The routine leukocyte differential flow cytometry HematoFlow™ method: A new flagging system for automatic validation.

Abstract

The complete blood cell count and white blood cell differential are the first step in the biological diagnosis of hematological diseases. Both are currently performed by automated instruments which control data and produce alerts. If such flags are activated, the automated differential cannot be validated and the operator must activate a visual blood smear review. Microscopic examination is still today the reference method despite its lack of sensitivity and reproducibility. The HematoFlow™ (Beckman Coulter) system is the first flow cytometry commercialized method designed for the routine differential. Using six markers in five colors and an automated gating strategy, it provides differentials proven to be reliable for 17 leukocyte subpopulations detection. Relying first on a retrospective analysis of 6,462 blood samples processed by HematoFlow™, thresholds were determined to detect the presence of immature granulocytes and/or blast cells. All possible gating strategy misclassifications of leukocyte subpopulations were then summarized in a systematic nomenclature leading to the development of an original flag system based on the detection of aberrant localization of cell events in specific new bivariate histograms. Ultimately, more than 50% of the results could be automatically validated using the HematoFlow™ system, without any false negative, thereby dramatically contributing to an important decrease of technicians' workload. Moreover a noticeable help was given for smear review interpretation and new immunological flags led to the confirmation of blood disease after classical immunophenotyping. These results were confirmed in a second prospective study including 15,335 cases, where more than 50% of the results were automatically validated by this new flag system. MFC stands as being more and more essential for analyzing differentials in routine and this new flag system could greatly improve its implementation.