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.