Development and validation of a sensitive flow cytometric method for determining CECs in RBC products

Abstract

Background and AimsSubpopulations of immature red blood cells (RBCs) named CD71+ erythroid cells (CECs) with different properties may contribute to RBC transfusion outcomes. However, it is challenging to quantify CECs in leukoreduced RCCs and whole blood due to their rarity and fragility. Current flow cytometry methods are not applicable to leukoreduced RCCs as there is limited peripheral blood mononuclear cells (PBMCs) to use for determination of CECs. We have developed and validated a flow cytometry method for quantifying CECs in WB. MethodsWe determined optimal PE-Cy7-CD235a, BV711-CD71, APC-CD45 concentrations and instrument setting by titration. Linearity and level of detection was determined by spiking labelled PBMCs from cord blood units into unlabeled WB. Low, medium, high levels of CECs were used to determine intra- and inter -run precision. ResultsDetection of CECs was linear (R2 = 0.98) over the range of concentrations assessed with a limit of detection of 0.005%. The overall CVs for the intra- and inter-run precision were 6.9% and 9.7%. ConclusionWe developed a simple, sensitive, and cost-effective flow cytometric method for quantifying the proportion of CECs in non-manipulated WB, which could help understand the impact of RBC products on recipient transfusion outcomes.