Proposed flow cytometric reference method for the determination of erythroid F-cell counts.
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 (r(2) = 0.994, slope = 1. 019, intercept = 0.24), values obtained using an isotype control (r(2) = 0.996, slope = 1.012, intercept = -0.17), and microscopic immunofluorescence counts (r(2) = 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 (r(2) = 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%).


CONCLUSIONS
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.
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan, USA.
J C
JC
Chen
N
N
Bigelow
B H
BH
Davis
D000328:Adult; D004906:Erythrocyte Count / Q000379:methods*; D004912:Erythrocytes / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005260:Female; D005319:Fetal Hemoglobin / Q000378:metabolism*; D005331:Fetomaternal Transfusion / Q000097:blood / Q000175:diagnosis; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence; D011247:Pregnancy; D011786:Quality Control
D005456:Fluorescent Dyes; D005319:Fetal Hemoglobin; D016650:Fluorescein-5-isothiocyanate