Screening by allele-specific primer PCR (ASP-PCR) for these mutations among 100 Caucasian and 100 Black random blood donors indicated allele frequencies of 2 . 5% and 0%, respectively. Ala100Thr alone was present in 33% of the Caucasians (but none of the Blacks) with no weakening of FY expression.A novel allele at the FY locus associated with the Fy x phenotype was studied. Mistyping of this weak Fy b antigen in clinical transfusion medicine may lead to delayed haemolytic transfusion reactions in immunized patients. A potential role for genomic typing is proposed.

The Rh blood group system is the next most important to the ABO system in terms of its clinical significance in blood transfusion. It is vital to the safe, efficient practice of transfusion medicine that Rh D phenotyping tests are selected, executed and interpreted correctly. However, the Rh D blood group antigen has been shown to be subject to many phenotypic variations, and different reagents and typing techniques vary in their ability to detect these variants. The range of D-positive phenotypes are reviewed in terms of their reactivity with monoclonal antibody reagents and their clinical significance. In view of the available evidence, it is suggested that patient typing can be safely achieved by the duplicate use of one high-avidity or two very similar IgM monoclonal anti-D reagents that detect most variants except category DVI in simple tube or microplate saline tests. Antiglobulin testing for weak D should not be carried out on patient samples. Donor typing can be safely achieved by the use of the same monoclonal, used in parallel with a polyclonal anti-D reagent that detects DVI on sensitive automated equipment.

Information on the number of D sites on weak D (Du) and D variant cells is limited and incomplete. The aim of this study was to use a simple non-isotopic technique utilising a combination of flow cytometry and ELISA to quantitate the number of D sites on an extensive range of these cells. Five human monoclonal IgG anti-D (BRAD-7 (JAC10), BRAD-5, 2B6, BRAD-3, H27) and one affinity-purified polyclonal IgG anti-D were each used at a saturating concentration of 20 micrograms/ml. In general, BRAD-3, BRAD-5 and 2B6 gave the highest number of D sites per cell (SPC), H27 and the polyclonal anti-D were slightly lower, while BRAD-7 gave the lowest SPC with all D-positive cells tested except DFR. Interestingly, BRAD-7 gave the highest SPC with DFR cells. Rh D antigen density for R2R2 cells was approximately double that seen with either R2r or Rzero (presumed R0r) cells. R1R1 cells gave only moderately higher SPC than R1r cells.Higher SPC were obtained with the R1 haplotype if the CW antigen was present. Weak D, Va and VI cells of the R1 haplotype had higher SPC than those of the Rzero or R2 haplotypes. The majority of D variant cells were found to have lower SPC than normal cells, and for polyclonal anti-D, which was the only anti-D to react with all D variants, SPC decreased in the order IIIc > IIIa > HMii > IVa > Va > DFR > DBT > IVb > VII > II > HMi > VI. The number of molecules of IgG anti-D bound to D variant cells varied by up to 10 times with reactive monoclonal antibodies. The highest SPC on weak D and D variant cells were obtained with BRAD-7 (DFR, 9,500), BRAD-3 (Va, 12,500), BRAD-5 (II, 5,500; VII, 5,300; weak D, 1,300), H27 (III, 24,000; VI, 2,900; HMi, 3,000; HMii, 16,800) and polyclonal anti-D (IVa, 9,300; IVb, 4,000; DBT, 4,300).

No abstract

The Rh D antigen is the most clinically important protein blood group antigen of the erythrocyte. It is expressed as a collection of at least 37 different epitopes. The external domains of the Rh D protein involved in epitope presentation have been predicted based on the analysis of variant Rh D protein structures inferred from their cDNA sequences and their D epitope expression. This analysis can never be absolute because (1) most partial D phenotypes involve multiple amino acid changes in the Rh D protein and (2) deficiency for 1 or more epitopes may be due to gross structural alteration in the variant Rh D protein structure. We report here the amino acid requirements for the majority of D epitopes. They have been defined by generating a series of novel Rh mutant constructs by mutagenesis using an Rh cE cDNA as template and mutagenic oligonucleotide primers. When transfected into K562 cells, the D epitope expression of the derived mutant clones was then assessed by flow cytometry. The introduction of 9 externally predicted Rh D-specific amino acids on the Rh cE protein was sufficient to express 80% of all tested D epitopes, whereas other clones expressed none. We concluded from our data that the D epitope expression is consistent with at least 6 different epitope clusters localized on external regions of the Rh D protein, most involving overlapping regions within external loops 3, 4, and 6.