Automated typing of red blood cell and platelet antigens: a whole-genome sequencing study.

BACKGROUND: There are more than 300 known red blood cell (RBC) antigens and 33 platelet antigens that differ between individuals. Sensitisation to antigens is a serious complication that can occur in prenatal medicine and after blood transfusion, particularly for patients who require multiple transfusions. Although pre-transfusion compatibility testing largely relies on serological methods, reagents are not available for many antigens. Methods based on single-nucleotide polymorphism (SNP) arrays have been used, but typing for ABO and Rh-the most important blood groups-cannot be done with SNP typing alone. We aimed to develop a novel method based on whole-genome sequencing to identify RBC and platelet antigens. METHODS: This whole-genome sequencing study is a subanalysis of data from patients in the whole-genome sequencing arm of the MedSeq Project randomised controlled trial (NCT01736566) with no measured patient outcomes. We created a database of molecular changes in RBC and platelet antigens and developed an automated antigen-typing algorithm based on whole-genome sequencing (bloodTyper). This algorithm was iteratively improved to address cis-trans haplotype ambiguities and homologous gene alignments. Whole-genome sequencing data from 110 MedSeq participants (30 × depth) were used to initially validate bloodTyper through comparison with conventional serology and SNP methods for typing of 38 RBC antigens in 12 blood-group systems and 22 human platelet antigens. bloodTyper was further validated with whole-genome sequencing data from 200 INTERVAL trial participants (15 × depth) with serological comparisons. FINDINGS: We iteratively improved bloodTyper by comparing its typing results with conventional serological and SNP typing in three rounds of testing. The initial whole-genome sequencing typing algorithm was 99·5% concordant across the first 20 MedSeq genomes. Addressing discordances led to development of an improved algorithm that was 99·8% concordant for the remaining 90 MedSeq genomes. Additional modifications led to the final algorithm, which was 99·2% concordant across 200 INTERVAL genomes (or 99·9% after adjustment for the lower depth of coverage). INTERPRETATION: By enabling more precise antigen-matching of patients with blood donors, antigen typing based on whole-genome sequencing provides a novel approach to improve transfusion outcomes with the potential to transform the practice of transfusion medicine. FUNDING: National Human Genome Research Institute, Doris Duke Charitable Foundation, National Health Service Blood and Transplant, National Institute for Health Research, and Wellcome Trust.

Sensitive detection of platelet-specific antibodies with a modified MAIPA using biotinylated antibodies and streptavidin-coated beads.

We have developed a modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) with enhanced sensitivity in detecting antibodies against human platelet antigens (HPA), using biotinylated monoclonal antibodies, streptavidin-coated beads and detection by flow cytometry. The beads-MAIPA gave superior signal-to-noise resolution (>10-fold higher) for detection of anti-HPA-1a and anti-HPA-5b compared with the in-house standard MAIPA. Also, efficient and reproducible detection of anti-HPA-15 (CD109) was shown. The enhanced sensitivity was confirmed using WHO International Reference Reagents for anti-HPA-1a, anti-HPA-3a and anti-HPA-5b, which allowed comparison of detection endpoints with other laboratories. Finally, the beads-MAIPA was validated for quantification of anti-HPA-1a. The lower limit of quantification was 0.4IU/mL for beads-MAIPA, compared to 1IU/mL previously reported for standard MAIPA. Based on improved performance against all HPA-antibodies tested, the beads-MAIPA has replaced the standard MAIPA in our laboratory in diagnostics of conditions due to HPA-immunization, such as fetal and neonatal alloimmune thrombocytopenia (FNAIT).

Consortium for Blood Group Genes (CBGG): 2009 report.

Consortium for Blood Group Genes is a worldwide organization whose goal is to have a vehicle to interact, establish guidelines, operate a proficiency program, and provide education for laboratories involved in DNA and RNA testing for the prediction of blood group, platelet, and neutrophil antigens. Currently, the consortium operates with representatives from Brazil, Canada, and the United States. Membership is voluntary with the expectation that members actively contribute to discussions involving blood group genetics. This year witnessed a change in the standing committee membership and the institution of a representative for the human platelet antigens group. Looking forward, the consortium sees challenges for the nomenclature of blood group alleles and user-required specifications for laboratory information systems to store genotype information.

Establishment of an HPA-1- to -16-typed platelet donor registry in China.

In order to determine gene frequencies of human platelet antigen (HPA) and establish a panel of accredited HPA-1a, -2a, -4a, -5a and -6a-negative donors as well as an HPA-typed platelet donor registry, a total of 1000 Chinese donors of Han nationality (500 from north China and 500 from south China) were typed for HPA-1 through -16 using a DNA-based polymerase chain reaction with sequence-specific primers genotyping method. The gene frequencies of HPA-1b, -2b, -3b, -4b, -5b, -6bw, -10bw and -15b were 0.0060, 0.0485, 0.4055, 0.0045, 0.0140, 0.0135, 0.0005 and 0.4680, respectively. The HPA-7bw, -8bw, -9bw, -11bw, -12bw, -13bw, -14bw and -16bw alleles were not found. The HPA-2b and -5b homozygous donors were detected at low frequencies. The HPA mismatch probabilities potentially leading to alloimmunization in random platelet transfusion vary with a region from 0.1% to 37% depending on the distribution patterns of common and less common alleles in each system. This study provides a useful HPA-typed plateletpheresis donor registry in China and could improve platelet antibody detection and HPA-matched platelet transfusion in alloimmune thrombocytopenic patients.

Gene frequencies of human platelet antigens in the Macedonian population.

Human platelet antigen (HPA) systems consist of more than 12 bi-allelic antigen polymorphisms. Due to these polymorphisms, platelet-membrane glycoproteins can be recognized as alloantigens or autoantigens and can cause conditions such as fetomaternal alloimmune thrombocytopenia, post-transfusion refractoriness to platelets, and post-transfusion throbocytopenic purpura. The purpose of this study was to investigate the distribution of HPA-1, -2, -3, and -5 in Macedonian population by using the polymerase chain reaction and restriction fragment length polymorphism. The allele frequencies were 0.865 for HPA-1a, 0.135 for HPA-1b, 0.852 for HPA-2a, 0.148 for HPA-2b, 0.578 for HPA-3a, 0.422 for HPA-3b, 0.909 for HPA-5a, and 0.091 for HPA-5b. Results of our study were not significantly different from those reported in the other European studies. Our population displayed the highest frequency for HPA-2b allele (0.148) reported among European population.

[Advances in the studies on human platelet alloantigen--review].

Human platelet alloantigens (HPA) are specific antigens carried by platelet glycoproteins, which genes showing single nucleotide polymorphism. HPA can induce alloantibodies bringing about alloimmune response. They play important roles in post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura, fetomaternal alloimmune thrombocytopenia, and graft-versus-host disease. Because of their side effects in clinical blood-transfusion, there were a great deal of studies on HPA during last few decades. This review focuses on the nomenclature of HPA, the polymorphisms of platelet glycoproteins, HPA typing of the serological and molecular technology, as well as the mechanism of alloimmunization to HPA and correlated diseases.

[Analysis of genetic polymorphism in randomized donor's HPA 1-16 antigens and establishment of typed platelet donor data bank].

To study the genetic polymorphism of HPA 1-16 platelet antigen alleles among unrelated volunteer donors and establish a typed platelet donor panel in Handan, typing was perfomed by polymerase chain reaction using sequence-specific primers (SSP-PCR); 148 random unrelated blood donors in Handan were genotyped for each of the HPA 1-16 antigen. The gene frequencies were analyzed and the genetype frequencies were determined by direct counting, and these data were compared with HPA distribution among various population by the chi-square test. The results indicated that HPA-1a, 2a, 4a-14a, 16a genes were found among the 16 HPAs in every sample tested. Monomorphic HPA-4a, 7a-14a, 16a were found in the samples. For HPA-1, 2, 5 and 6, a/a homozygosity was predominant with frequencies of 0.9595, 0.8108, 0.9865, 0.9797, respectively, and none of HPA b/b was found in the samples. HPA-1b, 2b, 5b, 6b were rarely found among subjects. HPA-15 had the greatest heterozygosity with a gene frequency of 0.2230, 0.5270, 0.2500 for HPA15a/15a, HPA15a/15b, HPA15b/15b, respectively. HPA-3 showed the second greatest heterozygosity with a gene frequency of 0.3851, 0.5135, 0.1014 for HPA3a/3a, HPA3a/3b, HPA3b/3b, respectively. HPA genotype frequencies showed a good fit to Hardy-Weinberg equilibrium. HPA1-5 gene frequencies for Chinese people in Handan were consistent with those of Chinese people in Shijiazhuang (P > 0.05). Among the HPA1-13, -15, the frequencies of HPA-1, -2, -6 for Chinese people in Handan differed appreciably from those for Chinese people in Taiwan (P < 0.05), others were similar to those of Chinese people in Taiwan. Among the HPA 1 - 8, a similarity was noted between Chinese people in Handan and Koreans (P > 0.05), except for HPA-3. Frequencies of HPA-1, -2, -5 significantly were differed from those in African Americans, as compared with HPA 1-5 (P < 0.05). Comparison of gene frequencies from HPA-1 and -5 showed significant differences between Chinese people in Handan and people in UK (P < 0.05). It is concluded that HPA-2, -3, -5, -15 of people in Western region of China have polymorphism, incompatible frequency of HPA antigen distribution is higher, which inevitably results in the increase of immunologic exposure, therefore attention must be paid to the importance of HPA-2, -3, -5, -15 in clinical disorders. This study for the first time completely analyses HPA1-16 gene frequencies in China, and provides data for establishing a typed platelet donor panel in Handan, China.

[Study on the simultaneous genotyping of human platelet antigens of 1, 2, 3, 4, 5, 6 system by PCR-SSP and its applications].

To set up the simultaneous genotyping of human platelet antigens of 1,2,3,4,5,6 system by PCR-SSP assay and use the genotyping method for the study of platelet antigens. In this study, 18 sequence-specific primers were designed and synthesized. The annealing temperature for all sequence-specific primer pair, the concentration of each primer pair and the concentration of Mg2+ were adjusted to the optimum so that HPA-1 to 6 systems could be amplified simultaneously under the same PCR cycling parameters. The electrophoresis of PCR products was conducted simultaneously on the same agarose gel. Control DNA samples that genotypes known were used to confirm the sensitivity and specificity of each sequence-specific primer. 15 coded samples (including 2 blood samples and 13 DNA samples) distributed by 10TH Platelet Genotyping and Serology Workshop of the International Society of Blood Transfusion (ISBT) were typed for HPA-1 to 6 systems by this method. A concordance rate of 100 percent was observed between the results of control DNA samples typed by our PCR-SSP assay and the data of known specificity of control DNA. The results of 15 coded samples tested by our method agreed well with the results provided by ISBT report.

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms.

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.

[Fetal-neonatal alloimmune thrombocytopenia]. / Trombocitopenia aloinmune en el feto y en el recién nacido.

Fetal-neonatal alloimmune thrombocytopenia is the commonest cause of severe thrombocytopenia in the newborn. This disorder is due to the destruction of fetal platelets by a maternal platelet-specific antibody caused by fetal-maternal incompatibility. The most serious complication is intracranial hemorrhage (10-30 % of newborns), which may cause death (10 % of the reported cases) or irreversible neurological sequelae (20 %). The diagnosis is usually made after birth when most affected neonates have petechiae, purpura or overt bleeding. The degree of severity varies according to platelet count. Current methods allow detection of maternal platelet alloantibodies (usually HPA-1a). Clinical grounds and the exclusion of other causes of neonatal thrombocytopenia are required to establish an accurate diagnosis. Recurrence of this disease is very high and has prompted clinicians to develop antenatal prophylactic programs in subsequent pregnancies. However, the optimal treatment of at-risk pregnancies remains controversial. The early diagnosis of this process allows effective therapy based on the infusion of compatible platelets and IgG immunoglobulins when hemorrhage is not obvious. Antenatal management of subsequent pregnancies can prevent recurrence of thrombocytopenia and intracranial hemorrhage. The aim of this review is to draw pediatricians' attention to the importance of this probably under-diagnosed disease in which early diagnosis can prevent potentially severe complications.

Gene frequencies of human platelet antigens 1-5 in indigenous Australians in Western Australia.

The frequencies of human platelet antigen (HPA) systems vary between different racial groups; however, HPA frequency data for some racial groups are still incomplete. We report the distribution of HPA 1-5 systems in Australian Aborigines from a remote community in the north-west of Australia and compare our findings with HPA observed in a Western Australian blood donor population. Using a polymerase chain reaction (PCR) with sequence-specific primers, 185 indigenous Australians and 1000 Western Australian blood donors were genotyped for each of the HPA 1-5 systems. Comparison of gene frequencies of alleles from HPA-1, -2, -3 and -5 systems showed significant differences between Aboriginal people and Western Australian blood donors (P < 0.001). In particular, the frequency of HPA-3b (0.068) in the Australian Aboriginals, from this study, was one of the lowest reported, whilst the frequency of HPA-5b (0.246) was one of the highest for this allele. Gene frequencies were similar to those reported for central Australian Aborigines but with no other ethnic group. In conclusion, this study confirms significant differences in HPA distributions between indigenous Australians, Australian blood donors and other racial groups. These results indicate a higher potential risk of alloimmunization to HPA-1, -2 and -3 in Australian Aborigines receiving transfusion therapy from a Caucasian blood donor population, thereby having practical implications for transfusion and pregnancy risks in people of Aboriginal origin.

Analysis of human platelet antigen systems in a Moroccan Berber population.

The human platelet alloantigen systems HPA-1, -2, -3, -4, -5 and -6 in a Moroccan Berber population from the Amizmiz region were determined by polymerase chain reaction using sequence-specific primers (PCR-SSP). The gene frequencies obtained from these unrelated Berbers were 0 x 747 and 0 x 252 for HPA-1a and -1b; 0 x 817 and 0 x 182 for HPA-2a and -2b; 0 x 682 and 0 x 317 for HPA-3a and -3b; 1 and 0 x 0 for HPA-4a and -4b; 0 x 8616 and 0 x 1383 for HPA-5a and -5b; 1 and 0 x 0 for HPA-6a and -6b. The Berbers have the highest frequency for the 1b, 2b and 5b alleles of all the populations reported to date and also the lowest frequency for the 3b allele.

[Platelet alloantigenic systems]. / Les systèmes alloantigéniques plaquettaires.

Platelet-specific alloantigen systems are located on glycoproteins expressed at the surface of platelet membrane. These di-allelic systems are due to an amino acid substitution. An official nomenclature was adopted in 1990 by the Working Party on Platelet Serology ICST/ISBT which defined the following rules: (1) platelet-specific antigen systems are called HPA for "Human Platelet Antigens"; (2) they are numbered chronologically according to the date of publication; (3) the most frequent allele is designated by the letter a and the less frequent by the letter b (i.e., HPA-1a 1b). Since the introduction of molecular biology techniques, the nomenclature has become insufficiently precise and an adjustment will probably be required in a near future.

HLA and HPA typing in idiopathic thrombocytopenic purpura patients treated with Kami-kihi-to.

We performed human leukocyte antigen (HLA) and human platelet antigen (HPA) in patients with Kami-kihi-to-responsive idiopathic thrombocytopenic purpura. The HLA-A2, A61 and Cw1 were significantly increased in responders compared with nonresponders, as were HLA DRB1 *0901, DRB1 *1502, and DPB1 *0501. In contrast, HLA DPB1 *0201 and DPB1 *0901 were significantly decreased in responders. The a/b genotype of HPA-2 and a/a genotype of HPA-3 were markedly increased in nonresponders, and anti-GPIb antibody was also increased. These results suggest that HLA, HPA, and anti-GP antibody studies may predict the response of idiopathic thrombocytopenic purpura to Kami-kihi-to.

Distribution of human platelet antigens in a Chinese population.

We report the distribution of the human platelet antigens HPA-1, -2, -3, -4 and -5 as determined using the polymerase chain reaction with sequence-specific primers in 100 random, healthy Chinese in Hong Kong. The HPA-1a, -2a, -4a and -5a genes were present in every sample tested, HPA-1b, -2b and -5b were rare, and the sample was monomorphic for HPA-4a. HPA-3a and -3b genes showed frequencies of 0.525 and 0.475 respectively. There was no departure from Hardy-Weinberg equilibrium in any of the five HPA systems studied.