Validation of a simple method to count very low white cell concentrations in filtered red cells or platelets

The increased performance of white cell (WBC) filters makes it difficult to count precisely the number of residual WBCs. Concentrations as low as 0.01 WBC per microL cannot be determined with electronic cell counters, conventional hemocytometers, or the flow cytometric techniques currently being used. This article describes a simple, manual method using a Nageotte hemocytometer with a large-volume chamber (50 microL) to count the number of WBCs contained in red cell (RBC) suspensions (preparations A, B, and C) and in platelet suspensions (preparation D) diluted 1 in 10 pure, or concentrated two fold. To validate the method, several reference ranges, prepared by successively adding mononuclear cells to a suspension of pure RBCs or platelets, were used. Among the different series, validation ranges varied from 0.2 to 12 to 0.01 to 0.5 WBCs per microL and correlation coefficients ranged from 0.929 to 0.996. To determine the limit of accurate detection, accuracy tests (n = 160) were carried out by two experienced operators on samples with WBC concentrations of about 5, 10, and 120 times the concentration at the theoretical limit of detection (1 WBC/chamber). No significant difference was observed in the various types of preparations (A, B, C, D) in the tests performed by the two operators. However, intra-assay coefficients of variation were 18, 9.5, and 2.2 percent, respectively, at WBC concentrations of 5, 10, and 120 times that at the theoretical limit of detection. These observations show that a limit of accurate detection (10%) seems to be reached when 10 cells are observed in a Nageotte hemocytometer.(ABSTRACT TRUNCATED AT 250 WORDS)