White Blood Cell Enumeration and Differential by Flow Cytometry: The ICSH WBC Reference Method.

Introduction: The current reference method for the white blood cell (WBC) differential is manual smear review as outlined in CLSI H20-A2. As with many manual methods, it suffers from a number of challenges including dependence upon the expertise of the interpreter, the quality of the smear and stain, when dysplastic features make cell identification difficult, imprecision with leucopenia, and enumeration bias due to non-uniform cell distribution.

Methods: This study describes an alternative method for establishing the leucocyte differential using a single-tube, 8-color flow cytometric reference method.

Results: Data presented is from an international comparison of normal (based on analyzer counts, N = 120) and abnormal (N = 496) clinical samples performed at four institutions using four different models of flow cytometers. Here we demonstrate equivalent performance between the flow cytometric method and the current manual reference method, but show improved performance of the proposed reference method for low/infrequent cell populations, for example, monocytes and basophils.

Conclusion: The flow cytometric method also performs well in comparison with hematology analyzers in current clinical use, including good correlation for total white blood cell enumeration. The findings indicate that the flow cytometric method, deemed the "ICSH WBC reference," could be used in lieu of CLSI H20-A2 as a reference for white blood cell enumeration and differential counting and specifically for the evaluation of automated differential counters.