Clin. exp. Immunol. (1988) 72, 351-356 An analysis of myeloma plasma cell phenotype using antibodies defined at the IIrd international workshop on human leucocyte differentiation antigens N. JACKSON, N.R. LING,* JENNIFER BALL, ELAINE BROMIDGE, P.D. NATHAN* & I.M. FRANKLIN Departments of Haematology and *Immunology, The Medical School, Birmingham (Acceptedfor publication 12 January 1988) SUMMARY Fresh bone marrow from 43 cases of myeloma and three cases of plasma cell leukaemia has been phenotyped both by indirect immune-rosetting and, on fixed cytospin preparations, by indirect immunofluorescence. Both clustered and unclustered B cell associated antibodies from the IIrd International Workshop on Human Leucocyte Differentiation Antigens were used. The results confirm the lack of many pan-B antigens on the surface of myeloma plasma cells, i.e. CD19-23, 37, 39, w40. Strong surface reactivity is seen with CD38 antibodies and with one CD24 antibody (HB8). Weak reactions are sometimes obtained with CD9, 10 and 45R. On cytospin preparations CD37, 39 and w40 are sometimes weakly positive, and anti-rough endoplasmic reticulum antibodies are always strongly positive. Specific and surface-reacting antiplasma cell antibodies are still lacking. Keywords myeloma phenotype antibody cluster INTRODUCTION antibodies from CD24 (HB8 and VIB-E3). Before and after the IIrd workshop, we carried out a phenotypic analysis of fresh Multiple myeloma (MM) is a neoplastic disorder of the B cell MM bone marrow cells using both clustered and unclustered lineage. It is characterized by an excess ofneoplastic plasma cells monoclonal antibodies (MoAb) supplied for the workshop, in the bone marrow, although the nature of the clonogenic cell including one from each ofthe new and the previously defined B remains uncertain. On terminal differentiation to plasma cells, cell associated clusters. Some of these results were presented at cells of the B lineage cease to express many characteristic B cell the Workshop (Nathan et al., 1987). antigens. These include antigens detected by antibodies of clusters CD19-23 as defined at the 1Ind International Work- shop on Human Leucocyte Differentiation Antigens (Nadler, METHODS AND PATIENTS 1986) and surface immunoglobulin (sIg) (Katagiri et al., 1984; Foon, Schroff & Gale, 1982). The majority of plasma cells also Patients lack major histocompatibility complex (MHC) class II antigens Forty-six patients who fulfilled the Medical Research Council (Wu et al., 1976; Kuritani & Cooper 1982; Katagiri et al., 1984), criteria for entry into the IVth Myelomatosis trial (Medical and Fc and C3 receptors (Stashenko et al., 1980). Recently, Research Council, 1985) were studied, including three with some cases of myeloma have been found to express CDIO plasma cell leukaemia (i.e. peripheral blood plasma cell count (CALLA; common acute lymphoblastic leukaemia antigen) on .2 x 109/1). Their paraprotein type and treatment status are some of the cells in blood and bone marrow (Ruiz-Arguelles et shown in Table 1. al., 1984; Durie & Grogan, 1985; Caligaris-Cappio et al., 1985). The I1Ird International Workshop (McMichael et al., 1987) Surface antigen detection: indirect immune-rosetting defined five new B cell associated antibody clusters (CD 37-w40 Indirect immune-rosetting was used for surface antigen detec- and 45R), and also included studies on other antibodies which tion, a technique well established in this laboratory (Ling, were not clustered but which were found to react with plasma Bishop & Jefferis, 1977; Kalland, 1977). The MoAb, as listed in cells. Plasma cell reactivity was tested mainly against plasmacy- Table 2, were diluted (1 Ml in 50 pi of phosphate-buffered saline toid cell lines (JVM-3, U-266, HFB-1, RPMI-8226). Cells from (PBS) containing 10% fetal calf serum (FCS) and 0-1 % sodium only two cases of MM were investigated and they showed azide. An anti-idiotype MoAb generated against an unrelated moderate reactivity with antibodies of CD38, CD39 and two myeloma paraprotein was used as a control. Then, 2 5 x I05 cells to be analysed were added (in 5-20 p1 of culture medium) and Correspondence: Dr N. Jackson, Department of Haematology, incubated at 4°C for 30 min. After washing twice in Hepes- Royal Free Hospital, Pond Street, London NW3 2QG, UK. buffered RPMI 1640 medium with 2% FCS, the cells were mixed 351 352 N. Jackson et al. with approximately 10 x 106 sheep erythrocytes coated with a Table 1. Patient characteristics with their paraprotein sheep anti-mouse immunoglobulin in 150 p1 of the same types medium. This mixture was left to stand at room temperature for 10 min, centrifuged at 100 g for 30 s, and left to stand for a Myeloma Plasma cell leukaemia further 10 min. One drop of Acridine Orange (18 75 pg/ml) was added and after gently resuspending the cells, one drop was Paraprotein Untreated Treated Untreated spread under a coverslip on a multi-spot Polytetrafluoroethy- lene-coated slide. The percentage of rosetted non-myeloid cells IgG 15 11 1 was enumerated by counting the number of white cells with at IgA 4 7 2 least four SRBC attached out of a total of 100 or 200 cells. A Light chain 1 5 fluorescence microscope (Leitz dialux, Leitz Instruments, Bed- only ford UK) was used, set for Fluorescein excitation by epi- illumination to visualize the fluorescing nuclei, with slight background illumination with ordinary light to visualize the Cytoplasmic antigen detection: indirect immunofluorescence erythrocytes. As Acridine Orange makes myeloid cell cytoplasm Approximately 1-2 x 105 cells were cytospun onto slides, and fluoresce red under these circumstances, these cells were elimi- fixed in acetone for 10 min. The slide was first incubated with 1 nated from the counting in order to concentrate on the plasma p1 ofMoAb in 50 pi PBS with 10% FCS at room temperature for cells, which were also recognized by their characteristic morpho- 30 min, and then washed for 30 min in PBS. The slide was then logy. However, this led to some other non-myeloid cells being incubated with a I-in-100 dilution of fluorescein-conjugated counted, e.g. lymphocytes. Hence, small percentage values sheep anti-mouse Ig (prepared in this department by Dr G. D. (< 10%) were regarded as negative; counts between 10-30% as Johnson) for 30 min, and washed in PBS for 30 min. The slides weak positivity of plasma cells with that antibody; counts were then dipped in propidium iodide (1 pg/ml) for 5 min for > 30% as definite positivity with that antibody. nuclear counterstaining, and mounted using polyvinyl alcohol Table 2. Antibodies used, their cluster designation (CD) where applicable, and the sources from which they were obtained Antibody CD Source control An anti-idiotype MoAb produced in the Immunology lab, Birmingham University BU 16 9 Immunology lab, Birmingham University Anti-CALLA 10 Becton-Dickinson* BU12 19 Immunology lab, Birmingham University BCI 20 Gallart, Barcelona BA5 21 Le Bien, Minnesota 29-110 22 Kraft, Melbourne MHM6 23 McMichael, Oxford ALB9 24 Boucheix, Villejuif, France HB8 24 Cooper, Alabama HB9 24 Cooper, Alabama VIB C5 24 Knapp, Vienna VIB E3 24 Knapp, Vienna ALIa 24 Ravoet, Brussels LC66 24 Ravoet, Brussels CLB/granBly I 24 Tetteroo, Amsterdam Anti-IL2 receptor 25 Becton-Dickinson* WR-17 37 Moore, Southampton OKTIO 38 American Type Culture Collection HB7 38 Tedder, Boston T16 38 Bourel, Rennes, France T168 38 Bourel, Rennes, France AC2 39 Rowe, Birmingham G28.5 w40 Ledbetter, Seattle 4KB5 45R Pulford, Oxford PCA-l Nadler, Boston BU25 Anti-MHC class II Immunology lab, Birmingham University KB61 Pulford, Oxford 7F7 Schulz, Innsbruck BU 11,18 Anti-RER Immunology lab, Birmingham University RFD6 Anti-RER Janossy, London * Purified antibody; others were ascitic fluid. Myeloma phenotype 353 WeaNs | W*-90r-.ve positive 1_ Definitely positiVe CD 10 20 30 40 50 60 70 80 90 100 _~~~,.~~ I 9 V 10 ,i Dea so l 19 20 I 21 I t 22 la I 23 I 24 (HB8) 24 (except HB8) s 25 o3 , 37 38 39 w40 it 45R Fig. 1. Surface reactivity ofmyeloma plasma cells with clustered antibodies: percentage ofpositive cells by indirect immune-rosetting. (0) Untreated; (0) treated; CD, cluster designation. with 1, 4-diazobicyclo-(2,2,2)-octane (Johnson et al., 1982). The Table 3. Cytoplasmic reactivity of myeloma slides were examined using a fluorescence microscope set for plasma cells with clustered antibodies fluorescein excitation (fluorescein, green; propidium iodide, red), and results graded as negative, weak, moderate, or strong. Cytoplasmic reactivity This method will also stain surface antigens that are not destroyed by acetone fixation. CD No. Negative Weak Moderate Strong 9 1 8 I RESULTS 10 8 2 19 6 4 20 8 Reactivity ofmyeloma plasma cells with clustered antibodies 21 6 Bone marrow mononuclear cells from 46 patients have been 22 9 1 analysed with various combinations of antibodies. The 16 most 23 4 2 recent samples have been tested against the full spread of B cell 24 (HB8) 6 6 2 24 (other) 6 antibody clusters, as defined by the IlIrd International Work- 25 12 shop. The results for antibodies from these clusters are shown in 37 7 1 Fig. 1 (surface typing), and Table 3 (cytoplasmic typing). In the 38 6 11 13 majority of cases the plasma cells were HB8+, but most other 39 5 3 antibodies from CD24 gave negative results. Most cases (26/36) 40 3 3 I were CD38 (OKT10)+, but some were negative by indirect resetting. In eight out of the 10 CD38- cases, the pattern of CD, cluster designation. positivity ofthe immunofluorescence on the cytospins suggested that the cells were in fact surface CD38+. One case was CDl0+ CDl9+, CD20+, and CD23+, and occasional weak surface Reactivity ofmyeloma plasma cells with unclustered antibodies reactivity was also obtained with CD9 (three cases), CD25 (two The results using unclustered antibodies are shown in Fig.