Toward a reference method for leukocyte differential counts in blood: comparison of three flow cytometric candidate methods.

A Complete Blood Count performed by an automated hematology analyzer frequently requires a microscopic slide review. Recently, we and others have proposed combinations of monoclonal antibodies for an extended leukocyte differential by flow cytometry. The aim of this study was to compare the performance of these proposals. Ninety-two samples were analyzed at 2 sites to compare the accuracy of three published methods. Reference methods used were i) cell counter for leukocyte count and ii) microscopic review as defined by CSLI H20-A2 for cell subsets. Comparison of flow cytometers from 2 manufacturers (FC500 and CANTO/LSRII) was performed. Published protocols were adapted to three different models of flow cytometer and each provided similar results in leukocyte subset enumeration, although some discrepancies were noted for each protocol in comparison with the reference method. The conclusion is that each protocol carries advantages and disadvantages and there is no clear "winner". This study supports the fact that flow cytometry is a candidate to become a reference method for the leukocyte differential. None of the tested protocols clearly demonstrated superiority and each had demonstrable deficiencies. Additional work to develop a consensual 8 to 10 color panel is concluded to be necessary for a satisfactory reference method.