儿童急、慢性特发性血小板减少性紫癜HPA基因多态性分析

目的:探讨广西地区儿童急、慢性特发性血小板减少性紫癜(ITP)的HPA-2和HPA-15等位基因频率分布情况及HPA-2和HPA-15基因多态性与儿童ITP发病的潜在联系。方法:收集2010年1月至2014年12月确诊为急性和慢性ITP的各46例患儿的临床资料,运用聚合酶链反应技术(PCR)结合直接测序方法,对急、慢性ITP两组各46例患儿及对照组48例健康儿童进行HPA-2和HPA-15基因分型。结果:3组之间HPA-2和HPA-15等位基因频率比较均有显著性差异(P0.05),每2组比较分析显示,HPA-2和HPA-15等位基因频率在慢性ITP组与对照组间差异显著,有统计学意义(P0.05),而急性ITP组与对照组及慢性ITP组与急性ITP组相比较差异均无统计学意义(P0.05)。结论:HPA-2与HPA-15基因多态性与儿童慢性ITP发病有关,与儿童急性ITP发病无关。     

血小板特异性抗原基因与职业性慢性苯中毒的相关性分析

目的探讨血小板特异性抗原HPA-1-6,15基因多态性与职业性慢性苯中毒的相关性。方法采用FLOW-SSO法和PCR-SSP法对32名职业性慢性苯中毒患者(病例组)做HPA-1-6,15基因分型,并将之与中国汉族人群(对照组,n=245 8)的数据比较。结果 HPA-3b等位基因频率、HPA-3基因型分布,病例组与对照组分别为0.593 7 vs 0.441 4(P0.05)及0.750 0 vs 0.506 9(P0.01);HPA-3b等位基因相关ab/bb基因型,实验组与对照组分别为96.88%(31/32)vs 69.49%(1 708/2 458)(P0.01)。HPA-1,2,4,5,6,15等位基因频率和基因型分布,2组比较均无明显差异(P0.05)。结论职业性慢性苯中毒可能与HPA-3基因多态性有关。     

拉萨藏族献血人群HPA-1～6、15基因多态性

目的 了解血小板特异性抗原(HPA)-1～6、15基因多态性在拉萨藏族献血人群中的分布特征.方法 采用聚合酶链式反应-序列特异性引物(PCR-SSP)技术对405名藏族无关献血者作HPA-1～6、15基因分型.结果 405名无关献血者HPA各等位基因的频率分别为:HPA-1a:0.9383,HPA-1b:0.061 7;HPA-2a:0.971 6,HPA-2b:0.028 4;HPA-3a:0.617 3,HPA-3b:0.382 7;HPA-4a:0.992 6,HPA-4b:0.007 4; HPA-5a:0.949 4,HPA-5b:0.050 6; HPA-6a:0.991 4,HPA-6b:0.008 6;HPA-15a:0.603 7,HPA-15b:0.396 3.人群资料比对结果显示,与南方汉族人群(n=1 554)相比,拉萨藏族献血人群HPA-1、HPA-2、HPA-3、HPA-4、HPA-5、HPA-15系统基因频率具有明显差异;与新疆柯尔克孜族人群(n =105)相比,仅HPA-2系统基因频率具有明显差异.拉萨藏族献血人群HPA-1b等位基因频率明显低于高加索人群(n=119),而与新疆柯尔克孜族人群(n =105)相近,但明显高于亚洲其他黄种人群.结论 拉萨藏族献血人群的HPA各位点基因频率显示出明显的民族和地域性特征.     

海南黎族人血小板1—17抗原基因与随机输血不配合率研究

目的调查海南岛黎族人群血小板抗原(HPA)-1—17等位基因多态性及其特点,评估其在随机输血中血小板输注无效的风险。方法采用PCR-SSP方法对海南岛180名黎族人群做HPA-1—17基因分型检测>计算各系统对偶抗原不配合率。结果在海南黎族人群的HPA-1—17系统中,呈多态性分布的等位基因及其频率为HPA-2a(0.9972)、2b(0.0028),HPA-3a(0.4889)、3b(0.5111),HPA-5a(0.9667)、5b(0.0333),HPA-6a(0.9972)、6b(0.0028),HPA-15a(0.4250)、15b(0.5750);其余HPA-1、4、7—14、16、17等位基因均呈单线性分布。在HPA-3、15等位基因中出现bb纯合子基因型,频率分别为0.2834和0.3667;其余系统均未见bb纯合子基因型。随机输血中,海南岛黎族人群HPA不配合的发生率依次为:HPA-3(37.49%)、-15(36.93%)和-5(6.23%)。结论揭示了海南岛黎族人HPA-1—17基因型和等位基因频率的分布及特点;立足人群的随机血小板输注,只需检测供、受者HPA-2,-3、-5和-15基因相合,就可基本达到血小板匹配性输注。     

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。     

CD109表达量及保存方法的相关性研究

目的分析中国南方人群HPA-15基因型分布频率,研究血小板上HPA-15抗原表达量。寻找较好的血小板保存方法以维持HPA-15的高表达量,为单克隆抗体俘获血小板抗原技术(MAIPA)检测HPA-15同种抗体提供合适的供者血小板。方法 1)随机选取广州血液中心的健康血小板单采献血者109名,留取EDTA抗凝管全血。SSPPCR方法检测HPA-15基因型。流式细胞术检测血小板上HPA-15抗原表达量。2)另随机选取健康献血者的单采血小板67例,将每份血小板分为2份,1份重悬于EDTA-PBS中放于4℃保存作为检测组A,另1份留于血小板辫子中放于血小板振荡仪中作为检测组B,在d 0、1、2、3、7、14检测2组血小板上HPA-15的检测含量,比较2组HPA-15含量的变化。结果 1)109名随机健康献血者HPA-15a和HPA-15b的基因频率是0.495和0.505。流式细胞术检测HPA-15抗原的MFI值的范围2.44-20.4,阳性率3.2%-86.7%。不同HPA-15基因型之间,HPA-15抗原表达量有统计学差异(P0.05)。2)分析比较AB这2个组,随着测量时间点的推移,2组HPA-15的表达量均有降低,但检测组B中HPA-15的表达含量高于检测组A,且2组的MFI值有统计学差异(P0.05)。结论 HPA-15a和HPA-15b的基因分布频率相近,HPA-15抗原表达含量整体水平偏低且个体差异性大。体外保存方式下,HPA-15的表达含量随着时间推移逐渐降低,4℃保存方式下HPA-15含量降解尤为明显。血小板振荡仪中保存血小板的方法为MAIPA检测HPA-15抗体提供更合适的血小板抗原。     

人类血小板抗原1—16基因与血小板输注无效风险研究

目的探讨HPA-1—16基因多态性分布与血小板输注无效相关性。方法应用PCR-SSP法对上海地区268名汉族人群行HPA-1—16基因检测;应用ELISA法对49名反复输血的恶性血液病患者行血小板抗-HLA-Ⅰ与抗-HPA筛查试验。结果上海地区汉族人群HPA-1—16系统中,HPA-1—6,15系统等位基因频率1a=0.9889,1b=0.0111,2a=0.8881,2b=0.1119,3a=0.5989,3b=0.4011,4a=0.9963,4b=0.0037,5a=0.9907,5b=0.0093,6a=0.9832,6b=0.0168,15a=0.6418,15b=0.3582,均呈多态性分布;其余HPA-7—14,16系统等位基因均呈单线性分布。HPA-1,2,4—6,15系统主要以aa纯合子基因型频率分别为0.9776,0.7799,0.9925,0.9813,0.9664,0.4328。在HPA-2、3、15系统中出现bb纯合子基因型,其频率均为0.0037,0.1530,0.1492外,其余系统均未出现bb纯合子基因型。另外,在HPA-1—6,15系统中出现ab杂合子基因型,其频率分别为0.0224,0.2164,0.4963,0.0075,0.0187,0.0336,0.4180,以HPA-3杂合度最高,其次次序为HPA-15、HPA-2。在随机输血中,HPA不合发生率以HPA-3为最高(0.3650),其次分别为HPA-15(0.3541)、HPA-2(0.1790)。49名反复输血的恶性血液病患者中,有61.22%(30名)输血后相继出现抗-HLA-Ⅰ,而始终未检出抗-HPA。结论上海地区汉族人群血小板输注无效的主要原因是抗-HLA-Ⅰ所致;只需检测供者与受(患)者的HPA-2、-3、-15基因相合,就可基本达到血小板匹配性输注。     

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.     

应用实时荧光PCR法建立献血者血小板特异性抗原HPA-1～6/10/15/21基因分型资料库

目的 研究中山地区单采血小板献血者人类血小板特异性抗原HPA-1～6/10/15/21共9对抗原基因多态性区域分布特点，建立中山地区血小板献血者HPA基因分型资料库。方法 用实时荧光定量PCR-TaqMan探针法对中山地区192名单采血小板献血者进行HPA-1～6/10/15/21基因分型，计算基因型频率、基因频率。结果 HPA-1～6/10/15/21中，HPA-4、10系统呈单特异性，只检测出aa基因型，未检出等位基因HPA-b;HPA-1、2、5、6、21基因型以aa占绝大多数；HPA-3、15具有较高的杂合度，基因型HPA-3aa、HPA-3ab、HPA-3bb频率分别是0.307 3、0.494 8、0.197 9;基因型HPA-15aa、HPA-15ab、HPA-15bb频率分别是0.270 8、0.505 2、0.224 0。结论 中山地区血小板献血者HPA-1～6/10/15/21基因与其他地区的同类资料相比显示出地域差异性，建立HPA基因分型资料库有助于解决血小板不配合输注产生同种免疫的问题。     

血小板特异性糖蛋白抗体在血小板输注无效患者中分布的研究

目的探讨血小板输注无效(PTR)患者的血小板特异性糖蛋白抗体的表达及PTR与血小板特异性抗原的相关性。方法采用ELISA方法检测27名临床上确诊为PTR患者的血小板特异性糖蛋白抗体,应用PCR-SSP的方法检测其HPA-1～17基因分型。结果 27名PTR患者的HPA基因频率为HPA-1a(0.900),1b(0.100);HPA-2a(0.926),2b(0.074);HPA-3a(0.648),3b(0.352);HPA-4a(0.981),4b(0.019);HPA-5a(0.940),5b(0.060);HPA-6a(0.815),6b(0.185);HPA-7a～14a、16a、17a(1.000),7～14、16、17b(0.000);HPA-15a(0.463),15b(0.537);PTR患者的HPA抗原分布与健康献血者的基因频率无统计学差异(P>0.05)。血小板抗体阳性的27名PTR患者中,血小板特异性糖蛋白GPⅡb/Ⅲa抗体阳性表达者占78%(21/27)、GPⅠa/Ⅱa抗体阳性占70%(19/27)。对3名PTR患者作血小板特异性抗原分析发现:患者产生的血小板特异性糖蛋白抗体与HPA基因型密切相关,以抗-HPA-3b、-5b为常见。结论多次输注血小板无效的患者,应选择与其HPA基因型一致的血小板供者,以提高血小板输注疗效,避免血液资源的浪费。     

Platelet alloantigens HPA-1, -2, -3, -5 and -6b in Finns

SUMMARY. The human platelet alloantigens HPA-1, -2, -3, -5 and -6b in the Finnish population were determined using allele specific restriction analysis (PCR-ASRA) for HPA-1, -2, -3 and -5 and monoclonal antibody immobilized platelet antigen (MAIPA) assay for HPA-1, -3a, -5b and -6b. No discrepancies were observed between the results obtained with the PCR-method and those obtained serologically. The gene frequencies obtained from 200 unrelated Finns were 0.86 and 0.14 for HPA-1a and -1b, 0.91 and 0.09 for HPA-2a and -2b, 0.59 and 0.41 for HPA.3a and -3b and 0.95 and 0.05 for HPA-5a and -5b. The frequency of the HPA-5b allele (10%) is lower in Finns than in Central- or South-European populations (20–30%). The HPA-1, -2 and -3 frequencies did not deviate from those observed in other European populations. The rare HPA-6b antigen was observed in three of 127 individuals from south-eastern Finland (2.4%), which suggests that the frequency of this allele in Finland is higher than previously thought.     

浙江汉族人群血小板抗原1-16多态性调查

目的了解浙江汉族人群血小板抗原1-16多态性分布。方法采用PCR-SSP方法对120份浙江无血缘关系汉族样本作HPA-1—16等位基因分型。结果HPA-1b、-2b、-3b、-6bw、-15b的基因频率分别为0.0125、0.0542、0.3833、0.0125、0.4833,未检出HPA-4b、-5b、-7bw、-8bw、-9bw、-10bw、-11bw、-12bw、-13bw、-14bw、-16bw等位基因。浙江汉族人群HPA-3、15抗原系统血小板随机输注错配概率分别为0.36、0.37。结论浙江汉族人群HPA多态性分布与中国其他地区汉族人群比较没有差异,HPA-3和15系统多态性较丰富,因此在随机血小板输注或胎母之间比较容易发生同种免疫反应。     

The use of genotyping to predict the phenotypes of human platelet antigens 1 through 5 and of neutrophil antigens in Taiwan

BACKGROUND: The human platelet antigen (HPA) 1 through 5 and the human neutrophil antigen (HNA-1) systems are relevant to immune-related thrombocytopenia and neutropenia. The alloantigen distribution profiles in the population will aid in estimating the risk of alloimmunization. STUDY DESIGN AND METHODS: Genotyping of the genes that control the expression of the HPA-1 through -5 and HNA-1 systems in Taiwanese (n = 326) and Taiwan's indigenous peoples (n = 608) was performed by PCR with the sequence-specific primer (PCR-SSP) method. RESULTS: In the HPA system, HPA-1b and HPA-4b were absent among Taiwan's indigenous tribes and detected among other Taiwanese only with frequencies of <0.2 percent and <0.5 percent, respectively. The GP1BA*2 (HPA-2b) and GP1A*2 (HPA-5b) allele frequencies range from 1 percent to 7 percent and 0.4 percent to 3.5 percent among the two ethnic groups, respectively. GP2B*1 (HPA-3a) and GP2B*2 (HPA-3b) showed similar allele frequencies. In the HNA-1 system, the FCGR3B*1 (HNA-1a) allele frequency was about twice that of FCGR3B*2 (HNA-1b) in Taiwanese and also in most of the indigenous tribes. Three FCGR3B (HNA-1) null persons were found in one indigenous tribe (Ami tribe), for an FCGR3B null frequency of 19.8 percent. However, no FCGR3B*3 (HNA-1c) allele was detected in Taiwan. CONCLUSION: The frequencies of HPA-1b, -2b, and -5b in the Taiwanese population were much lower than those among whites. In Taiwan, all of the HNA-1 null found was due to the deletion of the FCGR3B gene, and this deletion may be widely distributed in the Ami tribe.     

云南汉族血小板献血者HLA-A、B和HPA基因多态性的分析

目的研究云南地区汉族血小板献血者HLA-A、B基因和HPA1-17基因多态性,建立已知型血小板献血者基因数据库。方法分别采用PCR-SSOP和PCR-SSP方法对1 000名云南地区无血缘关系的汉族血小板献血者做HLA-A、B和HPA1-17系统基因分型,计算基因型频率、基因频率,并与其他人群进行比较。结果在云南地区人群中共检出HLA-A位点14个,HLA-B位点35个。A*02(35.81%)、A*11(35.42%)和A*24(17.72%)3个等位基因为云南汉族人群HLA-A座位的主要等位基因;B*46(12.87%)、B*40(60)(10.83%)和B*15(62)(9.78%)3个等位基因为云南汉族人群HLA-B座位的主要等位基因。每个样本均检测到HPA-1a、2a、4a-14a、16a、17a基因;HPA-4a、7a-14a、16a、17a呈现单态性,未检测出相应的等位基因HPA-b;对于HPA-1、2、5、6主要以a/a纯合子居多,a/a基因型频率分别是0.989、0.960、0.990、0.980,没有b/b纯合子出现。在HPA1-17中,具有最高杂合度的是HPA-15,基因型HPA15a/15a、HPA15a/15b、HPA15b/15b频率分别是0.392、0.360、0.248;HPA-3在其次,基因型HPA3a/3a、HPA3a/3b、HPA3b/3b频率分别是0.339、0.467、0.194。经χ2检验,结果符合Hardy-Weinberg遗传平衡定律。结论云南地区汉族血小板献血者HLA-A、B和HPA1-17基因频率与南方汉族接近,也呈现其自身特点。应建立本地区的血小板供者分型数据库。     

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.     

深圳地区汉族人类血小板抗原1-6系统基因多态性分析

目的 研究人类血小板抗原基因多态性,为人类学研究及临床输血实践提供依据。方法 采用PCR-SSP方法对深圳地区222名汉族随机献血者HPA1-6系统进行基因分型研究,对其基因及基因型频率进行统计,并与HPA在不同人群中的分布进行对比分析。结果 在6个HPA系统中,HPA-3基因型的杂合程度最高,HPA-3a/3a、HPA-3a/3b、HPA-3b/3b的频率分别为0.265 8,0.518 0,0.216 2;其余5个HPA系统均以a/a纯合子为主,a基因的频率范围为0.997 7～0.955 0,且均未发现b/b纯合子。1b、4b的基因频率很低,分别为0.009 O和0.002 3。结论 深圳汉族人群HPA1-6系统的基因频率与中国台湾人及中国香港人均很相似(P>0.05)。HPA-1、HPA-5与美国黑人、白人及荷兰人差异显著(P<0.05);HPA-2、HPA-3与日本人、韩国人、美国黑人、白人差异显著(P<0.05);HPA-4与日本人差异显著(P<0.05)。在未来的临床实践中要警惕HPA-2,3,5,6系统同种抗体导致的同种免疫血小板减少综合征的可能。     

Genotyping of Eight Human Platelet Antigen Systems in Serbian Blood Donors: Foundation for Platelet Apheresis Registry

IntroductionThe aim of this study was to investigate the allele and genotype frequencies of 8 human platelet antigen (HPA) systems among blood donors from the Blood Transfusion Institute of Serbia and to compare them with published studies. These data would be useful to establish the basis for a platelet apheresis donor registry.Material and MethodsSeventy-two unrelated male platelet apheresis/blood donors from Serbia were typed for 8 HPA systems (HPA-1 to HPA-6, HPA-9, and HPA-15) via the FluoGene method, based on polymerase chain reaction-sequence-specific amplification (PCR-SSP; PCR using sequence-specific primers) with fluorometric signal detection. Allele and genotype frequencies were estimated by direct counting and compared to the expected genotype frequencies according to the Hardy-Weinberg principle. The transfusion mismatch probability was calculated for every HPA specificity.ResultsThe allele frequencies were: HPA-1a, 0.868; HPA-1b, 0.132; HPA-2a, 0.917; HPA-2b, 0.083; HPA-3a, 0.611; HPA-3b, 0.389; HPA-5a, 0.903; HPA-5b, 0.097; HPA-9a, 0.993; HPA-9b, 0.007; HPA-15a, 0.472; and HPA-15b, 0.528. For HPA-4 and HPA-6 only allele a was detected.DiscussionThe HPA allele frequencies of European populations showed no significant differences in comparison with our results. Statistically significant differences were revealed in comparison with some populations of non-European origin. In the tested donors no HPA-2 bb genotype was detected, but we found 1 donor with the rare HPA-9b allele. The biggest transfusion mismatch probability in the Serbian population is for systems HPA-15 (37.4%) and HPA-3 (36.2%), which means that more than a third of random transfusions could cause mismatch in these systems. This study was enabled by the introduction of molecular HPA typing, and it provides initial results of the HPA allele and genotype frequencies in the population of blood donors in Serbia. They will be used to provide a compatible blood supply on demand for treating patients with alloimmune thrombocytopenic disorders. The successful implementation of PCR-SSP with fluorometric signal detection could be further complemented in the future by the introduction of high-throughput methods, which will largely depend on the available financial resources.