Validation of a flow-cytometry-based red blood cell antigen phenotyping method

Area of Science:

• Hematology
• Immunology
• Biotechnology

Background:

• Current red blood cell (RBC) phenotyping relies on visual detection of antigen-antibody interactions.
• Existing methods require large volumes of reagents and patient cells, limiting throughput and increasing costs.
• Limitations include the need for significant reagent and cell volumes for visually detectable reactions.

Purpose of the Study:

• To develop and validate flow cytometry for high-throughput RBC phenotyping.
• To enable fluorescence-based detection of antibody binding using minimal reagent volumes.
• To establish an automated, quantitative method for RBC phenotyping.

Main Methods:

• RBC phenotyping was performed using flow cytometry with commercially available monoclonal direct and indirect typing antisera.
• Seventy samples were analyzed using both flow cytometry and standard manual tube agglutination assays.
• Antisera included anti-C, -E, -c, -e, -K, -Jk ^a, -Jk ^b, -k, -Fy ^a, -Fy ^b, -S, and -s.

Main Results:

• Flow cytometry-based phenotyping achieved 100% concordance with manual tube agglutination assays across all tested antigens.
• The flow cytometry method required significantly lower reagent volumes (0.5-1 μl per antigen) compared to manual methods (50 μl per antigen).
• High throughput and automation potential were demonstrated.

Conclusions:

• Flow cytometry is a validated and effective method for RBC phenotyping.
• This technique offers advantages such as automation, quantitative results, and minimal reagent use.
• The method is suitable for high-throughput, low-cost phenotyping in blood suppliers and hospital blood transfusion services.