The Leukocytes and Platelets of a Marsupial, Trichosurus Vulpecula. a Comparative Morphological, Metrical and Cytochemical Study
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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.