Clonality assessment of lymphoproliferative disorders by multiparameter flow cytometry of paraffin-embedded tissue: An additional diagnostic tool in surgical pathology

A major drawback of immunohistochemical detection of monoclonality in B-cel l lymphoproliferative disorders is the lack of contrast between surface-imm unoglobulin staining and extracellular immunoglobulin staining. To bypass t his drawback, immunophenotyping of single-cell suspensions by flow cytometr y is commonly used. Although the expression of immunoglobulin light chain s ubtype can be quantified rapidly and reliably, the technique is hampered by the requirement of fresh unfixed material. We applied a recently developed technique for the isolation of single cells from formalin-fixed, paraffin- embedded material to measure clonality in B-cell lymphoproliferative disord ers (lymphoid tissue (n = 10) and non-Hodgkin's B-cell lymphoma (n = 10). I mmunocytochemistry indicated that common cell surface markers as well as th e immunoglobulin light chains could be detected in the cell suspensions der ived from archival material. In addition, the technique also allowed combin ed high-resolution DNA flow cytometric analysis. To investigate the effect of formalin fixation on cross-linking of extracellular immunoglobulins to l ymphocytes, a double-immunostaining experiment for both light chain immunog lobulins (kappa and lambda) was performed. This experiment showed that this crosslinking was minimal (less than 2%). All cases of reactive lymphoid hy perplasia were DNA diploid and showed a polyclonal expression of immunoglob ulin light chains. In contrast, in 9 of 10 non-Hodgkin's B-cell lymphomas, monoclonality was established on the basis of light chain expression, where as only 6 of 9 cases were conclusive by immunohistochemistry. Four of the 9 cases were DNA aneuploid. One case did not show light chain expression at all by both techniques. However, this case could be classified as malignant by flow cytometric analysis because of the DNA-aneuploid nature of the B-c ell subpopulation. The average S-phase fraction (SPF) of the B cells in the reactive lymphoid tissues was 3.5%. The mean SPF values for B cells in DNA -diploid cases of lymphomas was 3.0%, whereas the mean SPF of B cells in DN A-aneuploid cases was 6.1%. The presented technique is superior to immunohi stochemistry for the detection of monoclonality in B-cell lymphoproliferati ve disorders and therefore provides a powerful tool to support the diagnosi s of malignant lymphoma in routinely processed archival samples of lymphoid tissues. HUM PATHOL 31:422-427. Copyright (C) 2000 by W.B. Saunders Compan y.