Proposed flow cytometric reference method for the determination of erythroid F‐cell counts

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BACKGROUND Quantitation of adult erythrocytes (RBC) containing fetal hemoglobin (F cells) is of potential clinical utility in evaluating erythropoietic disorders, such as myelodysplasia and hemoglobinopathies, and in monitoring F-cell augmenting therapy. F-cell counting methodologies include fluorescence microscopy and flow cytometry. Previous flow cytometric methods have employed an isotype antibody control to distinguish F cells from non-F cells. We investigated the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde-fixed RBC to substitute for fluorescein isothiocyanate (FITC)-labeled isotype control antibody use in F-cell quantitation. METHODS Our previously published method for fetal red cell detection in fetomaternal hemorrhage was used, employing a FITC-labeled anti-hemoglobin F (HbF) monoclonal antibody reagent. Blood samples with varying F-cell counts were quantitated for F cells using both immunofluorescence microscopy and flow cytometry comparing FITC-labeled isotype to FL1 thresholding defined by FL2 autofluorescence. RESULTS F cell percentages obtained by using an FL2 defined threshold for FL1 gating correlated well with expected values in diluted blood samples (r2 = 0.994, slope = 1.019, intercept = 0.24), values obtained using an isotype control (r2 = 0.996, slope = 1.012, intercept = −0.17), and microscopic immunofluorescence counts (r2 = 0.989, slope = 0.999, intercept = −0.72). F-cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r2 = 0.994, slope = 1.014, intercept = 0.03). Intra-assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation <14%). CONCLUSION This novel method is a more objective and less laborious alternative for F-cell quantitation by flow cytometry compared to using an isotype control or microscopy, thereby providing a more robust methodology for clinical studies and consideration as a laboratory reference method for F-cell counting. Cytometry (Comm. Clin. Cytometry) 42:239–246, 2000. © 2000 Wiley-Liss, Inc.