Demonstration by Flow Cytometry of the Numbers of Residual White Blood Cells and Platelets in Filtered Red Blood Cell Concentrates and Plasma Preparations

Abstract

Background and objectives: New-generation polyester filters provide significant depletion of white blood cells (WBC) and platelets (PLT) in filtered red blood cell concentrates (FRCC) and in filtered plasma preparations (FP). The aim of this study was to elaborate a sensitive flow cytometric method for monitoring residual WBC and PLT in FRCC and FP. Materials and methods: We determined the number of WBC in 500 µl FRCC of FP using 50 µl of a combination of monoclonal antibodies (MAB) against CD45 (FITC labeled) and CD19 (PE labeled). After lysis of red blood cells, we mixed a specific number of reference beads with the remaining WBC. The number of residual WBC related to the acquisition volume was defined by the acquired reference beads. Using this method, the detection limit (DL) was 3 WBC/µl. Alternative methods used MAB against CD45 (FITC and PerCP labeled) and CD14 (PE labeled) or lymphocyte subsets such as CD3 (FITC labeled) and CD19, CD4, CD8, CD16 and CD56 (PE labeled) in combination with CD45 (PerCP labeled). The DL values were 10 WBC/µl for the CD45/CD14 staining and 0.1 WBC/µl for the determination of both CD3+ and CD19+ lymphocytes. For residual PLT in FRCC or FP, we used an FITC-conjugated MAB against CD41, with reference beads to determine the acquisition volume. PLT were demonstrated in a green-fluorescence (FL1) single histogram after gating in the forward light scatter x 90° light scatter signal dot plot. PLT counting was as described for WBC. The DL value was about 2 PLT/µl. Results: Filtration with Pall WBF-1 filters reduces WBC by 4 log and PLT by 3- 4 log, resulting in cell counts which are below the critical limit for causing adverse transfusion reactions. Conclusions: Flow cytometry techniques provide a reproducible and objective tool for counting residual WBC and PLT in blood preparations compared with the Nageotte hemocytometer. Absolute numbers of leukocyte and lymphocyte subpopulations are obtainable.

The origin and identification of unknown events associated with low‐level leucocyte counting by flow cytometry

“…The National Blood Service (NBS), England, has implemented universal leucocyte depletion of the blood supply as a precautionary measure against the possible risk of transmission of variant Creutzfeldt-Jacob disease (vCJD). To assess the performance of leucocyte-depletion systems, different methods, such as Nageotte [1], Imagn 2000 microvolume fluorimetry [2] and flow cytometry [3], have been developed to ensure accurate and precise counting of residual leucocytes in leucocyte-depleted products [4].…”

Flow cytometric determination of residual white blood cell levels in preserved samples from leukoreduced blood products

“…Several reports in the literature have indicated that for low concentrations of WBCs in LR RBC and PLT products, counts determined by Nageotte hemocytometer and microscopy provided results with either comparable precision 16,30 or less favorable than those observed by automated methods such as microfluorometry or flow cytometry. 1,17,18 While the coefficients of variation determined by flow cytometry in the present study are similar to those reported in the literature, 7,15,19,31 those determined by the Nageotte method were smaller than previously reported.…”

“…These values compare favorably to those reported in the literature. 7,14,[16][17][18][19]30,31,[35][36][37] Given that the pass or fail criteria for permitted rWBCs in RBC or apheresis PLT components in North America is less than 5 ¥ 10 6 per unit and less than 0.83 ¥ 10 6 per single-donor PLT in North America, the absolute WBC count per mL would fall in the range of approximately 15 Ϯ 3 WBCs per mL. The test sensitivity of 0.1 WBC per mL is approximately 1/150 of this value.…”

Why ‘Safer than Ever’ May Not Be Quite Safe Enough

“…Since 1998, 11 developed countries have introduced routine leukocyte filtration with the aim of reducing some of these risks: Austria, Canada, France, Ireland (Eire), Germany, New Zealand, Norway, Portugal, United Arab Emirates, UK and the United States. Filtration with the most effective filters currently available results in approximately 4 log 10 reduction of the number of leukocytes [41]. It is uncertain whether, without further processing, residual leukocytes that are present in leukoreduced platelet concentrates might still present transfusion-associated risks to the recipient.…”