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

Abstract

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. Whole blood was stained with a combination of antibodies including HLA-DR-FITC/CD19-PE/CD45-ECD/CD16-PC5 + CD71-PC5/CD5-PC7. 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. 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.