Flow cytometric quantification of antigen D sites on red blood cells of partial D and weak D variants in India

Abstract

The D antigenic sites are diminished in weak D and partial D variants. The aim of this study was to estimate D antigen on RBC in weak D and partial D variants in Indian population by using flow cytometric method. Blood samples of 42 cases of partial D, eight cases of weak D and 123 normal Rh phenotypes identified by serological methods were used in the study. An indirect immunofluorescence method was employed using 29 monoclonal anti-D as primary antibody. The D antigenic sites were calculated using control RBC as internal standard. The D antigenic sites in weak D and partial D variants are less than that found in normal Rh phenotypes. In weak D, the D antigenic sites were between 1500 and 7000 D antigens per cell. Among partial D variants, DVI had minimum and DVa had maximum number of D sites. Flow cytometry is a very good tool for demonstrating minor variation in D antigen when serological methods are inconclusive. The D antigenic sites per RBC in partial D variants identified in Indian population are reported for the first time.

Weak D caused by a founder deletion in the RHD gene

“…The mean RhD antigen density at the surface of RBCs from two unrelated deletion carriers was determined to be only 30. This number is only slightly higher than that associated with most of the well‐defined DEL alleles (usually <22) 12 but much lower than that associated with most of the weak D alleles (from 992 to 7000) 8,12 . To the best of our knowledge, the previously reported weak D allele–associated lowest D antigen site per cell was observed in two subjects carrying the weak D Type 26 allele, with the mean number being 50 13 .…”

“…This number is only slightly higher than that asso-ciated with most of the well-defined DEL alleles (usually <22) 12 but much lower than that associated with most of the weak D alleles (from 992 to 7000). 8,12 To the best of our knowledge, the previously reported weak D allele-associated lowest D antigen site per cell was observed in two subjects carrying the weak D Type 26 allele, with the mean number being 50. 13 Consequently, the deletion identified here appears to represent the weakest of weak D alleles reported so far.…”

Alloimmunization risk associated with amino acid 223 substitution in the RhD protein: analysis in the light of molecular modeling

“…No standard RBC sample was available. We therefore used the range of values determined by Kulkarni and coworkers for R1R2 RBCs. We determined the mean value for D antigen on RHD*52 RBCs and our R1R2 or weak D control RBCs (data not shown).…”

Molecular characterization of a new D‐ ‐ haplotype in a Comorian man

“…Specific anti‐D antibody‐binding capacity per RBC (RhD antigen density) was determined by flow cytometry according to the established workshop protocol and Kulkarni’s report [9,10]. RBC staining was performed using LHM76/55 and LHM169/80 (DiaMed, Cressier sur Morat, Switzerland) as primary anti‐D antibodies and FITC‐conjugated AffiniPure Fab fragment goat anti‐Human IgG(H + L) (Beckmann Coulter, Villepinte, France) as secondary antibody.…”