In humans with B-cell malignancies, the presence of monoclonal B lymphocytes (clonal proliferation) can be detected by comparing the fluorescence intensity distributions of lymphocytes stained with anti- kappa and anti-lambda reagents. The sensitivity of previously described single-color immunofluorescence techniques to low levels of clonal excess is limited by background from cytophilic immunoglobulins on non- B cells and by the low proportion of circulating B cells in individuals with minimal disease. We have used two-color immunofluorescence and B- cell gating to develop an improved assay that avoids false positives due to non-B cells, without requiring restrictive light scatter gates that may exclude true positives. This method is sensitive to 0.2% monoclonal B cells admixed with fresh normal lymphocytes, to 0.6% monoclonal B cells admixed with normal lymphocytes that have been stored for up to 72 hours, and readily detects 1% monoclonal cells in patient specimens. The two color B-cell gated assay offers sensitivity equivalent to the single-color assay and improved specificity for detection of low levels of clonal excess.

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