49047 Clin Pathol 1997;50:490-493 CD3 1 JC70) expression in plasma cells: an immunohistochemical analysis of reactive and neoplastic plasma cells J Clin Pathol: first published as 10.1136/jcp.50.6.490 on 1 June 1997. Downloaded from Dhirendra Govender, Pranitha Harilal, Mahomed Dada, Runjan Chetty Abstract that in fixed tissue sections JC70 staining is Aims-To investigate the immunohisto- restricted to endothelial cells and occasional chemical expression of CD31 (JC70) in bone marrow plasma cells.2 normal and neoplastic plasma cells. Although several cell adhesion molecules Methods-Plasma cells in bone marrow have been identified in normal and neoplastic biopsies and extramedullary locations plasma cells, CD31 expression has not previ- were examined. All extramedullary biop- ously been reported in plasma cells in formalin sies were formalin fixed and paraffin fixed, paraffin embedded tissues outside of the embedded. The bone marrow biopsies bone marrow. were fixed in formal acetic acid and We noticed during diagnostic use that embedded in paraffin wax. Twenty multi- reactive (polyclonal) plasma cells showed ple myelomas (12 bone marrow and eight intense membrane staining with the anti-CD3 1 extramedullary deposits), 10 extramedul- antibody, JC70. This prompted an immuno- lary plasmacytomas, and 30 biopsies with histochemical study of CD3 1 expression in reactive plasma cells (10 bone marrow, 20 reactive and neoplastic plasma cells. extramedullary biopsies) were stained with anti-CD31 (JC70) using the streptavidin-biotin detection system with Methods diaminobenzidine as a chromogen. Anti- The study consisted of a cohort of 60 cases gen retrieval in bone marrow biopsies was which comprised 20 cases of multiple myel- achieved by pressure cooking. In all other oma, 10 cases of plasmacytoma, and 30 cases biopsies, antigen retrieval was achieved by with reactive plasma cells. Conditions in which microwave pretreatment. reactive plasma cells were encountered in- cluded Castleman's disease, Hodgkin's disease, Results-All 20 extramedullary cases with http://jcp.bmj.com/ reactive plasma cells showed intense and mixed chronic inflammation. membrane staining. Focal staining was Formalin fixed, paraffin wax embedded detected in reactive plasma cells in bone tissue blocks of cases with reactive and marrow biopsies. Five of 10 plasmacyto- neoplastic plasma cells were obtained from the mas showed membrane staining. None of archives of the department of anatomical the cases of multiple myeloma, either pathology, University ofNatal Medical School. These consisted ofeight extramedullary depos- medullary or extramedullary, showed any on September 26, 2021 by guest. Protected copyright. immunoreactivity for CD31. its of multiple myeloma, 10 plasmacytomas, Conclusions-CD31, a member of the and 20 cases with reactive plasma cells. There immunoglobulin supergene family of cell were biopsies from various extramedullary Department of adhesion molecules, is strongly expressed sites, including soft tissue abscesses, granula- Anatomical Pathology, tion tissue, reactive lymph nodes, perivascular University of Natal in extramedullary reactive plasma cells, Medical School, focally in bone marrow reactive plasma chronic inflammatory infiltrates in skin, and so Durban, South Africa cells, and occasionally in extramedullary on. Formal acetic acid fixed, paraffin wax D Govender plasmacytomas. embedded tissue blocks of bone marrow P Harilal (3 Clin Pathol 1997;50:490 493) trephine biopsies were retrieved from the files R Chetty ofthe university department ofcellular science, University of Oxford. These comprised 12 University Keywords: CD3 1; cell adhesion molecules; plasma cells Department of cases of multiple myeloma and 10 cases with Cellular Science, reactive plasma cells, including five cases that University of Oxford, Platelet-endothelial cell adhesion molecule-1 had normal haematological indices. None of United Kingdom (PECAM-1 or CD31) belongs to the immuno- the 12 multiple myeloma cases had evidence of M Dada globulin supergene family (IgSF) of adhesion concurrent extramedullary deposits. molecules. Molecules belonging to the IgSF Tissue sections of 2 ,m thickness were Correspondence to: Dr D Govender, Department share the immunoglobulin structural homol- mounted onto poly-L-lysine coated glass slides of Anatomical Pathology, ogy unit.' (Sigma Diagnostics, St Louis, Missouri, USA). University of Natal Medical CD31 is expressed abundantly on endothe- The sections were then heat fixed for 10 School, Private Bag 7, Congella 4013, Durban, lial cells, and a monoclonal antibody to CD31 minutes at 60°C and deparaffinised. Antigen South Africa. has been used as a marker of these cells. Anti- retrieval was achieved by microwave pretreat- email: govendhG body JC70 detects a formalin resistant epitope ment (H2500 microwave processor, Energy med.und.ac.za which is widely distributed on a variety of Beam Sciences, Agawam, Massachusetts, Accepted for publication benign endothelial cells. In detailing the range USA) in a 0.01 M trisodium citrate solution 12 March 1997 of tissues stained by the antibody, it was noted (pH 6.0) for 10 minutes at 85°C. The bone CD31 (7C70) expression in plasma cells 491 Table 1 Plasma cell immunoreactivity with CD31 row sections and extramedullary deposits, showed any reactivity (fig 2). Internal controls Extramedullary Medullary Total in these cases were positive. Reactive 20/20 10/10 30 When applying X2 and Fisher's exact test to J Clin Pathol: first published as 10.1136/jcp.50.6.490 on 1 June 1997. Downloaded from Plasmacytoma 5/10 - 10 compare CD3 1 expression in reactive (polyclo- Multiple myeloma 0/8 0/12 20 nal) and neoplastic (monoclonal) plasma cells, statistically significant p values of < 0.01 and marrow sections were mounted on silane < 0.05 respectively, were obtained. coated glass slides. They were processed in a Monoclonality was confirmed in all plasma- similar way to that described above, but antigen cytomas and multiple myelomas. In reactive retrieval in these cases was facilitated by plasma cells, polytypic light chain expression pressure cooking. was noted. All reactive plasma cells and Endogenous peroxidase activity was blocked myeloma cells in the bone marrow stained with using an aqueous solution of 3% hydrogen VS38c. peroxide. Sections were stained with mono- clonal anti-CD31 antibody (JC70: Dako A/S, Discussion Copenhagen, Denmark; dilution 1/30) using The molecules belonging to the immunoglobu- the peroxidase labelled streptavidin-biotin kit lin supergene family show diversity in cellular (Dako). The reaction was visualised using distribution and function. All members of the diaminobenzidine (Liquid DAB, Dako) as a IgSF share a common structure, the immuno- chromogen. Endothelial cells and megakaryo- globulin homology unit.' Molecules belonging cytes in the tissue sections were used as positive to this family include the major histocompat- controls. Negative controls, in which the ibility molecules, the T cell receptor, the plate- primary antibody was withheld, were also run let derived growth factor receptor, colony simultaneously. In addition, all bone marrow stimulating factor-I receptor, VCAM, sections were stained with monoclonal anti- ICAM-1 (CD54), NCAM (CD56), carcino- body VS38c (Dako, High Wycombe, UK; pre- embryonic antigen (CEA), and PECAM-1 diluted) to assess the number of plasma cells (CD3 1).3 Their diverse functions consisting of present. In all cases, light chain immunostain- a number of homophilic and heterophilic cell ing was reassessed using kappa and lambda interactions3 include those occurring during light chains (Signet Laboratories, Dedham, development (NCAM),4 inflammation and Massachusetts, USA; prediluted, polyconal). wound healing (ICAM-1, VCAM-1),6 7 and possibly in neoplasia (CEA).8 Results CD3 1 is a transmembrane glycoprotein The main results are shown in table 1. found on the surface of platelets and its All 20 extramedullary biopsies with reactive precursors, monocytes, granulocytes (neu- plasma cells showed intense cytoplasmic mem- trophils, basophils, and eosinophils), some T http://jcp.bmj.com/ brane staining with CD3 1 (fig 1). The number cells and B cells, mantle zone lymphocytes, ofreactive plasma cells that stained varied from synovial lining cells, and at endothelial intercel- 75% to 100% of at least 100 cells counted per lular junctions.91-3 Although the precise func- slide. In contrast, reactive plasma cells in the 10 tions of CD31 are not clear, evidence suggests bone marrow sections showed focal membrane that it is an important vascular cell adhesion staining only. molecule involved in endothelial cell-cell Five of the 10 plasmacytomas also showed adhesion. CD3 1 mediates cell adhesion by on September 26, 2021 by guest. Protected copyright. membrane immunoreactivity for CD3 1. The participating in a number of complex ligand remainder were negative. None of the cases of interactions. It can interact both with itself multiple myeloma, including both bone mar- (homophilic interaction) and with other non- CD3 1 molecules (heterophilic inter- , ; ..S actions).'4 15 The JC70 antibody detects an antigen iden- .!,. tical to that detected by CD3 1 antibodies. -4: .,, ¢ +X JC70 was found to be a more reliable marker of '(A ra -- malignant endothelial cells than the i It; .,-. B - ,,v ..,..9'..:v ,,*{ **.^ it e 4 traditionally used endothelial markers, factor .N. ,@ s t VIII related antigen and Ulex europaeus. kgi,y tt$ : w0.o ** t* , w_ _ st i' i. (t* Furthermore, JC70