Rh弱D及Del样本的检测研究

为了研究初筛为RhD(-)样本中弱D及Del的检测，采用常规血清学方法初筛RhD，用间接抗人球蛋白试验(IAT)检测弱D，应用三氯甲烷/三氯乙烯吸收放散法检测Del。结果表明：在26200例献血者中，常规血清学初筛D(-)者56例(2．14％)，其中经IAT实验确认为弱D型者5例，吸收放散试验确认Del型者9例，且与CE表型相关。结论：为了临床输血安全，经常规血清学检测RhD阴性者，应当再用间接抗球蛋白试验及吸收放散实验检测是否为弱D及Del。     

Rh弱D抗原的临床检测意义

目的 探讨初筛为RhD阴性的样本进一步检测弱D抗原的临床意义。方法 对初筛为RhD阴性的样本进行间接抗人球蛋白试验（IAT）和三氯甲烷／三氯乙烯吸收放散试验。结果 49例初筛为RhD阴性样本中,IAT检出不完全D者4例,吸收放散试验检出D洗脱型（Delusion,Del）者6例,弱D抗原的检出与CE表型相关。结论 为保障输血安全,经常规血型血清学检测的RhD阴性样本需进一步进行IAT和吸收放散试验检测是否为弱D及Del。     

上海地区稀有血型Duffy系统Fy~a阴性献血者筛查和临床应用

目的了解上海地区献血者中Fya阴性的红细胞表型情况,以解决临床Fya抗原阴性患者急救用血的问题。方法利用间接抗球蛋白试验从上海地区献血者中筛查稀有血型Duffy系统Fya抗原阴性献血者;用盐水和抗球蛋白法对1名白种人疑难输血患者的血清做抗体筛选和鉴定,并使用乙醚放散结合抗球蛋白方法检测放散液中抗体,同时对其Duffy血型进行检测。配血用盐水、polybrene和抗球蛋白介质3种方法。结果从9479名献血者中筛查到Fy(a-)14例;从该名白种人患者血清中未检出不规则抗体,直接抗球蛋白试验阳性,红细胞放散液中检出抗-Fya,输注配合型的红细胞后,其血红蛋白明显升高。结论上海地区献血者中Fya阴性的红细胞表型极低。对于大量输血后临床症状未有改善患者,如果血清中未能检出红细胞抗体,应考虑检测患者红细胞上可能存在血型同种抗体。     

湖北荆门地区RhD阴性无偿献血者表型分布调查

目的 了解湖北荆门地区无偿献血者中RhD阴性表型分布情况.方法 常规血清学方法初筛RhD阴性标本,用间接抗球蛋白试验确认弱D,用吸收放散试验检测Del,RhC,c,E和e抗原使用血清学方法进行检测.结果 38 921名无偿献血者中初筛为RhD阴性124例;用抗人球蛋白试验确认弱D 11例;除11例弱D和10例假阴性后,103例确认为Rh阴性标本中共检出Del型25例;103例确认为Rh阴性的标本,血清学表型分布为dccee 63例,dCcee 28例,dCCee 6例,dccEe 4例,dccEE 1例,dCcEe 1例.结论 湖北荆门地区RhD阴性献血者中存在相当比例人群携带弱D表达抗原,为确保临床用血的安全,应加强弱D或Del型的确认.     

长春地区献血者RH Del血清学检测

Rh血型D抗原是引起严重新生儿溶血病的主要红细胞抗原.D抗原有多种变异体,如弱D型、部分D型、D放散型(Del)等.Del红细胞膜D抗原非常弱,常规间接抗人球蛋白试验(IAT)测定为阴性,只有通过敏感的吸收放散技术才能检出.Del在不同民族中表型频率差别较大.目前,全国大部分血站提供给临床的RhD阴性血液,都没有检测Del或用PCR方法检测RhD基因及Del基因型,因此对RhDel血液的继续研究就显得由为重要.     

RhD阴性献血者鉴定结果分析

目的探讨血站确认为RhD阴性的献血者中弱D、部分D、Del表型的比例及对安全输血的影响。方法血站使用单克隆IgM抗D试剂检测D抗原,用间接抗人球蛋白试验确认弱D或部分D;将血站确认为RhD阴性献血者标本送专业机构,用吸收放散试验和基因分型做进一步鉴定。结果 50例在血站确认为RhD阴性的献血者中,检出弱D 4例,占8%;检出Del表型13例,占26%;33例被确认为RhD阴性,占66%。结论弱D、Del表型作为阴性血液输给阴性受血者,可刺激产生抗体,给临床输血带来安全隐患。     

20例输注D~(el)型红细胞的RhD阴性受者的回顾性分析

目的探讨Del型红细胞是否诱导RhD阴性受者产生同种免疫反应。方法用吸收放散试验鉴定Del型血液,对临床输注Del型红细胞的患者进行追踪,采集输血后1周～1年的患者血液标本,RhD抗原初筛阴性的标本采用间接抗球蛋白试验(IAT)进行阴性确认。对患者血清采用凝聚胺法、间接抗球蛋白试验和微柱凝胶法筛查不规则抗体,然后对筛查阳性标本用谱细胞进行不规则抗体鉴定,并进行效价测定。结果采集到输注Del红细胞的RhD阴性受血者样本20例,男性10例,女性10例,女性均有孕产史。Del型受血者5例,男性2例,女性3例,输注Del红细胞后未发生输血反应,不规则抗体筛查阴性。真实RhD阴性受血者15例,男性8例,女性7例,8例男性和5例女性患者输注Del红细胞后未发生输血反应,不规则抗体筛查阴性;2例妊娠女性抗-D为阳性,未出现输血反应。结论 Del型个体未检出抗-D,产生抗-D的个体未检出Del型。Del红细胞导致RhD阴性受者产生同种免疫反应的概率可能很低。     

Rh阴性个体输入D~(el)红细胞产生抗-D免疫的研究

目的探讨Rh阴性个体输血中Del型红细胞的D抗原免疫原性。方法对临床输血的Rh阴性患者进行回顾性跟踪检测,采用PCR-SSP法检测标本RHD基因,间接抗球蛋白法检测抗-D效价,流式细胞术对比输血前后抗体强度的变化。结果 2名输注Del型血液的Rh阴性患者,抗-D效价升高,流式细胞术检测结果显示抗-D强度明显升高,说明Del表型红细胞具有一定的免疫原性。结论为了保障临床输血安全,常规血清学检测Rh阴性的供血者,应进一步排除弱D及Del型。     

单克隆IgM、IgG混合抗D血清在Del鉴定中的应用

Del即D洗脱阳性,是指部分D抗原阴性者的红细胞和抗D抗体做吸收放散试验时,证实这些D阴性的红细胞上带有微弱的D抗原。如将他们的红细胞输给Rh（D）阴性者,后者可能会产生抗D抗体。我们用3种不同厂家的单克隆混合抗D血清与人源性抗D血清作对比,对40例Rh（D）阴性者行Del鉴定,以确定单克隆IgM、IgG混合抗D血清鉴定Del的价值,现报告如下。     

凝胶微柱法与试管法检测放散液中抗体的比较

目的比较凝胶微柱法和试管法检测红细胞释放液抗体的能力。方法将30例外周血和41例脐血红细胞用两种方法测定直接抗人球蛋白试验(DAT)并进行酸放散,将放散液与三种谱细胞、A1、B细胞反应检测抗体的特异性。71例中12例作为阴性对照样本(DAT均为阴性),其中6例为健康献血者外周血,6例为不发生新生儿溶血病(Heamolytic disease of thenewborn,HDN)的婴儿脐血样本。结果24例外周血放散液中有10例两种方法均阳性,12例均为阴性,2例自身免疫性溶血性贫血(AIHA)患者样本凝胶微柱法阳性而试管凝集法阴性,6例健康捐献者的样本DAT试验阴性且两种方法检测放散液均为阴性;41例脐血样本中33例放散液两种方法均反应,2例放散液在试管法中与A1和B细胞反应而同样的放散液做凝胶微柱法时一个和A1细胞反应,另一个只与B细胞反应。结论两种方法检测放散液结果基本是一致的,检测AIHA患者红细胞释放液抗体时用微柱法比较好,检测脐血细胞释放的同种血凝素时试管凝集法更好—些。     

D~(el)红细胞膜表面D抗原表位及抗原强度分析

目的分析Del红细胞膜表面D抗原表位并测定其抗原强度。方法采用16种针对D抗原不同表位的单克隆抗体,通过吸收放散方法检测74例Del个体红细胞膜表面D抗原表位,应用流式细胞术检测其相对抗原强度。结果 74例Del个体D抗原1~9表位检测均为阳性,D阴性、Del及D阳性红细胞相对D抗原阳性率分别为(0.97±0.18)%、(3.06±0.31)%和(99.65±0.71)%,3种表型红细胞D抗原平均荧光强度分别为13.89±2.45,24.18±3.38,223.32±13.05。结论 Del红细胞膜表面D抗原表位表达基本完整,其D抗原强度极低,仅含有微量的抗原分子数目。     

Novel weak D types 31 and 32: adsorption-elution-supported D antigen analysis and comparison to prevalent weak D types

BACKGROUND: Weak D types are thought to express rather quantitative than qualitative D antigen variants. Distinct type-specific phenotypes and weak D cases with anti-D alloimmunization, however, suggest a variable degree of D antigen alteration. STUDY DESIGN AND METHODS: Variant D types were investigated by use of molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen density determination. RESULTS: Two novel weak D types were discovered, termed weak D type 31 and 32 with single RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches, with antigen densities of approximately 130 and 50 D sites per red blood cell, respectively. Adsorption-elution technique-supported D epitope mapping of these two weak D types, the recently described weak D type 26, and of the most common Central European weak D types (weak D types 1, 2, 3, 4.0, and 4.1) demonstrated the expression of all tested D epitopes. In contrast, a distinct D epitope loss was detected in weak D type 15 and partial D control samples. CONCLUSION: All novel and prevalent weak D types expressed all tested D epitopes. Our results indicate that adsorption-elution techniques may be of advantage whenever D epitope loss is suspected in extremely weak D variants.     

Removing IgG antibodies from intact red cells: comparison of acid and EDTA, heat, and chloroquine elution methods

BACKGROUND: To accurately phenotype red cell from patients with a positive direct antiglobulin test (DAT), nonlytic elution procedures were assessed for their ability to dissociate IgG from antibody-coated red cells without altering red cell antigen expression. STUDY DESIGN AND METHODS: Antibodies coating red cells that were sensitized in vivo (warm-reactive autoantibodies: 8 patients) or in vitro (42 alloantibodies) were eluted by using glycine-HCl and EDTA (acid/ EDTA), heat (56 degrees C, 10 min), or chloroquine method. RESULTS: Acid/EDTA elution gave the best results, reducing DAT positivity to microscopic levels or rendering the DAT negative in 48 of 50 instances, whereas 4 samples remained resistant to heat elution and 24 to chloroquine. Standard DAT agglutination scores demonstrated that both acid/EDTA and heat elution were superior to the chloroquine method (p < 0.0001). With the gel low-ionic-strength saline indirect antiglobulin test, acid/ EDTA was superior to heat (p < 0.001). Overall, acid/ EDTA elution dissociated more antibodies than heat (p < 0.0001), especially for Kell system (K, k, Kpa, Kpb) alloantibodies. Common red cell antigens, other than Kell system antigens, were unaffected by acid/EDTA elution. In contrast, the expression of most blood group antigens was diminished after heat elution. However, it was possible to type red cell antigens by using gel low-ionic-strength saline indirect antiglobulin tests or tube agglutination methods. CONCLUSION: Although heat elution may be used on a limited basis, the acid/EDTA method appears to be the procedure of choice for typing red cell coated with warm-reactive IgG alloantibodies or autoantibodies.     

BCSH-NIBSC anti-D reference reagent for antiglobulin tests: the in-house assessment of red cell washing centrifuges and of operator variability in the detection of weak, macroscopic agglutination

Summary. A batch of an anti-D preparation, reference 91/608, has been prepared for the preparation of red cells weakly sensitized with IgG that can reveal inhibition of the antiglobulin test by one volume of human serum, diluted 1:1000. The preparation provides an objective assessment of red cell washer efficacy and the confidential, in-house assessment of operator variability in detecting weak but definite macroscopic agglutination by blind, replicate tests.
Red cell washer efficacy and poor operator reading procedures causing disruption of weak agglutination are two major causes of false-negative antiglobulin tests; neither are adequately detected by the common quality-control procedure of adding strongly IgG-sensitized red cells ('Coombs control cells') to apparently negative antiglobulin tests. However, weakly IgG-sensitized red cells do offer a valuable control function that can detect some degree of cell washer inefficiency and reading errors although such cells are not a substitute for the more sensitive replicate testing.
Test protocols are provided to assess the efficacy of cell washing machines and operator skills in the detection of weak but definite macroscopic agglutination.     

Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques

BACKGROUND: RhD discrepancies between current and historical results are problematic to resolve. The investigation of 10 discrepancies is reported here. STUDY DESIGN: Samples identified were those that reacted by automated gel technology and were negative with an FDA-approved reagent. Reactivity with a commercially available panel of monoclonal anti-D was performed. Genomic DNA was evaluated for RHD alleles with multiplex RHD exon polymerase chain reaction (PCR), weak D PCR-restriction fragment length polymorphism, and RHD exon 5 and 7 sequence analyses. RESULTS: The monoclonal anti-D panel identified two samples as DVa, yet possessed the DAR allele. Two weak D Type 1 samples had a similar monoclonal anti-D profile, but only one reacted directly with one of two FDA-approved anti-D. Only two of four weak D Type 2 samples reacted directly with one FDA-approved anti-D, and their D epitope profile differed. CONCLUSIONS: The monoclonal anti-D reagents did not distinguish between partial and weak D Types 1 and 2. Weak D Types 1 and 2 do not show consistent reactivity with FDA-approved reagents and technology. To limit anti-D alloimmunization, it is recommended that samples yielding an immediate-spin tube test cutoff score of not more than 5 (i.e., < or =1+ agglutination) or a score of not more than 8 (i.e., < or =2+ hemagglutination) by gel technology be considered D- for transfusion and Rh immune globulin prophylaxis. That tube test anti-D reagents react poorly with some Weak D Types 1 and 2 red cells is problematic, inasmuch as they should be considered D+ for transfusion and prenatal care. Molecular tests that distinguish common partial and Weak D types provide the solution to resolving D antigen discrepancies.     

The role of antigen mobility in anti-Rh0(D)-induced agglutination.

Intact human erythrocytes were cross-linked with glutaraldehyde (GA) or dimethyladipimidate (DMA) and tested for their ability to bind [125I]-IgG anti-Rh0(D) and to undergo antibody-mediated hemagglutination. There was no decrease in antibody binding after treatment with GA concentrations up to 1.25% and DMA concentrations up to 1%. Red cells treated with these concentrations of GA and DMA did not agglutinate. The techniques employed to induce agglutination of the cross-linked red cells involved "incomplete" IgG anti-Rho (D) in albumin, "complete" IgM anti-D Rho (D) in saline, and the antiglobulin (Coombs) reaction. The agglutinability of the chemically modified red cells was inversely correlated with the extent of fixation. The dissociation of antibody binding from agglutinability in cross-linked erythrocytes suggests that Rho (D) antigen mobility is required for red cell agglutination. Antigen mobility was manifested by the transition from a relatively monodisperse distribution pattern of Rho (D) antigen sites to one of large aggregates or clusters when agglutination was induced by IgM anti-Rho (D), IgG anti-Rho (D) agglutination of protease modified red cells, and by anti-IgG agglutination of IgG anti-Rho (D)-coated red cells. Antigen clustering was not as prominent in red cells agglutinated by IgG anti-Rho (D) in the presence of albumin. Even though antigen mobility is a prerequisite for antibody-mediated hemagglutination, clustering does not appear to be an absolute requirement. The degree of antigen clustering differs with varying types of agglutination.     

Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers

D_el (D-elute) in the Rh blood group system is a variant with very weak D antigen and no agglutination is found by the indirect antiglobulin test. This variant is characterized by the presence of anti-D eluate obtained after an adsorption-elution test using anti-D antibodies. We studied here the molecular genetic status of D_el by using polymerase chain reaction with sequence-specific primers (PCR-SSP).
We screened 306 serologically apparent D-negative Japanese donors comprising 102 D_el types for exons 7, 4 and 10 of the RHD gene. No PCR product was found in all 204 non-D_el samples from the D-seronegative donors. However, PCR products were found in all 102 D_el samples and all 70 D-seropositive samples tested by the three PCR methods for exons 7, 4 and 10 analysis.
D_el was found with CCee, CcEe and Ccee, but not with CCEe, CcEE, ccEE, ccEe or ccee phenotype. The incidences of D_el in the samples with the serological phenotypes CCee, CcEe and Ccee were 80.0% (4/5), 68.2% (45/66) and 61.6% (53/86), respectively.
The results provide evidence that D_el samples have exons 4, 7 and 10 of an RHD gene present in some form. This is consistent with the evidence that D antigen is present on the cells although only detected by antibody adsorption and elution. The PCR-SSP method in the present study is useful to confirm D_el among serologically apparent D-negative samples.     

Rh phenotypes of Chinese blood donors in Hong Kong, with special reference to weak D antigens

Among Hong Kong Chinese blood donors, 99.71 percent were found to be D+. Of these, 55.02 percent were of the phenotype CCDee. The Du phenotype was found to be present in 0.016 percent. Among the 0.27 percent who were apparently D-, 0.079 percent were of the Del phenotype, while the remaining 0.19 percent were "true D-," as defined by a nonreactive eluate obtained by an adsorption and elution procedure using anti-D. The ccdee phenotype constitutes 56.77 percent of the "apparent D-" and 80.24 percent of the true D-. Data show that anti-D rarely occurs in Hong Kong Chinese, and it is postulated that this could be due to the presence of a very weak form of the D antigen among many of those who appear to be D-.     

RhD variants in Caucasians: consequences for checking clinically relevant alleles

BACKGROUND: Weak D type carriers cannot be immunized against D except when antigen density is below 400 antigens per RBC, whereas partial D carriers can produce anti-D. STUDY DESIGN AND METHODS: A total of 168 blood samples from Caucasian individuals were studied because of weak D expression and/or anti-D production. Serologic analysis and molecular analysis were performed. RESULTS: In total, 70 partial D and 62 weak D were identified. Among weak D samples, 30 weak D Type 1 and 21 weak D Type 2 alleles were found. Five new alleles were characterized carrying 399G > T, 680T > C, 833G > A, 851C > T, and 1015G > A, respectively. According to previous studies, antigen density was up to 500 for weak D Type 1 and 2, except when there was a dCe haplotype in trans. Antigen density was below 400 antigens per red blood cell for the new variants and most other weak D variants. CONCLUSION: These results provide molecular characterization of five new D variants. They also suggest that it would be advantageous to develop in routine laboratories weak D Type 1 and 2 genotyping for serologically depressed D antigen. It will help to avoid wasting of D- red blood cell units because carriers may safely receive D+ units.