P.A. Maaskant-van Wijk

The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis

BACKGROUND: VS and V are common red cell antigens in persons of African origin. The molecular background of these Rh system antigens is poorly understood.

Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons

BACKGROUND: Qualitative RHD variants are the result of the replacement of RHD exons by their RHCE counterparts or of point mutations in RHD causing amino acid substitutions. For RHD typing, the use of at least two RHD typing polymerase chain reaction (PCR) assays directed at different regions of RHD is advised to prevent discrepancies between phenotyping and genotyping results, but even then discrepancies occur. A multiplex RHD PCR based on amplification of six RHD‐specific exons in one reaction mixture is described. STUDY DESIGN AND METHODS: Six RHD‐ specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7, and 9. DNA from 119 donors (87 D+, 14 D‐ and 18 with known D variants; whites and nonwhites) with known Rh phenotypes was analyzed. RESULTS: All six RHD‐specific exons from 85 D+ individuals were amplified, whereas none of the RHD exons from 13 D‐ individuals were amplified. Multiplex PCR analysis showed that the genotypes of two donors typed as D+ were DIVa and DVa. Red cell typing confirmed these findings. From all D variants tested (DIIIc, DIVa, DIVb, DVa, DVI, DDFR, DDBT) and from RoHar, RHD‐specific exons were amplified as expected from the proposed genotypes. CONCLUSION: The multiplex PCR assay is reliable in determining genotypes in people who have the D+ and partial D phenotypes as well as in discovering people with new D variants. Because the multiplex PCR is directed at six regions of RHD, the chance of discrepancies is markedly reduced. The entire analysis can be performed in one reaction mixture, which results in higher speed, higher accuracy, and the need for smaller samples. This technique might be of great value in prenatal RHD genotyping.

Systemic analysis and zygosity determination of the RHD gene in a D‐negative Chinese Han population reveals a novel D‐negative RHD gene

Background and Objectives  The aim of this study was to systemically analyse the genetic background of D negativity in a Chinese Han population.

The applicability of different PCR‐based methods for fetal RHD and K1 genotyping: a prospective study

The applicability of different PCR‐based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3′‐non‐coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly‐developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3′‐non‐coding region or intron 4 often yielded false‐negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD‐specific exons simultaneously. For K1 genotyping, two different PCR‐based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740‐bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1‐specific PCR, amplifying a fragment of 91 bp only in cases of K1‐positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells. Copyright

SI07 High-Throughput Genotyping for Red Cell and Platelet Blood Group Antigens by DNA Micro Arrays

In the Netherlands, to provide antigen-negative red cells for certain groups of patients, the majority of donors is serologically typed for ABO, RhCcDEe and K and a subset of donors for clinically relevant systems (e.g. Fy, Jk, MNSs). Complete phenotyping of all blood donors, for all blood group antigens including high frequency antigens, is too laborious and simply not feasible, because of the lack of sufficient and high-quality typing reagents. For most blood group systems the molecular basis is known and found to be a single nucleotide polymorphism (SNP). In the last years, methods were developed facilitating high-throughput blood group genotyping by glass-based DNA microarrays. By this so-called chiptechnology the phenotype of a donor for all clinically relevant (including HFA) red cell antigens can be simply predicted by running a single assay. Typing of platelet antigens and perhaps also HLA can be included as well. Therefore, high-throughput genotyping or chiptechnology promises the future availability of a completely typed red cell and platelet inventory. A disadvantage of genotyping is false positive or negative results, due to currently unidentified or newly occurring mutations leading to null alleles or mispriming. Large-scale studies of blood group antigen typing including different ethnic groups are required to determine the error rate. An advantage of the use of blood group genotyping assays is improved detection of ‘weak phenotypic signals’, for example weak-D or Fyx antigens will be correctly identified by genotyping. If all red cells units are routinely completely typed a less restricted policy on preventive matching for clinically relevant blood group systems can be implement. Thus, in more patients or even in all blood recipients it may become possible to avoid alloimmunization with the benefit of a reduction in the occurrence of severe transfusion reactions (for example delayed haemolytic transfusion reactions by Jka alloantibodies) or a decreased survival time of transfused cells. Furthermore it will reduce costs and delays in provision of antigen-negative red cells. Thus, if the challenge can be met to develop the current microarrays or chiptechnology into systems that meet the throughput and quality demands of the blood bank, this can lead to an important change in transfusion policy.In the Netherlands, to provide antigen‐negative red cells for certain groups of patients, the majority of donors is serologically typed for ABO, RhCcDEe and K and a subset of donors for clinically relevant systems (e.g. Fy, Jk, MNSs). Complete phenotyping of all blood donors, for all blood group antigens including high frequency antigens, is too laborious and simply not feasible, because of the lack of sufficient and high‐quality typing reagents. For most blood group systems the molecular basis is known and found to be a single nucleotide polymorphism (SNP). In the last years, methods were developed facilitating high‐throughput blood group genotyping by glass‐based DNA microarrays. By this so‐called chiptechnology the phenotype of a donor for all clinically relevant (including HFA) red cell antigens can be simply predicted by running a single assay. Typing of platelet antigens and perhaps also HLA can be included as well. Therefore, high‐throughput genotyping or chiptechnology promises the future availability of a completely typed red cell and platelet inventory. A disadvantage of genotyping is false positive or negative results, due to currently unidentified or newly occurring mutations leading to null alleles or mispriming. Large‐scale studies of blood group antigen typing including different ethnic groups are required to determine the error rate. An advantage of the use of blood group genotyping assays is improved detection of ‘weak phenotypic signals’, for example weak‐D or Fyx antigens will be correctly identified by genotyping. If all red cells units are routinely completely typed a less restricted policy on preventive matching for clinically relevant blood group systems can be implement. Thus, in more patients or even in all blood recipients it may become possible to avoid alloimmunization with the benefit of a reduction in the occurrence of severe transfusion reactions (for example delayed haemolytic transfusion reactions by Jka alloantibodies) or a decreased survival time of transfused cells. Furthermore it will reduce costs and delays in provision of antigen‐negative red cells. Thus, if the challenge can be met to develop the current microarrays or chiptechnology into systems that meet the throughput and quality demands of the blood bank, this can lead to an important change in transfusion policy.