Rapid and reliable genotyping of human platelet antigen (HPA)-1, -2, -3, -4, and -5 a/b and Gov a/b by melting curve analysis

BACKGROUND: The probability for occurrence of neonatal alloimmune thrombocytopenic purpura (NAITP) depends largely on the frequency of each individual phenotype in various populations. In caucasians, antibodies to human platelet antigen (HPA)-1a are the major cause of neonatal alloimmune thrombocytopenic purpura, whereas in the Japanese population, antibodies to HPA-4b is most frequently involved in NAITP. Conventional PCR techniques for platelet antigen genotyping rely on sequence-specific primers (SSPs) and detection by gel electrophoresis, a method which is laborious and time consuming. New PCR technology, measuring the match of a hybridization probe with its target and thereby allowing simultaneous detection of both alleles, provides an efficient tool for genotyping of the HPA systems. STUDY DESIGN AND METHODS: A total of 105 healthy blood donors were genotyped for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b with new primers and probes designed for mutation detection by melting curve analysis (using LightCycler technology). Donor DNA was independently genotyped by an allele-specific assay, using SSPs, in a reference laboratory. RESULTS: There was full concordance between the two genotyping methods, and genotype frequencies were comparable with previous studies in caucasians. CONCLUSION: We present rapid and reliable detection systems for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b based on mutation detection of both alleles simultaneously by melting curve analysis. As the Gov system has been reported to have similar frequency of involvement in alloimmune thrombocytopenia as HPA-5, the opportunity for genotyping should aid the diagnosis of such patients.     

使用Taqman PCR技术建立人类血小板抗原-1～5、15分型体系

目的了解上海地区单采血小板献血人群HPA-1～5、15多态性分布,分析评估新的分型技术。方法利用TaqMan PCR技术对500份上海地区单采血小板供者标本进行HPA-1～5、15抗原系统等位基因分型,并随机抽取100份标本使用PCR-SSP技术进行比对。结果 HPA各等位基因频率分别为HPA-1a:0.999,HPA-1b:0.001,HPA-2a:0.953,HPA-2b:0.047,HPA-3a:0.582,HPA-3b:0.418,HPA-4a:0.999,HPA-4b:0.001,HPA-5a:0.988,HPA-5b:0.012,HPA-15a:0.524,HPA-15b:0.476;有1份标本HPA-5等位基因与SSP检测结果产生差异。结论上海地区HPA各等位基因频率与国内各地区人群分布无明显差异,与中国汉族人群HPA分布情况基本吻合,实验数据经验证符合Hardy-Weinberg平衡定律,实验结果准确可靠;HPA-5差异经测序验证判断可能由PCR-SSP非特异扩增所致;TaqMan技术在HPA抗原系统分型应用中特异性高,反应时间短,具有良好的应用前景,是现有技术方法的一种重要补充。     

Multicolor real-time polymerase chain reaction genotyping of six human platelet antigens using displacing probes

BACKGROUND: Several genotyping methods for six clinically relevant human platelet antigens (HPAs) have been reported. A four-color real-time polymerase chain reaction (PCR) method using displacing probes for genotyping of the six HPAs is described. STUDY DESIGN AND METHODS: Primers and four differently fluorophor-labeled displacing probes were designed and synthesized to detect single-nucleotide polymorphisms responsible for each of the HPA-1, -2, -3, -4, -5, and -15 genotypes. Two HPA systems were analyzed in a single PCR procedure. After validation with samples of known genotypes, a total of 150 blood samples from healthy donors were genotyped. The results were compared with PCR with sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), and/or direct DNA sequencing. The frequencies of each HPA allele were calculated. RESULTS: Unequivocal real-time PCR genotyping results were obtained with minimal manual manipulation and carryover contamination. All 150 blood samples were correctly genotyped as confirmed by PCR-SSP, PCR-RFLP, and/or direct DNA sequencing. The allelic frequencies of HPA-1 through -5 and -15 among the Chinese population in Xiamen were comparable with those previously reported with Chinese living in other territories. For each specimen, genotyping of all six HPA biallelic systems was achieved in three tubes of PCR within 90 minutes and with material cost of no more than $1. CONCLUSION: Genotyping of HPA with real-time PCR using displacing probes is more rapid and reliable compared with PCR-SSP and PCR-RFLP methods and is more affordable than existing real-time PCR-based HPA genotyping assays. Thus, our approach is more suitable for routine HPA analysis and ideal for both urgent clinical testing and high-throughput screening.     

人类血小板抗原HPA-15系统PCR-SSP基因分型技术的建立

本研究目的是采用PCR-SSP技术建立人类血小板抗原HPA-15系统的基因分型方法,并应用于血小板供者库的HPA基因定型。采用第11届国际输血协会（ISBT）血小板血清学与基因分型协作组推荐的序列特异性引物,调节引物浓度、Mg2＋离子浓度和探索最佳PCR扩增条件,建立HPA-15系统基因分型技术。该分型技术的准确性和可靠性,采用第11届ISBT血小板协作组提供的质控样本进行验证,同时采用本研究合成的引物及商品化试剂盒,对50名随机的汉族血小板捐献者进行HPA-15系统基因分型,作为平行对照。应用本研究的方法,对第11届ISBT送检的10份考核样本进行基因分型。结果表明：基因分型结果与ISBT公布的结果完全一致。50名随机的血小板志愿捐献者,经本研究的方法及美国G＆T公司的试剂检测,基因分型的结果相符合;观察到的基因频率：HPA-15a和-15b分别为0.5100和0.4900。结论：本研究建立的HPA基因分型技术具有简便、快速、准确的特点,适合于常规HPA基因分型,具有广泛的应用前景。     

PCR with sequence-specific primer-based simultaneous genotyping of human platelet antigen-1 to -13w

BACKGROUND: Accurate human platelet antigen (HPA) typing is important for patients with diagnosis of alloimmune thrombocytopenic syndromes and provision of HPA-matched blood components for these patients. STUDY DESIGN AND METHODS: Thirteen sequence-specific primers (SSPs) designed on the basis of known published polymorphisms for HPA-1 to HPA-13w, respectively, were employed for simultaneous HPA genotyping. All PCR amplifications were carried out with identical cycling conditions in 96-well plates containing primer mixtures. A total of 300 blood samples from unrelated volunteer donors in Taiwan were included in the study. RESULTS: All primers had specific amplification products. The typing results were available within 4 hours each time for up to four blood samples tested. Among the 13 HPAs, HPA-3 had the greatest heterozygosity with a gene frequency of 0.3267, 0.4967, and 0.1767 for HPA-3a/HPA-3a, HPA-3a/HPA-3b, and HPA-3b/HPA3-b, respectively. For the remaining 12 HPAs, the predominance of a/a homozygosity was noted for HPA-1, -2, -4, -5, and -6, with a frequency ranging from 0.9200 to 0.9967. The frequency of a/a homozygosity was 1.0000 for HPA-7w to -13w, except for HPA-10w, for which one case was observed to be HPA-10aw/HPA-10bw heterozygous. Excluding HPA-3, b/b homozygosity was noted in only one case (HPA-6b/HPA-6b). The prevalence rates of HPA-1 to -13w in this study were consistent with previous reports using different methods. CONCLUSION: An extended, streamlined PCR-SSP protocol for simultaneous genotyping of HPA-1 to HPA-13w was established. This allows fast and reliable diagnosis of alloimmune thrombocytopenia, and is readily applicable to large-scale genetic population studies.     

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.     

A naturally occurring LeuVal mutation in beta3-integrin impairs the HPA-1a epitope: the third allele of HPA-1

BACKGROUND: Single-amino-acid substitution Leu33Pro in the beta3-integrin is responsible for the formation of the human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (beta3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura. STUDY DESIGN AND METHODS: While HPA-1 genotyping a large cohort of patients with thromboembolic disease with a thermal cycler (LightCycler), one patient was identified with a unique HPA-1a melting curve. RESULTS: Sequence analysis revealed a C-to-G transversion at nucleotide 175 in the beta3-integrin (ITGB3) gene that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n = 2950) identified one nonrelated Pro33Val33-positive individual. To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, transfectants were generated expressing recombinant beta3-Leu33 or beta3-Val33. Interestingly, differences in the reactivity of anti-HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with beta3-Val33 compared to beta3-Leu33 and others reacting equally with both types. Similar findings were observed with recombinant human HPA-1a antibodies, with one of the three not binding to beta3-Val33. CONCLUSIONS: Our results demonstrate that the naturally occurring Leu33Val mutation in the beta3-integrin can disrupt some HPA-1a epitopes. These findings provide evidence for a heterogeneous humoral response against HPA-1a that may have potential clinical implications for alloimmune thrombocytopenia disorders.     

多重聚合酶链反应用于人血小板抗原-2、-4、-5系统基因分型的研究

目的建立用于检测人血小板抗原(HPA)-2、-4、-5系统基因型的多重聚合酶链反应(PCR)。方法在一个反应体系中同时扩增HPA-2、-4、-5系统特异性目的基因片段。用琼脂糖凝胶电泳确定HPA-2、-4、-5系统基因型;再用多重PCR对75名健康的单采血小板者进行HPA-2、-4-、5系统基因分型,分型结果与PCR-序列特异性引物(PCR-SSP)获得的结果进行比较。结果HPA-2、-4、-5系统分型的结果为:70名为2 a/2 a型,5名为2 a/2b型;73名为4 a/4 a型,2名为4 a/4b型;66名为5 a/5 a型,9名为5 a/5b型。未发现2b/2b、4b/4b及5b/5b纯合子个体。多重PCR结果与PCR-SSP方法获得的结果一致。结论多重PCR具有操作简便、快速、准确等特点,可以用于血小板血型抗原基因分型。     

Genotyping of the human platelet antigen systems 1 through 5 by multiplex polymerase chain reaction and ligation-based typing

BACKGROUND: Platelet-specific antibodies may be involved in refractoriness to platelet transfusions, disorders such as neonatal alloimmune thrombocytopenia, and post-transfusion purpura. Genotyping for the major human platelet antigen (HPA) systems HPA-1 through HPA-5 is of considerable help in establishing the diagnoses of these diseases. STUDY DESIGN AND METHODS: A new genotyping method is described. Alleles of all five systems are amplified in a multiplex polymerase chain reaction. Subsequently, aliquots of the amplification products are thermocycled in the presence of a pair of allele-specific oligonucleotide probes and a heat-stable ligase. After heat denaturation, the probes hybridize adjacent to complementary sequences of the amplification product. In a perfect match, the two probes become covalently joined. Detection of the ligation product is performed with an enzyme-linked immunosorbent assay. RESULTS: Complete concordance of genotypes between the ligation-based typing and established genotyping methods was determined in 54 Austrian (HPA-1, -2, -3, and -5) and 56 Japanese (HPA-4) individuals. Ligation-based genotyping of HPA-1 polymorphism using platelet-derived RNA as starting material gave concordant results in all 15 cases tested. CONCLUSION: Multiplex polymerase chain reaction in combination with ligation-based typing allows fast typing of large numbers of platelet donors and screening for critical antigens in pregnant women.     

青岛地区汉族人群HPA-1—5,15多态性分布研究

目的研究青岛地区汉族人群人类血小板抗原(HPA)1-5,15抗原分布多态性。方法采用PCR-SSP方法对青岛地区918名无血缘关系固定血小板无偿捐献者进行HPA1-5及HPA-15系统的基因分型.结果各被检系统等位基因频率分别是1a=0.9940,1b=0.0060,2a=0.9319,2b=0.0681,3a=0.5822,3b=0.4178,4a=0.9897,4b=0.0104,5a=0.9804,5b=0.0196,15a=0.4913,15b=0.5087;HPA基因频率分布与国内资料比较,HPA-1与北方人群(河南),HPA-2与南方人群(四川)差异有统计学意义;与台湾人群HPA-2,-4,与日本人群HPA-2,-3,-5,与美国黑人HPA-1,-2,-5,与白人HPA-1,-4,-5,-15分别有统计学显著性差异。结论青岛地区汉族人群HPA分布具有本地人群特点。本组HPA数据分布符合Hardy-Weinberg平衡定律,可以作为北方汉族人群HPA基因分布频率数据库和青岛本地化血小板供者HPA资料库。     

南宁地区人群血小板抗原HPA1—16bw基因分布频率的研究

目的研究南宁地区人类血小板抗原基因分布频率,为临床同种免疫血小板减少症患者提供诊断依据和HPA相容的成分血。方法采用序列特异性引物聚合酶链反应(PCR-SSP)对南宁地区1 500名无血缘关系的成年人做HPA1—16bw基因分型研究。在包含引物混合液的96孔反应板中,按照相同的循环条件扩增PCR产物。结果每份血液标本均可在4 h内获得分型结果。在16个HPA系统中,HPA-15基因型杂合频率最高,HPA15a/15a,HPA15a/15b和HPA15b/15b的频率分别为0.252 7,0.500 7和0.246 7。HPA-3的杂合程度仅次之,HPA3a/3a,HPA3a/3b和HPA3b/3b的频率分别为0.284 0,0.508 0和0.208 0。其余14个HPA系统均以a/a纯合子为主,其a基因频率范围为0.916 7—0.999 3。除了HPA-3b/3b和HPA-15b/15b的纯合子以外,还检测出了HPA-2b/2b5例和HPA-1b/1b1例。HPA-7bw至-14bw,-16bw的a/a等位基因纯合率为1.000 0。结论在临床工作中我们仍须警惕可能由HPA-1,-2,-3,-5,-6和-15同种抗体引起的疾病,HPA基因分型将有助于更好地诊断同种免疫血小板减少症和建立相容的HPA血小板供者库,提供更有效的血小板输注方法。     

IgG titer, subclass, and light-chain phenotype of pregnancy-induced HPA-5b antibodies that cause or do not cause neonatal alloimmune thrombocytopenia

BACKGROUND: The most common pregnancy-induced platelet-specific antibody is HPA-5b. Neonatal alloimmune thrombocytopenia that results from anti-HPA-5b may cause severe hemorrhage in only a few infants, but the sequelae for the affected children can be severe. It is therefore essential that infants at risk for neonatal alloimmune thrombocytopenia are identified. STUDY DESIGN AND METHODS: The IgG titer, subclass, and light-chain composition of pregnancy-induced anti-HPA-5b were determined by the monoclonal antibody-specific immobilization of platelet antigens assay. Sera were from 12 mothers, who among them had 16 pregnancies that resulted in an HPA-5-mismatched fetus (positive for HPA-5b). Eight mothers gave birth to an infant with a normal platelet count. Three maternal sera were obtained after delivery of a severely thrombocytopenic infant. Three alloimmunized women were followed repeatedly during the course of a subsequent pregnancy, again with an HPA-5-mismatched infant. RESULTS: There was no difference in the antibody titer or its subclass in mothers who had a thrombocytopenic child and the titer or subclass in mothers compared with those who had a child with a normal platelet count. All pregnancy-induced HPA-5b antibodies were of a predominant, lambda, light-chain type. The IgG subclass did not change during pregnancy. CONCLUSION: Neither the antibody titer nor the subclass composition predict the occurrence of thrombocytopenia in a newborn whose mother is alloimmunized against HPA-5b.     

国内首例抗HPA-5b引发的新生儿同种免疫性血小板减少性紫癜的检测、诊断及分析

本研究旨在探讨抗HPA-5b抗体引发的新生儿同种免疫性血小板减少性紫癜（NAITP）的实验检测及临床诊断。临床监测患儿血小板计数及其他临床表现;多重PCR及DNA测序检测患儿及其父母HPA-1—21bw系统基因型;流式细胞术（FCM）和血小板抗原单克隆抗体特异性免疫固定检测技术（MAIPA）检测及鉴定患儿及其母亲血清中的血小板特异性抗体。结果表明：患儿临床症状较轻,基因分析显示患儿为HPA-5ab,父亲为HPA-5ab,母亲为HPA-5aa;患儿母亲血清中含与父亲血小板反应的血小板特异性抗体为抗HPA-5b抗体。结论：该病例为抗HPA-5b抗体引起的新生儿同种免疫性血小板减少性紫癜。     

熔解曲线分析法用于HPA-15基因分型的研究

目的探讨熔解曲线分析法用于HPA-15基因分型,了解大连地区汉族人群HPA-15基因多态性。方法设计合成HPA-15等位基因特异性引物和1条公共引物,其中在2条等位基因特异性引物的5'末端分别加入具有不同长度并富含GC碱基的序列。使用这3条引物,进行实时PCR反应。在PCR反应后,进行熔解曲线分析,依据PCR产物熔解温度的差异,进行基因分型。应用熔解曲线分析法和常规PCR-SSP法对100名大连地区汉族人群的血液标本进行HPA-15基因分型。此外对熔解曲线分析法的重复性进行评价。结果 100个血液标本的熔解曲线分析法和常规PCR-SSP法的基因分型结果是完全一致的。10个血标本的2次熔解曲线分析结果是一致的。大连地区汉族人群HPA-15a、-15b基因频率分别为0.58和0.42。大连地区汉族人群与国内部分地区汉族人群相比,HPA-15等位基因频率的差异无统计学意义(P>0.05)。与新疆维吾尔族人群相比,HPA-15等位基因频率差异有统计学意义(P<0.05)。结论 HPA的熔解曲线基因分型方法具有快速、简单、准确、通量高、重复性好等优点,要优于常规的PCR-SSP法。在国内HPA-15等位基因分布存在着种族差异。     

血小板谱抗原的建立及其在鉴定血小板特异性抗体中的应用

目的 建立血小板谱抗原,鉴定引起血小板输注无效和新生儿血小板减少性紫癜的血小板特异性抗体,为血小板血型研究和临床治疗提供依据。方法根据中国人群人类血小板同种抗原(HPA)-1-HPA-16等位基因频率分布资料,利用聚合酶链反应-序列特异引物(PCR-SSP)技术对O型血小板供者进行HPA-1-HPA-6、HPA-15分型,筛选合适的供者,组成血小板谱抗原。通过建立的血小板谱抗原,利用简易致敏红细胞血小板血清学技术(SEPSA)鉴定同种免疫反应产生的血小板抗体的特异性。结果从O型血小板供者中筛选出11名供者,建立了血小板特异性抗体鉴定谱抗原。其可鉴定HPA-1-HPA-6,HPA-15抗体的特异性。在所筛检1 120份样本中,有3例患者检出HPA抗体,其中HPA-4b(Penb)抗体1例,HPA-15a(Govb)抗体2例。结论通过血小板谱抗原鉴定血小板抗体的特异性,对提高临床输注血小板的安全性和有效性,以及预防新生儿血小板减少性紫癜有积极的意义.     

Recent advances in non-invasive fetal HPA-1a typing

Non-invasive fetal HPA-1a typing is a valuable tool to identify the pregnancies at risk of fetal and neonatal alloimmune thrombocytopenia (FNAIT). At present, prenatal determination of the fetus HPA-1a type is performed for diagnostic purposes in pregnancies of HPA-1 alloimmunized women with history of a previous fetus or child with FNAIT. Different approaches have been used to determine the fetal HPA-1a genotype from cell-free fetal DNA (cffDNA) in the mother’s plasma, mainly based on real-time PCR. Due to the single nucleotide polymorphism (SNP) between the HPA-1a and HPA-1b allelic sequences, a robust and accurate detection of the fetal genotype is challenging, and the sensitivity of most assays is still limited early in pregnancy. Nowadays, the availability of technologies such as next generation sequencing (NGS) or digital PCR offers unprecedented possibilities of analyzing cell-free DNA (cfDNA)-amplified sequences with very high coverage and high sensitivity. In addition, other interesting approaches using variant sequence enrichment strategies have been recently described. In particular, coamplification at lower denaturation temperature PCR (COLD-PCR) offers a simple and sensitive strategy for noninvasive fetal HPA-1 typing. These novel approaches are explained in more detail in this review. Despite no population-based FNAIT screening programs have so far been implemented, the perspectives in terms of treatment and prevention are changing and less costly high-throughput maternal HPA-1a typing methods have been developed. Altogether, this may lead to the implementation of fetal HPA-1a typing with a broader scope in the future, playing a critical role within FNAIT screening programs.     

New mutation in the platelet beta3-integrin gene: implication for the diagnosis of fetomaternal alloimmunization

BACKGROUND: Fetal ventriculomegaly is a relatively common finding and fetomaternal alloimmune thrombocytopenia may be one of the causes. STUDY DESIGN AND METHODS: Such a case discovered at 21 weeks of gestation leading to platelet (PLT) immunologic testing is reported here. PLT genotyping was performed by polymerase chain reaction (PCR)-sequence-specific primers (SSPs) method and PCR-restriction fragment length polymorphism (RFLP) analysis. Serologic investigation was done with the monoclonal antibody-specific immobilization of PLT antigens technique. RESULTS: The mother was found to be HPA-1b homozygous and the father HPA-1a homozygous with PCR-SSP, but the mother was found to be HPA-1 heterozygous by phenotyping. This result was confirmed by PCR-RFLP. Sequencing of the glycoprotein IIIa exon 3 revealed a heterozygous mutation 262T > C, which does not induce an amino acid change. It is localized in the sequence of the antisense primer of the HPA-1 PCR-SSP, inducing the sole amplification of the DNA copy bearing the HPA-1b allele. CONCLUSION: Even if such mutations are a rare event, PLT phenotyping is still of interest to avoid rare false PLT typing assignation, the unknown polymorphism being only discovered by such a combination of techniques.     

HPA-13bw neonatal alloimmune thrombocytopenia and low frequency alloantigens: case report and review of the literature

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) linked to rare or private antigens is not a rare event. STUDY DESIGN AND METHODS: Such a case discovered during the follow-up of a second child with jaundice with mild thrombocytopenia is reported here. Platelet (PLT) genotyping was performed by polymerase chain reaction (PCR)-sequence-specific primers method and PCR-restriction fragment length polymorphism (RFLP) analysis. Serologic investigation was done with the monoclonal antibody-specific immobilization of PLT antigens technique. Glycoprotein Ia-specific amplification and sequencing were performed for the polymorphism 807 (exon 7). RESULTS: The mother was found to be HPA-13aaw, and the father HPA-13abw. A maternal alloantibody directed against HPA-13bw has been characterized, leading to the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: This report provides further evidence that NAIT associated with low-frequency antigens is not restricted to single families. Therefore, laboratory investigation of a suspected case should be carried out in a specialist laboratory well experienced in optimal testing to propose appropriate management for the index case and subsequent pregnancies.     

多重聚合酶链反应-微流芯片检测人血小板抗原系统基因型方法的建立与应用

目的建立人血小板抗原（HPA）-2、4、5系统基因型的检测方法。方法用多重聚合酶链反应和微流芯片检测方法，通过设计9条特异性引物、优化反应体系和反应条件，在一个体系中同时扩增HPA-2、4、5系统特异性的目的基因片段，利用微流芯片快速检测HPA-2、4、5系统基因型；并把分型结果与采用聚合酶链反应．序列特异性引物（PCR-SSP）获得的结果进行比较。结果对35名健康单采血小板者进行HPA-2、4、5系统分型的结果为：33名为2a／2a型，2名为2a／2b型；34名为4a／4a型，1名为4a／4b型；29名为5a／5a型，6名为5a／5b型。未发现2b／2b、4b／4b及5b／5b纯合子个体。该结果与PCR．SSP方法获得的结果完全一致。结论该方法可快速、准确地用于血小板血型抗原基因分型，尤其适合于大样本检测。     

应用PCR—SSP法分析中国人血小板抗原基因型频率

本研究建立了在同一PCR反应条件下同时检测人血小板抗原（human platelet antigen,HPA）系统HPA-1到HPA-5的PCR-SSP检测法。应用该方法分析了110例健康献血员的HPA-1到HPA-5的基因型,并以此为依据推算了中国人HPA-1到HPA-5各亚型的基因频率。结果表明HPA-1a和HPA-1b的基因频率分别为0.91和0.09,HPA-2a和HPA-2的基因频率分别为0.86和0.14,HPA-3a和HPA-3b的基因频率分别为0.60和0.40,HPA-4a和HPA-4b的基因频率分别为0.92和0.08,HPA-5a和HPA-5b的基因频率分别为0.85和0.15。结论：基因组DNA的血小板抗原PCR-SSP分型法切实可行,可广泛应用于临床血小板抗原的分型。