New rapid flow cytometric method for the enumeration of nucleated red blood cells

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Abstract
Background: Nucleated red blood cells (NRBC) in blood specimens compromise the automated white blood cell (WBC) count on most hematology analyzers. This makes it necessary to correct the WBC count by subtracting separately counted NRBC by manual microscopy. In addition, it is clinically important to establish the non‐physiological presence of NRBC in blood specimens because of their association with significant hematological and non‐hematological disease. Unfortunately, manual microscopic methods lack sensitivity, specificity and reproducibility required for both. Methods: We have developed a new, rapid flow cytometric method for the detection and enumeration of NRBC, based on two‐color staining with anti‐CD45‐fluorescein‐isothiocyanate (CD45‐FITC) and propidium iodide (PI). EDTA anticoagulated blood samples are incubated for 30 min with CD45‐FITC, followed by 30 sec acid‐hypotonic lysis, containing PI and subsequent addition of an alkaline‐hypertonic solution. The samples are thus ready for flow cytometric analysis. Results: The method typically yields up to four populations, (1) red blood cell (RBC) ghosts, debris, lyse‐resistant RBC, reticulocytes and platelets, (2) CD45+ WBC unstained by PI, (3) CD45+ WBC stained by PI, and (4) CD45‐/PIbright NRBC. Manual microscopic reference NRBC counts of 25 patient specimens showed excellent correlation with flow cytometric NRBC determinations (y = 0.943x+0.66; r2 = 0.982). Performance for precision showed a mean coefficient of variation (CV) for the flow cytometric method of =10%, with a mean CV for manual NRBC counts of 40%. Conclusions: We conclude that this method is suitable for NRBC counting in peripheral blood specimens with improved performance in terms of accuracy, reproducibility when compared to manual microscopic methods. Cytometry 37: 291–301, 1999.