ORIGINAL ARTICLE

Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry

P Jain¹, G M Giustolisi¹, S Atkinson¹, M O Elnenaei¹, R Morilla¹, K Owusu-Ankomah¹, F Rafiq-Mohammed¹, E Matutes¹, A Wotherspoon² and D Catovsky¹

¹ Department of Academic Haematology and Cytogenetics, The Royal Marsden NHS Trust, Fulham Road, London SW3 6JJ, UK
² Department of Pathology, The Royal Marsden NHS Trust

Correspondence to:
Correspondence to:
Professor D Catovsky, Department of Academic Haematology and Cytogenetics, The Royal Marsden NHS Trust, Fulham Road, London SW3 6JJ, UK;
d.catovsky{at}icr.ac.uk

Aims: To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs).

Methods: Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1–120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients.

Results: A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC.

Conclusion: Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay.

Keywords: cyclin D1; flow cytometry; mantle cell lymphoma

Abbreviations: B-LPD, B cell lymphoproliferative disorder; B-NHL, B cell lymphoma; BSA, bovine serum albumin; CLL, chronic lymphocytic leukaemia; CSB, cyclin staining buffer; FISH, fluorescence in situ hybridisation; FITC, fluorescein isothiocyanate; FL, follicular lymphoma; IHC, immunohistochemistry; MFF, mean fluorescein isothiocyanate fluorescence; MFIR, mean fluorescence intensity ratio; MNC, mononuclear cells; MCL, mantle cell lymphoma; PBS, phosphate buffered saline; PI, propidium iodide; SLVL, splenic lymphomas with villous lymphocytes

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