Arch. histol. jap., Vol. 34, No. 4 (1972) p. 311-360
Department of Anatomy and Histology, University of Adelaide, Adelaide, South Australia
The Leukocytes and Platelets of a Marsupial, Trichosurus vulpecula. A Comparative Morphological, Metrical and Cytochemical Study
R.A. BARBOUR
Received February 3, 1972
Summary. The leukocytes and platelets of Trichosurus vulpecula are in many res- pects typically mammalian. Relatively unusual morphological features are paucity of granules in neutrophils, elongated form of eosinophil granules, abundance and uniformity of basophil granules, and, in monocytes, high frequency of irregular nuclei (some annular) and coarseness of chromatin. Mean leukocyte count per cmm was 13,380 and mean counts for individual cell types were: neutrophils 5,790, eosinophils 221, basophils 31, lymphocytes 6,720, monocytes 619. The last two of these are fairly high amongst mammals, those for eosinophils and basophils fairly low. The mean number of nuclear segments per neutrophil was 2.41, per eosinophil 1.34. Mean diameters (in μ) of cell types in stained smears were: neutrophils 13.00, eosino- phils 13.77, basophils 14.24, lymphocytes 10.32, monocytes 14.99. Most platelets are similar in size to human ones but large examples are more common. Neutrophil granules are coloured intensely by sudan black B, eosinophil granules less so. Basophils and platelets, and most monocytes and lymphocytes, are sudanophobic. Amylase-labile PAS-positive material is abundant in neutrophils; there is some in plate- lets and, probably, also in basophil cytoplasmic matrix. Basophil granules and the peri- phery of eosinophil granules exhibit amylase-resistant PAS-positivity. No peroxidase is demonstrable in leukocytes using o-tolidine as substrate, but with benzidine a positive reaction occurs in neutrophils and eosinophils. Alkaline phosphatase is demonstrable only in basophils-mostly in their specific granules, a location not previously reported for any mammal. The simultaneous coupling azo-dye method used was more sensitive and gave more precise localization than the cobalt sulphide technique. Succinate dehydrogenase activity is exhibited by some lymphocytes, monocytes and platelets. Lactate dehydrogenase activity is more intense in these cells and, especially, in platelets; a weak reaction occurs in some neutrophils and eosinophils.
During the last twenty five years leukocytes and blood platelets have been the subject of many cytochemical investigations that not only have provided academic information but also have assisted in the understanding of their activities, relation- ships and origins and have added diagnostic tools useful in the assessment of some pathological states. Much less attention has been paid to comparative aspects of leukocyte and platelet cytochemistry and in this regard the marsupials remain vir- tually untouched. To start to fill this gap in knowledge is one of the prime aims of this study. The numerous earlier works, and some more recent studies, on comparative haematology were confined mainly to cell counts and morphology. Such reports have been reviewed from time to time, notably by SCARBOROUGH (1931/2), JORDAN (1938), ALBRITTON (1952), ANDREW (1965), SCHERMER (1967) and WINTROBE (1907). Even in this 311 312 R.A. BARBOUR:
field marsupials have received little attention-PONDER, YEAGER and CHARIPPER (1929c) and KNOLL (1932) reported briefly on several, while JORDAN (1938) and WINTROBE (1967) have given some figures for the American opossum, Didelphis, and BOLLIGER and BACKHOUSE (1960a) for the koala, Phascolarctos. The last mentioned authors have also published some figures for a monotreme, Tachyglossus (the echidna) (BOLLIGER, 1959; BOLLIGER and BACKHOUSE, 1960b). More recently PACKER (1968) has presented some eosinophil counts for the quokka (Setonix). As no-one has dealt with the species chosen for this work, the common Australian brush-tailed possum or phalanger, Tri- chosurus vulpecula, it seems desirable to begin with a morphological and metrical study of its leukocytes as a prelude to the cytochemical investigations. The findings of an investigation of this sort achieve their full meaning only when seen in perspective with results of similar investigations on other animals. Accordingly a comparative discussion of the findings is presented and this necessari- ly involves a fairly extensive review of the relevant literature which is, I believe, not out of place at the present time.
Material and Methods Twenty three animals were used; fifteen females and eight males. All were fully grown or nearly so. All had been captured recently from the wild state in or near the suburbs of Adelaide; some were kept for a short time in the University animal house before blood was taken for study. All animals appeared to be healthy. Six of the females had pouch-young at the time of examination. Blood was taken by cardiac puncture (with the animal under ether anaesthesia), transferred to a bottle containing potassium sequestrene as anticoagulant and then used for the cell counts and for the immediate preparation of smears for the various staining procedures. In a few cases some smears of fresh blood were made to see if their performance in the cytochemical tests differed from those made from anticoa- gulated blood but, as no differences were ever observed, this was not done routinely. Morphological and metrical studies Cell morphology. The descriptions of cell morphology to be given are based on smears stained by a routine Leishman's technique and by the accelerated Giemsa method given by LILLIE (1965). Cell measurements. All cell measurements were done on blood smears, as far as possible, Leishman-stained. In some cases Giemsa-stained smears gave better defini- tion of the cells and were used instead. These stains did not always show up the cytoplasm of the neutrophil granulocytes very clearly and these cells were some- times measured on counterstained PAS or sudan black-stained smears. To standardize as far as possible the conditions of measurement, cells were measured only in thin parts of the smears where there was little or no overlap of erythrocytes. Measurements were made using a graduated microscope eyepiece with oil-immersion objective and were estimated to the nearest 0.2μ. In general only cells with a circular outline were measured, though some symmetrically oval ones were included in which case the recorded measurement was the value nearest to the square root of the product of the greatest and least diameters. Cells of irregular outline, cells showing any sign of degeneration and cells of doubtful identity were Marsupial Leukocytes and Platelets 313 excluded; otherwise no selection was exercised-all suitable cells encountered in systematic scanning of appropriate parts of the smears used were measured. The number of neutrophils (200) and lymphocytes (115) measured in all samples was the same. The numbers of the other three cell types measured varied widely between samples depending on the prevalence of suitable cells in the smears. For eosinophils the number measured ranged from 1 to 60 (total 702), for basophils up to 35 (total 190) and for monocytes from 2 to 65 (total 836). Cell counts. Total leukocyte counts were done in a standard brightline haemo- cytometer with Neubauer ruling, using a 1:20 dilution. Five counts were done, on each sample and the final count scored for each animal was the average of the five. Differential leukocyte counts were done on Leishman or Giemsa-stained smears using at least 400 leukocytes in each case. From the total and differential counts actual counts for the various types of leukocyte were calculated; it is realized that this is not an accurate way of assessing the absolute counts of the types present in small numbers, especially the basophils, but it should be adequate to demonstrate any major differences from the figures for other animals (many of which have been cal- culated in the same fashion anyway). The degree of segmentation exhibited by the nuclei of all the measured neutrophils and eosinophils was noted, and these observa- tions provide the basis of the Arneth counts to be discussed. From the Arneth counts the mean number of segments per 100 cells has been calculated and this will be referred to as the Arneth score. All cell counts are given in the results as figures expressing the number of cells per cmm of blood. Cytochemical studies All cytochemical studies were done on blood smears and in all cases some parallel human smears (usually of the author's own blood) were stained simultaneously for comparison. As the human blood was low in content of leukocyte alkaline phos- phatase, rat blood was used for the comparative parallel smears in a couple of cases where this enzyme was being sought. Sudan black B stain for lipids. Smears from ten animals were stained by the sudan black method of SHEEHAN and STOREY (1947) as presented by HAYHOE, QUAGLINO and DOLL (1964). In two cases Leishman's was used, as recommended, as the coun- terstain but nuclear fast red was found to be preferable and was used for all the others. For the special examination of basophils, in six cases some smears were stained for one min in 0.5% aqueous toluidine blue immediately after fixation and the position of a number of basophils recorded with a vernier mechanical stage. The toluidine blue is totally removed during the subsequent sudan black staining. Periodic acid-Schiff (PAS) reaction. Eight samples were subjected to the PAS staining procedure, in each case some smears being exposed to 30min salivary diges- tion immediately after fixation to act as controls in the assessment of the glycogen content of the cells. The method used was that given by HAYHOE, QUAGLINO and DOLL (1964) with the exceptions that the Schiff's solution and the sulphur dioxide water were prepared as recommended by CARLETON and DRURY (1957) and Mayer's haematoxylin was used on those smears that were counterstained. As in the instance of sudan black staining, in four of the samples some smears were stained, after fixa- tion, with 0.5% aqueous toluidine blue so that the position of some basophils could 314 R. A. BARBOUR:
be recorded. Again the toluidine blue is completely removed by the following stain- ing procedure. Peroxidase. Two different methods were used for the demonstration of peroxi- dase activity. The first, that of QUAGLINO and FLEMANS (1958) which uses o-tolidine as the chromogen, was applied to four samples: counterstaining was with Leishman's. The second method was that recommended by LILLIE (1965); this is a more conven- tional method employing benzidine instead of o-tolidine and his "accelerated Giemsa" as counterstain; it was applied to nine samples. In all cases some control smears were incubated without hydrogen peroxide. Alkaline phosphatase. Alkaline phosphatase was also sought using two methods. One, applied to four samples, was that of WYLLIE (1964). This is a modified Gomori- Takamatsu cobalt sulphide technique with sodium β-glycerophosphate as the sub- strate; Leishman's was the coun terstain used. The other method used was the simultaneous coupling azo-dye method of HAYHOE and QUAGLINO (1958) which uses fast garnet GBC salt as the diazonium component and sodium α-naphthyl phosphate as substrate. This was applied to nine samples. HAYHOE and QUAGLINO (1958) recommend methyl green as the counterstain and this was used on some samples; other counterstains employed were Mayer's and Weigert's haematoxylin and nuclear fast red. It was found that the minutest amount of methyl violet left in the chloro- form-extracted methyl green solution produced staining of the basophil granules so rendering these cells unsuitable for assessment of their phosphatase content. In some smears from four of the samples the position of some phosphatase-positive cells was noted (before any counterstain was applied) and the smears then stained with toluidine blue to determine whether any of these cells were basophils. In all cases some smears were incubated in the absence of the organic phosphate substrate as controls. Dehydrogenases. Two dehydrogenases have been studied-succinate and lac- tate. The latter was sought in five samples, the former in seven. The method em- ployed was that of HAYHOE, QUAGLINO and DOLL (1964), a tetrazolium technique using acetone fixation, nitro BT as the chromogenic H+ acceptor, sodium succinate or lac- tate as substrate and nuclear fast red as counterstain.
Results Morphological and Metrical Studies Cell morphology The five types of leukocyte characteristic of all vertebrates are present in the blood of Trichosurus and, in general, are easily identifiable from one another. The main difficulty arises in distinguishing between some of the monocytes and large lymphocytes. Occasionally distinguishing monocytes from juvenile neutrophils is a problem. Neutrophils (Fig. 1-3). After Leishman or Giemsa staining the cytoplasm of neutrophils often shows little or no sign of granulation, the overall impression being that the granules are small and sparsely scattered in most of these cells. The stain- ing reaction of the cytoplasm is generally pink to mauve but very pale-sometimes so pale that the limits of the cytoplasm are very difficult to define. Marsupial Leukocytes and Platelets 315
The nuclei of neutrophils are dense with large chromatin clumps and present a range of segmented forms, having quite frequently up to five segments and rarely six or seven. Nuclear portions were regarded as separate segments only if it was clear that they were joined by no more than a very fine strand of nuclear substance. Using this definition of segments many of the single segments have a rather irregu- lar outline exhibiting indentations, projections and bends while others present a simpler round or oval shape. The nuclei of many of the non-segmented cells (i.e. many of the band forms) also present irregular U or S shapes or other similarly complex configurations. In many cases band nuclei of simpler outline exhibit a paler nuclear staining and, presumably, represent the youngest members of the population. In segmented forms individual segments sometimes differ considerably from one another in size. Convincing sex chromatin bodies (Fig. 4) are observed only very rarely. Eosinophils (Fig. 5, 6). Eosinophils are easily recognized by their prominent pink-staining cytoplasmic granules, about the same size as those of human cells, that appear to fill or almost fill the cytoplasm. In many cells a small region of lightly basophilic cytoplasm devoid of granules can be seen, usually adjacent to the nucleus. In intact cells the form of the granules is not evident but in cells that have broken open and allowed some granules to escape it can often be seen that they vary some- what in size and possess a plump fusiforn shape (Fig. 7). The nuclei of these cells have coarse chromatin granules but present a somewhat less intense staining reaction than those of neutrophils. The majority of eosinophil nuclei are non-segmented but many have a horse-shoe outline suggestive of a late band form of cell. Nuclei with two segments are common and tri-segmented forms are found; in these cases the individual segments usually present a simple round or oval outline. Basophils (Fig. 8, 9). The specific granules of the basophils stain intensely with Leishman's or Giemsa stain and are metachromatic to toluidine blue. They are uniformly round in shape and rather smaller than the granules of the eosinophils. In intact cells they appear fairly uniform in size but in cells that have ruptured and spilled out some granules, considerable variation is evident (Fig. 10). The granules are usually not distributed evenly throughout the cytoplasm but are more densely aggregated in some areas than others and leave some irregularly shaped and incon- stantly placed areas free of any granules at all; such regions of cytoplasm remain virtually unstained. Basophil nuclei are relatively large and present an irregular shape, apparently sometimes being segmented-though obscuration by the granules usually makes the outline of the nucleus difficult to see. The chromatin pattern is coarse, the large chromatin clumps presenting an ill-defined smudged appearance. Lymphocytes (Fig. 11-13). Lymphocytes exhibit a wide range of sizes, extend- ing from very small ones with little if any evident cytoplasm to large ones with much more abundant cytoplasm. The sizes cover a continuous range from small cells to large and it is not possible to recognize distinct categories on the basis of size. The nuclei of lymphocytes are round or, in some cases, may show some flatten- ing or a small indentation on one side. The chromatin pattern presents coarse 316 R.A. BARBOUR:
clumps and the overall density is lower in the larger cells. The cytoplasm is basophilic and fairly uniform in staining reaction though, in the small to medium cells particularly, there is usually a paler zone in the immediate perinuclear region. The nucleus is usually placed eccentrically in the cell-a fea- ture that is also much more evident in the smaller cells. Azurophil granules are seen in a very small proportion of cells and then exist only in small numbers in each cell.
Fig. 1. Typical possum neutrophil; Leishman stain. Fig. 2. Possum neutrophil with prominent granules; Leishman stain. Fig. 3. Human neutrophil; Leishman stain. Fig. 4. Possum neutrophil with sex chromatin body; Giemsa stain. Fig. 5. Possum eosinophil; Leishman stain. Fig. 6. Human eosinophil; Leishman stain. Fig. 7. Possum eosinophil-cell broken and spilling granules; Leishman stain. Fig. 8. Possum basophil; Leishman stain. Fig. 9. Human basophil; Leishman stain. Fig. 10. Possum basophil-cell broken and spilling granules; Leishman stain. Fig. 1-10: ×1,650 Marsupial Leukocytes and Platelets 317
Monocytes (Fig. 14, 15). Some monocyte nuclei are classically reniform but many present a more irregular outline and a few have a perforated, dough-nut shape. In some cases the finer chromatin pattern of the nucleus is a useful feature in distin- guishing monocytes from large lymphocytes but in many the nuclear chromatin is coarse and not noticably different from that of the medium to large lymphocytes. Monocyte cytoplasm is abundant, basophilic and sometimes has large vacuoles within it. Azurophil granules, often staining a quite pale pink, occur in some cells. Platelets (Fig. 16, 17). Platelets look very much like human ones, presenting the typical more densely-staining and centrally placed chromomere and the paler-staining peripheral hyalomere. Most platelets are also in about the same size range as those of human blood but there are more larger ones.
Cell counts Total white cell count. The mean total count for all the animals examined was 13,380 and the S.D. 6,480, the range being 2,600 to 26,700.
Fig. 11. Possum small lymphocyte; Leishman stain. Fig. 12. Possum medium lymphocyte with azurophil granules; Leishman stain. Fig. 13. Possum large lymphocyte; Leishman stain. Fig. 14. Possum monocyte with reniform nucleus and coarse chromatin; Leishman stain. Fig. 15. Possum monocyte with doughnut-shaped nucleus; Leishman stain. Fig. 16. Possum platelets; Leishman stain. Fig. 17. Very large possum platelet; Leishman stain. Fig. 11-17: ×1,650 318 R.A. HARBOUR:
Two animals had counts of more than 26,000 (in one case with a very high number of neutrophils, in the other of lymphocytes), the next highest being 18,600. Assessed by statistical calculation the observed counts exhibit a scatter that shows no significant departure from the normal distribution pattern. The difference be- tween the mean counts for females (15,340) and males (9,720) is probably significant (0.02 The overall mean diameters and S.D.S (in brackets) of the different nuclear- segmental types of neutrophil were as follows: non-segmented 13.05μ (0.99μ), two segments 12.94μ (0.92μ), three segments 12.98μ (0.95μ), four segments 13.08μ (0.93μ), five segments 13.16μ (1.02μ), six segments 13.66μ (1.07μ) and seven segments (one cell only) 13.2μ. However, since there was such variation in mean cell size between animals and since the number of cells of each segmental type measured in the differ- ent animals was inconstant, it would eliminate some bias to compare the means of the mean values for each animal of each segmental type. These figures are less wide- ly scattered than the above and are: non-segmented 12.98μ, two segments 12.97μ, three segments 13.02μ, four segments 13.08μ, five segments 13.02μ and six segments 13.67μ. Comparison of these in consecutive pairs shows a significant change in size at only one step-the five to six segment stage (0.001 non-segmented 13.69μ (S.D. 1.09μ), two segments 13.91μ (S.D. 1.04μ), and three seg- ments 14.56μ (S.D. 1.15μ). Because of the variation in the number of cells measured in different animals and in the mean eosinophil diameters of different animals the means of the means for the individual animals are probably more useful figures for comparison; these are: non-segmented 13.78μ, two segments 13.92μ and three seg- ments 14.74μ, and they show a significant increase in size from the two to the three segment stage (0.001 Basophils. The mean diameter of the 190 basophils measured was 14.24μ, the S.D. being 1.08μ and the range 11.0 to 17.8μ. The measurements are distributed in a normal manner. The mean diameters for the 21 animals where one or more baso- phils were measured varied from 12.90μ (based on 2 cells) to 15.30μ (based on 4 cells). The mean of these means is not very different from the mean of all the cells taken individually-14.28μ (S.D. 0.70μ). These means, too, are scattered in a normal fashion. Analysis of variance shows a highly significant inter-animal variation in basophil size (p<0.001). No correlation was detected between the size of the basophils and their cell counts (r=-0.007). Lymphocytes. The size of the lymphocytes covered a wide range, from 7.0μ to 15.2μ. The mean was 10.32μ and the S.D. 1.275μ-the smallest diameter and largest S.D. for all the five types of leukocyte. The diameters are not distributed in a normal manner (in χ2 test p<10-7) and this is apparently due to a very pronounced positive skewness in the distribution (p<10-9); no significant kurtosis was detected. The mean diameters for the separate animals varied from 8.74μ to 11.32μ; the mean of these is, of course, again 10.32μ and the S.D. is 0.66μ. Statistical calculation indicates no significant departure from normal in the scatter of these mean diame- ters. Analysis of variance again shows a very highly significant variation in cell size between animals (the value of e2z is far beyond that indicating the 0.1% level of significance). No significant correlation between cell size and cell count is apparent (r=-0.08). Monocytes. The sizes of the monocytes were scattered in a normal manner between 11.6μ and 18.2μ with a mean of 14.99μ and a S.D. of 1.12μ. As with the other cell types, the mean diameters of the monocytes in different animals showed great variation-from 13.83μ to 15.89; the mean of these means is also (coincidentally) 14.99μ and the S.D. 0.58μ. These mean values, too, are distributed in a normal man- ner. Analysis of variance shows, once more, a very highly significant inter-animal 322 R.A. BARBOUR: variation in cell size (p<0.001). There is no significant correlation between mean cell diameters and cell counts (r=0.17). Correlation of cell sizes. The correlation coefficient for the 10 different two-way comparisons of mean diameters of cell types in the various animals are as follows: neutrophil/eosinophil 0.21 (not sig.); neutrophil/lymphocyte 0.29 (not sig.); neutro- phil/monocyte 0.48 (probably significant, p is close to 0.02); eosinophil/lymphocyte 0.28 (not sig.); eosinophil/monocyte 0.35 (not sig., but p close to 0.1); lymphocyte/ monocyte 0.62. (significant, 0.001 Cytochemical Studies Sudan black B staining After sudan black staining the neutrophils stand out prominently, their cyto- plasm exhibiting granules of irregular size and distribution (Fig. 18). By comparison with the human neutrophils (Fig. 19) stained in parallel those of Trichosurus have fewer stained granules, occupying less of the cytoplasm and scattered less uniformly through the cell. Eosinophils of both man and Trichosurus show moderate staining at the peri- phery of the specific granules (Fig. 20, 21), but the staining in Trichosurus is often less obviously peripheral and appears to stain the whole granule to some extent. In general human cells show the more intense reaction. In no case have I been able to demonstrate any sudanophilia in basophils, either of man or Trichosurus. The cytoplasm of basophils (like that of other leukocytes and platelets) often exhibits an extremely pale grey staining reaction, that I take to be non-specific, in which the sites occupied by the specific granules (previously iden- tified by staining with toluidine blue) are apparent as completely unstained spots. Lymphocytes, monocytes and platelets of Trichosurus are almost always negative to sudan black staining. An occasional lymphocyte (Fig. 22) shows two or three black-stained granules and a few monocytes show some smaller and less well defined ones. No cells like the human monocytes (Fig. 23) with their plentiful small sudano- philic granules were seen. PAS staining The neutrophils are the prominently stained cells, exhibiting a generalized staining of their cytoplasm with, in a minority of cases, some more intensely stained areas of irregular shape superimposed upon this. There is considerable variation in staining intensity from cell to cell. In many cases cells identifiable by their nuclei as band cells show the weakest staining. In general the neutrophils of Trichosurus exhibit less intense PAS reaction than the human ones processed with them (Fig. 24, 25). In both human and Trichosurus smears the PAS reaction can be completely Marsupial Leukocytes and Platelets 323 prevented by previous amylase digestion and is presumed to be due to glycogen. Eosinophils show a weak PAS reaction, apparently at the surface of, or between, the specific granules (Fig. 26): human parallels show similar, but slightly more intense, staining. After amylase digestion staining is sometimes a little less intense-but not enough to indicate convincingly the presence of glycogen. The specific granules of the basophils are PAS-positive whether or not there has been amylase digestion. Basophils possess more agranular areas in their cytoplasm than eosinophils and it is possible to study the PAS reaction of these areas to some extent too. I have compared photographs of basophils taken when they were stained with toluidine blue with photographs of the same cells when they were subsequently stained by the PAS technique: this was done partly by comparing positive prints of the cells at the two stages of staining (Fig. 27, 28) and partly by projecting the nega- tive photograph of one stage onto the positive print of the other and observing to what extent the light and dark areas cancel out one another. Such comparisons leave little doubt that basophils do exhibit a positive PAS reaction apart from their granules. This occurs to some extent both in the cells that have been amylase- treated and in those that have not but, overall, I am convinced that more is present in the undigested cells thus suggesting that at least some of the basophils contain glycogen. In the digested cells the PAS-positive material is less widely spread throughout the cytoplasm and often occurs among tightly packed clumps of specific Fig. 18. Possum neutrophil; Sudan black and nuclear fast red. Fig. 19. Human neutrophil; Sudan black and nuclear fast red. Fig. 20. Possum eosinophil; Sudan black and nuclear fast red. Fig. 21. Human eosinophil; Sudan black and nuclear fast red. Fig. 22. Possum lymphocyte with several sudanophil granules; Sudan black and nuclear fast red. Fig. 23. Human monocyte; Sudan black and nuclear fast red. Fig. 24. Possum neutrophil; PAS and Mayer's haematoxylin. Fig. 25. Human neutrophil; PAS and Mayer's haematoxylin. Fig. 18-25: ×1,650 324 R.A. BARBOUR: granules or at the edge of the cell where it is closely applied to an adjacent erythro- cyte-sites where one might suggest the amylase failed to gain access to the glycogen. The appearance of human basophils stained simultaneously with those of Trichosurus is quite different (Fig. 29, 30). The human cells exhibit the presence of glycogen in the form of granules of varying size (on average smaller than the specific granules), and sometimes areas of diffuse staining, in the intergranular cyto- plasm. The specific granules of the human basophils show no sign of positive PAS reaction at any stage though, in the undigested smears, the granules can be restained Fig. 26. Possum eosinophil: PAS and Mayer's haematoxylin. Fig. 27. Possum basophil; toluidine blue. Fig. 28. Possum basophil, same cell as Fig. 27; PAS. Fig. 29. Human basophil; toluidine blue. Fig. 30. Human basophil, same cell as Fig. 29; PAS. Fig. 31. Possum platelets; PAS and Mayer's haematoxylin. Fig. 32. Human platelets; PAS and Mayer's haematoxylin. Fig. 33. Possum neutrophil; peroxidase (Lillie) and Giemsa. Fig. 34. Human neutrophil; peroxidase (Lillie) and Giemsa. Fig. 35. Possum eosinophil; peroxidase (Lillie) and Giemsa. Fig. 36. Human eosinophil; peroxidase (Lillie) and Giemsa. Fig. 26-36: ×1,650 Marsupial Leukocytes and Platelets 325 with toluidine blue after the PAS procedure showing that they are still intact. Fol- lowing amylase digestion the granules of human basophils cannot be restained with toluidine blue whereas those of Trichosurus can: apparently the fixation procedure used gives better preservation of the granules of Trichosurus than those of the human. Lymphocytes and monocytes are always PAS-negative, unlike those of the human where a few such cells in the smears that had not been treated with amylase show some positively stained granules. Compared with those of man the platelets of Trichosurus exhibit a very feeble PAS reaction (Fig. 31, 32). The staining occurs mainly in the granulomere and, as it is completely amylase-labile, is presumably due to glycogen. Peroxidase The first method employed for the demonstration of peroxidase (that using o- tolidine) produces consistently negative results with blood from Trichosurus though human smears stained simultaneously regularly show distinct staining of neutrophils and eosinophils. The second method (employing benzidine) produces positive staining of the neutrophils and eosinophils (Fig. 33-36)-in both cases the result appearing as blue granules in the cytoplasm of the cells, in general less prominent in eosinophils than in neutrophils and considerably less intense and less numerous in the cells of Tricho- surus than in those of the parallel human smears. Monocytes, lymphocytes and platelets are consistently negative in both human and Trichosurus. Basophils, after staining with Giemsa, are not suitable for assess- ment and no special study has been made to determine their peroxidase content. Alkaline phosphatase In three of the four samples stained by WYLLIE's (1964) method no staining at all was observed. In the other one most of the basophils showed some poorly defined black staining in one or more patches between the parts of the nucleus (Fig. 37) (and the specific granules were poorly stained by Leishman's stain suggesting poor preservation). No staining was seen in cells of any other type. In smears stained by the azo-dye technique and then counterstained with methyl green without preliminary examination no positively stained cells can be found. In these smears, however, the basophil granules are stained purple (presumably by some remaining methyl violet contaminating the methyl green stain) and positive staining might not be recognizable in these cell. In those smears that were examined before counterstaining a small number of positively stained cells was found, cells in which the cytoplasm exhibited brownish-red granules of varying size and depth of colour and in which the staining showed considerable variation from cell to cell and from animal to animal. On subsequent staining with toluidine blue all these cells proved to be basophils and, on comparing photographs of these cells before and after tolui- dine blue counter-staining (Fig. 38-41), it is apparent that most of the phosphatase reaction corresponds in position to that of some or most (or in some cells perhaps even all) of the specific granules. It has not been ascertained whether there are any basophils that do not stain for alkaline phosphatase by this method only that all the positive cells are basophils. 326 R.A. BARBOUR: Dehydrogenases A positive reaction for both the dehydrogenases sought occurs in lymphocytes, monocytes and platelets. The staining appears as small dark blue spots or, some- times (especially in lymphocytes), small irregular smudged areas and some pale diffuse greying of the cytoplasm. In general the staining after the lactate dehydrogenase technique is considerably more intense than following that for succinate dehydro- genase. Evidence of the latter enzyme appears in most lymphocytes and monocytes (Fig. 42, 43) but in only a few platelets and, overall, the intensity of staining is similar to that observed in the human parallels. Lactate dehydrogenase is demon- strated in virtually all lymphocytes and monocytes (Fig. 44, 45) with about the same Fig. 37. Possum basophil; alkaline phosphatase (Wyllie) and Leishman. Fig. 38. Possum basophil; alkaline phosphatase (Hayhoe and Quaglino)-strong reaction. Fig. 39. Possum basophil, same cell as Fig. 38; after subsequent toluidine blue staining. Fig. 40. Possum basophil; alkaline phosphatase (Hayhoe and Quaglino)-weak reaction. Fig. 41. Possum basophil, same cell as Fig. 40; after subsequent toluidine blue staining. Fig. 42. Possum lymphocyte; succinate dehydrogenase and nuclear fast red. Fig. 43. Possum monocyte; succinate dehydrogenase and nuclear fast red. Fig. 44. Possum lymphocyte; lactate dehydrogenase and nuclear fast red. Fig. 46. Possum platelets; lactate dehyrogenase and nuclear fast red. Fig. 47. Possum platelet; lactate dehydrogenase and nuclear fast red. Fig. 37-47: ×1,650 Marsupial Leukocytes and Platelets 327 intensity as in human cells, and in all platelets (Fig. 46, 47) where the reaction is much more intense than in the human. In fact, by far the most intense dehydro- genase staining observed is that for lactate dehydrogenase in Trichosurus platelets. In the human smears some staining for lactate dehydrogenase is observed also in most eosinophils, while in the smears of Trichosurus blood some neutrophils and eosinophils give a very weak reaction. Basophils were not identified in the smears stained for dehydrogenases. Discussion Morphological and Metrical Studies Cell morphology Difficulty in distinguishing some monocytes from large lymphocytes also exists in man (BLOOM and FAWCETT, 1968; HAM, 1969), monkey (SCHERMER, 1967), cattle (WINQVIST, 1954), guinea pig (SCHERMER, 1967), rat (JORDAN, 1938; SCHERMER, 1967) and Didelphis (JORDAN, 1938), and is probably of fairly general occurrence; it can even be a problem in electron microscopic studies (ANDERSON, 1966). Distinguishing some monocytes from juvenile neutrophils can also be a problem in human blood (HAM, 1969). Neutrophils. The neutrophil nuclei of Trichosurus conform to the usual mam- malian pattern and show no unusual features such as the ring shape seen in rat (VAUGHAN and GUNN, 1930; SCARBOROUGH, 1931/2; JORDAN, 1938),mouse (PETRI, 1933; SCHERMER, 1967) and golden hamster (STEWART, FRORIO and MARGRAGE, 1944; SCHERMER, 1967), or the extreme degree of segmentation noted in some monkeys (KRUMBHAAR and MUSSER, 1920; PONDER, YEAGER and CHARIPPER, 1929b; SUARES, RIVERA and MORALES, 1942; SCHERMER, 1967). The cytoplasm, likewise, shows nothing as outstanding as the relatively large pseudoeosinophil granules found in rabbit and guinea pig (SCARBOROUGH, 1931/2; LOEWENTHAL, 1933; SCHERMER, 1967; BLOOM and FAWCETT, 1968). The neutrophil granules in man are usually described as fine (DITTRICH, 1962; BLOOM and FAWCETT, 1968; HAM,1969), and they seem to be of similar size or smaller in most other mam- male-including dog (SCHERMER, 1967; BLOOM and FAWCETT, 1968), Cat (GILMORE, GILMORE and JONES, 1964; SCHERMER, 1967), rat (SCARBOROUGH, 1931/2; SCHERMER, 1967; BLOOM and FAWCETT, 1968),mouse (SCARBOROUGH, 1931/2),golden hamster (STEWART, FRORIO and. MUGRAGE, 1944), some monkeys and apes (KLIENEBERGER, 19271; PONDER, YEAGER and CHARIPPER, 1929b; SCHERMER, 1967),cattle (DIMOCK and THOMPSON, 1906/ 71), horse, pie, sheep and goat (SCARBOROUGH, 1931/2), a species of llama (PONDER, YEAGER and CHARIPPER, 1929a), and some marsupials, Didelphis virginiana (JORDAN, 1938), Macropus robustus and the wombat (PONDER, YEAGER and CHARIPPER, 1929c). Difficulty in visualization of the granules due to small size or numbers or to weak staining has also been noted in dog (SCARBOROUGH, 1931/2; BLOOM and FAWCETT, 1968), cat (LOEWENTHAL, 1933; GILMORE, GILMORE and JONES, 1964),rat (SCHERMER, 1967; BLOOM and FAWCETT, 1968), mouse (SCARBOROUGH, 1931/2; SCHERMER, 1967), pig (SCARBOROUGH, 1931/2) and wombat (PONDER, YEAGER and CHARIPPER, 1929c). Mam- malian neutrophil granules have not often been described as large or coarse; PONDER, YEAGER and CHARIPPER have so described those of some Camelidae (1929a) and Lemur 328 R.A. BARBOUR: catta (1929b). Eosinophils. The nuclei of the eosinophils of Trichosurus show no conspicuous features such as the annular shape seen in some cells or rat (KLIENESERGER, 19272; JORDAN, 1938; SCHERMER, 1967; BLOOM and FAWCETT, 1968), mouse (PETRI, 1933; ANDREW, 1965; SCHERMER, 1967; BLOOM and FAWCETT, 1968) and field mouse (LOEWENTHAL, 1933), or the extreme degrees of segmentation of some eosinophils of the rhesus monkey (SUAREZ, RIVERA and MORALES, 1942) and Didelphis virginiana (JORDAN, 1938). The size of eosinophil granules in different mammals appears to cover a consider- able range extending up to the very large ones in horse (SCARBOROUGH, 1931/2; ARCHER, 1963) and in some cells in dog (ARCHER, 1963). Those of Trichosurus are pro- bably about average in this range-they are about the same size as in man where they are similar to those in cow, goat, sheep and pig while they are larger in rabbit and smaller in rat and mouse (ARCHER,1963). They are relatively small in Didelphis (JORDAN, 1938). The elongated shape of the granules in Trichosurus is rather more unusual but certainly not unique; the occurrence of elongated granules in at least some eosinophils has been noted especially in cat (BUSCH and VAN BERGEN, 1902; KLIENEBERGER, 19271,2; SCARBOROUGH,1931/2; MITSUI et al., 1956; IRFAN, 1961; GILMORE, GILMORE and JONES, 1964; SCHERMER, 1967), and also in rabbit (SCARBOROUGH, 1931/2), guinea pig (LUCIA and LUCIA, 1928; SCARBOROUGH, 1931/2),golden hamster (SCHERMER, 1967), dog, horse (SCARBOROUGH, 1931/2) and in some members from all four classes of non-mammalian vertebrates (JORDAN, 1938; MITSUI et al., 1956). Light basophilia of the intergranular cytoplasm is a common feature having been described in man (ARCHER, 1963; BLOOM and FAWCETT, 1968), some types of monkey (PONDER, YEAGER and CHARIPPER, 1929b; SCHERMER 1967), horse (ARCHER, 1963), camel PONDER, YEAGER and CHARIPPER, 1929a), mouse (PETRI, 1933) and dog where it is vacuo- lated (SCHERMER, 1967). Acidophilia of the cytoplasm has been noted in these cells in brown lemur (PONDER, YEAGER and CHARIPPER, 1929b) and in Cattle (WINQVIST, 1954). Basophils. Judging from the literature (LUCIA and LUCIA, 1928; PONDER, YEAGER and CHARIPPER, 1929a, b, c; SCARBOROUGH, 1931/2; JORDAN, 1938; WINQVIST, 1954; FREDERICKS and MOLONEY, 1959; SCHERMER, 1967; BLOOM and FAWCETT, 1968; HAM, 1969) the nuclei of mammalian basophils vary little in form, usually presenting an irregular shape with, often, some degree of real segmentation. Trichosurus fits well into this general pattern. The granules, however, seem to vary considerably amongst different mammals (PONDER, YEAGER and CHARIPPER, 1929a, b,c; SCARBOROUGH, 1931/2; KNOLL, 1932; JORDAN, 1938; SMITH, 1947; ACKERMAN, 1963; SCHERMER, 1967; BLOOM and FAWCETT, 1968) in actual size, consistency of size, shape, consistency of shape and density and uni- formity of distribution within the cytoplasm-but not much in staining reaction. Basophil granules usually stain intensely with blood stains and are metachromatic, in fact ACKERMAN (1963) says that metachromasia is the only reliable criterion of basophils in various animals. THONNARD-NEUMANN (1963) has studied the basophils of man and rabbit and, on the basis of differences of intracellular distribution of gran- ules, has been able to divide the cells in each species into three categories that he believes are related to cell age; I do not think such a categorization would be possible in Trichosurus. As far as one can compare the basophil granules with those of other Marsupial Leukocytes and Platelets 329 animals (some accounts in the literature are very sketchy and photographs are rarely presented) the granules in Trichosurus are of moderate size, relatively uniform size and shape, and relatively numerous and densely packed in the cytoplasm. In pig (SCARBOROUGH, 1931/2) the granules are plentiful and probably about the same size as in Trichosurus; in camels and llamas (PONDER, YEAGER and CHARIPPER, 1929a) and in cattle (DIMOCK and THOMPSON, 1906/71) they are large and numerous; in a number of Primates (PONDER, YEAGER and CHARIPPER, 1929b) they are coarse but the number varies with the species; in man himself (ACKERMAN, 1963; BLOOM and FAWCETT, 1968) they vary much in size and also in shape and, from my own observations, are less densely packed than in the cells of Trichosurus; in cat (SCARBOROUGH, 1931/2; GILMORE, GILMORE and JONES, 1964) the granules are large and sparse; in guinea pig (LUCIA and LUCIA, 1928; SCARBOROUGH, 1931/2; SMITH, 1947; ACKERMAN, 1963; SCHERMER, 1967; BLOOM and FAWCETT, 1968) they appear to be abundant and predominantly large and round though showing some variation in size and shape; in rabbit (SCARBOROUGH, 1931/2; ACKERMAN, 1963; SCHERMER, 1967), dog (SCARBOROUGH, 1931/2; BLOOM and FAWCETT, 1968) and sheep (SCARBOROUGH, 1931/2; SCHERMER, 1967) they are small and variable and, probably, less abundant than in Trichosurus. As far as other marsupials are concerned, JORDAN (1938) describes the basophil granules of Didelphis virginiana as spherical or angular in form and readily soluble; in Macropus robustus (PONDER, YEAGER and CHARIPPER, 1929c) they are apparently plentiful as, also, in Macropus robustus Woodwardii (PONDER, YEAGER and CHARIPPER, 1929c) where they vary con- siderably in size; in Macropus fulginosus they are coarse, in Phascolomys wombat small (PONDER, YEAGER and CHARIPPER, 1929c). The staining reaction of the intergranular cytoplasm of basophils has received little attention. This part of the cell remains relatively unstained in Macropus robustus (PONDER, YEAGER and CHARIPPER, 1929c), exhibits some basophilia in camels (PONDER, YEAGER and CHARIPPER, 1929a) and stains pink with blood stains in Macropus fulginosus (PONDER, YEAGER and CHARIPPER, 1929c). Lymphocytes. The nuclei of mammalian lymphocytes generally present a fairly standard appearance of a round or slightly indented outline with coarse and densely packed chromatin clumps, especially in the smaller cells. The cells of Trichosurus fit this pattern. Some irregularity of outline was noted in some lymphocytes in a few of the Primates studied by PONDER, YEAGER and CHARIPPER (1929b) and also, to- gether with a fine chromatin pattern, in some cells that had lymphocyte-like cyto- plasm in guinea pig by SMITH (1947). Occasional apparently binucleate lymphocytes have been described in Didelphis virginiana, rat (JORDAN, 1938) and monkey (SCHERMER, 1967) and I have seen, very rarely, a cell in Trichosurus that may be in this category. Although large and small lymphocytes have often been described and listed separately in differential leukocyte counts it seems doubtful whether they ever costitute easily definable groups rather than simply being at different levels along a continuous range of sizes. There is a continuous size range in Trichosurus and in man (AMSS, 1967) and in most of the species dealt with by SCHERMER (1967); only in sheep does SCHERMER suggest that there are two fairly clearcut types based on size, as does LOEWENTHAL(1933) for mouse. The paler perinuclear region in stained lymphocytes has been noted in other species (man: ACKERMAN, 1967; mouse: SCHERMER, 1967; guinea pig: LUCIA and LUCIA, 330 R.A. BARBOUR: 1928) and is possibly of fairly general occurrence. There appear to be considerable differences between various mammals, however, in the average size of lymphocytes (q.v.) and in the abundance and size of azurophil granules. The lymphocytes of Llama pocas have been stated to have no azurophil granules (PONDER, YEAGER and CHARIPPER, 1929a). Estimates of the number of cells possessing such granules in normal human blood vary: BRAUNSTEINER and ZUCKER- FRANKLIN (1962) give 25%, whereas HAM (1969) 10%. They are present in varying numbers in at least some of the cells in several species of monkey (PONDER, YEAGER and CHARIPPER, 1929b; SUAREZ, RIVERA and MORALES, 1942; SCHERMER, 1967), cat (LOEWENTHAL, 1933; GILMORE, GILMORE and JONES, 1964; SCHERMER, 1967), dog (SCARBOROUGH, 1931/2; SCHERMER, 1967), rabbit (JORDAN, 1938), golden hamster (MUGRAGE, 1944; SCHERMER, 1967),sheep (SCARBOROUGH, 1931/2; SCHERMER, 1967),camel (PONDER, YEAGER and CHARIPPER, 1929a) and cattle(WINQVIST, 1954). In Macropus robustus (PONDER, YEAGER and CHARIPPER, 1929c), rat (JORDAN, 1938; SCHERMER 1967),mouse (SCARBOROUGH, 1931/2; PETRI, 1933; JORDAN, 1938),field mouse (LOEWENTHAL, 1933),guinea pig (LUCIA and LUCIA, 1928; SCARBOROUGH, 1931/2; JORDAN, 1938; SMITH, 1947; SCHERMER, 1967) and horse (SCARBOROUGH, 1931/2) they are also present but, according to some of the authors, are found solely or more predominantly in the larger lymphocytes. In man, also, ELVES(1966) says they are commoner in the large cells. Among the numerous mammals he studied KNOLL (1932) noted that azurophil granules were sparse or absent in the carnivores, but plentiful in echidna and in marsupials, Xenarthra, rodents and ungulates. Monocytes. The nuclei of monocytes in man (BRUCHER, 1962; BLOOM and FAWCETT, 1968; HAM, 1969) are usually oval or exhibit some degree of indentation leading to a kidney-shaped or horse-shoe-shaped outline. In various other eutherians (PONDER, YEAGER and CHARIPPER, 1929a, b; SCARBOROUGH, 1931/2) and in several marsupials (PONDER, YEAGER and CHARIPPER, 1929c) the monocyte nuclei are similar. The more frequent irregular forms I found in Trichosurus are probably an unusual feature of this species, though from the descriptions of GILMORE, GILMORE and JONES (1964) for cat, LUCIA and LUCIA (1928) for guinea pig and WINQVIST (1954) for cattle there is, per- haps, some parallel in those animals. I have found no other account of annular nuc- lei in monocytes though PETRI (1933) has described small perforations in some mono- cyte nuclei in mouse. Similarly the presence of coarse chromatin in some of the monocytes in Trichosurus is apparently another unusual feature-SMITH (1947) has described a similar appearance in some of the cells in guinea pig. The possession of azurophil granules seems to be the rule rather than the excep- tion for at least some of the monocytes in mammalian blood; they are present in man (BRUCHER, 1962; HAM, 1969), gorilla and marmoset (PONDER, YEAGER and CHARIPPER, 1929b) Cat (SCARBOROUGH, 1931/2; GILMORE, GILMORE and JONES, 1964), mouse (PETRI, 1933), rat, dog, horse (SCARBOROUGH, 1931/2), guinea pig (LUCIA and LUCIA, 1928; SCARBOROUGH, 1931/2), four species of Camelidae (PONDER, YEAGER and CHARIPPER, 1929a), and the marsupial, Macropus robustus (PONDER, YEAGER and CHARIPPER, 1929c). But they have been stated to be absent from the monocytes of rabbit (SCARBOROUGH, 1931/2), the monkey, Ateles geoffrei (PONDER, YEAGER and CHARIPPER, 1929b), and Macropus ruficollis (PONDER, YEAGER and CHARIPPER, 1929c). Marsupial Leukocytes and Platelets 331 Platelets. The platelets of Trichosurus have the appearance of those of other mammals where the main difference appears to be in size (q.v.). They certainly are not nucleated thrombocytes as have been reported in some other marsupials (KNOLL, 1957). Cell counts Total white cell count. The observed mean count of over 13,000 cells is higher than the upper limit of the generally accepted normal range in man, but it is not at all unusual when compared with counts found in other species. Mean counts in ex- cess of 10,000 (sometimes much in excess) have been found in many other mammals -several different apes and monkeys (KRUMBHAAR and MUSSER, 1920; KLIENEBERGER, 19271; HALL, 19291; PONDER, YEAGER and CARIPPER, 1929b; SCARBOROUGH, 1931/2; JONES et al., 1947; SCHERMER, 1967; WINTROEE, 1967), lemur (PONDER, YEAGER and CHARIPPER, 1929b),cat (WELLS and SUTTON, 1916; SCARBOROUGH, 1931/2; JORDAN, 1938; ALBRITTON, 1952; GILMORE, GILMORE and JONES, 1964; SCHERMER, 1967; WINTROBE, 1967),dog (WELLS and SUTTON, 1916; SCARBOROUGH, 1931/2; BRUNER and WAKERLIN, 1937; JORDAN, 1938; REKERS and COULTER, 1948; ALBRITTON, 1952; WINTROBE, 1967), rat (SCARBOROUGH, 1931/2; JORDAN, 1938; CAMERON and WATSON, 1949; ALBRITTON, 1952; EVERITT and WEBB, 1958; RIGDON, CRASS and RICHARDSON, 1960; SCHERMER, 1967; WINTROBE, 1967), guinea pig (SCARBOROUGH, 1931/2; KING and LUCAS, 1941),badger (WELLS and SUTTON, 1916), woodchuck (WELLS and SUTTON, 1916; JORDAN, 1938), mink, skunk, sloth (WINTROBE, 1967), pig (WELLS and SUTTON, 1916; SCARBOROUGH, 1931/2; JORDAN, 1938; ALBRITTON, 1952; ANDREW, 1965), camel and llama (PONDER, YEAGER and CHARIPPER, 1929a), goat (SCARBOROUGH, 1931/2) and bottle-nose dolphin (MEDWAY and GERACI, 1964). In other marsupials JORDAN (1938) and WINTROBE (1967) give counts of 15,300 and 12,000 respectively for Didelphis virginiana, while PONDER, MEAGER and CHARIPPER (1929c) give no figure above 8,600 for any of the several marsupials they studied (which included Didelphis virginiana). BOLLIGER and BACKHOUSE (1960a) give the mean white cell count for normal koalas as 6,200. GODWIN, FRASER and IBBOTSON (1964) have suggested that many of the high counts found in rats have been the result of the use of potentially diseased animals; while it is a possibility I do not think there is any real evidence that this is the case in my specimens of Trichosurus. JONES et al. (1947), working specifically with monkeys, have suggested that excitement associated with blood removal causes some increase in the leukocyte count; this factor could have played some part in the determination of the figure presented here for Trichosurus. Neutrophil count. Mean neutrophil counts for many eutherian species can be found in the literature or can be simply worked out from total and differential counts (KRUMBHAAR and MUSSER, 1920; PONDER, YEAGER and CHARIPPER, 1929a, b; VAUGHAN and GUNN, 1930; SCARBOROUGH, 1931/2; PETRI, 1933; JORDAN, 1938; KING and LUCAS, 1941; SUAREZ, RIVERA and MORALES, 1942; STEWART, FRORIO and MUGRAGE, 1944; JONES et al.,1947; REKERS and COULTER, 1948; CAMERON and WATSON, 1949; ALBRITTON, 1952; BOLLIGER and BACKHOUSE, 1960b; RIGDON, CRASS and RICHARDSON, 1960; GILMORE, GILMORE and JONES, 1964; MEDWAY and GERACI, 1964; SCHERMER, 1967; WINTROBE, 1967; BLOOM and FAWCETT, 1968; HAM, 1969). The mean count for Trichosurus comes well within the range of these figures being not remarkably high or low. Compared with 332 R.A. BARBOUR: other marsupials (PONDER, YEAGER and CHARIPPER, 1929c; JORDAN, 1938; BOLLIGER and BACKHOUSE, 1960a; WINTROBE, 1967) the count is fairly high but, from PONDER, YEAGER and CHARIPPER's (1929c) figures, is exceeded in Macropus robustus, Macropus agilis and Petrogale xanthopus. The Arneth count in Trichosurus shows a distinct displacement to the left com- pared with that of man where the normal number of non-segmented cells is only about 5% and the three-segment form has the highest value (UNDRITZ, 19521; BLAKISTON's Medical Dictionary, 1956; DORLAND's Medical Dictionary, 1965; STEDMAN's Medical Dictionary, 1966; WINTROBE, 1967): the normal "scores" derived from these references are 276 (BLAKISTON's Medical Dictionary, 1956; DORLAND's Medical Dictionary, 1965; STEDMAN's Medical Dictionary, 1966; WINTROBE, 1967) or 298 (UNDRITZ, 19521). Com- parison with the Arneth figures for other species is difficult because in some cases where a species has been studied by two or more investigators the results are wildly different. The figures given by KENNEDY and CLIMENKO (1932) give mean scores within the range 210 to 270 (i.e. fairly close to my figure for Trichosurus) for rabbit, hare, rat, mouse, guinea pig, sheep, cattle, goat, rhesus monkey and dog; and the values for PONDER, YEAGER and CHARIPPER's (1929a) four Camelidae also come within this range. TREADGOLD's (1920) and DANZER's (1930) figures for rabbit correspond closely with the above, but those of CORSY (1911), giving a score of 310, and HARM (1955), 364, are markedly higher. Likewise TREADGOLD's (1920) figures for guinea pig agree with those of KENNEDY and CLIMENKO (1932), but CORSY's (1911) score of 381 and SMITH's (1947) 427 are much more right-handed. CORSY's (1911) figure of 372 for dog is also far from KENNEDY and CLIMENKO's (1932), while MORRIS, ALLISON and GREEN (1940) say this animal shows a more left-handed pattern than man. In rat estimates of the Arneth score vary from 109 (YEAGER and HATERIUS, 1930) to 434 (CORSY, 1911);in this species the ring-shaped nuclei would make classification difficult (VAUGHAN and GUNN, 1930) and different criteria of segmentation have undoubtedly been used by different authors. The same problem would probably obtain in the hamster where more than one third of the cells are non-segmented (STEWART, FRORIO and MUGRAGE, 1944). For sheep (1929a) and cattle (1929b) SIMPSON gives figures that indicate scores (124 and 128 respectively) considerably lower than those of KENNEDY and CLIMENKO (1932).The latter authors, however, give quite low figures for cat (179) and pig (143)-but from CORSY (1911) the value for cat is 311. Among the various Primates studied by PONDER, YEAGER and CHARIPPER (1929b) the rhesus monkey had a score close to that from KENNEDY and CLIMENKO (1932), most of the others being rather widely scattered (partly because of small numbers of animals?) between 200 and 350; the only surprisingly aberrant one being the black spider monkey, Ateles ater, with a score of 614. With regard to the marsupials they studied PONDER, YEAGER and CHARIPPER (1929c) say that in general the counts are more right-handed than in man, and the mean scores derived from their paper are mostly scattered (rather widely, again perhaps because of small numbers of specimens) over the range 251 to 351- but Didelphis virginiana has a mean score of 516. The nearest of these to Trichosurus, with a score of 251 and 20% of non-segmented cells, is Macropus rufus but, as with most of the others, the predominant cells were those with three segments; in wom- bat they were the ones with four segments and in Didelphis those with six. Coming further down the vertebrate scale, in Tachyglossus the Arneth count shows a shift to Marsupical Leukocytes and Platelets 333 the right compared with man (BOLLIGER and BACKHOUSE, 1960b) while in many of the fishes and amphibians (especially fishes) that FEY (1966b) examined the count is very markedly left-handed. PONDER and FLINT (1927) showed that ether anaesthesia in rabbits produced a considerable shift to the left in the Arneth count but not for some minutes after administration-as blood was taken almost immediately from my specimens of Trichosurus this factor is not likely to have influenced the count. Eosinophil count. The mean eosinophil count found in Trichosurus, 221, appears to be towards the lower end of the range for mammals generally, and various ac- counts (RUD, 1947; CODE, MITCHELL and KENNEDY, 1954; GROSS, 1962; WINTROBE, 1967; MCDONALD, DODDS and CRUICKSHANK, 1968; HAM, 1969) suggest that the normal count in man is very similar. One of the lowest mammalian eosinophil counts is indicated in BOLLIGER and BACKHOUSE's (1960b) figures for echidna where they found eosino- phils (1% only) in only one of eight animals. Among other marsupials, figures given by PONDER, YEAGER and CHARIPPER (1929c), JORDAN (1938) and WINTROBE (1967) indicate counts between 200 and 600 for most species studied, though the first authors' figures put Macropus ruficollis (about 1,300) and Macropus rufus (under 100) outside this range. In the koala a mean count of only about 120 has been found (BOLLIGER and BACKHOUSE, 1960a). On the other hand PACKER's (1968) counts in Setonix ranged as high as 4656. In a variety of eutherian species (PONDER, YEAGER and CHARIPPER, 1929b; MAYERSON, 1930; SCARBOROUGH, 1931/2; JORDAN, 1938; DELAUNE, 1939; MORRIS, ALLISON and GREEN, 1940; KING and LUCAS, 1941; THEWLIS and MEYER, 1942; REKERS and COULTER, 1948; CAMERON and WATSON, 1949; ALBRITTON, 1952; ANDREW, 1965; SCHERMER, 1967; WINTROBE, 1967) the normal eosinophil count lies between about 150 and 800. In gol- den hamster (STEWART, FRORIO and MUGRAGE, 1944; TRINCAO, NOGUEIRA and GOUVEIA, 1949) and mink (WINTROBE, 1967), however, it is well below 100. Various figures for cat (SCARBOROUGH, 1931/2; LANDSBERG, 1940; ALBRITTON, 1952; IRFAN, 1961; SCHALM, 19611; GILMORE, GILMORE and JONES, 1964; SCHERMER, 1967), taken together, suggest a normal count for this species of 900 or so, while some published figures point to counts over 1,000 in some species of monkey (PONDER, YEAGER and CHARIPPER, 1929b; JONES, et al., 1947) (though most accounts of monkeys give lower figures, and SUAREZ, RIVERA and MORALES (1942)suggest that even their lower counts are elevated as the result of intestinal parasitism), ring-tailed lemur (PONDER, YEAGER and CHARIPPER, 1929b), skunk (WINTROBE, 1967), bactrian camel and llama (PONDER, YEAGER and CHARIPPER, 1929a).Figures published for dromedary (PONDER, YEAGER and CHARIPPER, 1929a) and bottle-nose dolphin (MEDWAY and GERACI, 1964) indicate eosinophil counts in excess of 3,000. The Arneth score of 134 is markedly lower than the normal figure of 224 (calcu- lated from the Arneth count given by BESSIS, 1954) for man and reflects the high proportion of non-segmented cells (69%) in Trichosurus compared with man where only 6% are non-segmented and 68% have two segments (BESSIS, 1954). I have not been able to find Arneth counts of eosinophils of other species for comparison. In rabbit (SCARBOROUGH, 1931/2; SCHERMER, 1967), cattle (SCARBOROUGH, 1931/2) and another marsupial, Macropus robustus (PONDER, YEAGER and CHARIPPER, 1929c), the nuclei are described as usually segmented so presumably resemble more closely the human pattern. In cat, on the other hand, SCHERMER (1967) says the nuclei are usually horse-shoe shaped and less often segmented-a description that would fit the cells 334 R.A. BARBOUR: of Trichosurus. In those mammals where the eosinophil nuclei are usually annular in form (rat: KLIENEBERGER, 19272; JORDAN, 1938; SCHERMER, 1967; BLOOM and FAWCETT, 1968; mouse: PETRI, 1933; ANDREW, 1965; SCHERMER, 1967; BLOOM and FAWCETT, 1968; field mouse: LOEWENTHAL, 1933) a similar or even more pronounced lack of segmen- tation would obtain. In many fishes and amphibians (FEY, 1966b) the eosinophils present a much more left-handed Arneth distribution than is found in man, some to the extent of having 100% of non-segmented forms. Basophil count. Basophils are relatively rare cells in the blood of most mammals. In normal echidnas BOLLIGER and BACKHOUSE (1960b) found no circulating basophils at all. Among the marsupials, the last authors report that the cells are very rare in koala (BOLLIGER and BACKHOUSE, 1960a), their figures indicating a mean basophil count of only 7 or 8 cells. In some other marsupials (PONDER, YEAGER and CHARIPPER, 1929c; JORDAN, 1938; WINTROBE, 1967), thepercentage of these cells has been given as either one, two or three per cent, indicating mean cell counts in the range 70 to 260 -somewhat higher than I have found in Trichosurus. In man various published figures (ALDER, 1922/3; MOORE and JAMES, 1953; CODE, MITCHELL and KENNEDY, 1954; BOSEILA, 1959; FREDERICKS and MOLONEY, 1959; THONNARD-NEUMANN, 1963; SHELLEY and PARNES, 1965; WINTROBE, 1967; MCDONALD, DODDS and CRUICKSHANK, 1968) suggest overall a normal mean count very close to that of Trichosurus. In many other eutherians the mean count appears to be well under 100-rat (SCARBOROUGH, 1931/2; THEWLIS and MEYER, 1942; ALBRITTON, 1952; RIGDON, CRASS and RICHARDSON, 1960), mouse (SCARBOROUGH, 1931/2; ALBRITTON, 1952), cat (SCARBOROUGH, 1931/2; ALBRITTON, 1952; GILMORE, GILMORE and JONES, 1964; SCHERMER, 1967), dog (MAYERSON, 1930; SCARBOROUGH, 1931/2; REKERS and COULTER, 1948; ALBRITTON, 1952),guinea pig (SCARBOROUGH, 1931/2; KING and LUCAS, 1941; ALBRITTON, 1952), woodchuck (JORDAN, 1938), mink and coatimundi (WINTROSE, 1967), some mon- keys (HALL, 1929; SCARBOROUGH, 1931/2; SUAREZ, RIVERA and MORALES, 1942; JONES et al.,1947), cow (SCARBOROUGH, 1931/2; ALBRITTON, 1952; SCHALM, 19612), sheep (SCARBOROUGH, 1931/2; ALBRITTON, 1952; SCHALM, 19612; SCHERMER, 1967) and horse (SCARBOROUGH, 1931/2; ALBRITTON, 1952). In golden hamster (STEWART, FRORIO and MUGRAGE, 1944; TRINCAO, NOGUEIRA and GOUVEIA, 1949; SCHERMER, 1967) basophils are apparently absent from the circulation, while a species of mammal that consistently shows an unusually high count is rabbit; from various accounts (KELLUM and FORKNER, 1923; SCARBOROUGH, 1931/2; CASEY, et al.,1936; ALBRITTON, 1952; BOSEILA, 1959) the normal mean count in rabbit is probably about 500). PONDER, YEAGER and CHARIPPER give figures that indicate much higher basophil counts in Llama glama and Llama pocas (1929a) (up to more than 4,000 in Llama pocas), and in chimpanzee, orang utan and the monkey, Macacus cynomolgus (1929b), but these figures are apparently based on small numbers of animals and have not been confirmed by other observers. Lymphocyte count. The existence of a lymphocyte: neutrophil ratio greater than unity in Trichosurus is, of course, a point of difference from the human but, as BOLLIGER (1959) and BOLLIGER and BACKHOUSE (1960a) have stated, this is a recognized normal feature of many other mammals, and in some the ratio is considerably higher than I have found in Trichosurus. It has been noted in echidna (KNOLL, 1932; BOLLIGER, 1959; BOLLIGER and BACKHOUSE, 1960b), koala (BOLLIGER and BACKHOUSE, 1960a), Marsupial Leukocytes and Platelets 335 Didelphis virginiana (JORDAN, 1938; WINTROBE, 1967) and Didelphis paraguayensis (KNOLL, 1932) (but not by PONDER, YEAGER and CHARIPPER, 1929c, in any of the ten species of marsupial that they studied, including Didelphis virginiana), and in a variety of eutherians-e.g. rat (KLIENEBERGER, 19271,2; SCARBOROUGH, 1931/2; JORDAN, 1938; THEWLIS and MEYER, 1942; CAMERON and WATSON, 1949; ALBRITTON, 1952; RIGDON, CRASS and RICHARDSON, 1960; LAWKOWICZ and CZERSKI, 1966; SCHERMER, 1967), mouse (KLIENEBERGER, 19271,2; SCARBOROUGH, 1931/2; KNOLL, 1932; PETRI, 1933; RUSSELL, NEUFELD and HIGGINS, 1951; ALBRITTON, 1952; LAWKOWICZ and CZERSKI, 1966; SCHERMER, 1967), guinea pig (BURNETT, 1917; LUCIA and LUCIA, 1928; SCARBOROUGH, 1931/2; KNOLL, 1932; KING and LUCAS, 1941; ALBRITTON, 1952; LAWKOWICZ and CZERSKI, 1966; SCHERMER, 1967),hamster (STEWART, FRORIO and MUGRAGE, 1944; TRINCAO, NOGUEIRA and GOUVEIA, 1949; LAWKOWICZ and CZERSKI, 1966; SCHERMER, 1967),cow (DIMOCK and THOMPSON, 1906/72,3;SCARBOROUGH, 1931/2; ALBRITTON, 1952; SCHALM, 19612), sheep (SCARBOROUGH, 1931/2; ALBRITTON, 1952; SCHALM, 19612; SCHERMER, 1967), pig (KING and WILSON, 1910; SCARBOROUGH, 1931/2; KNOLL, 1932; ALBRITTON, 1952; SCHALM, 19612) and some apes and monkeys (KLIENEBERGER, 19271; HALL, 19291; SCARBOROUGH, 1931/2; KNOLL, 1932; SUAREZ, RIVERA and MORALES, 1942; SCHERMER, 1967), just to mention those most frequently reported. In rhesus monkey JONES et al. (1947) found that this high lymphocyte: neutrophil ratio was maintained only on diets that were adequate in various vitamins but that it fell below unity on some natural and artificial diets that they believed to be inadequate; these authors also quote some earlier works on the rhesus monkey in which a ratio lower than one was found and regarded as normal. With a high lymphocyte: neutrophil ratio and a fairly high total leukocyte count it is not surprising that the mean lymphocyte count in Trichosurus is quite high also. Nevertheless published figures suggest that the count in some other mammals is higher-Didelphis virginiana (JORDAN, 1938), rat (THEWLIS and MEYER, 1942; CAMERON and WATSON, 1949; ALBRITTON, 1952; RIGDON, CRASS and RICHARDSON, 1960; WINTROBE, 1967),mouse (PETRI,1933), sloth (WINTROBE, 1967),pig (SCARBOROUGH, 1931/2; SCHALM, 19612) and monkey (HALL, 19292; SCARBOROUGH, 1931/2; SHUKERS, LANGSTON and DAY, 1938). Monocyte Count. The mean monocyte count of 619 found here is higher than that obtainable from the literature for most other mammals. Figures presented by BOLLIGER and BACKHOUSE (1960b) for echidna give a mean count of only about 140, and for koala (1960a) about 200. In other marsupials PONDER, YEAGER and CHARIPPER (1929c) give differential percentages that indicate mean counts mostly below 100 and the highest only a little over 300 (in Macropus fulginosus). On the other hand both JORDAN (1938) and WINTROBE (1967)indicate mean counts of about 1,100 in Didelphis virginiana (one of the highest figures I have encountered for any mammal)-but PONDER, YEAGER and CHARIPPER (1929c) found only 1% out of a total leukocyte count of 7,200 in that species. In eutherian mammals different estimates for a species often vary a lot but in many the mean monocyte count appears to be no more than about 400, and in some cases much lower than that. Others, going by some published figures at least, have mean monocyte counts of the order of that found in Trichosurus or even a little higher-e.g. rat (SCARBOROUGH, 1931/2; THEWLIS and MEYER, 1942; CAMERON and WATSON, 1949), mouse, guinea pig (SCARBOQOUGH, 1931/2), rabbit (SCARBOROUGH, 1931/2; CASEY et al., 1936; ALBRITTON, 1952), cat (SCARBOROUGH, 1931/2; ALBRITTON, 336 R.A. BARBOUR: 1952; SCHALM, 19611),dog (SCARBOROUGH, 1931/2; ALBRITTON, 1952),monkey (KRUMB- HAAR and MUSSER, 1920; JONES et al.,1947; SCHERMER, 1967), cow (DELAUNE, 1939; ALBRITTON, 1952),pig (SCARBOROUGH, 1931/2; ALBRITTON, 1952; SCHALM, 19612),horse (SCARBOROUGH, 1931/2; ALBRITTON, 1952) and dromedary (PONDER, YEAGER and CHARIPPER, 1929a)-although in most of these some much lower figures have been presented too. The positive correlation between the monocyte and neutrophil counts in Tricho- surus could presumably be due to the defence functions of these cells and the presence of unrecognized infection in some of the animals. Differential counts. Differential counts do not, of course, run parallel with cell counts for different types of leukocyte in comparing different species because the total leukocyte count is also a factor in transposing one to the other. Nevertheless they are related and further extensive comparisons in this part of the discussion may be avoided by showing a simple tabulation of the findings for Trichosurus and other non-eutherian mammals (Table 1). Table 1. Total and differential leukocyte counts for various marsupials and a monotreme. a BOLLINGER and BACKHOUSE (1960b);b KNOLL (1932);c BOLLIGER and BACKHOUSE (1960a); d PONDER, YEAGER and CHARIPPER (1929c) e JORDAN (1938);f WINTROBE (1967). Cell sizes A note of caution should be sounded here in relation to comparing figures from Marsupial Leukocytes and Platelets 337 different accounts as there is no certainty that uniform conditions of measurement have been used. In particular one wonders about the measurements given by PONDER, YEAGER and CHARIPPER (1929a, b, c), many of which, although stated to have been made on stained smears, are surprisingly small (while a few, however, are quite large) -this is especially obvious in Didelphis virginiana for which measurements have also been given by JORDAN (1938) and in the rhesus monkey for which SCHERMER (1967) has given the sizes of the granulocytes. JORDAN's and SCHERMER's figures are much greater for each type of leukocyte and are more like measurements that have been given for other mammals. And JORDAN's figures for Didelphis are, overall, remark- ably similar to mine for Trichosurus. The only other measurements I have found for marsupial leukocytes are those of PONDER, YEAGER and CHARIPPER (1929c) and they all indicate much smaller cells than I have found in Trichosurus except in wombat where they are of the same order (though somewhat different for the various cell types). No reason for the great inter-animal variations I found in the mean diameters of the various cell types is apparent and no attempt to explain this is being made here. Nor am I offering any explanation of the positive correlation of sizes between some leukocyte varieties. Neutrophils. The mean diameter of 13μ found for Trichosurus is within the range for mammals generally. JORDAN (1938) has given 14μ for Didelphis and the figures of PONDER, YEAGER and CHARIPPER suggest larger sizes still in bactrian camel (1929a), black spider-monkey (1929b), and wombat (1929c). A size similar to that in Trichosurus has been given for cattle (WINQVIST, 1954), horse (SCARBOROUGH, 1931/2), dromedary (PONDER, MEAGER and CHARIPPER, 1929a) and two monkeys (rhesus: SCHERMER, 1967 and sapijou: PONDER, YEAGER and CHARIPPER, 1929b). Various figures (DITTRICH,1962; WINTROBE, 1967; BLOOM and FAWCETT, 1968; MCDONALD, DODDS and CRUICKSHANK, 1968; HAM, 1969) indicate a similar or somewhat smaller size in man and smaller measurements have also been given for a variety of other mammals (PONDER, YEAGER and CHARIPPER, 1929a, b, c; SCARBOROUGH, 1931/2; PETRI, 1933; SCHERMER 1967). Eosinophils. The eosinophils of Trichosurus appear to be larger than those of most other mammals (PONDER, YEAGER and CHARIPPER, 1929a, b, c; SCARBOROUGH, 1931/2; PETRI, 1933; WINQVIST, 1954; GILMORE, GILMORE and JONES, 1964; SCHERMER, 1967). A similar diameter has been given for these cells in Didelphis virginiana (JORDAN, 1938) and wombat (PONDER, YEAGER and CHARIPPER, 1929c), and SCHERMER's (1967) size-range for sheep is similar too. PONDER, YEAGER and CHARIPPER (1929b) give a larger figure (17μ) for Papio babuin. Estimates of the average size of eosino- phils in human blood smears vary from 12μ (ARCHER, 1963; BLOOM and FAWCETT, 1968) to 16μ (MCDONALD, DODDS and CRUICKSHANK, 1968); GALLO's (1947) figures for the human give a mean similar to mine for Trichosurus. The size of eosinophils relative to that of neutrophils varies in different mam- mals; the eosinophils are somewhat the larger, as in Trichosurus, in man (GALLO, 1947; BLOOM and FAWCETT, 1968; MCDONALD, DODDS and CRUICKSHANK, 1968; HAM, 1969), a few of the other Primates Studied by PONDER, YEAGER and CHARIPPER (1929b), cat (SCARBOROUGH, 1931/2; GILMORE, GILMORE and JONES, 1964),mouse (PETRI,1933), dog, guinea pig, rabbit (SCARBOROUGH, 1931/2; SCHERMER, 1967),pig (SCARBOROUGH, 1931/2) and sheep (SCHERMER, 1967), alsoin Macropus fulginosus and Macropus ruficollis among 338 R.A. BARBOUR: the marsupials studied by PONDER, YEAGER and CHARIPPER (1929c)-but in their others the eosinophils were similar in size to or smaller than the neutrophils. Basophils. A mean diameter in excess of 14μ makes the basophils, too, rather large amongst those of other mammals (PONDER, YEAGER and CHARIPPER, 1929a, b, c; SCARBOROUGH, 1931/2; THONNARD-NEUMANN, 1963; SCHERMER, 1967). The basophils in Didelphis (JORDAN, 1938) and cattle (WINQVIST, 1954) are of similar size, while those in mouse (PETRI, 1933) are larger still (16μ). In man estimates vary from 10μ (BLOOM and FAWCETT, 1968) to over 14μ (MCDONALD, DODDS and CRUICKSHANK, 1968)-my impression being that the lower part of this range is more appropriate. The basophils are the largest of the granulocytes in Trichosurus, a state of affairs that also exists in monkey (SCHERMER, 1967), guinea pig, cat (SCARBOROUGH, 1931/2), mouse (PETRI, 1933) and in three of the nine species of marsupial (Macropus fulginosus, Macropus rufus and Macropus agilis) examined by PONDER, YEAGER and CHARIPPER (1929c). In many other mammals published measurements indicate that the basophils are similar in size to one of the other types, or intermediate between them, or even the smallest of the granulocytes-especially in many of the species treated by PONDER, YEAGER and CHARIPPER (1929a, b, c). The last condition is that prevailing in man according to the measurements of fresh cells made by ACKERMAN and BELLIOS (1955) and also the figures given by BLOOM and FAWCETT (1968), though others (WINTROBE, 1967; MCDONALD, DODDS and CRUICKSHANK, 1968; HAM, 1969) suggest that they are as large as or larger than the neutrophils. Lymphocytes. The range of sizes found in Trichosurus is probably little different from that in man (BRAUNSTEINER and ZUCKER-FRANKLIN, 1962; ELVES, 1966; WINTROBE, 1967; MCDONALD, DODDS and CRUICKSHANK, 1968). But, although I have not found a distribution curve or histogram or a mean lymphocyte diameter given for man, there is not much doubt that the peak of the curve is further to the right and the mean diameter larger in Trichosurus. Figures given for many other mammals (PONDER; YEAGER and CHARIPPER, 1929a, b, c; SCARBOROUGH, 1931/2) suggest that lymphocytes conform to a pattern similar to that in man or, in some, are even smaller. But there are some where the "average" lymphocyte is larger. Most notable among these is Didelphis virginiana where JORDAN (1938) gives the sizes as: small 7μ, medium 10μ and large up to 15μ, and states that the medium.-sized ones predominate-altoge- ther a picture closely resembling that in Trichosurus. For cattle WINQVIST (1954) gives a mean diameter of 10.7μ and DIMOCK and THOMPSON (1906/71) indicate an even larger size; for rhesus monkey KRUMSHAAR and MUSSER (1920) note that the lympho- cytes are usually large compared with those of man; and PONDER, YEAGER and CHARIPPER give a mean diameter of 10μ for Macacus cynomolgus (1929b) and a range of 12 to 16μ for Camelus bactriens (1929a). Monocytes. Many published accounts give wide ranges for sizes of monocytes in various species thus making comparison rather difficult but it is fairly clear that the size of these cells in Trichosurus is similar to that in some other mammals (PONDER, YEAGER and CHARIPPER, 1929b; SCARBOROUGH, 1931/2; WINQVIST, 1954; SCHERMER, 1967) including man (BRUCHER, 1962; WINTROBE, 1967; BLOOM and FAWCETT, 1968; HAM, 1969) and the marsupial, Didelphis virginiana (where JORDAN (1938) gives the size as 14 to 16μ). For some mammals figures suggest that the cells are rather smaller Marsupial Leukocytes and Platelets 339 (mouse: PETRI, 1933; rat and cat: SCARBOROUGH, 1931/2; several monkeys: PONDER, YEAGER and CHARIPPER, 1929b; several marsupials: PONDER, YEAGER and CHARIPPER, 1929c) or larger (bactrian camel: PONDER, YEAGER and CHARIPPER, 1929a; gorilla, yellow baboon and brown lemur: PONDER, YEAGER and CHARIPPER, 1929b; guinea pig: SCARBOROUGH, 1931/2; and wombat: PONDER, YEAGER, and CHARIPPER, 1929c). Platelets. In man most blood platelets are within the size range 1 to 5μ with a mean of about 3μ (GUDE, UPTON and ODELL, 1956; WINTROBE, 1967; BLOOM and FAWCETT, 1968; MCDONALD, DODDS and CRUICKSHANK, 1968; HAM, 1969). The average size of platelets is about the same in cat (SCARBOROUGH, 1931/2),dog, guinea pig (SCHERMER, 1967), rabbit (SCARBOROUGH, 1931/2; SCHERMER, 1907) and cow (WINQVIST, 1954) but they are considerably smaller in rat (GUDE, UPTON and ODELL, 1956). Tricho- surus thus conforms with the commonest finding in this regard. The fairly frequent occurrence of large platelets (up to 8 or 10μ) is also a feature of cat and dog (SCHERMER, 1967). Cytochemical Studies Sudan black B staining Neutrophils. Most other studies on the sudanophilia of neutrophils have used human blood which has given uniformly positive results (SHEEHAN, 1939; MCMANUS, 1945; DISCOMBE, 1946; BAILLIF and KIMBROUGH, 1947; SHEEHAN and STOREY, 1947; RHEINGOLD and WISLOCKI, 1948; BLOOM and WISLOCKI, 1950; ERANKO, 1950; STORTI and PERUGINI, 1951; ALBRITTON, 1952; HAYHOE, 1953; WACHSTEIN, 1955; LAMBERS and BAUER-SIC, 1962; WANG, 1963; ACKERMAN, 1964; BAKALOS and FRAGISKOS, 1964; HAYHOE, QUAGLINO and DOLL, 1964; HERMANSKY, 1965, 1966; HERMANSKY, LODROVA and POSSNEROVA, 1970), as has also that of several other mammals (WISLOCKI and DEMPSEY, 1946; RHEINGOLD and WISLOCKI, 1948; WINQVIST, 1954; BAUER-SIC, 1963, 1965; HERMANSKY, LODROVA and POSSNEROVA, 1970; JAIN, 1970). In many of these studies the conclusion has been reached that it is the specific granules themselves that give the positive staining reaction, and KAWABATA (1960) found that isolated neutrophilic granules from horse blood stained with sudan black. The staining of neutrophils in Trichosurus as compared with man is consistent with the theory of identity of the sudanophilic granules with the specific granules since in Trichosurus the cytoplasm of the neutrophils shows a less prominent reaction with both the sudan black and Romanowsky-type stains. Eosinophils. Other studies on sudanophilia of eosinophils, almost entirely on human cells, have demonstrated a positive reaction in the granules and in most cases the peripheral staining, such as I have demonstrated in both human and possum blood, has been observed (SHEEHAN, 1939; BAILLIF and KIMBROUGH, 1947; SHEEHAN and STOREY, 1947; RHEINOLD and WISLOCKI, 1948; BLOOM and WISLOCKI, 1950; STORTI and PERUGINI, 1951; ALBRITTON, 1952; HAYHOE, 1953; WACHSTEIN, 1955; ARCHER, 1960, 1963; BAUER-SIC, 1963; WANG, 1963; HERMANSKY, LODROVA and POSSNEROVA, 1970). The pattern of staining observed is belived to be due to the presence of phospholipid at the periphery of the granules (GEDIGK, 1956; ARCHER, 1960; GEDIGK and GROSS, 1962). Sudanophobia of eosinophils has been reported for cat (HERMANSKY, LODROVA and POSSNEROVA, 1970; JAIN, 1970). 340 R.A. BARBOUR: Basophils. My finding of sudanophobia of basophil granules in the human is in accord with that of BAILLIF and KIMBROUGH (1947), HAYHOE (1953) and ACKERMAN (1963). ACKERMAN (1963) has also found the basophil granules to be negative to sudan black in rabbit and guinea pig. There have, on the other hand, been reports of posi- tive sudan black staining of human basophlls (BLOOM and WISLOKI, 1950; WACHSTEIN, 1955; LENNERT, 1961; WANG, 1963: INAGAKI, 1966; SHIBATA et al., 1966). Lymphocytes. Though ACKERMAN (1967) says that many lymphocytes exhibit sudanophil granules due to neutral fat providing precautions are taken to prevent solution of the fat in organic solvents, routine procedures do not demonstrate posi- tivity to sudan black in human lymphocytes (SHEEHAN, 1939; McMANUS, 1945; BAILLIF and KIMBROUGH, 1947; BLOOM and WISLOCKI, 1950; HAYHOE, 1953; LAMBERS and BAUER-SIC, 1962; WANG, 1963; BAKALOS and FRAGISKOS, 1964; WINTROBE, 1967). These cells have given negative results also in several other mammals (WISLOCKI and DEMPSEY, 1946; WINQVIST, 1954; BAUER-SIC, 1963; JAIN, 1970). My positive findings for even a few cells in Trichosurus are, thus, unusual. Monocytes. Although McMANUS (1945) and BAILLIF and KIMBROUGH (1947)obtain- ed negative results with sudan black on human monocytes, the majority of accounts suggest that in man most of the monocytes exhibit rumerous small sudanophilic gran- ules (SHEEHAN and STOREY, 1947; BLOOM and WISLOCKI, 1950; STORTI and PERUGINI, 1951; ALBRITTON, 1952; HAYHOE, 1953; WACHSTEIN, 1955; CAMBERS and BAUER-SIC, 1962; WANG, 1963; BAKALOS and FRAGISKOS, 1964; HAYHOE, QUAGLINO and DOLL, 1964; WINTROBE, 1967; HERMANKY, LODROVA and POSSNEROVA, 1970); MACARIO (1965) found that over 90% of monocytes were positive. In rhesus monkey WISLOCKI and DEMPSEY (1946) report that the monocytes contain a few positive droplets and BAUER-SIC (1963) found some positive cells in cow. JAIN (1970) reports some positive monocytes in dog, cat, cow, horse and goat, but not in sheep. My almost negative findings for the monocytes of Trichosurus stand in some contrast to most of these descriptions. Platelets. My observations of lack of staining of platelets in man and in Tri- chosurus concur with other reports for man (GUDE, UPTON and ODELL, 1956; WANG, 1963; BAKALOS and FRAGISKOS, 1964) and one for rat (GUDE, UPTON and ODELL,1956). In rhesus monkey, however, WISLOCKI and DEMPSEY (1946) found small black dots in the platelets after sudan black staining. PAS staining Neutrophils. Most or all of the neutrophils are PAS-positive in practically all of the many vertebrate species that have been studied in this regard including man (GIBB and STOWELL, 1949; WACHSTEIN, 1949, 1955; WISLOCKI, RHEINGOLD and DEMPSEY, 1949; ERANKO, 1950; FRIEDERICI, 1955; HAYHOE, 1960; CAMBERS and BAUER-SIC, 1962; WULFF, 1962; WANG, 1963; ACKERMAN, 1964; GAHRTON, 1964; HAYHOE, QUAGLINO and DOLL, 1964; BAUER-SIC, 1965; HERMANSKY, LODROVA and POSSNEROVA, 1970), vari- ous other eutherian mammals (WACHSTEIN, 1949; WISLOCKI, RHEINGOLD and DEMPSEY, 1949; WINQVIST, 1954; FRIEDERICI, 1955; SUGIYAMA, 1955; MITSUI et al.,1956; BAUER- SIC, 1963, 1965; JAIN, 1969, 1970; HERMANSKY, LODROVA and POSSNEROVA, 1970) and a variety of non-mammalian species (SUGIYAMA, 1955; MITSUI et al.,1956; FEY, 1966b). There is considerable variation in the staining reaction of neutrophils between spe- cies (WACHSTEIN, 1949; SUGIYAMA, 1955; BAUER-SIC, 1965; FEY, 1966b; JAIN, 1969, 1970); Marsupial Leukocytes and Platelets 341 amongst mammals SUGIYAMA (1955)found that the most intense reaction is to be observed in horse, man and monkey and, less consistently, in cow and pig-it is thus not surprising that I found a less intense reaction in possum than human. Most investigations on PAS reaction of neutrophils have found that the cellular content involved in the reaction is virtually completely labile to amylase, indicating that it is glycogen. In the neutrophils of cattle WINQVIST (1954) found some diffuse staining after amylase digestion, and SUGIYAMA (1955) found that the positive material was not amylase-labile in three non-mammalian species. After detailed investigations GAHRTON (1964, 1966) and MAHR (1964) have concluded that the PAS-positive material in human neutrophils is mainly glycogen; but ACKERMAN (1964) and SHUBICH (1965) mention some amylase-resistant material in the granules which the former considers to be a glycoprotein, the latter lipid. Similarly KAWABATA (1960), using isolated gran- ules from fractionated horse leukocytes, found some stable PAS-positive material present in the granules. By biochemical analysis, however, it has been found that only a small part of the PAS-positive content of leukocytes is glycogen (OLSSON and DAHLQVIST, 1967). It seems generally accepted that the glycogen of neutrophils occupies an intergranular position, though ACKERMAN (1964) saysthat much of it may be associated with the specific granules. Working with fractionated cells, VERCAUTEREN (1965) concluded that most was associated with the smooth endoplasmic reticulum though the specific granules may contain some. Glycogen visualized in electron micrographs of human cells lies in the cytoplasmic matrix (ANDERSON, 1966). Eosinophils. The pattern of staining of eosinophils I have noted in Trichosurus has been widely observed in man (GIBE and STOWELL, 1949; WACHSTEIN, 1949, 1955; WISLOCKI, RHEINGOLD and DEMPSEY, 1949; ERANKO, 1950; FRIEDERICI, 1955; HAYHOE, 1960; GEDIGK and GROSS, 1962; LAMBERS and BAUER-SIC, 1962; WANG, 1963; BRZEZINSKI, 1965), a variety of other mammals (WINQVIST, 1954; FRIEDERICI, 1955; SUGIYAMA, 1955; MITSUI et al., 1956; ARCHER, 1963; BAUER-SIC, 1963; JAIN, 1969, 1970; HERMANSKY, LODROVA and POSSNEROVA, 1970) and many non-mammalian vertebrates (SUGIYAMA, 1955; MITSUI et al., 1956; FEY, 1966b). There appears to be no general agreement on the amylase-stability of the staining material or on the presence of glycogen in the cells; GEDIGK and GROSS (1962) believe that the existence of glycogen in eosino- phils has not been proved cytochemically and suggest that the amylase-stable stain- ing may be due to lipids. Glycogen has, however, been clearly shown in human eosinophils by electron microscopy (ANDERSON, 1966). Positivestaining of granules (presumably the specific granules, though not so stated in all cases) has been described in dog, cat and sheep (JAIN, 1969), horse and cow (SUGIYAMA, 1955; MITSUI et al., 1956), monkey (WISLOCKI, RHEINGOLD and DEMPSEY, 1949), and in a few lower vertebrates (SUGIYAMA, 1955; MITSUI et al. 1956; FEY, 1966b). Basophils. Previous observations on the PAS staining of basophils have been principally on human cells. Considering first the specific granules of these, some observers have noted that they exhibit a positive (generally amylase-resistant) reac- tion (ALBRITTON, 1952; LAMBERS and BAUER-SIC, 1962; ACKERMAN, 1963),while SMITH (1949), INAGAKI (1968) and KAUNG (1969) found, like I, that they do not stain. ASTALDI, RONDANELLI and BERNARDELLI (1953) and WANG (1963) found some amylase-resistant positive granules in human basophils but it is not clear that they were the specific 342 R.A. BARBOUR: granules. My finding that the specific granules are positive and resistant to amylase in Trichosurus parallels ACKERMAN's (1963) results with rabbit and guinea pig, and those of FEY (1966b) for some lower vertebrates. The presence of amylase-labile, PAS-positive material (either granular or diffuse or both) in the cytoplasm between the specific granules in human basophils, such as I have demonstrated, has been previously described by SMITH (1949), WISLOCKI, RHEINGOLD and DEMPSEY (1949), ACKERMAN (1963) and KAUNG (1969); and cytoplasmic glycogen has been noted in electron micrographs (ANDERSON, 1966; KAUNG, 1969). I have found no account stating clearly that glycogen can or cannot be demonstrated in the basophils of any other mammals, the implication is that ACKERMAN (1963) found none in rabbit or guinea pig. Lymphocytes. Most investigations have found that some of the lymphocytes in human blood contain a small amount of glycogen, mainly in granular form, demon- strable by the PAS method (GIBB and STOWELL, 1939; WACHSTEIN, 1949; ACKERMAN, KNOUFF and ROSTER, 1951; FRIEDERICI, 1955; ASTALDI and VERGA, 1957; MITUS et al., 1958a; QUAGLINO and HAYHOE, 1959; ACKERMAN, 1960a, 1967; HAYHOE, 1960; LAMBERS and BAUER-SIC, 1962; WULFF, 1962; BJORNBERG, 1963; OLSSON, DAHLQVIST and NORDEN, 1963; WANG, 1963; HAYHOE, QUAGLINO and DOLL, 1964; ASAKAWA, 1966; ELVES, 1966; UNDRITZ,1966a; WINTROBE,1967), though estimates of the proportion of cells showing a positive reaction vary greatly. ACKERMAN (1960a, 1967) says that some amylase- resistant granules may also be present. PAS studies on the lymphocytes of other mammals have given varied results. Positive findings have been reported for cow (WINQVIST, 1954; BAUER-SIC, 1963; JAIN,1969, 1970; HERMANSKY, LODROVA, POSSNEROVA, 1970),sheep, dog (JAIN, 1969, 1970; HERMANSKY, LODROVA and POSSNEROVA, 1970),pig (HERMANSKY, LODROVA and POSSNEROVA, 1970) and horse (FRIEDERICI, 1955; JAIN, 1970), negative for mouse, hamster (HERMANSKY, LODROVA and POSSNEROVA, 1970),cat (JAIN, 1969, 1970; HERMANSKY, LODROVA and POSSNEROVA, 1970), monkey (JAIN, 1969; HERMANSKY, LODROVA and POSSNEROVA, 1970) and goat (JAIN, 1970). Both positive and negative findings have been presented for rabbit (JAIN, 1969; HERMANSKY, LODROVA and POSSNEROVA, 1970), guinea pig and rat (FRIEDERICI, 1955; JAIN, 1969; HERMANSKY, LODROVA and POSSNEROVA, 1970). My negative results for Trichosurus are thus not without parallel in other mammals. In some lower vertebrates FEY (1966a) found some positive cells in all species studied. Monocytes. Absence of PAS-demonstrable glycogen from all monocytes, as I found in Trichosurus, was also found by JAIN in cat (1969, 1970), guinea pig, rabbit and rat (1969); some positive cells have been found in cow (BAYER-SIC, 1963; JAIN, 1969, 1970), horse, sheep, goat, dog (JAIN, 1969, 1970) and monkey (JAIN, 1969). In human monocytes GIBB and STOWELL (1949), HAYHOE (1960), WANG (1963), HAYHOE, QUAGLINO and DOLL (1964) and WINTROBE (1967) report the presence of some positive reaction in many (or even all) cells, but WACHSTEIN (1949), WISLOCKI, RHEINGOLD and DEMPSEY (1949) and WULFF (1962) obtained little staining in these cells. FEY (1966c) found positive cells in all the lower vertebrates he studied. Platelets. Platelets (or thrombocytes) have been found to be PAS-positive in all vertebrate species studied (man: GIBB and STOWELL, 1949; WACHSTEIN, 1949; WISLOCKI, RHEINGOLD and DEMPSEY, 1949; STORTI, PERUGINI and SOLDATI, 1953; FRIEDERICI, Marsupial Leukocytes and Platelets 343 1955; GUDE, UPTON and ODELL, 1956; LAMBERS and BAUER-SIC, 1962, WANG, 1963, ALEKSANDROWICZ, GAERTNER and URBANCZYK, 1965; several other mammals: FRIEDERICI, 1955; GUDE, UPTON and ODELL, 1956; JAIN, 1969; and some lower vertebrates: FEY, 1966a) and in most cases the material responsible has been noted to be partly or wholly labile to amylase, indicating the presence of glycogen. In cattle WINQVIST (1954) reports that the material stained in platelets is stable to amylase digestion, as does SUGIYAMA (1955) for some other vertebrates. JAIN (1969) states that the inten- sity of reaction of platelets is fairly weak in all the species of mammal he examined -this is certainly the case in Trichosurus too. Peroxidase ARCHER (1963) has stated that o-tolidine, while being a suitable agent for the demonstration of peroxidase in human blood cells, is not satisfactory for this purpose in some other animals. The negative results achieved with Trichosurus cells using o-tolidine, while positive findings were obtained with benzidine, then, are not sur- prising, and Trichosurus can apparently be added to the list of animals for which o-tolidine is not suitable. Neutrophils. The neutrophlls of man (ALBRITTON, 1952; HATTORI, 1958; HAYHOE, 1960; DITTRICH, 1962; LAMBERS and BAUER-SIC., 1962; WANG, 1963; BAUER-SIC, 1965; POZO-OLANO, 1967; WINTROBE, 1967) contain peroxidase, though a very rare condition of absence of this enzyme has been described (UNDRITZ, 1966a, b). A considerable variety of other mammals has been investigated in this regard also (SMITH, ROBINSON and TYSON, 1937; SATO and SUZUKI, 1938; VERCAUTEREN and BLONDE, 1954; WINQVIST, 1954; MITSUI and KAMEZAWA, 1955; NAKAMURA, 1955; MITSUI et al., 1956; BAUER-SIC, 1963, 1965; SCHAEFER and FISCHER, 1964; JAIN, 1967, 1970; SCHERMER, 1967; FEY and KUNTZE, 1970) and all have given positive, though variable, results. In many cases the stain deposit has been described as granular or in granules but only recently has it been elucidated, mainly by the use of electron microscopy, just what the granules are. Electron microscope studies on these cells in man (ENOMOTO and KITANI, 1966), horse (TAKIKAWA and OHTA, 1906) and rat (BEHNKE, 1969) have shown that the enzyme is present in only one of the kinds of granule in the cytoplasm, while other studies on man (BRETON-GORIUS and GUICHARD, 1969), rabbit (BAINTON and FARQUHAR, 1966, 1968a, b; DUNN, HARDIN and SPICER, 1968; BAGGIOLINI, HIRSCH and DEDUVE, 1969) and cat (ACKERMAN, 1968) have produced the further suggestion that it is the azurophil granules, not the specific granules, that contain the enzyme. Among non-mammalian vertebrates birds have been found generally to have per- oxidase-negative neutrophils (MITSUI and KAMEZAWA, 1955; NAKAMURA, 1955; MITSUI, et al., 1956; ATWAL and McFARLAND, 1966), while other classes have produced positive results for most species studied (SATO and SUZUKI, 1938; MITSUI and KAMEZAWA, 1955; NAKAMURA, 1955; MITSUI, et al.,1956; FEY, 1966b; FEY and KUNTZE, 1970). Among the 32 species of vertebrates (including 18 mammals) studied by NAKAMURA (1955) the cells of man gave the strongest reaction-my finding of a weaker reaction in Trichosurus than in man is in keeping with this. Eosinophils. Human eosinophils give a positive cytochemical reaction for per- oxidase (ALBRITTON, 1952; HATTORI, 1958; GEDIGK and GROSS, 1962; LAMBERS and BAUER-SIC, 1962; WANG, 1963; POZO-OLANO, 1967; WINTROBE, 1967) and so do those of 344 R.A. BARBOUR: most other mammals that have been studied (SMITH, ROBINSON and TYSON, 1937 SATO and SUZUKI, 1938; VERCAUTEREN and BLONDE, 1954; MITSUI and KAMEZAWA, 1955 NAKAMURA, 1955; MITSUI et al., 1956; BAUER-SIC, 1963; SCHAEFER and FISCHER, 1964 JAIN, 1967, 1970; SCHERMER, 1967; FEY and KUNTZE, 1970). In some mammals, how- ever, the eosinophils have been found to be negative, notably in cat (SATO and SUZUKI, 1938; UNDRITZ, 19522; MITSUI and KAMEZAWA, 1955; NAKAMURA, 1955; MITSUI et al., 1956; UNDRITZ, LANG and VAN OYE, 1956; JAIN, 1967, 1970; SCHERMER, 1967), but also in numerous other species belonging to several orders (UNDRITZ, 19522; NAKA- MURA, 1955; MITSUI et al., 1956; UNDRITZ, LANG and VAN OYE, 1956; FEY and KUNTZE, 1970). Among the non-mammalian vertebrates peroxidasepositive eosinophils have been demonstrated in most birds and reptiles that have been studied (MITSUI and KAMEZAWA, 1955; NAKAMURA, 1955; MITSUI et al., 1956), but negative reactions occur in these cells in many amphibians (especially urodeles) (UNDRITZ, 19522; MITSUI and KAMEZAWA, 1955; NAKAMURA, 1955; MITSUI et al.,1956; MITSUI, 1965; FEY, 1966b; FEY and KUNTZE, 1970) and all or most fish (MITSUI and KAMEZAWA, 1955; MITSUI et al., 1956; MITSUI, 1965; FEY, 1966b; FEY and KUNTZE, 1970). In many reports it has been noted that the reaction appears to be in the specific granules and recent electron microscopic studies of eosinophils from man (ENOMOTO and KITANI, 1966), rabbit (BAINTON and FARQUHAR, 1967; MILLER and HERZOG, 1969 and rat (KELENYI, ZOMBAI and NEMETH, 1965; BAINTON and FARQUHAR, 1967; BEHNKE, 1969; MILLER and HERZOG, 1969) have shown that the activity is, in fact, in the extra-crystalloid matrix of the granules. In the wide variety of vertebrates whose eosinophils were examined by NAKAMURA (1955) (which included 15 species of mammals) the strongest reaction was found in man-a result with which my finding of a weaker reaction in Trichosurus than in man is in keeping. Basophils. In man basophils are usually considered to be peroxidase negative (GRAHAM, 1920; TOKUE, 1928/91; ASTALDI, RONDANELLI and BERNARDELLI, 1953; HATTORI, 1958; FREDERICKS and MOLONEY, 1959; ACKERMAN, 1963, 1967; WANG, 1963; WINTROBE, 1967), though some positive findings have been reported (KLAUSNER and KREIBICH, 1913; LENNERT and SCHUBERT, 1960; LENNERT, 1961; CAMBERS and BAUER- SIC, 1962; OIKAWA and SATO, 1966; POZO-OLANO, 1967). MICHELS (1938) cites some other early findings, some positive, some negative, but points out their unreliability. Basophils have also been found to be negative or mostly so in a number of other mammals (TOKUE, 1928/92; SATO and SUZUKI 1938; ACKERMAN, 1963; JAIN, 1967, 1970; SCHERMER,1967) and in some fish and amphibians (SATO and SUZUKI, 1938; FEY, 1966b). Lymphocytes. Lymphocytes are peroxidase-negative in man (JACOBY, 1944; ALBRITTON, 1952; HATTORI, 1958; ACKERMAN, 1960a; HAYHOE, 1960; POZO-OLANO, 1967; WINTROBE, 1967), in various other mammals that have been examined (SATO and SUZUKI, 1938; WINQVIST, 1954; BAUER-SIC, 1963; JAIN, 1967, 1970; FEY and KUNTZE, 1970) and in a number of fish and amphibians subjected to study (SATO and SUZUKI, 1938; FEY, 1966c; FEY and KUNTZE, 1970). My negative findings in Trichosurus thus conform to the general rule. Monocytes. Contrary to my findings, some degree of positive peroxidase reaction (albeit sometimes weak and occasional) seems to have been the usual experience with human monocytes (JACOBY, 1944; HATTORI, 1958; HAYHOE, 1960; WANG, 1963; Marsupial Leukocytes and Platelets 345 HAYHOE, QUAGLINO and DOLL, 1964; POZO-OLANO, 1967; WINTROBE, 1967; HAM, 1969). Positive monocytes have also been found in rat (SMITH, ROBINSON and TYSON, 1937), horse (JAIN, 1967, 1970), cow (BAUER-SIC, 1963), dog (SATO and SUZUKI, 1938; JAIN, 1967, 1970), mouse, guinea pig, cat, rabbit and goat (SATO and SUZUKI, 1938) and in most of the fish and amphibians studied by FEY (1966a). Electron microscopic studies (ENOMOTO and KITANI, 1966; DUNN, HARDIN and SPICER, 1968; BRETON-GORIUS and GUICHARD, 1969)have shown that the enzyme is in cytoplasmic granules of human monocytes. Negative findings have, however, also been reported in cow (WINQVIST, 1954; BAUER-SIC, 1963; JAIN, 1967, 1970), cat and goat (JAIN, 1967, 1970), as well as in monkey (JAIN, 1967) and sheep (JAIN, 1967, 1970). My negative results in Trichosurus are thus not unique. Platelets. In agreement with my findings, platelets have been found to be peroxidase-negative in man (GUDE, LIPTON and ODELL, 1956), several other mammals (SMITH, ROBINSON and TYSON, 1937; GUDE, UPTON and ODELL, 1956; FEY and KUNTZE, 1970; JAIN, 1970) and various fish and amphibians (FEY, 1966a; FEY and KUNTZE, 1970). Alkaline phosphatase My experience with the two different types of staining method for alkaline phosphatase (Gomori-type and azo-dye) confirms the previously noted (TODERAN, 1964; ELVES, 1966) difference that azo methods give greater sensitivity and precision of localization of the enzyme. With the Gomori-type method I used, however, I did not observe any nuclear staining (which is now believed to be false (KAPLOW, 1955)) such as was obtained in some of the earlier studies on this enzyme (FELL and DANIELLI, 1943; WACHSTEIN, 1946; WISLOCKI and DEMPSEY, 1946; RHEINGOLD and WISLOCKI, 1948; RABINOVITCH and ANDREUCCI, 1949; ACKERMAN, KNOUFF and HOSTER, 1951). Neutrophils. The alkaline phosphatase reaction of human neutrophils must surely be the most thoroughly studied aspect of leukocytochemistry and there are many published accounts of the demonstration of this enzyme in normal cells (WACHSTEIN, 1946; RABINOVITCH and ANDREUCCI, 1949; PLUM, 1950; KERPPOLA, 1951; ALBRITTON, 1952; VERCAUTEREN, 1954; FROST, 1955; KAPLOW, 1955, 1963; WILTSHAW and MOLONEY, 1955; KENNY and MOLONEY, 1957; HAYHOE and QUAGLINO, 1958; KOLER et al., 1958; LEONARD, 1958; MITUS et al. 1958b; MEISLIN, LEE and WASSERMAN, 1959; MERKER and HEILMEYER, 1959; MITUS, MEDNICOFF and DAMESHEK, 1959; MONIS and RUTENBERG, 1959; TRUBOWITZ et al. 1959; KNOBLAUCH, 1962; KNUDTSON and EVANGER, 1962; LAMBERS and BAUER-SIC, 1962; RANLOV, 1962; BAXTER, 1963; ELVES, ROATH and ISRAELS, 1963; NOWAK-RESZEL, 1963; TANZER, 1963; WANG, 1963; WHITT, 1963; WULFF, 1963c; ACKERMAN, 1964; LOH, 1964; TODERAN, 1964; ALEKSANDROWICZ, GAERTNER and URBANCZYK, 1965; BAUER-SIC, 1965; ASAKAWA, 1966; MARTINEZ-MALDONADO, MENENDEZ- CORRADA and DE SALA, 1966; UBERLA, TILLING and GRATZEL, 1967; WINTROBE, 1967; HERMANSKY, LODROVA and POSSNEROVA, 1970). In general, investigators have noted that in individual cases the number of positive neutrophils is usually a minority but most have found that every individual has at least a few positive cells. Only a few workers have reported finding persons whose neutrophils were completely negative -notably NOWAK-RESZEL (1963) who found 20% negative individuals among his group of 300. Several have commented that the neutrophils are the only cells in the 346 R.A. BARBOUR: blood that stain. Positively staining neutrophils are also found in most other mam- mals (WACHSTEIN, 1946; WISLOCKI and DEMPSEY, 1946; RHEINGOLD and WISLOCKI, 1948; PLUM, 1950; VERCAUTEREN, 1950; WINQVIST, 1954; MONIS and RUTENBERG, 1959; BELL, 1962; BAUER-SIC, 1963, 1965; SCHAEFER and FISCHER, 1964; LAWKOWICZ and CZERSKI, 1966; SHUBICH, 1966; ATWAL and McFARLAND, 1967; FEY and KUNTZE, 1970; HERMANSKY, LODROVA and POSSNEROVA, 1970; JAIN, 1970). In some the proportion of positive cells and the intensity of staining in individual cells is much greater than in normal human blood. On the other hand, as in Trichosurus, the neutrophils contain no cyto- chemically demonstrable alkaline phosphatase in mouse (WACHSTEIN, 1946; ROSIEK, CZERSKI and SABLINSKI, 1962; SCHAEFER and FISCHER, 1964; SHUBICH, 1966; FEY and KUNTZE, 1970; HERMANSKY, LODROVA and POSSNEROVA, 1970), cat (BAUER-SIC, 1963, 1965; SHUBICH, 1966; ATWAL and McFARLAND, 1967; ACKERMAN, 1968; HERMANSKY, LODROVA and POSSNEROVA, 1970; JAIN, 1970), tiger and alpaca (FEY and KUNTZE, 1970). In dog both positive (PLUM, 1950; BAUER-SIC, 1965; FEY and KUNTZE, 1970) and nega- tive (WACHSTEIN, 1946; BELL, 1962; SHUBICH, 1966; ATWAL and McFARLAND, 1967; HERMANSKY, LODROVA and POSSNEROVA, 1970; JAIN, 1970) findings have been reported. Among non-mammals WACHSTEIN (1946) and BELL (1962) obtained negative results with chicken as did ATWAL and McFARLAND (1966) with the Japanese quail. FEY (1966b) and FEY and KUNTZE (1970) found that the neutrophils were negative in a few of the lower vertebrates they studied. Special studies, mainly using electron microscopy, on rabbit leukocytes (WETZEL, HORN and SPICER, 1963; BAINTON and FARQUHAR, 1966, 1968a, b; WETZEL, SPICER and HORN, 1967; BAGGIOLINI, HIRSCH and DE DUVE, 1969) have shown that the alkaline phosphatase is in the specific granules of these cells, a conclusion also reached regarding human cells by ACKERMAN (1964) from his cytochemical studies and by NAKATSUI (1969) using electron microscopy. Eosinophils. In contrast to the findings in neutrophils, human eosinophils have generally been found to lack demonstrable alkaline phosphatase (WACHSTEIN, 1946; PLUM, 1950; KERPPOLA, 1951; KAPLOW, 1955, 1963; LEONARD, 1958; MONIS and RUTENBERG, 1959; RANLOV 1962; WULFF, 1963c; WINTROBE, 1967); only VERCAUTEREN (1954, 1955) and, in occasional cells, HAYHOE and QUAGLINO (1958) have reported posi- tive findings. Positive eosinophils have been reported in various other mammals (VERCAUTEREN, 195; BELL, 1962; SCHAEFER and FISCHER, 1964; FISCHER and SCHAEFER, 1966; ATWAL and McFARLAND, 1967; FEY and KUNTZE, 1970; HERMANSKY, LODROVA and POSSNEROVA, 1970; JAIN, 1970) but these cells have been found to give no reaction in mouse (SCHAEFER and FISCHER, 1964; FISCHER and SCHAEFER, 1966; SHUBICH, 1966; FEY and KUNTZE, 1970), guinea pig, hamster, tiger and alpaca (FEY and KUNTZE, 1970), sheep and goat (JAIN, 1970): published findings for rabbit (MONIS and RUTENBERG, 1959; FEY and KUNTZE, 1970) and rat (MONIS and RUTENBERG, 1959; SCHAEFER and FISCHER, 1964; FISCHER and SCHAEFER, 1966; FEY and KUNTZE, 1970) disagree. Basophils. Alkaline phosphatase has not been demonstrated in the basophils of man (PLUM, 1950; KERPPOLA, 1951; MONIS and RUTENBERG, 1959; LENNERT, 1961; RANLOV, 1962; ACKERMAN, 1963; WINTROBE, 1967); nor, until JAIN (1970) recently found it in the intergranular cytoplasm in cat and cow, of any other mammal-it is not present in these cells in rat (MONIS and RUTENSERG, 1959; ACKERMAN, 1963), guinea pig (ACKERMAN, 1963), rabbit (MONIS and RUTENBERG, 1959),or horse (JAIN,1970). It was Marsupial Leukocytes and Platelets 347 found, though, in most of the fish and amphibians studied by FEY (1966b). In only one of these, however, Bufo bufo, was it present in the specific granules as I found it in Trichosurus. Lymphocytes. Some early studies suggested the presence of some enzyme in lymphocytes from lymph nodes in man (ACKERMAN, KNOUFF and HOSTER, 1951) and monkey (WISLOCKI and DEMPSEY, 1946), but blood and marrow lymphocytes of man (WACHSTEIN, 1946; PLUM, 1950; KERPPOLA, 1951; VERCAUTEREN, 1954; KAPLOW, 1955; HAYHOE and QUAGLINO, 1958; LEONARD, 1958; MONIS and RUTENBERG, 1959; ACKERMAN, 1960a, 1964, 1967; WANG, 1963; WULFF, 1963c; ALEKSANDROWICZ, GAERTNER and URBANCZYK, 1965; WINTROBE, 1967) and numerous other mammals (WINQVIST, 1954; MONIS and RUTENBERG, 1959; SHUBICH, 1966; FEY and KUNTZE, 1970; JAIN, 1970) have no cytochemically demonstrable alkaline phosphatase. Trichosurus thus conforms in this respect to the general mammalian pattern. In a few of the lower vertebrates they studied FEY (1966c) and FEY and KUNTZE (1970) found a positive reaction. Monocytes. The monocytes of man (WACHSTEIN, 1946; WISLOCKI and DEMPSEY, 1946; PLUM, 1950; KERPPOLA, 1951; HAYHOE and QUAGLINO, 1958; LEONARD, 1958; MONIS and RUTENBERG, 1959; LENNERT and LEDER, 1963; WANG, 1963; WULFF, 1963c; WINTROBE, 1967) and other mammals (MONIS and RUTENBERG, 1959; SHUBICH, 1966; JAIN, 1970) exhibit no alkaline phosphatase, and again Trichosurus fits the mamma- lian pattern. In some of the lower vertebrates he studied FEY (1966c) found that the monocytes gave a positive reaction. Platelets. The negative findings I obtained with Trichosurus are in accord with most studies on the platelets of man (KERPPOLA, 1951; ALEXANDER, 1953; DUDE, UPTON and ODELL, 1956; HAYHOE and QUAGLINO, 1958; MONIS and RUTENBERG, 1959) and various other mammals (CHEVILLARD, 1945; WAGNER and YOURKE, 1955; GUDE, UPTON and ODELL, 1956; MIZUNO, SAUTTER and SCHULTZE, 1958; MONIS and RUTENBERG, 1959; FEY and KUNTZE, 1970; JAIN, 1970). Some positive findings have, however, been reported with human platelets using cytochemical (STORTI, PERUGINI and ROSSI, 1953) and biochemical (PEDRAZZINI and SALVIDIO, 1957; MAUPIN and SAINT-BLANCARD, 1966) techniques. FEY (1966a) and FEY and KUNTZE (1970) also achieved positive results with some of the lower vertebrates they studied. Dehydrogenases Neutrophils. Other findings on the cytochemically demonstrable succinate dehydrogenase content of mature human neutrophils have not been in agreement; some investigators have achieved positive results (BALOGH and COHEN, 1961; WANG, 1963; ACKERMAN, 1964; ROZENSZAJN and SHORAN, 1967) while others have found, as I did in human and Trichosurus, that neutrophils give a negative reaction (MAHR, 1962; HAYHOE, QUAGLINO and DOLL, 1964; ASAKAWA, 1966) or usually so (QUAGLINO and HAYHOE, 1960; WULFF, 1963a; BAUER-SIC, 1965). No doubt the variations in results have been, to some extent at least, due to differences in method. The neutrophils of rat marrow (KETELS-HARKEN, 1964) and mouse (MORRISON and KRONHEIM, 1962) have also been reported as negative, but some positive staining has been achieved with cells from some other mammals (BAUER-SIC, 1963, 1965) and with the mito- chondria of fractionated neutrophils of horse (KAWABATA, 1960). ACKERMAN (1964) 348 R.A. BARBOUR: found in fixed intact cells that the enzyme was localized in the mitochondria, though he had previously shown (1960b) that in living cells the only staining achievable was intermitochondrial in position, the living mitochondrial membranes apparently pro- viding a barrier to the free penetration of the tetrazolium salt (as pointed out by PEARSE, 1960). Lactate dehydrogenase has been demonstrated in human neutrophils (BALOGH and COHEN, 1961; WULFF, 1963a, b; ACKERMAN, 1964; HAYHOE, QUAGLINO and DOLL, 1964; ROZENSZAJN and SHOHAM, 1967), but in some investigations only a weak reaction was obtained (WULFF, 1963b; ACKERMAN, 1964) or only a small proportion of the cells stained (HAYHOE, QUAGLINO and DOLL, 1964). Eosinophils. As in the case of neutrophils, findings on the succinate dehydro- genase content of human eosinophils have not been in agreement, positive staining having been achieved by some (BALOGH and COHEN, 1961; GEDIGK and GROSS, 1962; WANG, 1963; HAYHOE, QUAGLINO and DOLL, 1964; ROZENSZAJN and SHOHAM, 1967), while others have obtained negative (MAHR, 1962; ARCHER, 1963) or usually negative (WULFF, 1963a) results. KETELS-HARKEN (1964) obtained positive results with marrow cells from rat, BAUER-SIC (1963) got negative results with cow. Findings on the lactate dehydrogenase content of human eosinophils have been more-uniformiy positive (BALOGH and COHEN, 1961; QUAGLINO, 1961; HAYHOE, QUAGLINO and DOLL, 1964; ROZENSZAJN and SHOHAM, 1967). Basophils. In human basophils both succinate dehydrogenase (BALOGH and COHEN, 1961; ACKERMAN, 1963) and lactate dehydrogenase (BALOGH and COHEN, 1961; ACKERMAN, 1963; SHIBATA et al., 1966) have been demonstrated cytochemically, as have both these enzymes in basophils of rabbit and guinea pig (ACKERMAN, 1963). Lymphocytes. Consistently positive results have been obtained with the demos- tration of both succinate (QUAGLINO and HAYHOE, 1960; BALOGH and COHEN, 1961; LAMBERS and BAUER-SIC, 1962; MAHR, 1962; WANG, 1963; WULFF, 1963a; HAYHOE, QUAGLINO and DOLL, 1964; ASAKAWA, 1966; ACKERMAN, 1967; ROZENSZAJN and SHOHAM, 1967) and lactate (BALOGH and COHEN, 1961; WULFF, 1963b; HAYHOE, QUAGLINO and DOLL, 1964; ACKERMAN, 1967; ROZENSZAJN and SHOHAM, 1967; HERMANSKY, LODROVA and POSSNEROVA, 1970) dehydrogenases in human lymphocytes from blood and mar- row. Succinate dehydrogenase has been shown in lymphocytes from mouse spleen (MORRISON and KRONHEIM, 1962) and lactate dehydrogenase in blood lymphocytes of several other mammals (HERMANSKY, LODROVA and POSSNEROVA, 1970). The positive result found in Trichosurus in this study is thus in keeping with findings for mam- mals otherwise. Monocytes. As in the case of lymphocytes, human monocytes have been found to give positive staining for both succinate (QUAGLINO and HAYHOE, 1960; BALOGH and COHEN, 1961; WANG, 1963; WULFF, 1963a; HAYHOE, QUAGLINO and DOLL, 1964; ROZENSZAJN and SHOHAM, 1967) and lactate (BALOGH and COHEN, 1961; WULFF, 1963b; HAYHOE, QUAGLINO and DOLL, 1964; RUZENSZAJN and SHOHAM, 1967) dehydrogenases. Again Trichosurus conforms. Platelets. Human platelets have been shown by cytochemical means to contain succinate dehydrogenase (ACKERMAN, 1960b; BALOGH and COHEN, 1961; HAYHOE, QUAGLINO and DOLL, 1964; ROZENSZAJN and SHOHAM, 1967) and lactate dehydrogenase Marsupial Leukocytes and Platelets 349 (BALOGH and COHEN, 1961; HAYHOE, QUAGLINO and DOLL, 1964; ROZENSZAJN and SHOHAM, 1967), and both these enzymes have been demonstrated using biochemical techniques (KOPPEL and OLWIN, 1954a, b; MAUPIN and SAINT-BLANCARD, 1966). Bio- chemical means have also been successful in demonstrating succinate dehydro- genase in horse platelets (WAGNER, MEYERRIECKS and SPARACO, 1956). The presence of these enzymes in demonstrable form in Trichosurus is thus not unexpected. My finding of a greater reaction for lactate than for succinate dehydrogenase in the various blood elements of both man and Trichosurus is in accord with the observations of HAYHOE, QUAGLINO and DOLL (1964) on lymphocytes and monocytes and of these same authors and ROZENSZAJN and SHOHAM (1967) on platelets. ACKERMAN (1963) has noted this point in relation to granular leukocytes. KOPPEL and OLWIN'S (1954a, b) biochemical studies on platelets, however, showed the opposite relationship between these two enzymes. Conclusion Taken overall, the leukocytes and platelets of Trichosurus vulpecula are similar to those of other mammals. They present a number of features that are more or less unusual but not unique among mammals as a class. The most outstanding and the only really unique feature is the presence of demonstrable alkaline phosphatase in the specific granules of basophils, and this feature is, perhaps, more surprising since this enzyme cannot be demonstrated in any other type of leukocyte. The relatively unusual features in the morphology of the cells are the elongated form of the eosinophil granules, the abundance and uniformity of the basophil granules and, in the monocytes, the frequency of irregular forms of nucleus (includ- ing occasional annular examples) and the coarseness of the chromatin in many nuclei. The abundance of large platelets also seems to exceed that in most other mammals. In comparison with other mammals the counts of lymphocytes and monocytes are fairly high while those of eosinophils and basophils are low-none of these, however, is extreme. The mean diameters of eosinophils, basophils and lymphocytes, and, to a lesser degree, neutrophils are probably somewhat greater than in most other mammals. The absence of any staining by sudan black in almost all monocytes and, on the other hand, the presence of a few positively staining lymphocytes are some other relatively unusual features. In the results of PAS staining the leukocytes and platelets of Trichosurus exhibit no very outstanding features, while in the examination for peroxidase the negative result observed in the monocytes is the most noteworthy point. The absence of alkaline phosphatase from neutrophils is relatively unusual, while, as noted above, the presence of this enzyme in basophil granules is possibly unique in this species among mammals (though not many have been studied in this regard), although it has been reported in a sub-mammalian vertebrate. 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