Automated red blood cell differential analysis on a multi-angle light scatter/fluorescence hematology analyzer

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Abstract
Most hematology analyzers today are capable of performing white blood cell differential analysis but only limited red blood cell (RBC) differential parameters are available. Because of incomplete morphological information on RBC abnormalities, 5-10% of samples in hematology laboratories routinely undergo smear review. A more complete automated RBC differential capability is desired. Abbott CELL DYN 4000 analyzer was modified to perform three-dimensional (3D) RBC differential analysis (RBC/diff) on cell-by-cell basis at 488 nm as well as at 633 nm. Immature RBCs and all nucleated cells are labeled with a fluorescent nuclear stain, prior to RBC/diff using light loss and forward scatter (FSC) signal or two FSC signals and a third side scatter signal, projecting the cytogram onto a precalibrated 3D surface containing grid lines of volume (V) and hemoglobin concentration (HC), to determine the V and HC of a cell. Abnormally shaped RBCs (AbnRBC) are quantitated by the distance of each event to the 3D surface. A total of 154 normal and 484 clinical samples with various hemoglobinopathies have been analyzed. The 3D cytogram and the bivariate distribution of V and HC of individual event provided information to extract quantitative data on V, HC, schistocytes, and AbnRBC of mature and immature RBCs. The accuracy level of the 3D data is difficult to assess because of the semiquantitative nature of smear review, the only available reference method. The 3D RBC cytograms provide a wealth of morphologic information useful for diagnosis and treatment of patients. With powerful computer programs available today, it is evident that the rate of smear review can be significantly reduced as a result.