Willy A. Flegel

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.

Norovirus gastroenteritis causes severe and lethal complications after chemotherapy and hematopoietic stem cell transplantation

Norovirus (NV) infections are a frequent cause of gastroenteritis (GE), but data on this disease in immunocompromised patients are limited. We analyzed an NV outbreak, which affected immunosuppressed patients in the context of chemotherapy or HSCT. On recognition, 7 days after admission of the index patient, preventive measures were implemented. Attack rates were only 3% (11/334) and 10% (11/105) among patients and staff members, respectively. The median duration of symptoms was 7 days in patients compared with only 3 days in staff members (P = .02). Three patients died of the NV infection. Commonly used clinical diagnostic criteria (Kaplan-criteria) were unsuitable because they applied to 11 patients with proven NV-GE but also to 15 patients without NV-GE. With respect to the therapeutic management, it is important to differentiate intestinal GVHD from NV-GE. Therefore, we analyzed the histopathologic patterns in duodenal biopsies, which were distinctive in both conditions. Stool specimens in patients remained positive for NV-RNA for a median of 30 days, but no transmission was observed beyond an asymptomatic interval of 48 hours. NV-GE is a major threat to patients with chemotherapy or HSCT, and meticulous measures are warranted to prevent transmission of NV to these patients.

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.

International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Berlin report.

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

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.

How I manage donors and patients with a weak D phenotype.

Purpose of reviewSince the adoption of molecular blood-group typing, the considerable heterogeneity of the serologic entities weak D and DEL at the molecular level has come to light. I offer an approach to the management of donors and patients expressing D antigen weakly and carrying any of the various molecular types of weak D and DEL. Recent findingsMore than 50 distinct weak D alleles have been described. An internet-based survey of anti-D immunizations occurring in D-positive transfusion recipients reveals that no allo-anti-D has been observed in patients carrying prevalent weak D types. Allo-immunizations are documented for weak D types 4.2 (also known as DAR), 11 and 15. Anti-D immunizations have been reported in D-negative persons transfused with weak D and DEL red blood cells. SummaryPatients carrying any of the prevalent weak D types 1, 2, 3 or 4.1 are not prone to allo-anti-D immunization and may safely be transfused with D-positive red blood cells. Pregnant women with these weak D types need not receive RhIg. We should pay attention to weak D- or DEL-positive blood units that are labelled D-negative. The clinical benefit of removing DEL blood units from our supply of D-negative red blood cell units should be determined.

Molecular genetics and clinical applications for RH

Rhesus is the clinically most important protein-based blood group system. It represents the largest number of antigens and the most complex genetics of the 30 known blood group systems. The RHD and RHCE genes are strongly homologous. Some genetic complexity is explained by their close chromosomal proximity and unusual orientation, with their tail ends facing each other. The antigens are expressed by the RhD and the RhCE proteins. Rhesus exemplifies the correlation of genotype and phenotype, facilitating the understanding of general genetic mechanisms. For clinical purposes, genetic diagnostics of Rhesus antigens will improve the cost-effective development of transfusion medicine.

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.

International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report.

The Committee met in Cape Town during the 2006 Inter-national Society of Blood Transfusion (ISBT) Congress (seeAppendix 1 for Committee members). Some changes to theclassification documented in Blood Group Terminology 2004[1] were agreed and are described below. The full updatedclassification can be found on the Blood Group Terminologywebsite at http://www.blood.co.uk/ibgrl. New antigens wereadded to the MNS, Kell, Scianna, Cromer, Indian, Knops,and JMH systems (Table 1). In line with convention, aminoacid positions are numbered with the translation-initiatingmethionine as 1, although the more traditional numberingfor glycophorin A, with number 1 representing the first aminoacid of the mature protein, is also provided.