Franz F. Wagner

RHD positive haplotypes in D negative Europeans

BackgroundBlood group genotyping is increasingly utilized for prenatal diagnosis and after recent transfusions, but still lacks the specificity of serology. In whites, the presence of antigen D is predicted, if two or more properly selected RHD-specific polymorphism are detected. This prediction must fail, if an antigen D negative RHD positive allele is encountered. Excluding RHDψ and CdeS frequent only in individuals of African descent, most of these alleles are unknown and the population frequency of any such allele has not been determined.MethodsWe screened 8,442 antigen D negative blood donations by RHD PCR-SSP. RHD PCR positive samples were further characterized by RHD exon specific PCR-SSP or sequencing. The phenotype of the identified alleles was checked and their frequencies in Germans were determined.ResultsWe detected 50 RHD positive samples. Fifteen samples harbored one of three new Del alleles. Thirty samples were due to 14 different D negative alleles, only 5 of which were previously known. Nine of the 14 alleles may have been generated by gene conversion in cis, for which we proposed a mechanism triggered by hairpin formation of chromosomal DNA. The cumulative population frequency of the 14 D negative alleles was 1:1,500. Five samples represented a D+/- chimera, a weak D and three partial D, which had been missed by routine serology; two recipients transfused with blood of the D+/- chimera donor became anti-D immunized.ConclusionThe results of this study allowed to devise an improved RHD genotyping strategy, the false-positive rate of which was lower than 1:10,000. The number of characterized RHD positive antigen D negative and Del alleles was more than doubled and their population frequencies in Europe were defined.

RHD/CE typing by polymerase chain reaction using sequence-specific primers

BACKGROUND: Current DNA‐based Rh system typing strategies may detect the two RH genes and their prevalent alleles, but they are known to fail sometimes, when rare RH alleles (e.g., D category phenotypes) are encountered. It is almost impossible to find a single DNA‐based method that can accommodate the great heterogeneity within the human Rh system. STUDY DESIGN AND METHODS: An easy‐to‐perform DNA‐based method for the detection of the two RH genes and their alleles, including variant RHD alleles, was developed. By the use of one RHD/C‐, seven RHD‐, and four RHCE‐specific polymerase chain reactions, all triggered to work at identical thermocycling conditions, the DNA of 77 blood donors carrying weak D and that of 200 random donors with common D phenotype was investigated. In addition, 77 selected samples of ccDee and rare Rh system phenotypes were examined. RESULTS: Among 77 samples of weak D, one Rh33 and six DVI categories were detected, one of which showed new RHD‐specific nucleotide patterns. In DFR and CCee samples, novel variant RHD alleles were found. RHD DNA types of 200 random donors were found to be concordant with their D phenotype. For RHE and RHe genotyping, a full correlation with serologic phenotypes was found. Our method for genotyping RHC and RHc failed in some cases, because of an already published RHc allelic variation, which we have called RHc(cyt48). An estimate of the frequency of this RHc(cyt48) allele in a white population was made. CONCLUSION: The presented exon‐scanning RHD/CE polymerase chain reaction using sequence‐specific primers complements current DNA‐based Rh system typing strategies and is superior in the detection of variant RHD alleles.

Partial D, weak D types, and novel RHD alleles among 33,864 multiethnic patients : implications for anti-D alloimmunization and prevention

BACKGROUND: The D antigen includes category D, partial D, and weak D types, which are important because anti‐D alloimmunization can occur in some but not all persons that express a variant RHD allele. At present, there is little prospective information on the prevalence of D variants among obstetric patients and potential transfusion recipients.

Six years' experience performing RHD genotyping to confirm D- red blood cell units in Germany for preventing anti-D immunizations.

BACKGROUND: Red blood cell (RBC) units of D+ donors are falsely labeled D− if regular serologic typing fails to detect low D antigen expression or chimerism. The limitations of serology can be overcome by molecular typing.

PCR screening for common weak D types shows different distributions in three Central European populations

BACKGROUND: DNA sequencing showed RHD mutations for all weak D phenotypes investigated in a study from Southwestern Germany. Molecular classification of weak D offers a more reliable basis than serotyping and is relevant for optimal D transfusion strategies.

Primary anti‐D immunization by weak D type 2 RBCs

BACKGROUND: D is the most immunogenic blood group antigen. In about 0.4 percent of whites, D is expressed on RBCs in a weak form. Recently, it was found that the weak D phenotypes are caused by a large number of distinct RHD alleles generally encoding altered D proteins. No particular molecular weak D type has yet been shown to induce anti‐D. The threshold of D antigen density required for anti‐D immunization is not known.

Antibodies to high-frequency antigens may decrease the quality of transfusion support: an observational study

BACKGROUND:  There is only little information on the transfusion support of patients with antibodies to high‐frequency RBC antigens.

RHD allele distribution in Africans of Mali

BackgroundAberrant and non-functional RHD alleles are much more frequent in Africans than in Europeans. The DAU cluster of RHD alleles exemplifies that the alleles frequent in Africans have evaded recognition until recently. A comprehensive survey of RHD alleles in any African population was lacking.ResultsWe surveyed the molecular structure and frequency of RHD alleles in Mali (West Africa) by evaluating 116 haplotypes. Only 69% could be attributed to standard RHD (55%) or the RHD deletion (14%). The aberrant RHD allele DAU-0 was predicted for 19%, RHDΨ for 7% and Ccdesfor 4% of all haplotypes. DAU-3 and the new RHD allele RHD(L207F), dubbed DMA, were found in one haplotype each. A PCR-RFLP for the detection of the hybrid Rhesus box diagnostic for the RHD deletion in Europeans was false positive in 9 individuals, including all carriers of RHDΨ . Including two silent mutations and the RHD deletion, a total of 9 alleles could be differentiated.ConclusionBesides standard RHD and the RHD deletion, DAU-0, RHDΨ and Ccdesare major alleles in Mali. Our survey proved that the most frequent alleles of West Africans have been recognized allowing to devise reliable genotyping and phenotyping strategies.

The RHCE allele ceRT: D epitope 6 expression does not require D‐specific amino acids

BACKGROUND:  False‐positive D typing in patients may lead to anti‐D immunization caused by D+ transfusions or by omission of anti‐D prophylaxis. Known causes of such errors are RhCE variants carrying RhD‐specific amino acids and cold agglutinin activity of some frequently used monoclonal anti‐D.

Genetic mechanisms of Rhesus box variation

BACKGROUND: The RHD gene is flanked by two highly homologous DNA segments of approximately 9000 bp, the upstream and downstream Rhesus boxes. In haplotypes with an RHD deletion, the fusion of the two Rhesus boxes generates the single‐hybrid Rhesus box, the detection of which has been applied for RHD zygosity determination. Aberrant Rhesus boxes can confound this application and appear to be frequent among African individuals.