Gating strategy for immunophenotyping of leukemia and lymphoma.

Clinical specimens of blood, bone marrow, lymph node, extranodal tissue, and body fluid were collected from 67 cases of hematologic neoplasms (including chronic lymphoid leukemias, T- and B-cell lymphomas, and acute lymphoblastic and myelogenous leukemias) for comparison between the right-angle light scatter (RALS)/CD45 and the forward-angle light scatter (FALS)/RALS gating combinations. One to three diagnostic markers were selected from each case, yielding 124 paired results for comparison. We found that the percentage of tumor cell isolation and the total cell count in the tumor cell gate were higher in RALS/CD45 than in FALS/RALS. When 20% was used as a cutoff point, 30 markers in FALS/RALS failed to identify the tumor population, while only 3 markers in RALS/CD45 failed to do so. The discriminative factor in the RALS/CD45 gating was mainly the CD45 intensity, whereas all cases except 3 showed low RALS. Although T-cell neoplasms showed a higher proportion of high CD45 intensity, other groups shared similar ranges of CD45 intensity, which is therefore of limited value for differential diagnosis. The RALS/CD45 combination produces higher recovery and purity for tumor cell isolation than the FALS/RALS combination and should replace the latter for routine immunophenotyping of lymphoma and leukemia.

Flow cytometric analysis of residual white blood cell concentration and platelet activation antigens in double filtered platelet concentrates.

Reduction of contaminating leukocytes in platelet products by filtration has been shown to decrease the incidence of human leukocyte antigen (HLA) alloimmunization. Nonetheless, prevention is not complete when using current techniques, and a significant number of patients continue to exhibit clinical refractoriness and to produce alloantibodies. Interest in preventing HLA alloimmunization and other complications of white blood cell (WBC) contamination of transfused cellular products has resulted in ongoing efforts to increase the efficiency of leukodepletion filters. As the efficiency of these filters increases, more accurate and precise methods for counting extremely low numbers of WBCs must be instituted to ensure quality control. We have validated a simple, rapid flow cytometric assay for quantitating low numbers of WBCs in platelet products. The assay is sensitive to a lower limit of 0.1 WBC/microliter without concentration of the platelet product sample and has an excellent correlation (R2 = 1.00) between calculated and expected WBC concentration over a range of 0.1 to 100.0 WBC/microliter. (R2 values over the concentration ranges of 0.1 to 1.0 WBC/microliter and 1.0 to 10.0 WBC/microliter were 0.988 and 0.996, respectively.) The intraassay coefficients of variation at WBC concentrations of 50.4/microliter, 0.9/microliter, and 0.1/microliter were 4%, 8%, and 18%, respectively. The flow cytometric counting technique was applied, in concert with a Nageotte chamber manual counting method, to the enumeration of residual WBCs in 20 apheresis and random donor platelet concentrates filtered through two leukodepletion filters sterile docked in series. A greater than four log10 WBC reduction capability was demonstrated when utilizing this double filtration procedure, and its clinical applicability is underscored by data that showed no statistically significant change in expression of activation-specific platelet antigens before versus after filtration.