J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

J. clin. Path., 1977, 30, 981-988

Surface marker and other characteristics of Gaucher's cells

G. F. BURNS, J. C. CAWLEY, R. J. FLEMANS, K. E. HIGGY, C. P. WORMAN, C. R. BARKER, B. E. ROBERTS, AND F. G. J. HAYHOE From the Department of Haematological Medicine, University of Cambridge and Department of Medicine, University ofLeeds, UK

SUMMARY A full surface marker study of the splenic storage cells in a case of Gaucher's disease largely substantiates the monocyte/histiocyte nature of Gaucher's cells. In addition, an apparent T-lymphocyte deficiency is demonstrated in the and peripheral , and the possible significance of this finding is discussed.

Gaucher's cells have been extensively investigated survey showed early, but definite, Ehrlenmeyer-flask morphologically and biochemically, and it is now deformity of the femora. There was a peripheral thought that they represent histiocytic cells in which pancytopenia (Hb 10-7 g/dl, WBC 3'8 x 109/l, large amounts of glucocerebroside have accumulated 60 x 109/l) and differential white count because of a deficiency of ,B-glucosidase (Brady, showed 44% neutrophils, 49% lymphocytes, and nature of Gaucher's cells 7 % monocytes. A aspirate was hyper- 1972). The histiocytic copyright. has been inferred from their phagocytic ability (Lee cellular and contained moderate numbers of typical et al., 1967), high lysosomal enzyme content Gaucher's cells; the cytology and cytochemistry of (Rozenszajn and Efrati, 1961), and distinctively these are described in detail below. A diagnosis of reticuloendothelial distribution. However, this view non-neuropathic Gaucher's disease with hyper- has not been universally accepted (Roos et al., 1961; splenism was made. Fisher and Reidbord, 1962) and, in addition, The only other sib, a younger sister aged 54 years, immunological mechanisms have been invoked in also has hepatosplenomegaly with peripheral the pathogenesis of the disease (Pennelli et al., 1969). pancytopenia and high serum acid phosphatase and http://jcp.bmj.com/ Immunological surface marker studies, which have almost certainly has Gaucher's disease. However, proved of great value in the characterisation of the since she is asymptomatic a bone marrow aspirate pathological cells of a wide variety of other haemato- has not been performed to confirm the diagnosis. logical proliferations, should therefore contribute to There was no other family history of Gaucher's further understanding of the nature of the pathog- disease. nomonic cells of Gaucher's disease. Because of the patient's thrombocytopenic

No previous surface-marker study of Gaucher's bleeding attributable to hypersplenism, splenectomy on September 30, 2021 by guest. Protected disease has been described, and in the present paper was performed in December 1976 and this splenic we report such a study in a case of the non-neurono- material formed the basis of our study. Postopera- pathic form ofthe disease. tively, the patient has done well, and the pancyto- penia has disappeared with cessation of the thrombo- Patient and methods cytopenic epistaxes.

CASE HISTORY CYTOLOGY AND CYTOCHEMISTRY This 8-year-old girl of Anglo-Saxon descent presen- Bone marrow and splenic impression smears were ted with recurrent epistaxes. Examination revealed examined by a variety of routine methods including massive splenomegaly and moderate hepatomegaly polarised and ultraviolet microscopy, Romanowsky, but no otherabnormality.Theserumacidphosphatase PAS, acid and alkaline phosphatase, iron, peroxidase, was greatly increased at 17-2 KA units, and skeletal Sudan black, a-naphthyl butyrate esterase with sodium fluoride inhibition, and naphthol AS-D chloroacetate esterase stains. Bone marrow and Received for publication 7 March 1977 splenic material, fixed and processed in the manner 981 J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

982 G. F. Burns et al. described below, were also examined by electron used as an Fc control. microscopy. EAIgM-rosettes PREPARATION OF CELL SUSPENSIONS Ox RBCs coated with rabbit anti-ox RBC IgM antibody (EAIgM) were used by a method previously Splenic cells described by us (Barker et al., 1976; Burns et al., A single cell suspension was obtained by forcing 1977). This test also served as a control for the C3 several pieces from different areas of freshly removed rosettes. spleen through a 120 gauge stainless-steel mesh into a Hepes-buffered Hanks balanced salt solution C3-rosettes (HBSS). The cell suspension was kept on ice until (a) C3bmo and C3dmol For C3bmo indicator cells, its return to the laboratory where the cells were IgM-coated ox RBCs (0 5 ml) were pre-warmed to washed in HBSS, and mononuclear cells were 37°C and an equal volume of whole AKR mouse separated by centrifugation over Ficoll-Hypaque serum (1/10 dilution) was added. These were mixed gradients (Boyum, 1968). The cells were washed a for 70 s at 37°C and 5 jil Antrypol(R) (50 mg/ml), further three times in HBSS before being resus- which stabilises C3b against the action of KAF, pended in HBSS + 0-2% bovine serum albumin was added and thoroughly mixed for 2 min (Lach- (BSA) to a final concentration of 2 x 106 cells/mi. mann etal., 1973). For comparison, suspensions from a 'normal' As a source of C3dmo-coated cells, the reaction spleen (removed during gastrectomy for carcinoma was allowed to proceed at 37°C without added of the stomach) and the spleen from a patient with Antrypol for 30 min during which time most C3b hereditary spherocytosis (HS) were prepared in a is converted to C3d by KAF. similar way. (b) C3bhu and C3dhu2 These two types of indicator RBC were prepared in an identical way except that Peripheral blood cells human serum previously exposed to zymosan (R3

The patient's peripheral blood was examined on reagent) was used as the source of complementcopyright. two occasions-initially, five days after splenectomy (McConnell and Hurd, 1976). and, on a second occasion, 35 days post-splenectomy. All the C3 rosette tests were performed by the On the latter occasion, the sister's blood was also method of Ross et al. (1973). examined. In each case, the blood was collected into heparin and the mononuclear cells were separated in Mouse (M-) rosettes the manner described above. The method of Gupta et al. (1976), using fresh, In both the splenic and the peripheral washed, unsensitised CBA mouse erythrocytes was suspensions viability was assessed by the cells' employed. http://jcp.bmj.com/ ability to convert fluorescein diacetate to fluorescein In all rosette tests, the indicator RBCs were (Celada and Rotman, 1967). Viability was greater washed three times and resuspended to 1% in than 95 % in the peripheral blood, HS, and normal HBSS + 0-2 % BSA before testing, and percentages cell preparations. However, in the suspension from of rosetting cells were assessed using the fluorescein the Gaucher's spleen preparation the overall viability diacetate method of Ramasamy (1974), scoring only was 80%, while the Gaucher's cells, which consti- the viable fluorescing cells under combined UV and tuted approximately 30 % ofthis preparation, showed phase contrast microscopy. on September 30, 2021 by guest. Protected some 50% viability, dead Gaucher's cells accounting for the great majority of the non-viable cells. SURFACE IMMUNOGLOBULIN (SMIG) An indirect method was employed which uses ROSETTE TESTS polyvalent rabbit anti-human gammaglobulin raised in the laboratory followed by fluoresceinated goat E-rosettes anti-rabbit IgG (Gibco Bio-Cult Ltd, Paisley, Sheep erythrocyte rosette tests (E) were carried out Scotland). using aminoethylisothiouronium bromide-treated erythrocytes according to the method of Kaplan and Clark (1974). 1C3bmo is the bound fragment of activated mouse complement component C3, and C3dmo is the residual Fc-rosettes fragment of the inactivation of C3b by conglutinogen The method of Hallberg et al. (1973), using ox RBCs activating factor (KAF). sensitised with rabbit anti-ox RBC IgG antibody, 2C3bhu and C3dhu are the corresponding human com- was employed. Unsensitised washed ox RBCs were plement components. J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

Surface marker and other characteristics of Gaucher's cells 983

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Fig. 1 EM ofGaucher spleen ( x 8500). A matureplasma cell (P) is seen to be intimately associated with two typical Gaucher's cells (G). J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

984 G. F. Burns et al.

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Fig. 2 Fc-rosettespreparation ofsplenic Gaucher's cellsuspension. The ability ofthe Gaucher cell toform Fc-rosettes is well shown at the level ofboth the light (inset) andelectron microscope. In the electron micrograph ( x 12 000) the

Gaucher celldisplays the imperfectfixation not infrequently observed in rosettepreparations: this ispresumably attributable http://jcp.bmj.com/ to handling beforefixation and wasparticularly marked in the case ofthe Gaucher cells. The cytocentrifugepreparation (inset) shows the strong Fc-rosette typical ofthe intact Gaucher cells. The associated plasmacytoid cell on the left is also clearly seen to beforming an Fc-rosette.

ELECTRON MICROSCOPY (EM) The cells showed weaker alkaline phosphatase, iron, Bulk preparations of rosettes were formed by and naphthol AS-D chloroacetate esterase reactivity allowing 1 ml of the splenic cell suspension at 2 x and were peroxidase and Sudan black negative. on September 30, 2021 by guest. Protected 106/ml to react with 1 ml indicator erythrocytes. Under polarised light the striatedinclusionsshowed After centrifugation, the pellets were fixed in 3% the form birefringence associated with some lipids glutaraldehyde and processed for EM by routine (Bennett, 1950); in contrast, we have noted that the methods (Cawley and Hayhoe, 1973). inclusions of sea-blue histiocytosis are not bire- fringent (R. J. Flemans; unpublished observation). Results Under UV illumination, the Gaucher's cells did not autofluoresce, again in contrast to both the storage CYTOLOGY AND CYTOCHEMISTRY cells of sea-blue histiocytosis and many bone Both by light and electron microscopy, the large marrow reticuloendothelial cells in a variety of non- storage cells contained the striated inclusions which storage conditions which fluoresce a strong pink characterise Gaucher's disease (Figs 1 and 2). The (R. J. Flemans; unpublished observations). cytochemistry was also typical of Gaucher's disease in showing strong PAS, acid phosphatase, and ROSETTE TESTS f3-naphthyl butyrate esterase activity (Fig. 3a), The results of the various splenic-cell rosette tests which was partially inhibited by sodium fluoride. are summarised in Table 1. It is seen that a high J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

Surface marker and other characteristics of Gaucher's cells 985 copyright.

Fig. 3 Gaucher's cells. (a) a-naphthyl butyrate esterasepreparation. The strongpositivity ofthe Gaucher's cell is well shown. (b) E-rosette preparation; phase contrast, illumination. This positive rosette is typical in showing relativelyfew adherent sheep RBCs. (c) Fc-rosetteformation; phase contrast illumination. Thiis type ofstrong rosette with many adherent cells is typical. (d) Mouse erythrocyte-rosette formation; phase contrast illumination. The moderate number of adherent mouse RBCs in this example is typical. As is discussed in the text, this type ofrosette, although definitely http://jcp.bmj.com/ positive, tended to be less strong than the Fc rosette. percentage of Gaucher's cells formed rosettes with The mouse rosettes were of intermediate appearance, unsensitised sheep (E) RBCs (Fig. 3b), and with ox and this may indicate intermediate receptor density. indicator cells coated with either the Fc of IgG It should be stressed that only viablecells were scored (Figs 2 and 3c) or with human C3 (both C3b and in all these rosette tests. C3d). A high percentage of the Gaucher's cells also The percentage total E- spleen cells forming on September 30, 2021 by guest. Protected formed rosettes with unsensitised mouse (M) rosettes in the Gaucher's preparation was remark- erythrocytes (Fig. 3d), while a much lower per- ably low: indeed, none of the many small lympho- centage formed rosettes with ox indicator cells cytes present was seen to form E-rosettes. This was coated with mouse C3d. No rosette formation was in marked contrast to the findings in the two other seen with C3bmo-coated indicator cells. The Fc examined (Table 1). control (unsensitised ox RBCs) and the C3 control The results of our studies in the T- and B-cell (EAIgM) were also completely negative. content of peripheral blood are summarised in As is illustrated in Fig. 3 (b-d) the number of Table 2. In both the patient and her sister, the adherent indicator cells varied in the different percentage of peripheral blood T-cells appeared rosette tests. Thus, in the Fc and C3hu (not illus- markedly reduced, whether estimated by E-rosette trated) rosettes, the large number of adherent RBCs formation or by an esterase method recently probably indicates high receptor density. In contrast, described in this laboratory (Higgy et al., 1977). most Gaucher's cells which formed rosettes with In both the whole spleen tissue and the splenic sheep RBCs had only four to five adherent RBCs, cell suspension, the Gaucher's cells were frequently although a number had more than 10 adherent cells. found to be closely associated with one or more J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

986 G. F. Burns et al. Table I Rosetting properties ofsplenic cell suspensions in keeping with a monocyte/histiocyte nature. The presence of a high density of receptor for human C3 Rosette test % Rosette formation by viable splenic leucocytes (both C3b and C3d) is again in accord with a monocytic/histiocytic origin of the Gaucher cell Gaucher's disease 'Normal' HS (Ross and Polley, 1975). The failure to form C3bmo Total Gaucher's rosettes, and only a low percentage of rosette cells cells formation with C3dmo, is difficult to interpret but E 5 54 51 45 may reflect either a lower density of C3 products on Fc 71 59 34 35 those indicator cells or the ability of the Gaucher's EAlgM 0 0 5 4 cells to distinguish between human and mouse C3. C3bmo 0 0 ND ND C3moC The ability of Gaucher's cells to form rosettes with C3dmo 32 8 ND I sheep erythrocytes is also difficult to interpret. C3bhu 68 60 ND ND C3hu Normally, E-rosettes formed by T-lymphocytes of C3dhu 58 34 ND ND less than 10 ,t diameter consist of a tight morula M 7 53 2 ND made up of several layers of indicator cells. In ND = not done contrast, the much larger Gaucher's cells (> 40 IL diameter) usually displayed only relatively few Table 2 Peripheral blood rosetting data adherent cells sparsely distributed over the cell Patient % Rosetteformation Esterase staining surface. Although it is possible that Gaucher's cells by mononuclear cells possess a receptor similar to that of T-lymphocytes, E Fc T-cell B-cell in view of the other characteristics of the cells, it is difficult to imagine that the formation of rosettes Propositus 5 days with sheep RBCs is a true expression of a T-lympho- post-splenectomy 24 44 26 71 Propositus 35 days cyte phenotype. Histiocytes are known to be capable post-splenectomy 40 58 45 34 of recognising and removing effete erythrocytes, and Sister with Gaucher's disease 53 45 54 35 the rosettes seen with sheep RBCs may be related tocopyright. Normal 7-year-old girl 71 24 ND ND this process. Some support is lent to this inter- pretation by our observation of weak E-res-tte ND = not done formation by the storage cells of a single case of sea-blue histiocytosis (Burns, unpublished obser- plasmacytoid cells (Figs 1 and 2 inset). These vations). Because the mouse rosette test is generally associated plasmacytoid cells formed strong Fc considered to identify specifically a subpopulation (Fig. 2 inset), and mouse, but no C3 or E rosettes. of B-lymphocytes (Forbes and Zalewski, 1976) and the leukaemic cells in CLL (Stathopoulos and http://jcp.bmj.com/ SURFACE IMMUNOGLOBULIN Elliott, 1974), the reactivity of the Gaucher's cell Whereas some small lymphocytes in the splenic cell may also be attributed to the adherence of effete preparation were positive for Smig, both the RBCs. However, in our hands, some normal mono- Gaucher's cells and the associated plasmacytoid cytes and neutrophils form M-rosettes, and thus the cells were uniformly negative for SmIg. ability of Gaucher's cells to form these rosettes may be fully compatible with a monocyte-histiocyte

Discussion lineage. on September 30, 2021 by guest. Protected In view of the immunological abnormalities that The morphology of Gaucher's cells is so distorted may be associated with Gaucher's disease (Pratt et by the accumulated glucocerebroside that firm al., 1968), the severe deficiency of splenic T-cells conclusions about the basic nature of the cell are not demonstrated by the strikingly low percentage of possible at the level of either the light or electron E-rosette formation by the numerous lymphocytes microscopes. In addition, unlike typical marrow present in the patient's spleen is of interest. This histiocytes, the Gaucher's cells do not autofluoresce deficiency is clearly not attributable to sampling under UV light. However, our cytochemical studies, error since our splenic suspension was prepared from like those of others (Rozenszajn and Efrati, 1961), several pieces of tissue from widely different areas are consistent with the assumed monocytic origin of of the spleen. This unexpected finding prompted us these cells; in this regard, the a-naphthyl butyrate to examine the T-cell content of the patient's esterase positivity, inhibited by sodium fluoride, is peripheral blood. Interestingly, both the patient and particularly suggestive of a monocytic origin. her sister displayed a definite reduction in the Our demonstration of strong Fc-rosette formation percentage of circulating T-cells. These results by Gaucher's cells, in the absence of Smlg, is also cannot be simply attributed to a defect in our J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

Surface marker and other characteristics of Gaucher's cells 987 E-rosette technique because it yielded an entirely F. G. J. (1976). IgM receptors on the surface of hairy normal result in a control child and because similar cells of leukaemic reticuloendotheliosis (Letter). results were obtained by a completely different Lancet, 1, 1303. method employing esterase cytochemistry (Higgy Bennett, H. S. (1950). The microscopical investigation of et al., 1977). biological materials with polarized light. In McClung's Furthermore, the detectionofT-lympho- Handbook of Microscopical Technique, 3rd edition, penia in the sister argues against the finding in this edited by R. McClung Jones, pp. 591-677. Hoeber, patient being simply a response to the operation New York. and/or its attendant anaesthetic. The possibility that Boyum, A. (1968). A one-stage procedure for isolation the T-lymphopenia represents a manifestation of of and lymphocytes from human blood. hypersplenism or an unrelated chance association Scandinavian Journal of Clinical and Laboratory Inves- cannot, however, be excluded. tigation, 21, Supplement 97, 51-76. The association between plasma cells and both Brady, R. 0. (1972). Biochemical and metabolic basis of normal reticuloendothelial cells and Gaucher's cells familial sphingolipidoses. Seminars in Haematology, is well recognised, and the latter observation is 9,273-284. confirmed in the Burns, G. F., Cawley, J. C, Barker, C. R., Goldstone, present study. However, since the A. H., and Hayhoe, F. G. J. (1977). New evidence re- associated cells closely resembled mature plasma lating to the nature and origin of the hairy cell of cells morphologically, and lacked SmIg, the presence leukaemic reticuloendotheliosis. British Journal of of an Fc receptor on their surface is an unusual Haematology, 36, 71-84. finding since plasma cells usually lack the Fc Cawley, J. C. and Hayhoe, F. G. J. (1973). Ultrastructure receptor (McConnell, 1975). We are unable to offer ofHaemic Cells. W. B. Saunders, London. any definitive explanation for this finding. However, Celada, F. and Rotman, B. (1967). A fluorochromatic it has been suggested that plasma cells actively test for immunocytotoxicity against tumor cells and secreting immunoglobulin lack the Fc leucocytes in agarose plates. Proceedings ofthe National receptor Academy ofSciences, 57, 630-636. while inactive plasma cells possess this marker Fisher, E. R. and Reidbord, H. (1962). Gaucher's disease: (Ramasamy et al., 1974); the Gaucher-associated pathogenetic considerations based on electron micro- plasma cells may therefore be inactive cells. scopic and histochemicalobservations. AmericanJournal copyright. To conclude, the present marker study largely ofPathology, 41, 679-691. substantiates the assumed relationship between Forbes, I. J. and Zalewski, P. D. (1976). A subpopulation Gaucher's cells and cells of the monocyte/histiocyte of human B lymphocytes that rosette with mouse series. However, some possible differences between erythrocytes. Clinical and Experimental Immunology, Gaucher's and monocytoid cells were observed, but 26,99-107. their significance awaits further investigations of Gupta, S., Good, R. A., and Siegal, F. P. (1976). Rosette- both more cases of Gaucher's disease and the formation with mouse erythrocytes. II. A marker for human B and non-T lymphocytes. Clinical andExperi- http://jcp.bmj.com/ surface marker properties of the reticuloendothelial mental Immunology, 25, 319-327. cells of normal spleen, for which no data are Hallberg, T., Gurner, B. W., and Coombs, R. R. A. currently available. Moreover, the demonstration (1973). Opsonic adherence of sensitized ox red cells to of an apparent T-cell deficiency in the present human lymphocytes as measured by rosette formation. patient's spleen and peripheral blood suggests that International Archives ofAllergy, 44, 500-513. examination of T-cell function in further cases of Higgy, K. E., Burns, G. F., and Hayhoe, F. G. J. (1977). Gaucher's disease should be undertaken. Discrimination of B, T and null lymphocytes by

esterase cytochemistry. Scandinavian Journal of on September 30, 2021 by guest. Protected It is a pleasure to the Haematology, 18, 437-448. acknowledge help and coopera- Kaplan, M. E. and Clark, C. (1974). An improved tion of Professor R. W. Smithells and Mr J. M. rosetting assay for detection of human T lymphocytes. Beck, of Leeds General Infirmary, and Dr R. J. M. Journal ofImmunological Methods, 5, 131-135. Bell, of Scunthorpe General Hospital, all of whom Lachmann, P. J., Hobart, M. H., and Aston, W. P. assisted us in studying this family under their care. (1973). Complement technology. In Handbook of We are grateful to Mr G. Binns for technical Experimental Immunology, 2nd edition, edited by assistance. D. M. Weir. Blackwell, Oxford. GFB and KEH gratefully acknowledge support Lee, R. E., Balcerzak, S. P., and Westerman, M. P. from the Medical Research Council (UK) and the (1967). Gaucher's disease. A morphologic study and of the measurements of iron metabolism. American Journal of government United Arab Republic of Egypt Medicine, 42, 891-898. respectively. McConnell, I. (1975). T and B lymphocytes. In The Immune System, edited by M. J. Hobart and I. Mc- References Connell, p. 98. Blackwell, Oxford. McConnell, I. and Hurd, C. M. (1976). Lymphocyte Barker, C. R., Bums, G. F., Cawley, J. C., and Hayhoe, receptors. It. Receptors for rabbit IgM on human T J Clin Pathol: first published as 10.1136/jcp.30.10.981 on 1 October 1977. Downloaded from

988 G. F. Burns et al. lymphocytes. Immunology, 30, 835-839. Ross, G. D., Rabellino, E. M., Polley, M. J., and Grey, Pennelli, N., Scaravilli, F., and Zacchello, F. (1969). H. M. (1973). Combined studies of complement receptor Th- morphogenesis of Gaucher cells investigated by and surface immunoglobulin-bearing cells and sheep electron microscopy. Blood, 34, 331-347. erythrocyte rosette-forming cells in normal and Pratt, P. W., Estren, S., and Kochwa, S. (1968). Immuno- leukemic human lymphocytes. Journal of Clinical globulin abnormalities in Gaucher's disease. Report Investigation, 52, 377-385. of 16 cases. Blood, 31, 633-640. Ross, G. D. and Polley, M. J. (1975). Specificity of Ramasamy, R. (1974). A fluorescent stain for viable human lymphocyte complement receptors. Journal of rosette-forming cells. JoiurnalofImmunological Methods, Experimental Medicine, 141, 1163-1180. 5, 305-306. Rozenszajn, L. and Efrati, P. (1961). Cytochemical and Ramasamy, R., Munro, A., and Milstein, C. (1974). phase-contrast observations on Gaucher cells. Acta Possible role for the Fc receptor on B lymphocytes. Haematologica, 25, 43-48. Nature, 249, 573-574. Stathopoulos, G. and Elliott, E. V. (1974). Formation of Roos, B., Cottier, H., and Rossi, E. (1961). Electron- mouse or sheep red-blood-cell rosettes by lymphocytes optical observations in storage diseases (Abstract). from normal and leukaemic individuals. Lancet, 1, Experientia, 17, 430. 600-601. copyright. http://jcp.bmj.com/ on September 30, 2021 by guest. Protected