A major drawback of immunohistochemical detection of monoclonality in B-cell
lymphoproliferative disorders is the lack of contrast between
surface-immunoglobulin staining and extracellular
immunoglobulin staining. To bypass this drawback, immunophenotyping of single-cell
suspensions by flow cytometry is commonly used. Although the expression of
immunoglobulin light chain subtype 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 disorders (lymphoid tissue (n = 10) and non-Hodgkin's
B-cell lymphoma (n = 10). Immunocytochemistry indicated that common cell surface markers as well as the
immunoglobulin light chains could be detected in the cell
suspensions derived from archival material. In addition, the technique also allowed combined high-resolution
DNA flow cytometric analysis. To investigate the effect of
formalin fixation on cross-linking of extracellular
immunoglobulins to lymphocytes, a double-immunostaining experiment for both
light chain immunoglobulins (kappa and lambda) was performed. This experiment showed that this cross-linking was minimal (less than 2%). All cases of
reactive lymphoid hyperplasia were
DNA diploid and showed a polyclonal expression of
immunoglobulin light chains. In contrast, in 9 of 10 non-Hodgkin's
B-cell lymphomas, monoclonality was established on the basis of light chain expression, whereas 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-cell 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
DNA-
aneuploid cases was 6.1%. The presented technique is superior to immunohistochemistry for the detection of monoclonality in B-cell
lymphoproliferative disorders and therefore provides a powerful tool to support the diagnosis of
malignant lymphoma in routinely processed archival samples of lymphoid tissues.