WBC Differential by Flow Cytometry: The First Routine Application in a Large University Hospital Clinical Laboratory.
Authors: Roussel Mikael , Benard Cyrille , Sunnaram Béatrice Ly , Gerard Danielle , Feuillard Jean , Fest Thierry 
Published: October 2019

Abstract
Hematology analyzers deliver high precision blood cell counts and a good leukocyte differential (WBCD) on normal samples. But their ability to identify and quantify abnormal cells is less good and generates a significant amount of false positive results. Routinely, about 10% to 30% of results must have manual blood film reviews, which requires considerable time and are prone to a high degree of inaccuracy, especially for the less frequent cell types (Rümke et al. 1975). In contrast, flow cytometry offers superior detection and quantification of these rare events. A Cyto Diff tube combining six antibodies (CD45, 16, 2, 36, 19 & CRTH2) analysed on a modern multicolor flow cytometer make very accurate automated WBCD feasible for abnormal samples (Feuillard J et al. ISLH 2007). The objective of the study was to evaluate the efficiency of the Cyto Diff process compared to the normal laboratory process as: The time for both methods, the labor and time savings, the relative costs of both methods including med tech time, consumables, number of residual manual review. Two Coulter LH750® hematology analysers were used for the analysis of CBC, WBCD and Reticulocyte counting. An immuno-phenotyping system, with an automatic preparator Coulter FP 1000 and an Coulter FC 500® flow cytometer were connected with a Hematology analyzer to the REMISOL data manager that requests a reflex CytoDiff tube on every sample flagged by the hematology analyzer according to the laboratory’s validation rules. The remaining samples are displayed for manual validation by an operator. The complete line is called HematoFlow. Among the 4896 non-selected CBC tests evaluated during the 10 working days of our study, 877 cases were flagged by the analyzers, reviewed manually following the normal procedure as well as analyzed on HematoFlow. Interestingly, this latter allowed: 68.8% of auto-validation by the REMISOL Data Manager, 12.8% validation directly by the operator after checking the auto-gating, 8.4% required a region readjustment before validation and finally, only 10.3% (91 of the 877-flagged samples) required further exploration because the presence of large amount of ImmGrans, Plt clumps, NRBCs, etc. In conclusion, the CytoDiff tube performs well in regular clinical lab workflow saving almost 90% of the samples flagged by the hematology analyzers for WBC abnormalities that need further exploration following current routine procedure. Our study confirmed our previous results and the fact that the standard auto-gating is set correctly needing only 8.4% of region readjustment by an operator who can be trained easily in few days. Basically, we are expecting that one operator well-trained for smear review and working on the HematoFlow line can handle the same workload as at least 3 operators at microscope stations following a current normal procedure. Furthermore, the CytoDiff approach provides additional information concerning the white blood cells in pathological context never obtained previously by cytomorphology including the detection of likely pro-inflammatory monocytes, several blast subsets, and multiple lymphocyte sub populations as well.