Rh flow cytometry: An updated methodology for D antigen density applied to weak D types 164 and 165

Area of Science:

• Immunology
• Hematology
• Biotechnology

Background:

• A previously established flow cytometry assay for D antigen density determination relied on outdated, unavailable monoclonal anti-Ds.
• The need for an updated methodology using current, commercially available reagents was identified.

Purpose of the Study:

• To update and validate a flow cytometry-based methodology for quantifying D antigen density on erythrocytes.
• To utilize microspheres and currently available monoclonal anti-Ds for improved D antigen site quantification.

Main Methods:

• Quantified absolute D antigen density using flow cytometry with fluorescence-labeled microspheres.
• Employed an internal control CcDEe cell with nine commercial anti-Ds to analyze various D antigen samples.
• Validated the updated assay's reproducibility against published D antigen density values.

Main Results:

• The updated assay demonstrated high reproducibility, closely matching previously published D antigen density results.
• Quantified D antigen densities for novel weak D types 164 and 165 as 4500 and 1505 D antigens/red cell, respectively.
• Observed a slight decrease in absolute D antigen density over 19 years in an individual.

Conclusions:

• The updated flow cytometry assay provides reproducible quantification of D antigen densities across different RhD phenotypes.
• The assay enables the characterization of weak D types and can be used to study the impact of amino acid substitutions on RhD phenotype.