood from em- blood are shown in the colored illustrations by I)ryos, in bone marrow of older birds, and espe- Balfour (1911). Neave (1906) pictured, in cially in bone marrow of embryos (fig. 322). figures b and c of plate XXI, two pointed objects The granules in the serum (figs. 69 and 72) tend found in his blood smears. Similar objects have to stain more readily with Wright's than with been found on rare occasions in smears made May-Griinwald Giemsa. Wlienever they stain from the blood of chick embryos. Neave de- they spread a veil or screen over the cells (fig. scribes these bodies as follows (pp. 196-197):

) it difficult to ". 72 and obviously make observe . . Length varies from 50-58m and it occurs cellular detail. In addition, they modify the plentifully. It would appear to consist of a color reaction of the underlying cells so that iden- sheath jDointed at each end which contains proto- tification of cells under these conditions becomes plasm segmented into two or more portions." unreliable. In some cases only the granules take Balfour (1911) considers these to be yeast cells the stain and in other cases both the fluid serum that have fallen on the slide and which have come and its contained granules take the basophilic from the air. We are in agreement that they dye. Extreme examples are shown in embryo are contaminants on the slide from outside the ])one marrow (fig. 322), spleen (fig. 329), and specimen itself. thymus (fig. 332). Sometimes the granules that fall on top of the cell and not between the cells will take the dye, which causes the cell to look as if it contains many THROMBOCYTES small organisms. Oftentimes the serum granules will remain uncolored except near a ruptured or The nucleated thrombocytes of birds, reptiles, smudged cell (figs. 175 and 176). In these fig- amphibians and fishes have the same function as ures the small, darkly stained particles are the the blood platelet in mammals. serum granules. Heterophil granules from the Bradley (1937) called these cells thigmocytes. rods may closely resemble those in the serum but "Spindle cells" is anotlier term used commonly, are larger and take a more reddish color. especially in the older literature. When blood Occasionally the serum bodies are larger than is drawn, tlirombocytes and platelets clump rap- usual, resemble cocci, and may cluster around idly and soon disintegrate. The disintegration the cells (fig. 69). This example was from a is part of the mechanism of blood coagulation. heated slide but similar reactions have been seen In mammals platelets are pinched off from the in unhealed specimens. cytosome of megakaryocytes located in the bone Another type of abnormality obsei^ved both in marrow and lungs. The parent cell is large, mammalian and in avian blood is the production and is easily recognized and identified. It has of slender, flexilsle, protoplasmic processes from a polymorphonucleus in contrast to the multinu- the surface of erythrocytes. They are not found cleated condition of giant cells in birds. The in fixed and dried smears but the projections on megakaryocyte is lacking in avian bone marrow cells A and B of figure 29 indicate their appear- and, instead, thrombocytes arise from antecedent ance, except that they are longer in the living cell mononucleated cells that have a blast stage like where they are usually seen. Shipley (1916) that of other blood cells. Blount (1939b) noted observed them in tissue culture from cells of the that thrombocytes of 5 x lOyu were smaller than area opaca, and found that they appeared to be erythrocytes of 7 x 12^, and Magath and Hig- beaded for part or all of their length and termi- gins ( 1934) gave the average size at 3.9 x 8.1 m- nated in a small round knob. They were ac- The distribution curves for width and length, tively motile and the end of the process whipped based on 10 cells measured from each of 9 chick- back and forth. ens, gave means of 4.7 and 8.5 for tliese two Knowles et al. (1929) have depicted in color dimensions (fig. 89). These data are discussed many artifacts, abnormal cells, and cellular para- further in chapter 6. Similar distribution

41 were of extravascular origin and thus thrombo- curves have been prepared for the pigeon l>y was not related to eryduocytogenesis. Schoger (1939) who gave the range in length cytogenesis 3.0-4.5 as 7.5-8.5 microns and the width as microns. mononu- In birds, thromljocytes develop as Normal mature thrombocytes (figs. 73, 74) cleated cells and remain so throughout their life The typical thrombocyte (fig. 73) has been span. Unfortunately, for purposes of study, described many times as a cell slightly smaller they acquire early in their development a trigger- than an erythrocyte, elongated with rounded like fragility that makes them as readily reac- ends, but not having the regular oval contour of tive to damage in the immature as in the mature the erythrocyte. The thrombocyte nucleus also stages. In contrast, during the process of mam- oval shape but is not as elongated as malian evolution a shift in fragility apparently has a slightly that of the erythrocyte. The cytosome usually arose, so that developmental stages represented consists of a framework with large spaces. Some by the megakaryocyte lost this high degree of have called them vacuoles but they do not have fragility and retained it only in the functional vacu- is the discrete nature and regular contour of platelet. It is because the avian thrombocyte oles. Sometimes the cytosome shows structural so easily and quickly damaged at all stages that uniformity. Considerable variability in color we know so little about its cytomorphosis. Fewer on by the cytoplasm pale blue as in than half a dozen papers have been written on is taken — figure 73 or pale purple as in figure 74. Often the subject. cell membrane appears as a distinctly pur- When the thrombocyte disintegrates, not only the cells ; is especially true in plisli line (fig. 85 ) this does the cytoplasm go to pieces but the nucleus which disintegration is just beginning. rapidly reaches a pyknotic condition. Because in Thrombocytes contain specific granules that the disintegration mechanism is held in such deli- take a pink to reddish purple color. The vari- cate balance the thrombocyte would seem to be ability in number, size, intensity of color, and an ideal tool for the physiologist in his study of figure die position in the cell is extremely great. In cell equilibrium and disruption. Because 73 there is a single compact granule at each avian thrombocyte has many points of association nuclear pole with a suggestion of diffuse orange and similarity with erythrocytes and with lym- material beside the lower one. In figure 74 phocytes they have been placed between these there is a definite single granule jjetween the nu- types in the arrangement of subject mat- two cell lower pole Blount cleus and the side wall, and at the ter in the Atlas. Bradley (1937) and are four granules surrounding a lighter (1939b) regard thrombocytes and erythrocytes there stained homogeneous mass of similar material. as closely related genealogically, and the latter In figure 81 there is a chain of four rings. The author mentions the existence of thromlioplas- intensity of staining is less than in the two cells tids, but enucleated thrombocytes have not been and previously described. observed in these studies. Hartman ( 1925) The outline drawings (figs. 88 as) have been Gordon (1926) extensively reviewed the various arranged so that the cells in the first horizontal theories of the origin of thrombocytes. From are examples illustrating a single granule. Gordon's own experiments, he concluded that row second row shows 2 granules per cell, the thrombocytes were derived from erythrocytes. The third row shows 3 granules, and the cells in This conclusion was based in part on the fact granules. The the the fourth row contain 4 or more that when he bled a heath-hen repeatedly granules may lie at the poles of the cell or at the of thrombocytes increased when the num- number or side. If multiple, they may be close together ber of erythrocytes decreased. The ratio of far apart, and they may be compact and dense or thrombocytes to erythrocytes was 15:1000 at the diffusely organized. beginning of the experiment and after 5 bleed- granulation is number of The significance of the specific ings it was 45:1000. The absolute known. Blount (1939b) is of the opinion thrombocytes had increased from 35,000 to not these granulations do not represent hemo- 86,000 and the number of erytluocytes had de- that Possibly it is part of the trigger mech- creased from 2,350,000 to 1,900,000. Hart- globin. anism that brings about the rapid disintegration man (1925) concluded from his studies that they

42 a J

J k

m n

r 5 Figure 88.- -Outlines of normal thrombocytes and their nuclei, showing variability in shape, and in number and size of granules. 2,470 X.

a-g One specific granule per cell. Size of granules ranges h-k Two specific granules per cell. from small to large. Cell tending g toward a re- 1-0 Three specific granules per cell, active thrombocyte. p-s Four or more specific granules per cell.

of the cell. When the specific granules are ab- refractile, and increased in size with long ex- sent or are liidden by the nucleus, the throm- posure. If the cells are elongated, like those in bocyte becomes a difficult cell to identify. Fork- figures 73 and 88, k, m, and q, they could pass ner (1929) observed that thrombocytes have a for poorly developed erythrocytes; on the other clear cytoplasm at first but during the progress of hand, if rounded like figures 88 g, i, and s, they staining with neutral red and janus green, vacu- would simulate lymphocytes. Usually the nu- oles developed at one or both ends of the nuclei. cleus is smaller than that of the lymphocyte. These stained a muddy, brown color, were non- The nucleus of figure 80 is an exception and is

43 THROMBOCYTES FREQUENCY of LENGTH and WIDTH of chromatin clumps of the fully mature cell. The various cytologic effects produced in each cell areas occupied by liniu network and basichro- type. Cellular reactions of the kind shown for matin are about equal. the thrombocytes appear so frequently in birds The late innnature stage (figs. 78 and 79) inoculated with lymphoid tumor material that shows certain changes from the pi'eceding phase. they deserve further study. The cell has usually attained a slightly oval shape Kasarinoff (1910) described hypertrophy of but this is not always the case (fig. 364 from bone thrombocytes with an increase in vacuolization marrow) ; the cytoplasm has nearly the same following the subcutaneous injection of sodium degree of coloration found in the mature cell and cantharidate. Schogar (1939) described the it shows extensive vacuolization; the nucleus may morphology of thrombocytes in pigeons and di- still have a round contour or may be slightly oval, vided them into four categories: Normal, youth- but more important is the increased density of ful forms, forms in old age, and forms under chromatin clumping. Specific granulation is stimulus. It seems probable that our reactive present, but a point to which reference will be thrombocytes include his last two categories. made later is the faintness of the granules and The aging of thrombocytes has received practi- the fact that there is seldom more than 1 or 2 cally no attention, except by Schogar, and yet it small granules present. is a subject that might be pursued with considerable profit. There seems little doubt that the reactive thrombocytes are the ones with vacuoles. Gray, Snoeyenbos and Reynolds (1954) have Abnormal cells (figs. 80—87) photographed this type of cell in their study on

Probably neither of the two types of cells in- the hemorrhagic syndrome of chickens. Aged cluded under this heading could be called patho- cells may possibly be of the types shown in our figures logical. The cells in the first group have been 81 and 84. classified as reactive cells (figs. 80-84) and In the amphibian, Nectitrus, Dawson (1933b) those in the second group illustrate the cellular found that thrombocytes had the power to phago- breakdown that begins as soon as the blood is cytose colloidal particles of carbon. These par- liberated from the body (figs. 85-87). The ticles tended to aggregate at the poles, like the latter might even be classed as a technic artifact specific acidophilic granules of thrombocytes. because it takes place outside the body. Clot- Later (1936b) he showed that the particles of ting, on the other hand, also takes place outside carbon persisted in the cytoplasm of circulating the body and yet is a normal function of blood, thrombocytes as long as a year, but he concluded but from the point of view of cytology this proc- that the individual cell probably sui-vived only ess is degeneration. aliout 5 months. The thrombocyte is a highly

The type of cell shown in figures 80-84 is labile cell and survival even this long seems often seen in blood smears from birds that have rather surprising, yet his conclusion is justified been inoculated with lymphoid tumor cells or by the data of his experiment. filtrate, and occasionally it will be found in large This property of phagocytosis may be peculiar numbers in seemingly normal birds. All the ex- to particular species. Hartman (1925) in his amples selected for illustration were taken from classification of diflferent types of thrombocytes apparently normal birds, but the chicken that among vertebrates, noted phagocytosis in the furnished the cell for figure 84 died with exten- thrombocytes of the amphibian Bathrachoceps at- sive neural lymphomatosis 44 days after the tenuatus (Eisen) and in the reptile, Iguana tuber- smear was taken. A 5-week-old chicken fur- culata. nished the material for figures 80, 82, and 83, Figures 80, 82, and 83 illustrate a structural and in the same slide there were an unusual num- series but it is not known whether progression in ber of smudged monocytes of which figure 149 is this series is from right to left or from left to an example. Not enough has been done to use right. As already mentioned, figure 80 closely the blood picture as an indicator of incipient resemliles a lymphocyte except for the identify- lymphomatosis. It is a problem that should be ing specific granules. Figure 90 is a lympho- investigated, not on the basis of a certain number cyte from the same slide; it has a similar cyto- of cells of each kind but rather on the basis of plasmic texture and nuclear structure. In fig-

45 figure is similar to that shown in our figure 85. lire 82 the cytosome is frothy and contains two accentuation of the edge of the cell irregularly shaped masses of specific granular Usually the a shift in cell and nuclear material, and in figure 83 the entire cytoplasm is accompanied by more rounded condition. has a pinkish color, and abundant specific ma- sliape from an oval to a often the perimeter of the cytosome breaks terial surrounds the vacuoles. Figure 84 shows More the cell and there remains only a rela- the specific substance scattered in clusters and away from cytoplasm drawn out into clumps throughout the cytosome, and the cyto- tively narrow rim of these irregular peaks (figs. 86 and 87) . In both plasm itself has a pinkish color. Tlie cell (fig. illustrations, specific granules served to identify 81) is probably least removed from the normal the cell type, but again there is considerable sim- in that it is not uncommon to find thrombocytes lymphocyte. This is especially true with four clusters of specific granules (figs. 88, ilarity to a granules are absent, as is usu- p-s). when the specific ally the case in dn-ombocytes reaching this stage When cells of the type exemplified by these of degeneration. When the thrombocyte is small figures were first observed, it was assumed that like figure 87, the possilnlity of confusion is not because the cells were large and tended to have very great because this shrunken pyknotic cell is a rounded shape, they were immature; but after smaller than even a small lymphocyte, but a large die developmental series had been worked out thrombocyte with a nucleus of corresponding jjoth in the embryo and in the adult, and it had magnitude can often raise a question of how it been repeatedly demonstrated that the specific should be classified. granules of thrombocytes come relatively late in The rate at which the thrombocyte arrives at the developmental process and that when they this terminal stage of degeneration is variable, appear only 1 or 2 small granules are present, it and the predisposing factors are known only in was concluded that the overproduction of such part. It has been observed that usually a smear granules must have some other explanation. Be- inade within a few seconds after pricking the cause they often occurred in birds suspected of wing vein will give well-preserved thrombocytes, being in ill health or known to have been inocu- birds, even a evenly distributed ; but with some lated with tumor transplants, they have been few seconds of delay, such as occurs in taking called reactive thrombocytes. the second drop of blood instead of the first, will A second type of abnormal cell, the degenerat- produce many cells showing early reactions sim- ing thrombocyte (figs. 85-87), is very common. ilar to figure 85. The taking of the first drop As previously stated, degeneration is part of the is contraindicated for good smears in mammals of the thrombocyte; yet degen- normal function laest l)ut has been found by experience to give the eration from the standpoint of the individual cell result in birds. On the other hand, in some can hardly be considered normal. Death and chickens one may be quite careless, even allow- the processes leading up to it are not normal for ing a pool of blood to accumulate on the feathers, the individual; yet from the standpoint of the and after waiting 20 to 45 seconds before making race and the replacement by successive genera- the smear one may yet obtain thrombocytes that tions, death must be considered as normal. are well preserved. Even a 2-minute delay may Clotting of the blood of birds proceeds at about not cause much more degeneration than may be the same rate as in mammals (Dorst and Mills, found in certain birds from which the blood is however, there 1923) . When smears are made, taken immediately. is not as rapid clumping of throml30cytes as there fol- is of platelets, and cytologic changes can be lowed in more detail in avian cells than in tlie a sliift in latter. The first reaction involves Technic artifacts staining color (fig. 85) of the cell membrane from figures have been made because nodiing a pale blue to a reddish violet or reddish orange. No classified ah lias been found that could be so Whether this is brought about by a condensation occur. is most probable that it does of cell substance or by shrinkage beginning at the though it there are many smudged erythrocytes, cell surface has never been determined. Diesem Just as proljably smudged thrombocytes. A (1956) has illustrated this affinity of the throm- there are thrombocyte would resemlde bocyte margin for eosin in his plate 1. His partially smudged

46 a smudged erythrocyte too closely to permit iden- ity—than about any other cell in avian blood. tification, and it has already been pointed out For the solution of some of these problems the that, after the cytoplasm is gone, all smudged phase microscope would be useful in that it re- nuclei look very much alike. veals in the fresh living cell nearly all the details In conclusion, it should again be emphasized that can be seen in the stained preparation, and that less is known about thrombocytes —their cy- with it many of the rapid changes in the cell could tology, physiology, development, and reactiv- be followed directly.

Nongranular Leukocytes

The term "nongranular leukocytes" groups have stated (p. 63), "When preparations of lymphocytes and monocytes under one heading. blood are examined and viewed ojjjectively, it is The term is applicable to these two cell types be- seen that the nongranular leukocytes consist of a cause stainable granules are usually absent from series of transition forms which begins with the the cytoplasm of these cells; however, this char- smaller lymphocytes and ends with larger cells of acteristic should not be applied too broadly, be- quite different appearance, the monocytes, . . . cause some lymphocytes contain magenta-colored But in the midportion of this series of transitions bodies and some monocytes contain azurophilic is a group of cells which cannot be classified as granules. In mammalian hematology these two either typical lymphocytes or typical mono- cell types in some theories of hematology are said cytes." The same statement is applicable to to arise within the lymph nodes, and have a com- avian blood, and this might be considered as mon cell of origin. A review of the theories of lending weight to the ideas expressed by the hematopoiesis of lymphocytes and monocytes was unitarian school, but it does not exclude assess- given by Bloom (1938) and Bessis (1956). ment of the problem in a different way, as fol- The nongranular leukocytes oifer many com- lows: Lymphocytes and monocytes show but little plex problems and some answers must be given structural differentiation—neither cell type is to them before any workable basis of identifica- far removed from the conventional generalized or tion can be set It is up. true that practically any typical resting cell so frequently depicted in answers given will be arbitrary or empirical; textbooks. nevertheless, they are indispensible and nuist be In the blood there are no topographic tissue presented and discussed before considering the relationships. It is as if a smear were to be pre- individual figures and the accompanying descrip- pared for study from cells of the respiratory tive text. mucosa, epithelium of the digestive tract, of the Kasarinoff (1910) has reviewed the earlier liver, connective tissues, reticular tissues, and literature on the numjjer of types of leukocytes thyroid, and these cells had all been isolated from in avian blood. He demonstrated by colored their usual haJiitat, allowed to round up and then drawings his division into six types: (1) Small spread out and dried for identification. Identi- lymphocyte, large (2) lymphocyte, (.3) lympho- fication in many instances would be difficult. leukocyte, mast cell, (4) (5) pseudoeosinophil, Another example could be cited that is probably and true eosinophil. (6) In this study, small more pertinent—the similarity of appearance be- and large lymphocytes have been grouped to- tween certain heterophils and eosinophils. This gether and his lympholeukocytes have been iden- similarity will be discussed more fully later but tified as monocytes. Mast cells are called baso- it may be said here that these cell types also phils, pseudoeosinophils are called eosinophils, could be arranged in a structural series from and true eosinophils, heterophils. heterophil to eosinophil, and the literature on Shall lymphocytes and monocytes be treated as avian blood reflects this confusion; yet at present a continuous series extending from lymphocyte there are no suggestions that the mature form of to monocyte? Maximow and Bloom (1931) one develops into the other. From what has been

47 forced to use to catalog with- ability ranges overlap, and one is said it is apparent that inability multiple characteristics with the confusion that out question, or the mere existence of a structural continuity. often results. Characteristics of monocytes and series, is not in itself proof of genetic lymphocytes have been presented in table 3. Having applied this reasoning to lymphocytes once Relatively few cells will show all the character- and monocytes, it must be pointed out at istics listed under each cell type. The confusion that studies made on spleen, bone marrow, and comes when cells show characteristics partly of early embryonic blood make use of a structural one cell type and partly of the other. Examples series of cells to trace developmental stages. will be pointed out when individual figures are On the basis of such series one is led to an opin- discussed 50-73). ion, but the diversity of opinions on hematologic (pp. table 3 the characteristics are set up on tlie principles of cell relationships is evidence In basis that all mature lymphocytes are either small enough that such an approach falls short of carry- or medium in size. A group referred to as ing the weight often assigned to it. lymphocytes" has not been considered in The most convincing evidence that lympho- "large the preparation of criteria for the table for cytes and monocytes are as distinctly different several reasons. It is only rarely that cells of as are any other two cell types came from their this type have been seen in the circulating blood treatment with X-rays (Lucas and Denington, of normal adult birds. Four of them are illus- 1957). Single total body irradiation from 50r ages trated (figs. 121-124) but it is misleading to call to 300r applied once to chickens at different them lymphocytes when obviously it is much has clearly demonstrated that lymphocytes are more probable that they are immature cells in highly susceptible to this treatment, and by 1 to the process of development toward some other 3 days after irradiation had reached a maximum particular cell type. To make the term "lym- deo^ree of depression in which the normal value of phocyte" so inclusive that it designates both a 20t000 to 30,000 cells had dropped to about fully mature functional cell of circulating blood 2,000 to 3,000 cells. The recovery was rapid and also the other extreme, a stem or blast cell and, depending upon age of the bird and severity from which other cell types arise, may be entirely of treatment, was half to fully completed by the justifiable when propounding a unitarian theory 15th day after irradiation. The monocytes re- of blood-cell interrelationships.* However, this acted entirely differently; only the higher doses is not workable when the term is used merely gave any indication that these cells had been af- to identify a definite structural cell type without fected at all, and the drop in number reached its to any hematopoietic theory. It is for maximum between the third and eighth days. reference this reason that the "large lymphocyte" is treated The typical lymphocyte and the typical mono- separately from the small and medium-sized cell. cyte are easily distinguished but to describe and To do this will not change the values obtained for illustrate merely the typical cells would defeat an differential counts since, as already pointed out, important function of an atlas on blood, that of the "large lymphocyte" occurs so rarely in the showing variability so that questionable and unidentified cells in differential counts are reduced unitarian point of view lias been clearly stated by to a minimum. As Lucas and Denington ( 1956) 'Tlie ". ist der kleine (1909a, p. 157): . . Speziell morphology of the liver, Dantschakoff noted in a study on the Lymphozyt im erwachsenen Organismus nicht eine reife Zell- ganz differente junge the "typical" is very rare. On the other hand, form, sondern, im Gegenteil, es ist eine Entwicklungspotenz. variability as Zelle mit sehr mannigfaltiger, prospektiver what is normal may include a wide Keimzentren Gewiss entstehen die kleinen Lymphozyten in den avian erythrocyte. sich dann was shown to be true for the durch Wucherung der grossen, aber sie konnen liingeren oder kUrzeren Ruhe- In the case of lymphocytes and monocytes there is •.pater, vielleicht nacli einer grosse Lym- periode wieder durch Hypertrophie in typische overlapping in respect to the various character- der phozyten zuruckverwandeln und zum Ausgangspunkte istics commonly associated with each of these Haniatopoese werden." the ma- Translation: In particular, the small lymphocyte in cells. but on the contrary ture organism is not a mature cell form, convenient to classify cells on cell with most manifold prospec- It is simple and it is a quite distinct youthful Certainly the small lymphocytes the basis of a chief and supporting characteristic tive developmental potential. proliferation of the large, arise in the germinal center through as was done for polychromatic erythrocytes. hypertrophy into but they can later change back again through short or long period Unfortunately, this method will not work on typical large lymphocytes, perhaps after a emergence of hematopoiesis. lymphocytes and monocytes because their vari- of rest, and become the point of

48 . . . . .

Table 3.—Characteristics of lymphocytes and monocytes ^

Characteristic Lymphocyte (small and medium) Monocyte Cell size Size is continuous from a small cell with almost A few cells are smaller than the average heteropliil no cytoplasm to one that is as large as the but most monocytes are larger, average heterophil.

Cell shape Lymphocytes may be round and regtdar or have Mature monocytes are often round with a smooth a contour made irregular by projecting blebs contour. Monocytes with a hyaline mantle are of protoplasm or broad protoplasmic lobes. usually irregular. Immature cells may show- lobes. Mature monocytes often acquire an irregular shape if squeezed between other cells at the time the smear is made. This distorts the cell and its contents and makes it difficult to identify.

Nuclear position nucleus is The centrally placed or nearly so in The nucleus is usually eccentric. many cases but is eccentric sufficiently often to offer a point of confusion with monocytes.

Nucleocytosomal ratio The nucleus relative to the cytoplasm is large. The monocyte nucleus occupies a proportionately latter The may vary from a narrow to a rather smaller area of the cell than does the lymphocyte rim. broad A larger proportion of cytoplasm nucleus. The difference in nucleocytosomal ratio associated with an eccentric nucleus may make appears to be greater than it actually is because the cell appear to be a monocyte. the monocyte nucleus has an eccentric position.

Cytoplasmic structure . . The cytoplasm may be relatively homogeneous The cytoplasm contains uniform alveolar spaces, or contain closely packed irregular basophilic especially well developed in the region of the Hof. masses. pathologic Under conditions the cyto- In the literature the cytoplasm is often described plasm may become conspicuously and exten- as having a ground-glass appearance. Many sively alveolar. monocytes show two structural regions in the cytosome—a hyaline mantle forming the distal end of broad protoplasmic lobes and a denser, darker staining, granular portion adjacent to the nucleus.

orange-staining Hof and There usually are no Hof-Xike areas in the cyto- Most monocytes show a rarefied area of alveolar spheres. plasm even when the nucleus is eccentrically cytoplasm adjacent to the indentation of the placed. nucleus. This Hof sometimes contains an orange-staining substance filling the alveolar spaces. A well-defined Hof is not common in the wild species studied.

Specific cell inclusions Intensely stained magenta granules are fre- The dark magenta bodies of lymphocytes are quently found in the cytoplasm of lymphocytes. rare in monocytes. Instead the azurophilic sub- Cells containing them are regarded as abnormal, stance in these cells stains a delicate pinkish even though these bodies are probably the orange and are dustlike flecks in the cytoplasm. counterpart of the azurophilic granules reported Usually the color appears at the denser inter- for mammalian lymphocytes. They are very stices of the protoplasmic ground substance and different structurally and tinctorially from the merges into the blue color of the reticulum. azurophilic granules of monocytes. In some Occasionally these pinkish-orange bodies are wild species of birds they are so conspicuous as punctate and discrete. to cause the cell to superficially resemble a basophil.

Nuclear shape The nucleus of the lymphocyte is round, or Nearly all monocyte nuclei arc either flat on one nearly so. It rarely shows an indentation and side or show a broadly curved depression in when it does it is the sort of constriction that which lies the Hof. Round nuclei are more fre- comes from folding the nucleus on itself. quently found in small monocytes than in large ones.

Nuclear structure. Small lymphocytes and some medium lympho- Monocytes show chromatin clumps that are small cytes show large blocks of basichromatin that and an integral part of the nuclear reticulum. occupy most of the nuclear space. In many The nucleo]>lasm is usually more nearly colorless medium lymphocytes, the nucleus shows a mix- than in lymphocytes. ture of chromatin clumps and a dehcate reticulum. The nucleoplasm is tinged with dissolved basichromatin.

Cell division Division of lymphocytes in the circulating blood Division of the monocyte nucleus in circulating is by mitosis. blood is bv constriction.

' The characteristics chosen are those that can be seen after using Wright's stain and thev do not include mitochondria, neutral red bodies or other cell structures that are revealed with vital stains.

49 ures 93-95 and 99-101, and otliers are medium circulating blood of a normal chicken that dif- in size. Whether a cell such as shown in figure ferential counts would have to be based on one 92 can reconstitute its cytosome is not known. or two thousand cells, instead of one or two if The range in size of lymphocytes is illustrated hundred, to find any to count. Of course, outline drawings (fig. 150) and Ijy the monocytes are included with lymphocytes, as has also in the graph (fig. 152). None of the cells in these been done sometimes in the literature, the total are as large as blast cells. More on agranulocyte count would be 3 to 10 percent examples of size will be given in chapter 6. higher than the lymphocyte count alone. Large the subject size and shape shown in the lymphocytes are present in fowl leukemias or The variations in but colored illustrations are further extended in fig- after cell transplants of lymphocytomas, ure 150. Each row represents samples of lym- these blast cells usually identify themselves by phocytes taken from a different breed or source, their association with some particular cell line. but there were no obvious differences; however, par- it has often been noted that on a slide from a ticular bird a small cell may be dominant and that LYMPHOCYTES in another bird the medium size may be more abundant; or in one, blebs may be common and lymphocytes (figs. 90- ISortnal mature in another a hyalin cytoplasm may appear fre- 101) quently. It is these points of difference, when their significance becomes known, that will make Cell size.—These 12 cells illustrate the various for critical studies on avian blood. characteristics of lymphocytes presented in table found occasionally in the of them Blast cells may be 3. No one cell shows them all, and some circulating blood and four are illustrated here reveal structures that are usually ascribed to (figs. 121-124), and in the legends suggestions monocytes. Figure 90 is probably as close to are given as to the line that each represents, but a typical lymphocyte as any; it is average in size, for error. The cells of fig- contour, the nu- there is nnxch room it has a fairly round and regular ures 121 and 124 have nucleoli and show a coarse cleus lies in the center of the cell, the cytoplasm nuclear pattern; the erythrocyte relationship is forms a narrow rim that has neither a distinctly moderately certain for figure 121 but is question- granular nor a hyaline texture, and the nucleus able for 124, and the intensely stained nucleus contains large dense clumps of chromatin and a and cytosome are indications that it belongs to tinged nucleoplasm. This cell comes from the the thrombocyte line. The general appearance same slide that produced the reactive thrombo- of figure 122, particularly the tendency of the cytes, figures 80, 82, and 83. Lymphocytes are cytoplasm to be frothy or vacuolated, and the usually classified as small, medium, and large, vagueness of nuclear pattern are suggestive of the but too often the subjective impression is based early lymphocyte as seen in the thymus ( figs. 335 on nuclear size rather than the whole cell, or, and 336). The narrow rim of dense l^lue cyto- stated differently, Ijy what is left of the cell after plasm of figure 123 with its moderately uniform the cytoplasm has lobulated and broken off. meshlike nuclear structure suggests a granulo- medium-size cell with its medium-size Thus, if a typical as those seen small blast, but this cell is not as nucleus loses its cytoplasm it is counted as a in the spleen and the bone marrow. lymphocyte. Since lymphocytes often throw off Cell shape.—Avian lymphocytes may have a blebs of cytoplasm, perhaps they should be classi- slides from ap- cell; regular contour but in certain fied by size of nucleus rather than by size of parently normal liirds the lymphocytes will ex- errors in estimating would certainly be less. hibit many protoplasmic blebs (figs. 92-94). The cells shown in figures 92, 102, 103, and This reaction in mammals has been identified as 104 are considered as small lymphocytes. In stimulation of the blood toward antibody for- figure 102 all cytoplasm is gone except for barely mation by pathogenic agents or other causes. perceptible bits on one side of the cell and several In view of this, the history of the three individ- lightly stained magenta granules. The cell in is interest- lost uals in which these cells were found figure 92 is somewhat larger, but after it has loljes, ino-. Figure 94, which shows a few small the blebs that are in the process of pinching off, killed at 619 days of age, cells of fig- was taken from a bird it will be about the same size. The

50 at which time it had a wryneck but no lesions of ticed on autopsy, budding of lymphocytes oc- lymphomatosis or other diseases to account for curred. In 10 of the 14 studied with symptoms the condition. The cell represented in figure 93 of paralysis of the limbs and lymphatic hyper- has several broad lobes widi restricted bases, as plasia of visceral organs, budding of lympho- if the cell had been taken just at the moment the cytes was marked. In only diree of the 15 para- cytoplasm was jjeing pinched off. The smear lyzed birds with tumors found on autopsy was was taken when the pullet was 113 days old, and budding of lymphocytes noted. Three of the when killed at 151 days she showed severe gasp- 12 liirds with iritis or gray eyes also had numer- ing and emaciation. At gross necropsy there ous lymphocytes." was atonicity of the crop and a slightly irregular For comparison, a study of budding phenom- pupil, suggestive of ocular lymphomatosis. In enon in normal birds and in birds with various figure 92 the lobes are numerous. At their tips types of diseased conditions is needed. are basophilic granules that in size and shape Frank and Dougherty (1953) were able to pro- look as if they were intracellular organisms, but duce budding in lymphocytes of man in vitro by tliis is incorrect; instead, they are some of the treatment of the buft'y coat with cortisone and hy- basophilic masses that are commonly found drocortisone. The mean percentage of lympho- throughout the cytoplasm of most lymphocytes cytes showing budding in normal controls was (figs. 90 and 96-98). The smear from which 1.2 ±4 percent, and for those treated with hydro- figure 92 was made was taken from a hen at 113 cortisone it was 11.63 ±1.25 percent. days. At 288 days she showed a prolapsed The outline drawings in figure 150 have al-

uterus from which an unexpelled egg was re- ready been mentioned. In some it appears that moved. She recovered and then was killed at lymphocytes are capalile of locomotion by throw- 312 days because of lack of holding space. The ing out broad pseudopodia, and many of the cells bird was still in production and apparently look as if they had been caught during amoeboid normal. movement. Others, however, have small pro- When the lobes are discharged into the plasma trusions more or less equally distributed around of the blood, there remain cells that look like the cell. These are proliably blebs of proto- figure 102, in which there is barely enough cytoplasm pincliing off. Many others, with almost

plasm around the nucleus to exclude it from be- no cytoplasm left, look as if they had already lost ing a naked nucleus. This cell came from a the blebs. smear taken at 108 days. Wlien the hen was killed at 668 days she was a strong vigorous jjird. Nuclear position.—The nucleus lies at one side At gross necropsy some urates in the kidney and of the cell (figs. 91, 94, 99, 101, and some of the white spots in the air sacs were found. cells in fig. 150) almost as often as it does at The evidence furnished by these few examples the center (figs. 90 and 104). Sometimes the is certainly not sufficient to permit us to draw any amount of cytoplasm is so small that the nucleus generalizations concerning the association of can only be in the center (figs. 92, 102, and 150 blebs and the health of the bird. No extensive D, c and F, b and e). quantitative study has ever been made along these lines; yet, obviously, this should he done Niicleocytosomal ratio.—Nucleocytosomal ra- and it might lead to some interesting and impor- tio is influenced not only by the pinching off of tant information concerning the actual health of lobes, which is a regressive type of change, but birds that seem normal. In any studv of this also by a change in character of the cytoplasm in sort it would ])e necessary to take blood samples which it takes on a hyaline appearance and flows at closely spaced intervals in order to catch the out like a thin fluid in all directions (fig. 100). rise and fall of blejj formation. The discharge This was found quite frequently in chicks alwut of jjlebs proljably occurs cjuickly. 5 weeks old. The cytoplasm was pale and as Johnson and Conner (1933) sought to asso- seen in the microscope gave an impression of ciate budding in lymphocytes with manifestations fluidity without adequate framework. These ot the avian leukosis complex. They state: lol^es are different from the type where the cyto-

"In 14 birds of the 31 studied with symptoms plasm is pinched off. Often it spreads out be- of paralysis of the limbs but no gross lesions no- tween the ])onndaries of nearby cells, and it was

51 in relation that were quantity of cytoplasm is great enough difficult to find examples for drawing to nuclear size to establish the cell as a monocyte, not misshapen beyond recognition. Even m ratio if only the one character of nucleocytosomal figure 100 two of the projections were flattened is used for identification. against adjacent erythrocytes. The example area is present in but its Sometimes a lightly stained shown is large enough to he a monocyte the cytoplasm adjacent to the nucleus. The cell structure is quite different from that of mono- classed as in which this is illustrated (fig. 97) is cytes taken from the same slide, an example of a lymphocyte, altliough in the table on lympho- which is shown in figure 125. cyte and monocyte characteristics, such a light In general there is less cytoplasm relative to for ex- area is more typical of a monocyte (see, the nuclear area than in monocytes, but again This is be ample, figs. 126, 127, 129, and 144). there is overlapping of their ranges, as may another example illustrating the point that the seen by comparing figures 150 and 151 and presence or absence of only a single characteris- graphs on figures 152 and 153. If the outline basis for a satisfactory separa- drawings on these two plates were cut apart and tic is insufficient over the separate tion of these two cell types. Looking and shuffled it would be impossible to group of lymphocytes from figure 90 to them correctly in many cases into the two cell entire cyto- figure 101, there is little question that the types; this fact emphasizes the point that separa- plasmic structure of these cells is highly diverse tion of lymphocytes and monocytes is based on and that no narrow characterization will cover more than just size, shape, nuclear position, and nucleocytosomal ratio. them all.

orange-stained spheres.—These are Cytoplasmic structure.—The cytoplasm may Hof and characteristic of monocytes and so will be dis- stain intensely or faintly. It may be granular or Specific cytoplasmic inclusions are nearly homogeneous. The granular condition cussed later. in cells, of not normally present in lymphocytes but is quite common and appears as a flocculation to be abnormal, they do exist as ma- basophilic material (figs. 95-99). These fig- considered the de- genta bodies (figs. 102-116) ; therefore ures illustrate variations in the size of the baso- of their structure will be deferred until ab- philic masses and in the intensity of staining. In tails cells are discussed. These magenta figure 98 the granules near the edge of the cell normal bodies are almost specifically associated with are large and dark, tliose near the nucleus are lymphocytes and thus are useful for identifica- smaller. In figure 96 the basophilic material purposes. gives texture to the cytoplasm but there are no tion distinct basophilic masses. The flocculent ap- Nuclear shape. The lymphocyte nucleus is pearance of the cytoplasm lies at the limit of — approximately round, as shown in figures 90, 93, microscopic visibility and sometimes the cyto- and in many of the cells in figure 150. plasm appears to be a reticulum with denser 105, Deviations from that shape are common and, as masses at the interstices, such as shown in figures would be expected, they occur by imperceptible 95, 99, and 101. structural differences in various directions. cells mentioned so far have been stained All the the When the nucleus is eccentrically placed, with Wright's stain. The cell in figure 91 was of the portion adjacent to the mass of with May-Griinwald Giemsa, which ac- contour stained remainder, cytoplasm is more flattened than the counts for a coloration different from the rest of (figs. 97, which is adjacent to the outside wall the cells. It also shows the basophilic masses in 101, and 107). If the cytosome of the cell is the cytoplasm but here they are more nearly often the contour of the nucleus is more definite than in the pre- lobulated, spherical and are with irregular also but not necessarily concentric vious examples. the cell outline. The cytoplasm in figure 90 is nearly homo- Indentation of the nucleus is not common in geneous with only slight irregularities in density. lymphocytes but does occur sometimes (figs. 91, Further change toward a hyaline structure is the depression is not 94, 111. and 114) . Usually shown in figure 100. When the cytoplasm deep and the margins cui-ve inward to a sharp reaches this hyaline condition it tends to flow out instead of forming a de- angle ( ^ ) away from the nucleus. In the hyaline type, the V

52 ;

pression with a rounded base as often occurs in arguments against the lymphocyte as an undif- monocytes ( ^•^ ^ ). In lymphocytes ferentiated blast cell has been the absence of there is usually no Hof opposite the depression mitosis, a process that has been observed only a the cytoplasm appears the same opposite the de- few times and then in birds that had been ir- pression as in the more lateral areas, but some- radiated. Cells undergoing mitosis are usually times the nucleus shows an indentation and there difficult to name because identifying characteris a rarefied area opposite. In figure 131 there is istics are lost in the process; however, in the such a cell and it has been classed as a monocyte, case of figure 108, magenta bodies were present but some might be of the opinion that this cell that have a high specificity for lymphocytes. should be included with the lymphocytes because Blood stains and air-dried fixation do not give a of its small size and high nucleocytoplasmic sharp delineation of the individual chromosomes ratio. Fortunately, borderline examples of this but there is no question that the cell is dividing type are not numerous. mitotically. It is interesting that the magenta bodies should arrange themselves approximately Nuclear structure.—The nuclei of lympho- midway between the poles. The division of cytes are typically described as filled with dense lymphocytes by mitosis is in contrast to that of chromatin clumps. Probably figure 102 fits the monocytes that divide by nuclear constriction. usual description best and a similar elfect is given in figure 90. Dense clumps of basichromatin are common in small lymphocytes but rare in Developmental stages found in circulating larger cells. In lymphocytes of medium size the blood reticular network with small chromatin masses at the interstices is the usual arrangement (figs. Probably less is known about the cytomor-

95-99 and 101 ) . As we study the change in cell phosis of lymphocytes than about any otlier leu- size from the larger to the smaller, it is easy to kocyte. Immature lymphocytes have been iden- find all corresponding degrees of change in chro- tified in the thymus where they are developed in matin clumping. Figure 98 shows two phases of large numbers, and it has been suggested that the process in the same nucleus—on one side figure 122 represents a young lymphocyte, but there is the reticular appearance of the larger in general they are difficult to identify. There cell and on the opposite side are dense clumps are identifying structural features, it is true, but commonly associated with the smaller cell. fitting them into a maturation series is not easy. When there is a delicate reticular pattern in The developmental series of lymphocytes de- the nucleus the spaces between are clear and picted in atlases of mammalian hematology are transparent—the nucleoplasm is colorless (figs. usually derived from cases of lymphoblastic 97-99 and 101). Wlien the nuclear pattern leukemia. Here the association with many other takes the form of dense clumps there is some dis- cells, all developing in the same direction, makes solution of basichromatin, which gives to the nu- a kind of pure culture, as it were, from which a clear sap a color almost as dense as the chro- developmental series can be constructed. After matin itself (figs. 100 and 104) . The wide vari- it has been put together there still is not much in ation in size of chromatin clumps and density of the way of specific cell identification by which an nuclear pattern is clearly demonstrated when a occasional immature lymphocyte, which one smear containing lymphocytes of small and me- might find in the circulating blood, can be satis- dium sizes has been fixed in Petrunkevitch No. 2 factorily distinguished from various types of and stained in May-Griinwald Giemsa (figs. 199 blast cells. and 200) . When this technic is used the nucleus The cuiTe for the sizes of avian lymphocytes of the medium-sized lymphocyte may resemble shows approximately a normal distribution (fig. closely the nucleus of the monocyte (compare 152). It is generally recognized that specific figs. 200 and 201). criteria useful for the identification of age and maturation are generally lacking in the lympho- Cell division.—Cell division is rare in cir- cyte series. Wiseman (1931b and 1932) con- culating blood and in fact its occurrence in sidered that the degree of basophilia of the cyto- lymphocytes has been questioned. One of the plasm could be used as such a measure and under

53 cells included in this section. It stress that there could l)e a shift toward greater normal all the but basophilia among the cells counted. This would is admitted that the evidence is not extensive opinion have the same significance for the lymphocytes it seems sufficient to tip the balance of noted that all that Arneth found for nuclear changes in granulo- toward the abnormal. It will be various kinds cytes. In a study of lymphocytogenesis in the cells from figures 102-116 show in table 3 these have been called thymus (chap. 4) it was concluded that lympho- of granules; are equivalent blasts are larger than the next stage in maturation magenta bodies and they probal^ly azurophilic granules in and that mature lymphocytes were relatively to what has been called is a small. With increasing age there was a loss of mammalian lymphocytes. "Magenta body" mitochondria and a change from delicate reticu- new term that does not carry any implication of expression has Ijeen lar pattern in the nucleus to dense chromatin function or origin. A new clumps. devised to replace the term "azurophilic granules" because the colors and affinities for the It is true that there are certain examples sug- granules in lymphocytes and in gestive of nuclear change from an open, lightly stain of these different that to give stained reticular type (fig. 99) to one showing monocytes of birds are so be confusing. This dense clumps of chromatin. Some nuclei, such both the same name would not occur in tlie mannnalian as the one shown in figure 98, sliow a transitional confusion does cell types the azuro- condition from reticulum to clumps. It is a agranulocyte because in both similar. It is not the question whether this nuclear variability is a philic granules appear designating measure of increasing cellular differentiation or presence of these granules that led to frequent an inseparable accompaniment of decreasing cell these cells as abnormal but rather their association with nuclei that are degenerating and size. There is no evasion of the fact that cell cytoplasm is blown up and size and nuclear structure are very closely asso- with cells whose magenta bodies stain intensely, ciated; and if chromatin clumping is a mark of vacuolated. The the mono- cytomorphosis, decreasing cell and nuclear size whereas the azurophilic granules of faintly, and have an must carry the same connotation. cytes are small, stain rather orange color. About the largest magenta liodies observed, as well as some small ones, are shown in figure 104. lymphocytes (figs. 102—120) Abnormal All of them are spherical, which is the usual they appear diploid It is anticipated that a number of hematologists shape, but not infrequently and 106. will question the accuracy of cataloging as ab- or crescent shaped as in figures 105

Figures 90-108.—Normal and al)normal lymphocytes. 2,470X.

Figures 90-101: Normal lymphocytes. 99 Nucleus largely filled with granular basichromatin. 100 Normal lymphocyte with hyaline cytoplasm. 90 A typical lymphocyte. 101 Nearly maximum size for a normal lymphocyte. 91 Lymphocyte stained with May-Gruiuvald Giemsa. 92 Lymphocyte pinching off cytoplasmic blebs containing basoplhlic granules. Figures 102-108: Abjiormal lymphocytes xcith magenta 9.3 Lymphocyte with hyaUne cytoplasm like figure 100, bodies in the cytosome. in the process of pinching off cytoplasmic blebs. of cytoplasm remaining after bleb 94 Lymphocyte with finely granular nucleus. 102 Small amount small magenta bodies are pres- 95 Typical lymphocyte with nucleus less dense than in formation. Several figure 90. ent. 103 Magenta bodies often in pairs. Slight autolysis Figures 96-99: Four cells that show stages between pachy- of the nucleus. chromatic and lepiochromatic nuclei. 104 Large magenta bodies. 105 Some of the magenta bodies are arranged in pairs. 96 Basichromatin of lower half of nucleus coarsely and Nucleus partially autolysed. finely granular, upper half in clumps. 106 Nucleus almost completely autolysed. 97 Granular and slightly clumped basichromatin bodies in a reactive lymphocyte. For intermingled. 107 Magenta the rest of this series, see figures 109-112. 98 Slightly clumped basiciiromatin in the right side and telephase of mitotic division, granular basichromatin in the left side of the 108 Lymphocyte in between poles in spindle region. nucleus. ^iagenta bodies

54 >

J.jr

90 91 92 93 94,

95 96 97 98

i». r 19'

102 103

99 100 101

v.? ^ ^

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104 105 106 107 108

55 Figures 109-124.—Abnormal and so-called large lymphocytes. 2,470X.

117-120: Abnormal lymphocytes that show varying Figures 109-112: Reactive bjmphocytes with magenta bodies Figures degrees chromophohia of the basichromalin. Same set and vacuolization of cytoplasm. of were taken. of slides from which figures 3 and 44-49 109 Vacuolization has begun but nuoleo-cytoplasmic 117 Lymphocyte with two chromophobio fractures. ratio is about like that of a normal lymphocyte. Chromophobic areas surrounded by normal basichro- 110 Lymphocyte with leptochromatic type nucleus; slight 118 hypertrophy of nucleus and oytosome. malin. 119 Normal basichromalin restricted to about one-third 111 Reactive lymphocyte. Typical lymphocyte nucleus. Magenta bodies and cytoplasmic vacuoles relatively of the nucleus. almost completely chromophobic nucleus. few. 120 An 112 Typical reactive lymphocyte; hypertrophy of the Figures 121-124: So-called large lymphocytes, which are cell cytoplasm, frayed and vacuolated and con- blood cells (blast cells). All found in taining abundant magenta bodies. early immature circulating blood of chickens 4 months to 5 years old. Figures 113-116; Questionable abnormal and reactive reticular nucleus type with nucleolus lymphocytes. These may be monocytes. 121 Delicate probably belongs to the erythrocyte or thrombocyte 113 Azurophilic granules, concentrated mostly above series. probably belongs to the lymphocyte the nucleus; lymphocyte type nucleus. 122 Blast cell; 114 Mixture of azurophilic and magenta bodies. series. probably belongs to the myelocyte series, 115 Magenta bodies and vacuolated and frayed 123 Blast cell; nucleolus is faintly visible. cytoplasm. although probably belongs to the erythrocyte or 116 Lymphocyte with a few pale magenta bodies. 124 Blast cell; thrombocyte series. From same slide as the preceding three cells.

56 ••I^ »

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109 110 111 112

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113 114 115 116

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117 118 119 120

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121 122 123 124

57 Figures 125-138.—Normal monocytes. 2,470X.

PiouRE.s 12.5-131: Variations in size and appearance of Figures 132-135: Monocytes with numerous azurophilic ?iormal monocytes. granules.

125 A typical monocyte. A hyaline mantle extends 132 Azurophilic granules concentrated on the .side of beyond the reticular portion of the endoplasm. The the nucleus opposite the Hof. cytopla.sm is a mixture of azurophilic and (basophilic 133 Hof absent. Aziu'ophilic bodies filling most of the substances. cell. 134 Bilobed nucleus. ."Vzurophilic granules conspicuous 126 Monocyte with well-developed Ilnf, or clear area, against the blue-stained cytoplasm. filling the nuclear indentation. 135 .\zurophilic granules larger and more abundant than 127 Monocyte nucleus but with a nucleocytoplasmic normal. ratio of the lymphocyte.

128 Deeply indented monocyte nucleus but no Hof. Figures 136-138: Bilobed and double nuclei in monocytes.

129 A type of monocyte frequently seen in smears of 130 Slightly constricted nucleus, similar to the one in avian blood. figure 134, which is a larger cell. No Hof present in either of these cells. 1.30 A monocyte that shows the polychromasia of the 137 Nucleus nearly completely constricted. Metachro- cytoplasmic reticulum. matic cytopLasm. A small questionable Hof present. 131 A monocyte having the size and nucleocytoplasmic 138 \ binucleated monocyte. A definite Hof on the ratio of the lymphocyte. See table 5, page 71. upper side in the angle between the nuclei.

58 t)

125 126 127 128

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129 130 131

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132 133 131 135

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136 137 138

59 Figures 139-149.—Developmental stages of monocytes as found in the circulating blood, also abnormal cells and artifacts. 2,470 X.

Figures 1.39-142: Developnienla! stages toilh intensely Figures 145-147: Abnormal monocytes. stained nuclei, ranging from leplochromatic to pachychromatic type. 145 The accumulation of basophilic granules around

the periphery of the cell is not typical. The faint cell of the series; 1.39 The young amoeboid monocyte reddish tinge of the cytoplasm is suggestive of early possibly a monoblast. A nucleolus is faintly shown. autolysis. 140 Amoeboid monocyte with both azurophilic granules 146 A monocyte definitely undergoing autolysis. and magenta bodies. 147 Some Hof vacuoles have become abnormally large. 141 A round young monocyte. Vacuolar content stains a light orange color. 142 A young monocyte with a rounded nucleus. The cell has a well-developed Hof, and a concentration of Figures 148, 149: Technic artifacts. Smudged monocytes. azurophilic granules around the periphery.

148 .\ monocyte in which the cytosome, but not the Figures 143, 144: Developmental stages with lightly stained nucleus, has broken. nuclei. 149 Identification of this as a squashed monocyte 143 Amoeboid monocyte, more differentiated than the nucleus is b.ased on the fact that this slide contained blast stage. many such cells with transitional stages from normal 144 Nearly mature monocyte. The contents of the Hof intact monocytes to this type of large basophilic vacuoles are slightly stained. mass.

60 A.

r-'

'*'i ^,

142 140 139 141

#-;

143 144 145 146

^ ,r

147

149

148

61 A

C a^ b ; d

p ^ h c d c FiGUKE 150.—Outline of normal lympliocytes and their nuclei to show variations in size and shape within and between breeds or stocks of chickens. 2,470 X. from State a-e Ancona, from Hy-Llne Poultry Farms, Iowa. D, a-e Single Comb White Legliorn, Iowa College, Genetics Department. Poultry a-e Barrod Plymouth Rock, from Hy-Line ^ ^^ Single Comb White Leghorn, from Hamilton ' Farms, Iowa. Farm Bureau, Michigan. Bureau, C, a-€ New Hampshire, from Hy-Line Poultry Farms, F, a-e Rhode Island Red, from Hamilton Farm Iowa. Jlichigau. 62 Figure 151. —Outlines of normal monocytes and their nuclei to show variations in size and shape within and between breeds and stocks of chickens. 2,470 X.

Same slides and presented in tlie same order as in figure 1.50. 63 .

resemblance to those of lymphocytes than Wlien the nucleus stains darkly, the overlying closer macrophages. The nucleus of the magenta bodies that have the same staining af- to those of was studied in blood spots (Lucas, finities are not clearly visible (figs. 104 and 109) macrophage and in cardiac punctures of the embryo When the nucleus is extensively lysed as in fig- 1946) where the typical chromatin pat- ures 105-107 the overlying magenta bodies are (figs. 309-317) uniform reticulum with evenly dis- usually conspicuous. tern was a bodies of chromatin at the inter- No one has yet associated the presence of ma- tributed, dotlike network. This punctate appear- genta bodies with any particular pathological stices of tlie macrophage nucleus is quite different condition in birds, but the impression has been ance of the delicate reticulum or massive irregular gained that they are often associated with birds from the of the typical lymphocyte. that have a disposition toward early mortalhy. clumps McKinlay (1923), and Blackfan, Olson (1952) noted them in lymphocytes taken Downey and and Leister (1944) illustrated and de- from cases of avian leukosis but he does not say Diamond the lymphocytes found in cases of in- that they are specifically associated with the dis- scribed fectious mononucleosis. The azurophilic bodies ease. There is much speculation concerning the vacuolated cytoplasm of human lympho- presence of a lymphomatosis agent of viral size and the in infectious mononucleosis simulate, in that, by acting upon the lymphocyte, ultimately cytes bodies and hypertrophied, stimulates the cell into a neoplastic condition. part, the magenta of avian lymphocytes, These magenta bodies may have no relationship vacuolated cytoplasm be seen in some seemingly normal to lymphomatosis infection but their sporadic ap- which may Although chickens do not carry the vims pearance in lymphocytes of chickens would seem birds. infectious mononucleosis, as far as we know, to justify further study in an effort to find out of not be ignored that lymphomatosis what they are and what they are doing in the cell. the fact should disease, and that it has a stim- Their presence does not necessarily indicate a is also an infectious effect on the mononuclear cells accumulat- dying cell; mitosis can occur (fig. 108). ulating the avian body (Lucas, Michaelis and Wolfe (1902) are said by Doan ing within the tissues of Lucas, Craig, and Oakberg, 1949; Lucas (1932) to be the first to describe azurophilic 1949; 1950; Lucas, Denington, Cottral granules in lymphocytes. They used blood from and Oakberg, Oakberg, 1949, 1951). man. Their description agrees closely with the and Burmester, 1954; described fenestration in lym- observations made on avian blood. They estab- Osgood (1935) phocytes from cases of mononucleosis. He ob- lished the fact that it was actually a lymphocyte vacuolization and canaliculization of that contained the granules. In circulating served a somewhat similar to, yet different blood about one-third of the lymphocytes con- the nucleus of nuclei of avian cells. tained these granules, but they were absent from from, the clefts further variant of the magenta bodies is the lymphocytes of lymph nodes. The signifi- A figures 113-116, in which the colors cance of these granules is still not adequately shown in a pale orange to the typical magenta established. range from of these cells show the slight nu- Another type of cell response that produces color. Some lysis that already has been mentioned as atypical lymphocytes is hypertrophy with vacu- clear in lymphocytes with magenta bodies. olization of the cytoplasm (figs. 109-112). The occurring cytoplasmic inclusions in figure 114 show stain taken by the cytoplasm is faint compared The gradation from the light to the dark type of with that of the typical normal lymphocyte. The but the direction in which the reaction vacuoles may be small as in figure 109, irregular granule are going is not known. Cells of this type in size as in figure 111, and large as in figure 112. is be noted that these cells came There may be few or many magenta bodies and rare and it should birds having the same parents. In these these may show considerable variation in size; from same slides monocytes were present that seemed they may lie either in the vacuoles or between have an unusually large quantity of azuro- them or at their margin. From the appearance to granules. The history of this family of of the cytoplasm in which many vacuoles are philic birds is interesting. All were hatched June present, a transformation toward a macrophage nine 1944. The blood smears were taken at 112 might be suggested. The nuclei of such vacu- 19, days of age, except from F205C5. olated cells as seen in figures 109-112 show a

64 F205W4—Female. At 66 and 115 days reactions were disappearance of basichromatin. Figures 117— normal but at 163 days there was a slight loss of 120 show a transitional series leading to an weight. Died at 166 days. Questionable vis- "empty" nucleus. ceral lymphomatosis. F205X4^MaIe. Showed gasping at 163 days. Died The cytoplasm of erythrocytes was not affected at 339 days. Extreme emaciation and atrophy when the nuclei showed chromophobic bands; of the musculature. about the only difference noted in lymphocytes Male. At 115 and 163 days bird showed F205Y4— was a somewhat more vacuolar organization than vigorous reactions. Died at 518 days. Bird is generally found in lymphocytes. The possi- appeared to be in good condition. Cause of death undetermined (figs. 113-115 and 134). bility diat these nonstaining effects are due to F205Z4—Female. At 163 days reactions fair. At teclmic is not excluded but for the present it seems 253 days ill and put in a separate cage. Died at more likely that they represent some abnormal 292 days. Emaciation and impaction of crop disturbance of the cell. If technic produces the (fig. 133). defect, we have the question of how it alone could F205A5—Female. At 110 days showed poor reactions and at 151 days ill and put in separate cage. cause the chromatin to disappear and yet leave In moribund condition and killed at 157 days. so sharp a boundary with the basichromatin that Neural and questionable visceral lymphomatosis. remains. If the defect truly represents cell pa- F205B5—Male. At 163 days reaction fair. Died at thology, tracing it back to the disease condition 658 days. Cause of death undetermined. that is producing it should be relatively simple. F205C5—Female. Moribund at 75 days. Killed at 77 days. Lymphomatosis, neural. F205D5—Male. At 115 days reaction was fair. Un- steady on feet at 155 days and moribund at 165 days. Killed. Lymphomatosis, neural and MONOCYTES ocular. F205E5 Female. At 65 days reactions were fair. — Normal mature monocytes (figs. 125—138) At 126 days atonicity of crop. Moribund at 151 days and killed. Emaciation and dehydra- The monocyte cytoplasm has been described in tion (figs. 93, 116, 130, 132, and 142). the literature as having the appearance of glass Various conditions were shown by the nine beads, and this effect is given as an important birds, both clinically and at necropsy, and all characteristic for separating monoc^ies from died before they were 2 years of age. At the lymphocytes. This cytoplasmic quality prob- time the smears were made the birds seemed to ably does aid in the identification of these cells

be reasonably healthy but in their subsequent but actually it is of little value as a sole point of history their performance was poor; thus it would reference. The necessity of using multiple seem inadvisable to represent some of these as characteristics has already been emphasized. normal cells from normal birds. It certainly Owing to the overlapping of characteristics of should be determined, however, whether cellular lymphocytes and monocytes, it is necessary to reactions of this type have any value in under- consider each of their 10 points of difference. standing the causes of death in birds that die from These will be taken up in the same order as they tumors or in a generally unhealthy condition. were for lymphocytes. Another type of abnormal cellular reaction was found in the slides that showed, in the nuclei Cell size.—The ranges in size of lymphocytes of erthyrocytes, chromophobic bands that usually and monocytes overlap as already seen in tlie had a uniform width. In lymphocytes these comparisons from outline drawings and graphs. spaces were quite irregular (figs. 117-120). It is obvious that on the average the monocyte is The clefts in figure 117 are irregular; yet, as in larger than the lymphocyte; in fact, the average the erythrocytes, there is no actual break in the area of the whole lymphocyte is slightly smaller cell membrane but rather a sharply demarked than the average area of the monocyte nucleus. transition from a densely stained area to one that The maximum, minimum, and average values did not take the stain; in the latter, a very faint in table 4 are based on 300 planimeter measure- reticulum can be seen. The lack of staining can ments of cells projected and traced with a camera aftect small lymphocytes with lobes (fig. 118) as lucida. Half of these were from our Labora- well as larger cells (figs. 119 and 120). What- tory chickens and half from two different breeds

ever the reaction may be, it can cause complete from a commercial laboratory. The possibility 65 of breed or inbred line differences is discussed in chapter 6. These curves indicate that nuclear size is much less variable than tlie area of nucleus plus cytosome (fig. 153). For the nucleus of l)oth lymphocytes and monocytes the size is sym- metrically distributed on each side of the mean; whereas, when cytoplasm is added, there is a definite tendency toward skewness with relatively few low values, but a trailing off in incidence of the high values. Approximately 25 percent of the lymphocyte nuclei overlap 22 percent of the monocyte nuclei in size, whereas only about 15 percent of the lymphocyte total cell area overlaps 7 percent of the monocyte cell area. Another interesting point brought out by table

4 is the broad range from minimum to maximum areas, which for lymphocytes is 9.2-fold and for

monocytes is 3.8-fold. In terms of diameters one could ex^ject, therefore, a 3-fold range for lymphocytes and about a 2-fold range for mono-

cytes. Since there is a 3-fold range in diameters, one may separate lymphocytes arbitrarily into 3 categories—small, medium, and large. Cells with a diameter up to 7.8m would be classed as small, those from 7.9 to 10.3/x as medium, and those above 10.4/x as large. Of the 300 lymphocytes measured and plotted, 55 percent would be classed as small, 36 percent as medium, and 9 percent as large.' Blast cells have not been in-

" Wiseman (1932) reported that in dried blood smears from man the range for small lymphocytes is considered to be from 6/j to 9/x; for medium, from 9/i to 12/j; and for large, 12^-|-.

Table 4.—Nuclear and cell areas and diameters for lymphocytes and monocytes

Cell measurement 100 MONOCYTES

cell an'Li

AREA m p'^

Figure 15.3. —Frequency dislriliiition curves of cell area and nuclear area foi' .300 nionocyles cliosen at random.

same usefulness for the agranulocytes that the len (1903) finds that monocyte diameters range Arneth counts do for the granulocytes. from 6.7 to 9.3^; l)olIi of these values are below Mainland et al. (1935) studied statistically the average given in table 4. Magath and Hig- the significance of lymphocyte size in the human gins found the average to be Jietween 11 and species and concluded that cell size was a uni- 13.5. modal curve, but with skewness in most cases, and tliat a small and a large lymphocyte did not Cell shape.—The variety of shapes exhil)ited exist as classes. From their data, there was no by the monocyte can be appreciated best by glanc- indication that age, sex, or state of health were ing over figures 125-138 and the cells outlined factors influencing the size of lymphocytes in in figure 151. In general, the monocyte has a man. round shape. Occasionally there may be small These opinions on size are in agreement with bleb projections such as seen in figures 126, 127, those of Magath and Higgins (1934), who and 138. Whether they serve the same function measured the diameters of lymphocytes from in monocytes that they are said to sei-ve in lym- each of eight ducks and concluded that they all piiocytes, namely, contributing globulins to the

I (('long to one series with a size range from 4.0 plasma, is not known. to 8.1 M and that all attempts to classify them There is another type of cytosomal protrusion into small, medium, and large were futile. Cul- that occurs rather frequently. It is a hyaline

67 Nucleocytosomal ratio. Not only are mono- mantle that extends iu irregular fashion beyond — larger than lymphocytes on the average but the denser endoplasmic portion of the cell (figs. cytes mantle in the latter the cytosome is proportionately larger than it is 125 and 143) . The hyaline in lymphocytes. This difference is not so great, figure is somewhat more lightly stained. In con- as superficial examination of cells might trast to the mantle, the central reticulum retains however, The eccentric position of an approximately round shape. lead one to believe. there is a Weiss and Fawcett (1953) mention the ex- the nucleus gives the impression that of cytoplasm; but when the same istence of a mantle in avian monocytes when large mass of cytoplasm is uniformly distributed grown in tissue culture. The mantle was found quantity a centrally placed nucleus there seems to to be visible when cells were examined under the around narrow rim. This false impression is phase microscope. It is not altogether clear that be only a the indentation of the monocyte nu- the type of mantle (thin undulating membrane) magnified by nucleocytosomal ratios for the two they obsei-ved, and the type we have described cleus. The are given in table 4. They average are identical. Weiss and Fawcett believed that cell types lymphocytes and 1:1.12 for mono- mantle formation is part of the process whereby 1: 0.47 for cytosomal area in monocytes become macrophages. Additional cytes. In other words, the area in monocytes is 58 per- support for the idea that monocytes may become relation to nuclear terms of macrophages comes from Dawson's (1933a) cent greater dian in lymphocytes. In diameters the ratio is 1:1.22 for lym- study of blood cell reactions to lead poisoning in nucleocell monocytes. Necturus. phocjrtes and 1 : 1.46 for Since monocytes are large cells they are distorted more readily during the process of making Cytoplasmic structure.—^The descriptive term dried smears than are small cells. The cyto- "ground-glass effect" has been used by numerous some and even the nucleus often are squeezed be- writers on avian blood. It is important to try to are that tween other cells, and so take various angular understand what the structural elements shapes, or they may overlap other cells. These create the ground-glass illusion. Actually, we variations occur most readily when there is a have failed to see what others call the "ground- following three hyaline mantle. If, however, they happen to fall glass effect." Any one of die responsible for in an open space free from other cells, they elements within the cell might be usually show a round contour and cells such as the optical effect: such as these are the ones usually selected for drawing. (1) The open reticular framework seen in figures 138 and 143, where there exist uniform spaces bounded by a delicate Nuclear position.—The nucleus in many numerous reticulum. These spaces, however, have low re- monocytes is eccentrically placed, as may be seen fractility; the term "ground-glass" suggests in figures 126-133 and 135; sometimes it forms conspicuous refractility. a bridge across the middle of the cell (figs. 134 rather orange-stained substance and 136). The wide diversity of position is (2) The delicate that sometimes fills the vacuolar spaces in the shown in the outline drawings. In the first row (fig. 144). Figure 126 shows similar only 1 of 5 could be said to have the typical or Hof the but the material that fills them eccentric position. The proportion is about the spaces in Hof not taken an orange color. When the orange- same in the other rows. The ratio of 1 eccentric has substance is present, it increases the con- nucleus to 5 centrally placed is probably not a stained reticulum and thus gives the illusion true one because a dispropoi-tionate number of trast with the greater refractility. This may be what has cells selected for making the outline drawings of ground-glass appearance of the were atypical but were selected to show as wide been called the cytoplasm. a range of morphologic expression as possible. monocyte polychromatic reaction of the proto- The eccentric position of the nucleus is of con- (3) The

and , which gives siderable value in distinguishing between a mono- plasmic network (figs. 130 137) effect to the cytoplasm. The inter- cyte and a lymphocyte but it must be borne in a textural network that in the previous figures mind that a monocyte can have a centrally-placed stices of the light or a dense basophilic stain nucleus (fig. 151, A, b and B, a), as do many took either a mentioned a shift in lymphocytes. show in the last two cells

68 staining of this substance in an azurophilic di- 142 is called a Hof also, although it is obviously rection. Because of the mixture of sahnon and not encircled or even bounded by an identation bhie colors, the cytoplasm has been given a tex- of the nucleus. The area, nevertheless, is clearly tural effect that does not occur when the same demarked from the rest of the cytoplasm. Fig- sti'ucture shows a uniform single hue. Dual ures 128 and 133 are good examples of cells that coloration of the reticulum gives the optical effect show indented nuclei but there is no Hof in the of thickness and density. Of the three structural sense in which the term is used here. The Hof elements listed, this is the one that offers the shown in figure 129 is rather indistinct, and in best explanation of the "ground-glass effect." figure 132 it is present as a broad space that fills There is only one other possibility—the pres- most of the cytosome on one side of the nucleus. ence of definite azurophilic granules. They, The next three figures contain none. The vacu- however, are relatively rare (figs. 132—135) olar space opposite the indentation of the nucleus and thus would not be mentioned as typical for in figure 131 could be called a Hof, as could the monocytes. Some of the cells shown in the draw- clear space in the lymphocyte (fig. 97). The ings came from the same parents that produced Hof is nearly always found in monocytes but the reactive lymphocytes, as has already been there may be exceptions, as already mentioned. stated. The azurophilic bodies may give to the Just how closely associated are the Hof and monocyte a tinged margin (fig. 142). The only the rosette obtained with neutral red vital stain other cell type that shows a tinged border of this is not known. It is assumed that they are closely sort is an immature thrombocyte (figs. 296-299 associated but it has not been determined whether and 303). This particular monocyte shows a cells that fail to show a Hof, in the sense in which well-defined Hof that helps to identify it as a die term is used here, would also fail to show a monocyte. In other examples drawn from the rosette. same family of birds (fig. 132, 134, and p. 65), there is an increasing number of azurophilic Specific cell inclusions.—^The vacuolar spaces granules in tlie cytoplasm. Sometimes they are of the Hof often contain a homogeneous sub- on the side of the nucleus opposite the Hof; at stance that takes a very faint orange color with other times tliey are on the same side; and they Wright's stain, a stain that is better for diis pur- may be more or less uniformly scattered over pose than May-Griinwald Giemsa. Perhaps tlie the whole cell. Figure 135 is an extreme ex- orange spheres could be classed as an azurophilic ample. Here the azurophilic bodies are larger substance also, although the coloration is dis- than normal. They show considerable variabil- tinctly more yellowish than in the small azuro- ity in staining intensity, and in some respects philic granules described under the heading resemble an early heterophil myelocyte. This "Cytoplasmic structure." It takes an excellent type of azurophilic granule is so atypical for light source and microscope correctly used to monocytes in general that only rarely indeed show any tinge of color in the Hof; yet it is a real would a cell of this appearance be picked up in substance, as may be demonstrated in the abnor- a differential count. mal cell, figure 147, where the Hof substance has become concentrated into large spheres. Hof and orange-staining spheres.—The Hof All three substances— (1) the azurophilic has been mentioned a number of times as a lightly granules, (2) die azurophilic tinge of the retic- stained, vacuolated area in the cytosome that has ulum, and (3) the orange spheres of the Hof— considerable value in the identification of mono- are useful in the identification of the monocyte cytes. It may or may not contain orange-stained and carry much weight in separating monocytes material. The meaning of the tenn as used here horn lymphocytes. The only specific cell in- is slightly different from that stated in the broad clusions found in lymphocytes are the magenta definition given in Borland's Medical Diction- bodies, which are nearly always darker and more ary—"The area of the cytoplasm of a cell en- intensely colored than any of the three listed for circled by the concavity of tlie nucleus." This monocytes. definition may fit quite well in some cases. Typical examples of a Hof are shown in figures Nuclear shape.—Nuclear shape needs very 126, 138, and 144. The clear space in figure little additional discussion. The difference be-

69 involving tween the shape of the indentation in lymphocytes each of these cells. Yet any sui-vey lymphocytes will and in monocytes has been mentioned, and the hundreds of monocytes and general examples shown in the various figures of mono- clearly reveal thai each type has its own nu- cytes bear out the observation that the nuclear pattern. It will also reveal that monocyte those with a delicate depression is usually broad with a rounded bot- clei fall into two groups— coarse blocks. tom, as diagramed on page 53. open reticulum and those with The presence of this type of indentation is The same differences may be found in the im- helpful in identifying monocytes but certainly mature stages (figs. 139-144). it cannot be relied upon entirely; many mono- indentation of the nucleus cytes have round or elongated nuclei without any Cell division. —The is often carried further, depression (figs. 151, B, d, C, c, D, c, and E, c). so common in monocytes Some have irregularly shaped nuclei (figs. 151, leading to various degrees of constriction that center from two sides (figs. A, d, B, b, D, b and e, and F, d) or double in- may approach the nucleus from one side only dentations (figs. 136, 137, and 151, C, e), and 136-138) or cut the complete di- sometimes the indentation cuts the nucleus into (fig. 151, B, e). This may lead to into parts. When they two equal or unequal parts (figs. 138, 151, B, e vision of the nucleus two cell division by and F,b). This variability must be kept in mind are equal in size, they suggest of unequal nuclei when a differential count is being made so that amitosis; but the occurrence whether some monocytes will not be omitted from the (fig. 151, F, b) raises the question of any more re- count because they have atypical nuclei. this type of nuclear duplication has lationship to amitosis than has the lobulation of the granulocyte nucleus. No actual pulling Nuclear structure.—Nuclear structure is a apart of the cystosome to form two cell l^odies has pattern that is often viewed impressionistically ever been observed. Constriction of the nucleus without giving delil^erate attention to tlie parts. is much more frequently found in monocytes than Wlien the pattern is carefully studied, it breaks in lymphocytes, and this is a useful morphologic down into a complex of interrelated details sucli feature that aids in separating these two as size, shape, and distribution of chromatin leukocytes. clumps, the character of the reticulum and its relation to the basichromatin and the tinctorial re- Conclusions derived from use of table 3.—Fre- actions of the nucleoplasm. When viewed quent mention has been made of the fact that one superficially the monocyte nucleus gives the im- cannot decide whether a particular cell is a mono- pression that it has a delicate lacelike reticular cyte or a lymphocyte without considering numer- pattern of chromatin and a transparency that ous characteristics, which must be balanced is not generally obsei-ved in the lymphocyte against one another. A few cells, questionable nucleus. ones as well as those that are obviously of one Upon close examination, it may lie observed type or the other, have been presented in talndar that the clumps of clu'omatin at the interstices of form in table 5 to show how the various charac- the network are often small, as in figure 127. given in table 3 have been applied. Ta- Sometimes they may be relatively large and teristics ble 5 should make clear why some questionable dense, as in figure 125, which has a highly have been classed as monocytes instead of colored nucleoplasm and thus would not give a cells lymphocytes and vice versa and it brings out that transparent effect. In lymphocytes a reticular will sliow a characteristic that is appearance was often found associated with oftentimes a cell as frequently found in monocytes as in lym- larger nuclei. Whether the more open reticulum just and tliere may be a "-!-" in both rows and smaller chromatin clumps commonly found phocytes, of the table. in monocytes represent a characteristic difference between tlie two cell types, or are nothing nuclear size, is more than a reflection of the larger Developmental stages found in circulating undetermined. Nuclear pattern carries relatively blood (figs. 139-144) little weight in the separation of the two cell monocytogenesis are types, chiefly because a definite type of chromatin Lymphocytogenesis and because organization cannot be considered specific for vague and controversial subjects, chiefly

ro Table 5.—Classification of individual cells into lymphocytes and m onocytes '

Cell Figure type 2

94 Abnormal monocytes (figs. 145—147) worth (1937) in speaking of them say (p. 20), "These cells are probably not artifacts made in The two cells shown in figures 145 and 146 smearing but remnants of dead cells." are classed as abnormal because they are under- Kracke and Carver (1937) mention (p. 84) going early autolysis. In the former there are that in mammalian literature smudged cells have only early manifestations of cell breakdown, the been divided into two types: "It has been stated vacuolization in the region of the Hof has become that smudge forms are degenerating lymphocytes amorphous, and the remainder of the cytosome and that basket cells . . . are degenerating has an atypical tinge of color. The concentra- granulocytes. . . . Nevertheless, it seems more tion of basophilic bodies at the cell margin is probable that the smudge cell is an eaily stage probably not indicative of abnormality. and the basket cell a late stage of the same A somewhat later stage in autolysis is shown process. in figure 146, in which the cytoplasm has taken "Crushing and iiipturing of monocytes, neu- on an overall reddish color and there is a de- trophils, eosinophils and basophils . . . occur creased color difference between nucleus and in improperly made smears, especially when too cytosome. Otherwise the structural breakdown mucli pressure is applied to the drop of blood. has not been extensive. There is no indication These cellular remnants are found in various ab- whether the nucleus will go in the direction of normal states where there is excessive destruc- lysis or of chromatin clumping. tion of leukocytes. In these cases their occur- Abnormality has been expressed in still an- rence is probably the result of toxic agents or of other way (fig. 147). The orange spheres of an increased fragility of the cellular elements." the Hof increased in size whereas ordinarily they This point of view is in agreement with our are relatively uniform. The increase might be opinion that the actual production of smudged due either to an abnormal growth of a sphere or cells comes at the time the smear is made and that to the coalescence of adjoining spheres accom- it is not a record of in vivo degeneration of leuko- panied by changes in the reticulum. Aside from cytes. The basis for this opinion is the conspic- this one point there is very little indication that uous difference between smears made from the the cell is abnormal. same bird at the same time and also the fact that large numbers of smudged cells often occur at thinned-out portions of the smear where presum- Technic artifacts (figs. 148, 149) ably the pressure is greater. This point of view is not in conflict with the idea that there are dif- Only type of technic artifact has been one ferences in cell fragility and that increased fra- found thus far in monocytes a squashing of the — gility may accompany diseased conditions. cell when the smear is made. A squashed mono- Osgood and Ashworth (1937) say (p. 20) cyte is rarely identified with certainty. In fig- that, "One should not, however, make the error ure 148 the large size and the indented nucleus of omitting to include the disintegrated cells in aided in the exclusion of other cells. The mono- the differential count as a large number of disin- cytes, for some reason, were the only cells in tlie tegrated cells is strongly suggestive of a diag- slide from which figure 149 was taken that showed nosis of leukemia and failure to include them fragility. This slide gave an excellent series of may give an eri'oneous impression of the true stages from a slightly squashed monocyte to the incidence of other cell types." extreme condition shown in figure 149. Wlien We have not found at this Laboratory that the it reaches the last condition there is nothing by presence of smudged cells aids in the diagnosis which the squashed cell may be identified as a of the avian leukosis complex, but it is agreed that monocyte. Its size so far exceeds the size the of smudged cells can influence the differential count. normal nucleus that it has very little monocyte A good example is the slide from which figure meaning. The significance of the configuration 149 was made. Smudged monocyte nuclei simi- of such cells has been discussed in mammalian lar to the one illustrated were found in abun- literature, where they are called basket cells and dance; yet intact monocytes were scarce and were disintegrating cells when they have the appear- actually fewer than the smudged nuclei. Should ance shown in figure 149. Osgood and Ash- they be counted? It has been our experience

72 with avian blood that it is impractical. In mam- by the increased source of error ; and if some can- malian blood only the leukocytes have nuclei, not be properly classified, those that can be iden- and so any smudged nucleus must at least be a tified, such as the three granulocytes, should not leukocyte. But in avian blood where the ery- be included either. On the other hand, if in a throcytes and thrombocytes are nucleated also, particular bird the smudging affects only one of the possibility of error in identification of a the types of white cells, some account must be smudged nucleus becomes so great that any pos- taken of the fact, or the difterential values will be sible value of its addition to a count is nullified biased.

Granular Leukocytes

There are three granular leukocytes in birds as of smaller cells may be liberated. Perhaps this in mammals—heterophils, eosinophils, and baso- reflects a factor of cytoplasmic growth, independ- phils. Eosinophils and basophils received their ent of seeming maturity that is indicated by nu- names because the cells contain granules that clear lobulation, in that smears made from bone have an affinity for eosin and for basic dyes. marrow show many more small heterophils than The term "heterophil," which was suggested by are found in circulating blood, and yet these cells Kyes (1929), applies to the third granulocyte show multilobed nuclei and in this respect are cell type, in which the specific inclusions of ho- considered to be mature. This fluctuation in size mologous cells among the various classes of ver- of heterophils, and also of eosinophils, is a factor tebrates show great diversity in reaction to stains. that may account for differences in size reported The heterophil of birds and reptiles is the by different investigators for these cells. If one equivalent of the neutrophil in man. The term emphasized the size of heterophils as some have "neutrophil" is based on the staining reactions; emphasized the size of lymphocytes, it might be the temi "heterophil" is not. that cell size would prove to be a criterion as use- Ryerson (1943), from his comparative ful as number of nuclear lobes in indicating the studies, suggests that the morphology of hetero- condition of health of the l)ird. phils and eosinophils has been influenced by two From these figures of heterophils it can be lines of evolution through the vertebrate classes seen that the dominant shape is a circle, which is

(p. 44)," . . . one line contains the selachians, sui-prising in view of the fact that in life heter- reptiles, and birds; the other contains the cyclo- ophils are actively amoeboid. stomes, teleosts, amphibians, and mammals." In a strict sense practically none of tlie heterophils from circulating blood should be regarded as representing their true structure and appear- HETEROPHILS ance. Nearly all these cells reveal an artifact involving the nucleus, and many reveal a second artifact that involves the rods. The artifact Normal mature heterophils (figs. 154— in the nucleus is so constant with Wright's stain 167) that it is of considerable value in identifying the Heterophils as usually seen in the circulating cell type, but the one in the rods is an ever-present blood show a low variability in size. Some in- cause of confusion between heterophils and dication of range may be obtained by comparing eosinophils. the various drawings and, particularly, the graph The rods, which are the specific inclusions for (fig. 197). Kennedy and Climenko (1928) heterophils, have typically the appearance shown gave a range of 4.2 to 9.0/u with an average of in figures 154 and 155. They are long fusiform 6.35m, which is nearly 2.3m less than the average bodies, they are pointed at each end, and they of 8.7m from these data. Under stimulation take the eosin stain brilliantly. Both Dant- such as replacement after irradiation, a shower schakoff (1909a and b and 1916b) and Hamre

73 ,

(personal communication) have obsei-ved that The research necessary to prove or disprove the in some cells the rods are grouped in a fanlike point has never been undertaken Init the results cluster. This arrangement has never been ob- obtained with Wright's stain gave no indication served in mature cells in any of our smears; in- that every rod has a central granule and the same stead the rods are always scattered like a pile of opinion is supported by evidence from a study short straws with pointed ends. Ryerson ( 1943) of developmental stages in bone marrow and in his study of heteropliils in tuilles, shows a spleen stained with May-Griinwald Giemsa. radial arrangement of the rods in the myelocytes Even in squashed cells where the individual rods but not in the mature cells. There may be con- are thrown apart from each other, central gran- siderable variability among cells in the length of ules may be absent in one case (fig. 175) and these rods. Some are long and narrow (fig. present in another (fig. 176). Additional evi- 154) and others short and relatively broad (fig. dence on this point comes from the fact that in 2, 1). In some cases these differences can be cells where the rods have disappeared there may characteristic of a species (figs. 400, 401, 403, remain numerous central granules (fig. 161) or and 406.) they may be absent (fig. 165). In many heterophils some rods may contain Sometimes instead of a granule there may be central granules. Hamre is of the opinion diat a vacuole or at least a nonstaining clear space in each rod contains a central granule that is re- the center of each rod (figs. 166 and 167). In vealed when Wright-Giemsa is used (p. 230). the second figure most of the rod substance has

Figures 154-176.—Heterophils from circulating blood—mature, immature, and smudged cells. Some evidence of technic artifact is apparent in practically all normal mature heterophils. (The terms "central bodies" and "central granules" as applied to heterophils are synonymous.) 2,470X.

Figures 154-167: Normal mature heterophils selected to 166 Heterophil in which the central bodies appear as shoiD variahility in structure and size of cells and in vacuoles within the rods. staining defects. 167 Heterophil in which the rods have contracted around the vacuolar-type central bodies. 154 Well-preserved rods, without central granules. Nuclear lobes are poorly stained. 155 Rods with a few central bodies. Variable staining Figures 168-173: Developmental stages of heterophils found of the nuclear lobes. in circulating blood. 156 Approximately one central granule per rod. Nuclear 168 Heterophil granuloblast. No nucleolus visible. lobes stained. are fully Found on same slide with heterophil mj'elocytes, bodies are large 157 Partial dissolution of rods; central figures 169-172. staining of and do not disappear. Variable the 169 Heterophil promyelocyte. Nucleus in lower two- nuclear lobes. thirds of cell appears to merge with the cytosome. 158 Nearly complete dissolution of rods; the rod sub- Magenta granules and rings identify this as a stance gives to the cytoplasm an overall pink color heterophil. with Wright's stain. Central bodies are small. 170 Heterophil promyelocyte. Approximately the same 159 Nearly complete dissolution of rods, which gives an stage of development as preceding cell, but smaller. intense color to the entire cytosome. The outer 171 Heterophil promyelocyte. portions of the nuclear lobes are well stained; inner 172 Heterophil promyelocyte. Greater vacuolization parts are poorly stained. of cytoplasm than in preceding cells. Nucleus is not 160 Small only central bodies of heterophil showing yet clearly separated from cytosome. varying size. 173 Heterophil mesomyelocyte. Early stage in the 161 Heterophil only central bodies visible. The with differentiation of the specific heterophil rods. large central bodies of this and the preceding cells cause these cells to resemble the eosinophils, but they Figures 174-176: Technic artifacts. can be separated by the difference in color of the

cytoplasmic ground substance. Compare with fig- 174 Mature heterophil. M. G. G. ; the central bodies ures 177-180. were retained as they are following Wright's stain 162 Completely dissolved rods with only relatively few but the rod substance was almost completely small central bodies. Nuclear lobes are weakly dissolved. Nucleus poorly stained. stained. 175 Smudged heterophil with two nuclear lobes. Rods 163 Central bodies few, with a range in size varying retained their form. from small to large. 176 Smudged heterophil with rods dissolved. The large 164 Heterophil with but few central bodies and no rods. spheres are the central granules and the small ones 165 Heterophil without rods or central bodies. are serum granules

74 4f -I A^

154 157 158 155 156

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• • -. ft* 159 160 162 161 it ifg/

164 165 166 167

.ft-'

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169 170 171 168 172

173

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174 175 176

75 Figures 177-187.—Eosinophils from circulating blood—mature, immature, and smudged cells. 2,470 X.

Figures 177-183: Normal mature eosinophils that show 182 Eosinophilic granules, small and closely aggregated. size range. 183 Small eosinophil with a high nucleocytoplasmic ratio. Figures 177-180: Typical eosinophils. The light blue- staining cytoplasmic background is an identifying charac- Figures 184-186: Developmental stages of eosinophils found teristic of these cells; so is the full staining of the nucleus. in circulating blood, 177 The eosinophilic granules can be composed of 4 smaller granules in a square. 184 Eosinophilic mesomyelocyte. Strong basophilic cy- 178 The eosinophilic granules can appear as scattered toplasm with specific granules in early stages of small granules on a reticulum. development. 179 The eosinophilic granules can have a size range of 185 Eosinophilic metamyelocyte. Later stage of de- large to small and be intermingled. velopment than the preceding cell. 180 The eosinophilic granules can appear as large spheres with a clear space in the center, with little or no 186 Late eosinophilic metamyelocyte, almost fully indication that they are made up of 4 smaller differentiated. granules.

Figure 187: Technic artifact. Figures 181-183: Small normal mature eosinophils, not so common as the larger size. 187 Smudged eosinophil. The grouping of small gran- large granules duplicates what was 181 Eosinophilic granules, closely aggregated. Cell ules to form figure 177. might be confused with a heterophil. observed in the intact cell,

76 7- •• *" . ... % \^

177 178 179 180

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181 182 183

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18t 185 186

187

77 Figures 188-196.—Basophils from circulating blood—mature, immature, and smudged cells. 2,470X.

Figures 188-192: Normal mature, basophils, all of which cell margin and those above the nucleus that show slight to extensive artifacts due to the water used with coalesced when the cell dried. Wright's staining technic. 192 Basophil showing extensive artifact. Magenta masses above the nucleus represent basophilic gran- 188 Basophil having an appearance approaching that ules of the cytosome, not basochromatin of the following methyl alcohol fixation, figure 390. nucleus. 189 Basophil showing early artifact of coalescence and 193 Basophil promyelocyte. dispersion of granules. 190 Basophil showing moderate artifact of coalescence Figures 194-196: Technic artifacts, smudged cells. of granules above the nucleus and discharge from the cell surface. 194 Slightly smudged basophil. 191 Basophil showing exten.sive artifact. Complete loss 195 Moderately smudged basophil. of cytoplasmic granules except for two pairs on the 196 Extensively smudged basophil.

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188 189 190 191

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192 193 194

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79 FREQUENCY of DIAMETERS of GRANULOCYTES 30 r Ijete roph' Is — eosi nophils 25 bo sophi Is

20 o

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DIAMETERS in MICRONS

Figure 197.—Frequency distribution curves for cell diameter of heterophils, eosinophils, and basophils.

Figures 198-223. -Effect of Petrunkevitch No. 2 fixation and M. G. G. stain. Granulocytes arranged to give a series of nuclear lobes for Arneth counts. 2,470 X.

198 Erythrocyte nucleus with this technic ahnost a solid 208 Heterophil, 3 nuclear lobes. dark blue. 209 Heterophil, 3 nuclear lobes. 199 Small lymphocyte witli cytoplasmic blebs and pachy- 210 Heterophil, 4 nuclear lobes. chromatic type nucleus. 211 Heterophil, 4 nuclear lobes. 200 Average lymphocyte with leptochromatic type lui- 212 Heterophil, 4 nuclear lobes. cleus. Perinuclear space in this and the preceding 213 Heterophil, 5 nuclear lobes.

cell. 214 Heterophil, 5 nuclear lobes. 201 Monocyte. This technic shows the stainability of 215 Eosinophil, 2 nuclear lobes. Specific granules are chicken. the substance filling the vacuoles of the f/o/. not destroyed by this technic in the 202 Thrombocyte. The specific granule of the throm- 216 Eosinophil, 1 nuclear lobe. booyte lies within a vacuole. 217 Eosinophil, 3 nuclear lobes. 203 Heterophil, 3 nuclear lobes. This technic dissolved 218 Eosinophil, 3 nuclear lobes. out completely all rods and granules, leaving only a 219 Eosinophil, 3 nuclear lobes. cytoplasmic framework. 220 Eosinophil, 4 nuclear lobes. 1 lobe. Specific granules well 204 Heterophil, 1 miclear lobe. 221 Basophil, nuclear

205 Heterophil, 1 nuclear lobe. preserved. 206 Heterophil, 2 nuclear lobes. 222 Basophil, 1 nuclear lobe. 207 Heterophil, 2 nuclear lobes. 223 Basophil, 2 nuclear lobes.

80 f 4^ ^fir-

199 200 202 201 198 i\ 206 20t 205 203

207 208 209 210

211 212 213 214

215 216 217 218

J. ^#>^

81 222 223 219 220 221 disappeared, leaving vacuoles with a narrow rim has been said that heterophils become eosino- of stainable material. The significance of dif- phils. This similarity of appearance under some ferent types of rods, some with granules, some conditions is probably the basis for more con- with vacuoles, and some with neither, is not fusion in making accurate differential counts known. Are they nothing more than differences than any other single factor. between individuals or are they handles that can How are such heterophils and eosinophils to jje used to gain an insight into the bird's condi- be distinguished? This will be discussed more tion of health and disease? fully after the eosinophils have been described, Information is needed also on the chemical but the most important fact is that the rods as nature of the rods and their granules. Whatever they break down or go into solution give the cyto- may be the significance of the granules, they are plasm an orange or pinkish color such as is read- part of the apparently normal development and ily seen in figures 158-162. The background may be found in heterophil myelocytes. Weid- color of the true eosinophil is practically always emeich ( 1911) according to Lundquist and Hed- light blue. That difference, coml)ined with the lung (1925) concluded that the uncolored spots fact that in most cases some part of the hetero- within the rods are merely optical phenomena. phil nucleus is pale and poorly stained, makes it Lundquist and Hedlung found central granules relatively simple to distinguish the two granulo- in vital preparations but not in stained smears cytes. This characteristic difference in back- and agreed with Weidenreich that the central ground color was observed in the heterophils and granules are not definite organelles. Burck- eosinophils of reptiles by Ryerson (1943). hardt (1912) expressed essentially the same Nuclear staining is extremely variable, as may ". ]>oint of view: . . In der Mitte der Spindel be seen in the series of figures 2, 1 and 154-167. wird oft ein ungefarliter Punkt sichtjjar. der je Sometimes the staining is vigorous as in figure nach der Stellung der Mikrometerschraube bald 2, 1. where the nuclear lobes are clearly defined. aiifblitzt, bald Schwarz erscheint. Ueber die In other examples the nuclei vary in their recep- Zusannnensetzung dieses Punktes wurde schon tivity to stain from those moderately well stained mehrfach diskutiert, mir scheint er am einfach- as in figures 156 and 167 to those in which there

sten als Lichtbrechungsphanomen zu erklaren.'"' is no trace of chromatin pattern or a distinct nu- Anyone studying avian blood soon recognizes clear boundary. In the same cell (figs. 157 and the fact that the rods show varying degrees of 158) one lobe of the nucleus may be stained and dissolution. Figures 154—16.5 show a progres- the more central ones may not be stained. Often sive series from clearly tlefined and well-devel- there will be a difference in staining of the same lobe, the central portion being faint and the oped rods to no trace of rods, but the story does more part adjacent to the cell wall fairly dark. Fail- not read in quite this sequence. The granules ure of the nucleus to stain is not a mark of de- are more resistant to the processes of dissolution generation as indicated by Emmel (1936) but ihan are the rods. If the rods without granules merely means that Wright's stain failed to pene- flisappear from figure 154 there is left a cell like trate the cell and color the nucleus properly. lliat shown in figure 165, but if a cell like that The normality of the heterophil nucleus can be shown in figure 1.55 loses its rods there remains ([uite easily and convincingly demonstrated if, a ct'll like figure 164. Figure 1.56 would result after the smear is made on the slide, the blood is in sometliing like figure 163, and figure 157 allowed to acquire a dull sheen and is then would result in something like figui-e 161. In dipped immediately into Petrunkevitch No. each case of the loss of rod substance, the cell has 2 (see ch. 7). If stained with May-Griinwald come to look more like an eosinophil; in some Giemsa the nucleus appears as shown in figure instances the resemblance is quite close (fig. 203. The treatment destroys the rods or distorts

161 ). However, there is no implication in what litem so that they run together as a network but reveals the nuclear structure in all its detail. "Translation: In the middle of the spindle there can often In none of these slides was there any indication he seen an unstained dot. and after the adjustment of the niierometric srrew this often sliows up as light, sometimes as of nuclear degeneration. It is obvious that an dark. Tliere has heen much discussion already over the com- Arneth index could not be obtained from a dry position of this dot: it seems to me simplest to explain it as a phenomenon of light diffraction. fixed Wright's or a May-Griinwald Giemsa 83 stained slide. Two slides are necessary for The spatulate portion is somewhat larger than bird's blood—one processed to show the struc- the small mass discussed for figure 207. ture of the cytosome and its inclusions, and the Four lobes would be counted in figures 210— other to show the nucleus. This may be the rea- 212. The fourth lobe in figure 210 is the long son why so few counts thus far have been pub- narrow structure that is separated from the lower lished for birds on the number of lobes in lobe by a complete constriction; no connecting heterophils. thread has yet been fonned. The constriction Avian blood has not as yet received the same near the tip of the left lobe is not sufficient to critical study as human and mammalian bloods warrant counting this body as 2 lobes. Figures nor have avian diseases been approached with the 211 and 212 have 4 lobes each, and figures 213 idea of closely correlating the physiology of the and 214 have 5 each. Sometimes the lobes diseased condition with the hematologic re- break apart and no trace of a connecting line be- sponse, although from the reviews given by Ol- tween is visible (fig. 213, upper lobe). son (19.37 and 1952) it is indicated that many Judgment in counting lobes of eosinophils and workers have made important contributions to of basophils is based on the same criteria already blood diseases of birds. described for heterophils. The eosinophil, fig- Differential counts serve a useful purpose but ure 216, is counted as one lobe. In this particu- actually hold only a small place in the total of lar cell, there actually may be two lobes, one blood reactions. The Arneth or Cook-Ponder overlying the other, but it is less confusing and counts can contribute additional information. probably sufficiently accurate to include this in Figures 203-214 sliow the range of variation in the single-lobe class. Figure 215 is an example number of nuclear lobes in heterophils. It is of a nucleus belonging to class II. Figures 217- obvious that a single nucleus will be separated 219 are examples of nuclei belonging to class III. into two lobes by a gradual process. Thus all In figure 217, the middle lobe, although small, is

stages will be found, and it becomes necessary sufficiently large and definite to be counted as one to decide arbitrarily when one nuclear mass has lobe. Figure 220 is an example of a nucleus be- been sufficiently separated from another to be longing to class IV. No examples of class V called a second lobe. Figures 204 and 205 were found among the eosinophils. would each be considered to have a single nu- Basophils usually have but a single nuclear cleus. In mammalian work cells of this type are lobe (figs. 221 and 222). Rarely the nucleus often called juveniles, nonsegmenters, and band may be divided into two lobes (fig. 223). cells. The Arneth index gives a useful statistic that There may be some question as to the proper measures the rate at which old cells are being re- designation for figui'e 206 but since there is a placed. There are 5 classes. Class I includes

deep constriction, rather than a broad band, it all cells in which the nucleus is composed of one has been called two-lobed. In chicken blood, a lobe. The remaining classes—II, III, IV, and small particle of nuclear material may be re- V—include cells that have the corresponding tained more frequently along the length of the number of nuclear lobes; class V includes, also, desmos as the lobes pull apart than in manmial- those of over 5 lobes. Cells with more than 5 ian blood. An example of tliis is seen in figure lobes are rare; none were found in a sample of

207; it meets the requirements of a distinct lobe 1,500 cells counted. The results of one Arneth

in that it is separated from adjacent lobes by a count on 1,500 heterophils, 142 eosinophils, and complete constriction but, to be arbitraiy, and to 295 Ijasophils are given in table 6. Ameth used

give some meaning to the Arneth count, it seems nuclear lobe counts on eosinophils and basophils inadvisable to count these small nuclear masses but they have never been found as useful as the as lobes. On this basis the figure referred to has counts made on the heterophils. In the litera- 2 instead of 3 lobes. Three lobes are shown in ture it is stated that heterophils have more lobes figure 203. than eosinophils. This observation is supported Three lobes are shown in figure 208, and if by the data in table 6 where the index for the the small spatulate portion still attached to the former is 2.44 and for the latter 1.97. How- upper lobe were completely separated by a con- ever, the presence of more lobes is definitely not

striction, it would be counted as a fourth lol)e. a characteristic by which the two cell types can

84 ^The heterophil Arneth index be separated. It should be noted that the largest Table 7.— class for both of these cell types is II. In some [Count taken for each bird at 70 days of age.] counts on a Canada goose the index for the eosinophils was considerably greater than for the heterophils (table 24). Since but few had previouely used nuclear lobe indices of this sort on birds, it was considered desirable to make a sample count to determine the index variability and to find any possible suggestion of a relationship to livaljility or to infection with the agent of lymphomatosis (table 7). Of the 13 cases that showed values above the mean of 2.44 (a shift to the right) there were 2, or 15 percent, that were grossly diagnosed as lymphomatosis and 12 of 17 cases, or 70 percent, witii index value below the mean (a shift to the left) were diagnosed as lymphomatosis. A correlation was run between age at death and index value. It resuhed in an /• value of +0.423, significant between the 2- and 1-percent level. One test made on another group of birds did not give the same results. Obviously this type of test should be repeated and carried out on a much larger scale.

Table 6.—Arnelh counts on granulocytes of chickens

Class (nuclear lobes) Cells

Heterophils

Eosinophils

ISasophils. . — .

mal; yet the average index for the 4 that went count. If the heterophil count is recovering after to the termination date and were fonnd grossly destructive irradiation, the immature heterophils negative was 2.65. Perhaps this is a more nearly may be quite numerous. normal index than the lower value. A group of A granuloblast found in the circulating blood chickens known to be free from the agent of lym- is shown in figure 168. There may be some phomatosis and other diseases is needed in order question of whether it is destined to be a heter- to arrive at a set of normal standard blood values. ophil or a basophil, but the large size of the cell, The question also arises, Are the birds that are the rim of basophilic cytoplasm broken by many destined to develop grossly visible tumors in- mitochondrial spaces, and the uniform reticular fected and fighting against the disease during the pattern of the nucleus, all identify it as a blast several hundred days before neoplasia appears? cell. When compared with the granuloblasts of Before leaving the subject of the normal hetero- figures 330, 1 and 2, 366, and 367, little doubt phil, the fact should be mentioned that the French remains as to its identification as a granuloblast. veterinarian, Lesbouyries, (1941) described a The metagranuloblast stage of development sixth type of white blood cell in chickens. He has not been seen as yet in the circulating blood, listed a neutrophil in addition to a heterophil. In ]:)ut is found in bone marrow ( figs. 368 and 369) his descriptions tliey are not synonymous. His Four examples of promyelocytes (figs. 169- sixth cell is the type shown in figure 165 and it 1 72 ) have been illustrated. The nuclear bound- does resemble superficially a mammalian neutro- ary in the earlier phase of development (figs. 169 phil. Our own studies have shown it to be a het- and 170) is even less distinct than in the bone erophil from which the rods (without granules) marrow, and the number and density of magenta have dissolved, and there is no justification for rings and granules are greater. Figures 171 and giving it a different name and creating a separate 1 72 are not good examples of late promyelocytes, class for it. Breusch (1928) listed 4 types of because the contents of the vacuoles did not take granulocytes—eosinophilic leukocytes, ampho- the stain. It is assumed that tlie same stage, had philic or pseudoeosinophilic leukocytes, baso- it been taken from the bone marrow, would have philic leukocytes, and neutrophilic leukocytes looked like figures 370-372. but descriljed only the first 3, and includes only These immature heterophils in circulating these 3 in his tables of differential counts. The blood have a different appearance from those in application of terminology whereby heterophils bone marrow. Perhaps this is due to the fact and eosinophils are correctly identified has not that different stains were used in tlie two situa- been a simple matter. Magath and Higgins tions, or it may be due to the effects of these en- (1934) have listed the various synonyms that vironments on the penetrability or selectivity of have been used from 1880 to 1931. Even sepa- the stains. Whatever the cause, the difference in ration of these cells on the basis of those with appearance should be kept in mind and not be al- eosinophilic rods and those with eosinophilic lowed to hamper identification. graiuiles leads to difficulty in identifying these 2 A typical mesomyelocyte (fig. 173) has less cell types in ducks (see p. 207). Loewenthal than half of the definitive granules. Many of (1930) also found what he called neutrophiloid the precursor orange spheres have attained a cells, which in his opinion were derived from dense coloration but none of them have elongated rods by a process of dedifferentiation; he sug- as in figure 373. Two sizes of granules are gested that in the course of evolution it was present, and there is a possibility that the small this type of cell that produced the mammalian ones become the central bodies for the rods that neutrophil. develop out of the large ones. The carryover of magenta rings and granules from the early to

the late stages sometimes occurs as it has in figure Developmental stages found in circulating 173. The whole process of myelopoiesis will blood (figs. 168-173) not be discussed at this time since a rather critical

Immature cells of the heterophil series are rare and detailed comparison, which is presented

in normal blood, but probably not more so than later, is necessary in order to relate what is found are the immature stages of red cells when the in birds to the named stages given for mammals. difference in relative numbers is taken into ac- See also the discussion by Lucas (1959). 86 Abnormal heterophils posure to aqueous solutions. Hence one finds in this series of slides more examples in which The only cell defect recognized thus far is the rods have disappeared than one usually finds in fractured nuclei such as shown in figure 3, 1. normal birds by using the staining rack technic. In this case, 2 of the 3 lobes showed a nonstaining No technical method has yet been found that will band. It is apparent that the same type of nu- hold the rods well enough to insure confidence clear chromophobia occurring in the erythro- that the appearance of the heterophil is really cytes, lymphocytes, and other cells can also oc- due to the jjird and not to technic. Such a cur in heterophils. The possibility that these method is urgently needed. Even the Petrunke- fracture lines and empty nuclei represent path- vitcli fixed smears show dissolution of the rods ologic conditions, rather than technic defects, (fig. 203) and in this case neither rods nor gran- has been discussed (pp. 32 and 65). ules were visible. Petrunkevitch No. 2 (p. 230) Undoubtedly other abnormal heterophils exist is an alcoholic solution with copper, ether, and in birds as they do in mammals but up to this time other sujjstances, and it would not be expected to sufficient knowledge of the normal by which act like an aqueous fixative. Bradley (1937), identification of the abnormal can be made is quoted on p. 88, observed that adequate fixation lacking. Toxic granules in human neutrophils would not hold the rods if followed by aqueous are considered abnormal and play an important stains. role in prognosis. Osgood and Ashworth ( 1937) In view of the high lability of the rods, it seems note three points of difference from normal appropriate to raise the question. How faith- cells—color of cytoplasm, presence of vacuoles, fully has the heterophil rod been preserved in and coarse granulation. Osgood goes on to say tissue section? If rods disappear but granules (p. 51), "The author predicted death of more remain, a heterophil can look like an eosinophil. than 100 consecutive patients from a three or four Thus, the descriptions of tissue infiltration or of plus change in two of these factors. Ninety per- myelopoiesis as worked out on tissue sections cent of these died within a week after the pre- may be open to question until it has been demon- diction was made. These patients died of a wide strated that the technics used do not destroy the variety of conditions, including malignant tumors rods. Dantschakofl^ (1909a and b) in her study and leukemias as well as infections." In view on the bone marrow of birds follows the processes of its value in human hematology, it would seem of myelopoiesis through the stages showing rods, desirable to further study avian blood in search and her beautiful colored plates show them well of this mammalian counterpart. Thus far, how- preserved. Our sections of hematopoietic em- ever, nothing has been observed that could be bryo tissues failed to show these rods. identified in avian blood as toxic granules. Per- Much that is seen in the dried smear from cir- haps this is because most of these studies have culating blood may be artifact; yet the opinion is been on birds that showed no obvious disease. maintained that bird differences are in part responsible for some of the deviations from the typical. Although all slides are handled alike Technic artifacts (fig. 174) in bulk staining, some birds show rods and in others hardly a rod can be found. It would go The effect of water on the rods was emphasized far toward solving some of these problems if by soaking the slide for 20 minutes after methyl fresh blood preparations were carefully studied alcohol fixation and before staining in May- under the phase microscope. The variabilitv Griinwald Giemsa. The result of such treatment due to technic and that due to bird differences is shown in figure 174. All trace of rods has could be separated. Additional evidence that disappeared; instead there is a cytoplasmic net- rods of heterophils can come to look like granules work with a granule in the center of each space. of true eosinophils in vivo is indicated in the ex- The technic used in figures 154-165 was Wright's amination of smears from blood spots of eggs. stain but it was applied by the bulk method (p. Here every heterophil simulated an eosinophil instead to individual slides a rack. 229) of on (Lucas, 1946) . Natt and Herrick (1954) have Since the bulk method requires somewhat longer reaffirmed what others have demonstrated, that

staining and slower drying, it involves longer ex- loss of rod substance leaves a central granule

87 that gives to the heterophil an appearance similar ing chamber and the changes in the cells were to that of the eosinophil. followed. Usually filter paper was touched to The literature on the suLject of heterophils and the fluid layer on the opposite side in order to eosinophils reflects some of our present un- hasten the exchange. When the preparation was certainties. The obsei-vation that aqueous diluted with water they found large, clear cells solution causes a degradation of rods is not new. with large, clear, scarcely visible round granula- Bizzozero and Torre (1881) stated that if one tions; when it was diluted with 0.7-percent treats a blood preparation with water the white NaCl solution, all the granulations were round corpuscles in question swell; the same thing hap- and were larger than in the previous solution. pens to the rods themselves, which liecome pale Preparations treated with 0.9-percent NaCl pro- and at last disappear. They also found that duced mostly round granulations in the cells, but upon treatment with dilute citric acid the proto- in a few cells these bodies took the rod form. At plasm of the white corpuscles laecomes swollen lV2-percent NaCl concentration, the cells con- and pale. The rods first draw together so that tained rod-form granulations, and now and then there is formed a shining heap that hides the spherical granules. They summarize by saying nucleus. This heap very suddenly l^ecomes pale that in water the cells and granules swell and and finally disappears. Although Bizzozero and become faint, but in 1.5-percent salt solution the Torre recognized only four kinds of white blood cells shrink and the round granulations are trans- cells, they did include the true granular eosino- formed into the rod form. If not carried too far phil as distinct from the rod eosinophil. Denys these are reversible reactions, and thus the rod eosinophil is an artifact ( 1887) writing on the Jjone marrow of pigeons brought about by osmotic is less definite about the numljer of types of white conditions. blood cells, does not distinguish between the two The chief reason why the interesting conclu- eosinophils, and says it seems probable that most, sions of Lundquist and Hedlung have relatively if not all, of those seen as dots are simply rods little value is that their study of cellular change viewed from the axis. was made at the relatively low magnification re- Lundquist and Hedlung (1925) have pre- quired in using a counting chamber, and, al- sented a review of the subject and mention those though they mention diese changes in the same who believe the eosinophils in birds represent two cell, it would seem rather difficult to keep a par- distinct types and those who consider that there ticular cell in the field during a rapid exchange are only four kinds of leukocytes in birds. They of fluids. quote S. Henschen, who concludes that there are Bradley (1937) examined the blood of numer- two distinct types but who also mentions that post ous birds and made observations similar to mortem processes can cause the rods to go over our own. He reports (p. 995) that, "When into granules. Lundquist and Hedlung's own the color of the rod bodies has been partially investigations led them to the unique opinion removed, . . . demonstration is made of darker that the granular eosinophil cell represents the or lighter parts, giving the impression of deeply true condition and that the rods are artifacts, stained granules along the paler length of the produced at the time the smears are made. They body." In regard to technic he says (p. 997), point out that the inclusions have the form of "Avian (fowl) blood is best stained by a method granules after methyl alcohol fixation and of which applies the eosin or related acid stain in rods after formalin or trichloracetic. In order alcoholic solution and when the use of water or to follow the transformation from granules to saline solution forms no part of the process until rods they diluted the blood in a white cell pipette after the alcoholic stain has acted. with 0.9-percent sodium chloride solution, which "Adequate fixation before treating with water was considered to be isotonic with chicken blood. or watery stain is no preventive for the destruc- The diluent was allowed to act for 2 minutes be- tive action of the latter on the rods. Water ap- fore the blood mixture was put into a counting plied in moderation after staining is not detri- chamber, then an eosinophil was obsei-ved in mental to the result, and when left in contact order to note the form of granulation present. with the stained preparation for a longer time After this, a solution of higher or lower concen- is the means of showing up details of structure of tration was added to the side canal of the count- tlie rod bodies not otherwise appreciated."

88 Interpretation of the loss of rods is sometimes terms for designating tliese two cell types, such influenced by the type of problem involved. as "rod eosinophils," "granular eosinophils," Hewitt (1940) attributes this loss to degenera- "pseudoeosinophils," and "true eosinophils." tion or phagocytosis due to the malarial parasite. Some have reversed the last two terms so that Difficulty in the identification of eosinophils and "true eosinophil" applies to the heterophils. For heterophils is not limited to the older literature; this reason, the use of "true" and "pseudo" can for example, Diesem (1956) found it difficult lead to confusion. The terms "rod" and "gran- to separate these two cell types, and combined ular" are not good either because rods often them in his cell counts. change to spheres. Therefore, the terms "heter- The most recent opinion on the subject of rod ophil" and "eosinophil" have been chosen. degradation has been expressed by Dr. Hamre These have the added advantage that the cells (personal communication). The opinion is that are each designated by one word instead of two. the rods themselves remain unchanged when acted The eosinophil of birds is homologous with the upon by various stains and aqueous solutions but cell receiving the same name in other classes of that the capacity of the rod substances to absorb vertebrates. the stain does change, and that if Wright-Giemsa

is used as he has modified it (the technic he rec- ommends is given on p. 230) rods will always ISornial stain and each rod will contain a central granule. mature eosinophils (figs. 177— One additional cellular defect remains to 183) be described—squashed cells. Squashed or The eosinophil shows a rather wide range in smudged heterophils are probably the easiest size; some are large, like figures 177-180, and cells to recognize because the specific cellular in- some small, like figures 181-183. Usually they clusions are preserved. Squashing takes place are about the size shown in figure 177. The at the time the smear is made; yet the same vari- range for size (fig. 197) is shown by a frequency ability is to be found in the rods broken out of distribution curve in which there is a minimum the cell as was found in the intact cell (figs. 175 of a little over 4/u to slightly over llyu. The and Figure 176). 175 shows rods without cen- average size of 7.3;U is approximately 1 micron tral granules and figure 176 shows central gran- less than for heterophils or basophils, and prob- ules with dissolved and almost completely faded ably was brought about by the occurrence of rods. These were taken from different jjirds but small eosinophils in circulating blood ; whereas, ])oth smears were made the same day. The birds cells of the size shown in figures 181-183 are were exactly the same age and the slides were rare for heterophils or basophils. The cell is stained together. Both cells show many fine nearly round as may be seen from the figures. magenta-colored granules, partly among the The cytoplasm stains a pale, clear blue color, broken fragments of the cell but mostly surround- which, of course, shows best when the granules ing the cell. These small, darkly stained bodies are not crowded together. The granules are are senmi granules. Something is liberated often crowded and there is not much cytoplasm from the broken cell that acts as a mordant on to be seen ; yet the background blue color is one the serum granules and causes those in the imme- of the best means of separating the two types of diate vicinity to take the stain. As may be seen eosin-staining cells when the rods of heterophils from figure 176 their color and size differ from have been degraded to granules. The blue- even the smallest of the central granules of the staining ground substance is readily apparent rods. The central granules show a variability in among the granules of large eosinophils, but the size among themselves similar to that found in blue color is often obscured in small cells. the intact cell. Small cells, such as those shown in figures 181-183, are relatively rare. Their identification is aided by the strong affinity of the nucleus EOSINOPHILS for stain. There is some variation in the structures of the The fact that both heterophils and eosinophils granules. Often they appear as homogeneous stain with eosin has led to the use of numerous bodies, Ijut sometimes when they are larger and

89 . .

not so crowded, the structure of each granule is differences in concentration of granules in dif- revealed. It is made up of 3 or 4 smaller bodies ferent parts of the cell, as shown in the two exam- held together in a ring. This is shown especially ples. It may be that these deficiencies represent well in figure 177. The small granules that immaturity of the cell; yet the fact that there are make up the ring give it an angular contour and four nuclear lobes is evidence that the cell in fig- in the center is a clear space. Sometimes one ure 179 is not an immature cell; perhaps the may have an impression of a clear space in the cytoplasm continued to increase in volume after center of the sphere (fig. 180) and yet the indi- the process of granule formation had ceased. vidual particles that go to make up the ring can- Further discussion of the problem of variation in not be separated. the size and arrangement of eosinophil granules A knowledge of the detailed structure of the appears later (ch. 6 and fig. 411). eosinophil granule (or ring) in the chicken is One characteristic of the granules in the eosin- necessary for the identification of the eosinophil ophils that aids in separating them from hetero- in various species of wild birds, especially the phils containing granules is the impei-viousness ducks. The space may be responsible for the to aqueous solutions; the granules of the eosino- general report that the eosinophil granules are re- phil are never affected in the chicken and the fractile. In formalin-fixed cells that are stained same difference is shown following Petrunkevitch with phloxine and floated on a slide, as they are No. 2 fixation (compare figs. 203 and 21.5). in the counting chamber when Wiseman's method The nucleus always stains well in the eosino- has been used in preparing tlie material, they can phil. Except for the masking of lobes by the readily be distinguished from heterophils by granules, it would be possible to count the nuclei their strong refractility. Had these differences quite accurately. No cell with more than 4 nu- been observed by Lundquist and Hedlung ( 1925) clear lobes has been illustrated, and the data for they probably would not have concluded that eosinophils in table 6, based on 142 cells counted, these two eosin-staining cells belong to the same also indicate that an eosinophil with 5 or more type. loJjes would lie rare in the chicken. The Arneth Usually, granules are uniformly stained but index for eosinophils, 1.97, is lower than for

it is fairly common, especially in medium to heterophils. large cells, to find that some of the granules stain To point up the differences between these two faintly, as shown in figures 178 and 179. Quite cell types for purposes of identification, table 8 often in association with this variation there are was prepared.

Table 8.—Characteristics of heterophils and eosinophils

Characteristic Heterophil Eosinophil Cell size Usually a relatively small range of variation in Wide range in size but usually not in the same circulating blood but a large range of variation slide, in the bone marrow.

Cytoplasm When rods are well formed the cytoplasm is The cytoplasm maintains a pale blue background colorless. If there is any degradation of rods to color for the red-staining granules. Only rarely spheres, the cytoplasm is tinged with the is there an exception to this, eosinophilic material.

Specific granules Contain eosinophilic rods that may be long and Contain eosinophilic bodies that are uniform m narrow, or short and plum]), or even spherical, size in the same cell and usually uniformly dis- Rods disintegrate in aqueous solutions and the tributed. Eosinophilic bodies may be homo- central body may be all thai remains. Central geneous spheres or rings, often with 4 granules in bodies are variable in number and size; they the ring, or scattered separate small granules, may be absent or may be represented by a Resistant to aqueous solutions. In some species vacuole. the rings may be flattened and elongated to give the superficial appearance of a rod.

Nuclear lobes The average Arneth index is 2.44 or higher for The average Arneth index is slightly less than chickens. 2.00. Cells with class V nuclei are rare in chickens, if they exist at all.

the Nuclear staining Wright's stain usually fails to stain the nucleus Wright's stain shows a strong affinity for completely or well. nucleus and brings out the details of chromatin pattern.

90 Developmental stttges found in circulating chickens this cell is more abundant than the blood (figs. 184-186) eosinophil, averaging about 2 percent in a differential count. There is no confusion in term- The low percentage of eosinophils in the dif- inology for this cell and basophils are homo- ferential count makes it difficult to find develop- logous through various classes of vertebrates. mental stages of eosinophils in the circulating The only confusion comes in the theoretical re- blood of normal birds. The youngest observed lationship between the blood basophil and the thus far is shown in figure 184 a mesomyelocyte — tissue mast cell. in which there are fewer than half the number of Aqueous solutions have a severe detrimental granules foinid in the mature stage. The cyto- effect here, as they have on heterophils; there- plasm is still strongly basophilic. The range in fore, all basophils in a dried smear show technic coloi^ation of the specific granules is not so great artifacts in varying degrees. This same reac- as has been observed in these cells in the bone tion exists in mannnalian cells and has been dis- maiTow (fig. 379). The large size of the gran- cussed by Michels ( 1938) . Because of this fact, ules in the metamyelocyte (fig. 185) stands in abnormal cells have not been identified. contrast to the groups of small granules in figure 186. These granules in figure 185 give the cell an appearance similar to that of the mesomyelocyte stage of the heterophil (fig. 374) and it is ISormal mature basophils (figs. 188—192) possible that this cell has been misplaced, espe- Basophils are only slightly smaller than cially since small granules are mixed with the heterophils, as shown in the graph (fig. 197). large ones. Diiference in granule size is a char- This slight difference would not be apparent acteristic found in heterophil, but not in eosino- visually. The impression has been gained dur- phil, myelocytes. There is no question al)out ing the routine examination of many slides that the identity of the cell in figure 186; the strongly when die technic defects are minimal the cell is stained nucleus and the small uniform granula- relatively small; whereas, when there has been tions establish it as an eosinophil. extensive washing out of granules the cell seems larger. Not only is the size of tlie cell less but even the granules are smaller when they are well Abnormal cells preserved. See, for example, figures 389 and 390, which show basophils from bone marrow; No cellscei: belonging to this classification have the cells were fixed in methyl alcohol and stained been seen with thionin in alcohol. Basophils are round, as are the other two granulocyte types, and they are not severely deformed when other cells press Technic artifacts (fig. 187) against them. Sometimes the cell membrane becomes irregular in contour owing to the ex- This is an example of a smudged eosinophil. trusion of particles (fig. 190). The characteristic of a strong affinity for stain by The cytoplasm of liasophils is colorless. Usu- the nucleus of the eosinophil, in contrast to the ally it is masked by the granules jjut when they tendency toward defective staining in the hetero- are washed out there is very little residual color phil, is still retained in the squashed cells. The (figs. 191 and 192). More convincing, per- scattered contents of the broken cell (fig. 187) haps, are the small breaks seen between the gran- demonstrate again that the large eosinophil granules when they have not been severely disturbed ule is composed of smaller granular units. (figs. 188 and 189). In the Petrunkevitch No. 2 fixed smear there is a basophilic tinge to the cytoplasm (fig. 221). The spaces between the granules seem larger than normal because the BASOPHILS granules are either shrunken or partially dissolved. Specific granules for these cells have an affinity The granules in the dried smear are basophilic for iiasic dyes and show metachromasia. In and metachromatic; that is, they have an affinity

91 .

for basic dyes and die resulting color produced cells run about 2 percent of the differential count, in the stained object is different from the color and in pheasants it may be 10 percent (table 20) of tlie dye in solution. Even with a group of that receive the same treatment, there is slides Developmental stages found in circulating considerable variability between birds in the re- blood (fig. 193) sistance of the granules to water. This may in part reflect differences in age of the cells but it has This is the only immature basophil that has not been proved that the granules developing in been seen in the circulating blood. It closely basophil myelocytes are more resistant to water resemldes in cellular and nuclear detail the dian they are in older cells. heterophil granuloblast (fig. 168) ; but resem- The nucleus of basophils is usually masked by l^lance is lacking in one respect—the cytoplasm granules, but occasionally it may be visible as a contains numerous magenta granules. These structureless, pale blue staining body lying in the ])odies are equivalent to the granules and rings center of the cell. The penetration of Wright's found in heterophils at this same stage of de- stain, found to be poor in the heterophil nucleus velopment, and often the two cells are hard to is practically nil in basophils. Sometimes it ap- separate. Usually the magenta rings of the pears as if chromatin clumps of the nucleus were heterophil are larger than those of the basophil, being stained (figs. 190, 191, and 192), but this and in the latter there is less vacuolization of the is due to the basophilic bodies of the cytoplasm cytoplasm and the nucleus tends to remain in the that are trapped above or below the nucleus when center of the cell more frequently than in the the cells are flattened in drying. For some heterophil. These points are made evident by reason the pressing of the nuclear membrane a comparison of immature granulocytes from against the cell membrane protects tlie granules bone marrow shown in figures 370-372, 382, to some extent from the action of water. Failure and 383. The magenta body in the basophil of the nucleus to stain is not evidence of de- promyelocyte is not the definitive basophilic generation—when fixed with Petrunkevitch No. granule and is not affected by aqueous solutions. 2, they stain as strongly as any normal nucleus (fig. 221). Technic artifacts (figs. 194-196) Variations in shape of nuclei and number of lobes are seen in figures 222 and 223. Usually Since aqueous staining methods dissolve the

tlie nucleus is centrally placed and lias a round Ijasophilic granules, every cell illustrated as shape. Constricted nuclei may be seen, but only typical of the normal is, in reality, an example of rarely. A nucleus in the condition shown in a technic artifact, and the same can be said for figure 222 is counted as one lobe; only when the the failure of the nucleus to take the stain. In isdnnus between is reduced to one or more deli- addition to these artifacts, squashed cells may be cate sti'ands is the nucleus regarded as bilobed found. Figures 194^196 show three degrees of (fig. 223). A trilobed nucleus has never been severity. In figure 194 the cell is only slightly observed in a basophil, and from the Arneth squashed; the granules are separated and they counts on these cells (table 6), the bilobed con- are larger than normal, and the nucleus of the dition occurs only about once in one hundred cell shows earlv autolysis. In figure 195 the cell cells. membrane wall is definitely broken and some of After one has seen a representative collection the granules are scattered. In this particular of basophils, this cell type becomes the most cell there is considerable variation in the way the easily recognized leukocyte of the blood. Yet granules take the stain. This, however, may errors have been made in the literature; Emmel have existed in the cell before it was broken. The (1936) labeled as "normal premyelocyte" (his granules from the cell shown in figure 196 have fig. 6E) a cell that is a typical basophil of cir- been widely scattered and for some reason retain culating blood, as nearly as can be determined a strong affinity for stain. Obviously, all the from his black-and-white drawing. This would factors responsible for dissolution of granules help to explain why he found only two basophils are not yet fully known. The clefts in die auto- in diflierential counts made on 50 chickens. In lysing nucleus are probably the spaces between normal chickens tested at this Laboratory, these the blocks of chromatin.

92 Hemokonia and Serum Granules

Hemokonia, or blood dust (die English equiv- 322). Even when they do not stain, they can alent), refers to the cell fragments, debris, and be seen by reducing the diaphragm of the micro- minute bodies floating in the serum. Most of scope to increase the apparent refractility. In these lie near the limits of microscopic visibility embryo smears and in bone-marrow and thymus When cell fragments are large they are called smears of the embryo, they are especially abun- plastids, but when small they are called hemo- dant (figs. 329 and 332). The typical appear- konia. ance is shown in figure 322. Among the serum Little has been written about cell debris. granules of this field are fragments of broken Downey does not have "hemokonia," "blood cells also. Most low-power drawings from dust," or "serum granules" in the index of his hematopoietic organs of both adults and embryos 4-volume work on hematology. This is true for have been made without including the serum some of the atlases on blood. Kracke and Car- granules. Cell identifications cannot be made ver (1937) give one paragraph on the subject in accurately where the granules lie on top of the which they say (p. 107), "These particles do not cells in large numbers. As may be seen in figure stain and their nature is unknown. They appear 322, the serum itself may take up the stain as similar in size to the granules in the cytoplasm well as the granules in it. of granular leukocytes. This has led to the sup- Two other granules should be mentioned— (1) position that they are extruded granules but no definite yolk granules found when blood is taken conclusive proof has been given for this identity." from the early embryo at 48 to 72 hours, and In avian blood occasional small fragments (2) chylomicrons, which Cage and Fish (1924) from broken cells may be found. These are descrilie. Yolk granules are larger than serum chiefly heterophil rods or myelocyte rings and granules and sometimes a whole yolk sphere is granules. Their presence is due to the breaking seen. Yolk is not present in adult circulating of cells that occurs when a smear is made. They blood. appear in practically any smear of bone marrow; Whether chylomicrons are present in birds has and recognizable rods from granulocytes, espe- never been determined. As the term was first cially heterophils, can be found here and there used it referred to the submicroscopic spheres over most slides. Since granulocytes are so found in lacteal drainage from the small intestine much more abundant in bone marrow than in of mammals. It has a broader meaning than circulating blood, it is not surprising that debris this and in general refeis to lipid spheres found of this sort is also more abundant. in blood, chyle, or lymph. Dark field is re- What should be included under the term quired to see them. It would be interesting and "hemokonia" has never been well defined; thus useful to determine whether chylomicrons of it is not possible to decide whether serum gran- mammals and serum granules of birds are the ules should be included under the term. For same thing. There is considerable difference the present they are considered to be different in size, the serum granule being larger. from hemokonia. They are found in great The serums of pigeons and chickens are said abundance in the blood of birds. Usually they to contain a lipochrome pigment (Halliburton, do not take the stain but when they do, they pro- 1886) that gives to them a color identical with duce a stippled mask over the cells (figs. 72 and that found in fat cells of these birds.

ADDENDUM

The irregularity in the shape of erythrocyte known, but Bessis (1956, fig. 195) photographed nuclei is described in the early part of chapter 2 similar irregularly shaped nuclei in chickens and is illustrated in figure 29 The cause is not after treatment with folic acid antagonists.

93 A

Figure 224.—Blood from dorsal aorta of early chick embryo. 1,370X. Slides of embryo blood stained with May-Grunwald Giemsa.

Incubation age, 1 day 23 hours. A Incubation age, 1 day 22 hours. B to the stage 1-10 Typical early primary erythroblasts with lobulated 1- 2 Large primary erythroblasts, similar younger. basophilic cytosome and 1 or 2 nucleoli. shown in A or 11-15 Small shrunken primary erythroblasts. Frayed 3-20 Late primary erythroblasts of various sizes from and shrunken appearance of some cells probably large (14 and 16) to small (18 and 19). delay in drying the cells; especially true of due to 21-22 Large primary erythroblasts in mitotic division. 11 and 12. 23-24 Small cells with lobulated cytosomes. Probably 16 Primary erythroblast in mitotic division. thrombocytes. 17 Slightly smudged erythroblast. early embryonic

94 224

95 Figure 225.—Blood from dorsal aorta of early chick embryo. Incubation age, 2 days 17 hours. 1,370X.

1-11 Early polychromatic primary erythrocytes. 23 A mid-polychromatic primary erythroplastid. 12-19 Mid-polychromatic primary erythrocytes. 24 Large embryonic thrombocyte. 20 Mitotic telephase of early polychromatic primary 25 Embryonic thromboblast. erythrocyte. 26 Medium embryonic thrombocyte. 21 Mitotic metaphase of early or mid-polychromatic 27 A group of 4 small embryonic thrombocytes clumped primary erythrocyte. together but fi.xed before disintegration set in. 22 Mitotic telephase of early or mid-polychromatic primary erythrocyte.

96 J

^itf**

t % W<

>.^iVM.-'''j>-

225

97 Figure 226.—Blood from heart of chick embryo. Incubation age, 4 days 12 hours. 1,370X.

1-2 Mid-polychromatic primary erythrocytes. 14 Late polychromatic primary erythrocyte in the 3-9 Late polychromatic primary erythrocytes. late telephase stage. 10-12 Late polychromatic erythrocytes showing early 15-16 Smudged primary erythrocytes. stages of cytosomal fracturing, an artifact. 17 An erythroblast of later embryonic generation, or 13 Late polychromatic primary erythrocyte near the an embryonic tliromboblast. metaphase stage. 18 A medium embryonic thrombocyte. 19 A small embryonic thrombocyte.

98 '0 ^ f w m ^.

^ iM^ # # W^ ^5fe

^iMi)

^g<. A

226

99 Figure 227.—Blood from heart of chick embryo. Incubation age, 5 days 21 hours. 1,370 X.

1-12 Mature primary erythrocytes ranging in size from 19-24 Late polychromatic erythrocytes in which the are present. large (5) to small (9 and 10). artifacts of cytosomal fracturing 13-15 Mid-polychromatic erythrocytes of later embryonic 25 Early polychromatic erythrocyte in mitosis, prob- red-cell generations. ably an early anaphase. 16-18 Late polychromatic erythrocytes of later embry- 26 Erythroblast of later embryonic cell generations. onic red-cell generations. Cell 17 is at the transi- 27 Medium embryonic thrombocyte. tion between mid- and late polychromatic stages. 28 Small embryonic thrombocyte.

100 '.ty

227

101 •ye r ^ ^ fi r

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^9 i^ « © ^ € ^- % « ^ «s JU!».

228

102 Figure 228. —Blood from heart of chick embryo. Incubation age, 9 days 15 hours. 1,370X.

1-3 Mature primary erythrocytes. 19, 20 Smudged erythrocytes.

4, 6-10 Late polychromatic erythrocytes from later 21 Small embrj'onic thrombocytes that have embrj'onic red-cell generations. clumped, accompanied by degeneration as 11-13 Late polychromatic erythrocytes in which the shown by loss of cytoplasm and by pyknosis artifacts of cytosomal fracturing are present. of the nucleus.

5, 14-18 Mature erythrocytes from later embryonic 22 Embrj'onio macrophage. red-cell generations.

103 : - — ——

CHAPTER 3

Circulating Blood of the Embryo

ERYTHROCYTE CHANGES DURING Blutbildung ausserhalb und innerhalb des em- INCUBATION bryonalen Korpers annehem. Die ersten Blut- zellen treten hier wie dort zuerst als Lymphozy- Early in the process of blood development, ten kleinerer mid grosserer Form auf, diese mesenchyme transforms into angioblasts or into mussen also als die Stammzellen aller Blutele- primitive erythroblasts. The red blood cells— mente betrachtet iverden und erzeugen durch Pro- in the first generation, at least—develop from the liferation und Differenzierung in verschiedenen walls and within the lumen of the newly formed Richungen die mannigfaltigen Formen der roten und weissen Blutkorperchen, die wir im erwach- vessels (Sabin, 1920) . This process of develop- ^ ment is well under way before the first circula- senen Organismus finden." tory arc through the embryo has been completed. Murray (1932) analyzed the observations of As soon as afferent and efferent channels have Sabin and of Dantschakoff by studying early been joined, the heart pumps through them the blood formation in tissue culture. He used parts cells that previously had accumulated in the yolk of the primitive streak of the chick embryo before sac. This event begins at 33 to 36 hours and is the head fold developed. From these undiffer- well established by 48 hours incubation, which entiated cells, he obtained cultures that went

is the age when the first smears were made in this through all the early stages of blood island for- study. Before this, the development of hemo- mation, with angioblasts enclosed within an endo- globin has caused the blood to take on a red thelial boundary. These early stages agreed color. In the primary generation of red cells, very closely with the observations made by Sabin the hemoglobin is acquired much more rapidly on the living chick. Murray obsei-ved in tissue than in the later generations, with the result that, culture all the steps in primary erythrocyte dif- although the cell has a red color as seen with the ferentiation up to cells that had an oval shape and unaided eye, the stained cytoplasm retains a contained hemoglobin, even up to a fully mature strong affinity for basophilic dyes. It is this fact cell. This entire process took place in his cul- that leads to misunderstanding in cell termin- tures within 24 hours. ology and to error in identification of the stage It has been pointed out by Dantschakoff of development. As successive generations ap- (1908b and 1909b) that whereas Maximow pear this source of error becomes less and less. (1909) found but two generation types in the Dantschakolf's (1907 and 1908a and b) ob- development of red blood cells in mammals the early transformation of mesen- servations of primary and definitive—she found that in chick- blood islands and thence into intra- chyme into ens there was a succession of generations by vascular and extravascular blood elements were which the transition from the primary to the de- based on celloidin-sectioned material. The finitive type was accomplished. Each genera- statements agree closely with those of Sabin tion of cells attained maturity within its life span. (1920) but the names and interpretations are The primary generation of red blood cells in different. Both authors agree that blood cells the chick embryo is so conspicuously different can be produced from the endothelium of the aorta of blood islands of yolk sac and the dorsal 'Translation: On the basis of the discussion we can assume the early embryo. a complete analogy between the first blood formation outside and inside the embryonal body. The first blood cells in both Dantschakoff summarized her basic (1907) places first appear as lymphocytes of large or small size, the blood ele- hematologic theory as follows (p. 166) these mast be considered the parent cells of all and through proliferation and differentiation in differ- Erorterten konnen wir also ments, "Auf Grund des ent directions they make the numerous forms of red and white eine vollstandige Analogic zwischen der ersten blood corpuscles such as we find in the mature organism.

104 from all subsequent generations that the occur- The early generations of embryonic erythrocytes i-ence of slight differences in these later genera- retained round forms, whereas later genei'ations tions is often overlooked. The existence of a took on elliptical forms. Yolk plates are absent succession of generations brings our terminology from avian emljryonic erythrocytes but the possi- into question. In both birds and mammals the bility exists that tlie pattern of reproduction in term "primary" for the first generation of red amphibians may be carried over to some extent cells is satisfactory, but in the chick the cells that in birds. follow cannot be called definitive generations Figure 232 was prepared so as to sliow the since actually it is not until near the end of the succession of forms. For the sake of simplicity, embryonic life that the definitive type of red the embryonic erythrocytes have been divided blood cells appears. Probably the term "em- into only two groups—the primary generation Ijryo erythrocytes" or "embryonic erythrocytes" and succeeding generations. Under the age would more accurately designate these cells in baseline the hours given are 3 less than the actual the chick. In the embryonic span there are nu- duration of the egg in the incubator. This pe- merous generations beginning with the primary, riod of time was estimated to be required for the so that first, second, third (and so on) genera- egg to become warm and for developmental proc- tions could be designated. Actually, the term esses to get under way. All embryonic ages "generations" in reference to red blood cells is given in captions or text are estimated on this somewhat misleading since the cells are being basis. Incubation age is given either in hours produced continuously and not periodically. or in days and hours. Table 9 has been included The term is applied in the same sense as it is used because it is useful in converting from one kind for human populations in that one speaks of dif- of time scale to the otlier. ferent generations; whereas, in fact, there is a Dawson (1936a) made a similar statistical complete and unbroken frequency distribution and cytological study on the shift of erythrocyte in age from youngest to oldest. Even the mi- stages during embryonic development. His totic periodicity known to exist in adult birds and table 1 should be compared with our figure 232, mammals is not present in the embryo. and his photographs of smears from embryos Evidence for the existence of successive gen- that ranged between the age of 4 days of incuba- erations of red cells following the primary gener- tion and liatching age should be compared with ation is based almost entirely on the appearance figures 226-230, which cover essentially the of the mature cell, the shade and hue of the cyto- same range. Under each group of erythrocytes, plasm, the shape of the nucleus and cell, the nu- whether primary or later generations, there are cleocytoplasmic ratio, and the tendency of the five subdivisions—erythroblasts; early, mid-, nucleus to become dense and pyknotic. Reali- and late polychromatic erythrocytes; and mature zation that there are successive generations is one erythrocytes. The data included in the differ- thing, but saying that a particular cell belongs ential counts included all these stages but the to a particular generation—such as the third or only stages selected for illustration in figure 232 fifth— is another. It has not been possible to say were those that during embryonic life became this. Moreover, it has not been possible to say dominant cells. Each curve was plotted as a for sure how many generations there are during hand-drawn average from a large number of dif- embryonic life. We know that there are more ferential counts made from closely spaced ages. than 2, and the total is probably about 4. Sometimes, among the differential counts for a Embryonic erythrocyte generations can be fol- particular cell type at a particular age, the range lowed quite well in Amblystoma. Cameron of variability would run almost 100 percent, but (1941) demonstrated that cells of the first gen- in other cases all the values wou'd '>p closely eration contained 128 yolk plates and each suc- clustered. ceeding generation held half the number con- The first samples of blood were taken from the

tained in the parent cell from which it arose by dorsal aorta of 46- and 47-hour embryos. The division. After eight generations the yolk plates technic employed for obtaining intraembryonic were reduced to one per cell and then the plates blood at this early age is given in chapter 7. degenerated. At the time of hatching, definitive Low-power drawings from such embryos are erythrocytes appeared that lacked yolk plates. shown in figure 224, A and B. Although these

105 days hours. 1,370X. Figure 229.—Blood from heart, of chick embryo. Incubation age, 13 16 17 Microcyte. 1 Mature primary erythrocyte. thromboblast. 2-4 Mid-polychromatic erythrocytes, all of which show 18 Ery thro blast or Small embryonic thrombocytes that have clumped artifacts of cytosomal fracturing. 19 disintegrated the cytoplasm. 5-13 Late polychromatic erythrocytes; some show and Basophil mesomyelocyte. The presence of any granu- artifacts of cytosomal fracturing. Two cells, 7 and 20 locyte in the circulating blood of the embryo is rare. 13, are nearly mature. 14-16 Mature erythrocytes.

106 f « & % ^ ^ ^ • §

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229

107 ^^j^ m.

Figure 230.—Circulating blood taken from basilic (cubital) vein of the chick about 4 hours after hatching. 1,.370X.

1-3 Mature erythrocytes. 6-8 Mature thrombocytes of varying size and shape.

4, 5 Late polychromatic erythrocytes.

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230

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110 Figure 231.—Blood from basilic (cubital) vein of a 7-day-old chick. 1,370X.

A region was selected for drawing that had more than the 6, 7 Mature thrombocyte. average number of leukocytes for this number of 8 Mature lymphocyte. erythrocytes. 9 Mature monocyte.

1-4 Mature erythrocytes. 10 Mature heterophil. basophil. .5 Slightly immature thrombocyte. 11 Mature

Ill Table 9 spicuous nucleoli of figure 251. It is generally sels does the process go to completion. The believed that some change has taken place in the cells of the primary erythrocyte series in the cell metabolism that prevents the normal process mesenchyme tissue outside the vessels are de- of differentiation. The great mass of data on stroyed, either by disintegration or by phagocy- the cytology and propagation of leukemic and tosis. other neoplastic cells amply substantiates the Let us return to a description of the changing idea that an intracellular change can and does picture of the circulating blood. The rapid occur; but this does not exclude the presence in shift between 48 and 65 hours has already been the plasma of a "differentiating" factor, and indicated. The mid-polychromatic primary the sequence of events observed in embryonic erythrocytes are present at a higher percentage blood before and after circulation is established level at 65 hours than are the early polychromatic suggests that such a factor does exist. Wliether eiythrocytes and, with variations, remain high such a factor is sufficiently potent to force the after the latter have declined to a low level. The differentiation of a leukemic cell to the point maximum reached in the differential counts was where it performs the equivalent function of the 71 percent at 93 hours. There is an irregular normal cell is, of course, unknown. decline in mid-polychromatic primary erythro- Dantschakoff (1909a) made an interesting ob- cytes at 97 to 120 hours, and there is a tapering servation on erythropoiesis. The phenomenon off at 120 to 142 hours. Late primary poly- she saw is undoubtedly an expression of the dif- chromatic erythrocytes are present only in small fei'ence between intravascular and extravascular numbers at 65 hours, and the number increases environment. In the early chick embryo, pri- gradually until it reaches a peak at about 120 mary erythroblasts are being rapidly produced hours (fig. 226). Beyond that age there is a both inside and outside the vessels of the vitelline rather rapid diminution in number for this stage membrane and of the embryo. In both en- of development and by 160 hours (fig. 227) all vironments, differentiation to the extent of taking have disappeared. It is not until after 100 hours up hemoglobin is started but only inside the ves- that the primary generation of erythrocytes

ER^ THR0C^TE5 .n CHICK EMBRYOS reaches maturity; then they differentiate rapidly the later embryo generations of mid-polychro- to become dominant cells at 140 to 142 hours; matic erythrocytes continue to be present at a from that time on these cells may be found in the low but fairly constant level until about 285 hours circulating blood in considerable numbers up to of incubation and then drop off gradually. 16 days of incubation. Occasionally they have The frequency distribution curve for the late been seen after hatching. polychromatic erythrocytes of later embryo gen- The tabulation shown in figure 232 gives us erations does not show a sharp peak. Late poly- some information aliout the processes of intra- chromatic erythrocytes are present in the blood vascular differentiation, but the picture of decline at a high level throughout the period from 212 is always masked by the rise of the next higher to 312 hours, after which they decline rapidly stage of development. Undoubtedly much of at first but later continue to be present at a lower the apparent decline in mature primary erythro- percentage level. The course for these cells cytes is due to the increase in the proportion of trails out up to the time of hatching. They are subsequent generations since the data for these still present in variable numbers after hatching curves have been collected as percentage values. and practically any slide, even from older birds, A study is definitely needed in which the abso- will always show a few. lute number of each cell type is oljtained, as Kin- Mature late embryo erythrocytes do not appear dred (1940) has done with the rat. Then the until after 210 hours. Later they rise rapidly true picture of the rise and decline of cell types and from about 375 hours until hatching main- can be visualized; in addition, rate of differentia- tain a high level in the blood. On aljout the tion and the true life span of the cells during 16th day of incubation, they constitute about 90 embryonic life will be known. percent of the cells present. On the day before Following the primary generation there is a hatching close to 100 percent of the erythrocytes succession of generations—secondary, tertiary, present belong to this stage of development and and others. Since it is practically impossible to this, of course, is the condition that continues separate them except in the fully differentiated after hatching (fig. 230). cell, and then only in a general way, all genera- No one, to our knowledge, has as yet attempted tions after the first have been grouped together. to prepare a table of hematologic values for the Graphically this produces long low curves in- chick embryo at various ages. The data pre- stead of a succession of sharp peaks. sented by Flemister and Cunningham (1940) Erythroblasts with nucleoli and basophilic are at least a beginning. They found that in the cytoplasm are picked up occasionally at between allantoic circulation at 8 days incubation there 141 and 214 hours (fig. 227). They may be were 1,210.000 erythrocytes/mm\ and at 10 seen even later but would not constitute a signifi- days, 1,880,000. The hemoglobin at 8 days was cant percentage in a differential count. Early 9.3 grams/100 cc. and at 10 days, it was 14.7. polychromatic erythrocytes are never abimdant; Their percentage values for the types of leuko- between 160 and 243 hours they vary from to cytes listed are such as to indicate that they did 11 percent, and occasionally cells may be found not identify the nonhemoglobin containing cells up through the second week of embryonic life. the same way we have in this study. Most of the cells of these two stages are retained Data for each day of the last week of incuba- at the site of origin in the yolk sac. Differentia- tion were given by Roberts, Severens and Card tion processes within the circulating blood are (1939). They presented erythrocyte, total limited largely to development from the mid- white cell, heterophil, and lymphocyte counts for polychromatic erythrocyte on to the mature form. two lines of chicks. Up to the time of hatching, The percentage of mid-polychromatic erythro- 75 to 96 percent of the white cells were het- cytes of the later generations never reaches a erophils. The total white cell counts were gen- high level—38 percent at 160 hours is the max- erally below 10,000 per cubic millimeter from imum in our data. The talmlar data from which the 15th through the 19th day of incubation. On the graphs of figure 232 were constructed sug- the 20th day the count reached about 11,000 and gested that there was a sharp rise in percentage on the 21st day al)out 16,000 cells. It has been level at about 160 hours, followed by a sharp our impression diat heterophils were not such a decline that does not continue downward because constant constituent of embryo blood, and that

114 lymphocytes appeared only sporadically in the (1925), are megaloblasts and are destined to circulating blood, even up through hatching. produce erythrocytes.

It is apparent that additional studies on the The structure of the early primary erythroblast nuniljer of cellular elements in the circulating is shown in cells 7-70 of figure 224: A. Probably blood of the embiyo are needed to give us a more the least differentiated cell is 2, which has a firmly established baseline. cytoplasm of uniform texture cjuite different from that of die other cells. This cell was added to the drawing from another part of the same also 11-14, and 17. Con- DESCRIPTION OF THE CELLS slide, as were 5, sideration of the significance of this cell is helped A great deal can be learned concerning blood- by looking ahead to figure 225. Within less inculcation these will cell lineage and morphogenesis from a study of than 24 hours of additional the circulating blood in embryos at various ages. have differentiated in the blood into two distinct cell lines the dominant primary erythrocytes When this is supplemented by studies on hema- — topoietic tissues, spleen, bone marrow, and thy- and the embryo thrombocytes. The latter are mus, a fairly complete picture can be obtained of said to come from the same precursor cells as the it is possible the interrelationship of cells. Without this back- erythi'ocytes. Therefore, quite ground the various circulating blood and hema- that these are early embryonic thromboblasts. topoietic tissues of the adult fall into numerous Had Ilalph's jjenzidine technic been applied cjuestion could unrelated series of cells. (p. 231 ), the point in have been settled easily; with this technic the cytoplasm of primary erythrocyte cells gives a positive yellow color when hemoglobin is present, while the cyto- Pritnary erythrocytes plasm of the thromliocytes is negative. The appearance of the cell differs also from that of Satisfactory preparation of the primary eryth- the early primary erythroblast shown in figure rocytes at 2 days incubation recjuires careful at- 233. The latter has a cytosome filled with mito- tention to technic. Most of the early smears chondrial spaces " surrounded by granular cyto- gave shrunken cells that carried numerous protoplasm that has type of texture characteristic of plasmic processes around their periphery. They a primary erythroblasts. The primary erythro- looked like cells A, 11-15, of figure 224 blasts in which tiiis texture is found include those but most of them were worse. Any contamina- undergoing mitosis (fig. 224, A 16. B 21, and tion of the pipette with saline used to float the 22:'- embryo or with albumen or yolk would produce In the cytosome of the primary erythroblast this effect, and if there was any delay in getting the cells on the slide, spreading them out into a are three types of spherical bodies. One of these thin layer, and drying them, they would be is probalily an artifact. It appears in the cells greatly distorted. of figure 224 A as clear spherical spaces. The At all early incubation stages, satisfactory spaces may be small as in 2 and 5 or large as in ])reparation was found to depend greatly on 15 and 16. Although they appear to lie within speed in making the smears. With a few^ refine- the luicleus this is an artifact. A vacuole or other ments in technic, blood could be taken from the

' Takagi ( 1931 ) showed that rod and filamentous mito- dorsal aorta of the embryo as soon as circulation chondria are present in the hlood cells of the yolk sac of the early embryo, but he was not concerned with the specific begins, or it could be taken from vitelline ves- problem of the identity of mitochondria with cytoplasmic sels without contaminating it with other fluids. spaces. Jones (19471, who was interested in the structure of primitive erythroblasts. concluded that (p. 317): considerable difference in degree of dif- A "Light areas in basophilic cytoplasm previously described ferentiation exists between A and B of figure 224; as hyaloplasjn or paraplasm represent, for the most part, the negative images of underlying mitochondria." difference in incubation yet there is only 1 hour of " Takagi (1932) studied the distribution of chondriosomes in of chick embryos age. It is suspected that the development of the dividing blood cells. He used the yolk sac and made his observations on material sectioned in paraffin. embryo represented by A was retarded. Prac- At the metaphase. the chondriosomes were groupeil around dividing cell: none were located either in or tically all the cells at this age, according to the the poles of the on the spindle. During anaphase and telophase, they moved terminology of Doan, Cunningham, and Sabin toward the region of the constricting cell walls. 115 body that has considerable rigidity will push up cleus. From such cells as these arose the later through the substance of the nucleus when the cell stages in the differentiation process. The is flattened, even though it is formed in the cyto- orange-stained area may well have consisted of some. With the nuclear substance displaced, vacuoles of this type. If additional study should the vacuole appears to be within the nucleus. An prove that the vacuoles seen beside the nucleus additional basis for suggesting that these granules both in sectioned material and in dried smears are artifacts is the fact that they tend to occur are the same, this would offer an excellent cell most abundantly in cells that are most distorted organelle that could be used as a common basis {A, 13, 15, and B, 23). for identifying the same cell under two different The second type of vacuole is a highly refrac- technics. Dantschakoff (1908b) observed that tory one. In the drawings each such vacuole is the light-staining spheres contained centrosomes. indicated by a blue ring (figs. 224 and 225). None of the three types of vacuoles or spheres The number within a cell varies widely from none observed in the cytosome of the primary genera- to as many as are seen in figure 225, 23, but three tion of erythroblasts were observed in the cyto- seems to be an average number. Only a few are some of later generations. Tliis difference be- present in the cells of figure 224 A, and by 69 tween the first and subsequent generations is in

hours incubation (fig. 225) they have practically agreement with observations ]jy Dantschakoff disappeared; but these vacuoles are typical for (1908b) on sectioned material. the cells of figure 224 B. The nature of the sub- Since none of these types of spheres have been stance they contain, their function, and their observed in later generations of erythroblasts in specificity are unknown. Microchemical stud- smears, it might be possible to establish whether ies, or the tracing of their course of appearance later generations are similar to the cells (megalo- and disappearance with the phase microscope in blasts) * of the primary generation and to the living cells, might be revealing. megaloblasts of anemia—a question that has been The third type of vacuole is small and round, ably discussed by Jones (1943). This author is not refractile, has a tinge of color, and tends has pointed out that there are differences in nu- to form clusters. At the stage represented by fig- clear cytology among primary and normal de- ure 224 A they are best shown in cell i, but in B finitive megaloblasts and megaloblasts of anemia. numerous cells show these bodies (4, 5, 8, and Studies by the smear method show that such 12-14). When they first appear, they are scat- differences certainly do exist in the primary and tered luit as they increase in number they form a normal definitive erythroblasts of the chicken. group of spheres. By 65 hours incubation the As an example of this difference, the nucleus of individual bodies of these clusters have coalesced the primary erythroblast generally contains two and are responsible for the light area in the cyto- nucleoli. No other blood cell included in this plasm adjacent to the nucleus. Dantschakoff study has been obseiTcd to have this numljer of (1908b and 1909a) observed in her sectioned nucleoli. The chromatin reticular pattern is material the presence of an orange-stained area somewhat coarser in primary erythroblasts than beside the nucleus of the early erythrocyte found in the cells of a corresponding degree of differen- in the yolk sac. Similar orange-staining spheres tiation in later embryonic generations. The nu- were illustrated and described by Maximow cleoli seem to be more prominent in the first gen- (1909) in his study of early formation of blood eration than in later ones but this is probably due cells in the mammalian embryo. The whole entirely to physical causes associated with the process of differentiation of primary erythro- coarseness of the screen which permits a better blasts from mesenchyme has been described by view of the interior of the nucleus than can be Murray (1932). He used tissue culture prep- obtained when looking through the fine screen arations of the primitive streak of the chick that is characteristic of later generations of embryo. It was obsei'ved that the immature erythroblasts. primitive erythrocytes were rather small cells Differences in size among primary erythro- with pseudopodia and with nuclei which con- blasts have not been specifically mentioned thus tained one or two nucleoli. The cytoplasm far. The differences may be seen by glancing was strongly basophilic and contained one * "Erythroblasts" and "megaloblasts" are used as synony- or more eosinophilic masses adjacent to the nu- mous terms.

116 .

at figures 224, 233, and 234, which were selected usually applied to the human species, these cells to show range in size and in nucleocytoplasmic would still be called erythroblasts, since the term ratio. Mitotic figures are abundant at this age, "polychromatic" is applied to cells after the nor- as shown by cell 16 in figure 224 A and cells 21 moblast stage has passed, but for the bird, the and 22 of B. For persons seeking a large tough usage followed here seems to give maximum uni- cell suitable for the study of the avian chromo- formity of nomenclature for comparative pur- somes, the writers know of no better material, poses with otlier species having nucleated red although they have not examined the neuroblast cells. These terms—early, mid-, and late—re- that has been used so frequently. In smears fer to the blue, gray, and orange phases of color. fixed in Petrunkevitch No. 2 and stained with As previously indicated, the blue represents a

May-Gioinwald Giemsa it has been possible to predominant stroma of basophilic material. count about 60 chromosomes and it is likely that The gray stage appears gray because there is a some of the problems in this fiekl could be solved mixture of two opposite colors—blue and with technics selected specifically for cytogenetic orange—and as more of the original Ijasophilic studies. stroma is lost, there is no longer a balance and the

There is perhaps one word of caution to be orange color predominates. In later generations given. Cytologic evidence indicates that the of cells this transition is a period of cytosomal prime function of this generation of erythrocytes weakness, and artifacts readily appear in poly- is to form hemoglobin. This is accomplished in- chromatic erythrocytes. In the first generation, dependently of the differentiation stages that however, the transition is so rapid that the cyto- usually accompany this process. As a result, some does not develop a weakened condition; mitosis is often abnormal, and frequently chro- hence artifacts are not commonly seen (fig. 225) mosomes lag behind in the anaphase stage. The Artifacts appear in some cells after they have abnormalities observed at mitosis in the primary reached the late polychromatic stage (fig. 226). generation of erythrocytes are similar to those Erythrocytes at 65 hours incubation have taken reported on mitosis in neoplastic cells. Abnor- on spherical shapes but they may be pressed into mally large size often occurs in neoplastic cells angular shapes when the smear is made, and there and an occasional giant cell may be found among may l)e overlapping of cells that produces clear primary erythroblasts (fig. 251) .^ Such mitotic bands and crescents in the cytosome (fig. 225, 15 abnormalities and giant cells probably mean with 17). little or nothing toward the production of later The cytosome is closely packed with mito- blood neoplasias in the bird but since some em- chondrial spaces that have undergone a change bryonic cells have certain points in common with from the rods of the earlier stage to innumerable neoplastic cells, the possibility always exists that small granules Uiat give to the cytosome a tex- a residual, dormant cell of this type, if not de- turial quality, and would be inaccurate if a homo- stroyed, could later be stimulated to reproduce geneous wash of color were applied to the draw- itself. Most primary erythrocytes degenerate ings of these cells. along conventional lines, as seen in figures 249, A peculiarity of these cells is the presence of a 250. 252, and 253. seemingly ectoplasmic mantle around the periph- Hemoglobin development proceeds very rap- ery of some cells (figs. 225, 4, and 235), the idly and by 65 hours incubation (fig. 225) has significance of which is unknown. Illustrations reached the early and mid-polychromatic stages. of immature human red cells from various

This terminology is based on the tinctorial qual- sources have not shown anything similar to it. ity of the cytosome and not on nuclear or cyto- This leads one to suspect that it may possibly be plasmic differentiation. On the basis of criteria an artifact associated with the large size of the avian cell. A tendency toward the same type of '^Dawson (1933a) observed that giant atypical erj'throcytes reaction is shown in some of the cells of figure were produced in Necturus in the regeneration process that followed a destruction of normal erythrocytes by lead poison- 226 but this reaction does not reappear in any ing. The occurrence of gigantism in mammalian erythro- of the later generations, nor do smaller cells of poiesis has been reviewed by Berman (1947). It may express itself as a large uninucleated cell, as shown here for the same age (fig. 236) show the marginal rim. the chicken, or as a giant multinucleated cell. The latter may At this age (altout 65 hours) the nucleoli, usu- undergo multipolar mitoses and Berman is of the opinion that some of these may return to normal, uninuclear cells. ally two in number, are even more distinct than 117 in the cells shown in the preceding plate. The small, densely stained nucleus and indications of greater distinctness is due to tlie contraction of cytoplasmic differentiation, yet the hemoglobin the chromatin network into larger hut more content is low in comparison with that of figure widely spaced clumps. Mitotic figures are still 238, which is not so well differentiated cyto- abundant, and in cells 20 and 22 of figure 22.5 logically.

it is evident that division reduces the size of the The primary erythroblasts at 65 to 93 hours cell to half of the original volume. Undoul^t- of incubation shown in figures 235, 236, and 238 edly cell growth occurs during the interkinetic may reveal a faint tinge of eosin coloration but period but average size decreases as the cells get in some preparations made at this age the cyto- older (compare figs. 225 and 227). plasm may take a completely basophilic colora- A statistical study on the percentage of mitosis tion. The benzidine test (p. 231) reveals that, present in the early embryo has been prepared nevertheless, hemoglobin is present (fig. 237). by Dawson (1936a), who says (p. 262). "In the Thus this test demonstrates tliat basophilic color- blood stream there is little restriction of mitotic ation of the cytoplasm does not preclude the ex- capacity in the differentiating primitive line until istence of hemoglobin and it is for this reason that the mature stage is reached." the stage of erythrocyte development called baso- Two types of cells are distinctly visible at 6.5 phil erythroblast has been omitted from tJie series hours. One is the primary erythrocyte line, and of erythrocyte stages presented in table 2.

the other is the emlnyo thrombocyte line. The Hemoglobin is present in erythrol)lasts of em- cytology of the latter will be discussed after the bryos incubated 48 hours, although the cytoplasm description of the red cell has been completed. is fully basophilic. No benzidine tests were After 4 days incubation little change is visilile in made at this age, but the blood flowing through the primary erythrocyte except additional hemo- the vessels of the embryo and area vasculosa is globin accumulation, a greater range in size, and clearly red. a sharper distinction between red-cell and throm- Additional hours of development produce bocyte lines. The characteristic intensity of definite changes, which are seen in figure 226 and

cytosomal hue is shown in figures 238-240 and in figures 241 and 242, where the cytoplasm has ranges from early to late polychromatic. A lost nearly all of its mitochondrial spaces and the comparison of figures 238 and 239 brings out an perinuclear, vacuolar space characteristic of interesting point—the latter is a small cell with a earlier stages, is nearly gone. Most of the cells

Figures 233-243.—Stages in the differentiation of primary erythrocytes selected to show variations in size and struc ture. 2,470 X.

Figures 233, 234: Primary erythroblast from dorsal aorta Figures 238, 239: Cells from an embryo incubated 3 days of an embryo incubated 1 day 22 hours. Same slide as 21 hours. figure 224. 238 Mid-polychromatic ])riniary erythrocyte. Still shows

233 Early primary er\'throblast. Large nucleolus fills a refractile granule and mitochondrial spaces char- upper half of nucleus. acteristic of erythroblasts. 234 Primary erythroblast. Lobulations are few and 239 Early polychromatic primary erythrocyte. Less small. hemoglobin than preceding cell but further differentiation. Figures 235-237: Primary erythrocytes from embryo in- 240 Late polychromatic primary erythrocyte in mitcsis. cubated 2 days 17 hours. Polar view of what is apparently metaphase. Em- bryo incubated 4 days. 235 Late primary erythroblast. Same slide as figure 225. Rounded cytosome. The effects of an eeto- plasmie mantle is characteristic of cells of this size Figures 241, 242: Late polychromatic primary erythrocytes and degree of differentiation. from an embryo incubated 5 days 3 hours. 236 Small early polychromatic erythrocyte. From same 241 Average size. slide as preceding figure. 242 Small size. 237 Late primary erythroblast. Ralph's benzidine polychromatic primary erythrocyte. Larger method and M. G. G. I^resence of hemoglobin in 243 I^ate cyto.some indicated by yellow color. Cell equivalent than average, almost a mature cell. From same to figure 235—taken from same embryo. slide as figure 227

]18 234

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236 237 235

^^y^ 240 239

238

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242 241 243

119 Figures 244-253.—Primary erythrocytes—mature and abnormal. 2,470X.

FiGURB.s 244-248: Mature primary erythrocyte.'^. cubated 1.3 days 15 hours. Many examples of degenerating primary erythrocytes are found in 244, 245 Same slide. Embryo incubated G days 22 hours. older embryos. The second cell has taken on a slightly oval 250 Poor stainability of the nucleus is characteristic of shape. aging primary erythrocytes. I'mibrj'o incubated 15 24(5 A rare example of constricted cytosome as well days 23 hours. as nucleus, characteristic of amitosis. Embryo incubated 9 days 14 hours. 251 A giant late polychromatic erythrocj'te—very rare. 247 The primary erythrocytes are distinguished from Xuclear chromatin less dense than normal, thereby later generations of red cells by the more intense revealing clearly the nucleolus. Found in same slide coloration of the cytoplasm. Embryo incubated from which figure 240 was taken. 8 days 21 hours. 248 A primary erythrocyte in which an oval shape 252 Karyorrhexis of nucleus in a primary erythroblast has developed. Embryo incubated 11 days 1 or an early polychromatic erythrocyte, observed hour. rather regularly at young ages. Embryo incubated 2 days 18 hours. Figures 249-25.3: Abnormal primary erythrocytes. 253 Polynuclear primary erythrocyte. Cell shows evi- incubated 9 249 A primary erythroplastid with a body of chro- dence of aging and decadence. Embryo matin material still remaining. Elmbryo in- days 14 hours.

120 I ^ ^.

244 245

24()

247 248 249 250

252 253

251

121 Figures 254-264.—Developmental stages of embryo erythrocytes following the primary generation, often called secondary or definitive erythrocytes. 2,470 X.

FinuRES 254-256: Enjlhroblasts. All from the same slide. 260 Slight .staining toward the right of the nucleus. Embryo incuhaled 5 days 21 hours. 261 Overall staining of the nucleus but not sufficiently intense to be fully useful for cell identification. 254 Early erythroblast. 255 Erythroblast. Figures 262-264: Early polychromatic erythrocytes. Same 256 Late erythroblast. slide as figures 359-361.

Figures 257, 258: Late erythroblasts or early polychromatic 262 The cytoplasm resembles that of the late erythro- erythrocytes in mitotic division. Embryos incubated 9 blast but the condensation of nuclear chromatin is days 18 hours. characteristic of the early polychromatic erythrocyte. 263 A more differentiated cell than figure 262. 257 Late anaphase. 264 A more differentiated cell than figure 263. 258 Late telephase.

Figures 259-261: Erythroblasts showing various degrees of failure of nuclear staining. All from the same slide. Embryo incubated 5 days 3 hours.

259 The nucleus has an empty appearance owing to its incomplete staining.

122 254 256

255

257 258

259 260 261

263 262

123 Figures 265-275.—Late developmental stages of embryo erythrocytes; also three cells that show technic artifacts. 2,470 X.

265 Early polychromatic erythrocyte, typical of this that this cell may belong to the primary erythrocyte stage as seen in the late embryo, and after hatching. series. Embryo incubated 11 days 1 hour. Embryo incubated 5 days 22 hours. 271 Mature erythrocyte typical of the older embrj'o. 266 Mid-polychromatic erythrocyte. Cytoplasm frac- Embryo incubated 13 days 5 hours. tured. Embryo incubated 6 days 22 hours. 272 Mature erythrocyte identical with the red cell after hatching. Embryo incubated 16 days. Figures 267, 268: Late -polychromatic erythrocytes. Same slide as figure 266. Figures 273-275: Cells shoioing the defect of cytoplasmic fracturing. This generally occurs in the polychromatic

267 Nearly round cell. stages. 268 Slightly ovoid cell. 273 Mid-polychromatic erythrocyte. Embryo incubated Figures 269-272: Mature embryo erythrocytes. 5 days 18 hours. 274 Late polychromatic erythrocyte. Disturbed stroma 269 Mature erythrocyte. Embryo incubated 8 days 21 but no unstained spaces. Same slide as preceding hours. figure. 270 Mature erythrocyte. The large cell size, the con- 275 Late polychromatic erythrocyte. Partial margina- densed nucleus, the small nuclear size relative to the tion of the hemoglobin-bearing stroma. Embryo in- cytosome, and the color of the cytoplasm, all suggest cubated 6 days 22 hours.

124 ^ c^

265 266 267 268

« 9

269 270 271 272

273 274 275

125

have reached the late polychromatic stage hut a what we have pictured for the reticulocytes in the few show the color typical of the mid-stage ( fig. definitive cells after hatching. 226, 1 and 2). A different type of cytosome The particles near the margin of cell 5 of figure rarefaction appears at this age—the fracture 227 are merely foreign bodies that have fallen on breaks (cells 10 to 12 and others) that in the pri- the surface. The nucleus now holds a central po- mary erythrocytes are not as severe as in later sition in the cell. There is considerable varia- generations. Some cells, 3, for example, show tion in the ratio of nucleus to cytoplasm and in change toward the oval form; the final nucleo- some of the cells—for example, 2 and 11 oi cytoplasmic ratio is not yet established but the figure 227—the micleus is smaller relative to the nucleus has undergone considerable contraction, size of the cell than in the definitive erythrocytes. and the chromatin now forms coarse blocks. The This is a characteristic of the mature primary red luicleoli have practically disappeared but mi- cell (figs. 244 and 245). In general the cells totic figures are still large and conspicuous. The and their nuclei are still round, only a few show- capability of a cell for mitosis is considered by ing a slight elongation. Osgood and Ashworth (1937) to have consider- Chromatin of the nucleus is not so intensel)' able value for the proper placement of a cell in stained in the primary erythiocytes as in the later its developmental series. generations of red cells in the same blood. This Maturity of primary erythroblasts is attained type of faint coloration of the nucleus is found during the fifth day of incubation and reaches the as a general characteristic of the primary cells peak percentage by about the sixth day (fig. 232) (figs. 244 and 250) ; where extreme, it has been Dantschakoft" (1908b) states that primitive eryth- classified as indicative of deterioration and de- roblasts attain their complete maturity on the generation, but as seen in figure 227, it hardly fifth day. Figure 227 shows a typical low-power seems likely that this is the case at this age. field in which there is a mixture of mature first- Later generations of erythrocytes first appeared generation red cells and early stages of succeed- at about 120 hours (5 days) and it is hardly to ing generations. be expected that the primary generation would

The primary erythrocytes still show a wide be disappearing 21 hours later at a time when range in size (fig. 227, 5 and 9) although they the succeeding generations of erythrocytes are are now all equally mature. Such variation, just making their appearance. It is true that were it to occur after hatching, would indicate a definite examples of degeneration can be seen at pathological condition and would probably be all ages, such as karyorrhexis (fig. 252) and classed as anisocytosis. Among these mature erythroplastid production (fig. 249) in early cells there are shades of color difference. Al- generations of erythrocytes, and multiple nuclei though all have a deep orange base color, some of (fig. 2.53) in later generations. them (cell 5 and the one just below 12) have Figure 228 shows that new generations have some gray mixed with them. The cytosome has been poured out into the blood and that they lost the coarse textural quality of the eryth- have become Uie dominant cells; only a few rocytes at earlier embryonic ages (figs. 235, 236, mature primary erythrocytes remain—6 to 17 238, and 239) and taken on a uniform, finely percent of the total number of cells. The nu- granular quality (fig. 243). This cytoplasmic cleus and cytoplasm have the same appearance texture, characteristic of a cell that is mature or that they had earlier. It is a question whether nearly mature, may be observed at 5 days of in- these cells ever become oval in large numbers. cubation (figs. 241 and 242). Cells typical of this age are shown in figures 247 Since the study of reticulocytes in the blood of and 248; the latter is slightly ovoid. Degenera- the hatched chick came rather late in the program tion appears in erythroplastid formation, as weak of work, there was no opportunity to reexamine affinity of chromatin for stain, and as pyknotic

the primary erythrocytes to determine whether nuclei ( lower left-hand cell of fig. 228) . Mature they passed through a reticulocyte stage; but primary erythrocytes become increasingly scarce Dawson (1936a) showed a series of figures of pri- as the embryo grows older; one is shown in figure mary erythroblasts and erythrocytes stained with 229 (cell 1) from an embryo 13 days 16 hours brilliant cresyl blue, and these closely resemble old. They may be seen the first day after hatch- 127 ing but not from every chick. None are shown cell serves both lines. A common intravascular in figure 230. origin has been suggested for these cell lines. Figure 246 was inchided because amitosis is (See Sugiyama, 1926, for review of the litera- said to occur in human, amphibian, and bird ture.) The cells shown in figure 226, 17 and in blood cells after reproduction by mitosis has figure 227, 26 cannot be classified with certainty; ended. Constricted cells, cells with indented either cell may belong to either of the lines men- nuclei, and cells with two nuclei can often be tioned. Blast cells of both lines contain nucleoli. found, but according to E. B. Wilson (1925) The l)last cells of the two lines can be sep- (quotation on p. 31), amitosis involves con- arated fairly accurately later—during incuba- striction of botli cytoplasm and nucleus. Of the tion and, in the adult, when bone marrow pro- hundreds of slides examined, this is the only cell ihices these cells. wliich entirely fulfilled the criteria of amitosis The erythroblast has a more abundant, more and, when a cell having this appearance occurs lightly stained cytoplasm than the thromboblast, so rarely, additional evidence is needed before in which the cytoplasm is a darkly stained rim we can consider that amitosis is a normal means aroinid the nucleus. In die erythroblast the of increasing cell number. chromatin pattern is reticulate, whereas in the

thrombocyte it is more particulate and punctate. Anything subsecjuent to the blast stage is easy to identify; for example, in figure 227, cells Later emhryonic erythrocyte generations 13-15 are mid-polychromatic eiythrocytes and The term "later embryonic erytlnocyle genera- cells 16-18 are late polychromatic erythrocytes.

tions"' is used in the legends because it fully dif- Many of the latter type (cells 19-24) reveal

ferentiates between the cells to which it refers cytosomal artifacts. Early polychromatic eryth- and the cells of the primary generation; but it rocytes are scarce; one is shown in mitosis

is a cumbersome term, and in this section the cells (cell 25) and even this approaches the color of will be referred to simply as erythrocytes. the mid-erythrocyte. Differentiation proceeds Cells that produce the later genei-ations of em- slowly and even by the nintli day there are only jjryonic erythrocytes undergo a spurt of multi- relatively few mature cells; at least there are only plication within the luniina of the venous retic- a few that give a good vigorous color. Most of ulum. According to Dantschakoff (1908b), them are late polychromatic erythrocytes. By this occurs on the fourth day of incubation, and the 13th day mature cells are considerably more the venous reticulum appears at the time the yolk abundant, but at hatching or within the first 24 sac membranes grow into the yolk substance. hours after hatching, the dominant cell is the ma- Cells of this series appear first about the fiftli ture erythrocyte, with a few scattered immature day, as observed by Fennel (1947) and by others cells (fig. 230). who have followed the changing blood picture in The steps involved in the differentiation proc- the endnyo. Dawson (1936a) reports that stem ess of the erythrocyte line as well as some typical cells of this series appear on tlie fourth day. It artifacts of the mid- and late polychromatic has been our observation that up to 96 hours in- erythrocytes are shown in figures 254-275. The cujjation the only cells present are tlie primary least differentiated cell observed is illustrated in erythrocytes and the embryonic thrombocytes, figure 254. It has a slight amoeboid shape and and that the blast cells seen on the fourth day be- a smooth-textured cytoplasm, wliicli has a few long to the latter series. These cjuickly differen- small spaces. The nucleus is a reticulum with tiate into tluombocytes, and die blast cells seen fine meshes, and individual chromatin granules on the fifth and sixth days are the precursors are indistinct. A nucleolus is very faintly vis- of the later erythrocytes. Cytologically there is ible at the upper side of die nucleus. no real difference between the tluomboblasts of A type of blast cell much more commonly 4 days and the erythroblasts of 6 days (compare found is that shown in figure 255, which is round. figs. 255 and 280). At 4 days incubation the The cytoplasm with its mitochondrial spaces is a structural series leads to the thrombocytes, but narrow, strongly basophilic rim around the nu-

at 5 days and later it leads to the erythrocytes. cleus. The latter is a coarse reticulum, still Further testing might show that the same stem without sharp distinction between chromatin and

128 ;

linin network. Another cell taken from the same first generation of erythrocytes, where chromatin slide (fig. 256) is smaller but is essentially at clumping and hemoglobin acquisition developed the same stage of development as the larger cell ahead of cellular differentiation. except for a slightly more condensed pattern of In the generation succeeding the primary one, nuclear chromatin. the precocious development of these two factors Mitosis continues. The cells in figures 257 does not get as far ahead of general cellular dif- and 258 were found in the circulating blood of ferentiation as before and, with each succeeding two embryos, both inculjated for 9 days 18 hours. generation, there is a gradual approach to the The clumps of basophilic cytoplasm are uni- condition found in the adult bone marrow, where formly distributed. In the process of division cellular differentiation and hemoglobin ac- the cell loses all its identifying marks and it can cumulation keep pace with each other. Thus, only be suggested that these two cells are either all three cells within this group have been desig- late erythroblasts or early polychromatic eryth- nated as early polychromatic erythrocytes, and rocytes. in figure 264 the tinctorial quality of the cyto- The next three cells illustrated (figs. 259-261) some clearly indicates that hemoglobin is present. probably should be included with examples of Figure 265 has been included under the same artifacts. They are presented with the idea category. In this case, however, all the features of showing how blast cells appear when they are that are used to measure cytologic differentiation improperly stained. The cytoplasm in each cell have shifted to indicate a more differentiated stained well and showed the mitochondrial spaces cell: the nucleus has become slightly eccentric, but, because the nucleus was colored only faintly, the cytosomal rim around the nucleus is greater the boundary between nucleus and cytosome was in proportion to nuclear size than in the preceding not definite and the vacuoles of nucleoplasm and cells, the mitochondrial spaces have become cytoplasm merged. It is interesting to note again small and the chromatin clumps have grown in that when the surface reticulum is lightly colored size and density and in their closeness to each the internal structures become visible; thus the other. existence of a nucleolus is revealed in figures 259 The mid-polychromatic eiythrocytes present and 260, but in 261, where the reticulum takes no difficulty in identification and several ex- the stain more energetically, the nucleolus is only amples are pointed out in figure 227, cells 13—15. vaguely indicated as lying to the left side of the Figure 266 is a cell at this stage, drawn at a nucleus. higher magnification. There is, of course, a The failure of the stain to penetrate the nu- range of color in the cytoplasm of various cells cleus in figures 259-261 is limited to the large at this stage as there is for the one before and the blast cells. In more difi^erentiated cells such one following, and so some mid-polychromatic as figures 262-264, taken from the same slide, eiythrocytes may appear gray and others nearly the surface chromatin of the nucleus is intensely blue. colored. The difference is not due to cell size The next stage of development is the late poly- since the larger and smaller are equally flattened chromatic erythrocyte (figs. 267-269). The

it seems more reasonable to suppose that there cytoplasm is more homogeneous than it was in the is a difference in the physical character of the previous stage, and at the beginning of this

nuclear membrane. phase it still contains considerable basophilic The cytoplasm in figures 262 and 263 shows material. Many of the cells in figure 228 are at the same intense, dark blue that is shown in the this stage of development. As hemoglobin ac-

blast-cell stage; it is still a narrow rim extending cumulates, the cell elongates and the ratio of only slightly beyond the nucleus, and mitochon- cytosome to nucleus increases. The nucleus also drial spaces are still present. The nucleus re- becomes slightly elongated and the chromatin

tains a nucleolus that is conspicuous even in pattern takes on the characteristics of the lepto- figure 264; yet differentiation has occured, as chromatic type of nucleus found in mature cells. clearly evidenced by the clumping of the chro- The mature embryo erythrocyte often con- matin pattern and the taking up of hemoglobin. tinues its hemoglobin synthesis until it reaches This sequence of differentiation is reminiscent a tinctorial level equal to that of the primary of the precocious development occurring in the generation of erythrocytes (figs. 269 and 271).

129 This makes it quite difficult to identify with cer- globin and reduction in cell size. Our studies tainty a cell of the type shown in figure 270, have not supported the idea that the thromjjocyte where the cytoplasm is slightly grayish, and the series arises from hemoglobin-bearing cells, al- nucleus is shrunken and has started toward de- though it is recognized that a close parallelism in generation. These are all features character- development exists between erythrocytes and istic of the primary erythrocyte. thrombocytes. Six other theories on the origin By the end of the second week of incubation of thrombocytes have been reviewed by Sugi- and from then on until hatching, the mature yama. Some have called these small clumped erythrocytes are indistinguishal)le from defini- cells of the embryo "lymphocytes," and Sugi- tive cells (figs. 228, 229^271, and 272). The yama (1926) presents convincing evidence that three cells showing artifacts (figs. 273-275) this is an error. Typical lymphocytes in his have already been mentioned and they illustrate preparations did not appear in the circulating the defects in greater detail than do the low- blood of the emijryo until 17 days' incubation. power drawings (figs. 227-229). Only the Our observations agree with his and further show cytosome is affected, never the nucleus. In the that lymphocytes, even in the late embryo, are in- cytoplasm any odd effect may be produced. constant, and that they do not Ijecome a constant, calculable component of the blood initil after hatching.

It is ol)vious from the papers of Dantschakoff Embryo thrombocytes (1908b) and Sugiyama (1926) that both au- The tlirombocytes of the embryo appear as a thors were studying the same cell, the embryonic definite cell line soon after the primary eryth- thrombocyte. Dantschakoff claimed that pri- roblasts are well established and are clearly mordial cells (large lymphocytes) of the early present by 68 hours incubation (fig. 225), but embryo, produced microblasts and then micro- whether they are present at 48 hours also (fig. cytes (dwarf lymphocytes). She suggested that the 224) is still undetermined. The cells of 224 the dwarf lymphocytes might be related to A, 2 and 224 B, 1 and 2 may be primary throm- spindle cells (thromJjocytes) and that the dwarf boblasts. When seen in its entirety, cell 224 B, lymphocytes were different from the small lym- 2 resembled in some respects a yolk sac macro- phocytes that appeared rather late in embryonic phage such as shown in figure 308. The eryth- life. According to Dantschakoff (1916a) the rocytes contain hemoglobin and the thrombo- latter appeared between the fifteenth and seven- cytes do not; so if the two cell types are mingled teenth day of incidjation. they could be sorted rather readily by Ralph's By 65 hours of incubation, the thrombocyte benzidine method. line is easily distinguishable from the erythro- to There is a peculiar behavior of the primary cyte line; the cells of the former tend clump erythrocytes at the 48-hour age that may have a readily but the latter no longer exhibit this prop- bearing on the problem. If the primary eryth- erty, and since the erythrocyte line is a group of rocytes taken up into the cannula are not dis- cells fairly well synchronized in development, a charged immediately onto the slide and there structural series does not logically lead back to spread so that they dry quickly, they will clump the blast cell such as shown in figure 225, 25: yet and degenerate, as the thrombocytes will at an a complete gradation of cells does lead from this older age. Many cells will clump together and to the small embryonic thrombocytes ( fig. 225, their appearance when partly degenerated is 27), which supports the conclusion that the blast similar to figure 224 A, 11 and 12. If eryth- cell shown in figure 225 Ijelongs to the thrombo- rocytes and thrombocytes come from the same cyte series. The same is true of the cell shown in primordial cell, perhaps erythrocytes for a short in figure 276 from the same slide. Cell 25 period of early embryonic life assume a function figure 225 appears to be discharging cytoplas- peculiar to thrombocytes. mic blebs; whereas, in figure 276 only large pro- Sugiyama (1926) concluded that thrombo- trusions are present. Both show cytoplasmic cytes of the early chick embryo first arose by a spaces but pro])ably the feature that most strongly transformation of megaloblasts into Unombo- suggests their thrombocytic affinity is the punc- blasts. The transition involved loss of hemo- tate character of the nuclear chromatin. This

130 ;

of abundant mito- cytologic detail of the immature thrombocyte nu- staining: full differentiation is attained, especially in figures cleus was not obsei-ved by Sugiyama (1926), chondrial spaces probably ])ecause Wright's stain was used. 283 and 284. Beyond the blast stage, thrombocytes can be Shifting of the nucleus to an eccentric position (figs. but, identified easily; thus even in one field ( fig. 225) may occur in throml)oblasts 283-285) two older stages are visible. Cells 24 and 26 as in the erythrocytes, the nucleus tends to remain are probably at about the same level of differen- in the center of the cell, as in figures 280 and tiation, and cells of this size do not tend to clump 286, and in the four small thrombocytes in fig- together as readily as do the smaller cells (27). ure 225, i.e. 27. By 4 days of incubation a wide In the clumping process, cytoplasmic IdeJjs are range in stages of development has appeared; the increas- thrown off. A duplicate slide, made from the cells form a series of decreasing size and same eml)ryo that was used for figure 22.5, was ing cytoplasmic differentiation (figs. 286-292) stained with Ralph's modification of the benzi- yet the impression is gained that this differentia- parallel that which dine test. Two cells ( figs. 277 and 278 ) demon- tion process does not exactly after strate that hemoglol)in is absent from the throm- takes place at older embryonic ages and bocyte even at an early stage of differentiation. hatching. Like the primary generation of eryth- Figure 277 is probably equivalent to cell 26 in roblasts, this primary generation of thrombo- figure 225; figure 278 is smaller and the nucleus cytes seldom produces an oval cell. Later, how- frequently is not visible. Dividing throndjocytes are some- ever, cells of this shape can be found times found. Unlike the erythrocytes which if the preparation is made quickly enough. A maintain a compact cytoplasm, the thrombocytes few cells with oval shape have been seen at 4 days twice at 5 days, but even during mitosis (fig. 279) can be stimulated ( fig. 287) and again once or typical. to give off protoplasmic fragments. these cases are too rare to be called Spreading and drying the cells is not always Sugiyama (1926) pictured a throml)Ocyte of oval rapid enough to prevent degeneration of the shape, taken from an emlnyo incubated only 2 thrombocytes. Many of the cells illustrated in days. We have never seen a thrombocyte having figures 276-295 are not typical of what one so this shape in an endn-yo this young. often finds on the slide, since the ones selected The first step in the differentiation process fol- were tliose least degenerated. A degenerated lowing the blast stage is the loss of staining af- throndjocyte looks like a lymphocyte. Not finity by the cytoplasm (figs. 286 and 288) and every slide made from a series of embryos is the next is extensive vacuolization (figs. 290 and single magenta granule equally productive of early stages, but the em- 291 ) . In figure 290 a bryo that contril)Uted figures 280 and 286 had appears to be present outside the nuclear margin, an abundance of them. and in figure 291 such jjodies are numerous. A typical throml)ol)last at 4 days of incuiiation They are not the specific granules of the definitive extruded cliromatin is shown in figure 280. It has a narrow rim of thrond)ocyte lint resemble delicate, cytoplasm that stains an intense dark blue or deep particles more than anything else. The de- violet. Within are large mitochondrial spaces. fine pink granules that are characteristic of in the first The reticulum at the surface of the nucleus in fig- finitive thromliocytes never appear ure 280 appears more clearly defined than that generation of thrombocytes; the two bodies shown days in the nucleus of the amoeboid thromboblast in in figure 293 from an embryo incubated 5 figure 276. Associated with this more open net- 21 hours are about the size of definitive granules work is a vague suggestion of a nucleolus. but belong to the chromidial type. the cir- Failureof the nuclear surface to stain is a com- Thromljoblasts soon disappear from which only mon feature of young cells with large nuclei, and culating blood of the embryo, after thrombocytes three degrees of incomplete staining are shown large, medium, and small embryo the occurrence of in figures 281-283. These duplicate the reac- are seen. By the ninth day The gran- tion that occurred in young erythrocytes (figs. specific granules is fairly common.

eighth day ( fig. 2.59-261) in that the nucleolus is more readily ules may appear as early as the still not show them visil)le than in the well-stained cell. In these 294) but many of the cells do are present in three examples, deficiency in nuclear affinities until later in development. They chiefly be- does not influence the (fuality of cytoplasmic those cells selected for illustration 131 cause such cells were choseu in order to make it, or (3) was under the control of a different set more convincing the evidence that these are of factors with little or no causal relationship to tlirombocytes and not lymphocytes. Lympho- cellular breakdown. For the present, only ex- cvtes, monocytes, and granulocytes are not nor- amples can be mentioned, and these do not al- xiialiy present in circulating hlood of the embryo, ways support the same hypothesis. Figures 302 although cells belonging to these types may be and 303 show a definite acidophilic margin and ^een occasionally, and they will be considered slight diffusion of the reaction into cell proto- more fully when the cells found within the hema- plasm; yet there is no indication of cell rupture. topoietic organs are described. On the other hand, figure 306 shows cytoplasmic It has been indicated already that mature disintegration well started, yet basophilic affin- stages from later generations of thrombocytes, ities have been retained, and figure 295 shows like later generations of erythrocytes, approach both rupture and acidophilic staining. These a higher level of terminal differentiation than did examples come from different slides. The cyto- earlier generations and thus during the second plasm of thrombocytes under the best conditions week of embryonic development, all cytologic stains so delicately that only with optimum illu- features that characterize the definitive cell can mination correctly aligned can these differences be seen but not exactly as they will appear in the in color and structure be detected. circulating blood of the adult bird. Specific The cell represented by figure 302 was selected granules of the type shown in figure 296 are to show that an intact, almost definitive, throm- atypical in that they are larger than normal and bocyte can be found as early as 9 days 15 hours. in that each lies within a vacuole. This vacuolar This cell was not seen until after a number of effect may possibly be due to technic since it is slides had been made. This degree of differenti- found also in mature thrombocytes from the cir- ation is not typical for this age. Usually small culating blood of the adult fowl, when the blood thrombocytes of mid-embryonic life look like smear has been fixed in Petrunkevitch No. 2 and figures 300, 301, and 306, or those of figures stained with May-Griinwald Giemsa (fig. 202). 228 and 229. Cells (figs. 300 and 306) were The general appearance of the cell (fig. 296), selected for drawing in which specific granules the lightly stained vacuolated cytoplasm, the were still visible; when these granules are not condensation of the nucleus, and the acidophilic visible, disintegrating thrombocytes so closely affinity of the cell margin, all establish this par- resemble small lymphocytes that have dis- ticular cell as an embryo thrombocyte. Typi- charged part of their c^tosome, that the identifi- cally tlie specific granules are distributed among cation might be questioned. Some rounded the strands of the cytoplasmic reticulum (figs. cells (fig. 304) in which specific granules are 297-299, 302, 303, and 305). absent but in which disintegration has not begun The last-mentioned point, the affinity of the cell can definitely be identified by the lightly stained perimeter for eosin, is especially significant. vacuolated cytosome. If the technic methods

This reaction by the cell margin is often seen in could be improved, probably most of the embryo embryo thrombocytes of this age. The same re- thrombocytes after 10 days of incubation would action occurs in thrombocytes of adult birds (fig. look like figure 302. 85), but after hatching, the disintegration proc- When a cell reaches a stage of degeneration ess is less frequently stopped at the exact moment such as shown in figure 301 and a specific gran- when this initial stage in cytoplasmic breakdown ule is not visible, one can only guess at its iden- would be revealed. tity: if it is part of a cluster (fig. 229, 19) it is Other examples of crumpling and acidophilic probably a thrombocyte, but if it is an isolated staining of the cell margin, an initial reaction in cell, it may be a lymphocyte. Generally- how- the disintegration process, are shown in figures ever, the nucleus of even the small lymphocyte 297 to 299. There is merely a single indenta- is larger than that of the thrombocyte. tion of the cell wall in the first two of these fig- Even at the end of the second week of incuba- ures, but in the third the entire margin is scal- tion, thrombocytes in general have not taken on loped. There is a question of whether the shift the definitive form and many, even with specific from a basophilic to an acidophilic affinity (1) granules, still have an appearance of immaturity was a result of cell disintegration, (2) preceded (fig. 305) . This may be true until nearly hatch-

132 ing time (fig. 307 j, hut by the 21st day (fig. 230, sible that the chick could be stinmlated to give a cells 7 and 8) cells having this appearance can be defensive response just as well before hatching considered to be truly definitive and mature. as after hatching. Chick embryos have been inoculated with so

many different viruses and bacteria that it is probably safe to say that growth or serial passage Cells occasionally found in circulating of every well-known disease has been attempted blood of the enibyro on this medium. In nearly every case, the em- Granulocytes may be found in the circulating bryo was used merely as a test tube because a blood during the last week of embryonic life and better, synthetic one has not yet been devised. occasionally during the second week. Their oc- Embryos have been used as a culture medium currence, however, is sporadic, and in many without nuicli regard for possible contamination smears none were present; yet a study of hemato- of the egg. The studies of embryo reactions are poietic organs during this same period—and even often based on grossly visible lesions, which do of organs like the pancreas, which is not a not tell the full story. Studies of egg-borne dis- hematopoietic organ in adult life—shows that eases must be carried back to the cells of the tremendous numbers of granulocytes are being embryo. These cells must be searched for in- produced, but apparently they are being held in conspicuous deviations from the normal, some storage and normally are not lil^erated until after of which may represent defense reactions to hatching, so that the typical blood smear even for pathogens. The meaning of the presence or ab- a few hours after hatching shows only eryth- sence of heterophils in the circulating blood is a rocytes and thrombocytes (fig. 230). case in point; there is certainly no a priori reason When granulocytes do appear in the circulat- why the chick embryo could not be studied in ing blood of the embryo, this question comes to health and in disease just as scientifically as the mind: Is it rough handling, or is it infection of adult fowl. Zuckerman (1946) has provided the egg, that caused these granulocytes to appear us with an example of blood response to infection in some embryos and not in others? Wlien the in the embryo. Introduction of Plasmodium egg is opened, the embryo with its membranes is gallinaceum stimulated the heterophils to become slid into a bowl of warm saline and then is lifted almost as numerous as thrombocytes. In the to a filter paper in a flat Petri dish. Here the differential count on one embryo, there were 44.5 amnion is opened, and often in the moving proc- percent thrombocytes; 19.0 percent mature ess the other membranes are ruptured or torn heterophils, and 24.0 percent heterophil myelo- away. If the ventral body wall has closed over, cytes, a total of 43.0 percent. In addition there the tissues in this region are laid apart in order to were 1.0 percent macrophages, 8.0 percent mono- expose the heart and then the cannula is inserted. cytes, and 3.5 percent lymphocytes. No matter how dexterously these steps are ex- Roberts, Severens, and Card (1939) found the ecuted, there is still enough roughness to dislodge white cells of the embryo composed of neutro- some granulocytes from embryonic depots. phils and lymphocytes. During the last week On the other hand, approximately 8 percent of hatching there was a fairly constant number of hens' eggs have been found to carry a bacterial of each of these cell types. Their values were contaminant after all precautions have been averages and the variability among individual taken to prevent entrance of organisms from out- birds was not indicated. No mention was made side the shell. From indirect evidence viruses of the thrombocytes, which in the embryo often also can be transmitted from the hen to the egg look like lympocytes. Whether some, or all, (Cottral, 1950 and 1952). Probably our best of the white cell types are constant components evidence comes from work done at this Labora- of embiyo circulating blood can be decided tory—evidence that lymphomatosis, a virus dis- definitely only by additional investigations. ease, is transmitted through the egg (Cottral, The macrophage is another cell sporadically Burmester, and Waters, 1954, and Burmester, found in smears of blood taken from the embryo

Gentry, and Waters, 1955) . If some eggs carry heart. The large cell shown in figure 308 came bacterial or virus infections, and if phagocytic from blood drawn from the dorsal aorta and

cells are already on hand, it seems entirely pos- differs in appearance from all the other cells

133 shown in figures 309-318. The nucleus is im- polar or Itipolar processes was found also in the mense and has a uniformly delicate granulation. embryo (figs. 314-316). Whether these pre- The cytosome carries clear vacuoles ranging cursor cells are reticular cells or fibroblasts needs from small to very large. There is much to to be determined, but at least it can be stated suggest that this cell is a yolk sac or endodermal that in the embryo and in blood spots of eggs macrophage. How so large a cell can flow the precursor cell is not a lymphocyte. In the through capillary channels is not understood. circulating blood of the adult, lymphocytes dif- Cells of similar size but of different structure ferentiate into a reactive type cell, and it has were found in blood taken from the heart, but as been suggested that this may be leading toward already mentioned diese were present in only a a macrophage, but never has a series of stages few embryos and absent from most of them, and jjeen observed spanning the gap from the reac- were found most commonly at about 10 days of tive lymphocyte to the type of macrophage shown

incidjation. This age ( 10 days) agrees with the in figures 310, 313, or 318. span from 7 to 12 days of incubation when In the same slides that contained the cells of Sugiyama (1926) found macrophages in the figure 309 were numerous protoplasmic spheres endnyo blood. (fig. 311 A to D). Although differing in size The cell shown in figure 310 is definitely a they all stained in the same way and resembled macrophage althougli the cytosome contains but closely in structure the cytoplasm seen in figure a few inclusions, which are represented by 309. These cytoplasmic spheres were vacuo- several magenta granules and numerous vacu- lated like the cytoplasm of macrophages and oles. The luicleus was pressed down on some of probably were pinched oft from cells of this the vacuoles when the cell dried; these vacuoles type—at least no other cell visible in the blood made the nucleus appear to be vacuolated also, at this time seemed large enough to have pro- but actually it was not. An earlier less dif- duced them. The cytoplasm of the macrophages stage is represented by the three cells ferentiated has a structure identical with that found in the together in figure 309. The nuclear clumped pinched-oft fragments. structure is identical with that shown in figure Later in the study, cells grouped in a mass were 310, but differentiation of the cytoplasm toward discovered (fig. 312). In nuclear and cytoplas- the phagocytic condition of the macrophage has mic structure they were identical with the small not progressed as far as in the latter cell. The group of three discovered earlier (fig. 309). nuclear structure of both of these cells is similar Many large macrophages had been observed up to that observed in macrophages seen so abun- but the bulkiness of this group pre- dantly in blood spots (Lucas, 1946). In blood to this time it get through spots they appeared to be derived from fibro- cluded the assumption that could blasts and the same type of cell with long uni- the smaller circulatory channels. It is entii'ely

Figures 276-287.—Early developmental stages of the embryo tlu-ombocytes. 2,470 X.

276 Early thromboblast, loljulated stage. Embryo incu- FiGTRES 281-283: Embryo thromboblasts showing incom- bated 2 days 17 hours. plete staining of the nucleus. 277 Either a late thromboblast or a large early thrombo- 281 This type of empty nucleus often seen in blast cells Duplicate slide from same embryo as pre- cyte. following Wright's stain, less often with M. G. G. ceding figure. Treated with Ralph's benzidine and 282 A partial staining of the nucleus. with G. G. trace of hemoglobin in stained M. No 283 Nuclear content well stained but nuclear boundary cytosome. not well delineated. 278 Medium embryo thrombocyte. Same slide as pre- 284 Embryo thromboblast that shows the punctate char- ceding figure. acter of the chromatin granules. 279 Thromboblast in the telephase of mitotic division. 285 Large embryo thrombocyte. Cytoplasm still shows Embryo incubated 2 days 18 hours. the structure of the thromboblast. Nucleolus present. 286 I^arge embryo thrombocyte. Rarely fixed in this round form. Figures 280-286: Drawn from the same slide. Embryo in- 287 T^arge embryo thrombocyte that shows precocious cubated 4 days. development to the oval form. Same age as pre- 280 I,arge embrj-onic thromboblast. ceding one.

134 278 277 m % 279 2«0 281

282 283 284

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285 286

135 FiGUKES 288-307.—Late developmental stages of the embryo thrombocyte 2,470X. thrombocyte. Acidophilia and Figures 288-291 : Large functional embryo Diroinbocyles. 297 Medium embryo Drawn, from same slide as figures 280-286. folding of cell margin are first steps in cell disintegration. Three specific granules present. 288 Large embryo thrombocyte showing blebs of early 298 Medium embryo thrombocyte. Less differentiated disintegration. than preceding cell. One specific granule. 289 Large embryo thrombocyte showing partial loss of 299 Medium embryo thrombocyte. Folding of cell basophilic cytoplasm. margin well started. Two specific granules. 290 Vacuolization of cytoplasm with loss of staining thrombocyte. Much of the cyto- affinity. A stage in the differentiation process. 300 Small embryo specific granule. 291 Discharge of chromatin granules into the cytoplasm. plasm lost. One Found only in well-preserved early embryo material. 301 Small embryo thrombocyte. Much of the cytoplasm lost. No specific granule visible. 302 Mature embryo thrombocyte. A specific granule at Figures 292-295: Medium and small embryo thrombocytes. each pole. and 292 Medium embryo thrombocyte. Vacuolization 303 A binuclear mature embryo thrombocyte. Nu- chromatin. some discharge of nuclear merous specific granules. 293 Medium embryo thrombocyte. Several chromatin bodies in cytoplasm. Pink color due to early dis- Figures 304-307: Developmental stages of thrombocyte integration of the cell. Embryo incubated 5 days 21 generations coming late in embryonic life. hours. 294 Small embryo thrombocyte. Same type shown in 304 Medium embryo thrombocyte. Early stage of hours. figure 229, 19. Embryo incubated 7 days 23 development. No specific granules. Embryo incu- 295 Medium embryo thrombocyte. Cytcsome partly bated 13 days 15 hours. disintegrated. Embryo incubated 8 days 21 hours. 305 Medium embryo thrombocyte. Later stage in differentiation than preceding cell. Same slide as Figures 296-303: Stages in development and breakdown of figure 304. late embryo thrombocytes. Embryo incubated 9 days 15 306 Small embryo thrombocyte. Nucleus and cyto- hours. plasm undergoing degeneration. One specific granule. 296 Medium embryo thrombocyte. Slight autolysis at mature. One spe- the cell margin. Cytosome contains more than the 307 Definitive thrombocyte, nearly usual number of specific granules. cific granule. Embryo incubated 17 days 14 hours.

136 .*/!.

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300 301 302 29S 299 9 #

304 305 306 307

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137 316 311!

138 possiljle that an embryo carrying a pathogenic an understanding of the occurrence of macro- organism could produce masses of reactive cells phages within the heart of the embryo chick. that, after leaving their point of origin, become It was noted, in the study of jjlood spots, that thrombi of the circulating channels, but a simpler macrophages underwent degeneration and only explanation seems more reasonable at present. some of the cells retained the punctate pattern of When the glass cannula penetrated the ventricle, chromatin of the type seen in figures 309, 310,

it was quite likely, if the tip were dull or jagged, 312, and 313. Often when karyolysis of the nu- tiiat some heart tissue, particularly the lining cleus occurred, the underlying nucleolus was cells, would he separated from the wall and would brought into view. There is a suggestion that nu- then be sucked up with the blood. Hearts from cleoli are present in figure 312. Nucleoli were embryos of the same age—10 days of incuba- conspicuous in some slides of chick embryos that tion—were used in a test of this hypothesis. were sent to this Laboratory for cell identifica- They were removed from the body and opened, tion; the embryos had been inoculated with a and the l^lood was washed out. A slide was pathogen from man and the circulating blood pressed against the endothelial surface and from showed a series of transitional stages from the it came the type of cells shown in figures 314^316. reticular cell to the reactive cell. Figure 316 was the longest cell obtained by this Some studies have been made on the defense- method. Most of the cells were of the size and reaction mechanism in lairds but perhaps the

shape shown in figure 314 and some were like greatest need at present is to identify by the smear 31.5. There is a measure of similarity of nu- method the cells that, in the celloidin sectioned clear and cytoplasmic pattern between these cells material, are called resting amoeboid and wan- and those of figures 309 and 312. but there is still dering cells. As the problem now stands, a room for douljt that they are all of the same type. study of the actual number of different types of The endothelial cells of the liver and spleen are macrophages is confused by differences in tech- phagocytic in pigeons (Kyes, 1915). The cells nics that make even identical cells look different.

are capaljle of ingesting erythrocytes under nor- It has been claimed jjy Dantschakoff (1931) mal conditions and after the contained erythro- that the endodermal wandering cell was actually cyte has been digested, the hemophage reverts to the primordial germ cell caught in its migra- a flattened endothelial cell. Kyes' observations tion from the extraembryonic splanchnopleure

are mentioned here because it may help toward to the gonadal ridge. Dr. Robert E. Smolker of

Figures 308-318. -Reticular or phagocytic cells found in blood and vascular organs of the embryo. 2,470 X.

308 Early embryo macrophage, yolk sac type. Blood nucleoli in each nucleus. Three embryo erythro- from dorsal aorta. Embryo incubated 1 day 22 cytes .shown in the field. hours. 313 Mature embryo macrophage containing acidophilic 309 Embryo macrophage, beginning differentiation from and basophilic inclusions. Vacuolization typical of

its stem cell. Circulating blood taken from the functional macrophages.

heart. Embryo incubated 9 days 1 6 hours. Figures 314-316: Cells from a touch preparation of imier heart. They resemble, but may not be Figures 310-313: Mature embryo macrophages and celt surface of embryo 309 and 312. Embryo fragments. Blood taken from the heart. Embryo incu- identical with, those of figures bated 9 days 16 hours. incubated 9 days 22 hours.

310 Mature embryo macrophage. Only a few granular 314 A nearly rounded cell. inclusions. 315 Cell with bipolar proce-sses. 316 Cell with a long unipolar process. 311 Cytoplasmic spheres ranging in size from large to 317 Postmortem degeneration of a macrophage, embryo small, A to D. Believed to be pinched-off pieces of (21st day of incubation) killed and held in the cold macrophage cytosome. 2 hours. Embryo still had a large external yolk sac. 312 A group of macrophage stem cells, essentially Cell from spleen impression smear. mesenchyme. Probably pushed into the cardiac 318 Macrophage from blood of basilic (cubital) vein. blood stream by the entrance of the cannula. Two Chick just hatched.

139 of a 2-day- the Department of Natural Science, Michigan pattern resemble the yolk macrophage State University, has demonstrated (personal old embryo more than they do the macrophages communication) sections of early embryos that of a 10-day-old embryo. clearly revealed the primordial germ cells. An impression smear from the spleen of a late tem- Even in sections they were considerably larger embryo that, after killing, was left at room still another than any of the lilood cells seen in the embryo, perature for about 2 hours produced including the primary generation of erythrocytes. type of phagocytic cell (fig. 317). The strong Very probably we have never seen primordial acidophilic reaction of the cytoplasm is charac- degeneration and this type germ cells in our smears. teristic of post mortem can be seen in tissue-culture A final bit of data is the macrophage shown in of vacuole formation unfavorable figure 318. It was not obtained by cardiac punc- cells when the culture is held under structure is entirely dif- ture but in a drop of blood from the Ijasilic vein conditions. The nuclear the 10-day-old embryo; of a chick hatched only a few hours before the ferent from that found in sample was taken. In some ways the high degree in fact, tlie entire cell is morphologically different thus far. of vacuolization and the rather delicate nuclear from any macrophage described

ADDENDUM

of the granu- Throughout this book and in a recent publications showing stages in development nucleolus appears first in the tion (Lucas, 1959), it has been repeatedly men- locyte series the size in tioned that in smears or touch preparations the metagraiudoblast, reaches its maximum during the nuclei of erythrojjlasts and thromboblasts usu- the promyelocyte and then disappears the riglit of the ally reveal their nucleoli, whereas the nuclei oi subsequent stages. The areas on granuloblasts and lymphoblasts rarely do. Dur- nuclei in figures 382 and 383 that stain a faint of the baso- ing the period following the completion of the bluish color are probal)ly nucleoli manuscript, an effort has been directed (1) to- phil promyelocytes. picture the ward the identification of the tissue components Ackei-man and Knouff (1959) Fabricius with a in different hematopoietic organs from which the lymphoblast in the bursa of that this is various blood cell lines take their origin, and (2) nucleolus. Our own studies indicate toward the identification of equivalent cells in probably true also in the thymus. l>ut it is still sectioned material with the named stages of de- imcertain that this is the case in spleen and bone velopment found in touch preparations. marrow. universally a imcleolus From our studies thus far it appears that the Regardless of how sections of presence or absence of a nucleolus is a variable may prove to be present, based on its variable visi- that is dependent more upon the size of the cell avian tissues, the usefulness of of smear prepara- and its level of metabolic activity than upon its bility as a tool in the study being a fixed morphologic structure. We are tions is not nullified; by this tool, erythroblasts not including here tlie pseudo-nucleoli that are and thromboblasts can be distinguished from chromocenters in some animal cells. From sec- granuloblasts and lymphoblasts.

140 CHAPTER 4

Blood Cells From Hematopoietic Organs of the Embryo

There is general agreement that dnring the life Were we to judge from information on mam- of the chicken, erythrocytes have an intravascular mals, we might expect the liver to be an active origin and myelocytes an extravascular origin. embryonic hematopoietic organ; but in the

From the present study, and the work of others, chicken it remains practically free of developing

it appears prohajjle that the broader generaliza- blood cells (Bizzozero, 1889, and Dantschakoff, tion can be made that viable erythrocytes and 1908a and b). The difference between birds thrombocytes have an intravascular origin and and mammals may be due to the existence of a all leukocytes have an extravascular origin. large yolk sac in birds and a reduction to a rudi- Basically, this agrees with Dantschakoff's (1908a mentary condition in mammals. Dantschakoff and 1909a) concept, except that she calls a prim- observed that the liver of the normal chick em- itive blood cell a lymphocyte; whereas, in this bryo was not a hematopoietic organ, but accord-

study a lymphocyte is considered to be the mature ing to Haff (1914) it does have such a function stage of a distinct leukocyte line and not a stem for jjotli erythrocytes and granulocytes. The cell or primitive blood cell. endothelial cells of the liver sinusoids proved to The erythrocytes and the thrombocytes are the be the point of origin by way of a "large lympho- only two cell types normally present in the circu- cyte" for the different cell types. Wislocki lating blood of the chick embiyo. All others that (194.3) observed that in a species of monkey that develop within hematopoietic organs are held had adapted the placenta to the function of a there until after hatching. Granulocytes appear hematopoietic organ, the liver had been relieved in great abundance in bone marrow, spleen, kid- of this function and showed no blood-cell de- ney, pancreas, and sometimes in the liver and velopment. The pancreas of the chick at 19 days other parenchymal organs. According to Dant- of incubation is packed with heterophils. schakoff (1908a and b) and Danschakoff (19]61j Wliether this organ serves as a hematopoietic and c), granulocytes also develop in the yolk sac center or as a storage depot for the granulocytes and in the thymus, and Nonidez (1920) found produced by spleen and bone marrow has not developmental stages in the ovary after hatching. been determined. It was noted by Mrs. Effie The bird in its hematology is reminiscent of some M. Denington, a member of the staff of this Lab- of its reptilian and amphibian ancestry where oratory who made these obsei-vations, that, among blood-cell development is widely scattered over different embryos, there is wide variability in the body (Dawson, 1932). Danschakoff the number of heterophils present. (1916c) observed that in embryo chicks there was a similar wide potency of the mesenchyme to form blood cells. She found that during normal EMBRYO BONE MARROW development this potency was restricted to certain tissues ])ut if transplants of pieces of adult organs Certain differences l^etween bone-marrow for- were made on the allantois of the early embryo, mation in birds and in mammals has been pointed the mesenchyme cells among the striated muscle out by Hamilton (1952), who says (p. 508): fibers, and among the cells of liver, kidney, and "There is never any independent epiphysial ovary, and in the walls of major vessels, took center of ossification in long bones of birds, as on hematopoietic functions. Often the circulat- there is in mammals. The ends of the bones re- ing blood simulated that of leukemia. main cartilaginous for awhile and provide for

141 Figure 319.—Embryo bone marrow. Composite from two slides. Embryos incubated 11 days 21 hours. 1,370X.

osteoclast showing a ma- Primordial osteogenic cell in mitosis, late anaphase. 12 Part of a multinuclear genta sphere near the upper edge. 2 This cell resembles a metagranuloblast except for 13 Granuloblast indenting an osteoclast. the Hof. Contains a few small magenta bodies. 14 Heterophil promyelocyte. 3,4 Small mature osteoblasts. 15 Heterophil metamyelocyte. 5 Large young osteoblast. 16-18 Mature granulocytes. Some show degradation 6 Two small young osteoblasts clumped closely of the rods. together. 19 Early polychromatic embryo erythrocyte. mononuclear osteoclast. Early 20 Late polychromatic embryo erythrocyte. osteoclast. Large mononuclear 21 Mature embryo erythrocyte of the primary 9 Binuclear osteoclast. erythrocyte type. 10 Multinuclear osteoclast indented by three cells 23 Mature eriibryo erythrocyte of the later genera- of the granulocyte series, tions type. mass. 11 Multinuclear osteoclast. 24 A pinched-off protoplasmic

J 42 319

143 FiGUKK 320. —Embryo bone marrow. Composite made from 'A places on the same slide— the long cluster of cells from 1 area, 6 cells from a nearby area, and 3 from another area. Emljryo incubated 12 days. Nearly the same age as embryo used for preceding plate, yet definitely more advanced in development. 1,370 X.

1-3 Primordial osteogenic cells. Cell 2 is more differ- 13, 14 Probably belong to the granulocyte series.

entiated than 1 and 3. 15 A cell with magenta spheres in the cytosome. Primordial osteogenic cell with 2 magenta granules 16 Heterophil mesomyelocyte. and 5 magenta spheres. 17-19 All these cells are mature heterophil granulocytes. 5-9 Large young osteoblasts. 20 Late polychromatic erythrocyte with fractured 10 A primordial osteogenic cell or granuloblast in cytosome, a technic artifact. mitosis, early anaphase. 21 Mature embryo erythrocyte of a later generation. 11, 12 Aletagranuloblasis. The identification could have been made more certain had they been fi.\ed in their spherical shape.

144 4. ,?^>v!>

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145 Figure 321. —Embryo bone marrow. Embryo incubated 20 days. 1,370X.

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147 FiGiTRES 322-328.—Embryo bone-marrow cells and serum. 2,470 X.

322 Stained serum fluid and granules tliat almost always Figures 326, 327: Mononuclear osteoclasis. Same slide as cover the cells in embryo bone-marrow smears. in figure 324. Embryo incubated 19 days 18 hours. Figures 319, 326 Faint acidophilic material filling the cytoplasmic 320, and 321 drawn with these granules and stained is charac- serum omitted. spaces on the upper left side of the nucleus 323 A large young osteoblast, not as typical as those teristic of osteoclasts from adult bone marrow. shown in figure 320. Embryo incubated 14 days 11 Compare with figure 398.

hours. 327 Two magenta spheres at the tip of the cell. 324 Primordial osteogenic cell. Some vacuoles with 328 Mononuclear osteoclast with numerous magenta magenta crescents. Enibr_vo incubated 11 days 21 spheres. At the left are acidophilic bodies charac- hours. of the osteoclast. Embryo incubated 16 days 325 Slightly smudged primordial osteogenic cell. Same teristic slide as in preceding figure. 19 hours.

148 . .'• ;;,;^*. . .r-v . ...• -.^-i *--v .•.•••.*'« »* ••••;. «..•. «•,•;••;

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growtii in length, but they are eventually ossified mark eine sehr charakteristische Lage. In der and united with the shaft as the marrow cavity Mitte, parallel der Langsachse der Markhohle, of the latter extends out into them. . . . verlauft eine Arterie, die sich in den aussersten ". . . The marrow cavity is enlarged by the Teilen der Hohle stark enveitert und dann in chondrolytic action of all the vascular elements, zwei noch breitere diinnwandige Venen iibergeht. but chiefly by the walls of the budding blood ves- Ein Unterschied im Inhalt der beiden Gefasse sels. As the chondrocytes are liberated from existiert vorlaufig nicht. Die Kapillaren sind " their enclosing matrix, they rapidly degenerate sparlich." and are apparently not converted into marrow The histogenesis of bone has been a secondaiy cells as previously believed; tlie cells of the mar- consideration in this study, but osteoblasts and row are presumably brought in from the mesen- osteoclasts were encountered during the search chyme and periosteum by the intrusive blood for early stages of blood-cell development. vessels. When the femur of a chicken, incubated about "In birds, calcification does not precede ab- 12 days, is split open so that a plug of marrow sorption of the cartilage, as it does in mammals, projects beyond the spicules of bone and this is luitil the greater part of the marrow cavity is touched to a slide, cells of the types shown in fig- formed. The cones of cartilage, referred to ures 319 and 320 are found. The embryo that above, that ar^e continuous with the articular produced the cells shown in figure 319 was a few cartilages, are absorbed about ten days after hours younger than the embryo that produced hatching." figure 320. In all bone-marrow smears made at The histologic details of cartilage and bone this age an abundance of serum granules as in formation in the appendages of the embryo chick figure 322, is always present. Numerous smears have been described by Dantschakoff (1909b). were made to get one sufficiently clear for de- The marrow cavity appears in the hvmierus, tibia tailed study. For this reason, the low-power and femur on the eighth to ninth day of incuba- drawings in figures 319 and 320 are composites. tion. She finds that the initial perforation into Serum granules were present in the fields from the region of the marrow cavity comes from the which these two plates were made but were action of osteoclasts breaking through the thin omitted in the drawing. wall of newly formed bone. The opening per- The least differentiated cell in bone-marrow mits the entrance of surrounding mesenchyme smears from embryos of 11 to 12 days of in- cells. cubation is the type shown in cell 1 of figure 319. She divides marrow cavity production into two Figure 320 shows three additional cells belong- phases (p. 876) ing to this category. Cells 1 and 3 of figure 320 "In der Entwicklung des Knochenmarks bei are typical of the early osteogenic cell and cell den Vogeln gibt es eine gewisse, ziemlich lange, is differentiated. The lightly vom 9, bis 12. Tage dauernde Periode, wah- 2 somewhat more rend welcher seine Struktur und seine Differen- stained cytoplasm is the most characteristic fea- zierungsprozesse sich so sehr von dem endgiil- ture, along with a pattern of scattered, basi- tigen Zustand am Ende des fetalen Lebens unter- chromatin granules that are delicately stained. scheiden, dass es wohl berechtigt ist, wenn ich ' : the development of tlie bone marrow in diese Periode von den iibrigen trenne, und das Translation In birds there is a certain rather long period, lasting from the Mark wahrend derselben als primitives Knochen- 9th to the 12th day, during which its structure and its process from the final circumstances mark bezeichne. Das hauptsachlichste Unter- of diflerentiation are so distinct at the end of the fetal life that there is good reason for me to scheidungsmerkmal dieser Periode ist das voU- separate this period from the rest, and to designate the marrow during this time as primitive bone marrow. The most standige Fehlen einer Blutbildung in den Gefas- important mark of distinction of this period is the complete sen, diese letzteren dienen zu dieser Zeit bloss zur lack of a hematopoietic process in the vessels; at this time these to nourish the tissue and contain circulating blood, Ernahrung des Gewebes und enthalten zirkuli- serve only which consists of more or less mature elements. . . . erendes Blut, welches aus mehr oder weniger 'Translation: The blood vessels have a very characteristic ." ^ location in the primary bone marrow. In the middle, parallel reifen Elementen besteht. . . to the longitudinal axis of the marrow cavity, there runs an The description of these first vessels is as fol- artery that widens greatly in the outermost portion of the hollow and then merges into two still wider thin-walled veins. lows (p. 877): At the time there is not any distinction in the contents of the "Die Blutgefasse haben im primaren Knochen- two vessels. The capillaries are scanty.

151 The nucleolus is visible in 2 of the 3 cells shown cytosome and some of these are pushed up into in figure 320. Since primordial osteogenic cells the nuclear substance but they are not so abun- are relatively rare, it has not been possible to dant as in the osteoclasts. study them in as wide a variety of circumstances Small mature osteoblasts (cells 3, 4, and 6 of as would be desirable. At one time it was thought fig. 319) are more differentiated than their pre- that they stained lightly because they were cursors, tlie large young osteoblasts. The cyto- partially squashed, and the nuclear staining in plasm stains more intensely than any other cell figure 325 and in cell 31 of figure 321 may be of the marrow. Mitochondrial spaces are not so evidence for this; but the idea has been given up distinct as they were previously and the clear because, if squashed, the chromatin should show area adjacent to the nucleus is often masked. some liquefaction. It is agreed that they are The nucleus retains the eccentric position it pre- probably delicate and easily broken, but they viously had but now it is smaller, and the chro- have been seen intact in sufficient numbers to matin pattern is condensed, which causes it to warrant the belief that they make up a distinct stain more intensely. All these features are cell type. shown in the high-power drawing of a cell from Cell 2 of figure 319 shows cytoplasmic dif- an embryo incubated 14 days 11 hours (fig. ferentiation leading toward the osteoblast or the 323). Osteoblasts may clump as in cell 6 of granulocyte. Had a nucleolus been visible, the figure 319 but this apparently binucleated con- possibility of development toward the granu- dition does not make this an osteoclast like cell locyte would have been excluded. Cell 2 of 9. figure 320 shows differentiation toward the osteo- These small cells are called mature because clast. It is believed that both osteoblasts and they are morphologically identical with cells ob- osteoclasts of these cell lines arise from a com- served adjacent to bone in sections. They have mon stem cell, called the primordial osteogenic a darkly stained cytoplasm and a distinct para- cell, which, as shown in these figures, is a large nuclear area. The texts on general histology cell with abundant pale-staining cytoplasm and call these blast cells, but, functionally, they are a definite nucleolus. At tlie same time osteo- mature.^ They form a row along the bone genic activity is taking place, myelocytes also are spicule and produce a bone matrix. Eventually making their first appearance. This cell has they will be surrounded by this matrix and will many features in common with the primordial rest within a lacuna; they will then be called osteogenic cell. The nuclear stiiicture of cell 2 osteocytes. of figure resembles that of myelocyte, 319 a but Cells 7 and 8 of figure 319 are mononuclear the strong mitochondrial spaces, the intense blue osteoclasts. The appearance of the cytoplasm staining, and especially the clear area beside the of one of these cells is quite different from the nucleus indicate that it is an early osteoblast. cytoplasm of the other; cell 8 has many orange- The magenta granules are not so large as they colored granules to the left of the nucleus and no typically are for myelocytes and there may be vacuoles; cell 7 has only a few orange-colored some question of whether they are on or in the bodies but has many vacuoles. The binucleate cell ; if they were in it, the fact would add weight cell, vacuoles but no orange to the idea that this was a metagranuloblast or an 9, also has numerous early promyelocyte. In figure 320 somewhat " This Atlas may not be a suitable work in which to propose similar cells {11 and 12) have been identified as changing the name of the functional bone-producing cell from osteoblast to something more appropriate, but it does seem metagranuloblasts and probably cells 13 and 14 somewhat confusing to retain the suffix "blast" through all are the same. the developmental stages of this cell. It is recognized that "large young osteoblast" is not an adequate substitute term Dantschakoff (1908b) states that the bone for the immature stage. Any attempt to standardize hema- marrow begins its blood-forming function on the tologic nomenilature should include all the connective-tissue 14th day of incubation. derivatives, the phagocytic cells, and the cells of the circulating blood and their developmental stages, and this must come from Large young osteoblasts (cell 5 of fig. 319 the united efforts of many scientists. The same confusion exists in assigning terms to the developmental stages of the and cells 5—9 of fig. 320) show their immaturity osteoclast. They could be called osteoclastblasts and osteo- by their large size, lightly stained cytoplasm, clastcytes, but these terms are awkward. For the present the basis of the number of nuclei, on open reticulate chromatin pattern, and clearly stages are separated on the the assumption that the number is a fair measure of the stage visible nucleoli. Vacuoles are present in the of differentiation.

152 — :

bodies; the multinucleate cell, 10, has both; cell case, however, and other cell types might show 11, which is similar to 10, has neither. The cell vacuoles with colored strands of this type. These in figure 326 is shown under high magnification magenta masses did not appear in sectioned ma- to reveal the orange bodies in greater detail. terial, which suggested that they are artifacts but

These characters are present in some cells, and did not prove it. are absent from others. This is true not only in Sometimes magenta masses occur in the senmi the embi-yo but also in the adult bone marrow outside the osteoclasts. Since they appear to

(figs. 398 and 399) . Regardless of these varia- merge with the serum, it is possible that serum tions the bone marrow of the bird contains but a condensations on the cells at the time the slide single type of giant cell, the osteoclast, whereas is made could account for all these irregular

in mammalian bone marrow there are two types magenta-stained bodies but certainly this is not a the one just mentioned and the megakaryocyte, convincing conclusion at present. which produces platelets. Nucleoli found in the Another possibility considered was that this binucleate and mononucleate stages of develop- substance represented a dissolved cartilaginous ment are still retained in the nuclei of the fully matrix taken up by the osteoclasts, or that cells differentiated osteoclasts of bone marrow from containing this substance represented hypertro- adult birds. phied and degenerating cartilage cells liberated A conspicuous nucleolus, characteristic of tlie into the expanding marrow cavity. Cartilage at osteoclast, may be found also in tissue culture of the end of the femur was pressed hard against tliis cell (Hancox, 1946). This author ex- the slide in making the smears but no cells were pressed the opinion that the nniltinucleated con- found with a morphology that would support the dition of this type of giant cell arose by a fusion idea that magenta-colored masses and strands of cells, rather than by multiple division of a nu- arose from cartilage cells. In fact no evidence cleus within a single cell. was obtained by the smear method that cartilage The cytoplasm of some multinucleate osteo- cells were liberated into the marrow cavity after clasts contains magenta-colored bodies. Some the cartilage was dissolved. By the section of these bodies have irregular shapes; some, like method, Dantschakoff (1909b) observed that the example in cell 4, of figure 320, are spher- viable cartilage cells survived after the lacunae ical; and some—see those between cells 12 and broke down. She stated (p. 874) 14 of figure 320—are masses of stainable sub- "Die Knorpelzellen, die dabei aus ihren Kap- stance without definite form. seln befreit werden, bieten besonders bei dem A series of mononuclear osteoclasts have been ersten Auftreten der Markliohle keinerlei An- drawn under high power in order to give a closer zeichen von Degeneration. Sie bleiben als runde view of this substance. Figure 326 is a young helle Zellen zwischen den Elementen des von aus- osteoclast showing the characteristic vacuoliza- sen eindringenden Mesenchymgewebes liegen. tion of the cytoplasm, but without magenta- Einige von ihnen, die dem Rande der Hohle uncolored strands. In figure 324 a slight amount mittelbar anliegen, zeigen sogar im Gegenteil * of this material exists in the form of cui-ved bars mitotische Teilungsfiguren. ..."

concentric to the vacuole walls. Two spheres Concerning their later history, she says (p. are shown in the lower tip of figure 327; the 882): magenta bodies contained within them are not as ". . . Die aus den Kapseln befreiten Knorpel- definitely an integral part of the cell as are the zellen vermischen sich in der engsten Weise mit bars in figure 324. rather extreme cui-ved A den Elementen des in dasselbe Gebiet eindringen- condition is shown in figure 328, where many den jungen Mesenchyms. Bei ihrer allmah- vacuoles contain many, twisted threads of this lichen Entfernung von der Knorpelgrenze schei- substance, but more important is the fact that nen sie sich zu strecken und Auslaufer zu bilden. at the left side of the cell this substance appears

* to be undergoing a transition to the typical orange Translation : The cartilage cells which thus become free of masses characteristic of osteoclasts generally. their capsule do not give any sign of degeneration especially at the first emergence of the marrow cavity. They persist as The magenta substance seemed to have a pre- round clear cells among the elements of the mesenchyme tissue dilection for osteoclasts even in the early stages that is penetrating from the outside. On the contrary, some of them, lying right against the edge of the hollow, exhibit (cell 15, fig. 320). This was not always the mitotic division forms.

153 Ahnliche Bilder lassen mich annehmen, dass die because the eiythrocytes are discharged into the sich befreienden Knorpelzellen an der Ausbil- circulating blood and the granulocytes are held dung des Knochenmarkstiomas aktiv teilnelimen in depots until after hatching. A study of the koiinen. Mit Sicherheit kann man bloss ihre un- whole matter of relative production rates in vari- mittelbare Teibiahme an der Blutbildung ver- ous hematopoietic organs should be carried out. neinen. Es ist aber sehr schwer, etwas be- It should include a study of the yolk sac. Hema- stimmtes iiber ihr weitei-es Schicksal auszusagen, topoiesis in the yolk sac has been omitted from weil sie ja wenigstens zum Teil, wie gesagt, all- this study for two reasons—first, it does not lend malilich alle histologischen Merkmale der umbe- itself to the smear method and, second, it has been benden Mesenchymzellen annehmen und infol- covered by the extensive writings of Dantschakoff ^ gedessen nicht mehr erkannt werden konnen." (1908b) on sectioned material and of Sabin Granulocytes in all stages of development from (1920) on living preparations. The importance granuloblast to mature heterophil are found in of using the smear method for a study of erythro- figures 319 and 320. The developmental stages poiesis and granulopoiesis in the case of the pig- will not be described here since they are to be eon has been emphasized by McDonald (1939), ". discussed fully under adult bone marrow, but one who says (p. 293), . . The chief advantage finer characteristic noted at this time is the tendency of of the imprint method is that it brings out the heterophils, whether they be immature or mature, structural features, especially those of the nu- studies to clump with other cells. Three heterophils lie cleus, which are so important in critical ." within marginal depressions of the osteoclast, of immature cells. . . in cell 10 of figure 319, and in figure 320 cells of Granuloblasts are much more abundant the various types, including heterophils, have bone marrow before hatching (cells 1-6, fig. clumped together. Developmental stages of 321) than after. Six of them are shown in one erythrocytes do not clump. Fennel (1947) re- field and they occur in the spleen at this age (fig. in the same high concentration as in bone ports (p. 237) that "Giant . . . cells frequently 330) the least give rise to one or more granulocytes by the pro- marrow. Actually cells 2 and 4 are the duction of cellular blebs. Such blebs ultimately differentiated; the nucleus lies in the center of pulled away from the surface and became free. cell and the rim of cytoplasm is narrow and stains defi- Giant cells under other conditions fragmented intensely blue. The remaining cells show metagranulo- to form thrombocytelike cells." The cellular de- nite changes leading toward the slightly eccentric posi- tails shown in his drawings of this process taken blast—the nuclei have a chromatin con- from vital-stained preparations are not sufficient tion, some of them show points of partially broken to permit determination of whether his giant cell densation, and the cytoplasm is At the metagranulo- was an osteoclast, a macrophage, or a clump of up by mitochondrial rods. blast stage, which is not represented on this plate, cells. Nothing has been observed in this study the cytosome shows vacuoles and inclusions to indicate that any type of giant cell ever pro- by which a reasonably accurate guess can duces thrombocytes or granulocytes. be made as to the type of granulocyte the cell The marrow, during embryonic life, is in- will be when it is mature. This is not the case volved primarily in the production of granulo- in the blast forms shown in cells 1 to 6. cytes and erjrthroc5^es, and although probably Cell 7 of figure 321 is classified as a heterophil more erythrocytes than granulocytes are actually mesomyelocyte but actually it has barely passed produced, it appears to be the other way around the promyelocyte stage. The magenta rings, the pale- ' indefinite boundary of the nucleus, and the Translation : The cartilage cells released from the capsule mingle in the most intimate way with the elements of the orange precursor bodies are all present in cell young mesenchyme that are penetrating into the same terri- addition there are a few darkly stained tory. As they gradually get farther from the cartilaginous 7, but in boundary they seem to stretch out and to form outrunners. orange bodies. Wlien they have elongated, the Similar pictures lead me to assume that the cartilage cells that will be the definitive rods. Their are freeing themselves can take an active part in the formation orange bodies of the bone marrow stroma. One can with surety only deny presence is the basis for calling the cell a meso- their direct participation in hematopoiesis. But it is very hard Cell is also a mesomyelocyte but to state anything certain about their future destiny, since, as myelocyte. S has been said, they gradually take on. at least to some extent, it is somewhat older than cell 7. All the charac- all the histological earmarks of the surrounding mesenchyme of the promyelocyte are still present but cells, and hence cannot be recognized any longer. teristics 154 more of the definitive bodies are visible. Some typical of the erythrocyte or the granulocyte line. of the bodies appear in the round form and some Previously it was stated that the erythrocjiies and in the rod form. The total number is less than the thrombocytes of an early embiyo were dif- half of the number found in the mature heter- ficult to separate; but with increasing age of tlie ophil. A metamyelocyte is shown in the heter- embryo, separation becomes easier, and in the ophil above cell 28 of figure 321. This cell adult marrow there was relatively little con- does not yet have a full complement of rods but fusion. the nucleus has condensed until its boundary Naked nuclei and smudged cells (fig. 321,

is distinct. 29 and 30) need no additional explanation, and Other cells {9-11) are mature, or nearly so, the primordial osteogenic cell {31) has already but since they contain but a single nuclear lobe been discussed. The most important cell re- they would be classed as juveniles or as band maining for consideration is 28, which has been cells in mammalian blood terminology. Mature called a lymphocyte. It appears to be a small heterophils are abundant throughout the smear cell undergoing l3leb formation. The nucleus

and some of those having more than one lobe are is too large for the definitive thrombocyte ; more- designated by the numbers 12—14. over, if it were a thrombocyte the structure of the Eosinophils also are held in depots until after nucleus would not have been so clear cut and hatching. Two cells each with a single nuclear definite at this stage of cytoplasmic disintegra- lobe are indicated by numbers 15 and 16 (fig. tion. 321) and, in the same field, part of one other cell is shown at the border. With the exception of cell 15, all these cells are of the small type that, in the bone marrow, is considered to be EMBRYO SPLEEN the source of the small eosinophils sometimes found in the circulating blood (figs. 181-183). The spleen is an organ that develops quite early The basophil (fig. 321, 17) is an adult cell in in embryonic life; according to Hamilton which the distortion due to action of water on the (1952), it appears on the last half of the fourth

granules is as great as in the circulating blood, day. Efforts to procure satisfactory impression and the poor stainability of the nucleus is as evi- smears before the beginning of the eighth day dent here in the bone marrow of the embryo as were unsuccessful because the cells were so frag-

it was found to be in the circulating blood of the ile that all broke, and the naked nuclei were cov- adult (fig. 190). ered by a layer of blue-stained tissue fluid. The In this field of bone marrow, no erythroblasts smears resembled that portion of figure 329 are present Imt there are three early polychro- where there are naked and ruptured nuclei and matic erythrocytes (fig. 321, 18-20). These strands of dissolved chromatin; therefore no in- examples show considerable range in size, yet tact, recognizable cells could be seen. By the all are already older than eiythroblasts. Cell eighth day some cells remained unbroken in care-

21 is a good example of a mid-polychromatic fully made smears. Why cells should be deli- erythrocyte. There are no late polychromatic cate in early embryonic life and much tougher at erythrocytes in this field, and tlie remaining older ages is not known; cytologically they ap-

erythrocytes are mature. It is assumed, not that pear identical at both ages, and the size does not these mature erythrocytes represent cells held in change much with age. All the serum granules storage, ready to be discharged later, but that and disturbing elements in the smear were in- they came into the bone marrow by way of nu- cluded when the drawing (fig. 329) from the trient vessels from outside. spleen of the 8-day-old embryo was prepared. Cell 26 (fig. 321) has been identified as a Danschakoff (1916a) reviewed the early de- thromboblast. It has the characteristics of the velopment of the avian spleen and reaffirmed her early stage of this cell line in its densely stained observations on the function of lymphocytes as nucleus and cytojjlasm. Had the cell not been the progenitors of other cell types. Antibodies pressed out of shape during the making of the against adult spleen tissue were produced by

smear, its identification would have been more transplanting pieces of the spleen to the allantois

certain, but it does not have the characteristics of the embryo.

155 A review of the differences in the histology of The interval between 193 and 299 hours of the avian and mammalian spleens has been given incubation has wrought developmental changes by Lucas et al. (1954). that advance the architecture of the spleen to a Whereas the embryo bone marrow produced level that will be maintained up through the both granulocytes and erythrocytes in about equal hatching process. Since a low-power drawing numbers, in the embryo spleen it is the granulo- had been made of the bone marrow both at 12 cyte that is the dominant cell. After hatching, days and at 20 days of incubation, it was orig- the spleen becomes predominately a lymphocyto- inally planned that a low power drawing of the genic and monocytogenic organ. The shift in spleen would also be made at these ages, but a the cell picture is shown graphically by a com- study of spleen impression smears made after 12 parison of figure 329 (8 days of incubation), days of incubation showed tliat no significant figure 330 (12^ days of incubation), and figure changes had taken place in cell types; therefore 331 (35 days after hatching). In the first of a drawing at 20 days of incubation has been this series of three plates are two typical meta- omitted. The spleen at 8 days of incubation is granuloblasts (cells 1 and 2) and in the same at about the same level of development as bone field are numerous others (cells 3-6) that are marrow 4 days later, as far as the general ap- covered too heavily with stained serum to show pearance of the cells in the smears is concerned. the details of their structures. When the spleen has reached its 12-day level of The next stage in development of the granulo- development, blast forms of granulocytes become relatively rare. two examples (cells 1 and cyte is the promyelocyte, in which vacuoles and Only magenta granules and rings are present in the 2) are shown in figure 330. One is shown only Init blue-stained cytoplasm adjacent to an eccentric nucleus. in part, the narrow, dense ring is typical of the structure Most of these characteristics are to be found in of cytoplasm in cell 2 of cells in the hatched chick. Most of the cell 7, and lightly stained orange spheres are such the present also. This cell has been classified as a cells of this line in figure 330 have reached promyelocyte. Had some of the spheres taken metagranuloblast and promyelocyte stages of de- on the dense coloration that occurs antecedent to velopment. However, figure 330 cannot be replica of the transformation into definitive rods, the cell taken as representing an exact what would have been classed as a mesomyelocyte. every slide examined at 299 hours of incubation will show because among smears from a dozen Cell 8 clearly fulfills the characteristic of the embryos there will be definite shifts in the dom- mesomyelocyte. This particular cell has no inant cell type of a particular series. This might definitive rods but now there are present numer- be accounted for on the basis of slight differences ous darkly stained orange spheres that represent in developmental rates that always exist among the precursor substance. In this cell the nucleus embryos or on the basis of cycles in cell produc- has become smaller and the chromatin more con- latter agree with the sugges- densed. Other developmental stages are not tion. The would tions given in the literature for blood-cell de- shown in the field; cell 9 and the cell below 8 velopment in the yolk sac and bone marrow. have already differentiated into the mature form. Cells 3-6 (fig. 330) are identified as meta- Cell ii of figure 329 is an erythroblast ; the granuloblasts of the heterophil line but they are chromatin pattern of the nucleus and the faint not so clearly typical of this stage as is the eosino- nucleolus are the features which most readily phil metagranuloblast (cell 11). A question identify it. The unnumbered blast cell at the might be raised regarding cells 3-6, in which the bottom of the plate is probably of the same type nuclear structure, instead of retaining the deli- but the details of its structure are masked in part cate, lightly stained pattern of the granuloblast, by the serum. There are at least five small em- shows an increased density of staining and clump- bryo thrombocytes in the field; two of them are ing of chromatin, which is characteristic of the

indicated at 12. One cell {10) , in the prophase early erythrocyte line; had nucleoli been visible of mitosis, cannot be identified because the cyto- in these cells as in cell 12, these 4 cells would plasmic structure is not sufficiently distinctive have been called erythroblasts. One expects and the nuclear pattern has been lost in the proc- to find in the metagranuloblast stage an eccentric, ess of cell division. faintly stained nucleus that has an indefinite

156 boundary between it and the adjacent highly less clumped. Typical small thrombocytes are vacuolated cytosome. shown in the cells numbered 30 in figure 330. Cells 7 to 10 show the preciusoi- spheres from Smudged cells are present in every smear and which develop the densely stained orange bodies. usually are more numerous than the one example In cell 7 they are barely visible as light orange shown in figure 330, 31. bodies from the vacuoles produced in the metagranuloblast stage. In cell 9 the spheres are slightly more intensely colored. In cells S and 10 a few orange spheres are almost as darkly BLOOD CHANGES AT HATCHING stained as in the mesomyelocyte stage, but in none of the four cells {7-10) have the orange During the 24 hours after the hatching of the spheres taken on the deep eosin coloration that chicks dramatic alterations occur in the circulat- precedes the elongation of a sphere into a definite ing blood, spleen, and bone marrow. The most rod. Description would probably be facilitated complete change appears in the spleen, where a if names were given to the lightly and the darkly general outpouring of heterophils is followed by stained orange spheres; when a thorough cyto- a massive development of lymphocytes and mono- logical and cytochemical study of rod produc- cytes. Within several days following hatching tion has been made, logical names will suggest the bone marrow becomes an organ that produces, tliemselves. predominately, erythrocytes, thrombocytes, and

Cell 12 of figure 330 is an erytlu'cblast and is granulocytes. No extensive series of illustra- clearly identified by its typical structure as be- tions covering these events has been prepared, longing to the blast stage of development. Such chiefly because in any period of rapid transition cells are relatively rare at this age. Numerous what may be seen at one moment in one chick early polychromatic erythrocytes are pi'esent. may be entirely different in another. Cells 13 and 14 are examples; so is the cell lo- Notes were taken on smears from a group of cated between 3 and 4. Cells i5 to 17 are ex- chicks ranging from a few hours to several days amples of mid-polychromatic erythrocytes, and after hatching. The actual protocol taken at the cells 18 to 21 of late polychromatic erythrocytes. time probably tells the story as well as a more There are cells in the field other than those desig- studied rewrite of the same thing. We recom- nated by number; they also belong to these vari- mend a similar study to anyone who has attained ous groups. Mitosis, at least in the embryo, can some proficiency in cell identification and is take place in late polychromatic eryljirocytes grasping for a feeling of the dynamics of develop- (cells 23-27) and, of course, at earlier stages ment and balance as it occurs hidden from our of development also (cell 22). It would be in- usual vision. teresting to follow the plasmosome nucleolus Throughout this transition period there was

during mitosis and note how it is reconstituted considerable variability between birds and it during the interkinetic period, but the technics would be necessary to use a larger number of used here are not suitable for such a study. chicks at each age to determine the exact typical Thrombocytes are less common in smears from sequence of events. At each period usually four chicks were used. hematopoietic organs than from circulating blood

and it may be that this is due to the delay in opening the embryo and dissecting out the organ be- 1 TO 3 HOURS POSTHATCHING fore the smear is made. It is quite possible that in fixed and sectioned material some thrombo- Circulating blood.—Only in an occasional chick was the number of white blood cells anywhere near a nor- cytes undergo degeneration and become cells that mal ratio; in most cases, the number was definitely be- resemble small lymphocytes or naked nuclei. A low normal. This was especially true in regard to the reinvestigation of thrombocyte development from lymphocytes, monocytes, and basophils. The predom- stained sections might help to clarify the stages in inant cell in the early chick blood, just after hatching, is the heterophil. The other cells come into the picture thrombocyte differentiation. Cell 29 (fig. 330) at a later age. Even the heterophil, immediately after is a of size looks thrombocyte medium and some- hatching, may be absent from the blood (fig. 230). what like figure 289, except that the chromatin is Immature stages of erythrocytes are usually present.

157 I

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FiGUEE 329. —Embryo spleen. Embryo incubated 8 days 1 hour. At this age it is difficult to obtain a good smear because of the great fragility of the cells and the masking by the stained serum fluid and granules. 1,370X.

1, 2 Metagranuloblasts. 10 Immature cell in mitosis, prophase. 3-6 Metagranuloblasts in which the cytoplasm is 11 Embryonic erythroblast. masked by the overlying stained serum. 12 Embryonic thrombocytes. 7 A heterophil promyelocyte. 13, 14 Broken cells with liberated naked nuclei. 8 Heterophil mesomyelocyte with many eosinophilic spheres, some light and some dark. 15 Strands of chromatin from the nuclei of cells 9 Mature heterophil. broken when the slide was made.

158 ^^ -:

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329

159 Figure 330.—Embryo spleen. Embryo incubated 12 clays 11 hours. 1,370X

23-27: -polychromatic erythrocytes in mitosis. 1, 2 Granuloblasts. Figures Late 3-6 Metagranuloblasts. 7-10 Heterophil promyelocytes. 23 Early prophase. 11 Eosinophil metagranuloblast. 24 Late prophase, nuclear membrane broken down. 12 Erythroblast. 25, 26 Late prophase, uncoiling of spireme thread. 13, 14 Early polychromatic erytlirocytes. 27 Late anaphase. 1.5-17 iViid-polychromatio erythrocytes. 28 Late embryo thromboblast or early embryo 18-21 Late polychromatic erythrocytes. thrombocyte. 22 Mid-polychromatic erythrocyte in mitosis, prob- 29 Medium embryo thrombocyte. ably early anaphase. 30 Small embryo thrombocytes. 31 Smudged cell.

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162 Figure 331.—Spleen of chick 35 days after hatching. 1,370 X.

1 Early immature plasmaoyte. 12-14 Naked thrombocyte nuclei. 2 Late immature plasraacyte. 15 Mature heterophil. 3 Immature lymphocyte. 16 Possibly a monoblast or early immature monocyte 4-6 Mature lymphocytes, medium size. 17, 18 Early immature monocytes. 7-10 Mature lymphocytes, small size. 11 Naked lymphocyte nucleus. 19 Late immature monocyte.

163 Spleen.—Apparently at the time of hatching, and the stages most commonly seen begin at about the mid- very rapidly thereafter, the spleen discharges its hetero- polychromatic erif-throcytt and continue on to maturity phils; thus, in this period of 1 to 3 hours, one can find (fig. 230) . One gathers the impression by comparing a spleen filled with numerous mature heterophils; in the development of red blood cells as seen in the bone other cases, at the same age, there will be very few marrow with that seen in the circulating blood that heterophils, and in their place will be early eosinophil there is a tendency for the bone marrow to throw out metagranuloblasts, and even the early lymphoid se- the cells at about the mid-polychromatic erythrocyte ries is making its appearance. At this period of transi- stage or a little earlier. Later stages are present in the tion it is quite common to see numerous macrophages bone marrow but cells of older ages do not dominate in the spleen. At this age and even up to 24 hours of the picture. age there are many opportunities to study the development of the eosinophil and the lymphocyte. Spleen.—In the 4 chicks used at this age (8 to 12 hours) there was a wide range in the number of hetero- Bone marrow.—Of the 4 chicks used at this age, 2 phils but in general they had almost completely dis- showed numerous late heterophils, and 2 did not. There appeared and in their place were numerous eosinophils appears to be a transitional change in the bone mar- in about the mid-stage of development. Many macro- row. It probably begins somewhat earlier than 1 to phages were present and there was an increase in num- 3 hours after hatching, because the bone marrow is ber of small lymphocytes. In another chick at this already taking over the hematopoietic function in re- same age there was a moderate number of mature het- gard to granulocytes that the spleen is giving up. In erophils with developing eosinophils and developing addition, in the bone marrow one finds numerous ex- basophils; in the third chick there were numerous early amples of the red cells from early to late stages of de- heterophils, practically no eosinophils, and some early velopment. Mononucleated osteoclasts were always basophils; and in the fourth there was a mixture of present in these early stages, and were particularly mid-heterophils and early eosinophils. In spite of this numerous in one case where very few heterophils were variability, there still exists an extensive discharge of present in the bone marrow. Osteoblasts also were the heterophils into the circulating blood. This is fol- visible, but in those smears where heterophils are nu- lowed by a wave of development of eosinophils and per- merous, osteoblasts are relatively rare. The third cell- haps in some cases an additional group of heterophils ular element coming up at this time is the thrombocyte and basophils. At this time, also, there is some indi- series. Just as early erythroblasts may be found, so cation that the lymphocytes are taking the dominant also may early thromboblasts be identified. These are place that they will hold later in the spleen. relatively large cells and have a characteristic punctate, Bone marrow. In the bone marrow of all these strongly granular nucleus that stains more intensely — chicks (8 to 12 hours) there were a great many mature than the erythroblasts. Most of these cells, however, heterophils. This seems to be characteristic of the contain a well-developed nucleolus. This structure bone marrow at this stage and later. Almost equally aids in the identification of the cell, but in the throm- conspicuous are the developing red blood cells and the boblast it is often masked and covered over by strongly scattered thrombocytes. Some small thrombocytes are stained chromatin spheres. The thrombocyte as it gets present also, and one can find the developmental stages older has, typically, a very dark blue cytoplasm and in the production of granulocytes, but these are mingled frayed edges. In these early bone marrow smears with the adult stages and are not so conspicuous as they the transitional stages down to the small thrombocyte are in the spleen. can be followed. The fully formed thrombocyte, however, does not have the form present in the circulating blood because of degeneration of the cytoplasm that 24 HOURS POSTHATCHING takes place during fixation. Cells of the thrombocyte series are never numerous in the bone marrow but Circulating blood.—At 24 hours the white blood cells some are always present. in the circulating blood are predominantly heterophils. Other cell t}'pes are present, of course, but they have not reached their usual ratio and, in many cases, lym- 8 12 TO HOURS POSTHATCHING phocytes or monocytes, or both, may be lacking or at of searching is re- Circulating blood.—At this stage there may be some least be so scarce that a great deal holdover of what was found at 1 to 3 hours, because the quired to find them. At this stage there are abundant dominant type of cell among the white blood cells is the examples of late-developing red cells. heterophil. Sometimes the white blood cells are few Spleen.—Only in 1 of the 4 chicks at this age was in relation to the red cells and sometimes they are at there even a moderate number of mature heterophils about the normal ratio. At this age the number of in this organ. At this stage the lymphocyte develop- heterophils is generally out of proportion to the other is coming into view. This is an excellent stage white blood cells and the lymphocytes and monocytes ment to study lymphopoiesis: some of the cells can are definitely fewer than they will be later. It is char- at which acteristic of the circulating blood of the recently be traced back to lymphoblasts and to the reticular hatched chick that the red blood cells show a wide range cells from which they appear to have come. Among of immature stages of development, but predominantly these early lymphoid cells are some eosinophil myelo-

164 cytes, usually quite early in their development. Nu- different types of white cells were present in their usual merous macrophages are still present. proportions. In one chick there was a monocytosis and many cells were smudged. This seems to be the Bone marroiv.—At this stage in the bone marrow stage at which monocytes make their appearance ; some there is a variation from a very high percentage of of these monocytes show the typical reticular appear- mature heterophils to relatively few, and red blood cells ance of the cytoplasm. are dominant. These two are always present simul- taneously but the ratio varies at this age. Spleen.—Occasionally there are some late heterophils in the spleen but it is now predominantly a lymphoid picture. 1 DAY 18 HOURS POSTHATCHING The circulating blood, 7 days posthatching, is Circulating blood.—At this age there is still a domi- illustrated in figure 231. The structural char- nance of heterophils and a general absence of the acter of a lymphocyte, a monocyte, a heterophil, agranulocytes in the circulating blood. and a basophil, as well as of some thrombocytes Spleen.—At this period the dominant picture is and erythrocytes at this age, is shown. This pro- lymphoid with a variable number of heterophils, both portion of leukocytes to erythrocytes is greater in early and late stages. Early stages of eosinophil de- the field selected for illustration than is usually velopment are present also. found. Bone marrow. In one of the specimens there were — The spleen after its reorganization produces still a few primordial osteogenic cells but otherwise blood smears of the type seen in figure 331, which there was a fairly constant picture of mature granulocytes and red-cell developmental stages. was from a chick 35 days posthatching. The statement has been made several times that granulocytes held in hematopoietic organs of 4 18 HOURS POSTHATCHENG DAYS the embryo are discharged into the circulation Circlatiug blood.—In one chick there was a normal soon after hatching. A contrary point of view blood picture and in the other there was still a pre- has been given by Nonidez (1920) from his dominance of granulocytes and a lag in lymphocyte study on eosin-staining cells in the gonads of development. bantams. They were abundant in the embryo Bone marrow.—In both cases the bone marrow con- and began to disappear after hatching but he contained a great many fully developed heterophils as cluded that the cells did not pass into the blood well as developmental stages of red cells. stream, but underwent disintegration and were taken up by special cell elements and endothelial 5 DAYS 18 HOURS AND 6 DAYS lining cells of blood vessels. In the protocol POSTHATCHING numerous macrophages were noted in the spleen at 1 to 3 hours, 8 hours, and 24 hours after hatch- Circulating blood.—The chick at 5 days 18 hours showed an abnormal number of monocytes; lympho- ing (which agrees with the observations made by cytes were relatively few; the heterophils were not pre- Nonidez on the tissues of the gonads), and 1 dominant; and there were some basophils. The 4 phagocytic cell was pictured in figure 317. From slides taken at 6 days show a fairly normal picture and the data thus far accumulated it would appear a fairly normal proportion of the different types of that both gonad and spleen tissues are acting white cells. similarly, but additional study is needed to de- Spleen.—In every case the dominant picture is lym- termine whedier the granulocytes of the embryo phocytogenesis with only a few granulocytes develop- organs after hatching are the definitive cells of ing. In some slides, stages in lymphocytogenesis are all the well shown. the circulating blood or whether masses of embryo cells are destroyed and only those de- Bone marrow.—All the bone-marrow slides show veloped in the bone marrow after hatching reach about the same cell types as do those for the preceding age. This is a good age for study of developmental the circulating blood. DanschakofE (1916a) stages along the various lines. In one slide a throm- observed in sectioned material what has been re- boblast was clearly seen. ported here that, after hatching, earlier leu- kopoietic functions are reduced and the spleen 8 DAYS POSTHATCHING becomes chiefly a lymphocyte-producing and an erythrocyte-destroying organ. Circulating blood.—In most of these smears there was the usual ratio of white cells to red cells and the Plasma cells are rare in the normal chicken

165 but may be seen in spleen and bone marrow oc- giee in birds than in mammals. At 35 days of casionally. According to T. Makinodan (per- age (fig. 331) developmental stages can be seen sonal commmiication), plasma cells are abun- in abundance. Previous to this study of the dant in the spleen under experimental conditions. spleen, the thymus had been examined by the These cells are only rarely observed in our stock smear method and drawings had been made of of untreated adult chickens. The pure light-blue the changes in cell morphology from lympho- color of the cytoplasm and the few mitochondrial blast to small, mature lymphocyte (figs. 334- spaces of the early immature plasmocyte sepa- 338). These cells will be described in more rate it from other cells. Other identifying fea- detail when the plates illustrating the thymus are tures are the clear area adjacent to the nucleus described, but several lymphocytes in figure 331 and the presence of vacuoles of uniform, small are worthy of mention here. size in the cytoplasm. An early immature plas- Cell 3 is an immature lymphocyte; the narrow mocyte with one vacuole is shown in cell 1 of fig- rim of cytoplasm in a cell of this size with this ure 331. Early in the differentiation process, peculiar nuclear pattern (partly reticular and the nucleus shows a contraction of chromatin into partly clumped) is typical for the young lympho- large, dense clumps and the cell shows an amount cyte. Somewhat similar examples of this were of cytoplasm relative to nuclear size that exceeds found in the circulating blood (figs. 96-98) and the proportion found in other cells, especially at under these conditions they were called mature the corresponding stage of differentiation. lymphocytes of medium size, but as found in the Other cells at the same stage of development young spleen they are called immature lympho- would show a reticulum of chromatin in the cytes. This certainly appears to be an inconsis- nucleus. tency in cell identification but it is more a re- From this early stage to the late immature plas- flection of the fact that in spite of a great deal mocyte there is a diminution in size of both nu- having been written about the lymphocyte, we its developmental cleus and cytosome (cell 2) . Usually the Hof is actually know less concerning definite but often the dense blue of the cytosome stages than we do of any other leukocyte. Per- masks it. A plasmocyte at this stage resembles haps this is in part due to the fact that those who a mature osteoblast and it has been suggested that have written most about the lymphocyte have they may have a common cell of origin. Plas- regarded all sizes from the largest to the smallest, mocytes and osteoblasts look so much alike that including all stages of cytoplasmic and nuclear they could be confused readily in the bone mar- differentiation, as insignificant in comparison the fact that the cell is totipotent in its row, but in the spleen it is assumed that osteo- with capacity to produce other cells, and that every blasts are not present. In the bone marrow it is is hemocytoblast, or common stem the clear blue color of the plasmocyte that aids lymphocyte a cell, to all other cells. in distinguishing it from the osteoblast, which In the circulating blood of one chicken the takes a violet hue. The observations on the cy- lymphocytes may be small and in another they tology of plasma cells, made by Dantschakoff may be predominantly of medium size. The (1909b) from sectioned material, agrees closely graph (fig. 152), based on numerous slides, with the description given here, based on impres- shows a typical distribution curve for size. More sion smears. From sections of bone marrow, on the size of lymphocytes will be given in chap- she observed that plasma cells are always located ter 6. Careful statistical studies on the relation especially inside vessels and that they became of size to the health of the individual are needed. numerous when the bird suffered from genei'al- From studies on the thymus at least three stages ized exhaustion. Irradiation by X-rays caused of development can be recognized—lympho- the formation of plasma cells in the thymus blast, immature, and mature. This represents (Danschakoff, 1916b). fewer subdivisions than for any other cell type. Lymphocytes are the dominant cells of the It is suggested that lymphocytes of the type spleen beginning with the second day posthatch- shown in figures 96-98 are immature and to re- ing. Mjassojedoff (1926) found the spleen of gard them as such makes the developmental se- the adult fowl to be predominately a lymphocyte- ries consistent, but we do not yet have experi- producing organ. This is true to a greater de- mental data for the circulating blood that enable

166 recognizable us to say that, when a differential count is made, immature stage (cell 19) is readily the lymphocytes of medium size with reticular as a monocyte and cells like this are commonly nuclei should be classed as immature and only seen in the circulating blood. The Hof, azuro- small lymphocytes with dense chromatin should philic staining of the cytoplasm, and nuclear be called mature. indentation have not yet appeared. The medium-sized lymphocytes of figure 331 The implication was given under the discus- (cells 4-6 as examples) show a degree of sion of thrombocytes in bone marrow that it was chromatin clumping intermediate between the difficult to see how such labile cells as throm- immature lymphocyte (cell 3) and the small bocytes could retain their normal morphology lymphocyte (cells 7-10) but the character of the during the procedure leading up to the fixation cytoplasm does not change significantly from of tissue. Impression smears fix the cells more than the average fixative, which pene- one stage to the other as seen in the spleen ; how- quickly ever, in the lymphocytes of the thymus some trates a block of tissue relatively slowly (Under- yet even in the differences in cytoplasmic structure did occur. bill, 1932, and Medawar, 1941) ; thrombocyte appears as It is generally agreed in mammalian hema- smear practically every Even tology that monocytes arise in the same organs a naked nucleus {12-14 are examples). where lymphocytes have their origin, and this 11, which has been labeled as a naked lympho- its larger size, actu- appears to be true in birds also, but in avian cyte nucleus because of may species there are no lymph nodes, which are the ally be the nucleus of an immature thrombocyte. seen in organs chiefly responsible for lymphocytes and A few mature heterophils may be repre- monocytes in mammals. The spleen of birds spleen smears at this age but whether they of the splenic arteries after hatching is a lymphogenic organ and it is sent cells brought in by way not surprising that developmental stages of mono- or are cells that developed in this locus during discharged cytes should also be present in this organ. In embryonic life, and have not yet been the protocol above, developmental stages of the into the circulation, cannot be determined. monocytes were mentioned as occurring in the circulating blood of the young chick. Jordan's (1938) statement (p. 731) concerning the development of blood cells in the lung fish (Pro- LYMPHOCYTOGENESIS IN THE topterus ethiopicus) is interesting: "While mono- THYMUS cytes are probably formed in the general circulation, they may also arise in the spleen." Cells of the thymus have been called thymo- The idea of the maturation of monocytes in the cytes but they are identical with lymphocytes, circulating blood was expressed also by Dawson and the latter term has been used here. Wise- (1933a). His studies of hematopoiesis were man (1931b and 1932) differentiated 5 stages based on the amphibian, Necturus. In this in the cytomorphosis of lymphocytes: lympho- species, some monocytes arose in the spleen but blast, young cell, mature cell, old cell, and de- most of them differentiated in the lymphogranu- generate cell. The classification presented for lopoietic aggregations scattered through the the lymphocyte series in table 2 (p. 10) includes tissues. the first 3 of these 5 stages. A superficial examination of the cells in figure Many smears were made before a satisfactory 331 might lead to the suggestion that large cells one was obtained; most of them failed to show as in of the type shown at 16 are metagranuloblasts, many intact cells as have been illustrated in- but a closer examination shows that this is a dif- figure 332 A, which came from an embryo the ferent cell. The nucleus is a typical monocyte cubated 9 days 4 hours. The fragility of nucleus even at this early stage and there is no cells continued well past the age of figure 332 B yet in the older ages large tendency toward vacuolization of the cytoplasm (11 days 10 hours) ; as in metagranuloblasts. In the early immature lymphoblasts were relatively rare. monocyte (cells 17-18) there is some fuither The high-power drawings (figs. 334-338) condensation of the nucleus to a condition seen in were made after searching the slide to find suit- many monocjiies but no bodies characteristic of able stages in an embryo that had been incubated granulocytes appear in tlie cytoplasm. The late 14 days 11 hours. At the earlier ages, because 167 there was so much distortion of the cells and fied. The thymus has a lesser granulopoietic because the serum precipitate was so dense, it function than the spleen. Cell 11 has many was difficult to distinguish between mesenchymal structural characters of the promyelocyte but the cells and young lymphocytes. In 332 A no at- magenta masses are not the sharply defined tempt has been made to place a label of lympho- rings and granules usually found, which might cyte on any of the cells, although some of them be due to the unfavorable environment in which appear to be undergoing a transition from pri- it was fixed. Cell 12 is definitely atypical. Some mordial (mesenchyme) tissue to lymphoblasts. might identify it as a macrophage, and cell ii as Cell 1, which is fairly well presei-ved, shows its early stage. It also is reminiscent of the pe- a faint nucleolus in the lower part of the nucleus, culiar defect noted in bone marrow where large and other cells in the same field show nucleoli spheres and masses of magenta material were vaguely. It is this type of cell in the older deposited in the cytosome of primordial osteo- chicken that would be called the reticular cell. genic cells (figs. 320 and 328). Cell 12 may Somewhere in the transition to the lymphoblast well be a naked nucleus surrounded by cyto- the nucleolus is lost, or at least it becomes hidden plasmic residue and serum artifacts. beneath the delicate reticular pattern of the Cell 1 in figure 332 B closely simulates the chromatin at the surface of the nucleus. In primordial cells of 332 A but, also, it is identical figure 334 this is true even when the chromatin in appearance with the type of cell called the is lightly stained. As stated before, whether a lymphoblast (fig. 334). The lymphoblast de- nucleolus is actually present must be determined creases in size during differentiation and, al- from sections but, if present, it definitely disap- though the chromatin is not clumped, it does be- pears before the stage of the small lymphocyte come coarser (cells 2 and 3) and forms what is has been reached. This has been confirmed in called an early phase of the immature lympho- sections by Dantschakoff (1908b and 1909a). cyte; soon the forming of blocklike clumps of In Sundberg's study of lymphocytogenesis chromatin begins, usually at one side of the (1947) in man and other mammals, obsei^va- nucleus, which indicates that the differentiation tions were made that were almost identical to process has reached the late phase of the im- those reported here for birds. She found that mature stage (cells 4 and 5). Cell 5 in the cir- a reticidar cell was the precursor for the lympho- culating blood would probably be called a ma- cyte line. The nucleus of the reticular cell ture lymphocyte of medium size. Small mature showed a nucleolus, whereas this organelle if lymphocytes are present at this age but none are present in the lymphoblast was not visible. The shown in the field that has been illustrated. reticular cell described by Sundberg closely re- The thymus at this age is not a suitable source sembles in appearance the primordial osteogenic of material for making smears and evidence for cell of the embryo chick bone marrow (fig. 320, this is shown by the distortion exliibited by di- cells i-4). viding cells 6 and 7; these may be compared Broken nuclei produce long strands of stained with dividing cells in the spleen at 12^/4 days,

basichromatin across the slide and sometimes it where the mitotic figures are well presei-ved. looks as if a particular strand could be traced The cells shown in figures 334^338 represent back to the granule in the nucleus out of which a developmental series taken from the thymus

it had formed a streamer (fig. 332 A, 8) . Some when the embryo had been incubated 14 days 11 cells (like the two to the right of cell 1) stain hours. Search was made for well-preserved intensely and the chromatin is clumped, but the cells. All cells as large as figure 334 had nuclei cells are crowded and thus fail to show clearly that took the stain poorly. This poor reaction the type to which they belong. They resemble in has happened before with May-Griinwald Giemsa a general way the amoeboid wandering cell de- but usually there were similar cells on the same scribed by Dantschakoff. slide or equivalent slides that showed the nuclear Cells from an embryo that has been incubated surface well stained. With Wright's stain, cells 11 days (fig. 332 B) were somewhat better pre- this large and immature rarely show the nucleus served in smears than were cells taken from em- properly stained. bryos of younger ages; hence the cells that By the methods used here a nucleolus is not are going to produce granulocytes can be identi- visible at any stage of lymphocytogenesis al-

168 though, as pointed out earlier, it is present in the smears considerable structure is visible, the most primordial embryonic cell from which the lym- conspicuous being the mitochondrial spaces. A phoblast is derived. Even if it can be demon- well-defined plasmosome nucleolus lies near the strated by sections that a nucleolus is present, center of the nucleus in cell 1 of figure 333. The this would not invalidate the usefuhiess of the color taken by the cytoplasm is similar to that of smear method as a means of differentiating be- the plasmacyte of the spleen (fig. 331). Cell 2 tween the early stages of various cell lines, and is a slightly crushed reticular cell. for practical purposes it can be stated that, in Not as many reticular cells are found in smears smears, erythroblasts and thromboblasts have nu- as might be expected from the large number seen cleoli and that lymphoblasts and granuloblasts in sections because in making them the loose cells do not. (For discussion of monoblasts, see in the meshes of the reticulum are given up more Lucas, 1959.) readily than the framework itself. The reticular The cells in figures are still suf- 335 and 336 cells seen here are very similar to the pri- ficiently large to be designated as immature mordial osteogenic cells of the embryo bone mar- lymphocytes, the cell (fig. and immature 122) row, and if the cells of figure 332 A could be laid found in circulating blood closely resembles out, separate from each other, it is probable that figure 336. Other cells somewhat more ad- both the reticular cells and the primordial osteo- vanced in development in which the chromatin genic cells would have an appearance quite sim- shows early stages clumping of have already ilar to the mesenchyme of the embryo. The been discussed in connection witli figures 331 and reticular tissue of the adult organism is said to 332, and the medium and small mature lympho- be more like the embryonic mesenchyme es- cytes (figs. 337 and will be mentioned 338) pecially in potentiality for producing other cell again in connection with figure 333. types than any other connective-tissue cell of the The general appearance of a smear from the body. thymus 35 days after hatching is in- practically Danchakoff (1916b) in her study of thymus distinguishable from a smear of the spleen at development in the chick embryo concluded that the same age. The medium to small lymphocyte the cells of the thymus cortex are derived from is the dominant cell of both organs, but the de- small lymphocytes that originally came from velopmental stages of the monocytes have not mesenchyme cells. The small lymphocytes are been identified in the thymus. Cell 3 (fig. 333) said to produce fibroblasts, macrophages, plasma is a rather small lymphoblast similar to figure cells, and myelocytes. 335; cells that are not much smaller reached a Granulocytes are common in the thymus, and stage of nuclear clumping equal to the immature in sectioned material the heterophils often lie in Ijnnphocyte (cells 4 and 5 and the cell below a mass of reticular cells and debris that form a 17). There are many medium and small mature Hassall's corpuscle. Other cells found in the lymphocytes, some of which are indicated by avian thymus but not in the mammalian gland cells 6—12. In these the chromatin is clumped are the isolated striated muscle cells. The eosin- into heavily stained blocks. As mentioned staining affinities of these cells cause them to earlier, at low magnification there are no visible stand out conspicuously from the blue nuclei of significant changes in cytoplasmic sti-ucture dur- the lymphocytes (Wassjutotschkin, 1913 and ing the differentiation of the lymphocyte, but 1914). It was expected that these cells would under high magnification (figs. 334—338) mito- be seen in smears but none were found. chondrial spaces may be seen in some of the young cells. The spaces disappear as the cell grows older. FEATHER SHEATH CELL The framework of the thymus is composed of

reticular cells and their fibers. These cells are This is not a blood cell and is not directly often called epithelial or epithelioid cells. In related to one. It is a contaminant that was section they are readily recognized by the acido- found on many slides made from late embryos.

philic cytoplasm, which is large compared with Before its significance was known, it caused a the small nucleus. Cells in section show very great deal of confusion and concern. It was little structural detail in the cytosome but in called an x-cell when discovered, and that name

169 A

Figure 332. —Embryo thymus.

Embryo incubated 9 days 4 houre. Mesenchymal B Embryo incubated 11 days 10 hours. 1,370X. cells of the embryo thymus early in its development. 1 Lymphoblast.

1,370X. 2, 3 Early immature lymphocytes.

4, 5 Late immature lymphocytes. 1 Primordial cell with a nucleolus. 6, 7 Mitosis, prophase. Cells probably are developing 2 A rounded primordial cell. lymphocytes.

3, 4 Cells in which the cytoplasm is merged with the 8-10 Smudged, naked nuclei. stained serum. 12: uncertain; either macrophages or heter- 5 Naked nucleus. 11, Identification ophil promyelocytes. 6 Broken, naked nucleus. 11 Closely resembles a promyelocyte. broken cells. 7 Shrunken, 12 A highly atypical cell. It contains magenta 8 Strands of basichromatin from broken n'.tclei. spheres like the primordial osteogenic cell.

170 SC^/ 't d^*

• (^i^;

332

171 Figure 333.—Thymus. Chick, 35 days after hatching. Composite drawing from 4 pLaces on same slide. 1,370 X.

1 Rounded thymus reticular cell. A primordial cell. G-9 Medium mature lymphocytes. 2 Slightly squashed reticular cell. 10-12 Small mature lymphocytes. 3 Lymphoblast. 13-15 Smudged nuclei. 4 Immature lymphocyte at transition to mature 16 Mature heterophil. lymphocyte. 17 Smudged heterophil. 5 Immature lymphocyte, slightly more differentiated 18 Mature basophil. than 4.

172 r%-s;^^ §B ^4 i

'• #.« w

.i?s^.

333

173 Figures 334-338.—Stages in the development of lymphocytes in the thymus. All figures from the same slide. Embryo incubated 14 days 11 hours. 2,470X.

334 Large lymphoblast. Nucleus incompletely stained. 337 Mature lymphocyte of medium size. 335 Lymphoblast at transition to immature lymphocyte. 338 INLature lymphocyte of small size. 336 Immature lymphocyte.

174 335 336

334

337 338

175 Figures 339-342.—Squamous epithelial sheath cells covering down feathers. These cells separate readily and as a contaminant frequently fall on impression smears made from embryonic and young chick organs. 2,470 X.

339 A relatively small cornified down-sheath cell found 341 A large and slightly smudged down-sheath cell found in bone-marrow smear. Oval area of dead nucleus in a spleen smear. Coalescence of keratin bodies. slightly to the left of center. No keratin granules. No nucleus visible. Orange body with a dense Chick just hatched. nucleus is a broken erythrocyte on the surface of the sheath cell. Embryo incubated 20 days 4 hours. 340 An elongated down-sheath cell found in bone-marrow 342 A final stage in the keratinization of a down-sheath smear. Dead nucleus in the center. Numerous cell found in bone-marrow smear. Remains of the keratin bodies formed in the cytoplasm. Chick just dead nucleus visible in the center. Embryo incu- hatched. bated 19 days 19 hours.

176 * > 4 y %

.^.-^

339

\''K 340

\ / .f - ^^

4... -,. -

341 342

177 Sheath cells

A r

Figure 343.

A Part of an emerged down feather in which is diagramed the down-sheath cells that enclose the barbs and barbules of the down feather. B The sheath forms a simple squamous layer of keratinized dead cells.

178 iiulicaled the i^tale of knowledge about this ceil After it had been learned that these cells for a considerable length of time. were specifically associated with the down of the Cells like figure 342 were most commonly young chick or the late embryo and that they were seen. They occurred in most bone-marrow readily separated and disseminated, it was de- smears and later in some spleen smears. Since cided to conduct an experiment with two of the all the cells in figures 339-342 were drawn at men wlio take off the chick hatch each week. the magnification used for other high-power This Laboratory maintains a quarantine, and a drawings, it is apparent that they were many man coming onto the premises changes his times larger than erythrocytes. When cells like clothes, washes his hands, and puts on boots and figure 340 were found, a protozoan parasite was coveralls furnished by the Laboratory. When suspected, but none were known that closely ap- men enter the incubator rooms, a second change proached this morphology and retained a large of clothing is made. After the two men had central nucleus. As different tissues were used taken off the hatch they went through the quaran- for smears, the more widely did these cells seem tine in reverse, so that they entered the Labora- al- to be distributed. Finally, after they had lieen tory in their street clothing. Previously 6 seen in smears from connective tissue of the groin buminized slides had been prepared. Now each region and from air sacs as well as from bone man was asked to brush his hair about 6 inches marrow, thymus, spleen, and bursa, it became from 3 of the slides. evident that this was a contaminant. All 3 slides of 1 group showed sheath cells A search for the source led to an examination when stained, and 1 of the other 3 slides showed of the surface of the late embryo and of the young them. Therefore, any truly effective quarantine chick, the scalps of the people who were making should eliminate the possible transfer of these the slides, and the towels used to wipe the hands cells from one group of chicks to another, or else and instruments. it should be shown that these desquamated cells of pathogenic organisms. The x-cells were obtained only from the sur- cannot serve as carriers illustration are intended face of the chick. If a slide was covered with The cells selected for albumen and the down was brushed with a pencil to show three steps in the process of keratiniza- atypical while the newly hatched chick was held several tion (figs. 339, 340, and 342) and one ghost is inches above the slide, many cells of the type cell (fig. 341). In figure 339 a nuclear its slight color and oval shape. shown in figures 339-342 were seen. It was indicated by orange then but a short step to locate the sheath of cells In this cell the cytosome is uniforndy vacuolated a light-blue stain. that encloses the emerging down. When they and the ground substance takes Often the tliin edges of these cells are folded and were seen in sheets, as in 343 B, it was evident whence they had come. The remarkable thing in this case there are 2 folds, 1 above and 1 be- about them was their ability to separate them- low the level of the nucleus. selves so easily and completely from other cells Usually the cells have greater length than of the same epithelium that nearly always they width. The nucleus is orientated with its long were found as isolated cells. Although their axis transversely placed across the middle of the source was now known, there was still the ques- cell (fig. 340). It, of course, is dead and no tion of how they came to be a contaminant on chromatin particles are visible, only a faint impression smears, since the down of many em- orange coloration without structure. Keratin particles develojj in the bryos was wet and it was unlikely that the cells cytosome from many could be spread by air. The crevices of the separate centers. Some grow larger and flow mouse-tooth forceps used to ])ull the tissues apart together with adjoining masses. The typical ap- carried these cells. Although the forceps were pearance of the last stage when no more keratin- not touched to the slide when the smear was ized material can be packed into the cell is made, they must have been the chief source lie- shown in figure 342. The nuclear ghost is visiljle cause, subsequent to this period of investigation, ill the center of the cell. The cytosome is divided no down-sheath cells appeared in smears if the into multisided angular compartments with a teeth of the forceps were carefully cleaned after small residue of Idue-staining cytoplasm between each step in the dissection process. them to mark their boundaries. The intense 179 affinity of the keratin for the stain makes cells at of a partially squashed cell but there is little this stage of development appear nearly black evidence beyond its atypical appearance to in- under low magnification. dicate that this was the case. The cell shown in figure 341 is atypical in that The barjjs and Ijarbules of the chick down are the keratin has flowed together into large, ir- lield by a thin layer of these epidermal cells regular masses. The nucleus does not show and (fig. 343 A). Upon drying, the sheath breaks the orange body with its blue nucleus is an ery- readily, allowing these structures to spread and throcyte on the surface of the cell. The general assume the fluffy appearance characteristic of appearance of the down-sheath cell is suggestive down.

180 CHAPTER 5

Blood Cells From Bone Marrow of the Hatched Chicken

Bone marrow of the adult chicken differs in variety of differentiated cell types from the small its general appearance from the bone marrow of lymphocyte. Cultures in a plasma medium the embryo or the recently hatched chick by its showed, first, a degeneration of mature and late abundance of mature erythrocytes. Blast and polychromatic erythrocytes and some maturation developmental stages are present Init are not so of granulocytes, but this was followed by de- numerous as in the younger ages. These shifts generation. There was no evidence of cell di- in the incidence of different cell types with age vision in myelocytes or in niicrolymphocytes. will be described more fully when table 10 is Hetherington and Pierce (1931) gave a confirm- discussed. atory observation when they noted that in ex- Jordan (1936 and 1937) has described the plants of the buffy coat of chicken blood, all of bone marrow of several species of birds. In the lymphocytes degenerated after 48 hours. the marrow of all young birds he found lymphoid Mention has been made of practically all the nodules. These he regarded as centers of hema- developmental stages tliat are to be found in the topoietic activity, especially of erythrocytes. He bone marrow of the hatched chicken jjut a special also observed small vessels plugged with lympho- effort has been made to bring them all together cytes. Jordan and Robeson (1942) oliserved in a series of drawings under high magnification after splenectomy in pigeons that the lymphoid (figs. 345-399) in order that studies of bone foci and plugged vessels in the bone marrow were marrow in the chicken can be made as useful for increased. Their interpretations need to be re- diagnosis and for following the course of dis- viewed rather critically in the light of observa- eases as bone marrow studies have been in human tions made since then that lymphoid foci are ab- medicine. normal in endocrine glands (Payne and Brene- man, 1952), in vessels of nerves and among nerve fd)ers (Oakberg, 1950), and in the pancreas (Lucas, 1949; Lucas and Oakberg, 1950; ERYTHROCYTES AND Lucas, Craig and Oakberg, 1949; Lucas and THROMBOCYTES Breitmayer, 1949; Lucas, 1950 and 1951; Oak- berg, 1949 and 1951) and in the liver (Dening- The erythroblast shown by cell 1 of figure 344 erythrocyte. ton and Lucas, 1960; Lucas et al., 1954). The is almost an early polychromatic in figures 345- spleen like the bone marrow is a hematopoietic Younger blast cells may be seen organ, and in addition to the white pulp, contains 347. The last of the three closely duplicates lymphoid foci. Statistically, these are related cell 1 of figure 344. Cell 2 is an early poly- less to the infection by the agent of avian lymphoma- chromatic erythrocyte but shows slightly con- of the cells tosis (Lucas et al., 1954). Before similar lym- densation of chromatin than either phoid foci and plugged vessels in the bone represented by figures 347 and 348. In the low- examples marrow can be accepted as normal for birds, it power field (fig. 344) there are no good should be demonstrated that these are not equiva- of mid-polychromatic erythrocytes, although cell lent to the abnormal lymphoid foci and plugged 4 has not passed far beyond this stage. Beside vessels found in other organs of the body. the two cells at 3, there are several additional late A study by Erdmann (1917) of chicken bone polychromatic erythrocytes in the field. Cell 5 which again demon- marrow in tissue culture failed to produce a is one of these in division, 181 FiGUEE 344.—Bone marrow from a chicken, 145 tlavs old. 1,370X.

1 Late erythroblast. 11 Disintegrating mature thrombocyte. 2 Early polychromatic erythrocyte. 12 Metagranuloblast. 3, 4 Late polychromatic erythrocytes. 13-15 Mature heterophils. 5 Dividing late polychromatic erythrocyte. 16 Basophil mesomyelocyte. 6-8 Mature erythrocytes. 17 Mature or nearly mature basophil. 9 Thromboblast. 18-21 Lymphocytes, medium and small. 10 Early immature thrombocyte. Typical cell at this stage for adult l^one marrow. Added to the draw- 22-25 Cells slightly smudged. ing from another Ijone-marrow smear. 26, 27 Smudged naked nuclei.

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183 Figures 345-356 —Cells of the erythrocyte series found in the bone marrow of adult or nearly adult chickens. 2,470 X

345 Large early erythroblast. A cell this large and 350, 351 Mid-polychromatic erythrocytes. immature is relatively rare in adult bone mar- 352-354 Late polychromatic erythrocytes. Each cell in row. Nucleolus occupies the right third of the the series is slightly more differentiated than the nucleus. preceding one. 346 Erythroblast. 355 Cell with full complement of hemoglobin as 347 Late erythroblast. This cell and the preceding judged by the color and a fully differentiated one are typical of the erythrocyte stem cells in nucleus but with a round shape. bone marrow. 356 Normal mature erythrocyte. 348-349 Early polychromatic erythrocytes. Both show faint indications of their nucleoli at the lower left of each nucleus.

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346

345 347

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348 349 350

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351 352 353

354 355 356

185 Figures 357-365.—Cells of the thrombocyte series found in bone marrow. Figures 357, 363, 364, and 365 from the adult bird and 358-362 from 6-day-old chick. 2,470 X.

357, 358 Thromboblasts. A nucleolus is present in each characteristic of thrombocytes at this age but may but is faint because masked by chromatin gran- represent an earty disintegration reaction, ules, which are dense. 363 Mid-immature thrombocyte. 359-362 Early immature thromljocytes. The lighter 364 Late immature thrombocyte, perijjheral cytosomal margin as in figure 362 is 365 Mature thrombocyte.

186 357 358 359

360 361 362

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364 365 363

187 Figures 366-377.—Cells of the heteropliil granulocyte series found in bone marrow of adult chicken, except figure 376, which was from a day-old chick. 2,470 X.

366 Large early granuloblast. This size is found .373, 374 Heterophil mesomyelocytes. The beginning but rarely. rods are indicated by the darkly stained bodies 367 Typical early granuloblast. It is not possible coming from the precursor spheres. in the granuloblast stage to determine ^vhich 375 Heterophil metamyelocyte. More than half of type of granulocyte will be produced from it. ,,,.,, ,",",, the full of nao orn tj 4 t.-i * 1 1 1 i complement rods have appeared. 3os, .369 Heterophil metagranuloblasts. ^^ S'*^ Heterophil division, 370-372 Heterophil promyelocytes. Magenta rings are metamyelocyte in mitotic present and also cytosomal vacuoles containing Early telephase. rod precursor spheres. 377 Mature heterophil.

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189 379 381 380 378

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.^ I stiates that mitosis can occur in Ijiids relatively myelocyte Ijecause it appeared to have less than late in the differentiation process, and in the half the normal number of basophilic granules, erythrocyte development of the human being this but in view of the disintegrating effect of aqueous would be equivalent to aijout the normoblast solutions it may be older. Cell 77 is either a stage. mature or nearly mature Ijasophil. All of the field of figure 344 was drawn as it In all bone-marrow smears there are smudged 22- appeared under the microscope except for one cells, some that are slightly crushed ( cells cell (10) and that was added from another slide. 25 of fig. 344) and others (cells 26 and 27) that Cells 9 and 10 form two stages in the thromlio- are destroyed beyond recognition. cyte series. The former is the blast stage and is Lymphocytes ( cells 18—21 ) show a range from relatively rare but is recognizable by its punctate medium to small but they are all mature and in pattern of nuclear chromatin. Cell 10 is the this particular field there is no evidence that the most conmionly seen early throml)ocyte and was bone marrow is a lymphocytogenic organ. A added to the drawing so that its size and colora- better measure of this function comes from a tion could be compared with those of young study of table 10. These mature lymphocytes erythrocytes and myelocytes in the same field. shown in figure 344 could have come into the Its rim of dark-violet cytoplasm with a tendency l)one marrow by way of the circulating blood, to stain more liglitly at the periphery is a charac- as most of the mature ervthrocytes presumably teristic of the early thrombocyte (fig. 362), at did. least as seen in bone marrow. In figures 345-390 and 391-397 an attempt Mature thromliocytes that have not disinte- has jjeen made to show each cell line as com- grated are rarely seen in smears from bone mar- pletely as possible. Obviously, many cells are row for reasons already discussed. One ruptured similar to those pictured in the circulating blood thrombocyte (cell 77) can be identified by the and in other hematopoietic organs, but even con- remnant of acidophilic cytoplasm that is still at- sidering all the cells pictured, relatively few ex- tached to it. A close study of the several naked amples are shown of each cell type, and no matter nuclei in the field reveals that some could have how extensive the illustrations may be they init undoubtedly coiue from thrombocytes some cannot su!)stitute for the actual examination of from broken erythrocytes. came slides where, in a short time, far more cells than When numerous fields of ])one marrow are those represented in this volume can be seen. examined it is not hard to find all stages in Three erythroblasts from bone marrow, dif- granulocytic development in the embryo, but the fering considerably in appearance, have been l)one marrow of the adult, unlike that of the

illustrated (figs. 34.5-347) . Two show nucleoli embryo, seldom shows more than 1 or 2 early and one does not; one has a narrow band of stages in a single field. Only 2 stages of de- two have extensive cytoplasm. velopment of the heterophil series are shown in cytoplasm and l)y figure 345 is rare. Most figure 34^1—the metagranuloblast (cell 12) and The type represented mature cells (13-15). A badly distorted of the erythroblasts seen in adult bone marrow basophil (cell 16) has been labeled as a meso- look like figure 346 or 347. The latter with its

Figures 378-390.—Cells of the eosinophil and basophil granulocyte series from l)one marrow of adult chicken, except figure 379, which was from a day-old chick. 2,470X.

378 Eosinophil metagranuloblast. 385 Mesomyelocyte with a moderate number of 379 Eosinophil mesomj-elocyte. basophilic granules. 380 An eosinophil mesomyelocyte more differenti- 386 Late phase of the mesomyelocyte. ated than the preceding cell. 387 Basophil metamj-elocyte. 381 A late eosinophil metamyelocyte. 382, 383 Basophil promyelocytes. Figure 383 is slightly Figures 388-390: Basophil granulocytes from hone marrow more differentiated than figure 382. fixed in vielhyl alcohol and slained in thionin.

388 Mesomyelocyte. FiGtjRES 384-386; Basophil mesomyelocytes. 389 Metamyelocyte. 384 Early phase of the mesomj-elocyte. 390 Mature basophil.

191 .

denser chromatin patleru is somewhat more dif- to di\'ide tlie range into equal parts. In spite ferentiated than the cell in figure 346. of full awareness that there were three stages in Erytlnoblasts have been seen in the circulating this range, and after considerable deliberation blood of the adult (figs. 9 and 121), in the cir- had been given to the criteria of color and cellular culating blood of the early embryo (figs. 224 morphology, there were times when immature and 233-235), in the later embryo (figs. 254- erythrocytes on a particular slide were not named 256), and in embryo spleen (figs. 329 and 330) as they should liave been. It was felt that the To all of these Sabin (1920) applied the term difficulty might be due to a tendency, inherent

"megaloblast," but it has been claimed that the in human nature, to divide any continuous varia- stem cells of all erythrocytes at all ages are not bility into smaller units. If such a tendency alike and also that those of normal blood are not were to influence an investigator when he had a identical with those of anemia (Jones, 1943). slide under examination, and if the slide did not Anemias have not been studied in the chicken have the full range of polychromatic erythrocytes but from the other observations it is evident that in the fields examined, the investigator might be all erythroblasts do not look alike cytologically. impelled to divide what range there was into three The differences are due in part to the fact that parts. Repeated surrender to this tendency—one the erythroblast is not a static cell covering one slide to the next, or one day to the next—might point of time in development, but is a cell that result in inconsistent determinations. After the during the differentiation process can often be drawings for the Atlas were completed and ar- divided into early and late phases, both of which ranged, a imiform scheme was set up and with are capable of mitosis and the production of the illustrations in hand there has been no recent progeny. difficulty in calling cells of the same type by the Another factor that modifies the cytology of the same name. erythroblast is the degree of urgency for hemo- Early polychromatic stages of the bone mar- globin. In the first generation of erythrocytes row are represented by figures 348 and 349, and it is evident that a high rate of hemoglobin ac- both show shadows of nucleoli against the lower quisition has been given priority over cytologic nuclear margins. The first of these two cells is difl^erentiation. With each generation of erythro- not far removed from the blast stage and the blasts there is a gradual change to the definitive second has a cytoplasmic coloration approaching condition where the taking on of hemoglobin the mid-polychromatic erythrocyte. In these becomes an integral part of cell differentiation. cells mitochondrial spaces are still visible; yet As this study shows, the erythroblasts under in some examples of early polychromatic eryth- these various conditions have a different appear- rocytes the textural quality of the cytosome may ance cytologically. Thus the names primary be as uniformly homogeneous as in later stages erythroblast, embryo erythroblast, and definitive of development. erythroblast have been used. It is conceivable Mid-polychromatic erythrocytes are repre- that under the stress of a severe drain on the sented l)y two drawings (figs. 350 and 351) ; the erj^hrocytes in the adult these cells might be one in the first drawing is the younger. In these pushed back to the same demand for new hemo- particular examples, the nucleus is still large rel- globin that occurred in the first embryonic gene- ative to the size of the cell. In some mid-poly- ration. Under these circumstances it might he chromatic erythroc}'tes the nuclei may show exjjected that they would look alike, but under greater chromatin condensation than in figures normal conditions the definitive erythroblast of 350 and 351. They may be more like the one the bone marrow does not look like the primary shown in figure 352. This figure shows a cell erythroblast, and thus there is less confusion if lying at the borderline between the mid- and late specific terms are used whenever possible. stages of erythrocyte development. The cell has Beyond the erythroblast there are three poly- been placed in the late stage group because the chromatic stages that cover the period of dif- baso]iliilic substance has almost disappeared and ferentiation from a cell with a small amount of tlie acidophilic materials in the stroma are begin- hemoglobin up to one with its full capacity. ning to dominate. The cell represented by figure These three stages are, of course, arbitrary ones, 353 is clearly more differentiated than the pre- and in setting them up an effort has been made ceding one and not as far along in its develop-

192 a

nient as the ceil in figure 354. Accompanying of the embryo thromboblast (figs. 280-284). ihe increase in hemoglobin is a slight elongation In both, the nucleolus may or may not be visible, of the cell and an increased nuclear condensation. depending upon the extent to which the plasmo- Tiie tawny color, so often oljserved in late poly- some is masked by the overlying chromatin. The chromatic erythrocytes just before maturity, may frayed appearance of the peripheral margin of

1)6 due to the diffusion of basophilic granular the cytoplasm seen in figure 362 may also occur material associated with the reticulocyte stage of in the blast stage. development. The term "early immature thrombocyte" is a Differentiated cells with a full complement of rather awkward one; perhaps other investigators, hemoglobin and a small nucleus with a condensed after they have reexamined the problem, will chromatin pattern of the mature cell, yet with a think of a shorter name. In the early immature round, rather than an oval shape, may be found thrombocyte, tiiere is some clumping of the nu- ill the bone marrow of the adult chicken (fig. clear chromatin and often the structure of the 355). When such cells were observed in the nucleus will appear similar to that of the par- embryo, tliey were interpreted as representing an tially autolysed cell where the chromatin clumps atypical condition associated perhaps with forced have vague indefinite boundaries like those development, and probably the same can be said shown in figure 363. Figures 358 to 362 were for sucli cells when they appear at older ages. A made from cells of the bone marrow of the 6-day- mature, normal, typical erythrocyte is illustrated old chick rather than from the adult because there by figure 356 and is identical with those seen in were a large number of early stages available circulating blood, even to the tone of cytoplasmic for observation. This made it possible to ar- color, in spite of the fact that the cells in the bone range them in their proper developmental marrow were stained with May-Griinwald Giemsa sequence. and those of circulating blood with Wright's. The mid-immature thrombocyte has already ac- Thromboblasts were not recognized in the bone quired the characteristics that definitely identify marrow until after the developing stages had it as a thrombocyte—namely, the shift in nuclear been worked out in the circulating blood of the staining from violet to purple, the appearance of embryo. One reason was that they resembled definitive granules (fig. 363) and the elongation the erythroblasts. In fact, one cell in this series of shape as in the immature embryo thrombo- (fig. 357) was originally placed among the eryth- cyte. The cell at this stage of development has roblasts, but its punctate nuclear stmcture soon a deep basophilic cytosome, which, during dif-

indicated that it was in the wrong series. Two ferentiation to the late immature stage, fades to stem cells of the adult bone marrow, the granu- a lightly stained cytoplasmic framework (fig. loblast and the thromboblast, have narrow cyto- 364). The cell lias not yet assumed its mature plasmic rims of cytoplasm around their nuclei. oval shape. The nucleus is still larger than in The cytosomes take an intense violet color, but the mature stage; it is still round, ratlier than these two cell types are readily distinguishable oval. Cells of this type are often found in the l)y their difference in nuclear structure. circulating blood, as shown in outline (fig. 88 i). The erythrocyte was divided into five levels of Late immature and mature throml)ocytes may be development and tlie same number of sida- found in bone marrow but they are not common; divisions has been proposed for the thrombocyte, usually they have undergone at least partial dis- but for the thrombocyte series these are based on integration. The cell shown in figure 365 is iden- structural and size changes without the assistance tical in appearance with that in figure 73— of significant tinctorial changes as in the erythro- thrombocyte from circulating blood.

cytes; thus it has been somewhat difficult to establish terminal criteria for each stage of development of tlie thrombocyte. The thromboblasts are large cells (figs. 357 and 358) with a densely GRANULOCYTES stained cytosome that has numerous vacuolar and mitochondrial spaces. The classification of granulocyte stages of de- The appearance of definitive thromboblasts of velopment has been outlined in table 2, which the bone marrow is not much different from that gives 6 steps for the heterophil, 5 for the eosino-

193 phil, and 5 for the basopliil. Each of the 3 has prepared a colored plate showing granu- granulocytes reveals the characteristics of each lopoiesis; it was based on the use of a different of the 6 steps in development; but in the eosino- stain but in the sequence of stages (Hamre, 1952 I phils and basophils, 2 of the stages have been there was nearly complete similarity with the telescoped and the name chosen for the combina- data given here. The privilege of examining

tion is the last one in the series.' his data before publication has been helpful in

The granuloblast is identified !)y its large nu- this complex problem. He did not set apart as cleus surrounded by a narrow rim of cytoplasm a separate stage what has been given here as the densely stained. Relatively few cytoplasmic metagranuloblast. spaces are in the cytoplasm (figs. 366 and 367), The metagranuloblast is derived from the and these lie adjacent to the nucleus. granulo])last Ijy a great expansion in the cyto- The intensely colored narrow rim of violet- plasm at one side of the nucleus, and by an in- stained cytoplasm, comljined with the delicately crease in the vacuolization of this portion of the stained nuclear reticulum and lack of visible cytosome. Often the remainder of the cytosome nucleolus, makes the granuloblast readily recog- is unchanged from the condition characteristic of nizable even under relatively low magnification. the granuloblast—that is, it stains an intense violet color. This was the case in figure 368 but not Figure 366 is the largest granuloblast observed, in 369. At this the and figure 367 represents the size usually seen. stage nucleus may remain round (fig. or it be partially collapsed These cells resemble very closely the lympho- 368) may

( fig. ) . reticular pattern of the blast, but are different from the thromboblast and 369 The nucleus the erythroblast. may be just as finely patterned and delicately stained as in the granuloblast stage, or it may The scheme presented in talkie 2 has been show clumping. The nucleus collapses or be- written under the assumption that the granulo- comes irregular in shape more readily in the blast has equal potentialities to produce a hetero- heterophil metagranulolilast than in either tlie phil, eosinophil, or basophil. Early in this study eosinophil metagranulojjlast or the basophil it was thought that the stem cell for each type of pi'omyelocyte. Also, the chromatin condensation granulocyte had a different appearance, but it has is greater in the last two cells. The metagranulo- since been decided that these were either meta- blast stage of the heterophil and of the eosinophil granuloblasts or promyelocyte stages. Hamre can be readily separated. The difference is ap- ' Six subdivisions of the sranulocyte series are suggested in parent when a comparison is made lietween figure Report of the Committee for Clarification of the the First 368 and 378. In the heterophil, the boundaries Nomenclature of Cells and Diseases of the Blood and Blood- Forming Organs (1948), and Dr. L. W. Diggs states (personal of the vacuoles are rather indefinite and they ". communication), . . it is my opinion that terminologies appear oftentimes as spaces in a reticulum, but used in human hematology and widely accepted and understood, should he used for lower animals when applicable." in the eosinophil they are round areas with clean- This is a commendable point of view; unfortunately it has been necessary to depart in some cases from the terminology pro- cut boundaries. This difference was the chief posed by the Committee because the definitions associated with factor in calling cell ii of figure 330 an eosino- certain terms used in studies on blood of mammals do not adequately cover the ol)servations made on birds. As pointed out phil metagranulol)last rather than a heterophil of embryologists, and physiologists by Jones (1949), zoologists, the same stage. all have a stake in these problems. "Myelo" is an inappropriate term for immature stages of One group of hematologists (Anonymous, granulocyte development because it means "marrow," and in the birds granulocytes develop in many organs other than the 1949) gives only the term "myelocyte" to cover hone marrow. To have substituted the term ''granulocyte" for the entire span of specific granule production. "myelocyte" would have been acceptable as far as the developmental stages are concerned but would have conflicted with In avian blood for the heterophil at least, there the term almost universally accepted for the adult cell. It is a definite stage of development between the seems confusing to use the term "myeloblast" for the earliest stage and "progranulocyte" for the next stage and then follow ntetagranuloblast and the stage where specific with two myelocyte stages. Therefore, for this study on avian hematology these terms have been changed to "granuloblasl" granules first appear. Microscopically, it is one Ian old term) and "metagranuloblast," respectively; these are of the most conspicuous of the entire progression followed by the pro-, meso-, and meta- stages of the myelocyte, and these are followed by the mature cell. The prefix "meso" and it would be awkward not to have a name for uas used so that each of these phases of developTiient could be it. Its two outstanding characteristics are the identified without confusion, and so that the term "myelocyte" without any prefix could be used as a general tenn signifying presence of (1) cytoplasmic rings and gramiles the full range of development from the metagranuloblast up In that take the same intense magenta stain as the the mature cell. Metagranuloblast is a new term. ]

194 nucleus and (2) light-staining orange spheres 372, where nuclear boundaries are vague, and at (figs. 370-372). In figure 370 only vacuoles tliis stage they appear the same as they do in the are present in the cytosome but in figure 371 some embryo spleen." vacuoles are filled with lightly stained orange Several steps are involved in the production of spheres, and in figure 372 one or two of these a rod. First a vacuole is formed, it is occupied spheres have taken on the dark-orange color that by a light-orange sphere, which progresses to a places them at the transition where the specific darkly stained orange sphere, and this in turn be- bodies make their appearance. Because the comes a rod. The development of a sphere and stages antecedent to specific granule production its transformation into a rod mark the beginning can be followed so readily in birds, it seems that of tlie mesomyelocyte stage (figs. 373 and 374). use of avian blood would be advantageous in The mesomyelocyte stage has a nucleus that further study of the early stages of granulopoie- may be large and indefinite (fig. 373) or small sis. The magenta rings and the granules vary in with definite chromatin clumps (fig. 374) . Also, number; in some cells there are but few and in some magenta rings and granules may persist others they are abundant. The nature of the (fig. 373) . The process of rod differentiation is stainable material inside the magneta ring is not more advanced in figure 373 than in 374; in the known. latter it is not much farther than the "dark- The small granules are of two types; one takes orange sphere" stage. When the rods form a magenta stain and the other, a deep orange. ' One clinical hematologist, after seeing colored reproduc- If figures 370 and 372 are compared, it will be tions of these avian myelocytes, said: noted that in the latter the magenta-stained "The myelocytic series may be drawn exactly as they appeared, but if that is true, they appear to be inadequately granules are scattered around the orange-stained stained. If we had a human bone marrow or blood smear that had nuclei which were as pale and the structures as ill defined spheres and not in them ; but in figure 371 many as painted, we would say that the stain was unsatisfactory, of the orange granules lie in the exact center of would not attempt a differential or diagnosis and would ask each sphere and others do not. These orange for better stained preparations or a restain of the same preparation. A combination of Giemsa as a counterstain for the granules are abimdant in figure 374. The ques- Wright's or a change of the buffer water or a longer staining tion has come to mind repeatedly, Are the orange time might give better results." Various technics, including Wright-Giemsa, have been tried, granules identical with the central bodies of the and although differences in the appearance of the cells may general picture mature heterophil granulocyte? It might seem occur—with the same or different technics—the of heterophil granulopoiesis as shown here is representative that this should be easy to determine by following for the normal chicken. Nothing has been seen that supports the development of the rods to see where the the idea that avian myelocytes look the way they do in impression smears because inappropriate technics were chosen or granules go, but the presence or absence of a cen- because appropriate technics were faultily applied. It is sug- they do tral body in the rods is not constant, as was seen gested that the promyelocytes often appear the way because the cell undergoes extensive hypertrophy, both of the in figures 154-165, and not a single rod in nucleus and cytosome, as one of the first steps in heterophil the firm vacuoles and the liglit- figures 373—377 shows a central body inside it. myelopoiesis and because orange bodies give a honeycomb appearance to the nucleus and In figure 372 the small granules lie at the pe- cytosome by puncturing these structures at the time the cell is rijihery of the orange spheres. flattened in the process of making the smear (fig. 371). A little later in development, nuclear detail becomes visible It was the opinion of Dantschakoff (1908b) again I fig. 374). In the interim the nucleus has not changed that there was metachromasia of the specific its appearance to any great extent (compare figs. 369 and 374). Therefore, not much has been lost by using a technic (May- granules of heterophils during the early stages of Griinwald Giemsa) that does not reveal all the details of the differentiation. This reaction would agree with nucleus at the promyelocyte stage. On the credit side, this stain reveals the subtleties of changing form and color in the the observations reported in the paragraph above, rod precursors, which seems important when studying granu- No. 2 was used to the extent that the first granules to appear are locyte development. When Petrunkevitch's on spleen myelocytes, followed by May-Griinwald Giemsa, the of two types, magenta-staining and pale orange- border of the nucleus could be traced readily, and the nucleus granulation, staining. The magenta rings appear to be a dif- appeared as a large body containing a delicate but the cytoplasmic inclusions suffered severely by this technic. ferent organelle than the magenta granules and The author of the paragraph quoted is not mentioned here were not observed by Dantschakoff in her sec- by name for the reason that it is not intended to make this volume a springboard for a controversy. He is respected as tioned material. In a study by Lucas and Den- an outstanding clinical hematologist of human blood and he ington (1956, unpublished data) magenta rings may be expressing an opinion that would be shared by all hematologists of mammalian blood who look at these illustra- were observed in sectioned material. tions. A common ground of understanding is, of course, neces- discussion and this can be reached more quickly The nucleus of the promyelocyte may be dis- sary for any when it is recognized that many details of human and avian tinct but often it appears as in figures 371 and blood are not the same.

195 they produce bodies of irregular shape—some any one time. Even in the older age (fig. 380) pointed, some rounded, and some multiangular. the full range—vacuole to faint sphere to dark After the cell has developed about half of its sphere—is clearly shown and the framework normal complement of rods, it is called a meta- cytoplasm has the same pale-blue color that it has myelocyte up to the stage where, at maturity, it in the adult cell. has the full number of rods. This is a purely In the legend for figure 380 this cell is called arbitrary division and admittedly not exact, but a mesomyelocyte, and this stage, as defined here, it would be still more difficult in avian blood to is one that has less than half of its granules. At follow the criterion "Bean or kidney shaped first glance this cell appears to have half of its nucleus" (Anonymous, 1949) because, with ap- granules, or more. But this cell and the hetero- proaching maturity, the typical artifact of the phil are dealt with in the same way—only those heterophil nucleus becomes apparent; therefore, granules are counted that have arrived at maturity in figure 377 it would be impossible to say and have their full tinctorial density. On that whether this cell had an indented or bean-shaped basis the cell definitely has less than half of its nucleus but it is quite evident that it has about as granules. many rods as the cell will hold. Had the late metamyelocyte (fig. 381) been Figures 375 and 376 are examples of meta- found in the circulating blood it would probably myelocytes. In both tliere are one or two ma- have been counted as a mature cell—similar cells genta rings still carried over to this late stage; from circulating blood, shown in figures 177— usually, of course, they have disappeared by this 180, have been so named. The difference is that time. From figure 376 it is obvious that mitosis the cytosome of the cell in figure 381 was not does not stop at the granuloblast stage or even fully packed with granules. Wlien the eosino- when the specific granules first appear. phil of the circulating blood was discussed, it Developmental stages of eosinophils are as was pointed out that the specific granule for this scarce in the bone marrow as are the mature cell varied from a large homogeneous body to a forms in the circulating blood. The finding of group of four small bodies in a cluster. At no developmental stages went slowly but eventually time have the small bodies been found in the de- after enough cells had been studied certain defi- veloping stages of eosinophils in embryo spleen nite features were established that helped to sepa- or adult bone marrow. This variation is an im- rate them from heterophils. The qualities of portant one, and will be discussed again when cytoi^lasm and nucleus that make the eosinophil the blood of ducks is described, but its significance metagranuloblast distinguishable from the heter- in the cytomorphosis of the cell is still unknown. ophil of the same age have already been dis- Studies of basophil and eosinophil differentia- cussed in part. The nucleus of the eosinophil tion are handicapped by the fact that so small a throughout its developmental stages generally is proportion of cells belong to these groups. As more intensely stained than that of the heterophil. with the eosinophil it has been assumed that the Usually the vacuoles are clear—almost refrac- granuloblast stage of the basophil has the same tile—but as shown in figure 378 a few faintly appearance as described for the heterophil. stained bodies are visible. These differentiate Wlien cells like tliose in figures 366 and 367 are directly into the specific granules, and the meso- found, there are no identifying cytologic land- myelocyte (fig. 379) shows a full scale of tinc- marks to indicate the direction in which they will torial range from the faintest to the darkest. It develop. It was not difficult to locate cells that is diis direct transition from seeming vacuole to resemble those in figures 384-386, but cells like definitive granules that eliminated the promye- figures 382 and 383 were classed at first as locyte stage. There appears to be nothing equi- heterophils because they showed magenta bodies. valent to the magenta rings. Further study proved, however, that they were not In the heterophil the developmental steps of quite identical with those in the heterophils. In the precursor spheres were moderately well the cells under study the magenta bodies were synchronized in their formation; whereas, in the predominantly granules ranging from small to eosinophil, development of the spheres begins at large; the large ring characteristic of the hetero- different times, so that all stages are present at phils was absent or relatively rare. There were

196 a few small rings as is shown in figure 382, but one preceding. Cell 384 represents the first step these are not like the large thin-walled rings in the production of fully differentiated basophil shown in figures 370-372. It was chiefly be- granules, and cells 385 and 386 show an in- cause of these characteristics that the decision creasing number. By this method of fixation and was made to place a cell found in the circulating staining they appear to lie on a reticulum, but in blood (fig. 193) among the basophils and not cells 388-390, which are fixed in methyl alcohol among the heterophils. and stained with thionin, no network joining the The question arises. At what stage of develop- granules is visible. It is probable that the net- ment are the cells that are represented by figures work is an artifact and the idea of differentiation 382 and 383? It is best that the magenta bodies of granules from vacuolar substance was stated be ignored for the time being and that attention only as an opinion because it is obvious that in be given to the nucleus and cytoplasm. The the bone marrow, as well as in the circulating cytoplasm of cell 382, slightly hypertrophied at blood, aqueous solutions tend to dissolve and one side of the nucleus, is vacuolated and is be- distort the basophil granules. The nucleus also ginning to lose the strong basophilia of the granu- seems to be adversely affected by the technic loblast. If it did not have the magenta granules treatment (figs. 385-387). Figure 387 is a late it would be classed as an early metagranuloblast, metamyelocyte. and figure 383 would be classed as a slightly There is need for additional search for a tech- more dilferentiated cell at the same stage, but the nic that will preserve the basophil granules and presence of the magenta bodies has been given still reveal the detailed structure of the cell. as characteristic of the promyelocyte. It is as if Methyl alcohol and thionin preserve the granules, two steps in development had been compressed but nucleus and cytoplasm stain alike and have into one; therefore it is identified by reference such a delicate blue color that no structural de- to its most conspicuous feature and is called a tails are visible; so when it is stated in the legend promyelocyte—this is simpler than contriving a that figure 388 is a mesomyelocyte, figure 389 a new name to cover the two phases of development. metamyelocyte, and figure 390 a mature baso- This immediately raises the question. Are phil granulocyte, this is based only on the num- these magenta bodies the specific granules charac- ber of granules present. teristic of basophils? If they are, these cells should be called mesomyelocytes. At present the opinion is held that these are not mature specific granules and it is suggested that the defin- PLASMA CELLS itive basophilic granules of the more mature cell develop out of the faintly stained substance of Plasma cells may be found in spleen and the vacuoles. Thus, when the granules first ap- bone marrow but are not common in normal pear, thev are the pale-magenta bodies shown in healthy chickens. A few examples from bone figure 384 and from this pale staining condition, marrow have been seen and are presented here, by a progressive transformation, they produce and some that were observed in the spleen are the dark-magenta bodies shown in figure 384 and illustrated in figure 331, but in no case has a more abundantly in the three succeeding figures. cell been found that could qualify structurally The nucleus of the basophil promyelocyte does as a plasmablast. The possibility that such a not lose its sharp boundary or the details of its cell might be the primordial osteogenic cell in internal structure. This is because the cj^o- bone marrow or the reticular cell in the spleen is plasmic vacuolization in the basophil is not so a reasonable assumption since the early imma- vigorous as in the heterophil. ture plasmacyte (fig. 331, 1) resembles these On the right side of the nucleus of figure 382 primordial cells. Mjassojedoff (1926) found is a bluish shadow that looks very much as if a numerous plasma cells in the loose connective nucleolus were present below the surface. This tissue of adult chickens. He considered this may be the case but in the granuloblast nucleus abundance to represent a point of difference from

it is not typical. (See addendvmi, p. 140.) mammals where they are said to be scarce. Figures 384—386 are all mesomyelocytes, yet Figure 391 represents the earliest stage that each of tlie cells in the series is older than the has been found. The cytoplasm form a much 197 larger area around the nucleus than is charac- large size (figs. 398-399) . Cells of this size are teristic of blast cells but is considerably less than multinucleated, as shown in figure 399. Search found in the mature plasmacyte, and this par- was made for a mononucleated osteoclast and it ticular cell has been called an early immature was thought that such a condition had been found plasmacyte. The cytosome shows a mixture of in figure 398, but beneath the pink-stained ma- mitochondrial spaces and the spherical vacuoles terial in the center of the cells were faint traces that are typical for the plasmacyte. both imma- of other nuclei. Small mononucleated cells like ture and mature. The bluish color taken by the those found in the bone marrow of the embryo cytoplasm has little or no red in it; thus it has an (figs. 319, 326, and 327) probably exist in adult azure quality usually not found in other cell bone marrow, also, but were not seen. lines. The nucleus, even at tliis stage of imma- The cytoplasm of osteoclasts in smears seems turity, stains intensely; the chromatin is uni- to merge into the surrounding serum and only formly distributed but is composed of blocks rarely can one identify with any accuracy the larger than commonly found at this early stage of exact boundary all the way around the cell; often diiferentiation. This was true also of the im- it appears as shown at the left end of figure 399. mature plasmacyte pictured in the spleen (cell 1. The cytoplasm forms a network and in it are fig. 331). vacuoles of various sizes and granules of various The next four cells (figs. 392-395) are classed sorts; most of the latter—for example, the rods heterophils fell the as late immature plasmacytes . They are almost and magenta rings from — on as large as the early immature plasmacyte but surface of the cell when die smear was made. proportional size of the nucleus has decreased. Sectioned material usually gives an indication The sequence in this progressive change is in- that the osteoclast has polarity—one end pressed dicated by the arrangement of figures. The most against the spicule of bone and the other free in differentiated cell of the group is figure 395, in the marrow cavity. The appearance of the cell which a small nucleus lies at one side of a large in figure 399 would suggest that the left side of cell having a strongly basophilic cytoplasm. the cell has been adjacent to the bone and the During this process the chromatin aggregates into basophilic right end toward the cavity. large clumps, but the clumps do not fill the entire nucleus. In figure 393 the cytosome is filled with small vacuoles; in the others there is a range in size from small to large. DIFFERENTIAL COUNTS ON BONE The mature plasmacyte (figs. 396 and 397) is MARROW smaller than the immature stages. The nucleus still holds an eccentric position in the cell. The Differential counts have been made on the cells Hof, which was present in some of the immature of bone marrow in tlie chicken at 3 ages before cells, usually persists. Thus, the Hof is present and at 4 ages after hatcliing. One hundred cells in figure 396 and absent from 397. Vacuoles were counted from each of five chicks at each age. characteristic of plasmacytes are still present in The average was based on 500 cells. This num- the cytosome. A drawing was made of a cell ber is small for bone-marrow studies but, even identified as a mature plasma cell in the circulat- in this preliminary survey, certain ratios and ing blood, but since the cell occurred only once trends are indicated. Late polychromatic and among many slides examined, the drawing has mature erythrocytes are abundant at all the ages

been omitted. It did not have a vacuolated cyto- covered in table 10, but it is questionable whether plasm, and this raised some doubt that it was a the fluctuations in the two cell types have sig- plasmacyte. nificance; for example, where there were 16.8 and 29.2 percent at 285 hours, 49.6 and 18.4 percent at 347 hours, and 6.0 and 29.4 percent at 481 hours. Stages younger than the late poly- OSTEOCLASTS chromatic erythrocyte are always scarce. Thrombocytes are present at all ages and are Osteoclasts can be found in most bone marrow distributed fairly uniformly among the various smears. They are readily recognized by their stages of development except for the thrombo-

198 at these same two ages is blast. None of these were found at any of the cells were found only macro- ages examined, and the same was true for lympho- probably due to more than chance. Two at hours blasts, monoblasts, and immature and mature phages were included in the counts 285 hatching, but it would plasma cells. and again at 8 days after Immature and mature lymphocytes were found be expected that this cell might be seen at any of cells was 1 percent at 285 hours, and appeared at all ages after age Mitosis for all types hatching. No monocytes were found in this or less. The number of squashed cells fluctuated survey until after hatching. irregularly. Cells that could not be identified hours. Following The heterophil is the dominant granulocyte and were most abundant at 285 gradient until the per- is present at all ages and the incidence of granulo- that there was a downward hours, blasts just before hatching (481 hours) is higher centage reached a level of 0.2 at 4 days 18 than after hatching. The data in table 10 indi- and a level of 0.4 at 8 and at 175 days. cell counts the data presented here cate that this is true also of the other immature In respect to Burmester, and mature stages of the heterophil. The same differ greatly from those presented by found tendencies are indicated in the eosinophils, where Severens, and Roberts (1941). They of lymphocytes, whereas we found tliey were found most abundantly at 347 and 481 large numbers for the hours incubation age; after hatching they re- only a few. The data by Forkner (1929) differences from that mained at a fairly low level. Perhaps the same adult chicken show some data show a higher heter- tendency is indicated also in the basophils but given in table 10. Our Forkner's the number of cells counted was too small to defi- ophil than eosinophil count, whereas did not in- nitely establish a picture of a prehatching rise. values have the reverse order. He tabulation. His Among miscellaneous cells, some primary clude thrombocytes in his higher than erythrocytes were obsei-ved at 285 hours and none lymphocyte count was considerably after that. This cell is one that is known to arise ours. total number of in the yolk sac; therefore it was probably brought Marvin (1954) calculated the tibia into the bone marrow through the embryonic cir- bone marrow cells in both femurs and both His culation. in a strain of young White Carneau pigeons. Osteogenic activity was indicated at 285 hours mean normal value on 4 pigeons was 10*'X32±5 of these four leg but none later except perhaps immediately after cells for the total marrow hatching, and the fact that the elongated reticular bones.

199 Table 10.—Differential counts of chicken bone marrow CHAPTER 6

Blood Cells of Other Avian Species

DESCRIPTION OF CELLS chicken blood could be readily located in the Atlas. Although this study has been concerned pri- Table 11 lists the birds from which specimens marily with the blood of the chicken, the authors were taken. The study included the circulating have not been unmindful of the requirement, im- blood of all these species and the bone marrow plicit in the title of this volume, that attention be of many of them. given to the blood of other species of birds. After the blood cells of the chicken, with all Specimens have been collected from other do- their variations, have been thoroughly studied, mestic birds and from wild birds. But cells no difficulty should be experienced in identifying from such specimens are illustrated and de- the various blood-cell types in other birds, be- scribed only if they differ appreciably from cells cause the same cell type is closely similar in ap- found in cliicken blood. There seemed to be no pearance in most avian species. The only pos- reason why a cell from another species should be sible points of confusion have been illustrated in illustrated and described if its counterpart in figures 400-410, and are to be found in tlie

Table 11.—Birds, oth 4 •m *' "• : wr^ * :

392 393 391

395 396 397 394

Figures 391-397.—Cells of the plasmacyte series found in bone marrow of adult ciiickeus. 2,470X

391 Early immature plasmacyte. 392-395 Late immature plasmacytes. 396, 397 Mature plasmacytes.

203 Figures 398, 399. —Osteoclasts from tlie bone marrow of adult chickens. 2,470X.

398 Osteoclast that appears to have but one nucleus, but others were present at lower focal levels. Acidophilic material in the central portion characteristic of osteoclasts. 399 Multinucleated osteoclast. Boundary of cell often difficult to determine.

204 r- * v«-

398

• • • .4

399

205 400 401 402

403 404 405

406 407 408

« ^^

409 410

206 .

heterophils and eosinophils of ducks and tur- was taken, the eosinophils, of which figure 402 keys. CuUen (1903) noted that in guinea hens is an example, looks somewhat like heterophils. and in kingfishers the percentage of heterophils This similiarity in appearance is a possible cause was lower than for eosinophils, a relationship of confusion. that was the reverse of that found for other The two types of specific inclusions are not species of birds, therefore, close attention was actually alike when examined under high magni- given to these cells in the blood of wild birds and fication. The specific eosinophilic granules are to lie it was readily apparent that the confusion arose composed of very small bodies that appear from the fact tliat heterophils and eosinophils are on a network. If the granules merge with the highly variable in their appearance; some heter- network, because they are small or because there ophils resemble eosinophils and some eosinophils is no color difference between tlie two, the net- resemble heterophils. Confusion has been cre- work may then simulate a mass of poorly pre- ated by the fact that in some species—the duck, served rods in a heterophil. for example—the eosinophils are often rare, and Wright's stain, when applied to chicken blood, chromatin of the it may be that, in making differential counts, cer- often incompletely colors the tain variants of the heterophils were classified as heterophil nucleus. Heterophils of many other eosinophils and the remainder as heterophils. species show the same artifact. The difference Figure 400 is a heterophil from a mallard in staining affinity between the nuclei of heter- duck taken in Michigan and figure 401 is from an ophils and eosinophils has been utilized to aid individual of the same species taken in Utah. in distinguishing the two cell types, and a search begin- It should not be assumed that the cytologic dif- for tliis difference is always made at the ferences are due to geographic habitat; this much ning of a study on blood from a species not pre- variation can be found in a group of slides from viously examined. a species within a particular locality. One of Incomplete nuclear staining of heterophils is these cells (fig. 400) has broad, short, rounded shown in figures 400, 401, and 403, and complete rods that look much like large eosinophil spheres nuclear staining of eosinophils in figures 402,

(compare with fig. 404) . The rods of figure 400 405, and 407. are not uniformly stained. The density of When a specimen is found where the hetero- staining is greater toward the edge of the rod phil nucleus stains as well as the eosinophil tlian toward the center. The clear area of the nucleus, considerable study may be required distinguished center is not sharply defined like the vacuole in before the two cell types can be the prob- the center of the rod in chicken blood (fig. 166) readily. Hewitt (1942) faced same for hetero- The rods in another specimen of mallard duck lem in the selection of proper terms were small, narrow, and tapering with pointed phils and eosinophils. Eosinophils with round in bone marrow. There- ends (fig. 401). It is this shape that is charac- granules occurred only the homologies teristic of heterophil rods in most birds. In the fore, without attempting to solve those specimen from which the heterophil (fig. 401) of heterophils and eosinophils in ducks with

Figures 400-410.—Granulocytes from ducks and turkeys. 2,470 X.

Granulocytes the turkey. Wright's Figures 400, 401: Heterophils from adult male mallard Figures 406-408: from ducks. Wright's stain. stain.

400 From Lowell, Mich. 406 Heterophil. shde as preceding one. 401 From Utah. 407 Eosinophil from same 402 Eosinophil from same slide as preceding one. 408 Basophil.

410: Turkey granulocytes from a smear fixed Figures 403, 404: Two granulocytes adjacent to each other. Figures 409, and stained with May-Grunwald From a baldpate drake. Wright's stain. in Petrunkevitch No. 2 Giemsa. Same bird as the one from which figures 406 and 403 Heterophil. 407 were taken. 404 Eosinophil. 405 Eosinophil. Ruddy duck. Juvenile male. May- 409 Heterophil. Griinwald Giemsa. 410 Eosinophil. 207 cells in other birds, he grouped all cells in which also in the same individual. In the chicken the specific granules stained with eosin into two there were small, fine granules on a reticulum types of heterophils: heterophils with ellipsoidal (fig. 179) and larger spheres, nearly homogene- rods and heterophils with bacillary rods. ous in structure (fig. 180). The condition in When the criteria set up in table 8 (p. 90) figure 402 is equivalent to that in figure 179. are applied, as far as they may be applicable, The important fact to note is that, at least in to Hewitt's colored figures of these two cell types, chickens, the large type eosinophilic body is ac- the cells with the ellipsoidal rods become hetero- tually composed of four small granules arranged phils and those with Ijacillary rods become eo- in a square (fig. 180). Therefore, it is par- sinophils. The close agreement between the dif- ticularly significant that in the sphere located at ferential counts on ducks that he used and the about 1 o'clock on figure 404 there should be 4 counts on the common mallard (p. 216) offer fur- small, distinct granules in the form of a square ther confimiatory evidence that his heterophils with homogeneous material around them. In with bacillary rods are eosinophils in spite of the figure 405 they have taken another form, and here fact that they superficially resemble the hetero- the unit granules have moved apart in pairs so phils of other species of birds. that a rod is produced. The percentage values for leukocytes obtained These variations and their possible relation- by Magath and Higgins (1934) for adult tame ships are presented in figure 411, in which the mallard. Anas platyrhynchos L., differ somewhat progression is from the homogeneous sphere, from tliose given in table 18. Like Hewitt, he stage A, to the development of an enclosed tetrad found that polymorphonuclears with granules of granules, B, which in ducks may pull apart by were more abundant than those with rods, namely, pairs to form pseudorod structures, B' and B". 24.3 and 2.1 percent, respectively. The homogeneous sphere. A, may develop into a Figures 400^02 are from the mallai-d, figures square of four granules surrounded by much 403 and 404 from the baldpate duck, and figure matrix, B, or only a little, C. Step D has never 405 from the ruddy duck. The three were se- been observed but its existence, as a transitional lected because they again illustrate the wide vari- configuration between the scattered tetrads of C ation in the appearance of the eosinophil. Fig- and the reticulum with granules at the interstices ures 403 and 404 are cells that were adjacent on of E, is assumed. Any such scheme of pro- the same slide. The rods of the heterophil are gression should agree with the development of the cigar shaped with a vacuole in the center of each cell through myelogenic stages, maturity, and rod. The spheres of the eosinophil are very aging. During myelopoiesis, the only expression large and most of them are homogeneous, but in of the specific eosinophilic granule in the chicken a few there are small granules on a reticulum; is in the form of homogeneous spheres (figs. 379- the granules are arranged in a square. 381 ) . The same was true in a case of eosinophil The type of eosinophil in figure 405 is a source myelogenous leukemia. Of the immature stages of confusion. It appears but little different from that were drawii from circulating blood (figs. 184 the heterophil in figure 401. The specific bodies and 186), the first had homogeneous spheres and of the eosinophil are present in the form of short the second a tetrad type of arrangement of small rods with granules. Sometimes these granules granules. Since the second is a more differen- are located at the end and sometimes in the middle tiated cell than the first, it might have been con- of each body. The eosinophil of the turkey (fig. cluded that the spheres were changed into gran- 407) stains almost the same color as the hetero- ules, but when figure 186, a young cell with phil rods of the baldpate duck. A clear, light- granules, is compared with figure 180, a mature staining space is present in the center of some of cell with three nuclear lobes, it could he con- the spheres. cluded equally well that the process of change Perhaps all these variations can be resolved was going in the opposite direction. into some definite overall plan. Although much Well executed color drawings of the blood study still remains, there seems to be some evi- from an African vulture have been presented by dence that the specific granule of the eosinophil Neave (1906). He illustrates three morpho- has two morphologic forms with transitional logic types of eosinophilic polymorphs, but does stages between, not only in the same species but not attempt to name them.

208 —

/ \ / \

-» i \ A B \ C D \ / \ /

Figure 411. —A diagram in which relationships have been suggested for the different types of eosinophil granules that liave been observed.

A A large homogeneous sphere seen during myelopoiesis. and B" From the type of granule in either B or C B A combination of sphere and small granules arranged elongation of the square into either a rod or oval shap, in a square (for example, see fig. 404). can be produced (fig. 405). The rod and oval forme The type of granule often seen in the eosinophil of the can be confused easily with the rods of heterophils bus chicken—four small granules in a square, joined by have no relationship. carrying lines (figs. 177, 178). The presence of a matrix around D A transition stage leading to E, a reticulum them is questionable. small granules at the interstices (fig. 402).

Figures 406-410 illustrate the granulocytes the latter method the nuclear lobes were clearly found in turkeys. The rods in the heterophil shown and were used for Arneth counts. In (fig. 406) can be identified readily for what they the chicken heterophil (fig. 203) the rods were are, but if they are much thinner than shown in completely dissolved and only the protoplasmic this cell, they take on the effect of a reticulum network remained. The same picture has been

an effect that may cause them to resemble the seen in the turkey (fig. 409) . This technic, how- fine-granule type of eosinophil. Actually, there ever, produced different effects on the eosinophils was no confusion in the identification of hetero- of these two species. In the chicken (fig. 215) phils and eosinophils on the slide from which tlie eosinophil granules are well presei-ved, but figures 406 and 407 were taken because the eosin- in the turkey (fig. 410) they are completely dis- ophil (407) was so strikingly different from the solved, with the result that heterophils and eosin- heterophil in both color and structure. This ophils appear quite similar in this species; a close again emphasizes the fact that among various examination of the two cell types in turkeys does species the tinctorial qualities of cells of the same reveal a difference. The protoplasmic frame- type are not always alike; for example, the rods work around the eosinophil granules is sharp and of the heterophil (fig. 403) are colored about the definite and the size of the spaces is equivalent to same hue as are the granules of the eosinophil the area of the large granules that fill them (fig. (fig. 407) and neither of these are greatly dif- 407). The spaces within the heterophil vary in ferent from the dark-magenta bodies of the baso- size and are irregular in shape, and the proto- phil (fig. 408). plasmic network is not sharply defined. Once Duplicate smears were made of blood taken these differences are recognized there should be from some of the species listed in table 11. One no difficulty in the separation of heterophils and was fixed dry and the other in Petrunkevitch No. eosinophils when making Arneth counts in the 2 and stained with May-Griinwald Giemsa. By turkey. 209 When smears were taken from pheasants, it pigeon. In this species the cytoplasm of throm- was noted that the blood had a surprisingly high bocytes takes a more intense coloration than it viscosity. When the drop collected on the pusher does in most avian species, and the cells are slide had been touched to the smear slide, it did nearly round. Moreover, the thrombocytes of not spread easily to the opposite corners, as does pigeons appear to disintegrate less readily when the blood of chickens and geese. Only after the smear is made than they do in most species; considerable moving around of the pusher slide thus the form of the cell is retained. This, of did the drop spread laterally to form a uniform course, aids in holding the specific granule of the column of substance. This property of the thrombocyte intact so that identification can be pheasant blood made it difficult to obtain a thin, made easily when neither lymphocytes nor throm- uniform smear. bocytes have disintegrated. If the cytosome of The erythrocytes of the species of ducks, these two cell types has been lost they can still be cuckoos and the hawk mentioned in table 11 were separated by the fact that the nucleus of the lym- larger than those of the chicken ; the erythrocytes phocyte is larger than that of the thrombocyte. of all the other species had approximately the Lymphocytes in all species of wild and domes- same size as those of the chicken. These state- tic birds examined appeared the same as in ments are based on visual comparisons and not chickens. Magenta bodies or reactive lympho- on measurements. Wintrobe (1933) has given cytes, or both, were found in nine species, includ- the length and width of erythrocytes for the ing a species of duck. chicken, guinea, goose, and pigeon. A further Monocytes in other species are the same as discussion of the size of erythrocytes in the found in the chicken. Those of the great horned chicken will be given when table 12 is considered. owl were large with round nuclei and large, dense Immature stages of development and erythro- chromatin clumps. plastids of all sizes were found in many of the Heterophils were variable within an individual species of wild birds; they were especially or a species, as they were in chickens. Differ- numerous in some of the slides from ducks and ences in the shape of the rods in ducks have al- from the indigo bunting. In mature cells, the ready been mentioned; in turkeys the I'ods dis- nucleus was slender and rodlike with dense chro- solve readily as they do in chicken cells; in the matin clumps like figure 5. In slides from some owl the rods were of the "typical" type, pointed specimens of mallard ducks the nuclei were so at both ends. In other species of birds, as well contracted and slender that the dense chromatin as in the chicken, central bodies exist inside the clumps bulged outward, giving it a mulberry ap- rods, but their occurrence is not constant. pearance. Scarcely any spaces could be seen be- The eosinophils for some ducks have been pic- tween the clumps. tured. Figure 402 represents the granular type, Smears from ducks and pigeons showed eryth- figure 404 the large sphere type, and figure 405 rocytes that were distributed in pairs and in a the rod type; in figure 411 these have been dia- crossed position. With each erythrocyte in a gramed as E, B, and B', respectively. In the pair crossing the other at 90 degrees, and with pintail duck the specific bodies are spheres and the centers coinciding, a pinwheel effect was pro- vary in size from medium to large; in the green- duced. This peculiarity has never been ob- winged teal, rods and granules are mixed; and served in chicken blood. in the shoveller duck small granules are clumped The thrombocytes in the shoveller duck, tur- to form rods. The Canada goose and the owl key, pheasant, pigeon, dove, owl, black-capped have small granules, and the pheasant and the chickadee, cuckoo, and white-breasted nuthatch dove have large ones. In many of the passerines were larger than those in the chicken. Except they are like those in the chicken but in the red- for these differences in size, the thrombocytes in eyed vireo and purple finch they have fine gi'an- all the species listed in table 11 were similar to ules and in the robin the bodies aie almost re- those of the chicken. The specific granules of fractile. the turkey thrombocyte were usually in vacuoles. Basophils in other avian species appear as they There is always a possibility that thrombocytes do in the chicken except that the cells of the tur- will be confused with lymphocytes. Confusion key are larger and those of the owl are smaller. is especially likely to occur in the case of the The nucleus of the pigeon basophil is often ec-

210 centiically placed so that the granules lie at one lar set of measurements ; for example, the erythro- side of the cell. This cell in the mallard duck cyte nucleus of the first Columbian Plymouth of the sec- is extremely susceptihle to aqueous stains; even Rock was 18 percent longer than that Wright's stain dissolves most of the granules and ond one. These data raise again the questions, when this happens so that the cell contains only Wliat is the form of a typical erythrocyte? Are a few granules, the nucleus becomes more in- the rounded cells with oval leptochromatic nuclei tensely stained and the cell closely resembles a less mature than the longer cells with rodlike lymphocyte with magenta bodies. Often it is pachychromatic nuclei? only the pinkish ground color of the basophil Keller (1933) studied cell and nuclear size cytoplasm and the blue of the lymphocyte that in dwarf and normal breeds of chickens. In em- distinguishes one cell from the other. Both May- bryos, during the incubation period of 4 to 8 Griinwald Giemsa and MacNeil's tetrachrome days, there was no significant difference in stains dissolve the cytosome and its granules to lengths of erythrocytes. At hatching, the aver- such an extent that basophils could not be found age dimensions of length and width for the dwarf in smears following these stains. breed was 8.05 x 3.88m and for the breed of normal size, 7.51 X 3.77m. In the grown birds, the dwarf showed 7.84 x 4.19m and the chicken of normal size, 8.19 x 4.50m. It was concluded SIZE OF CELLS that the size of the breed had no influence on the size of the erythrocytes. It should be noted that Graphs in chapter 2 (figs. 89, 152, 153, and the average values given in her work are in every 197) give the average sizes of thrombocytes, case less than the minimum of the range given lymphocytes, monocytes, and the three granulo- in table 12. cytes. With the possible exception of the cui-ves Kitaeva (1939) also studied the size of for the granulocytes, they are unimodal. Ac- erythrocytes from three European breeds of tually the data for these graphs represented a chickens—Langshans, Brown Leghorns, and Ben- composite from four sources: thams. The differences were slight. Averages all adult birds were, Stock from the U. S. Regional Poultry Research Lab- computed from his data on oratory: length 11 .Om and width 6.6m. These values fall 6 Single Comb White Leghorns, line —relatively within the ranges given in table 12. resistant to lymphomatosis. An extensive study of cell size in relation to Single Comb White Leghorns, line 15— relatively on 11 susceptible to lymphomatosis. body weight was made by Melmer (1938) Stock from a commercial breeder: breeds of chickens. He used epithelial cells, New Hampshires. striated muscle cells, and erythrocytes. The Columbian Plymouth Rocks. bird weights varied from 335 to 2900 grams. Slides were taken from individuals of each group The area of the erythrocytes varied from 62.3 to and the various cell types were measured. 77.2 sq. M and, in spite of considerable vari- The most challenging data came from differ- ability, he obtained a correlation of — 0.54±0.08 ences in size of erythrocytes. The length and between body weight and erythrocyte area. He width of the cytosomes and nuclei of 25 cells studied size of erythrocytes in the White Leghorn from each bird were measured. The ranges and from 1 day of age to 54 montlis of age. Wlien the averages are given in table 12. The average dividing the series into those younger than 9 length of the erythrocyte for our stock was 10.6m, months and those older than 9 months, he ob-

about ; the nucleus was and the width was 6.6m tained an average erythrocyte area of 70.0 sq. m 4.1 X There was little difference between 3.0m. for the former and 66.5 sq. m for the latter. lines 6 and 15. Both breeds of chickens pro- Kalabukhov and Rodionov (1934), who were cured from commercial sources had erythrocytes interested in the problem of changes in the blood that averaged about 1.6m longer and the nuclei with age, gave the following figures, based on the were about 1.0m longer, but the widths of each sparrows Passer montanus L. and P. domesticus were the same as for the smaller cells from our altricial young: age 1-5 days, birds. Even among the individuals of a group L., which have erythrocytes there may be considerable difference in a particu- hemoglobin 4.0 percent, number of 211 . .

Table 12.—Dimension of erythrocytes

Cytosome

Length Width Breed Source ' Bird 2

Range Aver- Range age M

Single Comb While Leg- RPL6. 9.6-11.8 10.8 6. 2-8.

horns. 10. 0-11. 6 10.8 .5. 9-7. 7 9. 7-11. 8 10.7 6. 0-7. 7

Average for group . . . 10.8 Single Comb White Leg- RPL 15 9. 2-11. 4 10.6 6. horns. 9.4-11.6 10.3 9. 3-11. 6 10.4 Average for group 10.4

New Hampshires . . . . -commercial 10. 9-14. 12.4 10. 6-13. 4 12.1 10. 9-13. 2 12.0

Average for group . . 12. 2 Columbian Plymouth Commercial 11.0-13.4 12! 4 Rocks. 11.2-12.9 12.0 10. 9-14. 2 12.6

Average for group . , 12.3 . . .

Table 13.—Dimensions of thrombocytes

Cytosome

Length Width Breed Source 1 Bird 2

Range Average Range m ,

1-4. 6. 7. 0- 8. 5 7.8 3. 6 Single Comb Wliite Leghorns . RPL 6. 6-10. 5 8.8 3. 3-6. 1 7. 0- 8. 9 7.8 4. 3-5. 9 Average for group 8.1 3. Single Corub Wbite Leghorns RPL 15. 7. 7- 9. 9 8.6 6. 7- 9. 5 7.7 6. 1- 9. 7.1 Average for group. 7.8 Commercial 8. 9-10. 8 9.8 New llanipsbires . . . 7. 7-11. 5 8.9 7. 8-10. 4 9.0 Average for group 9.2 Cohiinbian Plymouth Rocks. Commercial 8. 3-10. 1 9.2 6. 8-10. 9 8.6 7. 4- 9. 5 8.5 Average for group 8.8 .. — —

shii'es was definitely smaller 35.6m'. The New Hampshires. The data for the eosinophils same difference in size for this breed is reflected, emphasize a point that was brought out when the as would be expected, in the nuclear area and in eosinophil of circulating blood was discussed the diameters of the cells and nuclei. If the so often an individual chicken showed almost ex- lymphocytes and their nuclei when flattened had clusively a large or a small type of cell. This is been circular and the nucleus had been in the evident in table 16, where maximum size was center, the cytoplasm would have formed a rim 6.0m for bird 6 of RPL line 1.5, but the minimum around it hardly more than a half micron in for bird 4 was greater—6.4^. Almost the same width. situation existed in birds 4 and 6 of RPL line 6, The striking difference in size and nucleocell and in 1 and 4 of the Columbian Plymouth Rock. ratios of lymphocytes and of monocytes is well The average for the basophil diameter of 7.8m brought out by comparing the data of table 14 for Laboratoi-y birds is less than 8.1 and 9.1m with those of table 15. The monocyte area for for chickens from commercial sources, but the both lines of Laboratory stock was large, and for samples are probably too small to have much both the commercial lines it was small. In other significance in view of ranges in diameter as words, in 3 of the 4 groups lymphocyte and mono- great as 4.9 to 10.9m within a liird and rather cyte areas appeared to be positively correlated, wide variability in the averages among groups but in the Columbian Plymouth Rocks the as- of birds. sociation was negative. The area of the monocyte nucleus is approximately half the area of the total cell or, comparing nucleus with cytosome, the ratio is approximately 1: 1.1 in contrast to CELL COUNTS, HEMOGLOBIN LEVELS, the lymphocyte, where the ratio is approximately AND HEMATOCRITS 1:0.5. Heterophils show a greater variability among An important function of an atlas on blood is birds within a group than among groups (table to aid in cell identification so that accurate dif- 16) and from tliis there is probably relatively lit- ferential counts can be made. A review of tle significance to the difference in size from 7.9 m earlier cell counts in some cases, as well as tabu- diameter for RPL line 6 birds and 9.9m for the lation of new data of their own has been given by

Table 15.—Area and diameter of monocytes

Cytosome

2 Breed Bird Diameter fi

Range Aver- Range age

Single Comb While Leg- RPL 6. 107. 9-151. 5 129.0 11.7-15.9 horns. 102. 7-151. 5 125.4 11.4-15.9 97. 6-146. 4 124.2 11.1-13.6

Average for group . . . 126.2 Single Comb White Leg- RPL 15. 84. 7-146. 4 120.7 horns. 105. 3-226. 151.9 87. 3-138. 7 113.2 Average for group 128.6

New Hampshires. . . . Commercial 66. 8-179. 8 100.3 77. 0-169. 5 107.7 53. 1-136. 1 101. 1

Average for group . . 103.0 Columbian Plymouth jommercial 61. 6-148. 9 93.4 Rocks. 51.4-133.5 95.7 71.9-143.8 114. 7 Average for group 101.3 . . ' 1

Table 16.—Diameter of granulocytes

Heterophil Eosinophil Basophil

Breed Source Bird = Range Aver- Range Aver- Range age M age

2- 5. 3- 6. 8 6.1 6. Single Comb White Leghorns RPL6. 6. 7. 8 6.8 6. 0-10. 7.6 5. 5- 8. 8 6.7 8. 8-10. 1 9.4 6. 7-10. 9 8.3 7.0 Average for group 7.9 4-10. 8.0 Single Comb White Leghorns. RPL 15. 8. 6-10. 6 9.4 6. 5. 1- 9. 2 7.6 5. 7- 7. 6 6.6 6.6- 7.9 7.2 4. 8- 6. 5.4 6.7 Average for group. 8.1 Commercial. 9.8-11.4 10.6 6. 3- 8. 5 7.8 New Hanipshires. . . 8. 2-10. 5 9.4 6. 1- 8. 3 7.1 8. 4-10. 5 9.7 7. 5- 9. 4 8.4 7.8 Average for group 9.9 Columbian Plvmouth Rocks. Commercial 6. 5-10. 3 8.9 6.3-^ 7.8 7. 4- 8. 4 7.9 6.2- 7.3 6.7 9. 1-10. 6 9.6. 8.5- 10. 1 9.3 7.9 Average for group. .

Table 17.—Normal blood values for chickens

RPL 1 stock—Single Comb White Leghorns Farm stock

Blood component Female Single Comb Rhode Island Male adult White Leg- Reds female horns female — — adult 6 weeks 12 weeks Adult adult

Erythrocytes millions/mm.^ 3.02 3.02 3.00 3.78 2.96 2.88 Hemoglobin gms./lOO cc. 10. 10 9.80 9.70 13. 50 10.70 11.00 Hematocrit percent 30.90 30.40 30.80 40.00 31.90 30.80 Buffy coat percent 1.00 1.00 1. 00 .80 Thrombocytes mm.' 30. 457 26, 254 30, 856 27, 586 37,211 60,311 Total white cells mm.^ 28,612 31,256 29, 397 16, 615 28, 863 35, 787

Num- Num- Num - Num- Num- Num- ber Per. ber Per- ber Per- ber Per- ber Per- ber Per- per cent per cent per cent per cent per cent per cent cu. mm. cu. mm. cu. mm. cu. mm. cu. mm. cu. mm.

Lymphocytes 23, 328 81.5 24, 310 77.8 22,371 76.1 10, 626 64.0 20, 704 71.7 20, 794 58.1

Monocytes. . 1,286 4.5 1,542 4.9 1,663 5.7 1,065 6.4 326 1.1 880 2.5

Heterophils . . 2,898 10.1 3,654 11.7 3,917 13.3 4,288 25.8 6,831 23.7 12,551 35.1

Eosinophils . . 438 1.5 1,210 3.9 728 2.5 241 L4 410 1.4 440 1.2

Basophils . . . 662 2.3 540 1.7 718 2.4 395 2.4 592 2.1 1,121 3.1

RPL = stock from the L'. S. Regional Poultry Research Laboratory.

1 How much value is there in blood determi- This is evident not only in chickens but also nations based on a single bird? in other species of birds. Cook and Dearstyne 2. How many birds must be included in one (1934) arrived at an estimate of the importance population to give mean values that are signifi- of the value given by a single count by grouping cantly different? all counts into classes in a frequency distribution At present the opinion is held that some types table. This method is more informative than of blood values, for a single bird, offer rela- either an average or a range. tively little information about the health of that Some types of blood data on chickens gave a particular bird. This is particularly true for narrow range in values for the normal popula- total white cell and differential counts. tion. Under such conditions, a wide departure

Table 18.—Blood values for common mallard duck (adult males)

[Differential counts in percent]

Birdi from the average, even by a single bird, may indicate ill health. A shift of probably as little as a half million erythrocytes per cubic millimeter could be significant. Likewise, hemoglobin values show a relatively narrow range of variability, and a difference of as little as 1 gram per 100 cc. might be viewed with suspicion. Hematocrit values do not as a rule vary more than about 5 percent either way. The average hematocrit values in table 17 are only 1 to 2 percent higher than those given by Hamre and Mc- Henry (1942a) except for the males. Buffy coats were read in the Van Allen hematocrit tubes with a low-magnification hand lens and as long as there was no hemolysis the normal values usually did not vary more than ±0.2 percent. On the other hand, thrombocytes, total white cells, and individual cell types often varied from half to twice the average, and thus individual readings do not mean much. This was found to be the case whether the comparison was among different individuals or among repeated bleed- ings from the same bird. Palmer and Biely (1935a) studied the variability of cell counts in great statistical detail and concluded that when careful attention has been given to technics the fluctuations in normal erythrocytes can be reduced to 15 percent. In their data, the coefficient of variability was generally quite low. This brings us to the second question. An an-

Tahle 20.—Blood values for ring-necked pheasant (adult males)

[Differential counts in percent]

Bird I —

These calculations were based on normal adult male chickens showed a higher percentage value female Single Comb White Leghorn chickens. for lymphocytes than did male chickens. The It is evident, thei-efore, that the desirable number reverse was true for heterophils. of birds to be used in an experimental group is A higher erythrocyte number for adult males determined by the blood component that is of than for adult females agrees with the work of particular concern the to problem at hand. Juhn and Domm ( 1930) . Before maturity there Fewer birds would be needed in each group if was no difference between the sexes. The aver- red-cell counts were of chief concern than if age values given for males of 3,600,000 and for eosinophils were to be followed critically. The females of 2,700,000 given by Taber et al. high variability for some of the blood com- (1943) agree fairly well with those given in table ponents makes it necessary for practical reasons 17. The averages computed from Kitaeva's data to accept high coefficients of variability in order (1939) for adult birds were 3.40 million eryth- that the number of birds involved in each experi- rocytes per mm.^ for males and 2.92 million mental group can be small enough to make the per mm.^ for females. Domm and Taber (1946) experiment practical. obtained an average erythrocyte count for males Another problem studied by Lucas and Den- of 3,250,000 and for females of 2,610,000. ington (unpublished data) has been the number They sought to determine if a diurnal rhythm for of cells that should be tabulated from a slide erythrocytes in the circulating blood existed in when making a differential count. The accuracy chickens, comparable to that which had been increases by the square root of the multiples of found in some mammals. They took their cells 100 counted. In other words, 400 cells samples at noon, 6 p. m., midnight, and 6 a. m. give values that are twice as accurate as when They found a definite tendency in males to give 100 cells are counted, and 900 cells give values highest values at midnight and lowest at noon. three times as accurate as when 100 cells are The same tendencies were evident in females counted. also, but the difference in averages at these two The question arises, When is the point of di- times of the day was not as great in females as minishing returns in accuracy reached for the in males. Domm and Taber found a seasonal time spent in making the counts? In the studies variation in erythrocyte counts; the lowest counts made by Lucas and Denington, it was found that came at the period of highest reproductive ac- 100 cells were sufficient for lymphocytes and tivity and the highest counts at the time of lowest heterophils; but for monocytes, eosinophils, and activity. See also Domm, et al. (1943). basophils, 292 cells were needed to give the Kakara and Kawasima (1939) found that maximum accuracy for the time spent in making birds sitting on eggs had a lower red-cell count, the counts. In making the counts, 300 cells lower thrombocyte count, and lower total white were used. Lucas and Denington found also cells than did laying hens. birds in that the number of used an experiment Chickens moved to a high altitude, 6,000 feet, can be reduced if the number of cells counted showed a slight increase in hemoglobin and eryth- per bird is increased. The gain comes chiefly in rocyte count, according to Vezzani (1939). those components having high variability blood An extensive study of erythrocyte numbers for especially so when the variability within birds many species of birds was made by Nice et al. is as great as the variability between birds. (1935). Counts on wild birds ranged from In the there same experiment were males from 3,930,000 (tufted titmouse) to 7,645,000 the same source and they were killed at about (junco). The median count was 5,230,000. All 550 days of age. Thirty-three individuals went of the counts are higher than the average for into the averages presented in table 17. Labora- chickens. In the bobwhite, a gallinaceous bird, tory males showed higher values than Laboratory the average was 3,532,000. Erythrocyte counts females for number of erythrocytes, grams of and hemoglobin determinations on pigeons and hemoglobin, and volume of packed cells (hema- on doves by Riddle and Braucher (1934) gave tocrit percentage). The total white-cell count higher values for males than for females. They was lower for males than for females. The sexes observed seasonal differences also, with the high- did not differ widely in the percentage values est values occurring in the autumn and lowest for monocytes, eosinophils, and basophils. Fe- values in the summer.

218 —

Venzlaff (1911) made erythrocyte counts from only generalization that can be made is that these 45 species of birds. In collecting his material data further emphasize that there is high varia- he attempted to obtain a reasonably uniform bility among different groups of chickens. representation of most of the families of birds Cell counts on pigeons, more extensive than from the Struthioniformes to the Passeriformes. given here (table 21), were made by DeEds Body weight and size of the erythrocytes were (1927). He found a very high variability for given, also, but the interrelationship of these the counts of each cell type; for example, small variables is still open to question. lymphocytes varied from 5 to 53 percent, large Percentage values are often misleading—^the lymphocytes from 9 to 67 percent, heterophils real differences between sexes are seen more from to 25 percent and the remaining cell types clearly in the data giving the number of cells per showed a similar variability. Thrombocytes cubic millimeter (table 17). The Rhode varied from 8,000 to 89,000 per mm.' Island Reds had a high thrombocyte count, Less variability was experienced in a later and a slightly elevated total white-cell count in study on pigeon blood cells by Schoger (1939). comparison with the others. The monocytes of In his data, lymphocytes varied from 40.5 to both farm stocks were low both in aljsolute and 62.0 percent, monocytes from 4.0 to 6.5 percent, in percentage values. This may be the reason heterophils from 29.0 to 48.5 percent, eosino- why some investigators group monocytes with phils from 2 to 4 percent, and basophils from lymphocytes in their differential counts. Hetero- 0.5 to 2.0 percent. He used 16 mature, healthy phils were even more variable than lymphocytes, birds. The variability in his data was less than ranging from 10 percent to 25 percent, and on shown in table 21. Red cell counts, hemo- the basis of absolute numbers per cubic milli- globin, and differential counts for leukocytes meter, the differences are even greater—2,900 were made by Gauger et al. (1940) on normal with para- to 12,600—over a fourfold difference. The pigeons and on pigeons infected range in average number for the lymphocyte typhoid. Their counts on both normal and in- groups was slightly over twofold. fected birds were also highly variable. They The low heterophil count for our birds may concluded that, due to this variability, chronic be dis- be due to the fact that these chickens are held carriers of this pathogen could not indoors throughout their lives and are relatively tinguislied from noninfected pigeons by blood- free from parasites and common poultry in- cell counts. fections except lymphomatosis. The count was The values given by Hewitt (1942) of dif- (breed not the highest for Rhode Island Reds, yet in all ferential counts on laboratory ducks with our averages in table groups of chickens it was definitely less than in given) agree closely fact based on common mallards. Hewitt's aver- wild birds (tables 18, 19, 20, and 21) . In 18, percent; mono- there are several points of difference between ages were: Lymphocytes, 40.4 percent; the percentage values of leukocyte types in cytes, 5.3 percent; heterophils, 44.4 2.4 per- chickens and wild birds, the most striking being eosinophils, 7.1 percent; and basophils, counts for tur- the low value for lymphocytes, the high value cent. Normal differential blood Lange for heterophils, and a consistently high level for keys, as given by Johnson and (1939), monocytes, 1.9 monocytes. Eosinophils in the Canada goose are: Lymphocytes, 50.6 percent; percent; eosinophils, and the mallard duck ran 7 percent and in pigeon percent; heterophils, 43.4 percent. Mc- and pheasant the averages were low. Basophils 0.9 percent; and basophils, 3.2 gave the counts for ranged from 2 to 10 percent. Any comparison Guire and Cavett (1952) blood in cells per mm.' of these tables with the data on the chicken indi- normal values of turkey leukocytes, 38,700; lympho- cates that absolute values have greater usefulness as follows: Total heterophils, than percentage values, and any extensive studies cytes, 17,200; monocytes, 1,900; on comparative avian hematology should include 16,600; eosinophils, 40; and basophils, 1,700. the same percentage data on the actual number of cells per cubic These data give essentially and Lange. A re- millimeter. values as obtained by Johnson blood values for The values on counts made by Wickware view of earlier literature on Magath and Higgins (1934). (1947) should be compared with those given in ducks is given by for other anseriform spe- table 17 for the leukocyte types. About the They also determined 219 .

cies, the size of erythrocytes and the number per heterophils and eosinophils no more than 5 lobes cubic millimeter. were found. Likewise, no more than 5 were Perhaps some of the variability in heterophil found for turkeys (table 22), pheasants (table

count on pigeons experienced by different in- 23) and geese (table 24) . The index values for vestigators was due to diurnal rhythm, the ex- male turkeys ranged from 1.75 to 2.16, with an

istence of which was worked out by Shaw ( 1933) average of 1.95; for females, it was from 1.65 to

He found that heterophil counts on the average 2.45, with an average of 2.09. For pheasants it were 76 percent higher in the afternoon than in was from 1.84 to 2.58, with an average of 2.27. the morning. The afternoon rise for 7 birds Only two slides were obtained from the geese; was: 1, no change; 2, rise of 106 percent; 3, rise they read 2.06 and 2.38. Counts were not made of 38 percent; 4, rise of 55 percent; 5, rise of on eosinophils for any species except the Canada 143 percent; 6, rise of 54 percent; and 7, rise goose —after considerable searching 16 cells were of 138 percent. found on each slide. The indices were 3.75 and 4.25; thus there is a preponderance of cells with 3, 4, and 5 lobes—a situation that thus far has ARNETH COUNTS not been observed either in eosinophils or in heterophils for this or any other species studied. Arneth counts were given for a group of Sugiyama (1938) gave Arneth counts for 8 species of birds. His data for the chickens discussed in chapter 2, page 85; and in domestic

Table 22.—Domestic turkey (White Holland): Table 23.—Rin^-necked pheasant (adult Arneth counts on heterophils males) : Arneth counts on heterophils

Bird The found in the pigeons he studied. However, the chicken have already been reported ( p. 85) . different. values for the remaining 7 are given here, since breakdown into classes was somewhat I, percent; II, 42 percent; many libraries do not have the journal. The Shaw found: Class 33 2 percent. Shaw also classes are indicated by Roman numerals, fol- III, 23 percent; and IV, owl, and the percentages in the various lov^'ed by a figure that is the percentage having studied an follows: I, 34; II, 48; HI, 18; this number of nuclear lobes. At the end of the classes were as index was 1.84. This was some- series, the mean or index is given by an italicized IV, 0. The by number: what lower than found in the owl studied Sugiyama. Cockatoo: I, 36.0; II, 60.0; III, 4.0; 1.68 Shaw compared the Arneth counts of the bone Ouail: I. 27.0; II, 59.0; III, 13.5; IV, 0.5; 1.88 marrow with the counts of the blood of pigeons. Sparrow: I, 26.0; II, 60.0; III, 12.5; IV, 1.5; 1.90 three specimens were as Swallow: I, 12.5; II. 65.5; III, 22.0; 2.70 The averages based on Pigeon: I, 20.5; II, 63.0: III, 15.5; IV, 1.0; 1.97 follows: Owl: I, 20.0; II, 48.0; III, 29.0; IV, 3.0; 2.15 Blood: I, 36, II, 41; III, 22; IV, 1; 1.87 Bunting: I, 14.0; II, 57.0; III, 23.0; IV, 6.0; 2.21 Bone marrow: I, 75; II, 30; III, 5; IV, 0; 1.30 Perhaps the first investigator to compile It seems odd that during the twenty years, and Arneth counts on birds was Shaw (1933) in his more, since the publications of Sugiyama and study on pigeons. He took blood samples from of Shaw on Arneth counts for normal birds, this April through September. The index varied technic, so common in the mammalian field, has from 1.76 to 2.11 but there was no seasonal not applied to the study of blood diseases trend. The average index for the 6-month period been was 1.95, which is almost the same as Sugiyama in birds.

221 CHAPTER 7

Technics for Avian Blood

Lack of satisfactory technics is one of the rea- his own, the small structural and tinctorial dif- sons why studies of avian blood have not been ferences depicted here should not conclude that carried forward as energetically and successfully they do not exist; he should consider the possi- as have studies of mammalian blood. Students bility that he is not working under optimum con- of avian hematology have found that they fre- ditions. quently get unsatisfactory results when they at- The information that follows was obtained tempt to apply technics that are known to be suit- from the late Dr. Max Poser of the home office able for studying the blood of mammalian species. of Bausch & Lomb Optical Co. and from Mr. H. Over a period of years this Laboratory has L. Shippy of the Detroit office of that company. modified a number of commonly used technics to Similar information has been presented by Dr. suit the needs of avian hematology. Oscar W. Richards in Color and Illumination, published by the Spencer Lens Co. Two other useful reference items by Dr. THE MICROSCOPE AND LIGHT Richards will be found in Literature Cited (Richards, 1938 and 1949). Another reference (Spitta, 1920) has Perhaps the greatest deterrents to accurate and provided particularly helpful explanations of the critical study are ( 1 ) lack of a good microscope, differences between achromatic and apochro- (2) lack of a good light source, and (3) deficien- matic lenses, and of why Huyghenian eyepieces cies in setting up and using microscope and light. should he used with the former and compensating Here is what one often finds in a laboratory: oculars with the latter. The "good" microscope is tucked away in a box. An ideal light source is a small, brilliant point When a special occasion arises, it is brought of light that is passed through a lens designed to forth and placed on a table close to a 75- or 100- produce parallel or nearly parallel rays. All watt bulb or a lamp with a frosted glass in front. the drawings in this Atlas were made with a tung- Then the condenser is dropped below the level of sten arc light. the stage until the amount of light is right, or the The lamp should be placed about 2 feet in diaphragm is closed so that the object shows up front of the microscope and the image of the light well. source brought to a focus on a white card placed For a quick look with low magnification, such in front of the microscope mirror. Adjustments procedure may be satisfactory, but efforts are in focus can be made by moving the lamp con- often made to do critical studies under an oil im- denser in and out. Just in front of the con- mersion lens with this type of set-up. The efforts denser lens of the lamp is a leaf diaphragm, which are disappointing because the microscope is pre- should l>e wide open at this stage of setting up vented from giving top-quality perfomiance. the lamp and microscope. The next step is to Many good books have been written on the place a clear blue daylight filter in one of the use of the microscope. Nevertheless it is not three slots in front of the lamp diaphragm. The uncommon to see research workers, technicians, slots are constructed to hold 2" x 2" filters. One veterinarians, and physicians using the instru- may purchase a good lamp capable of giving ment as if there were no directions. Correct use critical or Kohler illumination,' then nullify its is emphasized here not only because the authors value by placing a ground glass in the path of the hope that their comments may be helpful to other light. workers, but also because it is desired to assure

the reader that the structures ' depicted in the il- The essential differences between critical and Kijhler illustrations have actually been seen in specimens. lumination are given by Richards ( 1954) in a booklet accompanying each research microscope when purchased. This The reader who fails to locate, in specimens of booklet also contains a helpful bibliography. 222 —.

readjust the mir- In order to do critical microscopic work, one center of the field. If it is not, the microscope, should have a box of neutral intensity filters, ror, look through the open tube of pinhole eyepiece. graded 0.3, 0.6, 0.9, and 1.2. These numbers and check again with the Wiien, finally, the condenser seems to be in per- represent the logarithms of the opacity ; each filter it be checked by moving the has double the opacity of the one preceding it. fect alignment, can down. The circle of light will en- By placing falters, one can reduce the light until tube up and and below the focal point. If these there is no danger of eye injury. Place a pre- large above on the same axis within the pared slide on the stage of the microscope. Ad- two light cones remain has been completed. But if the just the flat mirror so that, when the low-power tube the task side of the tube axis as objective focuses on the specimen, the light falls light beam shifts to one and down, the adjustment directly in the center of the microscope field. To the tube is moved up should be repeated. Books by Bell- ascertain that it is centered, close the lamp dia- procedure and by Beck (1938) should be con- phragm as far as it will go and then elevate the ing (1930) on testing the align- microscope condenser until the opening of the sulted for further refinements lamp diaphragm appears as a sharp ring. With ment of the condenser. After the tests on alignment have been com- the flat glass mirror usually present on research eyepiece and open the lamp microscopes, there will be three circles instead of pleted, put in the all the way. It probably has already one. Critical work can be carried out with the diaphragm the field of light from the lamp glass mirror but for these studies on avian blood, been noted that only a small circle in the center of this mirror was replaced with a flat, front-sur- illuminates that the marginal half faced mirror, which eliminated all images except the low-power field and the field is dark. The tendency one. or two-thirds of condenser until the field After manipulating the mirror to bring the light of many is to drop the is fully lighted. Doing this is unobjectionable to the center of the field —or to what appears to be if you wish only to examine large masses in the the center—and after focusing the condenser to objectionable if you are interested the level of the specimen, check to see that the slide, but it is The correct procedure for filling the circle of light really is in the center of the field. in details. low-power field with light will be given after ad- This is the time to ask the question. Is the con- to the use of the oil immersion denser perfectly in line with the optical axis of justments relating been described. the objective, tube, and eyepiece? To find the lens have the microscope condenser ele- answer, proceed as follows: Starting with the edge of the lamp diaphragm is Open the diaphragm on the lamp as far as it vated so that focus and wide open, shift to the ob- will go, then close the microscope diaphragm in sharp jective of next highest magnification and note all tiie way. Lift out the eyepiece and look of light is still in the center. through the tube. There will be a circle of light whether the field the same for the other objectives. If all the that does not fill the front lens of the objective. Do not show the circle of light in the center Is this circle of light in the center? It is difficult lenses do the field, at least one is not par centered. This to tell, because moving the head makes the light of should be made at the factory or with appear to move. Freedom from the uncertainty adjustment competent representative of the com- can be gained by using a pinhole eyepiece, which the help of a of the microscope work can be has a small hole in the exact center (but no lens) pany. If most with only one lens, all adjustments can be With this in place the position of the circle of done fit the particular lens. (Most of the light can be determined easily and accurately. made to work reported in this Atlas was done with the oil If it is not in the center there are two or three All the lenses screwed into screws with which adjustment can be made. The inmiersion lens.) nosepiece should be par focal: as each is screws are on most microscopes. They are not one position, only a slight movement of the on some of the less expensive student instruments. moved into fine-adjustment screen should be necessary to If an adjustment is necessary to bring the im- it into focus. Shifting lenses in this way age of light to the center of the front lens, make bring certainly is recommended for the "dry" lenses Imt it, then return the regular eyepiece to the tube. reconunended as standard procedure Now open the microscope diaphragm and close it cannot be oil immersion lenses. How to bring the the lamp diaphragm. Is the image still in the for the 223 oil immersion lens into focus is described in most and the N. A. of the objective; thus for an N. A. of the instruction booklets provided by manu- 1.3 the effective N. A. would be 1.15. facturers of microscopes. Ill selecting the most desirable filter, each After the oil immersion lens has been focused worker sliould consider tlie sensitivity of his eyes on the cells or tissues on the slide, the lamp dia- to light; he should try a number of filters and se- phragm is again closed and the mirror adjusted lect the one that enables him to study cellular slightly so that the circle of light is centered in details without suffering eye fatigue. the field. Then the lamp diaphragm is opened Do not close the microscope diaphragm or drop slowly until its margin barely passes out of the the condenser out of focus to reduce the intensity microscope field. If one goes much further a of light. Closing the diaphragm reduces the ef- flare is produced across the objects on the slides, fective N. A. and hence the resolving power, but which is disturbing. it increases the apparent refractivity of cells and Next the eyepiece lens is removed and the their parts. Sometimes individuals mistake this microscope condenser diaphragm is opened effect for what is described as "seeing the ob- slowly; this action enlarges the circle of light jects better." A worker must have considerable seen through the back lens of the objective. The experience in the correct use of the microscope movement should be stopped as soon as a point is before he realizes that he can see more with the reached where further opening of the diaphragm condenser focused and the diaphragm open. If no longer enlarges the circle. An unlighted preparations of unstained cells, either living or rim remains on the margin of the back surface dead, are to be studied satisfactorily, the dia- of the lens. This is due to the fact that air sepa- phragm must be closed nearly all the way. The rates the top of the condenser from the bottom cells are distinguished by differences in refrac- of the slide, setting a theoretical limit of a nu- tibility, and closing the diaphragm emphasizes merical aperture ( N. A. ) of 1.0 for the entire lens these differences. system. If oil is placed on the condenser and On page 223 it was said that the problem of fill- the condenser is again brought to a focus at the ing the field at low magnification with a lamp level of the objects on the slide, it will be found, giving collimated light would be discussed later. after the microscope diaphragm has been opened The procedure for the high dry (4 mm. focal still further, that the back lens of the objective length objective) lenses is the same as for the oil is fully illuminated. This will occur if the nu- immersion lenses. In general, there are lenses merical aperture of the condenser is as high as, manufactured at three numerical apertures— or higher than, the numerical aperture of oil the 0.66. 0.85, and 0.95. Each has its advantages immersion objective. If less—for example, an and disadvantages; only the N. A. 0.66 has suf- N. A. 1.25 condenser and an N. A. 1.3 ob- ficient working distance to be used with the usual jective there will still — remain a narrow dark thick cover glass of the blood-counting chamber. ring. When the 8 or 16 mm. objectives are used the Oil on the condenser and on the bottom of the light does not fill the field. If the work is not slide often makes a mess if the slide is moved critical, a quick examination of a section is suffi- around much. The use of oil between condenser cient to see whether it is flat and whether the and slide is necessary if a single cell or part of a stain and counterstains are balanced. For count- cell is to be photographed or if objects within ing objects in the sections, a frosted blue light the cell are much less than a micron in diameter. bulb in a gooseneck lamp (or similar adjustable The procedure outlined gives maximum resolv- lamp) brought close to the flat mirror of the mg power. The resolving power can be estimated by means of a monogram (Richards, microscope is adequate. If the lamp is close enough it will fill 1938). The objects usually studied in blood the field with light. Wlien cells are large enough to preclude the need for the condenser is focused, an image of the light oil between the condenser and slide. bulb appears in the field. To avoid the print-

It has been said that use of an air-space in- ing, take the light from the side of the bull) rather stead of oil gave a theoretical limit of N. A. 1.0 than from the end. The mirror should not be but Spitta (1920) suggests that in actual prac- moved when the frosted bulb is brought into posi- tice it is an average of the theoretical limit of 1.0 tion. If movement is limited to the lamp bulb 224 —

pulled apart quickly in the light from the microscope lamp behind it is flame. The ends were give a rapid taper and to keep tlie walls still in alignment when the bulb is taken away. order to pulled, a 60 angle was If critical studies are to be made under low thin. As the tubing was- portion and the un- magnification, or if photomicrographs are to be made between the thin center or so of these had taken, an ordinary light bulb is not adequate, and healed ends. After a dozen broken off sev- one returns to the arc or ribbon filament lamp or Ijeen made, the thin portion was point the taper other light source which will provide critical or eral centimeters from the where Kohler illumination. The upper and lower began. preparation of a micro- lenses of condensers on most research micro- The next step was the flame can be scopes can be separated—usually Ijy sliding or burner. Any burner will do if the glass screwing them apart. The upper element is reduced to a height of about 2 mm. A opening, was placed laid aside and the lower element is moved up or sleeve, tapered to a small microburner, and this down until the light source itself or the edge of over the metal tube of a though the flame was the diaphragm in front of it is sharply in focus gave a small flame. Even approached cautiously. While the at the level of the objects on the slide. After the small, it was tension condenser has been lowered as far as possible thin part of the tulte was heating a slight soon as the thin glass had (up to the point just short of touching the edge was exerted so that as it be pulled quickly to a thin, of the mirror) it may still be necessary to elevate softened could quickly the tip was the slide above the level of the stage by as much short tip. If pulled too stiff enough to push through as 1.5 cms., in order to bring the image of the long and thin and not embryo and, if one hesitated light source or its diaphragm into focus. With the tissues of the and the bore be- another make, the condenser is moved down close too long, the walls tliickened small. The best tips were se- to the mirror but the slide does not have to be came excessively the delicate, thin elevated above the stage in order to bring the lected from those made and off about a half centimeter light to a focus on the slide. tubing was broken thickness. Technicians accurately identified cell types from the portion of intermediate grind the end to a bevel and their developmental stages when microscopes The next step was to best provided with N. A. 1.3 apochromatic objectives tip. The method followed was not the breakage but and N. A. 1.4 condensers were furnished and there was a certain amount of — the principles set forth one can when the liglit sources were carefully aligned after studying will make the process easier. with the microscope. add refinements that highspeed motor that carried a three- The illustrations shown in this Atlas were used A small over the end of the shaft was used. during the training of the technicians. jawed chuck It was mounted vertically in a bracket and two rheostats were hooked in series to the current line. One rheostat of the correct type would delicately PROCURING BLOOD be sufficient if it could be regulated enough; the only requirement is that the motor taking Mood Preparation of caunulas for turn at a slow, constant speed. The speed was from early embryos not measured l)ut it was estimated to be 1 to 4 per second. Into the chuck was in- The problem of drawing out glass tubing to a revolutions a small spindle that carried near the end small diameter seemed on the face of it to be a serted diameter. a flat emery disk about an inch in rather simple one, but it was soon discovered emery disk was in place and rotating broken, jagged tip would not smoothly en- When the that a beneath old, slowly, a flat dish of water was lifted up ter the dorsal aorta of embiyos, 2 to 3 days the surface of it so that the disk turned just below or the tip of the heart of older embryos. The the water. With the set-up described, the in- task called for a delicate, almost microscopic tip creased friction often stopped the motor; prob- with a smooth-beveled point like that of a hypo- ably a wonn-gear reducing unit would have given dermic needle. greater power and a nearer constant speed. Pyrex tubing, 2 mm. inside diameter and The fine glass tip of the cannula was lightly 4.5 mm. outside diameter, was used. An area touched to the flat surface of the moving emery in the center was heated with an oxygen-gas 225 wheel. The first trial demonstrated that the particles of emery and glass would completely plug the small bore of the glass tip. A thin-walled flexible rubber tubing was attached to the large end of the tip. An effort was made to dislodge the particles by blowing through the tubing during the grinding process. But blowing by mouth did not keep the bore free. It was found necessary to make a connection to an air or oxygen pressure tank in order to keep a flow of air or oxygen tliat would prevent the entrance of particles into the glass tip. The progress of grinding was checked under the microscope and was considered finished when there was a beveled, smooth, sharp tip. When the grinding was finished the tubes were cleaned with alcohol and ether, and dried with air.

Method for taking blood from the dorsal aorta of the 48- to 72-hour embryo

Eggs that are presumed to be fertile are taken from the cool room where they have been held FiGUKE 412. —A tripod to hold an infrared lamp, at a temperature of about 55 degrees since they used to warm slides and to drive off moisture be- were laid. They are placed in the incubator fore the smear is made and to dry the blood with a record of the hour and date. In these after it is spread. studies the age has been taken as 3 hours less than the total time held in the incubator. When was lifted off and the filter ring was placed over the embryo has reached 48 hours, incubation the embryo, which usually adhered readily to age (51 hours actual time in the incubator), it is the surface. With fine curved scissors and for- removed, opened carefully, and slid into a bowl ceps the embryo was cut free from the yolk sac, of warm saline or Ringer's solution. Sugiyama washed in saline, and lifted to a Syracuse watch (1926) followed Sabin's suggestion and in- glass. It was then placed under a low-power creased the salt content of Locke-Lewis solution dissecting microscope. to L04-percent NaCl for an embryo on the second In the meantime clean microscope slides were day of incubation, to LO-percent for an embyro drying and warming under the infrared lamp on the third day of incubation, and to 0.9-percent (fig. 412). The slides should feel warm, not for an embryo on the fourth day of incubation hot, to the back of the hand or the cheek. A and older. These improvements in technic are flexible, thin ruljber tubing of the type used for useful if the embryos are to be held for study over taking blood in erythrocyte counts was attached a period of time. Wliere the whole procedure to the glass cannula, and under the low power can be completed within a few mimites, the use of the dissecting microscope the cannula was of 0.85-percent or 0.90-percent solution of so- guided into the dorsal aorta. The heart nmst dium chloride alone produces no ill effects. be beating and the blood flowing in order to ob- Prior to opening the embryo, rings from filter tain a satisfactory preparation. A slight posi- paper were cut. These had an outside diameter tive pressure is set up with air from the mouth slightly larger than the margin of the area vascu- as the tip approaches the moist surface of the losa, and the inner diameter was slightly less embryo. If this is not done capillary attraction than this margin. The saline was removed from tends to draw saline and embryonic fluids into the bowl until the embryo lay above the level of the tube and these fluids quickly distort the the surrounding fluid. The vitelline membrane embryo blood cells. 226 )

and this The blood was drawn quickly by suction and embryo than in the chick after hatching, influence the spread then immediately expelled from the tube onto the may be one of the factors that slide. The difficulty warmed glass slide. As the blood leaves the of the blood over the glass circumvented by placing the cannula the tip is moved back and forth so as to usually can be barely touching distribute the cells and prevent them from piling pusher slide at a low angle, by slide, and by giving it one up in heaps. If there is any delay in entering it against the smear opposite end. the aorta, in taking the blood, in making the quick movement to the preparation, or in drying it, the cells will be distorted. This is especially likely to happen if fluids have entered the cannula contaminating The infrared lamp and its use in drying them. Efforts ahead of the cells or along with and uarming slides to prevent distortion of cells by other means were bulb vertically made. Heparin and silicone coatings over the A tripod carrying an infrared used for many inside of the cannulas were tried. Neither suspended (fig. 412) has been method gave improvement over careful, rapid years in this Laboratory in making blood smears. were made from 1/^-inch use of a clean, dry tube. Essentially the same The legs and braces procedure was used for embryos a day older. wires welded together. On top was a flat tri- with a hole large enough The filter-paper 'rings used to hold the embryo angle of sheet metal infrared bulb was were slightly larger than those for 48-hour to receive a light socket. An or embryos. placed in the socket. Clean paper toweling cloth was spread on a table beneath the bulb, and on this the slides were stacked. Usually the slides were stacked around the margin of the cir- the heart Method of taking blood from of cle of light and then about a dozen were spread hours and older embryos of 96 under the light. from the lamp drives the moisture The same type of cannula was used as described The heat from the slides, and when the smear is made the previously except that it had a slightly larger film of cells dries quickly. It was necessary to bore. A cannula with a still larger bore was slides close to the point where used when embryo age increased. A different have the warmed they were being used; otherwise, the slide cooled technic was used to procure blood from the sec- before the film was spread. The pusher slides ond week of incubation to hatching. should not be heated. If the slide is too hot, After S to 6 days of incubation the procedure artifacts will appear. They will be similar to of moving the tip of the cannula back and forth those that appear in erythrocytes (see chapter across the slide, as the blood was being expelled,

. After the smear is made the slides may be was discontinued and a new procedure was be- 2 ) retuined to the lamp for complete drying of the gun—a drop of blood was placed on the end of the cells or they be put directly into a box. In smear slide and distributed with a pusher slide. may either case, they are gently heated again just be- In still older embryos, the tip of the heart was fore staining in order to drive off any moisture cut open and the drop collected directly onto the have accumulated when the slides were end of a pusher slide, but in doing this there must that may be freedom from fluids around the heart, and cooled. the heart should be elevated above the surrounding tissue. At about mid-embryonic age of in- this drop of blood will often be carried cubation Method for collecting and carrying blood across from one end of the slide to the other and samples will leave only a few scattered cells over the sur- have described face of the slide. The ways in which the physical Denington and Lucas (1955) carried con- properties of the blood at this age differ from a box in which 15 units can be

. tray hold- those that obtain just before hatching and after veniently and safely (fig. 413) The pipettes is removable. Upon re- hatching is not known, but obviously the blood ing the red-cell turning to the laboratory, the worker can separate has a poor affinity for glass. Schechtman ( 1952 and place showed that the surface tension was less in the the tray from the rest of the equipment 227 ^

Figure 413. —A box and cover suitat)le for carrying 15 tubes for the hemoglobin test, tlie same number of Van Allen hematocrit tubes, and red-cell pipettes. The tray holding the lf,st of these can be lifted out and stored in the refrigerator until they are ready for counting. Front compartments carry hemoglobin pipettes, knife, cotton bats, and rubber tubes for drawing up blood and fluids.

it in the refrigerator, where it remains until other Denington and Lucas (195.5). When dried procedures involving the hemoglobin and the Van- powder was used, 3.3 grams were added to a Allen hematocrit tubes have been completed. 500-cc. bottle of pure methyl alcohol, freshly The box with clean slides and the infrared lamp opened. This quantity of stain is approximately are separate items; they are not included in the double the cpiantity used in making Wright's

box used for collecting blood samples. stain for human-blood studies. The stain is ripened for several months either at room temperature or in the incubator at 38 to 40 degrees STAINING centigrade A great many samples of dye, both from pow- Selection, preparation, and use tFright^s of ders made up into solution at the Laboratory and stain from liottles of the dye that had been put into solution ])y a supply house, were tested. Some Solutions containing dried Wright's powder gave too red a color to the cytosomes of erythro- have been made at the Laboratory and used; cytes and gave pale, incompletely stained nuclei, commercially prepared solutions have also been and others stained the nuclei very intensely but used. The difficulty in finding a Wright's stain gave a pale bluish color to the cytosomes of suitable for avian blood has been discussed by erythrocytes. The color balance of other cell 228 types was also effected. It was sometimes pos- ing methods. Stainless steel racks that hold 25 steel trays sible to take a solution that produced too much to 100 slides are used, and 2 stainless covers, are ar- red and too little blue coloration of the cells and with covers, or glass dishes with follow this with a stain that was equally out of ranged conveniently. Wright's stain is poured into the balance in the opposite direction and so procure into one tray or dish and distilled water stain to 15 a reasonably satisfactory slide. Much the same other. The slides are left in the 5 in result could be obtained by using a mixture of the minutes and then dipped slowly 2 or 3 times two solutions. the water. The rack is shaken to remove the Wright's stain was obtained from various com- excess water and then placed on a towel in a mercial sources. One product was found that, warm place (such as on a radiator or in an in- that ad- as a single staining solution, gave satisfactory cubator) to dry. Small drops of water results. Anyone seeking a Wright's stain for here to the surface of the smear and afterward use on avian blood should test out samples from dry slowly often produce faded round spots. the different sources in the hope that one of them will These spots usually do not interfere with be properly balanced. Buffers of various sorts study of the smear. greater dam- were tried with samples of Wright's stain. These The bulk staining method causes of cells than manipulations still gave results that were too age to water-soluble components red or too blue. does the rack method. Damage is less if the sec- The slides are spread on a staining rack over ond solution is stain and water, equal parts. a sink or tray and flooded with stock Wright's stain. Use enough stain. If the stain is dry 5 May-Griinivald Gienisa minutes after it is applied, too little was used. Usually after this interval of time the surface of Although May-Griinwald Giemsa (M. G. G.) the solution has a metallic sheen and an equal is a more vigorous stain than Wright's, the colora-

quantity of distilled water is added. The slides tion that it gives to the cytosome and nucleus is are allowed to stand for another 5 minutes. much like that given by Wright's. Wright's

There is wide variation in the periods of time stain is preferred for circulating blood of the that can be allowed for the concentrated stain, hatched chicken. The azurophilic substances and the water that follows it, to stand on the slide. in the monocyte are differentiated better with it Different standing periods should be tried before than with M. G. G., and the heterophil rods are concluding that the stain is unsatisfactory. The preserved better. But if the blood is leukemic following are some of the periods that have been or for some other reason contains numerous im- used in this study for the first and second solu- mature cells, use M. G. G. or (if two slides are tions: 5 minutes and 2.5 minutes, 5 minutes and available) both stains. Because M. G. G. 5 minutes, and up to 9 minutes and 9 minutes. stains intensely, it is recommended for all im- It was found that as the stock solution of stain pression smears from hematopoietic organs, both aged, longer periods were necessary. adult and embryonic, as well as for the circulat- After the staining time has been completed, ing blood of the embryo. Both May-Griinwald the slides are drained and blotted. Before the and Giemsa were purchased as prepared solu- coverglass is put on, they are again warmed under tions and have been entirely satisfactory. The the infrared lamp and the coverglass is heated following procedure was used: over an alcohol lamp. 1. Dry slides under the infrared lamp and place on a Many investigators do not cover blood smears staining rack.

but it has been a procedure followed in these 2. Cover with May-Griinwald solution, 5 minutes. Dilute with equal quantity of distilled water and studies on blood, and it has seemed that doing so 3. mix 1-2 minutes. makes the colors slightly more brilliant and the 4. Pour off and without rinsing add diluted Giemsa," little sharper. small details a 15-20 minutes. 5. Methyl alcohol. 1-3 dips. 6. Blot immediately. stain Bulk staining with Wright's 7. Put on coverglass.

to done at one time, When many slides are be ' Add 40 drops of stock Giemsa solution to 40 cc. of distilled is weak, 80 drops to 40 cc. may be used. the job is usually speeded up by using bulk stain- water. If the color 229 )

Wright-Gienisa chiefly to show nuclear structure of heterophils for making Arneth counts. After the blood The technic for Wright-Giemsa was suggested was spread, it was held for a second or two until a to us by Dr. C. J. Hamre of the University of dull effect, which comes with the drying of the North Dakota, who has found this stain satis- slide, began to replace the high gloss of the wet factory for the study of myelopoiesis in birds. surface. At that instant the slide is put quickly It is a vahiable method and for any critical study into a Coplin jar of fixative. Since the fixative on myelopoiesis should be used in addition to contains ether, it May-Griinwald Giemsa. is better to use a Coplin jar with a screw top. Fix, 1 to 24 hours. Wash in 1. Clean slides in concentrated HNO3, 3 hours. several changes of 80-percent alcohol and run up 2. Wash slides in running tap water, in distilled water, to absolute methyl alcohol. Staining with May- and in 95-percent alcohol. Griinwald Giemsa is done in a 3. Make smear of blood and dry in air. Coplin jar rather 4. Use Wright's stain on rack, 4 minutes. (Proper than on a rack and the staining times for one or length of time is determined by appearance of me- both dyes should be increased, sometimes as much tallic sheen. as 50 to 100 percent over that given under the 5. Add distilled water, 4^5 minutes. technic for staining the usual dry smear with 6. Wash in stream of running water and without drying M. add Giemsa,^ 15-20 minutes. G. G. On one set of slides, May-Griinwald 7. Wash in running distilled water. If precipitate is was applied for 10 minutes and Giemsa (80 drops present run Wright's stain over the slide. in 40 cc. of distilled water) for 20 minutes. 8. Wash off with running water. When used this way on blood from the early 9. .Stand slides on edge—do not blot. embryo, sharply defined chromosomes are obtained in mitotically dividing cells.

Petrunhevitch ISo. 2 and M. G. G. Methyl alcohol and thionin Petrunkevitch No. 2 is primarily a tissue fix- The chief use in this study for the combina- ative. It penetrates rapidly and since it is made tion of methyl alcohol fixative and thionin stain up in alcohol it does not require washing. The has been to hold the granules of basophils in their original formula as given by Petrunkevitch normal size and relationships within tlie cell. (1933) has been modified slightly to adapt it for This requires that the film of blood or impression use with avian tissues. smear be fixed before it dries. 80-percent alcohol 3 liters 1. Fixation in absolute methyl alcohol, 5 minutes. Nitric acid 90 cc. 2. Stain in thionin.^ 24—48 hours. Cupric nitrate 60 gms. 3. Differentiate in 95-]iercent alcohol. Paranitrophenol 150 gms. 4. Clear in 100-percent alcohol, xylene, and mount Mix and filter with cover glass. Ether 150 cc.

Combine the chemicals in the order given. Store in a cold place and in a well-sealed bottle. Reticulocyte stain

In handling the paranitrophenol, do not inhale One-percent brilliant cresyl blue was made up the dust and do not allow it to remain on the wet in 25 cc. of 0.85-percent salt solution or in avian skin. Wear rubber gloves when weighing out Ringer's solution—NaCl, 8.5 gms., KCl, 0.42 the material and when handling the prepared fix- gms., CaClj, 0.25 gms.; and water, 1,000 cc.

ative; this precaution is especially pertinent if The solution should be kept cold and should be fil- there is danger that the prepared fixative may be tered before using. Various methods were tried, splashed on the skin. including (1) mixing a drop of blood and a drop The paranitrophenol should be chemically of stain on the slide for periods varying from 1 to pure. Before using it. note whether there is an 5 minutes and (2) drying a film of dye made indication of deterioration—brownish discolora- with 0.3-percent solution of dye in 95-percent tion of the clumps of powder. alcohol and making tlie blood smear over this. Petrunkevitch No. 2 fixative was used here In the second method, the films of blood dried

" Use 15 drops of Giemsa stock to 10 cc. of distilled water. ' Thionin. saturated solution in 50-percent alcohol, 230 quickly and thus the dye on the slide was in solu- flooded with 1 -percent solution of benzidine in tion for an insufficient period of time to give ade- absolute methyl alcohol and allowed to stand 1 quate staining. For avian blood the first method minute. The solution is poured off the slide worked best, but more brilliant staining was ac- and, without washing, it is replaced with a 25- complished when the mixture was held for at least percent solution of superoxol in 70-percent ethyl 2 minutes before the smear was made. The same alcohol, which is allowed to stand IV2 minutes. reticle used for making thrombocyte counts can This is poured off and the slide rinsed for 15 be used for making reticulocyte counts. seconds in distilled water, after which it is dried Graam (1934) used a method that is essen- by blotting and covered. Cells and other struc- tially the same as given in the preceding para- tures containing, hemoglobin stain dark or light graph, but added 0.3 percent sodium citrate. brown. The dried slide should be counter- Six drops of this solution were mixed with about stained with Wright's in order to reveal the cells two drops of fresh pigeon blood. The cells were and parts of cells that do not contain hemoglobin. allowed to stain for 10 to 30 minutes before a In this study it was found that fine, needle-shaped drop of the mixture was removed and spread crystals were sometimes deposited on the slide across a slide to dry. This long staining time but usually these did not interfere with study of may have been the basis for the statement the cells. (p. 202), "Practically every red cell of the pigeon's blood contained some 'basophilic' sub- protoplasm." She goes on to say, stance in the MISCELLANEOUS TECHNICS "The younger cells contain the most of this so- called 'basophilic' substance. One must learn Method for hemoglobin determination to be consistent and he must arbitrarily decide which of these cells to call reticulocytes. I arbi- This subject has been discussed by Denington trarily called those cells reticulocytes which had and Lucas (1955) and only a summarization is a complete chain of basophilic substance around presented here. the nucleus." Fill a test tube with 10 cc. of 0.4-percent solu- Peabody and Neale (1933) used Loffler's tion of concentrated ammonium hydroxide in dis- methylene blue in 0.85 percent solution of sodium tilled water. Take up a 0.02-cc. sample of blood chloride. Air-dried smears were immersed in with a hemoglobin pipette; immediately discharge this stain for about 3 minutes, washed briefly in it into the ammonia solution; after rinsing the water and again air-dried. This stain was use- pipette several times in the solution, seal the test ful for temporary mounts but the preparations tube with a cork that has been washed, dried, and faded after a few hours. infiltrated with paraffin. Invert the tube 2 or 3 The basic method for staining reticulocytes times; after an hour add 0.36 cc. of concentrated from which slight variations in procedure have HCl to the tube and invert the tube 1 or 2 more ll/> as arisen, was given by Osgood and Wilhelm times. This amount of HCl, namely, times (1931). They used, for mammalian blood, nuich as previously recommended, prevents for- equal parts of venous blood that had been mixed mation of cloudiness in the tube; the precipitate with 2 mg. potassium oxalate per cc. and 1 per- that does fonn, aggregates into large masses, or cent brilliant cresyl blue in 0.85 percent NaCl. which usually fall to the bottom of the tube These substances were mixed in a test tube and float on top. Hemoglobin is not bound to this after one minute a drop was removed and a smear precipitate. The density of color is determined wavelength of made. This is the technic used by Magath and colorimetrically at a 410mM- Higgins (1934) to stain the i-eticulocytes of These values are compared with those on a stand- is based mallard ducks. ard curve previously prepared ; the curve on a series of 16 dilutions of hemin. Standard cui-ves were made both from dried commercial benzidine tech- Ralpfl's modification of the hemin and from fowl hemin prepared according nic hemoglobin in cells for to the method given by Elvehjem (1931); the transmission The method given by Ralph (1941) is appli- results were the same. When the of hemin per cc. of cable to air-dried blood smears. The slide is value, transposed into grams 231 —

blood, is multiplied by a factor of 2.3736, the square had one-twentieth the area enclosed by product is the number of grams of hemoglobin the larger square. The latter was of such size per 100 cc. of Idood. The derivation of the con- that it would fit within the opening made by the stant is given by Bankowski (1942). diaphragm ring of the eyepiece. The thrombocytes in tlie large square and the erythrocytes in the small square are counted. Cells that touch two of the sides are counted; Method for heniatocrit determination those that are crossed by the lines on opposite

Van Allen hematocrit tubes were found suitable sides are not counted. If the small square is for embryos, small chicks, and the adult fowl. exactly one-twentieth of the large square, a tabu- They are simple to use, give accurate results, and lation of 250 erythrocytes in that square is the are easy to clean. From a small puncture about equivalent of 5,000 erythrocytes in the large one drop of blood was drawn into the tube. The square. The figures now collected are substi- column of blood should be stopped exactly at the tuted in the following formula: 100-percent mark; then the open tip of the tube , Throm. counted X ery./mm.^ Throm./mm.„, , = should be quickly immersed in the diluent ^^^ 1.6-percent solution of sodium oxalate in distilled The values for erythrocytes per culnc millimeter water—which is sucked into the tube until the were determined from the counts made with the bulb is half full. Then the springclip cap is hemocytometer. fluid is not to placed over the open end; allowed If the inner square is not exactly one-twentieth fall out of the open end as this is done. The of the area of the larger one, differences can be carefully springclip cap must be firmly and compensated for jjy varying the counts of ervthro- seated. cytes above and below 250 by an amount needed The tubes are placed in the brass shields of to give the equivalent of 5,000 in the large square. the centrifuge and the shields are filled to near the top with water and are l^alanced by the amount of water added. They are spun at 2000 r. p. m. Methods for white-cell counts for 20 minutes. The radius to the middle of the shield was 7V-2 inches. The level of packed cells The relative merits of three methods—direct, and the thickness of the buffy layer were read semi-indirect, and indirect—were discussed by with a hand lens. Denington and Lucas (1955) but no conclusion After they are used, the tubes must be carefully as to which is the best was given. Many authors cleaned and dried. The use of acid was avoided have suggested technics that fall into these three because, if not thoroughly washed out of the tube, categories. The indirect method is the sim- it will hemolyse tlie red cells the next time the plest—after a total erythrocyte count has been tube is used. made, the number of white cells relative to the It must be rememljered that the buffy coat is nuniiier of erythrocytes is counted on the stained composed of both leukocytes and thrombocytes, slide and from that ratio, the number of white and the latter constitute half or more than half of cells per cubic millimeter can be estimated. The the buffy coat. same reticle and procedure that were described for thrombocytes can be used.

The Wiseman (19.31a) method is classed as a semi-indirect method. It is based on the prin- Method for making thrombocyte counts ciple that the total number of white cells in a Although an indirect method was used to de- cubic millimeter can be calculated if (1) the termine the number of thrombocytes per cubic eosin-staining cells are counted directly in the millimeter, it seemed reasonably accurate. At counting chamber of a hemocytometer and (2) the time blood was taken for use in erythrocyte these values are used in conjunction with the per- counts, a dried smear was made. After the centage of eosin-staining cells tabulated in differ- counts had been recorded, the stained slide was ential counts obtained from a stained slide. examined with an ocular reticle that had been Wiseman based his method on the affinity of scribed with two concentric squares. The inner phloxine for cells he called eosinophils, but both

232 — :

heterophils and eosinophils take this dye; there- bined heterophils and eosinophils found in the fore, the extrapolation to give a total cell count differential count gives the total number of white from a differential count must include both cell cells per cubic millimeter. types. If this is kept in mind, accurate results When the total number of heterophils and can be obtained with the Wiseman table. For eosinophils counted in the chamber is high and convenience, the taJjle was increased to half the percentage level in the differential count is units so that the calculations could be made on also high, the Wiseman method has reliable ac- the basis of the average of cells counted in both curacy; but when both these variables are low, the chambers of the hemocytometer. probability of error is increased proportionately. The following formula, which calls for less The Rees-Ecker method probably is the best of than half as much phloxine as recommended by the direct methods for estimating total white-cell Wiseman, was used: counts. It is an adaptation of a method designed for counting platelet number in human blood Phloxine 20 mgm. ( Wintrobe, Formalin 5 cc. 1952) and has been used by students Ringer's solution 95 cc. in the Poultry Department at Cornell University. (Ram, 1949; Goodwin, 1950; and Machado, To each 100 cc. .of dye was added 0.5 cc. of 1951 ). The method was first used for making 0.1 N HCl, which gave a pH of 5.7 to the solu- cell counts on avian blood by DeEds (1927) who tion. The dilution in the red-cell pipette was 1 based his technic on the pujjlication of Rees and to 200. The pipette was held in the refrigerator Ecker (1923 ) . The only difference between this 1 to 3 hours and then placed on a slow-moving method and the one given here, based on Win- Bryan-Garrey (1935) rotating cylinder, which trobe's procedure, is in the amount of brilliant does not damage the cells. The fluid in the tip cresyl blue. Rees and Ecker (1923) used only of the pipette was drawn off onto a piece of gauze 0.1 grams of the dye instead of 0.5 grams. before the chambers of the hemocytometer were The diluent is composed of: filled. Heterophils and eosinophils in all Sodium citrate 3. 8 gms. squares of both chambers were counted and di- Neutral formalin 0.2 cc. vided Ijy 2. If one chamber gave results con- Brilliant cresyl blue 0.5 gms. siderably different from the other in the number Distilled water 100.0 cc. of eosin-stained cells present, a new preparation Keep in a glass-stoppered bottle in the refrigera- was made. The volume of fluid under all the tor and filter before using. If formic acid is squares of one side of the counting chamber is produced from the breakdown of formaldehyde, 0.9 cubic millimeters. Therefore, if only one erythrocytes will be hemolysed and the solution eosin-stained cell were seen, this would repre- should be discarded. sent Blood is drawn to the 0.05-cc. mark on the 1X200 , ,., . , , ^r-^r (dilution tactor) pipette and the diluent added to bring the fluids

up to the mark above the bulb. The tube is or 222.2 cells (heterophils and eosinophils) per shaken in the Bryan-Garrey pipette rotor to give cubic millimeter of blood. If in the differential an even distribution of cells; then a suitable count these two granulocytes were the only cells quantity of the mixture is placed on each side of present, the leukocytes would also total 222.2; the counting chamber. but if these two types represented 50 percent of the leukocytes present, the white cells would total Coates (1929) adapted a different direct method, using brilliant cresyl blue, for twice as much—444.4 cells. If the eosin-stained the count- cells represented 1 percent, the total cell count ing of chicken leukocytes. He used two solutions would be 22,222.2 cells. It is possible to pre- Brilliant cresyl blue in water, and potassium pare a table by which the total number of white cyanide in water. We have not tested the Coates cells per cubic millimeter can be determined, or method. the table given by Wiseman can be used for this One of the chief difficulties in using some of purpose. The number of eosin-stained cells the direct methods for avian blood was the con- counted in one side of the chamber multiplied by fusion that came in separating thrombocytes and the factor in the table for the percentage of com- lymphocytes. The brilliant cresyl blue stains 233 the specific granules of the thrombocytes. If the for its identity under the oil immersion lens. top lens of tlie microscope condenser is removed Both with the Wiseman and the Rees-Ecker and the light brought to a focus at the level of method, erythrocytes can be counted in the same the cells in the chamber—with the diaphragm preparation or. if some drying has occurred, the partially closed, if preferred—there is no dif- counting chamber can be cleaned and refilled. ficulty in separating thrombocytes from each Erythrocytes in millions per cubic millimeter of the leukocytes. It is reconmiended, however, are estimated in the same way as for human that differential counts not be attempted in the blood. The correct procedure for counting counting chamber. It is better to make them erythrocytes is given in numerous books on tech- on the slide, where each cell can be checked nic of hematology for the human.

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ADDENDUM

AcKERMAN, G. A., and Knouff, R. A. 1959. Lym- Fredrickson, T. N., Chute. H. L.. and O'Meara, D. C. phocytopoiesis in the bursa of Fabricius. Amer. 1957. Preliminary investigations on the hematology Anat. 163-177. Jour. 104: of broiler flocks. Avian Dis. 1: 67-74. Bessis, M. 1956. Cytology of the Blood and Blood- Gordon, A. S. 1959. Hemopoietine. Physiol. Rev. Forming Organs. 629 Grune and Stratton. pp. 39: 1-40. New York. Kanesada. A. 1956. Lymphoid tissues occurring in Biggs, P. M. 1957. The association of lymphoid tis- the liver and bone marrow of reptiles and birds. sue with the lymph vessels in the domestic chicken Arch. Hist. Jap. 10: 471-481. (Jap. with Eng. sum- (Callus domesticus) . Acta Anat. 29: 36-47. mary and legends.) BiGLAND, C. H., and Triantaphyllopoulos, D. C. 1960. A re-evaluation of the clotting time of 1956. Postnatal development of lymphoid tis- chicken blood. Nature 186: 644. sues in the liver and bone marrow in White Leghorn chickens. Arch. Hist. Jap. 10: 525-530. (Jap. Cleland, J. B., and Johnson, T. H. 1911. Relative with Eng. dimensions of the red blood cells of vertebrates, summary and legends.)

especially of birds. Emu 11 : 188-197. Lucas, A. M. 1959. A discussion of synonymy in Cook, F. W. 1959. Staining fixed preparations of avian and mammalian hematological nomenclature. chicken blood cells with combination May-Green- Amer. Jour. Vet. Res. 20: 887-897. wald-Wright-Phloxine B stain. Avian Dis. 3: 272- 290. and Denington. E. 1957. Effect of total body .x-ray irradiation on the blood of female Single Crass. G., and Rigdon, R. H. 1954. Histologic study Comb White Leghorn chickens. Poult. Sci. 36: of the bone marrow in normal White Pekin ducks. 1290-1310. A.M.A. Arch. Path. 58: 159-167.

Denington. E. M.. and Lucas, A. M. 1960. Influence and Denington, E. M. 1958. The statistical of heat treatment on the number of ectopic lymphoid reliability of differential counts of chicken blood. foci in chickens. Amer. Jour. Vet. Res. 21: 734^ Poult. Sci. 37: .544-549. 739. Medway, W., and Kare, M. R. 1959. Blood and DiESEM, C. D., Bletner, J. K., and Venzke, W. G. plasma volume, hematocrit, blood specific gravity 1957. The effect of estradiolcyclopentylproprionate and serum protein electrophoresis of the chicken. (ECP) on the blood cells of chickens. Avian Dis. Poult. Sci. 38:624-631. 2: 63-75.

Ohno, S., and Kinosita, R. 1956. Three-dimen- -, Venzke, W. G., and Moore, E. N. 1958. The hemograms of healthy chickens. Amer. Jour. sional observations on the intranuclear structure. Vet. Res. 19: 719-724. Exp. Cell Res. 10:569-574.

242 Acknowledgments

This study of avian hematology was initiated in 1944 and has received help and encouragement from many sources. We want to express our deepest appreciation in particular to the institutions and persons listed below for the contributions indicated:

Live birds: Dr. Donald W. Douglass, Dr. Lawrence Fay, Mr. Herbert J. Miller, Mr. Roy Hunt, and Mr. Martin Pollok, all in the Michigan Depart- ment of Conservation; Dr. M. R. Irwin, Department of Genetics, University

of Wisconsin, Madison, Wis. ; Dr. Charles C. Sheppard, Poultry Department, Michigan State University, East Lansing, Mich.; and Mr. A. G. Lohman, Manager Hamilton Farm Bureau Cooperative, Lie, Hamilton, Mich. Slides: Dr. John W. Gowen and Miss Janice Stadler, Department of

Genetics, Iowa State College, Ames, Iowa; Dr. Wade M. Smith, Jr., and Miss Enid B. Allbaugh, Hy-Line Poultry Farms, Des Moines, Iowa; Dr. E. N. Moore, Poultry Science Department, Ohio Agricultural Experiment Station, Wooster, Ohio; and Dr. George W. Sciple, Bear River Research Station, Brigham City, Utah. The library work was greatly facilitated by the many courtesies extended by the staff of the Medical Library, University of Michigan, Ann Arbor, Mich. The authors appreciate the help given to the project by all members of this Laboratory. Those most directly concerned were: Mr. Berley Winton, Director; Mrs. Hazel W. Garrison, secretary; Mr. Seymour Albert; Mrs. M. Gay Flokstra, Mrs. Isabelle M. Letts, Mrs. Georgiann S. McClure, Mrs. Ellen Reibeling, and Mrs. Carolyn Tull, technicians; Mrs. Anne C. Garrison, translator; and Mrs. Coletta S. Waggoner, librarian.

To Swift & Co., Chicago, 111., we want to express our sincere appreciation for its generous financial support of this research through grants to Michigan State University. Dr. 0. A. Newton, who represented the Company, fully appreciated the significance of the research and consistently encouraged its execution. We also desire to express to Michigan State University our thanks for administering the research grants made by Swift & Co.

243

Index

Abnormal cell (s) , 15 Arneth count (s) —Continued eosinophil, 91 nuclear lobe(s) —continued erythrocyte, 30-33, 34, 41 criteria of, 84 primary, 120 eosinophils, 80, 85 heterophil, 87 heterophils, 80, 84, 85 lymphocyte, 54, 56, 64 owl, 221 monocyte, 60, 72 pheasant, 220 separation from artifact (s), 16, 33 pigeon, 221 thrombocyte, 45—46 bone marrow, 221 See also Cell quail, 221 Accipiler cooperi. See Hawk, Cooper's sparrow, 221 Accipitriidae, 202 swallow, 221 Achromatic. See Nucleus, chromophobic turkey, 220 Aged cell(s) Arneth index erythrocyte, 23 bunting. 221 nucleus, 11, 30, 32 chicken primary, 120 basophil, 85 lymphocyte, 167 eosinophil, 85, 90 thrombocyte, 45 heterophil, 83, 84, 85, 90, 220 Albumen, effect on primary erythroblast, 115 mortality correlated with, 85 Allantois, spleen implants, 155 shift, 85 Amblystoma. See Amphibian cockatoo, 221 Amitosis goose, 220 definition, 31 owl, 221 erythrocyte, 23-24, 26, 31 pheasant, 220 primary, 120, 128 pigeon, 221 monocyte, 70 bone marrow, 221 Amphibian effect of season, 221 Amblystoma, embryonic erythrocyte, 105 quail, 221 Bathrachoceps altenuatus, thrombocyte, 45 sparrow, 221 erythrocyte swallow, 221 amitosis, 23-24 turkey, 220 reticulocyte, 28 effect of sex, 220 Neclurus Artifact (s) erythrocyte, 31, 40 basophil, 92 macrophage, 68 eosinophil, 76, 91 monocyte, 68 erythrocyte, 33-34, 36, 39-41 thrombocyte, 45 embryo, 98, 100, 115. 117. 124, 129 Anas platyrhrnchos. See Duck, mallard heat, 36, 40 Anatidae, 202 heterophil, 73, 74, 87-89 Anemia, megaloblast of, 192 magenta bodies, 40 Angioblasts, 104 mechanical, 36 Anisocytes, 34 monocyte, 60, 72-73 Anisocytosis, 26, 31, 127 nucleus Anonymous (1949), 8, 194, 196 clear area, 17 Anseriformes, 202 extrusion, 34, 36 Antibodies, against spleen, 155 polychromatic

( , Arneth count s ) 220-221, 230 late, 11, 33 bunting, 221 mid-, 11 chicken, 84 pressure, 34 classes, 84 separation from abnormal cells, 16, 33 cockatoo, 221 technic, 33-34, 36, 39-41, 46-47, 72-73, 87-89, goose, 85, 220 91,92 nuclear lobe(s) vacuolization, 36, 40 basophils, 80, 85 See also Smudged (squashed) cell(s)

245 Ascheim ( Dantschakotf , 1908b), 26 Basophil ( s ) —Continued Atomic Energy Commission, project, 215 series, 10, 13 Atypical cell(s) in bone marrow, 196-197, 200 category of, 16 size, 214 erythrocyte (s) , 34 frequency curve, 80 See also Cell Avian leukosis complex in owl, 210 lymphocytes, 51, 64 in turkey, 210 smudged cells, 72 range, 80 species, blood cells of, 202-221 Avian technic Azurophilic granule aqueous solutions, 91, 155 lymphocyte, 54, 56 monocyte, 49, 58, 69, 71 methyl alcohol and thionin, 191 immature, 60 Petrunkevitch No. 2 and M. G. C, 80 early, 12 Bathrachoceps attenuatus. See Amphibian late, 12 Bausch and Lomb Optical Co. mature, 12 Poser, Max, 222 relation to magenta granule, 54, 71 See also Magenta granule Shippy, H. L., 222 Beck (1938), 223 Balfour (1911). 41 Belling (1930), 223 Bankowski (1942), 232 Benzidine test, Ralph's, 115, 118, 130, 131, 134 Bartschetal. (1937), 212 technic, 231 Basichromatin Berman (1947), 117 dissolved in nucleoplasm, 34 Biely and Pabner ( 1935) , 215 outside cell, 34, 168 Basket cell, relation to smudged cell, 72 Bird(s). See under scientific or common name Basophil! si Bizzozero (1889). 141 artifact(s),78, 91. 92 and Torre ( 1881 ) , 27, 88 smudged, 78, 92 Blackfan, Diamond, and Leister (1944), III, 64 circulating blood, 78, 91-92 Blakemore (1934), 215 developmental stages in, 92 count (s) Blast cell(s), 9, 11, 24, 50, 56, 66, 128, 129 Arneth, 80, 85 See also specific cells chicken, 216 Blood duck, mallard. 216 goose, Canada, 216 cell pheasant, ring-necked, 217 count (s), 214-220 pigeon, 217 accuracy, 218 sex, 216. 218 Arneth, 80, 84, 85, 220-221 turkey, 220 bone marrow, 198, 199, 200 granule (s), 78, 91-92 chicken. 216 dissolution of. 78. 91, 155 diurnal eilect, 220 in duck, 211 effect of altitude, 218 magenta, 13, 78, 92, 197 embryo, 113-114 metachromasia, 91 pheasant, 215, 217 granuloblast, 13 10, pigeon, 217, 219 in spleen. 164 sexvalues, 216, 218, 219 mature, 10. 13, 78. 91-92, 111. 146, 165, 172, 182, value of a single count, 216-217 191, 200 variability, 215, 217, 218 mesomyelocyte, 10, 13, 106, 182, 191, 197, 200 wild birds. 218 metagranuloblast, 10, 13, 197 distortion, effect of delay, metamyelocyte, 10, 13, 191, 197,200 227 nucleus from bone marrow, hatched chicken, 181-201 embryo, 141-180 Arneth count, 84 from hematopoietic organs, from other avian species, 202-221 Arneth index, 85 See also Cytoplasm and cytosome; Differences; eccentric, pigeon, 210 Nucleus failure to stain, 91-92 changes at hatching promyelocyte, 10, 13, 92, 200 bone marrow, 164, 165 nucleolus, 197 circulating blood, 157, 164, 165 separation from heterophil j^romyelocyte, 92 spleen, 164, 165

246 ) — Blood—Continued Bone marrow, 157, 164, 165, 181-201 circulating basophil, 182 differentiation factor for er) tliroblasts, 112, 113 series, 191 embryo, 104-140 cavity cells normally blood absent, 132 beginning formation, 152 present, 133, 141 vessels, 151 cells occasionally present, 133, 139-140 cartilage cells in, 153 defense reaction in, 133 cells from mesenchyme, 151 dorsal aorta. 94, 96, 226-227 development phases, 151 heart, 98, 100, 103, 106, 139, 227 enlargement, 151 hatched chicken, 17-93, 111, 157, 164, 165 count low leukocyte level, 157, 164 Arneth, 221 ratio of hetero])hils to nongranular leukocytes, differential, 198-199, 200 164, 165 embryo, 141, 142, 144, 146, 148, 151-155 clotting, compared with mammal, 46 eosinophil, 146 from cubital vein, 18, 108, 111, 139 series, 191 serum erythroblast, 182 compared with mammal, 41 erythrocyte, 142, 144, 146, 164 granules, stained, 41, 93 discharge at mid-polychromatic stage, 164 smear, gross appearance, 4, 15 series, 184 spot granuloblast, 146 heterophil, rod to granule, 87 granulocyte. 144, 193-197 macrophage, 64, 139 heterophil, 142, 144, 146, 164, 165, 182 from fibroblast, 134 small size. 73 surface tension, 227 series, 188 technic lymphocyte, 182 avian, 222-234 lymphoid nodule, 181 cannula, 139, 225-226, 227 osteoblast, 142, 144, 148, 151, 152, 164, 200 procuring, 225-228 osteoclast, 13, 151, 152, 153, 198, 200, 204 carrying box, 227-228 samples, mononuclear, 13, 142, 148, 153, 164 smear multinuclear, 13, 142, 198 cockroaches and flies series, 13 damage from, 34 plasma cell, 166, 197-198, 203 protection against, 39 primordial osteogenic cell, 142, 144, 146, 148, 151, contaminant, 41. 169. 176, 179 152 cover glass on slide, 229 serum, stain in. 41, 148 distribution of cells, 15 193 embryo. 226-227 thromboblasts, 146, 164, 182, 193 infrared lamp, 226, 227, 229 thrombocyte, 146, 191, 186 number of slides needed, 84, 217 series, 164, 182, 88 relative value, 1-3, 154 Bradley (1937), 41, 42, 87, canadensis. See Goose, Canada values. See Buffy coat; Hematocrit; Hemoglobin; Branta etc. Breusch (1928), 23, 86, 215 Brilliant cresyl-blue. See Stain and staining vessel ( s erythroblasts Bryan and Garrey (1935), 233 Owl, great horned inside, 104, 112, 113 Bubo virginianus. See outside, 113 Buffy coat of bone marrow, 151 cell types in, 232 l>lugged line difference, 215 effect of splenectomy, 29, 181 sex, 216 in bone marrow, 181 significant variability, 217 viscosity, 210 Bunting See also specific cells Arneth count, 221 Bloom (1938), 47 indigo (Passerina cyanea) , 202 Blount (1939a). 215 erythrocyte, 210 (1939b), 41, 42 Burckhardt (1912), 28, 83, 215 Bobwhite, erythrocyte count, 218 Burmester, Gentry, and Waters (1955), 133 199 Bone Severens, and Roberts ( 1941 ), epiphyseal center, lacking in birds 141 Burnett (1908), 215 red-tailed vessels of marrow, 151 Buteo borealis. See Hawk, 247

349126—61- -14 Cabot's ring. See Erythrocyte Cell ( s j —Continued Camera lucida, 14, 212 life span Cameron (1941), 105 erythrocyte, 32 Canary, 33 embryonic, 114 Cannula, glass, 225-226 thrombocyte, 45 effect of entrance into heart, 139 permeability, weakened by disease, 7 grinding tip, 225 polarity, osteoclast, 198 plugging, prevention of, 226 primordial, in embryo thymus, 170, 172 Cardinal {Richmondena cardinalis) , 202 ruptured by pressure, 34, 72 Carpodacus purpureus. See Finch, purple shape Cartilage, 151 amoeboid (pseudopodia) , erythrocyte, embryonic, cell(s) 128 liberated into marrow, 153, 154 elongated produce stroma, 154 bipolar, 139 matrix in osteoclast, 153 erythrocyte, 31 mitosis, 153 fibroblast, 134 smear, 153 mononuclear osteoclast, 13 Cell(s) reticular cell, 134 border unipolar, 139 colored erythrocyte, 211, 212 azurophilic bodies in monocyte, 69 poor criterion of age, 26 thrombocyte, 11, 42, 132, 136 relation to size of bird, 211 poorly defined, multinuclear osteoclast, 13, 198, subsidiary criterion of maturation, 27 204 lymphocyte, 49, 50-51, 62 broken, erythrocyte, 33, 34 monocyte, 63 characteristic(s) immature, 71 basophil, 13 oval eosinophil, 12-13, 90 erythrocyte erythrocyte, 11, 27, 48 mature, 11, 17 heterophil, 12, 90 change to round, 26 lymphocyte, 12, 49, 71 primary, 120 monocyte, 12, 49, 71 thrombocyte, 12 multiple, 48, 65 embryo, 131, 134 osteogenic cells, 13 primordial osteogenic, 13 plasmocyte, 13 round thrombocyte, 11 basophil, 91 counts. See Blood, cell erythrocyte, 23 degeneration produced by lysins, 26 thrombocyte, 46. 182, 186, 191, 210 granuloblast, 12 embryo, 103, 106, 131, 132, 136 heterophil, 73 development, synchronism of parts, 25 lymphoblast, 12 division 5ee also specific cells erythroblast. primary, 94 thromboblast, 11, 131 erythrocyte, 26, 31, 118 size, 211-214 primary polychromatic, 96. 98 basophil, 80, 214 heterophil, 188, 196 effect of fixation, 14, 91 lymphocyte, 49, 53 eosinophil, 80, 89, 214 monocyte, 49, 70 erythroblast, primary, 116 primary osteogenic cell, 142 thrombocyte, embryo, 131 erythrocyte, 212 distortion, erythrocyte, 31, 33, 34 primary, 118, 127 endothelial, 139 heterophil, 73, 80, 214 epithelial, 211 impression of. 14, 66 epithelioid, 169 lymphocyte, 49, 50, 62, 66, 67, 212, 213, 214 fragments, 93, 134, 139, 142, 198 possible significance of, 66-67 See also Erythroplastid monocyte, 49, 63, 66, 212, 214 giant, 17, 41, 117, 120, 154 possible relation to age, 73, 118, 211 5ee also Osteoclast thrombocyte, 41, 42, 44, 213 identification variable interference from stained granules, 93, 148, 158, anisocyte, 26, 34, 127 168 plasmocyte, 14 value of size, 14 visual (mental) impression of, 14, 66

248 —

Cell I s ) —Coiitit'ut (1 Chromatin—Continued squashed. 5ee Smudged (squashed) cell dissolved striated muscle, 169, 211 by secretions of cockroaches and files, 34 type into nucleoplasm, 30 categories, 15 extruded from nucleus, 34 totipotent, 9, 166 granular unidentified in bone marrow, 199, 200 osteoblast, 13

See also Abnormal cell ; Aged cell ; Blood, cell ; Bone osteoclast, 13 marrow; Development of cells; Differences; spe- primordial osteogenic cell, 13 cific cells network (reticulum) Charipper and Dawson (1928), 23, 24, 31 coarse Chickadee erythroblast, 11, 116, 118, 128

black-capped (Penthestes atricapillus) , 202 basophil metamyelocyte, 13 thrombocyte size, 210 fine Carolina, erythrocyte size, 212 erythrocyte embryo, 128 Chicken granuloblast, 12, 194 breed lymphoblast, 12 Ancona, 62, 63 lymphocyte, 53 Bentham, 211 metagranuloblast, 12, 194 Langshan, 211 monocyte Leghorn, Brown, 211 early immature, 12 Leghorn, White, Single Comb, 17, 18, 20, 32, 62, mature, 12 63,211,212,213,214,216 punctate New Hampshire, 62, 63, 211, 212, 213, 214, 215 macrophage, 139 Plymouth Rock multinuclear osteoclast, 13 Barred, 62, 63 thromboblast, 11, 128, 130, 134, 164, 191 Columbian. 211, 212, 213, 214, 215 staining, primary greater than definitive, 127 Rhode Island Red, 62, 63, 216 strands'of, 34, 158, 168, 170 source See also Chromosome, avian ; Nucleus farm stock, 216 Chromophobic. See Nucleus Hamilton Farm Bureau, 62, 63 Chromosome, avian Hy-line Poultry Farms, 62, 63 lag in anaphase, 117 Iowa State College, Genetics Department, 62, 63 material for study, 117 Laboratory, 218 Petrunkevitch No. 2, 230 No. 2, 17, 32, 39, 40 poor in air-dried smear, 53 Regional Poultry Research, 17, 216 Iine6. 211, 212. 213, 215 Chylomicrons, relation to serum granules, 93

Coates ( 1929 , 233 line 15, 211, 212, 213, 215 ) method, 233 Chondrocyte(s) . See Cartilage Chromatin Coccyzus— clumped, 4 americanus. See Cuckoo, yellow-billed eosinophil erythropthalmus. See Cuckoo, black-billed mature, 13 Cockatoo, Arneth count, 221 metamyelocyte, 13 Cockroach. 5ee Blood, technic, smear erythrocyte Columbidae, 202 mature, 17, 30, 210 Columbiformes, 202 polychromatic, 11, 25, 26 Committee for Clarification of Nomenclature, etc. primary greater than definitive, 116, 129 (1948), 194 heterophil, 12 Compsothlypidae, 202 metagranuloblast, 154, 194 Cook (1937), 215 lymphocyte and Dearstyne ( 1934) , 215, 216 immature, 12 Cook-Ponder counts, 84 mature, 12, 49, 53, 54, 169 5ee also Arneth count monocyte, 12, 49, 70 Cottral (1950, 1952), 133 plasmocyte Burmester and Waters ( 1954) , 133 immature, 166, 198 Cuckoo mature, 14 black-billed {Coccyzus erythropthalmus), 202 thrombocyte erythrocyte, 210 size, 210 embryo. 11 thrombocyte yellow-billed {Coccyzus americanus), 202 immature, 11, 45 mature, 12 Cuculidae. 202 249 — —

Cuculifonnes. 202 Cytoplasm and cytosome—Continued Cullen (1903), 67, 207 shape Cytoplasm and cytosome bleb formation border lymphocyte, 50-51, 54 acidophilic, pink monocyte, 67 monocyte, 69 produces granulocytes and thrombocytes, 154 thrombocyte, 11, 42, 69 thrombocyte, embryo, 130, 131 embryo, 132, 136 crescent around nucleus, metagranuloblast cruni]iled. thrombocyte, embryo, 11, 132, 136 eosinophil, 12 poorly defined, multiniiclear osteoclast, 13, 198 heterophil, 12 centrosome sphere, 116 narrow rim around nucleus. 9 clear space around nucleus granuloblast. 12. 193, 194 erythrocyte, 17 lymphoblast, 12 lym]ihocyte, 80 lymphocyte, immature, 166 thromboblast, at side of nucleus embryo, 131, 193 spherical bodies in lymphocyte, 52 erythrocyte. 33 osteoblast primary, 115 immature. 13 stigmata, erythrocyte, 33 mature, 13, 152 structure plasmocyte ectoplasmic mantle, 117 immature, 14 fractured mature, 14, 166 erythrocyte, 11, 33, 34, 36 primary erythroblast. contains centrosome, 116 primary, 127 color after staining glass bead, monocyte, 65. 68 acidophilic, orange, pink granules erythrocyte basophil, 91-92 mature, 17 eosinophil. 90, 208, 209, 210 polychromatic, late, 25 granulated and basophilic hetero])hil, 74, 83 lymphocyte. 51, 52 spheres, primary erythroblast, 116 monocvte, late immature, 12 hyaline. 50, 51. 52. 67-68 See also Eosinophil ; Heterophil rarefied area, spindle erythrocytes. 31 basophilic, clear, light blue reticular, monocyte, 12, 68 eosinophil, mature, 89 texture monocyte, early immature, 12 fine granular, erythrocyte, primary, 127 plasmocyte, immature, 13, 198 uniform, erythrocyte, mature, 11 thrombocyte, immature, 11 See also Vacuole in cytoplasm and cytosome basophilic, dark, intense blue toxic granules, 87 granuloblast. 12 See also Artifact; Mitochondria: Vacuole in cyto- lymphoblast, 12 plasm and cytosome osteoblast, mature, 13 Cytosome. See Cytoplasm and cytosome plasmocyte, 198 thromboblast, embryo, 131 Dafila acuta. See Duck. ])intail. American colorless, basophil, 91 Danschakolf (1916a). 130, 155, 165 gray 1 1916b), 73, 141, 166, 169 erythrocyte (1916c), 141 mid-polychromatic, 25, 26 Dantschakoff (1907), 104 primary, 127 (1908a), 104, 141 range, 26 (1908b), 6, 8, 25-26, 104, 116. 127, 128. 130, mixture of blue and orange. 25 141, 152. 154, 168. 195 polychromasia (1909a), 48, 73, 87, 113. 116. 141, 168 erythrocyte, 26 (1909b). 73,87, 151. 153. 166 monocyte, 58 (1931). 139 transition Dawson (1931). 40 erythrocyte, 33 (1932), 141 thrombocyte, 132 (1933a). 68. 117. 118. 167 division. See Amitosis: Cell, division (1933b). 45 inclusions, 6 (1936a). 29. 105. 118. 127.128 macrophage, 139 (1936b). 45 result of flattening, 6 DeEds (1927). 219. 233 250

i Definition (s) Differences—Continued abnormal cell, 16 erythrocyte—continued artifact, 16 definitive and primary—continued ani])hinucleolus, 14 cytosome cell types, 11-14 fracture, 127 nucleolus size range, 127 aniphinucleolus, 14 nucleoli, number, 116 karyosome, 14 nuclear staining, 127 plasmosome, 14 vacuoles, 116 Dendroica— granules, heterophil and serum. 89 striata. See Warbler, black-poll heterophil and basophil jnomyelocyte, magenta virens. See Warbler, black-throated green granules, 92

Denington and Lucas (1955) , 227, 228, 231 heterophil and eosinophil, 83, 90, 196, 207, 208, 209 Denys (1887), 88 lymphocyte and monocyte, 48, 49 Development of cells lymphocyte and thrombocyte, nucleus, 46 characteristics, erythrocyte, 11, 25 megaloblasts, anemia, primary and definitive, 116 in bone marrow, 181-201 spleen, before and after hatching, 156 in circulation of hatched chicken Differential count, 84 basophil, 92 accuracy with number counted, 217 eosinophil, 91 basophil, 92 erythrocyte. 24—30 bone marrow, 198-199, 200 heterophil, 74, 86 chicken. 216 lymphocyte, 53-54 duck, 216 thrombocyte, 44^5 embryo, circulating blood, 133 in embryo. 104-140 embryonic erythrocyte, 105 in hematopoietic organs, 141-180 goose, 216 in vitro, erythrocyte. 29 high variability. 219 influenced by smudged nuclei, 72-73 Diesem (1956) , 46, 89, 215 Differences sex, 216 Disease, III, 33 abnormal cells and artifacts, 16, 33 bacterial infection in egg, 133 biids and mammals birds, 84 bone and bone marrow formation, 141, 151 poultry, 15 erythrocj^e See also Lymphomatosis generations in embryo, 104 Distribution of structures circulation, immature in 24 artifacts on slide, 33 nucleus, 30 during division physiology, 29 magenta granules in lymphocyte, 53 erythropoiesis, maturation. 30 yolk plates in erythrocyte, 105 giant cells, number of types, 153 See also Blood, technic, smear, distribution of cells hematopoietic reactivity, 29 Doan (1932), 8, 61 intravascular and extravascular, 113, 141 Cunningham, and Sabin (1925), 115 lymph nodes, 167 Dog. 26 stain. and staining recovery from injury, 29 Dominici See Stain

Domm et al. ( 1943 ) . 218 serum particles, 41 and Taber (1946), 218 smudged cells, number, 39 Dorsal aorta spleen, histology. 156 embryo blood from, 94, 96 thrombocyte, fragility, 42 technic for procuring, 225-227 thymus, striated muscle, 169 See also Blood, vessel yolk sac, 141 Dorst and Mills (1923), 46 blood values, sexes, 218 Dove early immature monocyte and metagranuloblast, 167 eosinophil, 210 embryo erythrocyte count, 218 erythroblast and thromboblast, 115, 128 hemoglobin, 218 thrombocyte, primary and late, 131, 132 Laboratory, 202 erythrocyte thrombocyte size, 210 mature and immature Down. 179 oxygen consumption, 29 sheath cell. 178, 180 specific gravity, 29 See also Feather, sheath cell definitive and primary, 104—105 Downey, 93

coarseness of chromatin pattern, 116 and McKinlay 1 1923) . 64

251 Drawingis) Endodermal wandering cell, 139 high power, 14 Endothelium limitations, 191 heart, 139 low power, 14, 15 phagocytic scale of measuring, 4 liver, 139 Dryobales— spleen, 139 pubescens. See Woodpecker, downy tissue, 9 villosiis. See Woodpecker, hairy yolk sac, 104

Duck Environment, effect on immature cells, 7, 14, 112, 113

baldpate [Mareca americana) , 202 Eosin-azure. See Stain and staining eosinophil, 207. 208 Eosinophil, 89 heterophil. 207. 208 Arneth count cell count, variability, 215 Canada goose. 85, 220 eosinophil. 207,208,210 chicken, 84, 85 erythrocyte, crossed cells, 210 lobes, 90 heterophil, 208 Arneth index lymphocyte Canada goose, 84, 85, 220 magenta bodies, 210 chicken, 85, 90 reactive cell. 210 abnormal cell. 91 size, 67 birds, except chickens, 207, 208, 210

green-winged leal [Nettion carnlinense) , 202 count! s) eosinophil. 210 chicken. 216 Laborator), dilferential count. 219 duck, 216, 219 mallard {Anas platyrhynchos) , 202 goose, 216, 219, 220 basophil, 211 in hemocytometer, 233 differential counts. 216 pheasant, 217 eosinophil, 207, 208, 219 pigeon, 219 erythrocyte, nucleus. 23 sex. 216. 218 heterophil. 207, 208 turkey. 219 reticulocyte, 27 cytoplasm, pale blue staining, 76, 89, 90 technic. 231 depot, 155 pintail, American (Dafila acuta) , 202 developmental stages in circulating blood, 76, 91 eosinophil. 210 differences from heterophil, 90, 91, 196, 207. 208

ruddy {ErismaluTa jamaicensis) . 202 dove. 210 eosinophil. 207 effect of Pelrunkevitch No. 2. 80. 90, 207 shoveller {Spatula clypeala) , 202 granules. 76. 89 eosinophil. 210 refract ile, 90 thrombocyte size, 210 relation to maturity of cell, 90 rod shape, 207, 209 structure. 90, 196, 208, 209 Ectoplasmic mantle of primary erythroblast, 117, 118 types. 209 Ehrlich (Dantschakoff. 1908bj, 26 granuloblast. 10. 12 Elvehjem (1931), 231 spleen, 164 Embryo embryo, 156. 160 allantois, 141 in bone marrow, 196 area opaca, 41 in spleen, 164, 165 area pellucida, 31 leukemia, 208 blood. 10H40 mature, 10, 13, 76, 89-90, 1 16, 158, 200 dorsalaorta. 94.96, 118 metagranuloblast, 10, 12 heart. 98. 100. 103 bone marrow, 191, 196, 200 island. 104 embryo, 156, 160 removal of, 226-227 mesomyelocyte, 10, 13. 76. 91. 196, 200 gonadal ridge, 139 metamyelocyte, 10, 13, 76, 91, 196, 200 incubation age, 105, 112, 226 promyelocyte, 10, 12, 200 primitive streak, 104 series, 10, 12 removal of, 226 in 200 splanchnopleure, 139 bone marrow, stained serum, 41 size, 76, 89, 90, 214 vitelline membrane. 113. 226 frequency curve, 80 yolk sac, 104. 128 spherical bodies, 33 See also Bone marrow; Spleen; Thymus; specific smudge cell, 76, 91 cells terminology, 89 Emmel (1936), 7, 83, 92 See also Granule

252 — —

Epiphysial center, 111 Erythrocyte! s) —Continued Erdmann (1917), 181 effect of Erismatiira jamaicensis. See Duck, ruddy antimalarial compounds, 33 Erythrohlast(s),10, 192 fixation, 94 blast cells compared, 9, 192 Petrunkevitch No. 2, 80 characteristics, 11, 26, 112, 192 splenectomy, 29 embryo, 31, 114, 158 embryo, 192 hemoglobin acquisition, 104, 112 artifact, 98, 100, 103, 124, 127 later generations, 98, 100, 114, 128 bone marrow, 142, 144, 146 in spleen, 156 cell, description. 115-130 mitosis, 122 changes during incubation, 113, 114 nucleus, poorly stained, 122 developmental stages in circulating blood, 94, 96, thromboblast, compared with, 128 98, 100, 104-130 in bone marrow, 181, 182, 184, 191, 192, 200 differential count, 113, 114 in circulating blood, 23, 24, 94 division, 94, 96 in spleen, 157, 160 generations, 104, 114, 128-130 intravascular and extravascular, 113 life span, 114 primary mature. 103, 113, 142, 144 basophil, 118 polychromatic cell size, difference, 116, 118 early, 100, 124 changes during incubation, 113, 114, 118 mitosis, 128 chromosome, 117 late, 100. 103. 106, 113, 114, 124, 128, 129, 142 disappearance from circulation, 112, 113 mid-, 100. 106. 113, 114 division. 118 spleen, 157. 160 mitosis, abnormal. 117 mitosis. 157 generations of, 9, 104-105, 114 first appearance, 112 giant, 17, 117 at 22 to 29 somites, 31 hemoglobin and basophilia, 29 giant, 117 immature, 23 hemoglobin in bone marrow, 182 little, 26 life span, 32 precocious, 112, 129 line differences, 215 like thrombocyte mature. 10, 11, 23, 108. Ill, 146, 182, 184 clumping, 130 cyto|)lasm texture. 11 origin, 130 in bone marrow. 200 smudged, 94 normal. 17-24, 29 term, synonymous with megaloblast, 116 typical, 17, 18, 20, 23, 211 resembles large lymphocyte, 24 deviations from, 17, 18 terminology, 192 multipolar. 17 Erythrocyte(s), 17-41, 155 nucleus. 20 abnormal, 20, 30-33, 34 age of, 11 aged, 29, 30 aged. 23. 30 artifact, 33, 36, 39-41 double. 23, 27 atypical, 27, 30-33, 34 size, 211, 212 bone marrow, 181, 182, 184, 192, 193 squashed, 18 141 Cabot's ring, 39, 40 origin, intravascular, 104, 113, 128, count (s) orthochromatic, 23, 26 birds, except chickens, 218, 219 percent rise and fall, 113 192, 198 brooding vs. laying hen, 218 polychromatic, 24, 25, 26, 27, chickens. 216 artifact, 33, 144 diurnal effect, 218 basophil, synonym, 24 23', 192, 200 effect of early. 10, 11, 181, 182, 184, 184, 192, 200 altitude, 218 late, 10, 11, 23, 26, 108, 182, season, 218 atypical, 23 sexes, 216, 218 mitosis, 157, 160 200 variability, 217 mid-, 10, 11. 23, 26, 184, 192, circulation, 164 crossed cells, 210 enters debris on, 146 mitosis, 160 127-128 developmental stages primary, 115-120, circulating blood, 24-30, 164, 210 abnormal, 120 tissue culture. 104, 116 amitosis. 128 253 —

Erythrocyte ( s ) —Continued Feather—Continued primary—continued barbules, 178, 180 artifact, 100 diagram, 178 cytoplasmic fractures, 127 sheath cell, 169 ectoplasinic mantle. 117 carrier of pathogens, 179 nuclear vacuole, 115 contaminant smudged, 98 in hair, 179 cytoplasm, texture, 127 on slides, 169, 179 development, precocious, 129 down, 176 disap]5earance, 113, 114 keratin, 178 dominant cell, 113 granules. 176 generations of yolk plates in Ambhstoma, 105 Fennel (1947), 128, 154 giant. 120 Ferguson. Irwin, and Beach (1945). 215

in bone marrow, 200 Finch, purple iCarpodacus purpareus) . 202 mature, 100, 103, 106, 113, 114. 120, 124. 127. eosinophil, 210 130 Fixation and fixatives mitosis. 115 air-dried fixation, 2, 4, 7, 14, 231 nucleus, 127 effect on size of cell, 14, 91 polychoinatic methyl alcohol, 14. 87, 91, 191. 197, 230 early, 96. 100. 113. 118 Petrunkevitch No. 2 late. 98. 100. 103, 113. 118. 127 effect on mid-, 96, 98,113, 118, 128 basophil, cytoplasm, 91 terminology, 117 chromosome, 117 series, 9 eosinophil, 90, 207, 209 size, 118, 127 heterophil, 83, 87, 195, 207, 209 structure, 115-118, 127, 128 thrombocyte. 132 technic, damage, 115 formula. 230 ]5rotoplasmic process, 41, 115 nucleus. 80, 83, 90, 92, 230 ratio with thrombocyte, 42, 44 perinuclear space, 17 method, 232 technic, 230 separating stages, 27 rate of penetration, 167 series,9, 10, 11, 15, 184 wet fixation, 1, 14, 230 in bone marrow, 200 with May-Griinwald Giemsa, 230 shape Zenker formol, 1 distorted, 20, 115 Flemister and Cunningham (19401, 114 round and mature, 184 Fly damage to blood smear, 34 spherical bodies, 33 Folic acid, effect on reticulocytes. 28 stigmata, 33 Foot (1913), 17 size, 210, 211,212 Forkner ( 1929) , 17, 43, 199, 215 terminology, 24, 25 Frank and Dougherty (1953), 51 types, Bizzozero and Torre, 27 Frequency distribution, thrombocyte, size, 44 typical, 17 Fringillidae. 202 5ee also Cytoplasm and cytosome; Erythroblast; Furth. Seibold. and Rathbone (1933), 39 Nucleus; Reticulocyte; etc. Erythroplastid, 20, 31, 34, 36 Gage and Fish (1924), 93 birds, other than chicken, 210 Gallifonnes. 202 mid-polychromatic erythrocyte, 96 Garrey and Brvan (1935), 215 primary erythrocyte, 120, 127

Ganger etal. 1 1940), 219 survival value for cell, 31 Evolution Giemsa stain. See Stain and staining granulocytes. 73 Goodall (1909), 17.215 neutrophiloid cell from heterophil, 86 Goodwin (1950), 233 process of erythroplastid formation. 31 Goose, Canada (Branta canadensis), 202 Extravascular origin count (s) all leukocytes. 141 Arneth, 220 erythrocytes, 113 differential. 216 thrombocytes, 42 variability. 215 Eyepiece. See Microscope, ocular eosinojihil. 210 erythrocyte, 210 Falconiformes, 202 Gordon (1926), 42 Feather Graam (1934), 28, 231 barbs, 178, 180 (1935), 32

254 Granular leukocyte, 73-92 Guinea hen in bone marrow, 193-197 eosinophil, 207 See also Basophil; Eosinophil; Hetero])liil erythrocyte, 210 Granule (s), body(ies), substance (sj heterophil, 207 azurophilic Gull lymphocyte, 54 cell size and age, 212 monocyte, 49, 54, 69 Larus ridihundus, 212 immature, 12, 60, 71 mature, 12 Haff (1914), 141 basophilic Halliburton (1886), 93 monocyte, immature, 12 Hamilton (1952), 141,155 osteoclast, multinuclear, 13 Hamre (1952), 194 central granule of heterophil rod. See Heterophil (personal communication), 73, 74, 89 cytosome andMcHenry (1942a), 217 basophil, 91 and McHenry (1942b), 215 eosinophil, 89, 208, 209 Hancox (1946), 153 in vacuole, 40 Harne, Lutz, Zimmerman, and Davis (1945), 32 plasmocyte, mature, 14 Hartman (1925), 42, 45 reticulocyte, 11, 27 Hassall's corpuscle, 169 eosinophil Hatching, blood changes, 157, 164—167 on a reticulum, 76 Hawk size range, 76 Cooper's (Accipiter cooperi), 202 See also Eosinophil, granules erythrocyte, 210 osteoclast, 13 eosinophilic red -tailed (Buteo borealis) , 202 hemokonia, 93 Hayden (1929), 215 keratin, 176 Heart metachromatic, 91 blood from, 98, 100, 103 nucleus. See Chromatin; Nucleus effect of glass cannula entering, 139 serum, stained. 36, 41, 89, 93, 148, 158 smear from endothelial surface, 139 soluble Heath-hen, erythrocyte-thrombocyte ratio, 42 basophil, 78, 91, 92 Heinz (Dantschakoff, 1908b), 26 mesomyelocyte, 13 Hematocrit, 214 heterophil after Petrunkevitch No. 2. 87 chicken, 216 toxic granules, man, 87 variability, 217 See also Magenta granule; Specific granule; specific line difference, 215 cells sex, 216 Granuloblast (s), 6, 9, 10, 50, 142, 146, 196 compared, 218 basophil, 10, 13 Van Allen tube, 217 bone marrow, 1.54, 156, 188, 194, 200 centrifugation, 232 eosinophil, 10, 12 cleaning. 232 granulocyte lines have common, 12, 194 diluent. 232 heterophil, 10, 12, 74, 86, 200 technic, 232 mitosis, 144 Hematology nucleolus not visible, 169 approach to study of, 7-8 nucleus, 197 field of, 32 number reduced, spleen, 156 schools of, 9 resembles lymphoblast, 194 Hematopoiesis cartilage cell, 154 spleen, 160 lymphocyte, 47 Granulocyte(s), 144, 193-197 large, 48 bone marrow. 92, 93, 181, 199 monocyte, 47 embryo, 142, 144, 146, 1.54, 156 theories circulating blood, 165 associated with technic, -3-4 development, number of stages, 193-194 unitarian, 9, 48 circulating blood, 133 embryo, yolk sac, 154 mature, 142, 165 Hematopoietic organ(s), embryo terminology, 194 bone marrow, 141—155, 164 thymus, 169 kidney, 141 .See also specific cells leukocytes retained until hatching, 141 Granulopoiesis, 194 liver, rarely hematopoietic, 141 less in thymus than in spleen, 168 ovary, 141 Gray, Snoeyenbos, and Reynolds (1954), 45 pancreas, 141 255 — ——

Hematopoietic organ (s) —Continued Heterophil (s) —Continued placenta in monkey, 141 count(s) —continued spleen, 141, 155-157, 163, 164 chicken, 216, 219 striated muscle fibers, between, 141 duck, 216, 219 thymus. 141 embryo, 114, 133 walls of major vessels, 141 goose, 216 Hematopoietic system, labile in birds, 29 hemocytometer, 233 Hemin, preparation, 231 pheasant, 217 Hemocytoblast, 166 pigeon, 217, 219 Hemocytoblastosis, 7 diurnal rhythm, 220 Hemocytometer, counting chamber, 31, 88 turkey, 219 Wiseman method, 232, 233 developmental stages, 74, 86, 193-197 Hemoglobin terminology, 194 benzidine test, 115 duck, 207, 208 Ralph's technic, 231 effect of development, tissue culture, 104 citric acid, 88 erythroblast, 24, 26 irradiation, 73, 86 erythrocyte stages Petrunkevitch No. 2, 80, 83, 195, 209 polychromatic, 26 evolution, 73 poor criterion for, 25 granules precocious accumulation, 129 difference from serum, 41, 89 independent of magenta, 86, 195 basophilic staining, 29, 118 mesomyelocyte, 154 cell differentiation, 117 on erythrocyte, 146 level, 214^220 granuloblast, 10, 12, 74, 86 chicken, 216 immature, 74 embryo blood, 114 index, Arneth, 84, 85. 90 sexes, 216 mature, 10, 12, 73-74. 80, 83-86. 111. 144. 146. 154, compared, 216 155, 163, 172, 182. 188, 191. 200 sparrow, 211-212 mesomyelocyte, 10. 12, 74, 86, 144, 146. 154. 158, variability, 217 160. 188. 191. 195. 200 primary erythroblast metagranuloblast, 10. 12, 188. 191. 200 extravascular, 113 metamyelocyte, 10, 12, 142, 155, 188, 195, 196, 200 precedes cell differentiation, 112 nucleus takes on, 112 chromophobic band, 20, 32, 87 primary erythrocyte, 117 criteria for counting lobes. 84 precocious accumulation, 104 lack of staining. 83. 90. 195, 207 similar to, 192 promyelocyte, 10, 12, 74, 86, 142, 154, 160, 170. 188, reduced after splenectomy, 29 195. 200 technic, 231-232 ratio to eosinophils conversion factor, 232 guinea hens, 207 wavelength, 231 kingfishers, 207 thrombocyte, absent from early embryo, 131 mallard ducks, 208 Hemokonia rod( s) relation to serum granules, 93 arrangement in cell. 74 what is included, 93 artifact, 73, 87-89 Hemorrhagic syndrome, 45 as debris Heterophil (s), 73-74, 83-89 erythrocyte, 146 abnormal cell, 87 osteoclast, 198 adequacy of staining, 195 central granule, body. 86. 89, 195. 210 artifact, 73, 74, 87-89 absent. 86, 89 bone marrow, 142, 144, 146, 154, 155, 182. 188. replaced by vacuole, 74 194-196 significance. 83 clumping, 154 development, 195 compared with eosinophil, 83, 87-88, 90, 207, 208 dissolved in cytoplasm, 74, 83, 87, 88. 89, 90 count(s) significance, 83, 88 Arneth, 84, 85 duck, 207 birds, except chickens. 220-221 effect of relation to mortality, 85 citric acid, 88 sex values, 220 fixation, 88 bone marrow, 200 formalin, 88 dominant cell, 199 malarial parasite, 89

256 —— — ———77

Heterophil (s) —Continued Immature cell(sj —Continued See also Embryo; Hematopoietic organ; specific rod ( s) —continued effect of—continued cells Petruni^evitch No. 2, 83, 87, 209 Inclusion (s) salt solution, 88 intracytosomal trichloracetic acid, 88 appear to be, 6 water, 88 in macrophage, 139 hemokonia, relation to, 93 See also Granule; Mitochondria; Specific gran- in tissue section, 87 ule; Vacuole in cytoplasm and cytosome literature on, 88 intranuclear metachromasia, 195 appear to be, 6 orange, eosinophilic spheres, 86, 154, 156 margination, 4 shape, 73. 74, 88. 208 vacuole, 23, 195 turkey, 210 Incubation age, 105 See also Eosinophil, granules rise and fall of erythrocytes, 113, 114 series, 10, 12, 188, 200 time table, 112 shape, 73 Infection size, 73, 90 embryo, Plasmodium, gallinaceum, effect on dif- frequency curve, 80 ferential count, 133 range. 80, 214 toxic granules, 87 smudged, 74, 89, 172 Infectious mononucleosis, 64 spleen, 163, 164, 165 Intravascular terminology, 86, 89 environment, 113 turkey, 207, 209.210 origin of Hetherington and Pierce (1931), 181 erythrocyte, 104, 113. 128, 141 141 Hevesy and Ottesen ( 1945) , 32 thrombocyte, 128, Hewitt (1940), 28,89 In vitro, 2 (1942), 207,219 differentiation of erythrocytes, 29 Hof, 71, 142 5ee also Tissue culture abnormal, 60, 72 In vivo, 2 absent from test for amitosis, 31 lymphocyte, 49 Irradiation monocyte, 58 effect on association with rosette, 69 blood, small heterophils increased, 73 definition, 69 erythrocyte, 24, 27 orange spheres in monocyte, 12, 49, 52, 60, 69, 71, 80 immature heterophils, 86 present in separating lymphocytes, and monocytes, 48 monocyte. 12, 49, 52, 58, 69 thymus, increased plasmocytes, 166 plasmocyte, 166, 197-198 Isaacs (1925), 33 present sometimes in (1928), late immature monocyte, 12 lymphocyte, 49, 71 Johnson and Conner (1932), 51 Homology andLange (1939), 219 basophil, bird and other vetebrates, 91 Jones (1943),8, 116, 192 erythrocyte, mature, bird and mammal, 28 (1947), 115,116 Hydrocortisone, increased lymphocytes, 51 (1948), (1949), 194 Jordan (1936), 181 Iguana tuberculata, phagocytic thrombocyte, 45 (1937), 181 Illumination, darkfield, 3 (1938), 8, 167 Immature cell(s) and Robeson (1942), 29, 181 appearance, different in organ of origin, 7, 16, 86 JuhnandDomm (1930), 218 circulating blood, post hatching, 23, 24 Junco all ages, 24 erythrocyte count, 218 compared with mature erythrocytes slate-colored {Junco hyemalis), 202 oxygen consumption, 29 specific gravity, 29 Kakara and Kawasima (1939) , 218 eosinophil, 76, 91 KalabukhovandRodionov (1934), 211 heterophil, 74, 86 Kasarinoff (1910), 45,47 in mammals, suggestive of pathology, 24 Keller (1933), 211 normal cells, 15 Kelly and Dearstyne (1935), 215 wide variety, 15 Kennedy and Climenko (1928), 73 257 —

Keratin Lucas 1 1940). 4, 30 bodies in down-sheath cells, 176, 180 (1946),64, 87, 134 process of keratinization, 179 (1949), 64, 181 Kindred (1940), 8, 114 (1950), 181 Kingfisher (1951), 181 eosinophil, 207 and Breitmaver (1949), 181 heterophil, 207 Craig, and Oakberg (1949), 64, 181 Kinglet, golden-crowned [Regulus siilrapa), 202 and Denington (unpublished data), 215, 21c

Kirschbaum and Downey (1937), 4. 8 and Denington ( 1956) , 48, 195

Kitaeva (1939), 211, 218 and Herrmann ( 1935) , 4 Kniselyetal. (1947), 31 and Oakberg (1950), 64, 181 Knowlesetal. (1929), 41 and Riser (1945), 4

Kracke and Garver ( 1937 , 72, 93 -etal. ) 8, (1954),64, 156, 181 Kyes (1915), 139 Lundquist and Hedlung (1925), 83. 88, 90 (1929), 39, 73 Lymphoblast(s), 9, 10, 12, 164 nucleolus Laboratory absent, 169 lost, 168 erythrocyte, 17, 23 thymus, quarantine, 179 54, 170, 172, 174 rare in older gland, 167 smudged cells, 72 transition from See also Chicken, source mesenchyme, 168 Lymphocvte(s), Larus ridibundus. See Gull 9. 10, 12, 18, 45, 47, 48, 50, 62, 146. Leishman's stain. 5ee Stain and staining 165, 216 abnormal, 64-65 Leptochromatic. See Nucleus 54, 56, chromophobic areas, 56 Lesbouyries (1941), 86 Leukemia hypertrophy and vacuolization, 64 magenta bodies, 54, 64 cell modified in, 113 age, 53-54 in fowl area, 66, 212, 213, 214 lymphocyte, large, 50 chicken smudged nucleus, not diagnostic, 72 different breeds and stocks, 62, 212, 213 in mammal similar to other avian species, 210 lymphocyte series from lymphoblastic leukemia, count (s) 53 birds, wild, 219 man, toxic granule, 87 chicken. 216 mouse, cell, smudged 39 duck. 216, 219 resemble immature cells, 112 goose, Canada, 216 simulation of, 141 pheasant, ring-necked, 217 See also Avian leukosis complex; Lymphomatosis pigeon, 219 Leukocyte (s) Laboratory, 217 count (s) sex, 216 218 chicken, 216, 219 turkey, 219 methods for, 232-234 cytoplasm sex, 216, 218 blebs, 31. 50,51,54, 80. 1.55 variability, 217 antibodv formation, 50 hyaline, 50, 51, 54 granular, 10, 73-92, 193-197, 207 hypertrophy, 56 in huffy coat, 232 magenta bodies, 54 nongranular, 10, 47-73 compared with azurojjhilic bodies, 54 origin, extravascular, 141 structure, 49, 52, 71 spherical bodies, 33 development. 174 types in avian blood, 47, 73, 86, 88 to macrophage, questionable, 64, 134 See also specific leukocytes to monocyte, questionable, 47, 48 Light See also Lyniphocytogenesis infrared, 226 developmental stages, 10 drying slides with, 227 in bone marrow, 199, 200 microscope, 222-225 in circulating blood, 53-54 tungsten arc, 222 in spleen, 165, 166 Liver in thymus, 168, 169, 172 effect of splenectomy, 29 effect of macrophage from, 139 fixation, 80 Loewenthal (1930), 86 splenectomy, 29 258 —

Lymphocyte (s) —Continued Lymphocyte ( s) —Continued immature, 10, 12, 53, 163, 166. 167, 169, 170, 172, small, 9, 10, 12, 48, 50. 66, 80, 163, 167, 169, 172, 174, 199, 200 174, 182, 191 in blood spot, not a macrophage precursor, 134 dwarf in bone marrow, 155, 199, 200 different from small lymphocyte, 130

formation due to splenectomy, 29 ' related to thrombocyte, 130 in spleen, 164, 165, 166 microlymphocyte, 181 in thymus, 166, 168, 169, 172, 174 produce other cell types, 169 in tissue cuhure stem cell, 48, 104, 155, 166, 169 degenerated, 181 thrombocyte, separation from, 45 no cell division, 181 Lymphocytogenesis, lymphopoiesis, 166, 168 no differentiation, 181 in spleen, 164, 165 infectious mononucleosis, 64 in thymus, 54, 167-169 irradiation, 48 nucleolus absent, 168 large, 9, 24, 48, 50, 56 reticular cell precursor, 168 range, 66 Lymphoid loci, abnormal, in various organs, 181 rare in differential count, 24, 48, 50 Lympholeukocyte, same as monocyte, 47 magenta granules, 47, 50, 54 Lymphomatosis, 86 in mitosis, 53, 54 blood picture, not an indicator of, 45 in wild birds, 210 in embryo, 133 mature, 9, 10, 12, 111, 163, 167, 168, 172, 174, neural, 45, 51, 85 199, 200 ocular, 51, 85 normal, 50-53 relation to not a blast cell, 48 Arneth count, 85 medium, 9, 10, 12, 48, 50, 163, 167, 168, 169, 172, lymphoid foci in spleen, 181 174, 182, 191 resistance, 211, 215 an immature cell, 166, 167 susceptibility, 211, 215 range, 66 visceral, 51, 85 mitosis, 49, 71, 170 See also Avian leukosis complex monocyte, comparison with, 47, 48, 52-53, 71, 214 Lysin, action on erythrocyte, 26 nucleocytosomal ratio, 49, 51-52, 53, 68, 71, 214 nucleus McDonald (1939), 154 chromophobic, 32, 40, 56 McGuire and Cavett (1952), 219 mitosis, 54 Machado (1951), 233 naked, 163 Macrophage larger than thrombocyte, 210 blood spot fibroblast, precursor for, 134 position in cell, 51, 71 lymphocyte, not a precursor for, 134 reticular, 56 cytoplasmic spheres, pinched off, 134 shape, 49, 52, 62, 71 embryonic, 103, 139 size, 50, 62, 214 common, 7 to 12 days' incubation, 134 frequency distribution, 66 in circulating blood staining poor, 168 magenta granules, 134 structure, 71 53, response to infection, 133 167 relation to differentiation, 54, 166, too large for capillaries, 134 reactive cell vacuoles, 134 cytoplasm vacuolated, 56 yolk sac, 130, 134, 139 macrophage, no transition to, 134 nucleus, large, 134 wild birds, present in, 210 granulation delicate, 134 series, 10, 12, 164, 200 endodermal, 134, 139 in bone marrow, 200 endothelium, 139 in thymus, 166, 174 hypertrophied lymphocyte, compared with, 64, 134 with monocyte, 47, 48 in bone marrow, 200 shape, 49, 50-51, 71 in spleen, 139, 140, 164, 165 blebs, 51, 54 in thymus, 170 lobes, 50, 51 inclusions, 139 pseudopodia, 51 nucleolus present, 139 size,49, 50,53,62, 71 post mortem effect, 139, 140 chicken, breeds, 212, 213, 214 See also Phagocyte distribution, 66 Magath and Higgins (1934), 17, 27, 41, 67, 86, 208, lack of significance, 66-67 215. 219. 231

259 — — — — —

Magenta granule, body, ring, sphere Mareca americana. See Duck, baldplate associated with Marvin (1954). 199 avian leukosis, 64 Mast cell, 47, 91 health of bird, 65 Maturation rate, erythrocyte, 29-30 lymphocyte, 52, 53, 64 Mature pathologic significance, 64, 65 basophil, 10, 13, 78, 91, 111. 146. 155. 172, 182, chroniidial type, 131 191, 200 granule, different from specific granule, 197 azurophilic granule, 49 eosinophil. 10, 13, 76, 89, 158. 200. 208 magenta ring, 195 erythrocyte, 10. 11. 17, 18, 20. 23. 25. 26, 28, 29. in myelocyte, description of, 195 108, 111, 146, 182. 184. 198. 200 new term. 54 later embryonic. 103. 106, 113, 114, 124. 142. 144 occasionally present in primary, 100, 103, 106, 113, 120, 127. 142 embryo thrombocyte, 131 heterophil. 10, 12, 18. 20, 47, 73. 74. Ill, 144. 146, heterophil mesomyelocyte, 12 154. 155, 163, 164, 167. 172. 182. 188. 199. 200 monocyte, 71 lymphocyte, 10, 12. 18. 48, 50, 54, 111. 163. 166, present in 167, 168, 172, 174, 191, 199, 200 basophil promyelocyte, 13, 196-197 macrophage, embryo, 139 heterophil monocyte.'lO, 12. 49. 58, 72. 111. 200 mesomyelocyte. 154 osteoblast. 10. 13. 142. 152. 166 promyelocyte, 12, 74, 156, 188, 194 plasmocyte, 10. 14. 198. 199, 203 lymphocyte thrombocyte, 10, 12. 18, 33, 42, 45, 111. 186, 191, abnormal, 49, 54 193, 200 in mitosis, 53. 54 embryo. 136

wild birds, 210 Maximow'l 1909 ) . 8, 104. 116 mononuclear osteoclast. 148 and Bloom ( 1931 ) , 47 mullinuclear osteoclast. 142, 153 May-Griinwald Giemsa. See Stain and staining primordial osteogenic cell, 142 Meda\var (1941). 167 relation to Megakaryocyte. 41. 42, 153 azurophilic body, 54, 71 Megaloblast, 115, 192 orange spheres, 195 produces thromboblast, 130

5ee also Heterophil : Lymphocyte synonymous with Magnification erythroblast, 116 hish power. 4, 14 karyoblast, 9 lowpower,4, 14, 15, 17, 32 Mehner (1938). 211 optical. 14 Meleagrididae, 202 projected, 14 M elect gris gallopavo. See Turkey, domestic scale, 4 Mesenchyme, 104 Mainland et al. (1935), 66 nucleolus present. 168 Makinodan (personal communication), 166 tissue, 9. 153, 168 Mammal, mammalian, man Mesomyelocyte, 156 Arneth count. 221 basophil, 10. 13, 106, 182, 191, 197. 200 basket cell. 72 eosinophil, 10. 13, 76, 91, 200 blood, 25, 73, 84 combined with promyelocyte, 10, 13 erythrocyte heterophil. 10, 12, 74,' 86, 91, 144, 146. 154, 157, aged cell, 30 158, 160, 188. 200 maturation delay, 30 Metachromatic granule, 91 protoplasmic process. 41 heterophil, 195 reticulocyte, 28 Metagranuloblast(s), 142, 144, 154, 156, 158, 160, giant cell, 153 182, 194, 197 leukemia cells resemble chick immature cells, 112 lymphocyte, 49 basophil, 10, 13 azurophilic granules, 64 combined with promyelocyte, 10, 13 stages from lymphoblastic leukemia, 53 cytoplasm, vacuolated, 194 monocyte and lymphocyte from same organ. 167 early immature monocyte, different from, 167 neutrophil, 86 eosinophil, 10, 12, 156, 194, 196, 200 band and juvenile cells, 155 granuloblast, changes from, 194 toxic granules, 87 heterophil, 10. 12, 86, 92, 156, 191, 194. 196, 200 platelets, 41 nucleus, eccentric, 154 serum, fewer granules than in birds, 41 Metamyelocyte, 182 smudged cells, mouse, 39 basophil. 10. 13. 200 spleen, 156 eosinophil. 10. 13, 76, 91, 160. 196. 200

260 —

Metamyelocyte—Continued Mitochondria, mitochondrial—Continued heterophil, 10, 12, 86, 142, 155, 188. 195, 200 spaces—continued term, 196 present—continued Methyl alcohol. See Fixation and fixatives plasmocyte

Michaelis and Wolfe (Doan, 1932 j , 64 early immature, 13, 198 Michels (1938), 91 late immature, 13 Microblasts, 130 primary erythroblast, 115 Microburner, making cannula, 225 primordial osteogenic cell, 13 Microcyte, 34 Mitosis dwarf lymphocyte, 130 cartilage cell, 153 related to throiTibocyte, 130 embryo Microscope erythrocyte, 129 condenser, 222, 224, 225 in circulating blood, 118, 129 alignment, 223 thromboblast, 134 effect of lowering, 223 thrombocyte, 131 separable lenses, 225 erythroblast, 156, 192 use in separating leukocytes and thrombocytes, heterophil metamyelocyte, 188 234 immature cell in spleen, 158 diaphragm, 222, 223 in bone marrow, 200 lamp late in differentiation process, 191 bulb, frosted blue, 224 lymphocyte, 53 diaphragm, 223, 224 polychromatic erythrocyte, early, 100 filter, 222, 223, 224 primary erythroblast, 94 illumination abnormal, 117 critical. 222 chromosomes, 117 Kohler, 222 primary polychromatic erythrocyte lens, 222 early, 96 li£?ht, 222-225 late. 98, 157. 160 "flare, 224 mid-, 96, 160 mirror, 222, 223 primordial osteogenic cell, 142 front surface, 223 reduction of yolk plates, Amblystoma, 105 objective, 14, 15 technic for, 230

numerical aperture ( N. A. , 224 Mitotic periodicity, lacking in embryo, 105 ) parcenter, 223 Mjassojedoff (19261, 166. 197 parfocal, 223 Monoblast, 169, 200 ocular, eyepiece, 14, 15, 222 not seen, 12 pinhole. 223 possible monoblast, 163

phase on heterophil. 87 Monocyte ( s ) . 10, 47, 65-73 use of, 222-225 artifacts, 60. 72-73 Microscopy cell darkfield illumination, 3 abnormal, 60 electron, 2 area, 66, 212 phase, 3 count(s) Mitochondria, mitochondrial chicken, 216 in mitosis, 115 farm stock. 216, 219 in reticular cells, 169 duck, 216, 219 loss in goose, Canada, 216 lymphocytogenesis, 54, 169 pheasant, ring-necked, 217 osteoblast, 152 pigeon, 217, 219 primary erythrocyte development, 118 sex. 216, 218 spaces turkey, 219 absent, erythrocyte division, 49, 70, 71 early polychromatic, 24 shape, 49, 63, 67-68, 71 mid-polychromatic, 11 size. 49. 63, 65-67, 71 present chickens, different breeds, 214 embryo thromboblast, 131 frequency distribution, 67 erythroblast, 11, 129 range, 66 erythrocyte cytoplasm early polychromatic. 11, 24, 192 azurophilic granules, 58, 69, 229 mid-polychromatic, 25, 26 blebs, 67 monocyte, early immature, 71 color, 58, 68 osteoclast, mononuclear, 13 ground-glass effect, 65, 68

261 —

Monocyte (s) —Continued Neltion carolinense. See Duck, green-winged teal cytoplasm—continued Neutrophil, heterophil without granules, 86 Hof, 49, 71, 80 Niceetal. (1935), 218 definition, 69 Nittis (1930), 33 orange spheres, 68, 69, 71 Nongranular leukocytes, 47-73 vacuolar spaces, 69 lymphocytes, 50-65 hyaline mantle, 58, 67-68 monocytes, 65-73 specific cell inclusion, 69 theory of origin, 47 structure, 68-69, 71 Nonidez (1920), 141, 165 developmental stages Normoblast, no equivalent stage in birds, 28 in circulating blood, 60, 70-72, 167 Nucleocytosomal ratio amoeboid cell, 60, 71 eosinophil, 76 in spleen, 163, 167 erythroblast, primary, range, 117 fixation, Petrunkevitch No. 2, 80 lyniphocjte, 49, 51-52, 66, 68, 214 immature, 70-72 monocyte, 49, 66, 68, 214 early, 10, 71, 163, 200 Nucleolus (i) late, 10, 71, 163, 200 absent or not visible lymphocyte erythrocyte arises in same organ, 167 definitive mid-polychromatic, 11 compared with, 49, 65, 66-67, 70, 71, 214 mature, 17 continuous series between, 47-48 granuloblast, 6, 12, 74, 169, 194 mature, 10, 111, 200 lymphoblast, 6, 12, 169 normal, 58, 63, 65-70 lymphocytogenesis, 168 typical, 58 monoblast, 169 metagranuloblast, different from, 167 thrombocyte, large embryo, 11 nucleocytoplasmic ratio, 49, 58, 68, 71, 214 multiple nucleus macrophage, 139 area, 66, 214 primary erythroblast, 9, 116, 117 bilobed, 58, 70 plasmosome, 4, 11, 157 double, 58, 70 in reticular cell, 169 indented, 58, 70 present position, 49, 68, 71 erythroblast, 6, 11, 24, 56, 169, 184 shape, 49, 63, 69-70, 71 embryo, 128 size, 63 primary, 9, 116, 118 frequency distribution, 67 macrophage, 139 range, 66 osteoblast structure, 49, 70, 71 immature, 13 owl, 210 mature, 13 series, 10 osteoclast continuous with lymphocyte, 47 mononuclear, 13 in bone marrow, 200 multinuclear, 13, 153 smudged, squashed, 45, 60, 72 reticular cell. 168 Monocytosis, 165 thromboblast. 6, 56, 164, 169, 186

Murray (1932) , 104, 116 embryo, 131 Muscle cell, striated, 211 present sometimes Myeloblast(s),9, 10, 12,163 erythrocyte, polychromatic Myelocyte (s) early, 11, 192 early thrombocyte, compared with, 191 late, 24 mid-, 24 eosinophil, 91, 164 thrombocyte, early immature, 11 extravascular origin, 141 visibility increased heterophil, 7, 83, 91 by karyolysis, 139 osteoblast, similar to, 152 by weak staining of nucleus, 129 terminology, 197 194, Nucleoplasm tissue culture, no division, 181 lymphocyte, colorless, 53 See also Mesomyelocyte; Metamyelocyte; Promye- monocyte, 70 locyte tinged by dissolved basichromatin, 34 See also Nucleus

Natt and Herrick (1954) , 87 Nucleus (i) Neave (1906),41, 208 age of cell, measured by, 23. 25, 30 Necturus, 31, 40, 45, 68, 117, 167 artifact Nesterow (1935). 26 basophil, 92

262 Nucleus ( ) Continued Nucleus (i) —Continued i — pachychromatic continued artifact—continued — heterophil, 73, 74, 195, 196 thrombocyte vacuoles in, 134 embryo, 11 boundary mature, 12 indistinct position in cell embryo central erythroblast, 129 basophil metamyelocyte, 13 thromboblast, 134 erythrocyte, 17 heterophil, promyelocyte, 12, 74, 195 granuloblast, 154 metagranuloblast, 156-157 lymphocyte, 52 mature, 68 indistinct sometimes, heterophil niesomyelocyte, monocyte, 12 osteoclast, mononuclear. 13 chromatin eccentric 13 extruded, 34 basophil promyelocyte, metagranuloblast, 12 magenta body, possibly related to, 131 eosinophil strand. 34. 158, 170 erythrocyte, 20 chromophobic heterophil metagranuloblast. 12 band lymphocyte, 52 156. 194 erythrocyte, 20, 32, 34, 36 metagranuloblast, 154, heterophil, 20, 87 monocyte lymphocyte, 40, 56 immature, 12 cause unknown, 32, 40 mature, 68 reaction, 32 osteoblast, 152 squashed, 40 immature, 13 comparison plasmacyte, 14, 198 chromatin networks, 6 thromboblast, 131 monocyte compared, 49 external and internal appearance, 4, 6, 7 lymphocyte and erythrocyte, 18, 20 shapes, 23 protrusion, 17, dead, 176, 179 pycnotic 30 elimination of, value to cell, 32 erythrocyte, 20, 23, erythrocyte, typical, 17, 23 primary, 127 deviation from, 17, 23, 34 thrombocyte, 103 fixation effect, 80 shape basophil, 92 aged erythrocyte, 23 with chromatin clumping, erythrocyte, ghost. 179 associated 17, 23, 211 granules of basophil mask, 7, 92 heterophil metamyelocyte, 12 inclusion, 4 bean shaped, vacuole, 23 constricted karyolysis, reveals nucleolus, 139 basophil, 92 13 karyorrhexis, 120, 127 eosinophil metamyelocyte, leptochromatic, lightly stained, open erythrocyte chromophobic, 32, 34 mature, 17, 18, 20 120 erythrocyte, mature, 11, 17, 23, 211 primary, monocyte, 49, 53, 70 monocyte, immature, 60 elongated, erythrocyte, mature, 23 multiple indented embryo thrombocyte, 136 eosinophil metamyelocyte, 13 erythrocyte, mature, 17, 23, 24, 27 erythrocyte monocyte, 70 late polychromatic, 26 osteoclast, 142, 152, 153, 198, 204 mature, 17, 18,20,23,24 primary erythrocyte, 127 lymphocyte. 52 naked, 146, 155, 158, 170 monocyte, mature, 12. 49. 58, 70 nonstaining, 7 lobed nucleoplasm Arneth counts. 80. 220-221 nonstaining, 26 basophil. 13. 80. 92 stained, dissolved basichromatin, 34 eosinophil, 13, 80 pachychromatic, darkly stained, dense erythrocyte. 27 erythrocyte, 11, 17. 20, 23, 210, 211 heterophil, 12, 74. 80 counting. 84 aged, 11 criteria for monocyte, immature, 60 monocyte, 70 erythrocyte, mature, 11, 17, 23 plasmocyte, late immature, 14 oval, 263 — )

Nucleus (i) —Continued Orten (1934), 28, 29

continued and Smith ( 1934 , shape— ) 27 round Osgood (1935), 64 erythrocyte, 18, 20 (1938), 25 late polychromatic, 11 and Ashworth (1937), 8, 9, 72. 87, 127 lymphocyte, 49, 52 Baker, and Wilhelm (1934), 27

1 , monocyte, 49 and Wilhelm 1931 ) 231 osteoclast, multinuclear, 13 Osprey, erythrocyte, 212 thrombocyte, mature, 12 Osteoblast! s), 164 variability of count, 200 eosinophil metamyelocyte, 13 development from primordial osteogenic cell, 152 erythrocyte, late polychromatic, 26 immature, 10 lymphocyte, 62 large young, 142, 144, 152 monocyte, 63 mature. 10, 142 size resembles plasmac\ te, 166 erythrocyte. 211. 212 small. 152 large, osteoclast, multinuclear, 13 term, 152 lymphocyte, 62, 66, 213 variation with heterophil, 164 monocyte, 63, 66, 67, 214 Osteoclast (s small acidophilic granules in, 148, 152, 204 erythrocyte. 18, 20 binuclear, 142 plasmacvte, late immature. 14 counts. 200 thrombocvte. smaller than lymphocyte. 43 development from primordial osteogenic cell. 152 smudged, squashed. 18, 34, 146, 170, 172, 182 giant cell, only one t)'pe in bird marrow, 153 appearance, 39-40 indented by granulocyte. 142 erythrocyte, 36, 39 magenta spheres. 148, 153, 198 heterophil, 74 mononuclear, 10, 13, 142. 148, 152, 153, 164 on adjacent cells, 39 multinuclear. 10. 13, 142. 153, 198, 204 stained poorly nucleolus. 153 erythroblast. embryo, 129 polarity, 198 revealed nucleolus, 129 series, 10, 13 erythrocyte term, 152 embryo, 122 Osteocyte(s) primary, 120 series, 10, 13 feather sheath cell. 179 term, 152 heterophil, 74, 83, 90 Osteogenic cell. See Primordial osteogenic cell lymphoblast, 168, 174 Owl thrombocyte, embryo, 131 Arneth count, 221 structure basophil smaller than in chicken, 210 differences among metagranuloblasts, 194 eosinophil, granule, 210 erythrocyte, embryo, 128 great horned {Bubo viriiinianus) ,202 impression of, 70 monocyte large, 210 inside and outside. 40 thrombocyte larger than in chicken, 210 lymphocyte, 49, 52, 70 Oxygen consumption, greater in immature cells than so-called large, 56 monocyte, 49, 70 in mature, 29 See a/50 Amitosis; Chromatin; Mitosis; Nucleocyto- somal ratio; Nucleolus; specific cells Pachvchromatic. See Nucleus

Numerical aperture (N. A.) , 224 Palmer and Bielv (1935a) , 215, 217 Nuthatch, white-breasted iSitta caroUnensis ), 202 (193.5b). 215 erythrocyte nucleus. 212 Pappenheim I Dantschakoff. 1908b), 26 thrombocyte size, 210 Paridae, 202 Passer— 181 Oakberg (1949). 64, domesticas. See Sparrow (1950), 181 montanus. See Sparrow (1951), 64, 181 Passeriformes. 202 Objective. 5ee Microscope Passerina cyanea. See Bunting, indigo Ocular. See Microscope eosinophil like that of chicken, 210 Olson (1937). 84,215 Passerine, (1952), 64, 84 Pathology Orange stained. See Heterophil; Monocyte; Osteo- air sac, white spots, 51 clast blood poisoning, 85

264 ) ——

Pathology—Continued Pigeon—Continued crop Laboratory, 202 atonicity, 51, 65 macrophage from endothelium, 139 impaction, 65 reticulocyte, 27, 28 dehydration, 65, 85 staining, 231 emaciation, 61, 65, o5 serum, lipochrome pigment, 93 enlarged heart, ascitic fluid, 85 thrombocyte, 42, 45 gasping, 51, 65 confusion with lymphocyte, 210 lymphomatosis value of smear technic, 154 neural, paralysis, 65, 85 Pipilo erythrophthalmus. See Towhee ocular, iritis, gray eye, 51, 85 Planimeter. 212 visceral, 65. 85 Plasma. See Serum prolapsed uterus. 51, 85 Plasma cell(s), 197-198 urates in kidney, 51 See also Plasmacyte

( s , 10. 13 See also Avian leukosis complex; Lymphomatosis Plasmablast )

, identity, 197 Pavne and Breneman (1952 1 181 Peabody and Neale (1933), 27, 231 Plasmacyte( s) Penlliestes atiicapillus. See Chickadee, black-capped characteristics, 166 Petrunkevitch (1933), 230 immature Petrunkevitch No. 2. 5ee Fixation and fixatives early, 10, 13, 163. 197, 198. 203 Phagocyte, phagocytic late, 10, 13, 163, 203 embryo, response to infection, 133 in bone marrow, 166, 197, 200, 203 endothelial cells, 139 in loose connective tissue, 197 thrombocyte. 45 in spleen. 166, 197 Sec also Macrophage mature. 10, 165. 203 Phasianidae. 202 resembles osteoblast. 166 PImsianus colchicus. See Pheasant, ring-necked series. 10, 13. 200, 203 Pheasant Plasmodium gallinaceum, increased heterophil num- 133 basophil, 92 ber, blood viscosity, 210 Platelets. 41 count (si clumping, 15 Arneth, 220 See also Megakaryocyte ; Thrombocyte differential, 217 Poikilocytosis. 31, 34 eosinophil, 219 Polychromatic variability, 215 color 24, eosinophil, 210 acidophilic and basophilic stains combined, 25, 117 ring-necked iPhasianiis colchicus) , 2()2 thrombocyte size, 210 terminology, 117 Phenylhydrazine hydrochloride, 29 measure of development, 25 Phloxine series. 26 affinity for hetero])hils and eosinophils, 233 5ee also Erythrocyte reticulocyte granules, 28 in Wiseman's method. 232. 233 Precipitate on slide, resembles Photomicrography, adjustment of condenser, 225 Premyelocyte, 92 Picidae, 202 Price-Jones (1910), 33 Piciformes, 202 Primary Pigeon erythroblast. See Erythroblast Erythroplastid basophil, 210 erythroplastid. See count (s Primordial Arneth, 221 cell. .See Cell differential, 217. 219 germ cell. 139 paratyphoid, 219 larger than blood cells, 140 eosinophil, 219 Primordial osteogenic cell(s), 9, 10, 13 erythrocyte, 218 count, 200 in bone marrow, 199 distinct cell type, 152 sexes compared, 218 in mitosis, 142, 144 thrombocyte. 219 magenta granules, 144, 148 variability, 215 nucleolus, 152 effect of splenectomy. 29, 181 produces erythrocyte, 210 osteoblast, 13. 152 crossed cells, 210 osteoclast, 13, 152 hemoglobin, sex difference, 218 plasmablast, 13, 197 hemolysis, 39 resembles reticular cell, 168 265 ——— — —— —

Primordial osteogenic celUs) —Continued Pycnosis, pycnotic smudged, squashed, 146, 148, 152 nucleus, 30 stem cell for erythrocyte, 20, 23, 30 plasmoblast, 13, 197 thrombocyte, 45 vacuoles, 148 vacuole formation. 30 Problems for study absolute numbers of cell types in embryo, 114. 115 Quail, Arneth count, 221 aging in erythrocytes, 30 Quarantine, Laboratory, 179 Arneth index for normal chicken, 85 cellular reactions and mortality, 65 Rabbit central granules (Always present in heterophil erythrocytes injected into. 26 rods?). S3 74, immature cells of chicken and, 29 chroniophobic nucleus, 32 Ralph (1941), 231 chromosomes of primary erythroblasts, 117 Ram (19491,233 common identity of vacuoles in smears and sections Rat, reticulocyte, 27 in primary erythroblasts, 116 Reactive cells contribution of plasma to differentiation of cells, 112, lymphocytes, 56, 134 113 wild species, 210 cytology of incipient lymphomatosis, 45 thrombocytes, 38, 45. 46. 47 defense mechanism of embryo, 139 Red blood cells. See Erythrocyte equivalent of toxic granules in avian heterophils, 87 Rees and Ecker ( 1923 1 . 233 extrusion of nuclear contents. 34 Rees-Ecker method. 233 granulocytes of embryo (Do they become definitive Regional Poultry Research Laboratory, 211, 212, 213, cells?), 165 214, 215, 216 heterophil rods (Are they faithfully preserved in Regulus satrapa. See Kinglet, golden-crowned tissue sections?), 87 Reptile (s) heterophils under phase microscope, 87 Iguana tuberciilata, phagocytic thrombocyte, 45 magenta granules, association with reticulocyte, 28 nucleus. 71 turtle pathologic conditions in lymphocytes, 64. arrangement of rods in heterophil, 74 microchemistry of vacuoles in primary erythrocytes, box. nucleolus in immature heterophil, 6 116 Resting amoeboid cell, 139 normal reticulocyte values, 27 wandering cell, 139 normalcy of leukocytes in embryo circulation. 133 Reticular cell(s) physiology of thrombocyte disruption, 42 acidophilic cytoplasm, 169 plasmosome nucleolus during mitosis. 157 in bone marrow. 200 preservation of basophil granules, 197 in embryo vessels, 139 production rates in hematopoietic organs, 154 in spleen. 164 reactive thrombocyte, significance, 45 in thymus, 169, 172 relationship of azurophilic and magenta granules, 71 precursor for separation of lymphocyte, 164. 168 abnormal cells from artifacts. 33 macrophage, 134 immature from medium lymphocytes, 166 plasmoblast, 13 significance of resembles erythroplastid formation, 31 mesenchyme. 168, 169 nuclear density in erythrocytes, 23 primordial osteogenic cell, 168, 169 size of heterophil, relationship to aging. 73 small nucleus. 169 technic to preserve thrombocytes, 132 Reticular tissue. 9. 169 thrombocyte degeneration under phase microscope. Reticulocytefs). 10. 11.23 47 chicken, 28, 29 thrombocyte development from sections, 157 color, tawny, 193 Progranulocyte. 9 effect of Promyelocyte. 9, 156, 194 folic acid, 28 atypical, 168 splenectomy, 29 basophil. 10, 13, 78, 92, 197, 200 fish, 28 combined with metagranuloblast, 10, 13 frog, 28 eccentric nucleus. 156 granules. 11,23,28 eosinophil, combined with mesomyelocyte. 10. 12 all stages of erythrocyte development, 28 heterophil, 10, 12, 74. 86, 142, 154, 157, 160. 188, change with maturity, 27 200 percent Protopterus ethiopicus, 167 chicks, 28 Pseudoeosinophil, 47 children, 27 266 — — )

Reticulocyte (s) —Continued Similarity (ies) percent—continued basophil and lymphocyte, 211 ducii, mallard, 27 birds and mammals pigeon, 27 erythrocyte rat, 27 protoplasmic processes, 41 pigeon, 28 specific gravity for mature and immature cells, primary erythrocyte, 127 29 reptile, 28 function of platelets and thrombocytes, 41 respiration of, 29 monocytes from lymphogenic organs, 167 separation from mature cells, 29 chromosome lag in primary erythroblasts and neo- staining, 230-231 plastic cells, 117 effect of time, 28, 231 eosinophil and heterophil, 83 Reticuloendothelial cells, spherical bodies, 33 erythroblast and thromboblast, 128 Rhian. Wilson, and Moxon (1944), 215 failure of nucleus to stain, 131 Richards (1938), 222, 224 primordial osteogenic cell and reticular cell, 168 (1949), 222 smear of spleen and thymus at 35 days, 169

• (1954), 222 thrombocyte, degenerated, and a lymphocyte, 131

Richardson (1937), 28 Siskin, pine (Spinus pinus . 202 Richmondena cardinalis. See Cardinal Sitta carolinensis. See Nuthatch, white-breasted Richter (1938), 85 Sittidae, 202

Riddle and Braucher (1934) , 218 Slide Roberts. Severens. and Card (1939), 114, 133 contaminant on. 41, 179 Robertson et al. (1947), 28 heated, 36, 40, 41

Robin (Turdus migratorius) , 202 infrared lamp, use of, 226, 227 eosinophil, 210 remove moisture, 227 Rod(s). .See Eosinophil; Heterophil Smear. 5ee Blood, smear Rouleaux formation, absent from bird blood. 15 Smolker (personal communication), 139

Ryerson ( 1943) , 6, 73, 74, 83 Smudged (squashed) cell(s), 39, 170, 182 down-sheath cell, 176, 180 erythroblast, primary, 94 Sabin (1920). 101, 112, 154, 192 erythrocyte, 36, 40, 103 Salt solution primary, 98 damage to with chromuphobic bands, 40 embryo blood cells, 226 in bone marrow, 155, 191, 199, 200 primary erythroblasts, 115 in spleen, 157, 160 chicken blood for influence on differential count, 72-73 different ages of embryo, 226 mature effect on heterophil, 88 basophil, 78, 92 Locke-Lewis, 226 eosinophil, 76 Scale, magnification, 4 heterophil, 74, 89 Schechtman (1952), 227 monocyte, 72-73 Schilling (1929), 85 Sodium cantharidate, 45 Schoger (19391,42,45,219 Spaces in cytoplasm. 5ee Mitochondria; Vacuole in Schwarz (1946), 17 cytoplasm and cytosome Separation Sparrow Arneth count, 221 abnormal and atypical cells, 16 eastern tree, erythrocyte size, 212 eosinophils and heterophils, 83 erythrocyte lymphocytes and monocytes, 70, 71 area. 212 Serum, plasma number, 212 differentiating factor in, 112, 113 hemoglobin, 21], 212 granules Passer— mask underlying cells, 41, 148, 156. 158 domesticus. 211 precipitate. 168 montanus, 211 relation to hemokonia, 93 Spatula clypeata. See Duck, shoveller size relative to yolk spheres, 93 Specific granulefs), body (ies), substance(s) stained. 36, 89, 148, 155, 156, 158, 170 absent lipochrome ])igment, 93 eosinophil promyelocyte, 12-13 Sevfarth (1927), 28 heterophil Shattuck (1928). 26 metagranuloblast, 12 Shaw (1933). 220.221 promyelocyte, 12 Shipley (1916), 41 thrombocyte, embryo, 131

267 —

Specific granule(s) —Continued Spleen—Continued basophil, 91-92 embryo—continued comparison of erythrocyte. ])olychromatic. 160 immature and reactive thrombocyte, 46 granuloblast. 160 lymphocyte and monocyte, 49 reduced, 156 dissolution of granulocyte, dominant cell, 156 in basophil. 91-92 hetero])hil in heterophil. 74, 87-89 mesomyelocyte, 158, 160 effect of fixation, 87, 88, 90 promyelocyte, 160 basophil, 80, 91 metagranuloblasts, 158, 160 eosinophil, 80 ruptured cells. 155. 158, 160 heterophil, 80 serum, stained. 158 thrombocyte, 80 thromlioblast. 160 metachromatic. 91 thrombocyte, 158 present time of development, 155 basophil eosinophil, 164. 165 mature, 13 granuloblast, 164 mesomyelocyte, 13 erythrocyte, degenerating. 30 metamyelocyte, 13 granuloblast. 154 eosinophil heterophil. 163. 164 mature, 13, 89. 90 discharge of. 164 variations in form. 76 lymphocyte mesomyelocyte, 13, 91 dominant cell. 164, 165, 166 metamyelocyte. 13, 91 immature. 163 heterophil mature. 163 mature, 12, 73, 74. 90 naked nucleus, 163 mesomyelocyte, 12 time of a])pearance, 164 metamyelocyte, 12 lymphoid foci. 181 thrombocyte macrophage. 139, 140, 164 late immature, 11, 45, 46 monoblast. 163 mature. 12 monocyte, inmiature, 163 descri]ition, 42 plasmocyte, 166 number, 43 immature, 163 reactive, 38 serum, stained. 41 present sometimes thrombocyte, naked nucleus. 163 azurophilic granules, monocyte, 49 thymus, similar at 35 days. 169 magenta bodies, lymphocyte, 49 Splenectomy thrombocyte effect on hematopoietic organs. 29 medium emlnyo. 11 pigeon, 29 mid-immature, 11 Squashed cell (s) . See Smudged (squashed) cell thrombocyte, embryo, 132 Stain (s) and staining. 228-231 atypical, 132 benzidrine test. 115. 118, 130. 131, 134 staining. 234 technic, 231 5ee also Basophil; Eosinophil: Heterophil brilliant cresyl blue Specific gravity, erythrocyte, mature and immature erythrocyte, stigmata, 33 compared, 29 reticulocyte, 230-231 Speidel (1932), 31 thrombocyte and lymphocyte separation, 233 Spencer Lens Co., Richards, 0. W., 222 white cell count, 233 Spindle cell(s) Dominici, 26 erythrocyte, 30, 31 eosin-azure. 26 bipolar, 34 Giemsa, 26 unipolar. 34 hematoxylin and azure II eosin. 1 thrombocyte, 41, 130 janus green stain on thrombocyte. 43

Spiniis pinus. See Siskin, pine Leishmann s, 33 Spitta (1920), 222, 224 LoefDer's methylene blue, reticulocytes, 231 Spleen,7, 50, 115 MacNeirs tetrachrome, 211 basophil, 164 May-Griinwald Giemsa changes after hatching, 157, 164, 165 after benzidine test, 118. 134 embryo, 155-157, 158. 160 after Petrunkevitch No. 2, 80, 83, 117. 132. 195, eosinophil, mature, 158 207. 209, 230 erythroblast, 158, 160 bone marrow. 193, 195

268 Stain! s) and staining—Continued Technic I sj —Continued May-Griinwald Giemsa—Continued smear method circulating blood drying blood slides, 226, 227 ' adult. 52, 53. 54, 69, 74, 80, 83, 87, 207, 209, value of, 154 211 warming slides before using, 226, 227 embryo, 3. 117, 118. 134, 193 staining, 228-231 immature cells. 7. 14, 168 thrombocyte count. 232 serum. 41 white cell counts. 232-234 technic, 229 Rees-Ecker method. 233-234 Wright's stain, compared with. 14. 17. 41. 134, Wiseman's method, 232-233 168, 193, 229 See also Blood: Microscope; Stain and staining neutral red. thrombocyte, 43. 49 Terminology, 8-14 ])enetration poor, 6, 7, 92 chromophobic nucleus, 32, 40 phloxine, Wiseman's method. 232, 233 eosinophil and heterophil, 86, 89 polychromatic effect. 24, 25 erythroblast. primary, complicated by hemoglobin, thionin. 91. 191, 197, 230 104 Wright-Giemsa, 74 erythrocyte hetero])hil. stages of development. 195, 230 based on one chief characteristic, 27 technic. 230 later embryonic generations, 105 Wright's stain polychromatic, 117 after benzidine test. 231 primary generation, 105 bulk method. 87. 229 granular leukocyte, 73 circulating blood granulocyte series, 194 adult, 7, 14. 23, 33, 49. 52. 69. 73. 74. 78, 83, heterophil, 73 87.90.92. 193.207.211 megaloblast, 8 embryo. 3. 131, 134, 168 niyelocvte, 194 preparation. 228 nongranular leukocyte, 47 rack method, 87, 229 standardized, 8, 194 serum, 41 See also specific cells variation in samples, 228-229 Thigmocytes, 41 See also Reticulocyte; specific cells Thionin. See Stain and staining 10, Starling (Sturnus vulgaris 1 , 202 Thromboblast(sl,9. 11, 146, 155 er) throcyte, nucleus, 23 cvtoplasm, basophilic, 11 Striated muscle cells in thymus, 169 embryo, 96, 98. 106, 115, 160 Strigidae, 202 chromatin, punctate, 128, 134 Strigifornies, 202 disappearance from circulation, 131 Sturgis and Bethell (1943). 215 endiryo erythroblast, compared with, 128 Sylviidae. 202 megaloblast, origin, 130 Sturnus vulgaris. See Starling mitosis, 134 Substantia granulo-filamentosa, 28 nucleus Sugiyama (1926). 31. 128, 130, 131. 134, 226 nucleolus present, 131 (1938), 85, 221 staining incomplete, 131, 134 Sundberg (1947), 168 primary generation, 131 Swallow, Arneth count, 221 series, structural, 130 Sylviidae, 202 typical, description, 131 in bone marrow. 16k 165. 182. 186. 191. 193. 200 Taberetal. (19431.218 erythroblast. resembles, 193 Takagi (1931). 115 nucleus (19321, 115 nucleolus present. 164 Tate and Vincent (1932), 33 punctate chromatin, 164, 191, 193 Technic (s) Thrombocyte(s). 18, 27. 38. 41-47. 108, 111, 133, 157 artifact(s). 33-34. 36, 39-41. 46-47. 60. 72. 74. age groups, 45 87-89, 91, 92 artifact, 46 avian species, 16, 222-234 count (s) blood samples, box for handling. 227-228 chicken breeds, 216, 219 dorsal aorta, blood from. 226-227 in bone marrow. 198, 200 erythrocyte, counting. 234 in circulating lilood, 216 heart, blood from, 227 method, 232 hematocrit, 232 ratio hemoglobin, 231-232 blood and hematopoietic organs, 157 infrared lamp, use of, 226, 227 to erythrocyte. 42. 44, 232 269 Thrombocyte (s) —Continued Thrombocyte (s) —Continued count (s) —continued phagocytosis, 45 sex, 216 reactive, 38, 45, 46, 50 variability, 217 series, 10, 11,200 degeneration, 38, 42, 46, 146, 164, 182, 191 shape, variability, 43 orange margin, 46 size, 41-42, 44 effect of fixation, 80, 167 chicken, 212, 213 embryo, 11. 96, 98, 100, 103, 106, 115, 128, 130-133, wild s])ecies, compared with, 210 158 specific granule(s), 42, 43, 80 binuclear, 136 comparison of immautre and reactive, 46 blebs pinched off, 131 in vacuole(s), turkey, 210 degeneration of, 131, 132 number, 42, 43 blebs, 136 staining of, 233-234 margin, 136 staining crumpled, 132, 136 jaims green, 43 stains with eosin, 132, 136 neutral red, 43 nucleus, 136 pigeon. 210 developmental stages, 11, 38, 134, 136 typical, 38, 42 large embryo, 11, 136 Thromboplastid. 42

medium embryo, 11, 134, 136, 160 Thymus, 7, 50, 115, 172, 174 small embryo, 11, 136, 160 embryo thymus, 170 differentiation, 131 serum stained from, 41 greater in later tlian in early embryo genera- granulopoietic function less than in spleen, 168 tions, 132 Hassall's corpuscles, 169 generations irradiation, plasma cells increased, 166 later embryo, 131-132 lymphojjlast, 166, 174 primary, 131 lymphocyte hemoglobin absent, 131 immature, 53, 172, 174 in spleen, 156 mature, 172, 174 intravascular origin, 128 lymphocytogenesis, 167-169, 174 large, 10, 11 monocyte development, not observed, 169 medium, 10, 11, 96, 98, 100, 157 smear from thymus like that of spleen, 169 mitosis, 131 striated nmscle cells, 169 primary erythroblasts, appear later than, 130 thymocytes, 167 erythrocytes, common origin with, 130 thymus cortex from lymphocytes, 169 series, 10, 11 Tissue culture, 2 structural, 130 sha])e, oval. 134 area opaca, 41 time of ajjpearance. 131 lymphocyte small, 10, 11. 98, 100, 103, 106, 157 degenerate, 181 specific granules, 136 mitosis absent, 181 atypical, 132 monocyte, 68 change with later generations, 132. 136 osteoclast, 153 first appearance, 131 primary erythroblast, differentiation, 116 erythrocyte, compared with, 41, 42 primitive streak, 104, 116 fragility, evolution of, 42 Toryii (1930), 29

immature. Ill, 198.200 • (1931), 29 early, 10, 11, 182, 186, 191, 193 Towhee (Pipilo erylhroplillialmiis) , 202 late, 10, 11, 186, 193 Trichloracetic acid, effect on heterophil rod, 88 mid-,10, 11,186, 193 Troje (Dantschakoff, 1908b), 26 in bone marrow, 164, 181, 186, 191-193, 198 Tufted titmouse, erythrocyte count. 218 in huffy coat, 232 Turdidae. 202 in circulating blood, 41-47, 96 Tiirdus migra/oriiis. Sec Rubin life span, 45 Turkey lymphocyte, separation from. 233. 234 basophil. 210 mature, 10, 12, 38. 42, 108, 111, 186. 191 count (s) naked, 163, 191 Arneth, 220 origin differential, 219

intravascular, 128, 141 domestic i Meleap;ris gallopavo) , 202 ' lymphocyte, not from, 130 eosinophil, 207. 209 theories, 42, 130 heterophil. 207, 209

270 Turkey—Continued Vezzani ( 1939], 218 thrombocyte Vireo, red-eyed (Vireo oUvaceus), 202 size, 210 eosinophil, 210 specific granule, 210 Vireonidae, 202 Twisselman (19391, 215 Vireo oUvaceus. See Vireo, red-eyed Vulture, eosinophilic polymorphs, 208

Underhill (1932j, 167 Warbler black-poll {Dendroica striata), 202 Vacuole (s) in cytoplasm and cytosome black-throated green [Dendroica virens), 202 artifact, 40 Tennessee (Vermivora peregrina) , 202 basophil erythrocyte nucleus, 212 metagranuloblast, 13 yellow-throated, 169 promyelocyte, 13 Wassjutotschkin (1913), 169 eosinophil, metagranuloblast. 12, 194 (1914), 215 erythroblast Waters (1945), Weidenreich 83 basophil, 24 (1911), . 68 primary, 116 Weiss and Fawcett (1953) cell (s) . See Leukocyte ; specific cells erythrocyte, mature White Wickware (1947), 219 effect of heat. 36 Wills (1932), 27 overlying objects, 39, 40 Wilson (1925), 31, 128 heterophil Wintrobe (1933), 210 metagranuloblast, 12, 194 (1952), 233 promyelocyte, 12, 74, 188 29, 32 rod, 74 Wirth (1950). (1931a), 232 lymphocyte, reactive, 56 Wiseman 167 macrophage, 134. 139, 140 (1931b), 53, 167 cytoplasmic spheres. 134 (1932), 53, 66, method, 232-233 osteoblast, immature, 13, 152 Wiseman's osteoclast reliability. 233 141 mononuclear, 13, 152. 153 Wislocki (1943), multinuclear, 13, 198 Woodpecker puhescens) , 202 plasmocyte downy (Dryobates villosus) 202 immature, 198 hairy {Dryobates , erythrocyte nucleus, 212 early, 13, 166 red-headed, (1930a), 29,30 late. 14 Wright mature, 198 (1930b), 29. 30 VanAlstyne (1931), 29 primordial osteogenic cell, 13 and thrombocyte Wright's stain. See Stain and staining embryo, 131, 132 large, 11 X-cell, 169, 179 medium, 11 See also Feather sheath cell small, 11 immature Yeast cell, contaminant on slide, 41 early, 11 Yolk granules, 93 late, 45 plate! s), in Aniblystoma erythrocytes, 105 mid-, 11. 44 sac, 104, 128, 226 reactive, 38, 44 macrophage, 130, 134, 139, 140 specific granule in. 210 Venzlaff (1911), 219 Vermivora peregrina. See Warbler, Tennessee Zuckerman (1946), 133 o

271