Special Articles

be -dried HEMATOLOGICAL technique spread; they should quickly by waving them to and fro under a fan (vide part VI). Part VIII Staining.?Combined staining with May- Griinwald and Giemsa stains gives the most (Lond.) By L. EVERARD NAPIER, f.b.c.p. satisfactory result?the nuclei stain deeply, the and connecting chromatin filaments are well shown, C. R. DAS GUPTA, Mi. (CaU. , while the granules also show characteristic ,n D-^'.Calcutta, and size. If and Giemsa (From the School of Tropical Medicine, colour May-Griinwald stains are not with counts available, proper staining The Arneth and Schilling or Leishman's not as K (13) Wright's stains, though the satisfactory, gives quite good results. Iron- or Arneth (1904) divided ? aCCOrding for these main c g1 hamiatoxylin haematoxylin staining into five iej which counts in to of the Romanowski ^orphonuclears m the preference any t? the number of is segments into groUps stains sometimes advocated; while these ln were the their turn classified -? TTnder stains out the nuclei and the chromatin of the lo) bring according to the shape c0m- filaments the colour of the is not the count in well, granules ?riginal classification i-cati0n distinctive so that differentiation between Phcated and had only limited PP, and (1927) and eosinophils is very difficult. general practice. Cooke lint taking the Arne and simplified, divisions .4. Arneth count _ y^odifiedlnto account the five original into the only going The nucleus the Arneth count without of the polymorpho- ?f be lntncate subdivisions of Arneth. ,vy,\ch the nuclear cells is divided into lobes which may * or of devised a nuclear separate joined together by fine filaments Schilling < - iinpie differential countmfetk?f and chromatin. Cooke's criterion of separate lobu- leucocyte Jpnroorated be l?be count of the'neutrophils.were lation is that either the lobes should distinctly and form of a ^c?rPO^ or should be a fine considered in the '? separate, joined together by but not broad bands of -Apparatus required (vide part Sctaiua0hilling B chromatin filament, by the Arneth nuclear material. Lobulation of the nucleus, it Smears.?Smears for andyd C0llnts should be very thin, 98 THE INDIAN MEDICAL GAZETTE [Feb., 1941

an is assumed, gives indication of the age of the & cell?the less the lobulation the is the class V by 5, and then adding together younger of ce cell. The youngest cell in the normal peripheral dividing the sum total by the number counted. circulation is one which has a nucleus shaped ^ ^ The means of the nuclear lobes like the letter C, while the cells with five or weighted these three are calculated in more distinct nuclei are the oldest. The prin- examples ciple of the method is to make a differential following way :? count of the to group the cells neutrophil cells, 3 according to the lobulation of the nuclei, and to Example 1 Example 2 Example calculate the percentage of cells in each group. The cells of the various types can be described IX 10 = 10 X 41 = 41 X 9 = = I as follows :? 2 X 25 = 50 X 44= 88 X 22 = 3 X 47 = 141 X 14 = 42 X 36 Class I. in which ? lg& Neutrophil 4 X 16 = 64 X 1 = 4 X 23 one or = 50 the nucleus has only definite lobe, two 5 X 2 = 10 X 0 = 0 X 10 or more lobes joined by a definite band of I chromatin and not by thin filaments. 100 275 100 175 100 301 Class II. When there are two separate lobes joined by one thin filament, or when there are = 3.0* two completely separate lobes. Weighted mean 2.75 1.75 Class III. When there are three separate & lobes connected by two thin filaments, or there Interpretation.?Immature granulocytes l are three completely separate lobes. cells of Arneth class I and some of class II) R, , Class IV. When there are four separate none of class III, IV or Y, are found in ^ I lobes connected by three thin filaments, or there bone marrow. If there be a sudden stimulus the bone marrow a<3 are four completely separate lobes. to produce granulocytes, ^ j an a Class V. When there are five or more infection, the leucocytosis is brought young cells in the blood, ? I lobes connected by four or more fila- by appearing separate number and the of these ments, or there are five or more completely proportion depend!? the nature of the stimulus and the reacts? separate lobes. upon power of the marrow of the individual, and the The count is made and & by identifying classify- is a shift to the count. left in the Arneth _ .g one hundred neutrophil granulocytes from ing leucocytosis as a result of muscular exercise of the and the result is I different parts slide, only due to redistribution of is not accoi1 form :? cells, in the following . usually reported panied by any increase of young leucocytes 11 Example 1, a normal count the peripheral circulation, and is therefore associated with ' Class I II III IV V Total any '. e in chronic infection or the Number .. 10 25 47 16 2 100 Again, toxaemia, is an increased leucocyte destruction, which nw there is an increase in the number of When or may not be balanced by new leucocyte form9 cells with one and two separate nuclear lobes, tion; in the former case there will be leucocyte? i.e., of class I or class II, at the expense of those or a normal count and in the latter leucopen1 with three or more lobes, there is said to be a In either case there is an increase in 1 y?Uw| left shift' in the Arnetli count, as in the forms, actual or relative, which results in a sn1 example given below :? to the left and a decrease in the weighted me? ^ It will be that this shift is Example 2, a 'left shift' count apparent quite ^ dependent of the total leucocyte or total gra1111 Class I II III IV V Total ' ' 0 locyte count, and provides another angle Number .. 41 44 14 1 0 100 the degree of the toxaemia. Conversely, when there is an increase in the In pernicious anaemia and certain other con^is 4- and multi-lobed nuclei, it is known as a tions of bone marrow dysfunction, there ^ ' right shift \ shift to the right in the Arneth count, &1! therefore an increase in the mean; ' weighted a count ^ Example 3, right shift' such conditions there are many multi-lobulate Class I II III IV V Total Normals Number 9 22 36 23 10 100 11 III IV Weight Various indices and methods of expressing mefiD more concisely the results of the Arneth count (Cooke's modification) have been proposed. Cooke and Ponder Probably the most suitable of these methods is (1927). the calculation of the weighted mean of the Kennedy (1933) (Iraq) nuclear this is obtained lobes; by multiplying Das Gupta (Calcutta) 15 2 the number of cells in class I by 1, the number in class II by 2, the number in class III 3, by cells and only very few young cells coming the number in class IV the number in by 4, the circulation. 1941] HEMATOLOGICAL TECHNIQUE : NAPIER & DAS GUPTA 99

phases, neutrophilic struggle, monocytic defence, B. Schilling count and lymphocytic cure, but it does not seem to take into account the nature of the infection In this count the neutrophil granulocytes classified and the variations in the divided into four groups and consequent response. there are so many directions in which follows :? Further, changes may take place, that the hsemogram *den- seems to facile >.ave a distinct peculiarly susceptible interpreta- 2. to the conscious or unconscious (. correspo tion suit wishes tation in the nucleus; they of the interpreter. a C- or We do not propose to give a full discussion 3. these have Stab?celisS'(St); on the interpretation of the hsemogram, as it has but no true shoe-shaped nucleus lo^latl^'count, anaJ only an indirect bearing on the subject of to class I of the Arneth correspond anaemia, but, as far as the neutrophil to our staff or cells. ?0r?tive elements are concerned, Schilling's contention is Stab cells be of the rege may that infection may cause two types of reaction, and are differentiated ^ ^generative type, of the ^^ the regenerative reaction and degenerative re- and staining reaction in the new leuco- an cn'0tic,, action; regenerative reaction, ^hapethe latter the nucleus is ribbon-like py . are formed in the bone marrow and this is With and usua cytes clumps of chromatin, y^ reflected in the circulation a rise may be vacuola peripheral by shaped, and the cytoplasm in the total white-cell count, an increase in the Arneth.Arneth. juveniles and regenerative forms of stab cells, M III IV while in the degenerative reaction, which is found in severe toxaemia, on account of the degenerative influence of the toxin on the marrow, the formation of new cells is retarded; this may be shown by a leucopenia. In the differential count of such a case there are few juvenile cells, and the stab cells that reach the circulation fail to segment at the usual rate, and may degenerate with the result that there is a preponderance of stab forms, including many degenerative forms, in the blood. The reaction, (Reg.) however, may not be purely regenerative or degenerative in character, but may be a mixture of the two, and the hsemogram may be inter- mediate, with slight leucopenia and a greater (Dej) preponderance of the younger cells than in a J St. Se^, purely degenerative process. Schilling. (It is surprising to the writers that no provi- sion is made in the classical Schilling hsemogram the of the 4- for separate counting regenerative . Segmented (S) polymorphonuclears, and degenerative forms of stab cell, when lng two or more distinct lobes. following aie obviously the presence or absence of the latter to Schilling the According is an important factor in the interpretation of ??nstitiients of a normal hsemogram. the hsemogram.) Conclusion.?The interpretation of the Arneth Total Percentages count is simpler, though the implications are more but it presents a minor Mon" limited, practical ^. L. in that more skilful is W-B.C. B. E. J- I St objection staining Ul.j | required to ensure accurate counting of the J separate lobes. 3'5 5,000 o-l 1-2 0 I 0-11 |57-6' The value of these counts is mainly in estab- : the 8,000 lishing presence of infective or toxsemic 60-70 factors, which may be important contributory causes of the ansemia, and/or may prevent res- to = = ponse appropriate hsematinics. The presence basophil. E. eosinophil. uciearS. ? 111 L. ? . Mon. large of multi-lobular cells, shown only in the Arneth count, is important in the diagnosis of pernicious is much ansemia. Schilling hsemogram Discussion.?The , than a differential granulocyte poun ^0re claims? tha* (14) The erythrocyte' sedimentation rate Arneth count, and Schilling Qn i P , The _ JeSlves far more information. The determination of the sedi- senes o erythrocyte on a imaginative mentation rate is not a test that gives depends highly that are (ESR) P?*ses to infection and toxsemia much direct information regarding the nature of do, occu in Posed and _ ^ the but it is of value differentiating to, possibly there ansemia, laemopoietic system in which 100 THE INDIAN MEDICAL GAZETTE [Feb., 1941

' functional' such as anaemia disorders, pernicious The tube is now set in a stand in in which the true ESR is within normal upright limits, a spring clip holds the of the tube from anaemia due to such causes as and points against a rubber cork at the bottom end. T& disease in which the ESR is > malignant markedly tube is now left to stand and the leve increased. upper of the red cell column, which is generally sharp' the ESR is affected in the tw Further, by changes is read at the end of one and after and size of the red in so far as these again shape cells, hours*. The of the column of affect and length plaSlI1,j changes formation, by 'from the zero mark to the of the red ce in the concentration of the red cells in top changes column is the amount of sedimentation and th1 the this last overshadow the other plasma; may is read off in this is a measurable must be _ millimetres; reported changes and, being factor, the sedimentation rate in millimetres for 011 allowed for in the true sedimentation estimating hour and for two hours. rate. Normal limits one as from the taken in (a]ter hour)? Finally, single specimen Men Women the normal course of a routine blood examina- Textbooks .. 3 to 5 mm. 4 to 7 mm- tion the ESR can be estimated without additional Our own experience in 3 to 15 mm. 5 to 40 mro* labour, it should always be included. Indians in Calcutta. The test suffers from the of the multiplicity The high figures that we obtained in appai'?1^ methods by which it is performed and by the normal subjects are partly explained by the hi? < of the forms in which the results are temperatures that prevailed, compared with those , variety the noi'i1, The most used places where the tests on which other expressed. commonly technique figures are based were carried out. is that of Westergren (1926), but, as it is important that the correction for anaemia should Wintrobe's method be made and as no correction chart has been Apparatus required? out for this this worked technique, method has I. Wintrobe's tubes?a fiat-bottomed glass tub? its limitation. 100 mm. long graduated from 0 to 100 1 mm. Wintrobe's method (1933) is really a modi- intervals; it has a uniform bore. II. Anti-coagulant?sodium and oxaia fication of this method; it has the advantage potassium ttt ? mi.xture (vide part I). that the cell volume can be estimated on the III. Electric centrifuge. 1 same sample of blood and also that a correction Together with a syringe and stop watch for anaemia can be made. Westergren's method. test.?A known Principle of the volume of Technique.?Wintrobe used his cell-voluI1!ie mixed with a definite blood is intimately amount tube for the determination of the sedimentati0'1 of anti-coagulant, and the mixture is drawn into rate. a tube of known calibre to a certain ' ' up point; 1 he blood is collected in an oxalated allowed to stand in an flaj^ the tube is then upright and is thoroughly mixed; the cell-volume tube at room and the level of position temperature is filled with the oxalated blood means of the red blood cells in the tube is noted at the by ^ capillary pipette up to the zero mark, which } of one hour and after two # end again hours. exa-ctly 100 mm. from the bottom. The tube The fall in the level of the red cells is expressed now ^ allowed to stand in a vertic*1 in millimetres. upright position on a special stand or on a tray and efl Westergren's method plasticine, the reading is taken at the of one and again after two in the sade required? hours*, Apparatus way as in the I. Sedimentation tubes. Westergren's method; cell-volufl1? tube is then put into a centrifuge and centrifuge Westergren tube. This is about 300 mm. long at 3,000 revolutions minute for half an and is graduated from 0 to 200 mm. in I per ho^ to obtain the 1 mm. intervals; it has a uniform bore of packed cell volume percentage) about 3 mm. and is open at both ends. thus both procedures are done in the same tube II. Special Westergren rack, or an improvised rack, with the same blood to the tubes in a vertical sample. keep position, and Normal limits to prevent the blood escaping. (after one hour)? Men Women III. Anti-coagulant:?3.8 per cent sodium citrate miii lextbooks .. solution. 0 to 9 mm. 0 to 20 nifli- Our own IV. for collection of blood. experience in 2 to 20 mm. 2 to 30 Syringe, etc., Indians in V. Stop watch. Calcutta. Correction . anosmia.?One facto}- Technique.?In this method 3.8 per cent for important m the rate of solution of sodium citrate is used as anti- sedimentation of bodies in is the concentration seo1' in the of one part of citrate of these bodies. The coagulant proportion mentation standards solution to four parts of the blood. for red cells in plasma ar

'Discussion.?There are a number of factors a showing the (1938) worked out chart involved in the phenomenon of erythrocyte of sedimentation r?^,^0-n. ship cell volume and sedimentation, but the most important is the normal blood and in blood size of the rouleaux formation and this is mainly chartBhowingjg is reproduced degrees of anamia; this determined by the composition of the plasma. below. Increase in plasma fibrinogen will cause an increase in the size of the rouleaux formation and therefore an increase in the sedimentation rate. It is probably on this fact that the value of this test depends, as, with the exception of the concentration of the red cells for which allowance can be made, this effect overshadows all others.

The sedimentation rate is increased in so many conditions that it is very dangerous to . attach any specific diagnostic significance to it. This must be quite obvious from the wide range of the ESR rates that has been recorded in apparently healthy in- dividuals. However, the ESR is a test of great value in estimating the progress in chronic infections, such ^0 as 10 20 30 40 tuberculosis and leprosy. A of Blood. Volume of packed red cells cjcm. per 100 ccn? standard should be ' technique adopt- means of Chartilai't forfnr correction rate for anajmia by and in the results the correction of sedimentation rate for anajmia by means of ed, reporting the volume. method must be stated or the corpuscular volume. employed To the information loses much of its value. an?miai find the junction of the lines theC?^eCteryG tu a^s.?lutely J-he specimen of blood should be col- lected during the fasting state and examined within one hour of

(iv) J-nT Collectio?- women it is advisable to avoid the Menstrual period and the few days anc* a^er it. Ti-l ore. he specimen should be rejected if there be a slight trace of coagulation or any mar^ed (vi) ti???he haemolysis. tubes should be set up in an absolutely vertical position in a quiet place, well away from any apparatus that vibrates. The temperature of the room should be between 72? and 80?F. whenever (Vfo J-nT possible. recording the results, correction for anaemia must always be applied.