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.

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. Acridine orange (AO) was used to identify leucocytes in serial diluted blood samples ranging from 0.65 to 1,000 cells/microL. 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. There was a good correlation in the number of leucocytes between our new method and the expected numbers from serially diluted blood samples (r2 = 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 = 1.31 x - 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 degrees C were stable for 48 hours and CV of the assay was at an acceptable level. 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.