Inconclusive flow cytometric surface light chain results; can cytoplasmic light chains, Bcl-2 expression and PCR clonality analysis improve accuracy of cytological diagnoses in B-cell lymphomas?

Abstract

Background: Flow cytometric immunophenotyping (FCI), is widely used in cytology for distinguishing between B-cell lymphoma (BCL) and reactive lymphocytic proliferations (RLP), mainly by identifying monotypic B-cell populations. Since this cannot always be determined by ratios of surface immunoglobulin light chains (sIg LCs) we wanted to assess if cytoplasmic immunoglobulin (cIg) LCs, Bcl-2 and polymerase chain reaction (PCR) based clonality analysis can improve accuracy of cytological diagnoses of BCL.

Methods: Our study included 98 fine needle aspiration biopsies from lymph nodes suspicious for BCL with inconclusive sIg LCs. In all cases PCR clonality analysis was performed in order to determine immunoglobulin heavy chain (IGH) gene and T-cell receptor (TRC) gene rearrangement. In selected cases expression of Bcl-2 and cIg LC were determined by FC.

Results: Thirty patients had lymphoma and 68 had reactive lymphocytic proliferations. Three patterns of sIg LCs staining were found: negative, dual positive and difficult to interpret. Percentage of lymphomas was highest in the dual positive group (75 %). Morphology coupled with cIg LCs determination and/or Bcl-2 expression was able to give a correct diagnosis in 83 % of cases. Molecular tests would have been misleading in 15 % of cases because 7/30 BCL were polyclonal and 8/68 RLP were monoclonal.

Conclusions: Determination of cIg LCs, Bcl-2 expression and PCR clonality analysis of B cells improved accuracy of cytological diagnoses in BCL with inconclusive sIg LC. However, clonality determined by PCR was misleading in some cases.

Fig. 1

Flow cytometric dot plots showing patterns of sIg LCs. Negative (a,b), dual positive (c,d), and difficult to interpret (e,f)

Fig. 2

Percentage of lymphomas in each group of sIg LCs. Cases with specific patterns of sIg LCs determined by FCI (blue columns, left axis) and the percentage of lymphomas within these three groups (red dots, right axis)

Fig. 3

Results of IGH gene rearrangement studies in three different sIg LC patterns. The red columns represent the proportion of cases with monoclonal B cells in BCL. The blue columns represent the proportion of cases with monoclonal B cells in RLP. Final diagnoses are considered