Multicenter Evaluation of Methods for Counting Residual White Cells in Leukocyte-Depleted Red Blood Cells

Abstract

Two wet workshops were conducted involving 20 laboratories to determine optimum methods for counting residual white blood cells (WBC) in leukocyte-depleted red blood cells (RBC). In both studies, calibration samples containing known concentrations of WBC were prepared by diluting 1-day-old EDTA blood in RBC virtually free of WBC. Study No.1 was aimed at determining the lower limit of detection and at examining advantages and disadvantages of two methods based on flow cytometry (FC) and on the Nageotte chamber (NC), respectively. This study showed that the lower detection limits for FC and NC were 0.1 and 1WBC/µl, respectively. Methods based on FC showed less variability at 0.1-1 WBC/µl but were equal in performance to counting procedures based on the NC above lWBC/µl. We then designed study No. 2 to evaluate three different protocols using the NC. In protocol 1, samples were diluted 1:10 with Plaxan and withTiirk’s solution; in protocol2 a mixture of Türk’s solution and Zap-oglobin, a reagent used in manual hemoglobin determinations, was used to dilute the samples 1:5; and protocol 3 involved initial 1:10 dilution of a 1-ml sample with Plaxan followed by WBC concentration into the original 1-ml volume by centrifugation. In each participating laboratory, two technologists independently processed the samples and read the results with an NC. Plaxan and Tiirk’s solution gave comparable results. Interobserver variability was not critical to the results. All three NC methods were valid for counting at concentrations of ≥ 1 WBC/µl. Protocol 3 showed the greatest precision, allowing the measurement of WBC at concentrations of 0.1-1/µl (30,000-300,000 WBC in a 300-ml RBC unit) with a coefficient of variation of approximately 50% or less. Based on these results the Biomedical Excellence for Safer Transfusion (BEST) Working Party considers all three NC protocols adequate for the routine evaluation of current leukocyte removal filters, i.e. at levels ≥ 1WBC/µl. The evaluation of new and more effective filters and validation of filter performance may require techniques with a greater precision at counts below 1 WBC/µl, such as protocol 3. For the latter purposes FC is also applicable. Because the counting techniques were not validated using stored blood, these conclusions only apply to samples obtained from blood stored for 1 day.