RHD genotyping of serological weak D phenotypes in the Iranian blood donors and patients

BackgroundMost prevalent weak D types in the Caucasians molecularly defined weak D types 1, 2 or 3 and can be managed safely as RhD-positive, conserving limited supplies of RhD-negative RBCs. Therefore, identification of RHD alleles prevalence in each population could improve the policies related to accuracy of RhD typing. The aim of this study was to determine the frequency of RHD variant alleles among donors and patients for the first time in Iran.Materials and methodsRHD genotyping was performed on 100 blood donor and patient samples with weak D phenotype. PCR-SSP and DNA sequencing were used to identify the RHD alleles.ResultsMolecular analysis showed only 15 samples were RHD*weak D 1(n = 13) and RHD*weak D 3(n = 2), and no cases of RHD*weak D 2 were detected. RHD*weak 15 (n = 43) was determined as the most prevalent D variants in our population and the other weak D types follows: RHD*weak 4, 5, 80 and one case of each one: RHD*weak 8, 11, 14, 100 and 105. Partial D variants also was identified in 18 samples as follows: RHD*partial DLO, DBT1, DV2, DHK and DAU-1.ConclusionThe results of this study highlight the specific pattern of RHD status in the Iranian population. The weak D types 15 was the most common weak D type in the Iranian population. However, the screening for weak D types 1, 2 and 3 with 15 % frequency is also necessary for accurate RhD typing and developing clinical strategy of blood transfusion in weak D patients.     

RHD alleles in Brazilian blood donors with weak D or D-negative phenotypes

The RHD gene is highly polymorphic and the existence of a large number of alleles results in RhD variant phenotypes. RHD genotyping has been used to distinguish normal D antigen from D variants due to limitations of serologic methods. The purpose of this study was to determine the phenotypic frequency of RhD and RhCE antigens and to investigate the RHD alleles present in samples with the weak D or D- phenotypes from Brazilian blood donors. A total of 2007 donors were phenotyped for D, C, c, E and e antigens. Samples phenotyped as D- were genotyped by polymerase chain reaction-sequence specific primers, and exon 10 and intron 4 of the RHD gene were analysed. D- samples containing the RHD gene or samples considered weak D were further characterised using genotyping platform or nucleotide sequencing. Using serologic methods we found that 87.3% of the donors were D+, 11.9% D- and 0.8% weak D. The frequency of RHD gene in D- individuals was 9.2%. Five RHD alleles from phenotypically D- donors were characterised in six molecular backgrounds: RHDΨ, RHD-CE-D(s), RHD-CE-(2-9)-D, RHD/RHDΨ, RHDΨ/RHD-CE-D(s) and RHD-CE(2)-D. The most common weak D antigens types found were 1, 3, 4.0/4.1 and 4.2, whereas the most prevalent weak D type was 4.2 (or DAR). The RHD genotyping proved to be a necessary tool to characterise RHD alleles in donors phenotyped as D- or weak D to increase the transfusion safety in highly racial mixed population.     

PCR检测Rh血型C/c基因型

目的：建立Rh血型系统C/c基因分型的方法，检测人群中C/c基因频率。方法：用快速盐析法抽提样本DNA，用PCR检测C/c基因型。结果：本组RhD阳性人群中，C基因型频率为0．674，c基因型频率为0．326。RhD阴性人群中，C基因频率为0．234，c基因频率为0．766。结论：RhD阳性与阴性人群C和c基因频率存在明显的差异。RhD阳性人群以C为主，RhD阴性人群中以c为主。     

Systematic RHD genotyping in Brazilians reveals a high frequency of partial D in transfused patients serologically typed as weak D

BackgroundThe discrimination between weak D types and partial D can be of clinical importance because carriers of partial D antigen may develop anti-D when transfused with D-positive red blood cell units. The aim of this study was to determine by molecular analysis the type of D variants among Brazilian patients requiring transfusions with serologic weak D phenotypes.Material and methodsSamples from 87 patients (53 with sickle cell disease, 10 with thalassemia and 24 with myelodysplastic syndrome), serologic typed as weak D by manual tube indirect antiglobulin test or gel test were first RHD genotyped by using the RHD BeadChip Kit (BioArray, Immucor). Sanger sequencing was performed when necessary.ResultsRHD molecular analysis revealed 32 (36.8 %) variant RHD alleles encoding weak D phenotypes and 55 (63.2 %) alleles encoding partial D antigens. RHD variant alleles were present in the homozygous state or as a single RHD allele, one variant RHD allele associated with the RHDΨ allele, or two different variant RHD alleles in compound heterozygosity with each other in 70 patients, 4 patients and 13 patients, respectively. Alloanti-D was found in 9 (16.4 %) cases with RHD alleles predicting a partial D.DiscussionThe frequency of partial D was higher than weak D types in Brazilian patients serologically typed as weak D, showing the importance to differentiate weak D types and partial D in transfused patients to establish a transfusion policy recommendation.     

RHD/CE typing by polymerase chain reaction using sequence-specific primers

BACKGROUND: Current DNA-based Rh system typing strategies may detect the two RH genes and their prevalent alleles, but they are known to fail sometimes, when rare RH alleles (e.g., D category phenotypes) are encountered. It is almost impossible to find a single DNA-based method that can accommodate the great heterogeneity within the human Rh system. STUDY DESIGN AND METHODS: An easy-to-perform DNA-based method for the detection of the two RH genes and their alleles, including variant RHD alleles, was developed. By the use of one RHD/C-, seven RHD-, and four RHCE-specific polymerase chain reactions, all triggered to work at identical thermocycling conditions, the DNA of 77 blood donors carrying weak D and that of 200 random donors with common D phenotype was investigated. In addition, 77 selected samples of ccDee and rare Rh system phenotypes were examined. RESULTS: Among 77 samples of weak D, one Rh33 and six DVI categories were detected, one of which showed new RHD-specific nucleotide patterns. In DFR and CCee samples, novel variant RHD alleles were found. RHD DNA types of 200 random donors were found to be concordant with their D phenotype. For RHE and RHe genotyping, a full correlation with serologic phenotypes was found. Our method for genotyping RHC and RHc failed in some cases, because of an already published RHc allelic variation, which we have called RHc(cyt48). An estimate of the frequency of this RHc(cyt48) allele in a white population was made. CONCLUSION: The presented exon-scanning RHD/CE polymerase chain reaction using sequence-specific primers complements current DNA-based Rh system typing strategies and is superior in the detection of variant RHD alleles.     

RHD基因测序方法的建立及其在弱D表型分子鉴定中的应用

目的 建立RHD基因的测序方法并对9例弱D表型作分子鉴定.方法用间接抗人球蛋白试验（IAT）筛选弱表达的D变异体,聚合酶链反应-序列特异性引物（PCR-SSP）方法 扩增RHD基因特异的外显子及其侧翼序列,PCR产物直接序列分析测定核苷酸的变异.结果 用PCR-SSP方法特异扩增RHD基因,50份随机Rh阴性（ccdee）样本呈阴性结果,51例随机Rh阳性样本呈阳性结果.扩增产物测序结果表明,15份代表性Rh阳性单倍体型的RHD基因序列和标准序列一致.用所建立的方法对9份弱D表型样本进行测序分析,发现5份有845G＞A突变,3份有1 227G＞A突变,1例有1013T＞C突变.结论 所建立的RHD基因测序方法可以用于弱D的分子鉴定.9份弱D表型的分子机理得到明确.     

微量聚合酶链反应对人类白细胞抗原的基因分型

目的 探讨应用于移植的人类白细胞抗原（ＨＬＡ）二类基因（ＤＲＢ／ＤＱＢ）快速分型新技术。方法 采用快速ＤＮＡ抽提技术,建立了ＨＬＡ＿ＤＲＢ／ＤＱＢ微量序列特异性引物聚合酶链反应基因分型方法,对１４２份供受者ＤＮＡ进行ＨＬＡ－ＤＲＢ／ＤＱＢ基因分型,并与单克隆抗体一叔法ＨＬＡ血清学分型技术进行对比研究。结果 应用微量ＰＣＲ－ＳＳＰ方法共检出１３种ＤＲＢ１等位基因和 ＤＱＢ１等位基因,与血清学分型结果     

Comprehensive analysis of RHD alleles in Argentineans with variant D phenotypes

BACKGROUND: The serologic assignment of the RhD status may be hindered in patients with weak D expression. A comprehensive study of RHD alleles occurring in the mixed population of Argentina is necessary to evaluate the most suitable DNA typing strategy. STUDY DESIGN AND METHODS: A total of 18,379 patients from two stratified groups, Group 1 (G1; public hospital) and Group 2 (G2; private laboratory), were RhD phenotyped, and 88 samples with reduced D expression underwent molecular characterization. RESULTS: The frequencies of D+, D–, and variant D phenotypes differed significantly (p < 0.001) between G1 and G2 (94.49% vs. 87.66%, 5.15% vs. 11.58%, and 0.36% vs. 0.75%, respectively). Eleven alleles were responsible for the weak D expression. Approximately 60% of the variant D phenotypes from G1 and G2 were weak D Types 1 through 4.0/4.2 and 25% were DVII. RHD alleles associated with African ancestry were encountered in G1. A new ?282G>A mutation within the promoter region of DAU‐4 and DOL alleles was identified. Three weak D Type 1 samples on R₀ haplotypes were found in G1. CONCLUSIONS: The D phenotype distribution in G2 resembles that in Europeans while the frequencies in G1 account for the Amerindian and African genetic contribution. The genotyping strategy described here is suitable to study D variants in the overall population and could allow a better use of the few available D– units and a rational administration of anti‐D immunoprophylaxis. The results also show that weak D Type 1 alleles do not exclusively segregate with a Ce allele, as assumed until present.     

Frequency of RHD variants in serologically weak D Turkish blood donors

BackgroundRhD typing has remained of primary importance, as being the leading cause of hemolytic disease of the newborn. Among Rh system’s 55 blood group antigens, RhD is the most immunogenic. We aimed with this study to determine weak D/partial D variant frequency in blood donors who were admitted to our blood center and have serologically designated blood group weak D.Materials and methodsWe screened blood donors who admitted between 2011 and 2017 to our blood center. Sixty-seven serologically weak D phenotyped donors have participated in the study. These donors' samples were studied further by Polymerase Chain Reaction Sequence- Specific Primers (PCR-SSP) for determining D variants.ResultsWeak D phenotype was detected in 228(0.12 %) out of 177,554 donors. Sixty-seven of them agreed to take part in the study. The frequency of weak D and partial D was 68.7 % (n = 46), and 22.4 % (n = 15), in order. The most encountered weak D and partial D variant was type 15 and DFR type, respectively.ConclusionsThe prevalence of serologically weak D phenotypes varies by race and ethnicity. Turkey is a country covering a mixture of European and Asian DNA with different ethnic groups. Thus, our research as giving the overall distribution of RHD variants from the largest city of Turkey, which may reflect the general ethnic background of the country, would help to the establishment of a databank for blood banking. This paper is the first molecular study on RHD variants in Turkey. New molecular research would be more reliable and precise.     

聚合酶链反应限制性片段长度多态性检测载脂蛋白E基因型

目的建立一种简便、准确、实用的人载脂蛋白Ｅ（ａｐｏＥ）基因型的检测方法。方法应用聚合酶链反应（ＰＣＲ）特异性扩增ａｐｏＥ基因含编码１１２位和１５８位氨基酸的基因序列，扩增产物用限制性内切酶ＨｈａⅠ酶切，聚丙烯酰胺凝胶电泳后，观察酶切位点的限制性片段长度多态性（ＲＦＩＰ）图谱。结果运用ＰＣＲ－ＲＦＬＰ法检测了１１３名健康人ａｐｏＥ基因型，频率分别为ε３／３６９．０％，ε３／２１６．８％，ε４／３１１．５％，ε４／２１．８％，ε４／４０．９％；ε２、ε３和ε４等位基因频率分别是９．３％、８３．２％和７．５％。结论该方法简单、快速、准确，对人体无害，适合于一般实验室开展及大规模人群调查     

Weak D type 1.1 exemplifies another complexity in weak D genotyping

BACKGROUND: Weak D expression is caused by a large number of RHD alleles. Increasingly recommendations for D+ or D- transfusions are based on polymerase chain reaction (PCR) identification of certain RHD alleles. Possible sources of error are rare D variants that are inadvertently carrying known polymorphisms of frequent weak D types. STUDY DESIGN AND METHODS: Weak D donors were checked by direct column agglutination. In donors with unusually weak expression of D, the molecular weak D type was determined by weak D PCR and nucleotide sequencing. The serologic profile of a weak D type 1 variant was determined by agglutination serology and flow cytometry. RESULTS: Several donors in whom direct agglutination barely revealed any D expression were shown to carry the new RHD(L18V,V270G) allele dubbed weak D type 1.1. Initially, such donors had been mistyped as weak D type 1 by PCR. In a systematic study, weak D type 1.1 was shown to be present in 7 of 23 donors with very weak D expression who all lived in a restricted area of Northern Germany. Although weak D type 1.1 was typed D- or barely D+ by direct agglutination, it was easily detected by antiglobulin technique and was shown to carry about 600 antigens D per red blood cell. CONCLUSION: The observation of weak D type 1.1 with its distinct phenotype pinpointed to two general problems of current RHD genotyping strategies: Mistyping of alleles with additional mutations and striking geographic variation of the allele distributions.     

Quantitation of RHD by real-time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism: an evaluation of four quantitative methods

BACKGROUND: Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti-D. RHD zygosity can be determined by quantitative real-time polymerase chain reaction (PCR) basically by determining RHD dosage, and this feature is relevant in investigating RHD mosaicism and chimerism. STUDY DESIGN AND METHODS: Monoplex and duplex real-time PCRs, uncalibrated and calibrated, were tested. RHD zygosity was determined for 72 samples and compared with serology-based prediction of RHD zygosity. Additionally, a range of constructed RHD dosages were compared with determined RHD dosages. Finally three samples, Days 1, 90, and 120, from a patient with loss of D were included. RESULTS: All setups enabled differentiation between hemi- and homozygous samples. Results from calibrated methods had the smallest variation and enabled differentiation at the mean +/- 3SD. Incongruity between determined and predicted RHD zygosity was found in three samples. PCR-sequence-specific priming specific for the hybrid downstream Rhesus box was performed on those samples confirming the real-time PCR results. Determined RHD dosage equaled constructed RHD dosage in both calibrated real-time PCRs. Patient samples showed RHD with a mean of 1, 5, and 8 percent relative to GAPDH in all samples. CONCLUSION: Duplex calibrated real-time PCR was a robust and exact method for determination of RHD zygosity and applicable in evaluation of RHD mosaicism and chimerism. This was illustrated with patient samples, where this assay revealed for the first time signs of mosaicism in both expression of D antigens and in RHD dosage.     

嗜肺军团菌毒力岛基因聚合酶链反应分型鉴定

目的建立嗜肺军团菌毒力岛基因分型鉴定方法，探讨其致病特性和分布状况。方法根据GenBank公布的嗜肺军团菌核苷酸序列设计和合成嗜肺军团菌种和毒力岛基因鉴定引物，采用PCR法对25株军团菌属标准参考株，3株国内和20株广东分离嗜肺军团菌，53份临床标本和57份水样、进行嗜肺军团菌种和毒力岛基因分型鉴定。结果5株嗜肺军团菌标准株只有嗜肺军团菌1型（Philadelphia-1 ATCC 33152）12个毒力岛基因全阳性，其他4株只检出11个毒力岛基因。20株军团菌属中其他菌株，分别检出2—11个毒力岛基因；3株国内嗜肺军团菌检出11个毒力岛基因；广东地区分离的20株嗜肺军团菌，7株检出12个毒力岛基因，12株检出11个毒力岛基因，1株未检到毒力岛基因。53例病源不明性肺炎患者支气管洗液和痰液，PCR检测嗜肺军团菌DNA阳性5例（9．4％）。结论广东地区嗜肺军团菌广泛存在水源环境中，而且是医院感染性肺炎的重要病源之一。不同血清型嗜肺军团菌所含毒力岛基因不同，毒力岛基因与致病性相关。