Variability in absolute lymphocyte counts obtained by automated cell counters

There is increasing interest in the absolute lymphocyte count. This is partly driven by the need to obtain absolute values for lymphocyte subsets such as absolute CD4+ counts in human immunodeficiency virus (HIV)-infected persons. The absolute total lymphocyte count is usually determined in the routine hematology laboratory on a separate sample from the same patient specimen and then combined with percentage results from flow cytometry to obtain the absolute value of the lymphocyte subsets. We have studied analytic variability in the absolute lymphocyte determination and compared it to the variability of the total white blood count (WBC). In a series of 524 specimens, four different automated methods were compared to each other and to the traditional eye count differential. The automated methods were four widely used automated cell counters (Technicon H*1, TOA NE8000, Coulter STKS, and Abbott CD3000). The results indicate that analytic variability in the absolute lymphocyte counts, due, primarily, to method variability, is significant and is larger than the variability typically observed on interlaboratory trials of relative CD4 counts. These method biases cannot easily be reduced by calibration, since the cell classification algorithms are built-in features of the various cell counters. Analytic variability of the absolute lymphocyte counts was found to be 12.4% compared with analytic variability of only 4.9% for total WBC counts on the same samples. Our data suggest that more precise results would be obtained if flow cytometry results expressed each phenotype as a fraction of the leukocytes as well as total lymphocytes. Conversion to absolute values could then be accomplished through determination of the total WBC in the routine hematology laboratory.