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Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay
Published on: August 5, 2011
Proposed flow cytometric reference method for the determination of erythroid F-cell counts
J C Chen1, N Bigelow, B H Davis
1Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan, USA.
Cytometry
|August 10, 2000
Summary
This study introduces a new flow cytometry method for counting fetal red blood cells (F cells) using red blood cell autofluorescence. This approach offers a more objective, efficient, and robust alternative to traditional methods for clinical applications.
Area of Science:
- Hematology
- Clinical Chemistry
- Immunology
Background:
- Quantifying adult erythrocytes (RBCs) with fetal hemoglobin (F cells) is crucial for diagnosing erythropoietic disorders and monitoring therapies.
- Current F-cell counting methods include fluorescence microscopy and flow cytometry, often relying on isotype antibody controls.
- Previous flow cytometry techniques used fluorescein isothiocyanate (FITC)-labeled isotype controls, which can be laborious.
Purpose of the Study:
- To investigate the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde-fixed RBCs as a substitute for FITC-labeled isotype control antibodies in F-cell quantitation.
- To develop a more objective and less laborious method for F-cell counting via flow cytometry.
Main Methods:
- A previously established method for fetal red cell detection was adapted, utilizing a FITC-labeled anti-hemoglobin F (HbF) monoclonal antibody.
- F-cell counts were determined using immunofluorescence microscopy and flow cytometry.
- Flow cytometry compared FITC-labeled isotype controls with FL1 thresholding defined by FL2 autofluorescence.
Main Results:
- F-cell percentages using FL2-defined thresholds correlated strongly with expected values in diluted samples (r² = 0.994) and microscopic counts (r² = 0.989).
- Results from the FL2 autofluorescence method closely matched those obtained using an isotype control (r² = 0.994).
- The autofluorescence gating method demonstrated low imprecision across intra-assay, interobserver, and interinstrument variations (CV <14%).
Conclusions:
- The novel method using FL2 autofluorescence is a more objective and less laborious alternative for F-cell quantitation by flow cytometry.
- This approach provides a more robust methodology for clinical studies compared to isotype controls or microscopy.
- The autofluorescence gating method is suitable for consideration as a laboratory reference method for F-cell counting.
