116例住院患者ABO血型正反定型不相符的原因和处理方法

目的探讨拟输血患者ABO血型正反定型不相符的原因及其处理方法。方法对116例ABO血型正反定型不相符的血标本,采用不规则抗体筛选和特异性鉴定、吸收放散试验及直接抗人球蛋白试验进行鉴别,找出其原因并提出不同的处理方法。结果在116例ABO血型正反定型不相符的患者血标本中,由生理原因引起8例(6.9%),由临床治疗原因引起10例(8.6%),由疾病原因引起98例(84.5%)。通过增加抗A1、抗AB及抗H定型血清,了解患者治疗前血型,37℃生理盐水洗涤患者红细胞,增加患者血清量及离心次数,吸收放散试验及抗体特异性鉴定等处理,准确鉴定了患者ABO血型。结论对ABO血型正反定型不相符的疑难血标本,根据系列血型血清学检测结果并结合临床病情资料进行综合分析,可准确鉴定患者ABO血型。     

扩增阻滞突变系统用于ABO血型基因分型研究

目的建立一种快速、低成本的ABO血型基因分型方法。方法设计6对引物,建立扩增阻滞突变系统(ARMS),对30例中国汉族无关个体外周血样本的ABO基因型进行鉴定,并与血清学方法和直接测序结果进行比较。结果 ARMS技术检测30例样本结果同血清学方法、直接测序结果吻合率达到100%。结论该研究建立的ABO血型基因型鉴定方法可快速检测出28种ABO基因型,具有一定的临床应用价值。     

疑难ABO血型标本的表型与相应等位基因检测在临床中的应用

目的探究疑难ABO血型标本的表型与相应等位基因检测在临床中的应用。方法本次研究选取2015年5月-2016年3月期间我院各科室送检的临床病人和献血者的45份血液样本。通过试管法检测45例患者及献血者的血清表型,同时采用sanger双碱基DNA分子测序的方法检测ABO等位基因。结果血清学检测的45例血液样本中,出现了A抗原减弱12例(Aw6例,A3 1例,Ael,5例);B型减弱6例(Bw);AB抗原减弱9例(AB表型);Cis AB:12例;B(A):6例;存在A101、A102、A311、Ae L02、Ax02、Ax13、Bl01,Bw,O01、O02、B(A)04、Cis AB01、Cis AB08等基因亚型。结论常规的血清学方法对疑难ABO血型,尤其是ABO血型的亚型鉴定存在一定的局限性,血型基因测序分析的方法可以较好地区分血清学定型模糊的结果。     

罕见Ael亚型的血清学和基因序列分析北大核心CSCD

目的:探讨A_(el)01亚型血清学和分子机制。方法:采用微柱凝胶法和试管法检测先证者ABO表型,多功能非放射性磷酸盐偶联法检测N-乙酰半乳糖胺基转移酶的反应活性,荧光PCR法进行ABO血型基因分型,Sanger一代测序法对第1~7外显子进行测序。结果:先证者血清学试验检测为A_(el),血清和红细胞上均无N-乙酰半乳糖胺基转移酶活性,ABO血型基因分型结果为AO2,基因测序结果确认为A_(el)01/O02,第1~7外显子测序结果为该样本在A101/O02的基础上发生了239G>A的突变,该突变为首次发现。结论:根据血型血清学结果推测,该突变导致了A抗原弱表达。基因分型预测ABO血型表型,基因测序是直接获得ABO亚型的证据,最终须结合ABO血清学方法来确定其ABO血型。     

罕见Bel01亚型的血清学和基因分析

目的:探讨Bel01亚型的血清学特征和分子机制.方法:采用血型血清学方法分析该献血者ABO表型,磷酸盐耦联法检测糖基转移酶活性,PCR-SSP法进行ABO亚型基因分型,对第1～7外显子进行测序.结果:血清学试验检测疑为Bel,血清和红细胞上均无D-半乳糖基转移酶活性,ABO血型基因分型结果为BO2,基因测序结果确认为B...     

1例B(A)亚型血型鉴定及分析

目的:对1例献血者ABO血型检测与初筛血型检测不符样本进行血清学及分子生物学鉴定并分析其原因。方法:纸片法初筛血型,应用PK7300血型仪进行ABO血型鉴定,试管法进行血型血清学复测ABO血型,使用测序技术对献血者ABO基因进行测序并比对测序结果。结果:纸片法血型鉴定为AB型,PK7300血型仪正反鉴定一致为B型,试管法血型检测该献血者为B(A)型,测序分析证实ABO基因型:B(A).02/O.01.02,在700位有CG杂合突变,测序结果与试管法血型血清学检测一致,证实该献血员为B(A)亚型。结论:PK7300血型仪鉴定血型存在亚型漏检,试管法ABO血型鉴定是实验室全自动血型检测方法很好的补充。     

抗Di^b抗体合并抗E抗体的鉴定及配合型血液筛选北大核心CSCD

目的对1例存在抗高频抗原抗体患者血标本进行抗体特异性鉴定,并为其筛选相配合血液。方法使用血型血清学试验进行血型鉴定、交叉配血、抗体筛选及鉴定,使用序列特异引物PCR(PCR-SSP)进行Diego血型基因分型。结果血型血清学试验结果显示患者血清中存在抗E抗体及抗高频抗原抗体抗Di^b抗体;PCR-SSP结果显示患者Diego基因型为Di^(a+b-);从其直系亲属中挑选相合血液输注,无不良反应,输血后病情缓解。结论对与谱细胞无反应格局的疑难抗体鉴定病例,可将基因分型与血型血清学实验相结合,进行疑难抗体鉴定。     

ABO血型正反定型不一致样本的分子机制及其临床输血分析

目的 分析ABO血型正反定型不一致样本的分子机制,为临床ABO血型正反不符样本的鉴定和输血提供参考.方法 收集ABO血型正反不符的样本,选取其中血清学反应格局相似的样本6份,应用血清学方法、序列特异性引物-聚合酶链反应、ABO基因直接测序及TA克隆单体型分析等方法分析其分子机制,对其进行分类,并回顾性调查临床输血情况.结果 导致该6份样本ABO血型正反不符情况有3种:抗原减弱(2份)、抗体减弱(3份)、ABO亚型(1份).遵循同型或相容性输注原则进行输血均取得满意效果.结论 ABO血型存在异质性,血清学反应格局相同的样本可由多种机制引起.     

ABO血型正反定型不符与交叉配血不合的原因及其处理方法

目的:探讨引起患者ABO血型正反定型不符与交叉配血不合的原因及其处理方法,确保输血安全.方法:应用血型血清学检测方法对ABO血型正反定型不符及交叉配血不合的血标本进行正反定型、不规则抗体筛选及特异性鉴定、吸收放散试验、唾液血型物质测定等系列检测,分析引起血型正反定型不符及交叉配血不合的原因,为患者选择同型及交叉配血相合的血液进行输注.结果:在输血前的血型血清学检测中发现ABO血型正反定型不符及交叉配血不合的血标本468例,其中自身冷抗体132例(28.2%),血型不规则抗体96例(20.5%),血型抗原性减弱68例(14.5%),血型抗体效价减弱60例(12.8%),自身抗体阳性54例(11.5%),血浆蛋白异常18例(3.8%),实验方法学原因16例(3.4%),ABO血型不合的干细胞移植13例(2.8%),肝素影响9例(1.9%),血标本污染2例(0.4%).结论:ABO血型正反定型不符及交叉配血不合的原因大多数是由自身抗体,不规则抗体,血型抗原抗体减弱及血浆蛋白异常等引起的.因此对ABO血型正反定型不符及交叉配血不合的血标本,应采用多种血型血清学检测方法进行互相验证,以保证血型鉴定及交叉配血无误,确保临床输血安全.     

Bw11亚型家系表现ABO等位基因竞争现象的研究

目的分析携带ABO*BW.11等位基因的家系成员ABO血清学和分子生物学特征。方法应用血清学方法检测先证者及其家系成员共9人的ABO血型表型。采用PCR方法扩增ABO基因第6、7外显子并对扩增产物直接测序,同时克隆测序先证者及其父亲的标本。结果血清学检测初步判断先证者及其弟弟为AB亚型,先证者的父亲及其两女儿为B亚型。克隆测序发现先证者ABO等位基因第7外显子在B101的基础上第695位碱基发生T>C变异,表明为ABO*BW.11等位基因。先证者的父亲、弟弟及其两个女儿均携带该变异等位基因。A基因与BW.11以及BW.11与O基因同时遗传时竞争现象存在明显差异。结论ABO基因c.695T>C变异可能会导致Bw11亚型存在等位基因竞争现象。分子生物学方法结合血清学方法有助于精准鉴定ABO疑难血型。     

献血者ABO血型反定型O细胞凝集原因分析

目的分析献血者ABO血型反定型O细胞凝集原因,以确保受血者输血安全。方法对江门市2000年1月至2008年6月间,采用微板法检测献血者ABO血型出现反定型O细胞凝集者进行冷凝集素效价测定、吸收放散试验、意外抗体鉴定等血型血清学检测。结果308410例献血者中有25例出现反定型O细胞凝集。其中血清中含有冷凝集素导致O细胞凝集16例：效价1：4～1：32者10例、效价≥1：64者6例：血清中存在意外抗体导致O细胞凝集9例：抗-M（IgM）5例、抗-Le^a（IgM）2例、抗-Le^b（IgM）1例、抗-P1（IgM）1例。结论采用微板法检测献血者ABO血型出现反定型O细胞凝集时,应进行相关的血型血清学检测;非血源紧缺的情况下,反定型O细胞凝集者不宜作为献血者。     

一例CisAB血型样本的血清学和遗传学鉴定

目的 对一份血清学检测为CisAB型的标本进行基因分型,并测序确认.方法 血清学方法采用试管正反定型法;基因分型方法为序列特异性引物聚合酶链反应(PCR-sequence specific primer,PCR-SSP); DNA测序采用双脱氧法.结果 血清学检测结果为:正定型为AB型,反定型检出抗B,抗H+++,自身不凝.PCR-SSP的结果是CisAB01型,家系调查符合CisAB型遗传规律.ABO基因测序结果:第6外显子出现c.261缺失G,297为AA纯合子;第7外显子检出c.467C＞T、c.803G＞C突变,同时发现1个新的杂合突变c.724G＞T.结论 该标本血清学表型为CisAB型,测序结果基因型为ABO*CisAB01与ABO* O01杂合,经提交GenBank确认ABO基因第7外显子c.724G＞T为新突变,序列号为JF304777.     

HLA-A位点PCR-SSP基因分型和血清学分型的比较

目的：建立优化ＨＬＡ－Ａ位点ＰＣＲ－ＳＰ分型方法。方法：采用普通扩增仪和Ｅｐｐｅｎｄｏｒｆ管对细胞系ＤＮＡ和３７个健康正常人进行基因分型,血清学检测采用标准淋巴细胞毒试验方法。结果：发现引物浓度和浓度比、ＤＮＡ的纯度及酶的选用,是影响ＨＬＡ－Ａ位点ＰＣＲ－ＳＳＰ分型准确性的重要因素。该方法对３７个标本的基因分型结果与血清学结果相符合。结论：ＰＣＲ－ＳＳＰ方法具有简便准确的优点,可以作为ＨＬＡ－Ａ位     

A rapid long PCR-direct sequencing analysis for ABO genotyping

ABO is the most clinically important blood group system in transfusion and transplantation medicine. The popular ABO genotyping methods, such as the sequencing of exons 6 and 7 and sequence-specific primer (SSP)-PCR, often lead to ambiguous typing results. Long PCR-sequencing method was designed to analyze two regulatory regions (promoter and CBF/NF-Y enhancer regions) and all genomic sequences (except for intron 1) of the ABO gene. Using rapid DNA polymerase with high-fidelity, we amplified 6.3 kb and 7.3 kb for sequencing of enhancer-exon 1 and exons 2-7, respectively. ABO genotyping was performed using this technique in the peripheral blood of three unrelated families. The time requirements of the PCR amplification and purification processes were about 2.0 hours and 15 minutes, respectively. Five different ABO alleles (ABO A102, ABO A105, ABO O01, ABO O02, and ABO B101) with allele-specific CBF/NF-Y minisatellite repeats from three families were analyzed. All genotyping results agreed with serologic findings and results expected by Mendelian inheritance. Compared to conventional PCR-direct sequencing for ABO genotyping, this method proves simple and fast for the analysis of ABO genotypes. Therefore, it might be valuable in clinical transfusion or forensic applications.     

Retrospective analysis of molecular biology mechanism of ABO blood group typing discrepancy among blood donors in Jinan blood station

BackgroundTo accurately identify ABO blood typing in pre-transfusion testing is very important to ensure blood transfusion safely, which is a major responsibility of blood station.MethodsEighty-one blood donors samples with ABO blood group typing discrepancy was collected among 61952 donor samples in our blood station from January 2019 to July 2020. Blood group serological method was used to detect ABO blood group. DNA Sequencing was used to determine the genotype. The antibody screening test detects antibodies other than ABO.ResultsIn total, 61,952 donor samples were analysed for ABO typing discrepancies. The incidence among blood donors was 0.13% (81/61952). The most common reason of ABO typing discrepancies was due to specific antibody or non-specific agglutination (54.32%, 44/81), mainly anti-M antibody, cold autoantibody, anti-D antibody, anti-N antibody and anti-Lea antibody. The major cause of forward typing discrepancies among blood donors was ABO subgroups (25.93%, 21/81), including 10 cases of A subtype (1 case of A2, 2 cases of A3, 2 cases of Ax, 3 cases of AxB, 1 case of Ael, 1 case of Ahm), 6 cases of B subtype (2 cases of B3, 1 case of Bel, 3 cases of AB3), 2 cases of B subtype (A), 1 case of cisAB, and 2 cases of acquired B. The serum antibody was weakened in 16 cases (19.75%).ConclusionsThe blood types should be correctly identified by combining serology with gene sequencing to ensure the safety of clinical blood transfusion, when the forward and reverse typing discrepancies among the blood donors.     

2例CisAB/B亚型的血清学和分子生物学研究

目的 结合ABO血型血清学结果,采用ABO基因测序技术鉴定CisAB血型.探讨CisAB/B亚型的血清学和基因型的关系.方法 对于送至青岛市中心血站ABO血型待定的献血者标本,采用经典试管法进行血清学分析.采用聚合酶链反应(PCR)直接测序的方法分析ABO基因第6,7外显子及侧翼序列、启动子和增强子序列.对于有突变的标...     

Application of real-time PCR and melting curve analysis in rapid detection of Ael and Bel blood types

The ABO blood group is the most important blood group system in transfusion medicine. In addition to the normal levels of ABO antigen expression, Ael and Bel represent the two major blood types that have a weak expression of the A or B antigens on red blood cells. Due to the fact that typing of Ael and Bel by conventional serological methods is time consuming and sometimes gives false-positive and false-negative results, it is warranted to develop an additional technique for the identification of Ael and Bel individuals. Through genetic analysis we have previously identified Ael as possessing an A allele with IVS6+5G-->A mutation (Transfusion 2003;43:1138-1144) and Bel as possessing a B gene with 502C-->T mutation in Taiwan (Vox Sanguinis 2003;85:216-220). Hence, real-time PCR-based genotyping methods were developed in this study to facilitate the detection of Ael and Bel. For genotyping of Ael and Bel, the region of mutations was PCR amplified and subjected to the LightCycler (LC) real-time PCR assay using LC Red640-labeled hybridization probe. Melting curve analysis was performed to determine the melting temperature Tm that was used for genotype detection of Ael and Bel blood types. For Ael genotyping, the melting curve of the normal control appears as one peak at 59.19+/-0.07 degrees C (mean+/-SE) and that of Ael appears as 2 peaks at 59.21+/-0.07 degrees C and 64.39+/-0.07 degrees C, corresponding to the O and Ael alleles, respectively. For Bel genotyping, the melting curve of the normal control appears as one peak at 67.99+/-0.11 degrees C and that of Bel appears as 2 peaks at 59.99+/-0.12 degrees C and 68.1+/-0.13 degrees C, corresponding to the Bel and O alleles, respectively. This genotyping method was shown to be accurate, based on automated sequencing of the PCR-amplified products. It takes only 90 min to perform this genotyping test. Detecting the Ael and Bel blood types by combined LC-PCR and melting-curve analysis is a rapid, reliable, and easy method.     

A rapid and reliable PCR method for genotyping the ABO blood group. II: A2 and O2 alleles

PCR permits direct genotyping of individuals at the ABO locus. Several methods have been reported for genotyping ABO that rely on differentiating the A, B, and O alleles at specific base substitutions. However, the O allele as defined by serology comprises at least two alleles (O¹ and O²) at the molecular level, and most current ABO genotyping methods only take into account the O¹ allele. Determining the presence of the O² allele is critical, as this not-infrequent allele would be mistyped as an A or a B allele by standard PCR typing methods. Furthermore, none of the methods to date distinguish between the A¹ and A² alleles, even though 10% of all white persons are blood group A². We have developed a method for genotyping the ABO locus that takes the O² and A² alleles into account. Typing for A² and O² by diagnostic restriction enzyme digestion is a sensitive, nonradioactive assay that provides a convenient method useful for forensic and paternity testing and for clarifying anomalous serological results. © 1996 Wiley-Liss, Inc.     

一例多位点突变导致的罕见B(A)型的血清学及分子遗传学研究

目的探讨1例ABO血型血清学正、反定型不符的血样标本的分子遗传学基础。方法采用试管法对此例血样进行ABO血型正、反定型,应用直接测序及单倍型测序的方法确定其基因型。结果此例样本血型血清学结果显示,其正定型为AwB型,反定型为B型;直接测序及单倍型测序最终结果显示,其中一条等位基因为O01,另一条等位基因与A101标准序列相比,存在c.297A>G、c.657C>T、c.796C>A、c.803G>C及c.930G>A碱基突变。结论经基因测序证实,此例血清学定型困难的样本基因型为O01/B(A)new型,此突变类型的B(A)型既往未见报道。     

HLA-B27基因检定技术

用聚合酶链反应和血清学方法检测ＨＬＡ－Ｂ２７，并进行比较。方法：采用ＰＣＲ技术７８例可疑为强直性脊椎炎患者样本的ＨＬＡ－Ｂ２７基因，并与血清学比较。结果：３６例具有Ｂ２７基因者中２４例同时作血清学试验，５例血清学难以判定结果，并与ＰＣＲ结果不一致。