DEL型个体抗体筛选的调查分析

目的探讨DEL型个体是否对D抗原产生体液免疫应答。方法对经间接抗球蛋白试验确认为Rh(D)阴性的1030名献血者和89名孕妇做抗体筛选,对抗体筛选为阳性的献血者用10个O型谱细胞和筛选特异细胞做抗体鉴定。对产生抗-D的Rh(D)阴性献血者和孕妇用DEL特异性引物鉴定检测DEL型;对117名Rh(D)阴性献血者以吸收放散试验检测DEL型,对DEL阳性的献血者做抗体筛选。结果10名献血者和5名孕妇产生抗-D,他们的DEL基因分型均为阴性;117名Rh(D)阴性献血者DEL吸收放散试验检测阳性24名,并且抗-D阴性。结论在产生抗-D的个体中未检出DEL型,在DEL型的个体亦未检出抗-D。     

Rh阴性孕妇血型D抗原同种免疫反应研究

目的研究Rh阴性孕妇血型D抗原同种免疫反应,为建立有区别的D亚型的输血规则提供参考。方法对RhD阴性孕妇进行基因分型,并检测是否有抗-D;总结分析孕妇妊娠次数以及胎儿红细胞的检出与产生抗-D之间的相关性。结果在122名RhD表型阴性的孕妇中,检出d/d基因型97例,Del型20例,弱D15型1例,RHD-CE(2—9)-D/d融合基因型3例,DⅥⅢ型1例。在97名RhD阴性(d/d)孕妇中,有38例产生抗-D;20名Del血型孕妇均未检出抗-D;3名RhD-CE(2—9)-D/d中有2例产生抗-D;1名弱D15型的孕妇未检出抗-D;1名DⅥⅢ型孕妇抗-D检出阳性。统计分析表明孕妇的妊娠次数以及外周血中检出胎儿红细胞的检出与抗-D的产生有相关性,这可以成为Rh阴性孕妇被D抗原免疫的证据。结论不同D亚型的个体可能会对D抗原有不同的免疫反应,我们认为在中国Del血型的受者可以接收D阳性血液。建立有区别的输血规则可以节约有限的Rh阴性血资源和提高输血安全。     

部分D表型同种抗-D1例及其基因型分析

目的分析1名部分D表型孕妇抗-D同种免疫反应及部分D表型基因的遗传情况。方法常规血清学方法鉴定1名部分D表型孕妇血清抗-D,然后通过RHD基因10个外显子的检测、RHD基因合子型分析、以及D抗原12个抗原表位的测定,系统鉴定该例部分D表型的类别和亚型,并对其1家3代进行遗传分析。结果该例部分D表型为DVI(Ⅲ)类型,孕妇血清抗-D是由于妊娠Rh阳性子女产生的,效价为32,抗-D不与其自身红细胞及其它DVI(Ⅲ)部分D表型红细胞反应;家系分析表明先证者弟亦为DVI(Ⅲ)部分D表型,其父亲为隐性携带DVI(Ⅲ)部分D表型基因,而其母亲1条染色体缺失RHD基因,先证者DVI(Ⅲ)部分D表型基因未遗传至其女。结论证实DVI(Ⅲ)部分D表型个体可被正常D抗原致同种免疫反应,同时显示个例的基因变异为家系遗传性,而非个体基因变异所致。     

1例部分D表型孕妇抗-D及基因型分析

目的分析1例孕妇抗-D同种免疫反应及部分D表型和基因型。方法分别采用盐水法和微柱凝胶卡法检测RhDCcEe抗原,以及常规血清学试管法和凝胶卡法鉴定孕妇血清抗-D抗体及其效价,通过检测RHD基因的10个外显子、RHD基因合子型,以及测定D抗原12个表位鉴定部分D表型。结果该个体盐水法测定为dCcee,但微柱凝胶卡法检测为DCcee表型,RHD基因外显子检测缺失第3~6外显子,合子型测定为D/d,D抗原表位分析显示缺失大部分D表位,提示该个体为部分D表型DVI-III型,基因型为DVICe/dce;孕妇血清抗-D抗体第20周第1次检测为阳性,效价为1∶32,至生产前为1:64,产后确认为Rh(D)新生儿溶血病,生下一子为Rh阳性。结论部分D表型DVI-III型过往国内亦有报道发生抗-D同种免疫反应和新生儿溶血病,我国汉族DVI-III型多见,产前检查时须防漏检。     

DEL红细胞膜D抗原表位分析

目的分析Rh血型D放散型(DEL)红细胞膜D抗原表位(epitopemapping)。方法采用微量吸收放散技术通过9种抗D抗原不同表位的人抗D单克隆抗体,检测3名已知Rh表型和RH基因型的D放散型个体的红细胞膜D抗原表位,分别以Rh阳性、Rh阴性、部分D表型DVa(Hus)和DVIⅢ型样本作为对照。结果3名携带RHD1227A等位基因的D放散型个体,红细胞膜D抗原9个抗原表位均检测为阳性,而对照样本检测结果各不相同。结论携带RHD1227A等位基因的中国汉族D放散型个体红细胞膜可能表达基本完整D抗原。     

RhD(-)孕产妇的RhD同种免疫分析及围产期孕妇新生儿溶血病的监测、预防和治疗

目的分析304名RhD(-)孕产妇RhD同种免疫发生情况,探讨RhD(-)孕产妇抗-D产生的影响因素,建立正确的围产期孕妇RhD新生儿溶血病的监测、预防和治疗方案。方法采用标准血清学方法对3975名孕产妇及其丈夫进行ABO及RhD抗原鉴定。对RhD抗原鉴定为阴性的标本,进一步采用间接抗球蛋白方法检测RhD抗原,以排除或确认弱D型或部分D表型。对所有RhD(-)孕产妇及其丈夫进行CcEe表型的血清学分型。采用抗球蛋白方法对所有RhD(-)孕产妇标本进行不规则抗体初筛,对初筛阳性者进一步用鉴定细胞做抗体鉴定及抗体效价测定并采用PCR-SSP方法确定是否为DEL型。结果 3975份孕产妇标本中304份经鉴定为RhD(-),其中29份产生了抗-D,本组调查中RhD(-)孕产妇抗-D产生的比例为9.54%。29名产生抗-D的RhD(-)孕产妇中,夫妇ABO血型相合者24名(82.76%),不合者5名(17.24%)。经分子生物学方法鉴定,29名产生抗-D的Rh(-)孕产妇均排除DEL表型。结论 RhD孕产妇RhD同种免疫的发生受多种因素的影响,DEL型孕产妇产生抗-D的几率较低。应及时、定期监测RhD围产期孕妇的抗-D水平,对未产生抗-D的孕妇应给予抗-D免疫球蛋白治疗,对已产生抗-D的孕妇,应密切监测其抗-D水平,并在孕期及新生儿出生后给予正确治疗。     

RhD阴性患者输注“亚洲型”DEL型红细胞短期内引起高效价抗-D的同种免疫研究

目的 通过对1例RhD阴性患者输注“亚洲型”DEL红细胞后诱发同种抗-D免疫的研究分析,探讨RhD阴性患者输注DEL型策略。方法 采用血清学方法进行ABO及Rh系统血型抗原检测、不规则抗体筛查及鉴定,高分辨熔解曲线和基因测序分析检测DEL型的RHD基因分型。结果 受血者为O型,RhD阴性。供血者为RHD 1227G>A突变的“亚洲型”DEL,R h表型为Ccee。受血者输血后抗体筛查阳性,并鉴定为抗D和抗C,抗D效价512,抗C效价128。结论 RhD阴性患者输注“亚洲型”DEL红细胞会引起抗-D同种免疫。因此对于RhD阴性献血者应加强DEL型的筛查,必要时可以增加RHD基因检测以保障输血安全。     

瓦房店地区Rh阴性血DEL血清学检测

目的将Rh阴性血液与DEL型分开,指导临床更加科学合理地应用Rh阴性血液。方法应用三氯甲烷／三氯乙烯吸收放散实验对Rh阴性血液进行DEL鉴定。首先对市中心血站从2003年3月～2007年11月所采集的血液标本进行筛选,采用IgG＋IgM混合抗-D试剂进行盐水法初筛,共收集Rh阴性标本88个,然后进行弱D筛选,采用抗人球法和盐水法对88个阴性标本进行弱D测定,排除弱D后,再将其余标本进行表型测定,采用IgM抗-E、抗-e、抗-c、抗-c试剂以盐水法检测Rh分型,确定其表型,最后将确定出的非D变异型的Rh阴性血液标本用三氯甲烷／三氯乙烯作吸收放散实验,确定是否为DEL型。结果经过对血液标本的初筛,共检测出有效Rh阴性标本88个,在进行弱D的测定中,共检测出2个D变异型,其余86个标本再做DEL检测,结果DEL阳性标本37个,真正的Rh阴性标本49个,DEL阳性标本占所检测Rh阴性标本的42．05％。37个DEL阳性标本的小类测定中,CCdEe表型1个,占总数的2．70％CCdee表型1个,占2．70％;CcdEe表型3个,占8．11％;Ccdee表型7个,占18．92％ccdEe表型2个,占5．41％ccdee表型23个,占62．16％。结论近1年采集的阴性标本中,DEL阳性标本所占比例高,应引起重视,DEL检测可以更好的指导临床输血工作,保证输血安全。     

山东汉族RhD阴性个体基因多态性研究

目的研究山东汉族人群RhD阴性个体RhD基因多态性。方法采用标准血清学方法和抗球蛋白实验筛选Rh阴性个体;对Rh阴性个体再用吸收放散实验确定DEL型。运用多重聚合酶链反应(PCR)方法分析RhD阴性个体基因存在情况。结果67例Rh阴性个体中DEL型16例,占Rh阴性个体的23.88%,其6个RhD基因特异性的第3、4、5、6、7、9外显子全部存在;剩下51例Rh阴性个体中1例缺失第5外显子,其余50例6个外显子全部缺失。结论山东汉族人群RhD阴性个体中存在一定比例的DEL型,且所有DEL样本均具有完整的RhD基因,在排除DEL型后的RhD阴性个体可能携带RhD基因,但其比例较低。     

RHCE(2-9)-D变异产生抗-D引起新生儿溶血病1例

目的 对1例有输血史多次分娩史的RhD阴性孕妇D抗原进行血清学和分子生物学检测,明确RhD表型和基因型。方法 微柱凝胶技术进行母亲和新生儿Rh血型初筛,母亲红细胞经典抗人球蛋白技术进行RhD阴性确认,吸收放散试验确定是否为Del。基因检测确定孕妇RHD基因型。结果 该孕妇结果为Ccdee, RhD确证试验为D阴性,吸收放散试验阴性,基因检测为RHCE(2-9)-D变异,产生抗-D,抗-E。新生儿为CCDee表型,游离抗体检出抗-D和抗-E,放散液检出抗-D。患儿诊断为新生儿溶血病,进行输血、光疗和换血治疗。结论 RHCE(2-9)-D变异型血清学可能表现为D阴性,确证试验阴性,有产生抗-D并引起新生儿溶血病的风险。     

Anti-D in pregnant women with the RHD(IVS3+1G>A)-associated DEL phenotype

BACKGROUND: Pregnant women with the DEL phenotype appear to be D– by routine serology. Women with DEL phenotypes that show a partial D‐like epitope loss may develop anti‐D. It has been proposed that this alloantibody could have a deleterious effect with respect to hemolytic disease in the fetus and newborn. CASE REPORTS: Two pregnant women, one in Australia and one in Germany, were serotyped as D– and were sensitized to the D antigen. Noninvasive fetal RHD genotyping was performed to plan pregnancy management. RESULTS: In both cases the fetal RHD status could not be assigned due to the presence of a maternal DEL allele. This was suspected through detection of high RHD amplicon levels during quantitative polymerase chain reaction. For both cases extended molecular typing of the maternal genomic DNA revealed a RHD(IVS3+1G>A) allele. For case one, the D+ infant developed a mild hemolytic disease requiring phototherapy. In the second case a D– (or DEL) newborn was unaffected. CONCLUSION: Fetal genotyping from maternal plasma reveals RHD variants in pregnant women with anti‐D. Fetuses and newborns of sensitized pregnant women carrying the RHD(IVS3+1G>A) allele are at risk of hemolytic disease.     

浙江汉族Rh DEL表型的分子机理研究

为了研究浙江汉族Rh DEL表型的分子机理，用吸收放散的血清学方法鉴定Rh DEL表型，然后用RHD基因特异的聚合酶链反应-序列特异性（PCR—SSP，polymerase chain reaction-sequence specific prime）和序列分析方法鉴定DEL表型RHD基因的10个外显子和外显子-内含子连接区域可能的变异。结果表明：在122例浙江汉族Rh阴性血型中共检测到35例DEL表型个体，其中RhCCdee、RhCcdee和RhCcdEe表型分别有6例（17．14％）、28例（80．00％）和1例（2．86％）。序列分析发现，Rh DEL表型个体的第9外显子都有1227G〉A突变。D杂合性试验发现，有29例（RhCcdee，28例；RhCcdEe，1例）存在RHD基因的缺失，有6例（RhCCdee）不存在RHD基因的缺失。结论：RHD1227A是浙江汉族RhDEL表型个体的重要遗传标记。     

RhD阴性献血者RhD弱抗原变异体的发生频率

目的了解RhD阴性献血者RhD弱抗原变异体的发生频率,为提高输血安全性提供理论依据。方法首先采用盐水试管法对初筛为RhD阴性的血样进行RhD表型鉴定;然后采用间接抗球蛋白试验筛查Partial D和Weak D,最后用吸收放散试验从间接抗球蛋白试验确认阴性的血样中筛选并确认DEL型。结果 RhD阴性献血者中最多的Rh表型为dccee,其次为dCcee,分别为57.34%（207/361）和32.13%（116/361）。共筛查出Partial D和Weak D 19例,总检出率为5.26%（19/361）,其中dccEe 8例（2.22%）、dCcee 5例（1.39%）、dCCee 4例（1.11%）、dccee和dCcEe各1例（0.28%）。共确认DEL型87例,总检出率为24.10%（87/361）,其中dCcee 72例（19.94%）、dCCee 8例（2.22%）和dccEe 7例（1.94%）。结论 RhD阴性献血者RhD弱抗原变异体发生频率较高。常规血清学检测Rh阴性的供血者,应进一步排除RhD弱抗原变异体,以保障临床输血安全。     

The risk assessment study for hemolytic disease of the fetus and newborn in a University Hospital in Turkey

Maternal red-cell alloimmunization occurs when a woman’s immune system is sensitized to foreign red-blood cell surface antigens, leading to the production of alloantibodies. The resulting antibodies often cross the placenta during pregnancies in sensitized women and, if the fetus is positive for red-blood-cell surface antigens, this will lead to hemolysis of fetal red-blood cells and anemia. The most severe cases of hemolytic disease in the fetus and newborn baby are caused by anti-D, anti-c, anti-E and anti-K antibodies. There are limited data available on immunization rates in pregnant women from Turkey. The aim of the present study was to provide data on the frequency and nature of maternal RBC alloimmunization in pregnant women in a tertiary care hospital. In this study, we retrospectively evaluated the indirect antiglobulin test results of Rh-negative pregnant women performed in our Blood Bank between 2006 and 2012. Indirect antiglobulin test positive women also underwent confirmatory antibody screening and identification. During the study period, 4840 women admitted to our antenatal clinics. With regards to the major blood group systems (ABO and Rh), the most common phenotype was O positive (38.67%). There were 4097 D-antigen-positive women (84.65%) and 743 women with D-antigen-negative phenotype (15.35%). The prevalence of alloimmunization was found to be 8.74% in D-antigen negative group. Despite prophylactic use of Rh immunglobulins, anti-D is still a common antibody identified as the major cause of alloimmunization in our study (anti-D antibody 68.57%, non-D antibody 31.42%). While alloimmunization rate to D antigen was 6.46%, non-D alloimmunization rate was 2.69% among Rh-negative pregnant women. Moreover, detailed identification facilities for antibodies other than anti-D are not available in most of centers across Turkey. However, large-scale studies on pregnant women need to be done in order to collect sufficient evidence to formulate guidelines and to define indications for alloantibody screening and identification.     

Secondary alloanti-D immunization post transfusion of “Asia type” DEL red blood cells

Background“Asia type” DEL red blood cells (RBCs) express a very weak D antigen and cannot be detected by routine RhD typing. Thus, it is routinely typed as D-negative (D–) blood group and transfused to D– recipients. Here we described a case of secondary alloanti-D immunization that was associated with transfusion of DEL RBCs to D– recipients and was initially considered as primary alloanti-D immunization.Case presentationA 44-year-old D– woman (G2P2) with adenomyosis and anemia underwent transabdominal hysterectomy. She received four units of D– RBCs before operation. Before transfusion, the alloantibody screening test was negative. Four days after the first transfusion, she needed another RBC transfusion. Unexpectedly, the routine pre-transfusion alloantibody screening test became positive and anti-D (titer, 128-fold) was identified, indicating an alloanti-D immunization. The anti-D developed four days after the first transfusion was unexplained, so alloantibody identification was performed on the sample collected before the first transfusion, and weak anti-D combined with anti-E, which was not detectable during the previous routine pre-transfusion alloantibody screening test with non-enzyme-treated screening cells, was identified using bromelain-treated panel cells. The remaining blood samples of first transfusion in bag tails from two donors were collected for RHD genotyping analysis. One donor was later identified as “Asia type” DEL having RHD* 1227 A/01 N.01 genotype.ConclusionCaution should be applied when we conclude that transfusion of “Asia type” DEL RBCs to true D– recipients could induce primary alloanti-D immunization, especially if the short time interval between transfusion and detection of anti-D is observed.     

Overestimation of fetomaternal haemorrhage by the acid-elution technique in mothers with β-thalassaemia minor

Summary. In Rh-negative women, it is important to quantify the magnitude of an Rh-positive fetomaternal haemorrhage (FMH) so that sufficient Rh immune globulin (RhIg) can be administered early in the postpartum period to prevent alloimmunization. The standard post-partum dose of Rhlg varies from 100 μg in the UK to 300 μg in North America. It is therefore important to identify all Rh-negative women who have had an FMH greater than 10 ml in the UK or greater than 30 ml in North America because an FMH greater than these amounts will affect the dose of RhIg that is administered. As acid-elution techniques can overestimate the magnitude of an FMH in the presence of an elevated maternal haemoglobin F level, we performed a prospective study to determine how often this occurred. Of 1,894 consecutive Rh-negative mothers who delivered Rh-positive infants, whose blood was screened for an FMH greater than 10 ml of fetal blood using an acid-elution procedure, 11 were found to have an FMH over 10 ml. In five of these 11 women, the volume of FMH was less than 10 ml using an alternative technique (rosette test) to assess the FMH size. Six of these women were found to have β-thalassaemia minor on the basis of a low MCV, and high haemoglobin A₂ and/or high haemoglobin F levels. In five of these the FMH was significantly overestimated by the acid-elution technique compared to the rosette technique. Therefore, in the presence of a maternal condition, which may result in an elevated haemoglobin F level, an FMH estimated to be over 10 ml in the UK or 30 ml in North America using an acid-elution procedure, should be confirmed by an alternative technique, which does not involve the estimation, directly or indirectly, of haemoglobin F.     

山东汉族RhD阴性个体基因多态性研究

目的研究山东汉族人群RhD阴性个体RhD基因多态性。方法采用标准血清学方法和抗球蛋白实验筛选Rh阴性个体;对Rh阴性个体再用吸收放散实验确定DEL型。运用多重聚合酶链反应（PCR）方法分析RhD阴性个体基因存在情况。结果67例Rh阴性个体中DEL型16例,占Rh阴性个体的23．88％,其6个RhD基因特异性的第3、4、5、6、7、9外显子全部存在;剩下51例Rh阴性个体中1例缺失第5外显子,其余50例6个外显子全部缺失。结论山东汉族人群RhD阴性个体中存在一定比例的DEL型,且所有DEL样本均具有完整的RhD基因,在排除DEL型后的RhD阴性个体可能携带RhD基因,但其比例较低。