Evaluation of an 8-color flow cytometric reference method for white blood cell differential enumeration.

Background: Manual microscopy is the current reference method for white blood cell (WBC) differential counts. However, manual counts are time and labor intensive, difficult in patients with low WBC counts, and can misclassify cells having difficult morphology. We investigated an 8-color, single-tube, lyse no-wash flow cytometric method to perform an extended 8-part differential as a potential replacement reference method for WBC differential enumeration.

Methods: Whole blood was stained using a panel of antibodies including CD45APC-Cy7, CD16+CD19FITC, CD33+CD64PE-Cy5, CD123PE, HLA-DRPE-Cy7, CD34+CD117APC, and CD38A594 with the membrane permeant DNA binding dye Hoechst 34580 to generate an 8-part differential (lymphocytes, granulocytes, monocytes, eosinophils, basophils, immature granulocytes, blasts, and nucleated RBCs) using TruCount beads to generate absolute counts for all populations. Manual and instrument differentials were generated for 300 blood samples ranging from normal to complex. Results were compared with the flow cytometric differential.

Results: The flow cytometric WBC and differential correlated well with the Sysmex XE2100 hematology analyzer and gave comparable results to the manual differential. Areas of greatest discordance included enumeration of populations present at low numbers and misclassification of cells with unusual morphology by the manual method. This study describes a novel single-tube flow cytometric method for performing a WBC count and 8-part differential that performs well with both normal and difficult patient samples. These findings confirm the results of prior studies supporting the use of a flow cytometric differential as an improved reference method for the WBC differential and extend prior efforts by allowing positive identification of most cell populations. © 2010 International Clinical Cytometry Society.