Performance of a Nageotte hemocytometer method and a flow cytometric assay for residual leukocyte quantification in leukoreduced canine packed red blood cells

Abstract

Objective: To evaluate the performances of a manual Nageotte hemocytometer method and commercial fluorescent bead-based flow cytometric assay for quantifying [rWBC] in leukoreduced canine packed red blood cell (pRBC) units.

Design: Prospective study. Five, commercially purchased, double leukoreduced canine pRBC units were spiked with canine leukocytes to create 6 pRBC standards with the following [rWBC]: < 0.1, 0.375, 1.5, 3.0, 6.0, and 24.0 WBC/µL. [rWBC] of each pRBC standard was measured with the Nageotte hemocytometer and flow cytometric techniques. Limit of detection (LoD), linearity, and bias were determined for each method. For each standard, accuracy and precision were calculated; the cumulative accuracy and mean precision for measurements between the LoD and 24.0 WBC/µL were also determined.

Setting: University veterinary blood bank and clinical pathology laboratory.

Measurements and main results: The Nageotte hemocytometer method had an LoD = 1.48 WBC/µL, inadequate linearity (R² = 0.92), and a significant negative proportional bias (slope best-fit line = 0.52 ± 0.03). Between [rWBC] 1.5-24 WBC/µL, the technique demonstrated poor cumulative accuracy (6.7%) but acceptable mean precision (17.3%). Relative to a 2 rWBC/µL threshold, at 1.5 WBC/µL the method was inaccurate (6.7%) with acceptable precision (16.6%). The flow cytometric assay had an LoD = 1.3 WBC/µL, acceptable linearity (R² = 0.99), and a mild positive proportional bias (slope best-fit line = 1.11 ± 0.01). The technique had acceptable cumulative accuracy (80%) and mean precision (10.7%) for measuring [rWBC] between 1.5 and 24 WBC/µL. At 1.5 WBC/µL, this method was acceptably accurate (86.7%) and precise (16.0%).

Conclusions: The flow cytometric assay demonstrated acceptable performance for quantification of [rWBC] in leukoreduced canine pRBC units. The Nageotte hemocytometer method should be used cautiously due to poor accuracy and significant negative bias.

Keywords: blood banking; flow cytometry; hematology; pRBC; transfusion.