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Gaucher's Cells 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 spleen and peripheral blood, 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 platelets 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 bone marrow 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 blood cell 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 Nr 4K*4. ,257k. copyright. http://jcp.bmj.com/ on September 30, 2021 by guest. Protected t. 1-,2! ,. ik 9,-- 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. 4 copyright. 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).
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