Comparative evaluation of the erythroblast count generated by three-color fluorescence flow cytometry, the Abbott CELL-DYN® 4000 hematology analyzer, and microscopy
G. Paterakis, L. Kossivas, R. Kendall, N. Anagnostopoulos, A. Stavropoulos-Giokas, A. Tassiopoulou
Erythroblasts (NRBCs) in peripheral blood and bone marrow aspirates could be enumerated by automated flow methods. The accuracy of such automated NRBC counts, however, should first be proven against microscopy. Thus we performed a comparative study, testing NRBC counts generated by a new three-color flow cytometry method and the Abbott CELL-DYN 4000 hematology analyzer against microscopy. Two sample populations were evaluated: 21 filtered bone marrow aspirates with hematological disorders and 22 peripheral blood samples derived from patients with thalassemia or sickle cell disease. NRBCs per 100 white blood cells were measured by: 1) microscopy, counting 1000 nucleated cells on stained bone marrows and 200 cells on peripheral blood films; 2) flow cytometry, using the Ortho Cytoron Absolute flow cytometer with a whole blood lysis procedure, and three-color fluorescence analysis, using conjugated monoclonal antibodies directed against the transferrin receptor, CD71-fluorescein isothiocyanate, CD45-phycoerythrin, and the nucleic acid dye LDS-751; and 3) the Abbott CELL-DYN 4000, which measures NRBCs according to size and DNA fluorescence. Automated NRBC counts were compared with manual NRBC counts by linear regression and Passing Bablok analysis. The following NRBC count results were obtained: 1) flow cytometry regressed against optical count for bone marrow samples (r2 = 0.775, slope = 0.803, y-intercept [int] = -3.33) and for peripheral blood (r2 = 0.836, slope = 1.166, int = -0.21); 2) Abbott CELL-DYN 4000 vs. optical count for bone marrow samples analyzed in resistant red blood cell mode (r2 = 0.667, slope = 0.639, int = 2.36) and for peripheral blood (r2 = 0.945, slope = 0.840, int = 0.00). Both automated methods evaluated for NRBC enumeration, the CELL-DYN 4000 and the three-color flow cytometry using CD71-CD45-LDS751, show strong correlations with the optical NRBC count in bone marrow specimens with hematological pathologies and in peripheral blood samples of patients with thalassemia and sickle cell disease.
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