A Simple Flow–Cytometric Method for Absolute Counting of Residual White Blood Cells in Leukocyte–Reduced Packed Red Cells

curate counting of white blood cells (WBC) in leukocytedepleted products [1–5]. We report on a flow-cytometric method based on the identification of WBC nuclei by propidium iodide. Also, a known amount of fluorescent beads are contained in the tube where the sample and reagents will be added. The availability of beads in the tube avoids errors produced by pipetting, ensuring that the number of beads is always constant. A calibration curve was prepared to validate the technique. A representative range of numbers of WBC (0,6–330 WBC/μl) was obtained by means of diluting EDTA-anticoagulated whole blood with fresh packed red cells in SAGMANITOL containing a residual number of 0.55 WBC/ μl. Leukocyte-depleted packed red cells were obtained by filtration three times with PALL RC XL1 filters and residual WBC number were counted with a Nageotte chamber. Using a tube with a known and fixed number of fluorescent beads (Trucount tubes, Becton-Dickinson), 200 μl of a permeabilizating solution (DNA-Prep LPR, Coulter) were added to 100 μl of a series of leukocyte-depleted red cell samples with a known WBC number. Each tube was vortexed for 8 s. There after, 1 ml of a propidium and RNAse solution (DNA-Prep Stain, Coulter) was added to the tubes and vortexed again for 8 s. Before being analyzed flow-cytometrically, the samples were incubated in the dark at room temperature for 15 min. Five readings were taken at each point of the curve, reading approximately 190 μl of volume each time. Short Communication