Molecular genotyping of Indian blood group antigens amongst regular voluntary blood donors of Surat city,Gujarat, India

BackgroundThere is paucity of data related to the prevalence of the rare blood group antigens amongst South Gujarat blood donor population due to unavailability and high cost of antisera. Therefore it is difficult to screen donors for such rare antigens by gold standard haemagglutination assay. The single nucleotide polymorphism (SNPs) of In^a and In^b antigens is the base of the PCR based detection methods that help to detect these alleles in regular voluntary blood donors.Materials & methodsBlood samples of 200 unrelated regular voluntary blood donors wee collected. DNA was extracted using phenol-chloroform method and genotyped for Indian (In^a/IN*01, In^b/IN*02) blood group alleles by Sequence Specific PCR. In^a antigen positivity was confirmed by serology test.ResultsFour donors were found heterozygous for In^a antigen i.e. In (a + b+) by SS-PCR and their In^a positivity were confirmed by in-house polyclonal Anti-In^a reagent. SS-PCR was standardized using known heterozygous sample of a blood donor. The frequency of In^a antigen (2.0 %) was higher than Caucasians, lower than Iranians and Arabs while comparable to those reported among Indians of Mumbai city.ConclusionIn absence or unavailability of antisera particularly for low frequency alleles like In^a, such PCR based method would be extremely helpful to prepare rare donor registry by screening blood donors’ at large scale. Red cells of In^a positive donors can be used as in-house reagent red cells for screening and identification of corresponding antibody.     

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.     

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.     

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.     

深圳献血人群Duffy血型基因多态性的研究

目的建立Duffy血型的分子生物学检测方法,研究深圳献血人群Duffy血型基因多态性。方法在2011年8～11月本中心900名无血源关系献血者标本中,用卡式微柱凝胶抗球蛋白方法鉴定Duffy血型表现型,建立Duffy血型的基因分型方法 PCR-SSP法及PCR产物直接测序法,对900例标本DNA进行PCR-SSP法检测,其中50例做Duffy血型基因编码区域序列测定。结果血清学结果为Fy(a+b-)855例,Fy(a+b+)43例,Fy(a-b+)2例,Fy(a-b-)0例。900例Duffy血型PCR-SSP法基因分型结果与血清学表现型完全一致。50例FY基因编码序列部分测序结果与血清学、PCR-SSP法吻合,表现型FY(a+b-)标本测序结果可见FY基因第2外显子第125位核苷酸碱基为纯合子G;表现型FY(a-b+)标本测序结果为第125位核苷酸碱基为纯合子A。结论 Duffy血型PCR-SSP基因分型方法及PCR产物直接测序法可以正确地鉴定Duffy基因型,适用于Duffy血型基因多态性的研究。深圳地区Fya的基因频率为0.973 9,Fyb的基因频率为0.026 1。     

聚合酶链式反应-特异性序列引物基因定型在新生儿溶血病ABO血型定型中的应用

目的研究聚合酶链式反应-特异性序列引物(PCR-SSP)基因定型在新生儿溶血病(CHDN)ABO血型定型中的应用。方法收集20例来自广东省肇庆市第一人民医院新生儿溶血病(HDN)的样本,对这些样本进行ABO血型鉴定等一系列血型血清学方法进行检测,然后用PCR-SSP基因定型技术进行样本的基因分型。结果与其血型的血清学分型结果进行比较,新生儿溶血病ABO血型的定型是新生儿输血的必要保障,而PCR-SSP基因分型较血型血清学方法更快捷、准确。结论 PCR-SSP基因正型技术将不再局限于疑难血型的鉴定中,并会越来越多地应用于HDN的鉴定中。     

Blood group antigens frequencies in the northeast of Iran

Background

Identification of blood group antigen frequencies in a population has various benefits in transfusion medicine. Most data in the literature include frequencies of these antigens in European and American countries. In this study for the first time we have reported frequencies of these antigens in the northeast of Iran.

Materials and methods

Blood group antigens were characterized in the 522 blood donors in Mashhad, Iran. The following antigens including ABO, Rh (D, C, E, c, e), MNSs (M, N, S, s), Lutheran (Lu^a, Lu^b), P (P, P₁), Kell (K, k, Kp^a, Kp^b), Lewis (Le^a, Le^b), Duffy (Fy^a, Fy^b) and Kidd (Jk^a, Jk^b) were typed and phenotypes frequencies were expressed as a percentage.

Results

In the ABO blood group, the most common phenotype was O (33.9%) followed by A, B and AB. In the Rh blood group, the most common antigen was e (97.9%) and R₁r (31.8%) being the most common phenotype. The most common phenotypes for MNSs, P, Lu and Kell blood group were M+N+, S−s+, P₁, Lu (a−b+), K−k+ and Kp (a−b+). A very rare phenotype of Lu (a−b−) was also observed in 2.7% of cases. We found rare phenotypes of Le (a+b+) and Fy (a−b−) in 7.9% and 3.4% of subjects, respectively.

Conclusion

We determined some differences in phenotype frequencies of blood group compared with other studies. We found higher frequencies of B blood group and also more frequencies of some rare phenotypes, Lu (a−b−), Le (a+b+) and Fy (a−b−).     

Molecular basis of glycophorin C variants and their associated blood group antigens

SUMMARY. The normal and variant forms of GPC and GPD molecules carry antigens of the Gerbich blood group system. This blood group system comprises three high-incidence antigens (Ge2, Ge3 and Ge4) and four low-incidence antigens (Wb, Ls^a, Dh^a and An^a). Erythrocytes of the Ge and Yus phenotypes lack normal GPC and GPD molecules but express variant molecules (denoted GPC.Ge, GPC.Yus, respectively) that functionally substitute for normal GPC and GPD in the membrane. Leach phenotype cells lack GPC and GPD molecules and are elliptocytic in shape with a membrane that is less deformable than that of normal cells. The Ls^a antigen is expressed on higher molecular-weight variants of GPC (GPC.Ls^a) and GPD (GPD.L3^a). Wb, Dh^a and An^a antigens arise from point mutations in the GYPC gene and are expressed on GPC.Wb, GPC.Dh^a and GPD.An^a, respectively. The structure of each of the variant GPC and GPD molecules and the location of the Gerbich blood group system antigens is discussed. The GYPC gene, located on chromosome 2q¹⁴-q²¹, is 13.5 kb long and comprises four exons. Exons 1,2 and most of exon 3 encode the N -terminal extracellular domain while the remainder of exon 3 and exon 4 encode transmembrane and cytoplasmic domains of GPC. Exons 2 and 3 are highly homologous, with less than 5% nucleotide divergence. The molecular basis of generation of variation GPC and GPD molecules, and the structure of the GYPC gene from different Leach phenotype individuals, is discussed.     

PCR-SSP与血清学技术用于ABO血型分型的比较

目的 探讨ABO血型基因分型技术应用于临床检验的可行性.方法 随机抽样100例江西健康汉族个体,并收集20例正反定型不符的标本,采用血清学和序列特异性引物PCR法(PCR-SSP)鉴定血型.结果 100例健康个体的基因分型结果与血清学分型结果一致,19例正反定型不符标本的基因分型结果准确.分型结果表明江西地区以O型为多,100例健康个体中,O型36例(36%),A型32例(32%),B型24例(24%),AB型8例(8%),基因频率:p(A)基因为0.225 3,q(B)基因为0.175 3,r(O)基因为0.599 8,且符合Hardy-Weinberg平衡.结论 基因分型技术应用于临床检验具有可行性,在鉴定疑难血型方面可以作为血清学分型的补充.     

The abundance and organization of polypeptides associated with antigens of the Rh blood group system

Summary. Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA 18.18, LA23.40) recognize related structures and 100,000–200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRTCs 32, 122,125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent el al. , 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M _r 30,000 and M _r 35,000–100,000, respectively, and suggest that there are 1–2 × 10⁵ copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex.     

RhD血型基因定型在新生儿溶血病产前诊断的应用

目的 检测血液RhD血型基因型并应用于新生儿溶血病(HDN)的产前诊断。方法 采用聚合酶链反应-序列特异性引物(PCR-SSP)方法,根据PCR产物的强弱判定RhD基因型。结果36名个体血液样本中,10名为RhD-／RhD-纯合子,3名为RhD ／RhD-杂合子,23名为RhD ／RhD-纯合子。结论 该方法可以准确检测出RhD血型基因型,并可用于RhD血型不合引起HDN的产前诊断。     

Classification of major and minor blood group antigens in the Kuwaiti Arab population

Ethnic differences in blood group frequencies might result in clinically important mismatches for transfusions. Arab people represent a large population for which no comprehensive database of red cell genotypes is available and Kuwaitis are no exception. For instance, the Rh blood group is the most elaborate blood group system that shows a high degree of polymorphism among different ethnic groups, there has been little classification of RH alleles in Arab people. Blood samples from 917 Kuwaiti Arab donors in the Kuwaiti Bone Marrow registry were tested with a single-nucleotide polymorphism DNA array. Blood group antigen prevalence were compared to known prevalence in European populations. Multiple subjects were found to be antigen negative for certain phenotypes that is considered rare by the American Rare Donor Program; (Fy(a-,b-) and Kell). In the minor blood group antigens, the FYA allele was predicted to be low in Kuwaitis, when compared to other published accounts. The frequencies of MNS blood antigens in the study population were not significantly different from those reported for European/Caucasian populations. The predicted frequency of the Diego blood group antigen was similar to that observed in a South Asian population. The weak D 1, 2, 3 phenotypes were not prevalent in the Kuwaiti Arab population; however, other RHD variants were detected. We provided information about blood group antigens in the Kuwaiti population that is important for guiding transfusion care. Several interesting findings demonstrated clinical importance, which could be useful in developing transfusion medicine policies and approaches.     

罕见B(A)血型的鉴定

目的观察2例罕见B(A)血型的血清学特征并研究其分子机制。方法用血型血清学鉴定2例献血者标本ABO血型,用序列特异性引物聚合酶链反应(PCR-SSP)基因分型和直接测序确定其基因型。结果 2例献血者血型血清学检测结果均表现为B(A)亚型的特点。标本1基因分型为BO2,测序结果为第7外显子B基因发生640AG突变,符合B(A)04/O02的基因型特点;标本2基因分型为BO1,测序结果为第7外显子B基因发生700CG突变,符合B(A)02/O01的基因型特点。结论2例标本均为B(A)表现型,基因型分别为B(A)04/O02和B(A)02/O01。     

Neopterin concentrations in blood donors differ between AB0 blood group phenotypes

OBJECTIVES: Neopterin is produced by human monocyte-derived macrophages upon stimulation with interferon-gamma and is therefore a sensitive indicator for cellular immune activation. Common factors like age, diastolic blood pressure, body mass index, or smoking habits were found to be associated with neopterin concentrations in humans. DESIGN AND METHODS: In order to find possible genetic determinants which might influence neopterin production, we investigated 8288 consecutive blood donors after exclusion of samples suspicious of infections. RESULTS: Donors with blood group phenotype 0 had moderately, but significantly (P < 0.0001) higher neopterin concentrations (mean +/- SD: 6.94 +/- 1.52 nmol/L) than those with phenotype A (6.75 +/- 1.50 nmol/L), phenotype B (6.73 +/- 1.48 nmol/L), and phenotype AB (6.68 +/- 1.57 nmol/L). CONCLUSIONS: Neopterin levels are higher in donors with blood group phenotype 0 than in other phenotypes. Data point to a genetic background of different neopterin concentrations. However, alterations of neopterin levels were much less expressed than the changes known to occur during diseases with an activated immune response.     

Pattern of distribution of 35 red cell antigens in regular voluntary blood donors of South Gujarat,India

Background

Extended phenotyping is one of the important method of reducing red cell alloimmunisation. Extended phenotyping of red cells from voluntary donors have many uses in addition to its application in population genetics. As there was very little data extended phenotyping on a cohort of Indian Voluntary blood donors this project was undertaken.

天津市滨海新区Rh+无偿献血者Rh血型表型的分布调查

目的 探讨天津市滨海新区Rh+献血者Rh血型表型的分布情况,建立Rh+血型表型数据库,确保临床输血安全,减少输血不良反应的发生.方法 采用简单随机抽样法选择2013年1月至2015年12月天津市第五中心医院输血科留存的2 672份Rh+库存悬浮红细胞为研究对象.研究对象纳入标准:①所有悬液红细胞均来自天津市滨海新区中心血站;②血液经Rh系统中抗-D检测,确定为Rh+血型;③血液入库时,严格核对运输条件、物理外观、血袋封闭及包装、标签等,均应符合血液入库标准.采用微柱凝胶法进行Rh血型系统的抗-D、抗-C、抗-c、抗-E、抗-e检测,并根据抗原检测结果计算Rh血型各表型频率.结果 本组2 672例Rh+无偿献血者的Rh抗原中,按照抗原阳性率由高至低排序,依次为抗-D(100.0％)、抗-e(90.8％)、抗-C(86.7％)、抗-c(58.2％)及抗-E(48.8％)抗原.本组2 672例Rh+无偿献血者中,共检测出9种Rh血型表型,按照抗原频率由高至低排序,依次为CCDee (40.5％)、CcDEe(35.5％)、CcDee(9.4％)、ccDEE(9.2％)、ccDEe(4.1％)血型表型,其他4种表型仅占1.3％(35/2 672).结论 天津市滨海新区Rh+无偿献血人群的Rh血型表型以CCDee为主.建立Rh+血型表型数据库,可为临床及时提供表型相合的Rh+血液,防止由于Rh血型系统不合引起的输血反应,确保临床输血安全.     

序列特异性引物PCR法检测MN血型基因型

目的　建立MN血型系统基因分型的方法 ,检测人群中MN基因的频率。方法　用快速盐析法抽提样本DNA ,序列特异性引物PCR法检测MN基因。结果　 115例汉族人群中MM基因型为 2 5例 ,MN基因型为 6 0例 ,NN基因型为 30例。M基因频率为 0 .4 78,N基因频率为 0 .5 2 2。结论　该方法可以检测MN血型基因型。浙江汉族中N基因频率大于M基因频率     

Frequency distribution of human platelet antigens in the Indian population

This study was undertaken with an aim of establishing the frequency distribution of various human platelet antigens (HPA) in Indian populations by means of DNA-based technology. A total of 1164 people belonging to various population groups were studied for the frequency distribution of HPA. DNA extraction was performed from peripheral venous blood samples. Polymerase chain reaction allele-specific amplification technique was used for HPA genotyping. The HPA bands were visualized by using ethidium bromide-stained agarose gel, after electrophoresis. The homozygosity of the HPA-1b/1b genotype was found to be significantly higher (P < 0.05) in the Parsi population group and Vatalia Prajapati population group, compared to Maharashtrians. Frequency distribution of HPA-1b in our populations was found to be slightly lower than that reported in some western populations. This study has established a DNA technique to diagnose cases of NAITP definitively and to treat these cases during the neonatal period, and also gives the frequency distribution of HPA in some of the Indian population.