Simplified flow cytometric method for fetal hemoglobin containing red blood cells

Area of Science:

• Hematology
• Clinical Chemistry
• Biotechnology

Background:

• Monitoring fetal hemoglobin (HbF) levels is crucial for patients with sickle cell disease and thalassemia, particularly those undergoing treatment with HbF stimulators.
• Existing flow cytometry methods for quantifying HbF-containing red blood cells (F+ cells) often involve multiple washing steps, increasing processing time.
• A need exists for a faster, simpler, and equally accurate method for F+ cell enumeration.

Purpose of the Study:

• To develop and validate a simplified, rapid, no-wash flow cytometric assay for quantifying F+ cells.
• To compare the performance of the simplified assay against a conventional washing method and high-performance liquid chromatography (HPLC).
• To assess the suitability of the simplified assay for clinical monitoring and fetal cell analysis.

Main Methods:

• A novel flow cytometry protocol was developed, eliminating the washing steps required in previous methods.
• The simplified assay was validated using mixtures of fetal and adult red blood cells and by comparing results from 12 patients with the original procedure.
• Intra- and inter-assay coefficients of variation (CVs) were determined, and sample stability with different anticoagulants (EDTA, citrate, heparin) was assessed.
• Correlation analysis was performed between the simplified flow cytometry assay and HPLC for %HbF quantification in 83 samples.

Main Results:

• The simplified no-wash flow cytometry method demonstrated no significant difference in %F+ cell counts compared to the original washing procedure.
• Intra-assay and inter-assay CVs for the simplified method were consistently low (≤3% and ≤7%, respectively), indicating high reproducibility.
• Samples remained stable for up to two weeks when stored at 4°C with various anticoagulants.
• A highly significant correlation was observed between the %F+ cells measured by the simplified flow cytometry assay and %HbF measured by HPLC across diverse patient diagnoses.

Conclusions:

• The simplified no-wash flow cytometry assay is a convenient, rapid, and reproducible method for quantifying F+ cells.
• This assay serves as a reliable alternative to HPLC for monitoring sickle cell and thalassemia patients, especially where HPLC is unavailable.
• The method is also applicable as a fetal cell assay in cases of fetomaternal hemorrhage.