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

Autor: Nancy C. Bigelow, Jenn C. Chen, Bruce H. Davis
Rok vydání: 2000
Předmět:
Zdroj: Cytometry. 42:239-246
ISSN: 1097-0320
0196-4763
DOI: 10.1002/1097-0320(20000815)42:4<239::aid-cyto4>3.0.co;2-z
Popis: 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 5 0.994, slope 5 1.019, intercept 5 0.24), values obtained using an isotype control (r 2 5 0.996, slope 5 1.012, intercept 52 0.17), and microscopic immunofluorescence counts (r 2 5 0.989, slope 5 0.999, intercept 52 0.72). F-cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r 2 5 0.994, slope 5 1.014, intercept 5 0.03). Intra-assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation
Databáze: OpenAIRE
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