THE CYTOCHEMICAL DEMONSTRATION OF LIPIDS IN BLOOD AND BONE-MARROW CELLS

F. G. J. HAYHOE Department of Medicine, University of Cambridge

(PLATES XCV AND XCVI)

THE distribution of lipids in cells and tissues has been studied for many years by the use of a number of inert dis-azo dyes which are soluble in neutral fats. The commonly employed compounds Sudan I11 and Sudan I V have not given very satisfactory results, and these have now been largely replaced by Sudan black B. This dye, which was &st introduced by Lison in 1934, stains neutral-fat droplets extremely well. It also stains phospholipids and sterols, and it has been employed in the study of mitochondria, the Golgi apparatus, the myelin sheaths of nerve fibres, and the lipids of such endocrine glands as the adrenal cortex, testis and corpus luteum.

Among the earlier observations on the lipid content of leucocytes were those of Petry (1908), who isolated eosinophil granules and stated that they showed no fat reaction, of Sehrt (1927), who described lipid granulas in neutrophil polymorphs stained with Sudan I11 and Nile-blue sulphate, and of Bacsich (1935-36). who stained lipid granules in leucocytes with Sudan 111 but found many samples of this dye unsatisfactory. The first report on the staining of leucocyte granules with Sudan black B waa by Sheehan (1939), who fixed air- dried blood films in methyl alcohol for half-a-minute and stained them for 30 minutes in a saturated solution of Sudan black B in 70 per cent. alcohol. He used an eosin-methylene blue counterstain to aid in cell identification and briefly reported that polymorphonuclear leucocytes were filled with deeply stained granules, eosinophil granules appeared to have only a surface layer of lipid, transitional cells showed a variable number of granules, while large and small lymphocytes were always quite free from lipid. Myelocytes showed many granules; myeloblasts had usually a small number of obvious granules but were sometimes clear ; lymphoblaats contained no granules at all. McManus (1945) studied films of human blood and bone marrow stained with Sudan black B and confirmed Sheehan’s findings of numerous sudanophilic granules in the cytoplasm of neutrophil polymorphs and the absence of staining in lymphocytes. He could find no positive granules in monocytes, but described sudanophilic granules in cells of the late myeloblast series. Wislocki and Dempsey (1946), in monkeys, confirmed previous observations on neutrophil and eosinophil polymorphs and on cells of the lymphocyte series. Myelocytes resembled the corresponding mature polymorphs in sudanophilia, and cells recognised as monocytes contained a few black granules in their cytoplasm. Megakaryocytes and blood platelets in the bone marrow of the monkey contained minute, finely scattered, lipid particles, and Wislocki, Bunting and Dempsey in a leter paper ( 1947) suggested that these Sudan-black-positive dots might represent mitochondria.

J. PATE. BACT.-VOL. LXV (1953) 413

414 F . G. J . HAYHOE

Further studies on leucocyte granules after staining with Sudan black B and May-Griinwald-Giemsa were reported by Baillif and Kimbrough (1947), who were able to confirm the results of McManw. They stated that monocytes, lymphocytes and basophil leucocytes were unstained.

Sheehan and Storey (1947) described an improved method of staining Ieucocytes with Sudan black B. The usual methyl-alcohol fixation is replaced by formaldehyde-vapour fixation, the Sudan-black solution is maintained at a neutral or slightly alkaline reaction, and a small proportion of phenol is added to act as a mordant.

Rheingold and Wislocki (1948) made detailed observations on the sudano- philia of cells of human peripheral blood and bone marrow. They stated that sudanophilic granules were present in neutrophil-leucocyte precursors from the time when specific granules first appear. These granules stained greyish-brown to black and appeared to be identical in number and sue with the specific neutrophil granules. The granules of eosinophil polymorphs and myelocytes were also sudanophil and showed the darker periphery and clear interior described by Sheehan and other investigators. Basophil granules were studied in the blood of patients with chronic myeloid leukaemia and were found to be positive, though ranging in intensity of staining from pale grey to deep black. The larger basophil granules were pale, with a clear centre, while smaller ones stained deeply and appeared solidly black. Since previous workers had differed over the sudanophilia of monocytes, Rheingold and Wislocki studied the blood of patients with a high monocyte count in which these cells were readily identifiable. Variable cytoplasmic sudanophilia was encountered, ranging from a few faint dots up to an obvious particulate dusting of the cytoplasm. The use of a counterstain tended to obscure these dots, and monocytes were studied after staining with Sudan black alone. Lymphocytes contained no lipids. Mega- karyocytes and platelets in human material were not found to contain sudano- philic granules, in contrast with the previous reports of these authors and others on the sudanophilia of comparable cells in the monkey. A faint diffuse colouration of the red cells and antecedent erythroblasts was observed, but the authors were not prepared to interpret this as definitely due to lipid.

Bloom and Wislocki (1950) studied blood and bone-marrow cells with the acid hsmatin stain for phospholipids described by Baker (1946) and compared the results wit,h those seen after staining with Sudan black B. They concluded that Baker’s method stains the finer particles more satisfactorily. These are probably mitochondria, present in myeloblasts, lymphoblasts, megakaryocytes and monocytes, but it is noteworthy that they could also be seen, albeit less distinctly, with Sudan black B. This increased sensitivity with Sudan b h k is explained by the use of formalin-calcium fixation instead of methyl-alcohol fixation and by not using a counterstain.

Storti and Perugini (1949, 1951) record their results obtained with Sudan black B and a Giemsa counterstain in normal and abnormal blood and marrow cells. They found that haemocytoblasts, all cells of the red and lymphatic series, plasma cells, megakaryocytes, platelets, most basophilic leucocytes and reticulaz histiocytes, and the so-called lymphoid haemohistioblasts, were completely sudanophobic. Neutrophil and eosinophil granuloblastic cells and monocytes showed reactions similar to those previously reported. These authors describe the lipid content of various cells in pathological conditions. The only abnormalities met with were of a quantitative kind and referred to grando- cytes. The lipid content of neutrophils was thought to be diminished in some cases of chronic myeloid leukzmia and of malignant lymphogranulomatosis and in most infectious diseases. There was no correlation between lipid content of granulocytes and the haemoglobin level, leucocyte number, temperature, or degree of nuclear segmentation. Tiirck cells, Sternberg cells, Howell-JollJ- bodies, Cabot rings, reticulocytes and the hyperbasophilic cells of infectious

CYTOCHEMISTRY OF LIPIDS 415

mononucleosis were dl sudanophobic. Auer bodies were sudanophilic. Storti and Perugini (1951) were also able to study 16 cases of acute leukzemh, and in three of these the findings with Sudan black B were unexpected. Two -88, diagnosed after May-Griinwald-Giema staining as para-hsemocytoblastic leukaemie, showed the cellular sudanophilia respectively of promyelocytic and myelocytic and of myeloblastic type. A third case appeared with Giemsa’s shin to be acute monocytic leukaemk but with Sudan black B resembled myeloblastic leukemia.

PERSONAL INVESTIQATIONS

Methob Air-dried smears of blood or bone marrow were made and fixed aa soon as

1. Fix by exposure to formalin vapour for 4-5 minutes. 2. Wash in running tap water for 15 minutes. 3. !I’reat with a saturated solution of Sudan black B in absolute alcohol

4. Wash well in 70 per cent. alcohol for 2-3 minutes. 5. Wash in tap water for 1 minute. 6. Counterstain with diluted Leishman’s stain for 15 minutes if desired.

possible.

for 30-90 minutes.

Results Normal bone marrow

Sternal-marrow aspirates were obtained from 15 healthy persons. Smears were made directly on clean glass slides and treated as described above. Positive staining was confined to the cytoplasm.

Cells of the erythropoietic series contained only minute quantities of granular sudanophilic material. In pro-erythroblasts and normo- blasts of all degrees of maturation there were perinuclear aggregations of fine rods and dots of positive material, usually more prominent in earlier cells of the series and tending to occupy a position predominantly on one side of the nucleus (fig. 9). Their localisation closely resembled that of the perinuclear “ hof” which is frequently conspicuous in pro-erythroblasts and early normoblasts in Romanowsky-stained preparations. Later normoblasts showed little, and mature red cells none, of this particulate sudanophilia, but the cytoplasm of late normoblasts and the whole of the mature erythrocytes were tinted a greyish-brown colour. The depth of this colouration varied in different preparations from a faint greyish-yellow to a deep brown, like that of the most conspicuous eosinophil granules and appeared to be related to the length of time during which the preparation was exposed to the Sudan black. Slides stained for 30-90 minutes showed light tinting of erythrocytes, while those stained for 2-10 hours showed much deeper colouration.

Cells of the myeloid seriu (figs. 1-6) showed sudanophilia increasing in degree with progressive maturity of the cells. The fully mature neutrophil polymorph invariably contained large numbers of sudano- philic granules, which were usually coarser and much more conspicuous

416 F. Q. J. HAYHOE

than the specific granules observed in Romanowsky-stained smears, filling the cell and often partly obscuring the nucleus. Neutrophil metamyelocytes and myelocytes showed less sudanophilic granulation, corresponding roughly with their content of specific granules. Pro- myelocytes contained a few coarse granules of the same type as later myelocytes, often aggregated in a single area of the cell, perhaps near the cell centre. Myeloblasts could not be identified in normal marrow without a counterstain, when they appeared negative, although fine particles might have been obscured by the Leishman’s stain.

Eosinophil polymorphonuclear cells showed greater avidity than any other cell for Sudan black B. Staining for so short a time as five minutes resulted in deep staining of eosinophil granules, while neutrophil granules remained scarcely tinged. The eosinophil granules are probably coated with a sudanophilic substance, since they nearly always presented a hollow appearance and were larger after Sudan- black staining than after ordinary hematological staining techniques. These large, deeply sudanophilic, hollow granules filled the cytoplasm, frequently overlay the nucleus, and could be seen scattered over large areas of the slide in the neighbourhood of the occasional disrupted eosinophil cell. Eosinophil metamyelocytes and myelocytes also contained these coarse granules, which were unmistakeably eosinophil specific granules with surface sudanophilia (fig. 3). In all eosinophil cells the background cytoplasm was unstained and the sudanophilic material surrounding the granules did not form a general matrix ; the granules with their black coating remained reasonably discrete and were indeed sometimes surrounded by a clear halo of unstained cytoplasm.

Basophil polymorphonuclear cells were readily recognised in smears counterstained with 1,eishrnan’s stain, though they could not be identified without counterstaining. They contained no positive material after Sudan black B, but their granules stained sharply and meta- chromatically with Leishman-methylene blue.

Lymphocytes were invariably negative. Monocytes were difficult to identify in normal marrow smears

and were sought for in smears of peripheral blood after fixation with formalin vapour and staining with Sudan black B and Leishman’s stain. Here they could be easily recognised and were found to contain moderate numbers of both coarse and fine granules scattered over the cell (fig. 6). Occasionally an undoubted monocyte was quite free from sudanophilic material.

Occasionally darker coarse granules could be distinguished in smears which had not been counterstained ; they were found only in platelet clumps, and the possibility that they might be artefacts produced by superposition of several platelets could not be excluded. No trace of sudanophilic material could be seen in platelets, singly or in clumps, in smeam counterstained with Leishman’s stain.

Platelets invariably stained a diffuse grey.

PLAm x m Fro. I.-* neutrophil polymorphe, 8 neutmphd metemyelocyte and a clump of

FIG. 2.-A p u p of neutrophil polymorpha and 8 neutmphil myelocyta.

FIO. 3 . - h eoeinophil myelocyte with corn strongly positive graulea. mntm are vieible in theee grenulee with slight dteretion in focus.

FIO. 4.-A neutmphil promyeloqta with mmty positive granulee. FIO. 6.-Neutrophil ceb 8t Merent etegee of development end, below, a myeloblest

FIO. 6.-A neutmpw polymorph, 8 monooyta and two negetive lymphwytea in 8

All films atabed with Sudan blaok B.

plateleta. x1000.

x 1OOO.

Umtained x 1OOO.

x 1OOO.

with finely pOeitive t h y roda and dots chiefly around the nuoleus.

peripheral blood amear lightly oountemtsined with Leishma's atein.

x 1OOO.

X 1OOO.

J. PATH. BACT.-VOL. LXV

FIO. 1.

FIO. 2.

FIO. 3.

CYTOCHEMISTRY OF LIPIDS

FIG. 4.

FIG. 5.

Fro. 6,

PLATE XCV

CYTOCHEMISTRY OF LIPIDS 417

Begakaryocytes showed diffuse greyish-brown staining, with innumerable tiny granules scattered throughout the cytoplasm and obscuring the nucleus (fig. 7). The positive effect was most marked in marrow preparations which had been st,ained for several hours and in which the erythrocytes were deeply tinged.

Plasma cells contained fine sudanophihc granules aggregated in the cytoplasm away fiom the nucleus; the whole cytoplasm was diffusely tinged a greyish colour.

Abnormal bone marrow and blood

Bone-marrow preparations obtained from a variety of pathological conditions were stained by Sudan black B. Peripheral blood smears taken from cases of leukaemia with a high leucocyte count were also included. Little difference in staining was observed between cells in abnormal blood conditions and cells of normal blood and bone marrow. The conditions investigated and the results obtained are listed below.

Pernicious ancemia. (a) In the stage of active megdoblastic hyperplasia, fine sudanophilic perinuclear granules were conspicuou~ in most megaloblasts in the early stages of maturation (fig. 8), and pro-erythroblasts showed sudanophilia exactly like that of pro- erythroblasts in normal marrow (fig. 9). Later megaloblasts showed few or no granules but the cytoplasm became diffusely tinted a pale grey colour, and late megaloblasts and mature megalocytes showed diffuse colouring similar to that of mature normal erythrocytes. The macropolycytes and giant metamyelocytes of pernicious-anaemia marrow contained normal amounts of large sudanophilic granules.

( b ) In the stage of active normoblastic hyperplasia during response to B12, all marrow cells gave reactions similar to those of normal marrow.

Hcernolytic ancemia. Cells stained normally, and no unusual findings were observed in erythroblastosis with very active normo- blastic hyperplasia.

Nutritional mcrocytic a n m i a of pregnancy. N.M.A. megaloblasts resembled pernicious-anaemia megaloblasts in sudanophilia.

Nutritional macrocytic antemia of idiopathic steatorrhma. Celi reactions presented no unusual features.

Iron-deJiciency ancemia. All cells resembled those of normal marrow in reaction to Sudan black.

Myeloblastic lezbkcemia (acute myeloid leukemia). Myeloblasts contained none of the coarse granules normally observed in later cells of the myeloid series, but they possessed many very fine and lightly stained greyish-brown dots and rods in the cytoplasm, particularly conspicuous in the juxta-nuclear areas. In paramyeloblasts the sudanophilic material tended to be concentrated centrally between the lobes of the nucleus.

418 F . G. J. HAYHOE

Myeloid leukcemia (chronic). Most granulocytes at all stages of development were indistinguishable in Sudan-black reaction from normal cells of similar maturity, but in several peripheral blood smears from cases of chronic myeloid leukamia some of the mature polymorphs showed no stained granules ; only a small proportion of mature leukamic granulocytes were involved-some 2 or 3 per cent.- but no comparable observation of granule-free polymorphs was made in normal blood or marrow.

Many mature lymphocytes were negative and almost invisible in simple Sudan-black B preparations. Lymphoblnsts and a variable proportion of the more mature lympho- cytes (about 10 per cent. in one case, 50 per cent. in another and none at all in a third) contained sudanophilic material (fig. 10). In the case of undoubted lymphoblasts the arrangement of this material resembled that found in myeloblasts and the particles were of a similar fine and lightly stained nature. The sudanophilic lymphocytes showed a dark, finely granular positive reaction in the narrow cyto- plasmic rim of the cell. Lymphocytes in normal blood and in all other pathological conditions examined were always negative, and nothing comparable with this sudanophilia of the lymphocytes in chronic lymphatic leukaemia has been encountered elsewhere.

Monooytic leukcemia (Naegeli type). Monocytes in monocytic leukamia were found to contain scattered sudanophilic granules, some coarse and others fine, and resembled the monocytes of normal blood (fig. 11). Pro-monocytes contained fewer granules and showed a transition from the most primitive pro-monocytes with monoblast- like sudanophilia and very few coarse granules to the more mature forms which resembled more closely mature monocytes.

Monoblastic leukmmia (acute). Monoblasts observed in this condition, and also in the previous one, resembled in general the myeloid and lymphatic parent cells but appeared to contain rather less sudano- philic material than these. although the positive granules were of similar type and distribution (fig. 12).

Myelbmatosis. Myeloma cells contained small quantities of sudano- philic material distributed in two forms. A diffuse colouration of the cytoplasm was present, and definite fine granules were scattered over the whole cell. There was no strict uniformity of reaction among different groups of myeloma cells or indeed among cells in the same clump, and sudanophilia ranged from a marked grey-brown general tint with clear-cut granules to a scarcely detectable reaction (fig. 13).

Glandular fever. Peripheral blood smears from two cases of infectious mononucleosis were examined. The percentage of cells with monocyte-type sudanophilia corresponded with the monocyte percentage in a Romanowsky preparation. The remaining mono- nuclear cells, including both normal lymphocytes and " glandular-fever cells ", were entirely negative.

Lymphatic l eukmia (chronic).

PLAm XCVI

FIG. 7 . - A m e g m . xIOOO.

FIa 8 . - h - 1 ~ megeloblaet. X 1OOO.

FIQ. 9.--Two pm-erythmbkte, an early normoblant and a negative 1ymphyt.e.

Fro. 10.-A group of 1ymphwyt.m and lymphoblaate from a 0888 of Chmnic lympMi0

FIG. 11.-A p u p of monooytee with OO(VBB grandee, a monoblast with a &re pOeitive

FIG. 12.-A group of monoblasta from a OBBB of mute monooytic leuksemie. x 1000.

FIG. 13.-A group of three myeloma cells.

x 1OOo.

1- showing a positive aytoplasmio &ion.

d o n in the juxbnuoleer zone, and a pleeme oell. x 1000.

x 1000.

x 1000.

All ~ ~ L U E steined with Buden blaok B.

J. PATH. BACT.-VOL IXV

CYTOCHEItUSTFtY OX' LIPIDS

FIG. 7.

FIG. 8.

FIG. 9.

FIQ. 10.

PLATE XCVI

FIG. 11.

FIG. 12.

Fin. 13

CYTOCHEMISTRY OF LIPIDS 419

DISCUSSION A considerable measure of agreement exists among most research

workers as to the lipid content of blood and marrow cells determined by staining cytochemically with Sudan black B. Differences recorded by various investigators may be of less significance than a t first appears. Thus Storti and Perugini found no sudanophilic material in cells of the erythroblast series and lymphatic series, whereas primitive cells of both these series are stated by Bloom and Wislocki to contain fine rods and granules. This apparent contradiction is probably due to the use of a Romanowsky counterstain by the former authors. Such a counterstain obscures entirely the fine granules found in primitive cells of all series.

Three major questions arise in relation to the sudanophilic reactions of harnopoietic cells. The first is whether the lipid granules present in the myeloid series are identical with the specific granules seen in Romanowsky-stained smears. This is almost certainly the case, since the occurrence, number and distribution of the lipid granules and of the specific granules in various cells of the neutrophil and eosinophil series run closely parallel. A further corroborative observa- tion is that the specific granules of a Leishman-stained preparation become obscured if, after short exposure to Leishman’s stain, the slide is stained with Sudan black B. Again, counterstaining Sudan- black preparations with Leishman’s stain reveals no specific granules distinct from t,he sudanophilic granules. Nevertheless there is a discrepancy in size between sudanophilic and specific granules, the former being very much larger and almost filling the mature neutrophil cell. This difference in appearance is discussed by Discombe (1946), who concludes that it is an optifact resulting from the absorption of one of the Leishman akures (probably Bernthsen’s violet) on the surface of the lipid granule with the production of a discoid image of much smaller diameter than the granule itself when light from the condenser is focussed by the granule.

The second question is whether the granules which stain with glycogen techniques are identical with the sudanophilic ones. Their distribution in the myeloid series is comparable as far as neutrophil cells are concerned, but in cells of the eosinophil and basophil series there are obvious differences between the distribution of sudanophil and glycogen-staining material. In neutrophil cells differences are less obvious, but the glycogen granules are usually much finer than the sudanophilic and very much more numerous, producing in the mature neutrophil pol ymorph an appearance of dense irregular fine stippling. Erank6 (1950) was able to show that the irregularity of the glycogen stippling in the neutrophils is due to unstained lipid granules and that these could be stained in the same preparation by a suitably modified combination of Sudan-black B and P.A.S. tech- niques applied successively to the smear. The glycogen and lipid

420 F . Q. J . HAYHOE

granules are therefore separate components of the neutrophil poly- morphonuclear cytoplasm.

Third, the relationship between sudanophilic granules and the granules seen in peroxidase preparations must be discussed. In every way the resemblance between these two types of granule is remarkable. In size, number, appearance and distribution they seem almost identical. This resemblance was observed by Sehrt (1927), who concluded that the oxidase reaction of blood cells was proportional to their lipid content, and by Lison (1936), who thought it possible that the oxidase reaction depended, not on the presence or absence of oxidases, but on the existence in the cell of lipids capable of dissolving and retaining oxidation products. It appears likely, therefore, that Sudan black B might be used in preference to oxidase stains for the routine hsmato- logical investigation of primitive cells in blood or marrow. This has been done by Storti and Perugini in a small series of cases with the interesting results already quoted. In 6 cases of acute leuksmia which I have studied with Romanowsky, Sudan-black B and peroxidase stains there were no discrepancies and this was so also in 13 of Storti and Perugini’s 16 patients. The unexpected Sudan-black findings in their other 3 cases suggest that this stain might produce valuable new diagnostic information, but it is unfortunate that parallel oxidase preparations were not made, since this information might also have been provided.

The fine perinuclear rods and dots visible in primitive cells in Sudan-black preparations unobscured by a counterstain are believed by most observers to be cytoplasmic organojds-mitochondria and components of the Golgi apparatus. They are said to be more con- spicuous after Baker’s acid hsmatin stain for phospholipids and to react negatively with the pyridine-ektraction control test. In shape, size and distribution they resemble the mitochondria visible in supravital-stained preparations (Bloom and Wislocki, 1950). Since these fine granules are present in primitive blood cells of all series and show no unexpected features in the ansmias or leucopathies, their demonstration is unlikely to be of any immediate practical value.

SUMMARY Reports of the use of Sudan black B as a cytochemical stain for

lipids in hsmatological material show substantial agreement on the major findings, but minor differences are reported concerning the sudanophilia of primitive cells and of monocytes, erythroblasts and megakaryocytes. Some of these discrepancies may be due to the use by certain authors of obscuring counterstains.

In the present series, sternal-marrow smears from 15 normal subjects were stained with Sudan black. In all cells positive staining was confined to the cytoplasm.

Cells of the erythropoietic series contained only minute quantities of finely granular sudanophilic material, probably mitochondria. A

CYTOCHEMISTRY OF LIPIDS 42 1

diffuse tinting of erythrocytes and of erythroblast cfioplasm was observed in overstained slides.

Cells of the myeloid series showed increasing numbers of positive granules with increasing cell maturity. Eosinophil granules showed a positive periphery with unstained centre. Basophil granules were negative.

Lymphocytes did not stain, but monocytes showed scattered fine and coarse particles. Megakaryocytes were moderately positive and platelets probably negative. Plasma cells showed diffuse and granular staining.

Marrow preparations from subjects with a variety of blood dis- orders were studied ; little difference from the staining reactions of cells in normal marrow could be seen. Positive material was, however, present in some lymphocytes from chronic lymphatic leukaemia. Glandular-fever cells were negative.

Most primitive cells showed fine perinuclear rods and dots and these were thought to be cytoplasmic organoids.

The relation of sudanophilic granules to specific granules, glycogen granules and peroxidase granules is discussed. Specific granules are sudanophilic; glycogen granules are not. Granules which give a positive peroxidase reaction are also positive to Sudan black.

My thanks are due to Sir Lionel Whitby for his advice and help and to Mr B. W. Gurner and Mr R. J. Flemans for assistance with the photomicrographs. Part of this work was done during the tenure of an Elmore Research Studentship and formed a contribution to a thesis for the degree of M.D. of Cambridge University. I am grateful t o the Regius Professor of Physic for permission to reproduce this work.

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BAKER, J. R. . . . . . . 1946. Quart. J . Micros. Sci., lxxxvii, 441. BLOOM, M. L., AND WISLOCKI, G. B. DISCOMBE, G. . . . . . . 1946. This Journal, lviii, 572. ERAKEG, 0. . . . . . . . 1950. Nature, clxv, 116. LISON, L. . . . . . . . 1934. C. R. SOC. Biol., cxv, 202.

MCMANUS, J. F. A. . . . . 1945. Nature, clvi, 173. PETRY, E. . . . . . . . 1908. Wien. klin. Wechr., xxi, 1360. RHEINGOLD, J. J., AND WISLOCKI, 1948. Blood, iii, 641.

SEHRT, E. . . . . . . . 1927. Mfinch. med. Fvechr., bxiv, 139. QHEEHAN, H. L. . . . . . 1939. This Journal, xlix, 580. SHEEEAN,H.L.,AND STOREY, G. W. 1947. This Journal, lix, 336. STORTI, E., AND ~ERUGINI, S. . 1949. Studia QhiSleriana, Studi Med. Biol.,

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1950. Blood, v, 79.

,, . . . . . . . 1936. Histochimie animale, Paris.

G. B.

i, 313. 3 , ,, 9 , . 1951. Acta hcemutol., v, 321.

WISLOCKI, G. B., BUNTING, H., 1947. Anat. Rec., xcviii, 527.

WISLOCIU, G. B., AND DEMPSEY, 1946, Ibid., xcvi, 249. AND DEMPSEY, E. W.

E. W.