Blood group genotyping facilitates transfusion of beta-thalassemia patients

We evaluated the usefulness of blood group genotyping as a supplement to hemagglutination to determine the red blood cell (RBC) antigen profile of polytransfused patients with beta-thalassemia. We selected 10 alloimmunized patients who were receiving antigen-matched RBCs based on phenotype, and had clinical evidence of delayed hemolytic transfusion reaction. DNA was prepared from blood samples and RH E/e, K1/K2, FY A/FY B, and JK A/JK B alleles were determined by PCR-RFLP. RH D/non-D was determined according to the PCR product size associated with the RHD gene sequence in intron 4 and exon 10/3'UTR. RH C/c was tested by multiplex PCR. The phenotypes and genotypes of nine of the 10 samples were discrepant. Five of the discrepancies occurred in the Rh system. One sample was phenotyped as Rhcc and genotyped as RH C/C, and two samples were phenotyped as RhCc and genotyped as RH C/C. Two other samples were phenotyped as RhEe and genotyped as RH e/e. Three samples had discrepancies in the Kidd system with phenotype Jk(a+b+) and were genotyped as homozygous for JK B. One sample had a discrepancy in the Duffy system: it was phenotyped as Fy(a+b-) and homozygous for FY B. Genotyping was very important in determining the true blood groups of many polytransfused patients with beta-thalassemia, and it assisted in the identification of suspected alloantibodies and the selection of antigen-negative RBCs for transfusion.     

Blood group genotyping in a population of highly diverse ancestry

Accurate phenotyping of red blood cells (RBCs) can be difficult in transfusion-dependent patients such as those with thalassemia and sickle cell anemia because of the presence of previously transfused RBCs in the patient's circulation. Recently, the molecular basis associated with the expression of many blood group antigens was established. This allowed the development of a plethora of polymerase chain reaction (PCR)-based tests for identification of the blood group antigens by testing DNA. The new technologies complement phenotyping and overcome some of the limitations of hemagglutination assays. These molecular assays were developed on the basis of DNA sequences of individuals of Caucasian ancestry. The present study addresses the concern that these genotyping assays may not be applicable to populations of highly diverse ancestry because of variability in intronic regions or because of unrecognized alleles. We determined both phenotype and genotype for RH D, K 1/K 2, JK A/JK B, FY A/ FY B-GATA in 250 normal blood donors using PCR. Phenotype and genotype results agreed in 100% of the cases, indicating that molecular genotyping protocols can be effectively applied to populations with a highly diverse genetic background. However, genotyping for Duffy antigens provided information that could not be obtained by phenotyping. Essentially, 30.5 % of the donors with the FY B gene typed as Fy(b-) because of mutations in the GATA box. This information is very useful for the management of transfusion dependent patients.     

Importance of extended blood group genotyping in multiply transfused patients

Blood group antigen systems are not limited to the ABO blood groups. There is increasing interest in the detection of extended blood group systems on the red cell surface. The conventional method used to determine extended blood group antigens or red cell phenotype is by serological testing, which is based on the detection of visible haemagglutination or the presence of haemolysis. However, this technique has many limitations due to recent exposure to donor red cell, certain drugs or medications or other diseases that may alter the red cell membrane. We aimed to determine the red cell blood group genotype by SNP real time PCR and to compare the results with the conventional serological methods in multiply transfused patients. Sixty-three patients participated in this study whose peripheral blood was collected and blood group phenotype was determined by serological tube method while the genotype was performed using TaqMan^® Single Nucleotide Polymorphism (SNP) RT-PCR assays for RHEe, RHCc, Kidd and Duffy blood group systems. Discrepancies were found between the phenotype and genotype results for all blood groups tested. Accurate red blood cell antigen profiling is important for patients requiring multiple transfusions. The SNP RT-PCR platform is a reliable alternative to the conventional method.     

Fetal blood group genotyping

Blood group genotyping using DNA extracted from fetal tissue is useful to identify fetuses at risk for hemolytic disease of the fetus and newborn (HDFN) due to maternal red cell alloantibodies. Four considerations are important for fetal blood group genotyping. First, paternal heterozygosity must be established, including tests that evaluate RHD hemizygosity. Second, the source of fetal tissue for DNA extraction requires certain considerations. Third, because the fetal genotype is used to predict the expressed phenotype, a thorough knowledge of blood group genetics is required. Moreover, the test algorithm should include the evaluation of the parental phenotypes and genotypes to help identify variant alleles. Fourth, the blood group antigen expression at birth should be evaluated to confirm the inheritance. The identification of an antigen-negative fetus on the basis of the blood group genotype provides significant advantages in managing the pregnancy at risk for HDFN. In the near future, fetal DNA in maternal plasma will likely replace fetal blood group genotyping for RHD . Significant challenges remain to detect other clinically significant blood group antigens using maternal plasma DNA.     

Emerging strategies of blood group genotyping for patients with hemoglobinopathies

Red cell alloimmunization is a serious problem in chronically transfused patients. A number of high-throughput DNA assays have been developed to extend or replace traditional serologic antigen typing. DNA-based typing methods may be easily automated and multiplexed, and provide reliable information on a patient. Molecular genotyping promises to become cheaper, being not dependent on serologic immunoglobulin reagents. Patients with hemoglobinopathies could benefit from receiving extended genomic typing. This could limit post transfusional complications depending on subtle antigenic differences between donors and patients. Patient/donor compatibility extended beyond the phenotype Rh/Kell may allows improved survival of transfused units of red blood cells (RBC) and lead to reduced need for blood transfusion and leading to less iron overload and reduced risk of alloimmunization. Here we discuss the advantages and limitations of current techniques, that detect only predefined genetic variants. In contrast, target enrichment next-generation sequencing (NGS) has been used to detect both known and de novo genetic polymorphisms, including single-nucleotide polymorphisms, indels (insertions/deletions), and structural variations. NGS approaches can be used to develop an extended blood group genotyping assay system.     

Application of RHD and RHCE genotyping for correct blood group determination in chronically transfused patients

BACKGROUND: In chronically transfused patients, conventional blood group typing may be impossible because of mixed-field agglutination. STUDY DESIGN AND METHODS: In 27 patients with congenital anemia and lifelong transfusion history, genotyping for D, RHD, and RHCE was performed with polymerase chain reactions. These results were compared with the blood group typing results documented in the medical record. RESULTS: Two of 27 cases had been typed D-negative by serologic tests and D-positive by genotyping. In 20 patients, the CDE formula had been determined serologically according to the medical record; 4 of these patients were Cc by serologic tests and C/C by genotyping. One patient typed ee by serologic tests, and genotyping revealed heterozygosity (E/e). CONCLUSION: In patients with a lifelong transfusion history, serologic blood group determination may be impossible, and pretransfusion test results are not always available or reliable. In whites, Rh-matched transfusions are possible with genotyping. The genetic background of the RH genes has to be elucidated in other ethnic groups, such as in black patients with sickle cell disease, before genotyping can be applied without restriction.     

Blood group genotyping by high‐throughput DNA analysis applied to 356 reagent red blood cell samples

BACKGROUND: DNA analysis for the prediction of blood group antigen expression has broad implications in transfusion medicine. It may be of particular interest especially to detect variants, when antigen expression is weak or altered. The use of high‐throughput DNA analysis has never been applied to donors whose red blood cells (RBCs) are selected for reagent RBCs. The aim of this study was to analyze the concordance between the serologic phenotype and that predicted from DNA analysis in panel donors, to determine the benefit of the use of DNA analysis in reagent RBC selection strategy. STUDY DESIGN AND METHODS: The “Panel National de Référence du Centre National de Référence sur les Groupes Sanguins” is a reference reagent mainly used for antibody identification. DNA genotyping of 356 panel donors was performed with BeadChips (human erythrocyte antigen v1.2 BeadChips, BioArray Solutions). The comparison between serologic phenotype and that predicted from DNA analysis held on 8876 paired results obtained from 10 blood group systems and 25 antigens. RESULTS: A 99.95% concordance was observed. Discrepancies in four cases (RH, KEL, LU, and DO systems) were analyzed. Genotyping precisions on the Duffy system were of particular interest. No new rare blood group was observed. CONCLUSION: Systematic DNA analysis of panel donors should unquestionably change the management of reagent RBC selection. The notion of “antigens in double dose” should evolve regarding data obtained from DNA analysis, allowing an improved quality of reagent RBCs for antibody screening and identification.     

Benefits of blood group genotyping in multi‐transfused patients from the south of Brazil

We evaluated the usefulness of blood group genotyping as a supplement to hemagglutination to determine the red blood cell (RBC) antigen profile of polytransfused patients with hematological diseases and renal failure. Seventy‐nine patients were selected. They all received more than three units of blood and eight (10%) had already clinical significant alloantibodies occurring alone or in combination against Rh, K, Fya, and Di antigens. DNA was prepared from blood samples and RHCE*E/e, KEL*01/KEL*02, FY*01/FY*02 and JK*01/JK*02 alleles were determined by using PCR‐RFLP. RHD*/RHD*Ψ and RHCE*C/c were tested using multiplex PCR. Discrepancies for Rh, Kell, Duffy, and Kidd systems were found between the phenotype and genotype‐derived phenotype in 16 of the 38 chronically transfused patients. The genotypes of these patients were confirmed by DNA array analysis (HEA Beadchip^?; Bioarray Solutions, Warren, NJ). Genotyping was very important for the determination of the true blood groups of the polytransfused patients, helped in the identification of suspected alloantibodies and in the selection of antigen‐negative RBCs for transfusion. J. Clin. Lab. Anal. 24:311–316, 2010. © 2010 Wiley‐Liss, Inc.     

Blood group phenotypes in Taiwan

This report describes a study of 31 red cell antigens in 13 blood group systems tested over a period of 3 years in the Chinese population of Taiwan. The study provides evidence that major differences exist between Taiwanese and whites or blacks in five blood group systems: Rh, MNSs, Duffy, P, and Xg.     

ABO blood group genotyping by quenching probe method

We developed a multiplex ABO genotyping method with quenching probes (Q-probe). In this method, it is possible to discriminate the mutations, not only frequently used positions 261 and 796 but also position 703 in a single PCR. Each probe was designed to have cytosine residue at 5' or 3' end and labeled with three different fluorescence dyes, enabling the triplex detections of these polymorphisms. All polymorphisms were successfully detected by using fluorescence labeled Q-probe in a specifically amplified PCR product. Each Q-probe showed unique dissociation patterns depending on the polymorphism types. All of the results obtained with Q-probe were compared with standard serotyping and TaqMan PCR method and resulted in complete match with each other. Consequently, these results indicated that multiplex ABO genotyping method is quite accurate and convenient method for the determination of ABO genotype.     

Massively parallel and multiplex blood group genotyping using next-generation-sequencing

ObjectivesThirty-six blood group systems are listed by the International Society of Blood Transfusion, containing almost 350 antigens. Most of these result from a single nucleotide polymorphism (SNP). Serology is the standard method for blood group typing. However, this technique has some limitations and cannot respond to the growing demand of blood product typing for a large number of antigens. Here we describe a blood group genotyping assay directly from whole blood samples using Next-Generation Sequencing (NGS), allowing the simultaneous identification of 15 SNPs associated with the blood group systems of 95 patients in a single run.Design and methodAfter an automated DNA extraction, targets are amplified by multiplex polymerase chain reaction (PCRm). Two panels addressing 9 groups have been developed (MNS, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, and Colton), one for 8 SNPs, the other for 7 SNPs. For each sample, both panels corresponding to 14 amplicons (1 amplicon containing 2 SNPs) are pooled. Then a dual-indexed library is generated from each pool by linking Illumina adaptors directly onto amplicons, followed by sequencing using the MiSeq platform (Illumina).ResultsIn a single experiment, 95 blood donor samples have been sequenced for the genes of interest. Among the 1425 targeted single nucleotide polymorphisms, 1420 were identified by sequencing, reflecting a coverage of 99.65%. The obtained data shows a good correlation (99% for all SNPs) with other blood group typing methods. Depending on the allele pairs analyzed, correlations vary between 97.12 and 100%.Conclusion: Next-Generation sequencing would supplement serological and molecular techniques and, in the near future, could replace it with complete and fast results acquisition for pre-screening and identification of rare blood bags.     

四川省汉族人群Duffy血型基因分型研究

目的研究四川地区Duffy血型基因型分布,为建立红细胞库奠定基础。方法对150名汉族健康献血者采用PCR-SSP方法进行Duffy血型系统进行基因分型检测。结果四川地区汉族人群Duffy基因分型检测结果,Fya基因频率为:0.929.Fyb:0.071;检出3例Fy(a-b-)个体。结论 PCR-SSP法检测Duffy血型简单、快捷、准确,为建立红细胞库奠定了基础。     

尖锐湿疣患者及高危人群HPV DNA分型检测结果分析

目的分析尖锐湿疣（CA）患者及高危人群人乳头瘤病毒（HPV）的DNA分型检测结果。方法应用荧光定量聚合酶链反应（FQ-PCR）对448倒CA患者及高危人群进行HPVDNA分型检测。结果448例检测总阳性率为47．77％,男、女阳性率分别为44．85％和50．00％,其中6／11型181例,16／18型20例,6／11与16／18型13例。126例CA疣体中HPV阳性率为98．41％,其中男、女HPV阳性率分别为98．33％和98．48%。322例高危人群HPV阳性率为27．95％,其中男、女HPV阳性率分别为20．90％和34．57％,二者差异有统计学意义（P〈0．05）。性伴侣或配偶患CA者、CA治疗后复查者、非CA患者及自检者的HPV阳性率分别为32．76％、36．25％、18．99％和25．71％。结论CA患者及高危人群检测中HPV以6／11型最多。荧光定量PCR可作为CA患者早期诊断的指标。CA高危人群HPV检出率均较高,且女性高于男性,应重视对这些高危人群的HPV常规检测。     

Molecular genotyping of Indian blood group system antigens in Indian blood donors

Background

Haemagglutination has been the gold standard for defining the blood group status. However, these tests depend upon the availability of specific and reliable antisera. Potent antisera for extended phenotyping are very costly, weakly reacting or available in limited stocks and unavailable for some blood group systems like Indian, Dombrock, Coltan, Diego etc. The Indian blood group system consists of two antithetical antigens, In^a and In^b. The In^a /In^b polymorphism arises from 252C?>?G missense mutation in the CD44 gene. This knowledge has allowed the development of molecular methods for genotyping IN alleles.

SNaPshot技术应用于西藏高原人群Duffy血型基因分型

目的应用SNaPshot技术对Duffy血型进行基因型检测,对西藏高原人群Duffy血型的分布进行研究,为临床输血工作提供指导依据。方法采集725份西藏高原人群静脉血,提取DNA,进行引物合成与PCR扩增,再通过SNaPshot检测技术对Duffy血型第125位的SNP位点进行检测,计算遗传学相关参数,并与文献公布的各地区人群及千人基因组计划公布的非洲、东亚、欧洲、南亚、美国等人群和遗传基因组数据库公布的非洲、东亚、其他、美国以及犹太人的基因频率数据进行比较。结果 725份血液标本中Fya+b-、Fya-b+和Fya+b+的表型分别为629例(86.76%)、16例(2.21%)和80例(11.03%),FYa和FYb的基因频率分别为92.28%和7.72%。与各人群基因频率相比,在国内各地区不同民族之间的血型等位基因频率没有明显差异,在亚洲人群中的差异不明显,但与非洲、欧洲、南亚、美国、犹太人相比有显著差异。结论西藏高原人群的Duffy血型分布与国内各地区人群分布一致,但与其他种族人群有明显差异。