4181 JClin Pathol 1994;47:418-422 VS38: a new monoclonal antibody for detecting plasma cell differentiation in routine sections J Clin Pathol: first published as 10.1136/jcp.47.5.418 on 1 May 1994. Downloaded from H Turley, M Jones, W Erber, K Mayne, M de Waele, K Gatter Abstract mouse monoclonal antibody, VS38, that is Aims-To characterise a new mouse reactive with an antigen expressed strongly on monoclonal antibody, VS38, which recog- normal and neoplastic plasma cells but is nises an intracytoplasmic antigen of 64 absent from haemopoietic cells of other lin- kilodaltons present in normal and neo- eages. This antigen is also expressed to some plastic plasma cells; and to establish its extent in both normal and neoplastic epithe- value as a diagnostic reagent for routine lial cells. VS38 was tested on a wide range of pathological practice. normal and neoplastic tissues including solid Methods-A range of normal and neo- tumours as well as blood and bone marrow plastic tissue sections, both frozen and specimens, with the aim of establishing its routinely fixed, were immunostained, value as a diagnostic reagent for pathological using the microwave method of antigen practice. retrieval for routinely fixed specimens. The antibody was also tested on blood and bone marrow specimens and a range Methods of human cell lines. The molecular PRODUCTION OF ANTIBODY VS38 weight of the antigen recognised by the The source of antigen was a membrane prepa- antibody was obtained by western blot ration of MCF-7 AdR cells. About 4 x 1O' analysis. FACS analysis was used to cells were injected intraperitoneally into a demonstrate the cellular location of the Balb/c mouse, together with Freund's com- antigen and its presence on tonsil cell plete adjuvant. Eleven days later, the mouse suspensions and myeloma cases. was given a booster injection of 1 6 x 106 cells, Results-VS38 recognised normal and with non-complete adjuvant. The booster was neoplastic plasma cells in all of the repeated again seven days later. Three days all tissues, including routinely fixed after the last injection the mouse was killed http://jcp.bmj.com/ plasma cell neoplasms tested. The anti- and the spleen removed. Cell suspensions body also weakly stained epithelial ele- were prepared from the spleen and were fused ments within the tissue but was absent with cells from the myeloma cell line NSIs, from haemopoietic cells of other using polyethylene glycol to induce cell lineages. fusion, as described by Mason and Conclusion-Antibody VS38 is of poten- colleagues.7 tial value in identifying myeloma or on September 27, 2021 by guest. Protected copyright. plasmacytoma in bone marrow or other BIOCHEMICAL ANALYSIS OF THE VS38 EPITOPE tissues. It differentiates lymphoplasma- Cell cultures were washed twice with phos- cytoid lymphoma from lymphocytic and phate buffered saline (PBS) and then har- follicular lymphoma. It also subdivides vested with 0 25% trypsin and 0 02% EDTA. large cell lymphomas into two groups The cell suspension was washed in culture University which may be a more reliable method medium and centrifuged for five minutes at Department of of separating these tumours than mor- 1000 and The cells were Cellular Science, John rpm 4°C. washed Radcliffe Hospital, phology alone. twice in PBS and centrifuged for a final spin Headington, Oxford at 2000 rpm for 10 minutes at 4°C. The cell OX3 9DU (J Clin Pathol 1994;47:418-422) pellet was solubilised with an equal volume of H Turley M Jones 1% (v/v) Triton X-100 (Sigma) in 10 mM K Gatter TRIS-HCL (pH 8) for 20 minutes on ice. K Mayne The terminal stage of B cell differentiation is This was then spun at 2000 rpm for 15 min- Haematology the plasma cell for which no unique antigens utes at 4°C and the supernatant fluid col- Department, Royal have been Perth Hospital, Perth, yet described. Apart from their lected. This was either used immediately for Australia morphology, plasma cells are usually distin- analysis by polyacrylamide gel electrophoresis W Erber guished from other B cells by their lack of sur- (PAGE), or stored as a membrane extract at Laboratorium face HLA class I and class II antigens, surface - 700C. Hematologie et immunoglobulin, Fc and C3 receptors, and Immunologie, Academisch the presence of intracytoplasmic immuno- SDS-PAGE Ziekenhuis, VUB globulin.' Most antibodies which stain plasma Sodium dodecyl sulphate (SDS) polyacry- Brussels, Belgium cells label other B cells as well as cells of other lamide gel electrophoresis was performed on M de Waele lineages.2 6 Most do not work on routinely the extract, according to the method of Correspondence to: Dr Kevin Gatter fixed material, thus further limiting their Laemmli.8 Samples were suspended in buffer Accepted for publication usefulness to pathologists. containing DL-dithiothreitol (Sigma) and 25 November 1993 We describe the characterisation of a boiled for two minutes, allowed to cool, and Plasma cell detection in routine sections 419 loaded on to a gel (5 5% stacking gel, 6-5% were then fixed in acetone/methanol (1 in 1) running gel). Molecular weight markers for 60 seconds at room temperature, placed (Sigma UK) were run at the same time. into TBS (05M TRIS, pH7-6, diluted 1 in 10 Proteins were transferred electrophoreti- in 0- 15M saline) and either stained using the J Clin Pathol: first published as 10.1136/jcp.47.5.418 on 1 May 1994. Downloaded from cally using a Transblot (Biorad UK) appara- alkaline phosphatase-antialkaline phosphatase tus to nitrocellulose membranes9 and the (APAAP) technique, or stored wrapped in protein detected by applying the antibody, aluminium foil at - 20°C until required. They followed by a rabbit-anti-mouse peroxidase were then allowed to reach room temperature conjugate (Dako UK) and visualised using before fixation, as described. diaminobenzidine with metal ion enhance- ment.'0 IMMUNOCYTOCHEMICAL STAINING The APAAP staining technique was per- TISSUES formed, as described before.'2 The enzyme A representative range of normal and neo- reaction was developed using napthol-AS- plastic tissues, including bone marrow biopsy MX-phosphate and fast red (TR-Salt, Sigma specimens, was obtained from the Histo- Chemical Co) as substrate. For immunoper- pathology Department at the John Radcliffe oxidase labelling tissue sections were incu- Hospital. Tissues were immediately snap bated first with the monoclonal antibody for frozen in liquid nitrogen and stored at 30 minutes, then with peroxidase conjugated - 70°C. Cryostat sections (5-8 ,um) were rabbit-anti-mouse (Dakopatts), and finally then cut and collected on to glass multiwell with peroxidase conjugated swine-anti-rabbit slides and dried overnight at room tempera- immunoglobulin."3 The peroxidase reaction ture. The slides were then fixed in acetone at was developed using diaminobenzidine room temperature. The sections were either (Sigma Chemical Co). The slides were then stained immediately or stored wrapped in alu- washed and mounted in aqueous mountant minium foil at - 20°C until staining. (Apathy's; BDH Poole, Dorset). Formalin fixed, paraffin wax embedded sec- tions were cut at approximately 5 pm and FACS ANALYSIS floated on to silane coated glass slides. These White cells and mononuclear cells were iso- were dewaxed, rehydrated, and microwaved lated from peripheral blood and bone marrow in a 700 Watt microwave for 8 minutes in aspirates by red cell lysis using ammonium 0-01 M sodium citrate, then into buffer before chloride'4 or density centrifugation on staining as normal." Lymphoprep (Nygaard, Oslo). The peripheral blood cells were permeabilised using buffered CELL LINES formol acetone (BFA), as described by The following cell lines were obtained from Slaper-Cortenbach et al.'5 After washing in A B C the Sir William Dunn School of Pathology, phosphate buffered saline (PBS) containing http://jcp.bmj.com/ Oxford: K562 (erythroleukaemia); HeLa 0-1% bovine serum albumin (BSA), perme- (cervical epithelial carcinoma); U937 (malig- abilised cells were incubated for 30 minutes at 205 - nant histiocytosis); HL60 (promyelocytic 4°C with the antibodies. Cells were then leukaemia); RVH421 (melanoma); A431 washed and incubated for 30 minutes with (vulval carcinoma); Daudi and Raji (Burkitt's fluorescein isothiocyanate (FITC) conjugated 116 - lymphoma); Nalm-1 (pre-B cell leukaemia); rabbit-anti-mouse F(ab')2 immunoglobulin 97 and Jurkat (T cell lymphoma). The cell lines (Dakopatts a/s). After a final wash the cells on September 27, 2021 by guest. Protected copyright. L428 (Hodgkin's disease) and Thiel (plasma were fixed in 1-5% formaldehyde in washing 68 - cell) were obtained from Dr V Diehl; SU- buffer and analysed in a FACScan flow DHL-1 (T cell leukaemia) from Dr ML cytometer (Becton Dickinson) using Consort Cleary; Karpas 299 (T cell lymphoma with a 30 and Paint-a-Gate software. 2:5 translocation) and Karpas M (plasma cell) from Dr A Karpas; and HT29 (colon carci- noma), MCF 7 ADR (breast carcinoma) from Results the Imperial Cancer Research Fund, London. BIOCHEMICAL ANALYSIS OF EPITOPE The plasma cell lines L363, JJN3, Karpas A strong band of molecular weight 64 kilodal- 29 707, OPM-1, and EJM were obtained from tons (reduced) was detected by VS38 from Professor K Nilsson, University of Uppsala, extracts of the cell lines Thiel (plasma cell Sweden. LB 84-4 (plasma cell), ARH 77 derived), MCF-7, and MCF-7 ADR (breast Figure 1 Western blot (plasma cell), and 8226 R10 (plasma cell) carcinoma derived) (fig 1). Occasionally a analysis of lysatesfrom were from Professor Brian GM Durie. All cell higher molecular weight band of 73 kilodal- three cell lin Lane A M(Ces. (breast lines were cultured in RPMI containing 10% tons was detected in the MCF-7 ADR cell carcinoma) ;F-7 fetal calf serum (Gibco Biocult Ltd) at 37°C.