The use of acridine orange and glutaraldehyde-fixed chicken red blood cells for absolute counting of residual white blood cells in leuco-depleted packed red blood cells

Abstract

Background: We have previously developed an affordable flow cytometric method for absolute cell count using glutaraldehyde-fixed chicken red blood cells. However, its use is limited to CD4+ T cells. In the current investigation, we studied the potential use of glutaraldehyde-fixed chicken RBCs to determine the number of residual white blood cells (rWBCs) in WBC-reduced blood component. Methods: Acridine orange (AO) was used to identify leucocytes in serial diluted blood samples ranging from 0.65 to 1,000 cells/μL. The absolute number of AO stained leucocytes were determined by using a known number of glutaraldehyde-fixed chicken RBCs on flow cytometer. The results were compared with the expected value and the absolute count determined by BD Leucocount™ (Becton Dickinson Bioscience). In addition, the stability of AO stained leucocytes and sample stability at various time points were measured. Reproducibility of the assay method was also addressed. Results: There was a good correlation in the number of leucocytes between our new method and the expected numbers from serially diluted blood samples (r 2 = 0.99, y = 1.04x + 0.50, p < 0.001).Furthermore, absolute leucocyte counts determined by the new method correlated well with those obtained from BD Leucocount™ (r2= 0.99, y = 1.31x - 6.37, p < 0.001). FL-1 intensity and the absolute number of AO stained leucocytes were stable for at least 24 hours after staining. Samples stored at 4 o C were stable for 48 hours and CV of the assay was at an acceptable level. Conclusion: This flow cytometric method for absolute leucocyte counts using AO and glutaraldehyde-fixed chicken RBCs is a simple, rapid, reliable and inexpensive method for routine monitoring of low levels of leucocytes in blood products.