A 5-color flow cytometric method for extended 8-part leukocyte differential

Abstract : Objectives Microscopic leukocyte differentials display many drawbacks. Several single 5 to 8-color tubes using multiparameter flow cytometry (MFC) are able to provide extended differentials with sequential gating-based analysis strategies. We investigated a new 5-color MFC method to perform an extended 8-part differential with a simplified gating strategy. Methods Whole blood was stained with a combination of antibodies including HLA-DR-FITC/CD19-PE/CD45-ECD/CD16-PC5 + CD71-PC5/CD5-PC7. Results An original approach was developed to exclude debris and straightforwardly gate the cells to identify sixteen populations. Strong correlations were obtained with the analyzer for neutrophils, lymphocytes, monocytes, and eosinophils (R2>0.9). Abnormal cells, such as immature granulocytes and blast cells were identified with a good sensitivity and excellent correlation against cytomorphologic review (R2=0.66 and 0.99, respectively). The choice of HLA-DR and CD5 improved specificity for the identification of activated T-lymphocytes and some lymphoid neoplastic cells, respectively. Conclusions Here a new cytometric differential is proposed with a robust gating strategy which may be used even by unskilled cytometrists and can be easily automated. © 2017 International Clinical Cytometry Society