77

Lymphology 10 (1977) 77-84 © Georg Thieme Verlag Stuttgart

Morphological and Cytochemical Identification of Lymphoid Cells

D. Catovsky, A. E~no M.R.C. Leukaemia Uriit, Royal- Postgraduate Medical School, London

Summary Morphology - Light and Electron Micro­ This paper reviews the contribution of: light and scopy (TEM) electron microscopy, surface structure .and cyto­ Morphological features of lymphocytes may chemistry (acid phosphatase, B-glucuronidase, a-naphthyl esterase, and PAS reactions) in the depend on the state of transformation and on recognition and classification of normcd and ab­ the degree of differentiation. The latter may normal lymphocyte populations be demonstrated better by surface markers. ''Mature" lymphocytes can readily be dis­ tinghished from other leukocytes, but it is In recent years important advances in the not possible to distinguish between T and B · methods for re~gnition of different popu­ lymphocytes with.either light microscopy or lations of lymphocytes have taken place {1 ). transmission electron microscopy. Normal anti­ At the same time new techniques in cell mor­ genically stimulated lymphocytes can o:casion­ phology and cytochemistry have been applied ally be difficult to distinguish from malignant successfully to the study of lymphoid cells. cells. Transitional forms between B lympho­ The two main problems in the identification cytes and plasma cells such as seen in W~den­ of lymphocytes are: strom 's macroglobulinemia are characterized 1. Distinguishing lymphoid from nonlymphoid by marked basophilic cytoplasm and increased cells, particularly when the features of amounts of rough endoplasmic reticulum differentiation and maturation ~e not obvi­ (Fig. 1). ous. This is apparent in the problem of The importance of lymphocyte morphology diagnosing and classifying leukemias (2) and its accurate description is particularly high­ and lymphomas in order to es~blish with lighted in the field of non-Hodgkin lympho­ certainty the nature of the malignant mas. In these lymphomas the terms ''well process. Frequently, studies of normal differentiated" (morphologically mature) and mononuclear cells, separated by density "poorly differentiated" (resembling lympho­ gradient techniques, require the clear identi­ blasts) have quite different prognostic signifi­ fication of lymphocytes from other mono­ cance. In our experience examination of the nuclear cells such as monocytes. nuclear chromatin condensation of lymphoid 2. Identifying the various lymphocyte popu­ cells under TEM permits a more accurate and lations (B, T, K, ''Null") by means other objective defmition of the terms ''well differ­ than the surface markers. This whole area entiated" and ''poorly differentiated." is at a very early stage of development, but the promises of the initial observations Lymphoblasts have almost no chromatin with scanning electron microscopy (1) have, condensation; mature lymphocytes, have a however, not been fulfilled (3, 4, 5, 6). predominance of coarse chromatin and inter­ We will review the contributions of mor­ mediate forms such as the prolymphocyte can phology, surface structure, and cyt~hemis­ readily be recognized (7). Follicular centre B try for the identification of l~phmd cells. cells which have a cleft (cleaved) or rounded (noncleaved) nucleus (8) also vary in morpho­ logical appearances depending their degree of transformation. In follicular lymphoma with 78 D. Catovsky, A. Enno

Fig. 1 Lymphoid cell intermediate between a B lym­ Fig. 2 Lymphocyte with electron-dense granules phocyte and a plasma cell, with relatively abundant from a case of T-CLL (x 11,000). rough endoplasmic reticulum. From a lymph node of a case of Waldenstrom macroglobulinemia (x9,000).

associated peripheral blood lymphocytosis, of the T-ceil type (T-CLL) (14). Ultrastructure the cells often have cleaved or notched nuclei of these cytoplasmic inclusions shows that (9). TEM studies suggest that the ceil seen in t.I-Jey are membrane-bound and consists, in follicular lymphoma are similar to those seen some cases, of parallel arrays of microtu­ in reactive follicles, although nuclear folding bule-like st.ructures ( 13). These structures is more marked in the neoplastic cleaved fol­ are also seen in a high proportion of infecti­ licular centre cells ( 1 0). Large noncleaved ous mononucleosis lymphocytes (13) and in blast cells with a finely stippled and homogen­ some T-CLL cells (15), but rarely in normal ous nucleus and deep basophilic (pyronino­ lymphocytes. philic) cytoplasm with many vacuolations con­ taining lipid droplets are characteristic of Af­ rican (Burkitt's) lymphoma. These cells are seen in the rare cases of sporadic non-African (non-Burkitt's lymphoma which present as '. or develop acute leukemia (L3 of the FAB classification) (2). Selective involvement of the germinal centers has been demonstrated (11) in this latter group; the cells have been found to have B-lymphocyte characteristics ( 11 , 12). These cells also appear to be larger • than those of the African type (8). Changes seen in disease suggest that T lympho­ cytes may often be characterized by the pres­ ence of numerous azuropllilic granules on Ro­ -, manowsky stains or seen as electron dense gra· nules under TEM (Fig. 2). These features are seen in reactive T lymphocytosis, particularly 3 in infectious mononucleosis (13) and in a rare Fig. 3 Sez:uy ceiJ from a peripheral-blood buffy form of chronic lymphocytic leukemia (CLL) coa t, with a highly convoluted nucleous (x 10,000). · Morphological and Cytochemical Identification of Lymphoid Cells 79

Another type of neoplastic T lymphocyte, the drying techniques have improved the preser- Sezary cell, characteristically has a highly vation of the lymphocyte surface morphology irregular, convoluted, or cerebriform nucleus (5, 6, 23). All these factors were, however, which is most prominent on TEM (16, 17) not considered in ti1e initial studies (3). (Fig. 3). A small variant of the Sezary cell is Lymphocytes are seen by SEM as spherical seen in cases with a high leucocyte count cells with numerous short microvilli randomly and may suggest CLL. Sezary cells are less distributed over the whole cell (23). Initial well recognized i.n paraffm-embedded . s~ctions studies by Polliack and co-workers (2 14) (16). _Con~o luted I_ymphoblasts have a~~ be~n - suggested that T lymphocytes had a s:noother ?escnbe? m a malignant l~p~oma ongmatmg surface and B lymphocytes, a villous one. The m thymtc cells (8, 18). T~s dtsea~e more fmding of a villous surface in B-CLL and a ?ften presents as a leukemta and IS character- smootil surface in thymocytes lends further tzed by_T-lymphocyte markers (18, _19, 20). support to this hypothesis. Later, however, it When ~ t agnose d as an acute_ l eukemta~ ~e became clear, even using the early techniques, name 'T acute lymphoblastic leukemm that about 20% of B lymphocytes were indis- (T-ALL) is common~y- ~ sed, although the . tinguishable from T lymphocytes (24). Studies convoluted nucleus mthally regarded as typt- on rosetted cells (B or T) also conflicted (25) cal may. not always be present. Other mor- . wtth. some reports that B cells were smother phologt~al features of the acute lympho~lastic than T cells (26). 1t has now been shown leukemm (ALL) cells and methods for dts- . f · ·shin. th f .d k . conclustvely that smooth cells were arte acts 1 1 tmgut g em rom. acute mye 0 1 ( eu ) emm resuIt" mg f rom ti1e cont ac t o f u nfiLXe d ce ll s (At\1L) cells are descnbed elsewhere 2 · with the silver membranes (23). Under opti- Hairy cells, characteristic of hairy cell leukemia mum conditions most lymphocytes are found (leukemic reticuloendotheliosis) have attracted to have short microvilli (4, 5, 6); the study of much interest because of the possibility of purified populations of B and T lymphocytes their beirtg an atypical lymphoid cell (7). Mor- has revealed no significant differences (23). phologically, the main features of these hairy In the formation of RBC rosettes with lympho­ cells are their long cytoplasmic villi, paucity cytes, the contact between the RBC and of lysosomal (electron-dense) granules, and lymphocyte occurs via the microvilli. This the presence of a cytoplasmic structure known raises the question of the significance of these as nbosome-lamella complex in half the cases. structures in cell recognition and communica­ This structure is also observed, although rarely, tion (23). The lymphocytes in CLL tend, in irt the cells of other B lymphoproliferative general, to have fewer microvilli than normal disorders (7). lymphocyt~s (23, 24), and ti1e blast cells in ALL often have a smooth surface. No differ­ Surface Stntcture ences are found between Null and T-ALL Scanning electron microscopy (SEM) has pro­ (23 , 27) or between B-CLL and Sezary cells vided a three-dimensional view of the cell (5). Hairy cells when viewed with SEM can surface and its processes. Although most have abundant microvilli such as in lympho­ lymphocytes can readily be distinguished from cytes, ridged and ruffled membranes such as other leukocytes (granulocytes and mono­ in monocytes (28, 29, 30, 31), or a combi­ cytes 3, 6), except probably eosinophiJs (21), nation of the two (28, 29). the evidence for surface differences between T and B lymphocytes has been controversial. Knowledge of the artefacts which can arise Cytochemistry from preparation techniques is inlportant Acid Phosphatase. This lysosomal enzyme can because fue cell membrane is highly suscepti­ be demonstrated in smears or tissue sections ble to many external factors, e.g. , temperature by several cytochemical methods (32, 33). (22 , 23). More recently, immediate fixation Granulocytes in all stages of development, of the cells in suspension and critical-point lymphocylr:s, plasma cells, monocyles, mega- 80 D. Catovsky, A. Enno

karyocytes and histiocytes have all been shown · cells (38). In our experience most B lympho­ to contain acid phosphatase. In sections of proliferative disorders, ie., B-CLL, prolympho­ lymph nodes and spleen of hwpan beings and cytic leukemia (B-PL), B-ALL, as well as Null- various rodents, Tamaoki and Essner (34) have .ALL, have only a weak or moderate acid shown a positive reaction in thb lymphocytes phosphatase reaction in a small proportion of of the T -dependent areas and ~ negative reac- cells, usually less than 30% (39). These fmd­

.tion in the lymphocytes of the I B-dependent ings in B-cell neoplasia are contrasted with areas. Wehinger and Mobius (35), althqugh not those in T cell neoplasia where strong acid able to demonstrate a clear difference between­ phosphatase activity in a majority of cells has T and B lymphocytes, found a '!higher propor­ been reported in T-CLL (14), T-PL (39), tion of T than B lymphocytes With a positive T-ALL (18, 20, 27, 36, 39, 40) and Sezary's reaction; Ba" and Perry (36) ~ere unable to syndrome (41) (Table 1). The acid phosphatase conf"mn tp.ese fmdings. Thus, ill would appear reaction in T-ALL is alniost always localized that a clear distinction betweert normal peri­ to a small paranuclear zone, similar to that pheral-blood T and B lymphocYtes cannot be observed in thymocytes of rats, mice, and made using the acid phosphatase reaction. This guine~ p~s (34). variation in results may, however, be a reflec­ A double-blind study of the value of acid tion of the different separation' techniques phosphatase reaction in immunological clas­ employed. sification of ALL and its relationship to prog­ Human fetal thymocytes have been shown to nosis, on material from the M.R.C. UKALL be strongly positive for acid phbsphatase (18); trials, has shown in a preliminary analysis, this corresponds with the fmdings of Tamaoki that the characteristic strong localized re­ and Essner in rodent thymus lymphocytes action is found almost exclusively in T-ALL (34). They also noted a difference in the intra­ and not in Null-ALL cases (42}. cellular distnbution of acid phqsphatase granu­ Electron microscopy of T-ALL cells has shown les between thymic and nonthyrttic lympho­ that most of the acid phosphatase activity is cytes in the mouse, the rat, anq the guinea localized to the membranes of the Golgi pig. Tpymic lymphocytes usually contained apparatus, but enzyme activity is also present one or a few coarse particles of1 positivity or in the few lysosomal granules (27). This an aggregate of small particles at one pole of characteristic pattern was also noted in some the cytoplasm; lymphocytes in the lymph normal T lymphocytes forming rosettes with nodes and spleen possessed many particles sheep RBC and in a relatively higher propor­ scattered throughout the cytopJasm. tion of PHA-transformed T lymphocytes {43). Mitogen-stimulated T lymphocytes and in­ In contrast to T-ALL, the acid phesphatase fectious mono-nucleosis cells knbwn to be T -de­ activity seen at the ultrastructunil ievel in rived have also been shown to have a strong T-CLL is, confmed predominantly to the acid phosphatase activity (37). A low acid numerous lysosomal granules (Fig. 4) with phosphatase activity was reported in CLL little or no reaction in the membranes of the Golgi zone.

Table 1 Cytochemistry of the Lymphoid Cell Leukemias. Cytochemical Reaction T-ALL 1.Null-ALL T-eLL; B-eLL I Sezary Acid Phosphatase +t+ ± +++ ± ~G lucuronidasc ++ ++ +++ ± Acid-a-Naphthyl Not Esterase + reported PAS + ++ +/++ ++

± Weak positivity, minority of cell~ reacting +/++ Moderate reaction +++ Stron~ ~caction and most cells positive Morphological and Cytochemical Identification of Lymphoid Cells 81 ·

A strong acid phosphatase reaction can also be found in hairy cells. This enzyme is L (+) tartrate resistant, and it corresponds to the isoenzyme 5 on disk acrylamide gel electro­ phoresis (44 ). Tartrate-resistant acid phospha­ tase activity is, however, not specific for hairy cells, as it has been demonstrated in G<1ucher's cells ( 45), PI:-IA-transformed lymphocyt~s, lymphocytes of infectious mononucleosis ( 45} B-PL (46), an occasional case of T-PL (47), and in T-CLL (42).

(J-G lucuronidase Lymphocytes, in contrast to granulocytes and monocytes, have a granular rather than a dif­ fuse reaction pattern for this enzyme (4). It has been suggested that the reaction is useful Fig. 4 Acid phosphatase reaction localized to for distinguishing Al\1L from ALL. Lympho­ the cytoplasmic granules in a lymphocyte, fiom blasts react with a granular pattern in contrast a case of T-CLL (x 15.000). to the myeloblasts which often to not react, or occasionally show a diffuse reaction ( 48, 49). found two-thirds of the cases from both T-ALL Barr and Perry (36) described two patterns of and Null-ALL to be equally reactive. It would granularity in peripheral blood lymphocytes: appear from this study that the {3-glucuronid­ type a: large blocks of reaction; type {3: ase reaction may not be as useful as the acid multiple small granules. They found that T phosphatase reaction in distinguishing between lymphocytes were almost always positive and the va rious types of ALL. half the cells had type a reaction. Less than three quarters of the B lymphocytes were po­ et-naphchyl Acetate Esterase sitive, but they always exhibited the type {3 The a-naphthyl acetate esterase reaction, in reaction. {3-glucuronidase has also been shown conjunction with NaF inhibition, can be used to be present in the lymphocytes of the T-cell to differentiate monocytes and monocyte pre­ dependent areas and absent from the lympho­ cursors from myeloid cells (53). Usi ng a modi­ cytes of the B-ceU dependent areas in the fied technique, Mueller et al. (54) demonstra­ spfeen and lymph nodes of human beings and ted acid a-naphthyl esterase activity in lympho­ rats (34). Jn contrast to the findings with acid cytes of the T-cell dependent areas of rat phosphatase, Tamaoki and Essner (34) found lymph nodes and spleens, but no activity in no {3-glucuronidase activity in thymus lympho­ the lymphocytes of the B-cell dependent areas. cytes of most rodents with the exception of More recently, Kulenkampff eta!. (55) used rats. this technique on human lymphocyte prepa­ While the morphologically well differentiated rations from blood and tonsils and demons­ B-cell neoplasms, i.e. , B-CLL and B-cell lym­ trated two specific patterns: 1) a localized phomas, have been shown to have a low (J­ strong reaction (T-like) in a majority ofT­ glucuronidase activity (50, 51) the well-differ­ lymphocytes; 2) a weak localized reaction (thy­ entiated T-cell neoplasms, i.e., T-CLL ( 14) mus-like) in a thymocyte subpopulation. Most and Sezary cells (52), have a strong {3-glucu­ thymocytes and all B lymphocytes did not re­ ronidase activity (Table 1). act. Mitogen-activated human T lymphocytes showed a decrease in activity; B-CLL cells and AJU10ugh the reaction pattern in ALL has not Null-ALL cells were negative (Table 1) . T-ALL been studied in great detail, Brauer et al. (20) cells were positive with the "thymus-like" re- 82 D. Catovsky, A. Enno

action. This reaction may help to distinguish al. {20) who found no difference between T­ T-ALL from Null-ALL, but it would seem ALL and Null-ALL with regard to the PAS more useful for distinguishing normal T from reaction. B lymphocytes. In some respe¢ts1 the pattern PAS positivity may not necessarily represent of reaction for this enzyme in T cells may be a difference in lymphocyte subpopulations similar to the (j-glucuronidase reaction, ie. a but may indicate a difference in proliferative strong reaction in mature T lymphocytes and activity. Using tritiated thymidine uptake a weak reaction in thymocyte$. In contrast, studies, Loffler has shown that a larger num- the acid phosphatase activity would· appear - ber of PAS-negative blasts were in the S-phase to be stronger in thymic cells and weaker in than PAS-positive blasts {61). Additional evi­ mature T lymphocytes. dence {62) suggests that T-ALL has a greater proliferative activity, i.e., ~gher mitotic and PAS Reaction labelling index, than blasts from Null-ALL. A positive reaction in blood cells usually de­ This may explain why T-ALL cells may have notes glycogen; this can be confrrmed by the a lower glycogen content than Null-ALL cells. sensitivity to diastase {33). Normal lympho­ Taswell and Winklemann (63) ftrst noted that cytes contain much less staining material than Sezary cells contained ~ules of PAS-positive granulocytes; but a few fme, or even coarse, material. More recently {41), it was noted that granules may often be demons~rated. ·A posi­ only 75% of the cases have PAS-positive cells. tive, granular PAS reaction has been consider­ The PAS material in these cellsJs often seen ed characteristic of lymphoid cells. Astaldi forming coarse granules or clumps. and Verga (29, 56) and Quaglino and Hayhoe (57) demonstrated that the strong PAS stain­ Acknowledgements ing in CLL cells was due to an increase in The author wishes to acknowledge the assistance of Miss Maureen O'Brien for the electron microscopy glycogen. In a study of a large number of and Miss Maria Cherchi for the cytochemistry. cases of acute leukemia, Hayhoe et al. {58) found that PAS staining (granules and blocks) References was the most reliable characteristic of ALL. 1. Report of WHO/IARC-sponsored Workshop on it seems that the PAS reaction •may not be human .B and T cells. Identification, enumeration, as specific for ALL as originally felt. PAS po­ and isolation of B and T lymphocytes from hu­ sitivity has been demonstrated in the blasts of man peripheral blood. Scan. J. ImmunoL 3 (1974) acute monocytic leukemia and erythroleuk­ 521-532 2. Bennett, J.M., D. Catovsky, Marie- Therese Daniel, emia {58, 59); "blocks" were eyen noted in G. Flandrin, D.A. G. Galton, H.R. Gralnick, C Sul­ acute monocytic leukemia {60)~ tan: FAB Co-operative group: Proposals for the Although some normal lymphocytes contain Oassification of the Acute Leukaemias. Brit. J. Haemat. 33 (1976) 451-458 a few PAS-positive gran':lies, it has been diffi­ 3. Polliack, A., N. Lampen, B.D. Clarkson, E. De· cult to determine whether these cells are B or Harven, z. Bentwich, F.P. Siegal, H. G. Kunkel: T lymphocytes because of the rapid changes Identification of human B and T lymphocytes by in intracellular glycogen when mcubated. We scanning electron microscopy. J. exp. Med. 138 (1973) 607-624 have observed {39) that Null-ALL, B-CLL, 4. Alexander, E.L., B. Wetzel: Human lymphocytes: and B-PL cells generally have a ~eater PAS similarity of B and T cell surface morphology. reactivity than T-PL and T-ALL cells (Table Science 188 (1975) 732-734 I); B-ALL cells have usually been found to s. Alexander, E., S. Sanders, R. Bray/an: Purported be negative. Other workers have noted that difference between human T- and B-cell surface morpho~ogy is an artefact. Nature 261 (1976) 239- ALL cases with a strong paranuclear acid ·241 phosphatase reaction in the blast cells and 6. Newell, D. G., S. Roath, J.L. Smith: The scanning clinical features suggestive of T-ALL had electron microscopy of normal human peripheral smaller amounts of PAS-positive material blood lymphocytes. Brit. J. Haemat. 32 (1976) 309-316 than the acid phosphatase-negative ALL's {61 ). 1. Oztovsky, D.: Hairy-cell leukaemia and prolym­ However, this was not confirmed by Brouet et phocytic leukaemia. Clinics in Haematology 6 (1977) 245-268 Morphological and Cytochemical Identification of Lymphoid Cells 83

8.Lukes, J., D. Collins: Immunologic characteriza­ 21 Polliack, A., S.D. Douglas: Surface features of tion of human malignant lymphomas. cancer 34 human eosinophils:a scanning and transmission (1974) 1488-1503 electron microscopic study of a case of eosincr 9 Spiro, S., D.A. G. Galton, E. Wilishaw, ~G. Loh­ philia. Brit 1. Haemat. 30 {1975) 303-306 man: Follicular lymphoma: a survey of 75 cases 22 Lin, P.S., D.F.H. Wallach, s. Tsai: Temperature­ with special reference to the syndrome resembling induced variations in the surface topology of chronic lymphocytic leukaemia. Brit. J •. cancer 31 cultured lymphocytes as revealed by scanning {SuppL II) {1975) 60-72 electron microscopy. Proc. Nat. Aead. Sci. U.S.A. 10 Glick, A.D., J.H. Leech, J.A. Waldron, J.M. Flex­ 70 {1973) 2492-2496 ner, ~G. Horn, ~D. Collins: MalignantTymphcr _ 23 Newell, D. G. E.: The fine surface and internal mas of follicular center cell origin in man. n. ID­ structure of normal and disordered lymphocytes. trastructural and cytochemical studies. J. Nat. Ph. D~ thesis, Faculty of Medicine, University of Cancer lnst. 54 {1975) 23-36 Southampton 1977. 11 Mann, ~B., E.S. Jaffee, ~C Braylan, K. Nanba, 24 Polliack, A., E. de Harven: Surface features of M.M. Frank, J.L. Ziegler, C W. Berard: Non-En­ normal and leukemic lymphocytes as seen by demic Burkitt's lymphoma: A B-cell tumor relat­ scanning electron microscopy. Ail interpretative ed to germinal centers. New Eng.; J. Med. 295 review. C1in. Immunol. Iriununopath. 3 {1975)

{1976) 685-691 I 412-430 12 Flandrin, G., J. C Brouet, M.-T. Daniel, J.L. 25 Lin, P.S., A.G. Cooper, H.H. Wortis: Scanning· Preud'homme: Acute Leukaemia:with Burkitt's electron microscopy of human T-een and B-cell Tumor cells: A study of six cases with special rosettes. New Eng. 1. Med. 289 {1973) 548-551 reference to lymphocyte surface markers. Blood 26 Kay, M..M.., B. Belohradsky, K. Yee, J. Vogel, D. 45 {1975) 183-188 Butcher, J. Wybran, H.H. Fudenberg: Cellular in­ 13 McKenna, ~ W., J. Parkin, ~D. Brunning: Cytcr teractions: scanning electron microscopy of hu­ plasmic inclusions of parallel tubular arrays in man thymus-derived rosette-forming lymphocytes. the atypical lymphocytes of infe~ious mononu­ C1in. lmmunoL Immunopath. 2 {1974) 301-309 cleosis. Blood 48 {1976) 1003 {Abstract). 27 Catovsky, D., B. Frisch, Susan van Noorden: B, . 14 Brouet, J. C, G. Flandrin, M. Sasportes, J.L. T and "Null" cellleukaemias. Electron cytoche­ Preud'homme, M. Seligman: Chronic lymphocy­ mistry and surface morphology. Blood Cells 1 tic leukaemia of T -cell origin. An immunological {1975) 115-124 and clinical evaluation in eleven patients. I..ancet 28 Roath, S., D. G. Newell: Cell surface characteris­ 1975/11, 890-893 tics in hairy-cell leukaemia. Lancet 1975/1, 283- 15 Kersey, J., P. Coccia, C Bloomfield, M. Nesbit, 284 ~ McKenna, ~ Brunning, H. Hallgren, K. Gajl­ 29 Golomb, H.M., ~ Braylan, A. Polliack: 'Hairy, Peczalska: Surface markers deime human lymph­ cell leukaemia Oeukaemic reticulo-endotheliosis): oid malignancy with differing prognosis. In: Im­ a scanning electron microscopic study of eight munological diagnosis of leukaenlias and lymphcr cases. Brit. J. Haemat. 29 {1975) 455-460 mas. Eds. s.· Thierfelder, IL Rodt, E. ThieL Leh­ 30 Catovsky, D., B. Frisch, A. Okos, Susan van Noor­ manns Verlag, Munich (in press). den: Scanning electron microscopy and the nature 16 Zucker-Franklin, D.: Properties of the Sezary of the 'hairy' celL Lancet 1975/1 462-463 lymphoid celL An ultrastructural. analysis. Mayo 31 Schnitzer, B., M..L. Mead: Nature of the hairy celL Oin. Proc. 49 {1974) 567-574 Lancet 1975/1 805 17 Lutzner, M., R. Edelson, P. Schein, I. Green, C 32 Goldberg, A.F., T. Barka: Acid Phosphatase reac­ Kirkpatrick, A. Ahmed: CutaneoUs T-celllym­ tion in leukocytes. Nature 195 {1962) 297 phomas. The Sezary syndrome, mycosis fungoi­ 33 Pearse, A.G.E.: Histochemistry; theoretical and des, and related disorders. Ann. Int. Med. 83 applied, J. & A. Churchill, Ltd. London (VoL I (1976) 534-552 and ll), Third Edition 1968 18 Stein, H., N. Petersen, G. Gaedicke, K. Lennert, 34 Tamaoki, N., E. Essner: Distribution of acid phos­ G. Landbeck: Lymphoblastic lymphoma of con­ phatase, ~ucuronidase and N-acetyl~ucosami­ voluted or acid phosphatase type; - a tumor of nidase activities in lymphocytes of lymphatic tis­ T precursor cells. Int. J. Cancer 17 {197 6) 292- sues of man and ro~ents. J. Histochem. Cytochem.

295 I i7 {l969) 238-243 19 Sen, L., L. Borella: Oinical importance of lym­ 35 Wehinger, H., W. Mobius: Cytochemical studies phoblasts with T markers in childhood acute on T and B lymphocytes and lymphoblasts with leukemia. New Eng. J. Med. 292 (1975) 828- special reference to acid phosphatase. Acta hae­ 832 mat. 56 (1976) 129-136 20 Brouet, J.-C., F. Valensi, M.-T. Daniel, G. Flan­ 36 Ban-, ~D., S. Perry: Lysosomal acid hydrob.ses drin, J.-L Preud'homme, M. Seligmann: Immu­ in human lymphocyte subpopulations. Brit. J. nological classification of acute lymphoblastic Haemat. 32 {1976) 565-572 leukemias. Evaluation of its clinical significance 37 Li, C Y., LT. Yam, K. W. Lam: Acid phosphatase in a hundred patients. Brit. J. ~mat. 33 {1976) isoenzyme in human leukocytes in normal and 319-328 pathological conditions. J. Histochem. Cytochem. 18 (1970) 473-481 84 D. Catovsky, A. Enno

38 Douglas, S.D., G. Cohen, E. Konig, G. Brlttinger: 51 Pangalis, G.A., X. Yataganas, P. Fessas: pglucuronid­ Lymphocyte lysosomes and lysosomal enzymes ase activity on lymph node imprints of malignant in chronic lymphocytic leukaemia. Blood 41 lymphomas and chronic lymphocytic leukaemia (To (1973) 511-518 be published) 39 Catovsky; D., J. Galetto, A. Okos, E. Miliani, D. 52 Flandrin, G., M.-T. Daniel: /3-glucuronidase activity A. G. Galton: Cytochemical proflle ~d B and T in Sezary cells. Scand. J. Haemat. 12 (1974) 23-31 leukaemic lymphocytes with special reference to 53 Schmalzl, F., H. Braunsteiner: The cytochemistry acute lymphoblastic leukaemia. J. Clin. Path. 27 of monocytes and macrophages. Ser. HaematoL VoL (1974) 767-771 - m (2) 1970, 93-131 40 Ritter, J., G. Gaedicke, K. Winkler, H. Beckmann, S4 Mueller, J., G. Brun del Re, H. Buerki, H.-U. Keller, G. lAndbeck: Possible T-cell origin of lympho­ M. W. Hess, H. Cottier: Non-specific esterase activity: ' blasts in acid phosphatase-positive acute lympha­ a criterion for differentiation of T and B lympho- tic leukaemia. Lancet 197 S/II 7S cytes in mouse lymph nodes. Eur. J. ImmunoL 5 41 Flandrin, G., J. C Brouet: The sezary cell: cyto­ (1975) 270-274 logic, cytochemical and immunologic studies. 55 Kulenkampft. J., G. Janossy, M.F. Greaves: Acid Mayo Qin. Proc. 49 (1974) 575-583 esterase in human lymphoid cells and leukaemic 42 Catovsky, D.: (Unpublished observation) blasts. A marker for peripheral T lymphocytes. 43 Catovsky, D.: Morphological recognition of lym­ Brit. J. Haemat. 1977 phocytes and macrophages. In Recent Results in 56 Astald~ G., L Verga: The glycogen content of the Cancer Research, Eds. G. Mathe, L ·Florentin, M.­ cells of lymphatic leukaemia. Acta haemat. 17 C Simmler, Springer Verlag, Berlin 1976,56,1-7 (1957) 129-135 44 Li, C Y., LT. Yam, K. W. lAm: Studies of acid 57 Quaglino, D., G. Hayhoe: Observations on the phosphatase isoenzymes in human leukocytes: periodic acid Schiff reaction in lymphoprolifera­ demonstration of isoenzyme cell sp~cificity. J. tive diseases. J. Path. Bact. 78 (1959) 521-532 Histochem. Cytochem. 18 (1970) 9,01-910 58 Hayhoe, F.J., D. Quaglino, R. Doll: The cytology 45 Mover, S., CY. Li, L.T. Yam: Semiquantitative and cytochemistry of acute leukaemias. Her Ma­ evaluation of tartrate-resistant acid phosphatase jesty's Stationary Office, London 1964 activity in human blood cells. J. Lab. Clin. Med. 59 Schmalz/, F., H. Braunsteiner: The application of 80 (1972) 711-717 ' cytochemical methods to the study of acute leuk­ 46 Buskard, N.A., D. Catovsky, A. Okos, J.M. Gold­ emias. A review. Acta haemat. 45 (1971) 209-217 man, D.A. G. Galton: Prolymphocytic leukaemia: 60 Bennet, J.M., CE. Reed: Acute leukaemia cyto­ cell studies and treatment by leucopheresis. In: chemical prof.tle: diagnostic and clinical implica­ IDimatologie und Bluttransfusion. Ed. lL Loffler, tions. Blood Cells 1 (1975) 101-108 Lehmans Verlag, Miinchen 1976, !'8, 237-253 61 LO!fler, H.: Biochemical properties of leukaemic 4 7 LOfjler, H., M. Graubner, J.F. Desago: Pro lympho­ blast cells revealed by cytochemical methods: their cytic leukaemia with T-cell properties and tartrate­ relation to prognosis. In: Advances in the Bioscien­ resistant acid phosphatase. In: Immunological dia­ ces. Eds. T.M. Fliedner, S. Perry. Pergammon Press, gnosis of leukaemias and lymphomas. Ed. S. Thier­ Oxford 1975, 14, 163-173 felder, H. Rodt, E. ThieL Lehmanns Verlag, 62 Murphy, S., R. J. A. Aur, S.S. George, A. Mauer: Munich (in press) Relationship of pretreatment cytokinetic studies 48 Mann, J.R., J.S. Simpson, R.M. Munkley, J. Stuart: in 94 children with acute leukaemia to other ini­ Lysosomal enzyme cytochemistry in acute leuk­ tial prognostic variables and to outcome of treat­ aemia. J. Clin. Path. 24 (1971) 831-836 ment. American Society of Hematology Meeting, 49 Wasastjerna, C, E. Vuorlnen, M. Lehtinen, E. Ik­ Boston, USA, 1976, (Abstract) kala, E. Huh tala: The cytochemical p.g!ucuronid­ 63 Taswell, H.F., R.K. Winklemann: Sezary syndrome: ase reaction in the differential diagnosis of acute a malignant reticulemic erythredema. J. Ainer. leukaemias. Acta haemat. 53 (1975) 277-284 Med. Ass. 177 (1961) 465-473 50 Zittoun, R., M. Cadiou, C Dao, J. 1M. Blanc, J. Bousser: Lymphocytic beta-glucur~nidase in the lymphoproliferatl.ve syndromes. Biqmedicine 18 (1973) 415-420

D. Catovsky, M.R.C Leukaemia Unit, Royal Postgraduate Medical School, London W 12 OHS