Serologic strategy in detecting RHD altered alleles in Brazilian blood donors

ABSTRACT Background: We evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting the largest numbers of D variants in blood donors. Methods: We selected 101 samples from Brazilian blood donors with different expressions of D in our donor routine. The tests were performed in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a tube, gel and solid-phase red blood cell adherence (SPRCA). All DNA samples were extracted from peripheral blood and the D variants were classified using different molecular assays. Results: The RHD variants identified by molecular analysis included weak D types (1, 2, 3, 11 and 38) and partial Ds (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII). The monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested. Conclusion: Our results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

Weak D Testing is not Required for D− Patients With C−E− Phenotype

BACKGROUND: Although testing to detect weak D antigens using the antihuman globulin reagent is not required for D− patients in many countries, it is routinely performed in Korea. However, weak D testing can be omitted in D− patients with a C−E− phenotype as this indicates complete deletion of the RHD gene, except in rare cases. We designed a new algorithm for weak D testing, which consisted of RhCE phenotyping followed by weak D testing in C+ or E+ samples, and compared it with the current algorithm with respect to time and cost-effectiveness. METHODS: In this retrospective study, 74,889 test results from January to July 2017 in a tertiary hospital in Korea were analyzed. Agreement between the current and proposed algorithms was evaluated, and total number of tests, time required for testing, and test costs were compared. With both algorithms, RHD genotyping was conducted for samples that were C+ or E+ and negative for weak D testing. RESULTS: The algorithms showed perfect agreement (agreement=100%; κ=1.00). By applying the proposed algorithm, 29.56% (115/389 tests/yr) of tests could be omitted, time required for testing could be reduced by 36% (8,672/24,084 min/yr), and the test cost could be reduced by 16.53% (536.11/3,241.08 USD/yr). CONCLUSIONS: Our algorithm omitting weak D testing in D− patients with C−E− phenotype may be a cost-effective testing strategy in Korea.

A Case of Primary Anti-D Alloimmunization by RHD (c.1227G>A) DEL Red Blood Cell Transfusion / 대한수혈학회지

The Rh blood group D antigen is the most immunogenic of all antigens, next to ABO antigens. Anti-D immunization is clinically important since it may cause clinical problems, such as severe hemolytic transfusion reactions and hemolytic disease of the newborn. DEL is an extremely weak D variant that cannot be detected by basic serologic typing and is typed as D-negative without the absorption-elution techniques and RHD genotyping. Of the DEL phenotype, RHD (c.1227G>A) allelic variant is the most common in Korea. The DEL phenotype has been considered to carry only a few D antigens to induce anti-D immunization, but a few cases have reported that this allelic variant is capable of inducing anti-D immunization in a D-negative recipient, for which it is clinical significant. Herein, we present a case of primary anti-D alloimmunization in a RhD negative patient after receiving RHD (c.1227G>A) DEL red cell transfusion identified by serological and molecular tests, including RHD genotyping.

Study of RHD genes polymorphism among RhD negative Han indivduals in Xuzhou area / 国际检验医学杂志

Objective To investigate the RHD gene polymorphism among the RhD negative Han individuals in Xuzhou.Methods The RhD antigen phenotypes were detected by routine serological techniques,and were identified with indirect antiglobulin test (IAT).The genotypes of RHD were detected by using sequence specific primer polymerase chain reaction(SSP-PCR).Results A-mong 110 RhD negative individuals,there were 5 cases carring RHD positive gene,47 cases carring RHD negative gene,22 cases carring RHD-CE(2-9)-D gene,17 cases carring DVI Ⅲ gene,2 cases carring weak D15 gene,and 17 carring DEL-1227A gene,re-spectively.Conclusion The RhD negative Han individuals present complex RHD gene polymorphism in Xuzhou region,and variant alleles such as RHD-CE(2-9)-D,DVI Ⅲ,DEL-1227A are given priority.

Prenatal determination of fetal RhD genotype by real-time PCR examination of fetal DNA in maternal plasma / 第二军医大学学报

Objective To set up a novel non-invasive prenatal determination technique of fetal RhD genotype by real-time PCR examination of fetal DNA in RhD negative maternal plasma. Methods Plasma fetal DNA was extracted by manual DNA micro-extraction method from the plasma of 22 RhD negative women (15-40 gestation week). We amplified the exons 7, 10 and intron 4 of RhD gene and sex-determining region Y gene (SRY) using real-time polymerase chain reaction. The presence of fetal DNA was confirmed by testing SRY. The results of genotyping and serum RhD status of the newborns were compared to evaluate the accuracy of the present method. Results Cell-free fetal DNA was detected in the maternal plasma samples. Among the 19 RhD negative specimens, 14 cases had the specifically amplified exons 7, 10 and intron 4 of RhD gene and SRY gene, and the results were confirmed by serological examination of fetal umbilical blood after delivery. Among the 19 specimens, 3 cases were not detected the specifically amplified in exons 7, 10 and intron 4 of RhD gene and SRY gene, and the results were also confirmed by serological examination of fetal umbilical blood. The accuracy of the present method was 89. 5%. SRY detection confirmed fetal DNA presence in maternal plasma in all boys. The other 2 cases only had specifically amplified exons 10 of RhD gene, and the results were confirmed as RhDel phenotype. Real-time PCR easily differentiated pregnant woman whose RBCs had a weak D phenotype (n = 3) from truly RhD-negative patients. However, in these cases fetal RhD status cannot be determined. Conclusion Real time PCR detection of fetal DNA in RhD-negative maternal plasma is simple, quick and specific for noninvasive prenatal diagnosis of fetal RhD blood type, which can help to prevent hemolytic disease of newborns.

Alelos RHD nulos en pacientes RhD negativo de una población hospitalaria de la ciudad de Rosario / RHD null alleles in RhD negative patients from a hospital of Rosario

El fenotipo RhD negativo en la población caucásica es causado por una deleción completa del gen RHD. Sin embargo, han sido reportadas regiones específicas de este gen en individuos RhD negativo de diferentes grupos étnicos. El objetivo de este trabajo fue investigar la presencia de alelos RHD nulos en pacientes RhD negativo que concurrieron al Hospital Provincial del Centenario. Se tipificaron 12672 individuos y se seleccionaron las muestras RhD negativo halladas. Se determinó el fenotipo Rh completo y posteriormente se investigó la presencia del gen RHD utilizando una estrategia de PCR multíplex. En las muestras que presentaron fragmentos RHD específicos se realizaron reacciones de PCR alelo específicas para determinar el origen de los exones. Se encontraron 653 (5.15%) muestras RhD negativo. Cincuenta y cinco (8.42%) presentaban al menos el antígeno RhC o RhE. Los estudios moleculares permitieron detectar 7 alelos RHD Psi, 5 alelos híbridos RHD-CE(3-7)-D, 2 alelos híbridos RHD­CE(3-9)-D y 1 alelo nuevo RHD (46 T>C). La frecuencia de individuos RhD negativo en la población estudiada fue significativamente menor a la reportada en caucásícos. Los resultados moleculares obtenidos indican que 2.30% (15/653) de los individuos que no expresan el antígeno D son portadores de alelos RHD nulos. Los alelos RHD-CE(3-7)-D, RHD-CE(3-9)-D y RHD (46 T>C) están presentes únicamente en individuos RhD negativo que expresan los antígenos RhC y/o RhE con una frecuencia del 14.50% (8/55). Por otro lado, el alelo RHD Psi está asociado exclusivamente al fenotipo dccee, siendo el 1.17% (7/598) de estos individuos portadores del pseudogen RHD Psi. Estos hallazgos señalan la importancia del estudio del polimorfismo molecular del locus RH para el desarrollo de estrategias de tipificación de ADN confiables, que permitan realizar la genotipificación RHD prenatal y optimizar la selección de unidades a transfundir en los Bancos de Sangre.

The RhD negative phenotype in Caucasians is mainly caused by a complete deletion of the RHD gene. However, specific regions of the RHD gene in RhD negative individuals have been reported in different ethnic groups. The purpose of this study was to analyse the presence of silent RHD alleles in RhD negative patients concurring to the Hospital Provincial del Cen­tenario. Blood samples from 12672 individuals were studied and the RhD negative phenotypes were selected. Initially, the complete Rh phenotype was determined and DNA samples were screened using a multiplex PCR strategy to detect the presence of an RHD allele. Samples carrying RHD specific fragments were further studied by RHD exon scanning with allele specific PCR. 653 samples out of the 12672 (5.15%) were found RhD negative. Within this group, 8.42 % were either RhC positive or RhE positive. Molecular studies detected 7 RHD Psi alleles, 5 RHD-CE(3-7)-D hybrid alleles, 2 RHD-CE(3-9)-D hybrid alleles and 1 RHD (46 T>C) novel allele.The frequency of RhD negative individuals observed in the population studied was lower than that reported for Caucasians. Molecular analysis showed that 2.30% (15/653) of the individuals with no expression of the D antigen carry RHD null alleles. RHD­CE(3-7)-D, RHD-CE(3-9)-D and RHD (46 T>C) alleles are present only in individuals expressing either RhC or RhE with a frequency of 14.55% (8/55). The RHD Psy is associated with the dccee phenotype and 1.17% (7/ 598) of these individuals carries the RHD Psi pseudogen. These findings highlight the importance of studying the molecular polymorphism of the RH locus so as to develop reliable DNA typing strategies.

RHD Genotyping in RhD-negative Korean Donors by Polymerase Chain Reaction: Sequence Specific Primers(PCR-SSP) Method / 대한임상병리학회지

BACKGROUND: In Caucacians, almost all RhD-negatives have deletion in RHD gene and the RHD genotyping by PCR-SSP is a valuable tool. The aim of this study is to investigate the frequency of RHD gene deletion in RhD-negative Korean donors and evaluate the clinical usefulness of various RHD genotyping methods in Korean. METHODS: Two hundred fifty RhD-positive blood obtained from Blood Transfusion Research Institute and 119 RhD-negative blood samples were obtained from Korea Red Cross Dong Bu Center. Phenotyping of RhD, RhC/c, and RhE/e antigen was performed using polyclonal and monoclonal antibodies(Dade AG, Switzerland). PCR-SSP was performed by primer sets, specific for exon 3, exon 4, exon 5, exon 6, exon 7, exon 9, and exon 10 of RHD gene and for exon 2, intron 4, and intron 4-exon 5 common to RHD and RHCE genes. RESULTS: The phenotypes of 250 RhD-positives consisted of 106(42.4%) CCee, 93(37.2%) CcEe, 26(10.4%) ccEE, 21(8.4%) Ccee, 3(1.2%) ccEe, and 1(0.4%) ccee. In RhD-negative donors, 62(52.1%) were ccee, 37(31.1%) Ccee, 10(8.4%) ccEE, 6(5.0%) CcEe, 2(1.7%) ccEE, and 2(1.7%) CCee. Twenty-two out of 101 RhD-negatives showed no deletion in all used methods. Deletion frequency of RhD negatives varied according to the methods: 76(75.3%) in intron 4-exon 5 boundary; 74(73.3%) in intron 4; 72(71.3%) in exon 4; 67(66.3%) in exon 7; 63(62.4%) in exon 10; 9(8.9%) in exon 5; no deletion in exon 3, exon 6, and exon 9. Different RhD phenotypes also showed different RHD gene deletion frequency: 80-90% deletion in ccee; 70% in ccEe; 40-50% in Ccee and CcEe; no deletion in CCee phenotypes. CONCLUSIONS: RHD gene deletion frequency varied according to the methods applied and individual's own RhD phenotypes. Therefore, RHD genotyping is not appropriate for a routine test in Blood Bank and individual variation should be considered in prenatal care of RhD-negative women.

Study of RHD genes among RhD negative individuals in Hainan province,China / 中国输血杂志

Objective To understand the RHD gene profiles of RhD negative individuals in Hainan Han population,and provide reference to establish the right method for RHD genotyping.Methods RhD was tested by anti-globulin test, and RhDel and genuine RhD-negative phenotype were identified by absorption/elution method. RhD negative samples were further tested for RHD exons by PCR-SSP.The RhD negative samples with intact RHD genes were further analyzed by PCR-SSP for RHD introns 2, 10 and RHD?gene. Results Thirty-one (29.25%)cases of RhDel individuals were identified among 106 apparent RhD-negative individuals. All RhDel samples had RHD genes;67 cases of genuine RhD-negative had no RHD genes and 8 cases were partial D. All 31 RhDel samples had Din2 and Din10 but none had RHD?. Additionally, We detected exons 1,3,4,6,7,9 and 10 in one case of ccdEe sample. Conclusion The proportion of RhDel phenotype is high among apparent RhD-negative Hainanese, and total RHD exons can be detected in all RhDel samples. Polymorphisms of RHD gene are present among genuine RhD-negative Hainanese. There is no exon 5 in all 8 cases of partial D, which suggests that exon 5 specific amplification may be very important in RHD genotyping for Hainan Han population.