title: An improved clonal excess assay using flow cytometry and B-cell gating.
abstract: 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.
authors.affiliation: Smith Kline & French Labs, King of Prussia, PA.
authors.forename: B W
authors.initials: BW
authors.lastname: Letwin
authors.forename: P K
authors.initials: PK
authors.lastname: Wallace
authors.forename: K A
authors.initials: KA
authors.lastname: Muirhead
authors.forename: G L
authors.initials: GL
authors.lastname: Hensler
authors.forename: W H
authors.initials: WH
authors.lastname: Kashatus
authors.forename: P K
authors.initials: PK
authors.lastname: Horan
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002999:Clone Cells; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000032:analysis; D007146:Immunoglobulin lambda-Chains / Q000032:analysis; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000276:immunology; D011947:Receptors, Antigen, B-Cell / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D011947:Receptors, Antigen, B-Cell; D006493:Heparin