Genotipificación del gen RHD en ADN fetal libre en plasma de embarazadas RhD negativo / Genotyping of the RHD gene in free fetal DNA in plasma of RhD negative pregnant women

Objetivo: Determinar el grupo RhD fetal a través del estudio del gen RHD en ADN fetal que se encuentra libre en plasma de embarazadas RhD negativo. Método: Se analizó la presencia de los genes RHD, SRY y BGLO en ADNfl obtenido de plasma de 51 embarazadas RhD negativo no sensibilizadas, utilizando una qPCR. Los resultados del estudio genético del gen RHD se compararon con el estudio del grupo sanguíneo RhD realizado por método serológico en muestras de sangre de cordón, y los resultados del estudio del gen SRY fueron cotejados con el sexo fetal determinado por ecografía. Se calcularon la sensibilidad, la especificidad, los valores predictivos y la capacidad discriminativa del método estandarizado. Resultados: El gen RHD estaba presente en el 72,5% de las muestras y el gen SRY en el 55,5%, coincidiendo en un 100% con los resultados del grupo RhD detectado en sangre de cordón y con el sexo fetal confirmado por ecografía, respectivamente. Conclusiones: Fue posible deducir el grupo sanguíneo RhD del feto mediante el estudio del ADN fetal que se encuentra libre en el plasma de embarazadas con un método molecular no invasivo desarrollado y validado para este fin. Este test no invasivo puede ser utilizado para tomar la decisión de administrar inmunoglobulina anti-D solo a embarazadas RhD negativo que portan un feto RhD positivo.

Objective: To determine the fetal RhD group through the study of the RHD gene in fetal DNA found free in plasma of RhD negative pregnant women. Method: The presence of the RHD, SRY and BGLO genes in fetal DNA obtained from plasma of 51 non-sensitized RhD negative pregnant women was analyzed using qPCR. The results of the genetic study of the RHD gene were compared with the RhD blood group study performed by serological method in cord blood samples, and the results of the SRY gene study were compared with the fetal sex determined by ultrasound. Sensitivity, specificity, predictive values and discriminative capacity of the standardized method were calculated. Results: The RHD gene was present in 72.5% of the samples and the SRY gene in 55.5%, coinciding 100% with the results of the RhD group detected in cord blood, and with the fetal sex confirmed by ultrasound, respectively. Conclusions: It was possible to deduce the RhD blood group of the fetus through the study of fetal DNA found free in the plasma of pregnant women with a non-invasive molecular method developed and validated for this purpose. This non-invasive test can be used to make the decision to administer anti-D immunoglobulin only to RhD-negative pregnant women carrying an RhD-positive fetus.

Expert consensus on RhD blood group testing strategies in blood transfusion compatibility testing laboratories / 中国输血杂志

RhD antigen is the most immunogenic antigens in the Rh blood group system and can cause hemolytic transfusion reactions (HTRs) and severe hemolytic disease of the fetus and newborn (HDFN). RhD-negative red blood cell (RBC) is a scarce resource in China, and RhD-negative patients face challenges in timely blood transfusion. For different populations that were initially negative for RhD (saline method), what techniques should be used, how to issue test report and when should " Asian type" DEL (RHD*01EL.01, c.1227G>A) be identified are the confusions of blood transfusion workers in the process of clinical blood supply. More than 200 technical experts discussed and reached a consensus on blood transfusion compatibility testing for Chinese population. The purpose of the consensus is to further standardize and refine the RhD blood group testing strategy, provide technical support for achieving accurate blood transfusion for patients with different RhD blood groups, continuously improve the levels of prevention and treatment for RhD-HTRs and RhD-HDFN, and lay a foundation for the future development of industry standards concerning RhD blood group testing strategy.

Molecular genetic analysis of a patient with double population of red blood cells for RhD antigen / 中国输血杂志

【Objective】 To solve the difficulty of RhD blood group typing in a patient with double population(DP) of red blood cells for RhD antigen by serological and genotyping analysis. 【Methods】 Separation of the two populations of red blood cells of the patient was performed using capillary centrifugation method. ABO, RhD and RhCE typing, direct anti-human globulin test (DAT), irregular antibody screening, antibody identification and blood crossmatching of the patient were conducted using the standard serological methods. The hybrid Rhesus zygosity analysis of the RHD gene was performed by PCR-RFLP method. RHD and RHCE genotype of the patients were identified by PCR-SSP method. 【Results】 The patient was B type but with DP of red blood cells for RhD, Rhc and RhE antigens. DAT of the patient was positive and the alloanti-D was detected in serum. The RHD zygosity was D-/D- homozygote. PCR-SSP testing showed the RHD gene deletion (RHD * 01N. 01/01N.01 genotype) and Ccee of RHCE genotype in the patient, which was consistent with RHD zygosity analysis. 【Conclusion】 This is a special case with D-negative phenotype which was wrongly detected as D-positive type after D-positive red blood cells transfusion in emergency. When the DP of red cells for D antigen encountered like this case, the RhD typing can be accurately determined by using RHD genotyping analysis to provide strong evidence to the clinical blood transfusion.

Discussion on the collection and supply mode of RhD negative blood suitable for prefecture-level city: a retrospective analysis / 中国输血杂志

【Objective】 To explore a RhD negative blood collection and supply mode suitable for Liaocheng area and improve the blood demand satisfaction rate of RhD negative patients. 【Methods】 Under different collection and supply modes (collection mode: in 2020, not advocate walk-in donation by RhD negative repeat blood donors, but in 2021 and 2022 were the opposite; supply mode: in 2020, type A, B, O and AB RBCs were frozen within 6 days of blood collection without inventory, but in 2021 and 2022, they were stored of 2-6 U and the remaining were frozen within 6 days of blood collection), RhD negative blood was divided into Type A, B, O and AB, appointment donation and walk-in donation, first donation and repeat donation, cold storage red blood cells(RBCs), frozen RBCs and frozen thawed deglycerolized RBCs, and the collection and supply data of each observation group from 2020 to 2022 were compared. Based on whether blood demand of RhD negative patients was met, the patients were divided into the group of going to other places for medical treatment, the group of RhD positive blood transfusion and the group of RhD negative blood transfusion to analyze the blood usage. 【Results】 From 2020 to 2022, the proportion of RhD negative blood donated by repeat appointment donors decreased year by year (P<0.05); the proportion of RhD negative blood donated by repeat walk-in donors increased year by year (P<0.05); the proportion of frozen thawed deglycerolized RBCs to RhD negative RBCs increased year by year (P<0.05); the proportion of cold storage RBCs distributed as RhD negative and RhD positive decreased year by year respectively (P<0.05); the proportion of the increase in frozen RBCs inventory to the current year's frozen RBCs inventory decreased year by year (P<0.05). The satisfaction rate of blood demand of RhD negative patients increased year by year (P<0.05), with the number of patients with RhD positive blood transfusion and going to other places for medical treatment decreased year by year respectively(P<0.05). 【Conclusion】 In Liaocheng area, it is appropriate to encourage RhD negative blood donors to donate blood randomly, and A, B, O and AB type cold storage RBCs kept in 2-6 U inventory respectively. When exceeding the inventory, frozen RBCs are prepared within 6 days, which can improve the blood demand satisfaction rate of RhD negative patients and avoid sending excessive RhD negative RBCs to clinical use as RhD positive RBCs.

Analysing The Correlation Of Lipid Profile Pattern In Chronic Rheumatic Heart Disease (RHD) Patients

Background:The global burden of ARF and RHD is significant, and is predominantly found in populations living in low-resource settings. This study was conducted with a view for analysing the lipid profile variation in patients with chronic Rheumatic Heart Disease (RHD) and correlating the same. The early management of RHD has to be started to prevent further cardiac complications. To date no hypothesis is established on the relation between the Lipid Profile and RHD. This study would be beneficial to Indian cardiologists in defining the optimal treatment by the choice of antibiotic therapy and Lipid lowering agents like Statins.Material And Methods:It was a cross-sectional study carried out on the patients visiting Cardiology Out Patient Department (OPD) at a tertiary care hospital in Ahmedabad and who were diagnosed with Rheumatic Heart Disease (RHD).The blood lipids levels of thepatients were evaluated with current classification schemes and treatment levels for hyperlipidemia based on the National Cholesterol Education Panel’s (NCEP) Adult Treatment Program-3 (ATP-III) guidelines.Result:Total 89 patients were included in the study. 76 patients were associated with dyslipidaemia and 13 patients had normal lipid values. As far as total cholesterol levels were concerned, 23 out of 76 patients (30.26%) had above target levels for total cholesterol. Also 46 patients out of 76(60.52%) had raised triglycerides levels and 47 patients (61.84%) had raised LDL levels. As far as HDL levels were concerned, 38 out of 76 patients (50.00%) had below desirable levels. The frequency of deranged LDL is higher as compared to HDL, triglycerides and total cholesterol levels. If these values are to be co-related to the disease, then LDL as a lipid parameter stands most significant.Conclusion:Morbidity and mortality in RHD is very high in Indian patients and one of the determinants of the cardiovascular morbidity is the increased lipid levels. The frequency of deranged LDL is higher as compared to HDL and total cholesterol levels in these patients. This study was planned to evaluate an entirely novel hypothesis that RHD is an independent risk factor for Atherosclerosis and CAD. The exact reason behind the occurrence is unknown but it can be environmental or genetic. RHD and Dyslipidaemia might engulf younger patients. The study is also aimed at evaluating all lipid parameters with RHD.

Frecuencia de grupos sanguíneos ABO y RhD, en universitarios de El Alto ­ Bolivia / Frequency of ABO and RhD blood groups in university students in El Alto - Bolivia

INTRODUCCIÓN: los sistemas sanguíneos continúan siendo de gran importancia en múltiples disciplinas como la medicina transfusional, genética poblacional, medicina forense, trasplante de órganos entre otras, estas se basan en la determinación de los grupos sanguíneos ABO y Rh. El siguiente trabajo analiza la fenotipificación eritrocitaria en un grupo de estudiantes de la carrera de Bioquímica y Farmacia de 5to, 6to y 7mo semestre, Universidad Franz Tamayo (UNIFRANZ) sede El Alto - Bolivia. MATERIALES Y MÉTODOS: se realizó un estudio descriptivo de serie de casos, determinando los sistemas ABO y RhD, para las pruebas directa e indirecta a fin de evitar discrepancias; utilizando como soporte la técnica de microplaca, regida a controles de calidad en Inmunohematología. RESULTADOS: los resultados mostraron que el 86% eran mujeres, el grupo etario dominante esta entre 18 a 25 años de edad. El sistema ABO determinó el mayor porcentaje al grupo O con un 93%, destacando que no se encontró el grupo AB en el grupo de estudio. El sistema RhD, tuvo 100% de positivos, no encontrándose fenotipos RhD negativo ni variantes ni parciales. CONCLUSIÓN: la fenotipificación de grupos sanguíneos tanto directa e indirecta, a través de la plataforma de microplaca ayuda a evitar errores y discrepancias, tal como lo recomienda el Manual de Inmunohematología en servicios de sangre en Bolivia.

INTRODUCTION Blood systems continue to be of great importance in multiple disciplines such as transfusion medicine, population genetics, forensic medicine and organ transplantation, which are based on the determination of ABO and Rh blood groups. The following work analyzes the erythrocyte phenotyping in a group of students of Biochemistry and Pharmacy of 5th, 6th and 7th semesters, UNIFRANZ, El Alto. MATERIALS AND METHODS A descriptive case series study was carried out, determining the ABO and RhD systems for direct and indirect tests in order to avoid discrepancies; using the microplate technique as support, according to quality controls in Immunohematology. RESULTS The results showed that 86% were women, the dominant age group was between 18 and 25 years of age. The ABO system determined the highest percentage of group O with 93%, highlighting that the AB group was not found in the study group. The RhD system was 100% positive, with no RhD negative phenotypes, neither variant nor partial. CONCLUSION The phenotyping of blood groups, both direct and indirect, through the microplate platform helps to avoid errors and discrepancies, as recommended by the Manual of Immunohematology in blood services in Bolivia.

Molecular basis of RhD-negative phenotype in North Indian blood donor population

Background & objectives: RHD gene typing is highly complex due to homology with RHCE genes. Molecular polymorphism of the RHCE and RHD genes have been characterized among various populations, but no studies have been undertaken among Indians. This study was undertaken to assess the genetic basis of RHD-negative phenotype in Indian blood donor population. Methods: Sample from a total of 200 phenotypically RhD-negative blood donors were analyzed for presence of RHD gene using polymerase chain reaction (PCR). RHD genotyping was done using three primer sets designed for exons 4 and 10 and one set for identification of pseudo (RHD?) gene between introns (int) 3 and 4. Amplified PCR products were analyzed by gel-electrophoresis (XY Loper, Uvitech, Cambridge) and confirmed by nucleotide sequencing (ABI 3730 xl 96 capillary system). Results: No PCR product was found in 195/200 (97.5%) of study samples indicating homozygous gene deletion. Of the 5/200 (2.5%) showing RHD gene polymorphisms, 4/200 (2%) were positive for presence of exon 10 only (RHD-CE-D hybrid). RHD? gene was not detected in any of the samples tested. One sample showed presence of all three tested regions and was negative for RHD? gene. Interpretation & conclusions: RHD gene deletion was found to be the most common cause of an RHD-negative phenotype while RHD? gene was, reported to be present in up to 39 per cent of various ethnic populations, but was not detected. RHD-CE-D hybrid gene (found in 2.5% individuals) is important for predicting the requirement of Rh prophylaxis during the antenatal period.

Identification of Weak D Type 1 in Rh Blood Group System and Discussion of Transfusion Strategy / 中国实验血液学杂志

OBJECTIVE@#To investigate the molecular mechanism of one patient with abnormal serological phenotype in RhD and discuss the transfusion strategy.@*METHODS@#The RhD variant sample was screened from a patient with IgM type anti-D antibody and further determined by three different sources of anti-D antibodies. Ten exons and the adjacent introns of the RHD gene were amplified, purified and sequenced. RhCE phenotypes and RHCE genotypes were detected.@*RESULTS@#The patient with Rh variant showed abnormal results of serological tests. The RHD gene sequence analysis showed that the RHD*01W.01 with a variation (c.809T>G, p.Val270Gly) in exon 6 of the RHD gene was found in the patient. The RhCE phenotype was CcEe. The genotyping results of RHCE were consistent with the serological typing results.@*CONCLUSION@#The Rh variant of the patient is RHD*01W.01, these findings indicate that RhD variants should be analyzed by molecular assays for the sake of safe transfusion.

Identification of pregnant women with DFR genotype and discussion of prenatal examination strategy / 中国输血杂志

【Objective】 To identify three cases of pregnant women with the D variant phenotype using serological and molecular tests, and discuss the strategy of prenatal examination. 【Methods】 The peripheral blood samples from three pregnant women with the D variant phenotype were collected. RhD variant phenotype was determined using routine serological methods with two different kinds of monoclonal anti-D. The serological characteristic for the epitope of D antigen was further analyzed using the commercial panel anti-D reagents (D-Screen, Diagast). The hybrid RHD-CE-D allele was analyzed by the Multiplex Ligation-dependent Probe Amplification (MLPA) assay and polymerase chain reaction with sequence specific primers (PCR-SSP) method. Further Sanger sequencing of RHD gene exons was also performed. 【Results】 DFR phenotype was primarily determined by serological characteristic for the epitope of D antigen. RHD*DFR2/01N.01(n=2) and RHD*DFR1/1227A(n=1) genotypes were identified by the MLPA assay, PCR-SSP and Sanger sequencing. 【Conclusion】 Two pregnant women with RHD*DFR2/01N.01 genotype should be treated as D negative patients clinically, while the pregnant woman with RHD*DFR1/1227A genotype can be treated as Asia type DEL to avoid unnecessary antibody screening and anti-D prophylaxis.

Phenotype and RHD gene polymorphism of 64 Rh negative individuals / 中国输血杂志

【Objective】 To collect blood samples of 64 RhD negative patients in our hospital for RHD genotyping and phenotype analysis (RhC/c/E/e), and analyze the distribution characteristics of different RHD genotypes. 【Methods】 The RHD gene of RhD negative patients was genotyped by fluorescence quantitative polymerase chain reaction (PCR) method. The Rh phenotype was identified by IgM anti-e, anti-c, anti-C and anti-E, respectively. 【Results】 Forty-two cases of RHD deletion were detected, dominated by ccee phenotype (88.1%); 9 of RHD1227A cases, dominated by Ccee phenotype(77.8%); 8 of RHD-CE(3-9)-D2 cases, dominated by Ccee phenotype (75%); 1 of RHD-CE(3-10)-D2 case with Ccee phenotype, 1 of RHD*711delC case; 1 of RHAG site invalid type were detected. The typing results could not be determined in 2 cases by PCR method. 【Conclusion】 RhD negative patients showed diversity in RHD genotype, dominated by RHD deletion, followed by RHD1227A, RHD-CE(2-9)-D2, RHD-CE(3-10)-D2, RHD*711delC and RHAG site deletions.

Molecular basis of Del phenotype in RhD negative population of Zhongshan / 中国输血杂志

【Objective】 To conduct serological and molecular study of Del type in RhD-negative donor population in Zhongshan area, so as to improve the diagnosis of Del type. 【Methods】 A total of 102 initially RhD-negative samples, collected from December 2017 to February 2019, were classified by RHCE and PCR-SSP genotyping. And 95 cases of truly negative RhD were confirmed by IAT, 28 cases of Del type were identified by absorption and elution test. The phenotype and genotyping characteristics of Del type in Zhongshan area were summarized based on domestic data of relative literature. 【Results】 Among 102 initially RhD-negative samples by serological test, 95 were truely RhD-negative, 28 were DELRHD 1227A without any other Del allele. Among them, RHCE antigen type were Ccee in 20(71.4%) cases, CCee in 8(28.6%), with no difference in comparison with other regions in China. The frequency of Del in RhD-negative blood donors was 29.5% (28/95), with difference between Shanghai, Taiwan, and Fuzhou, but no difference between Nanchang, Zhejiang, and Wuhan. 【Conclusion】 The study showed that the Del phenotype was closely related to Ce haplotype, and has no difference with other regions in China. The frequency of Del type in RhD negative donors was 29.5%, with regional differences. RHD1227A was the main allele of Del.

Study on the gene frequency and polymorphism of 11 RBC blood group systems in RhD negtive population in Hunan, China / 中国输血杂志

【Objective】 To investigate the gene frequency and polymorphism of RBC blood group systems in RhD negtive population in Hunan, so as to lay a foundation for clinical blood transfusion and construction of multiple rare blood group database. 【Methods】 Blood samples were taken from 300 RhD negative blood donors, confirmed by serological method, from June 2019 to June 2020,. RHD genotyping was performed by SSP-PCR. For blood donors with typing results as RhD negative plus RHD gene deletion, antigens genotyping of MNS, Duffy, Kell, Domrock, Diego, Kidd, Sciawnna, Colton, Lutheran and Yt RBC blood group systems were performed by SSP-PCR and analyzed by the chi square test of SPSS 20 statistical software. 【Results】 RHD gene deletions accounted for 58.67% (176 / 300) of serological D negative blood donors. The gene frequencies were as follows: MNS: GYPB*S=0.045 5(8/176), GYPB*s=0.954 5(168/176), GYP*Dane=0.039 8(7/176); Duffy: FY*A =0.965 6(170/176), FY*B=0.034 1(6/176); Dombrock: DO*A=0.082 4(14.5/176), DO*B=0.917 6(161.5/176); Diego: DI*A=0.025 6(4.5/176), DI*B =0.974 4(171.5/176); Kidd: JK*A=0.485 8(85.5/176), JK*B=0.514 2(90.5/176); Kell: KP*A=0.005 7(1/176), KP*B=0.994 3(175/176); Lutheran: LU*A=0.005 7(1/176), LU*B=0.994 3(175/176); Yt: YT*A=0.002 8(0.5/176), YT*B=0.997 2(175.5/176). The genotypes of Kell(K+ /k+ ), Scianna and Colton blood groups were KEL*02 /KEL*02, SC*01 /SC*01 and CO*A /CO*B, respectively. The expected frequencies of the combination of type O, RhD negative and other blood group systems were between 1/100 000 to 1/10 000. 【Conclusion】 Among RhD negative blood donors in Hunan, the gene profiles of MNS, Duffy, Domrock, Diego, Kidd, Kell and Lutheran blood group system were polymorphic, and Kell (K+ /k+ ), Colton and Scianna were homozygous. The data of other RBC blood group systems from RhD negative blood donors is of great significance to establish local database of rare blood groups.

Preliminary study of ssDNA aptamer cytotoxicity on RhD+ red blood cells / 中国输血杂志

【Objective】 To investigate the effect of ssDNA aptamer of RhD blood group antigen on erythrocyte toxicity. 【Methods】 Two full-length ssDNA aptamers(82 bp) of RhD blood group antigen were obtained by gene synthesis.Five samples of whole blood with EDTA anticoagulant were collected to prepare red blood cell suspensions (4×107/mL), which were split into 10 tubes(100 μL/tube), corresponding to 5 experimental groups and 5 controls.Two monospecific full-length ssDNA sequences (100 pmol/μL, 5μL each) were added into the experimental group, while the same amount of normal saline into the control.After treatment, the experimental group and the control were incubated for 60 min at 37℃.After washing, they were suspended in LISS solution and stored at 4℃.The experimental group and the control were set according to different time point during storage (1 h, 1 d, 3 d, 10 d and 17 d), with 5 tubes in each group.For erythrocytes in LISS suspension at different storage time, Annexin V labeled with FITC was used as a probe to label the phosphatidylserine (PS) content and Fluo-4 to label Ca2+ .The eversion of PS and the change of Ca2+ concentration in red blood cells in LISS suspensions were determined by flow cytometry. 【Results】 After incubation, all groups were examined under the light microscope.No agglutination occurred in the experimental group, while agglutination occurred in the control.Flow cytometry showed that the number of Annexin V-FITC staining cells of suspended erythrocytes at the same storage time-point was similar between the experimental group and the control, with no significant differences.In the experimental group, apoptosis rate of Annexin V cells at 10-day storage(6.06±1.38) was significantly higher than that at 1-hour storage(P<0.05), so as at 17-day storage(7.77±1.23) than 1-hour, 1-day and 3-day storage(P<0.05). The apoptosis rate of Fluo-4 AM cell in suspended RBCs at the same storage time-point was similar between the two groups(P>0.05). In the experimental group, the apoptosis rate of Fluo-4 AM cell at the 3-day, 10-day and 17-day storage was 20.84±4.16, 22.35±3.37 and 27.06±2.81, respectively(P<0.05). 【Conclusion】 ssDNA aptamer was not found to have any cytotoxic effects on red blood cells, and RhD ssDNA aptamer may be used as a material for the detection and preparation of universal blood.

Molecular mechanism and bioinformatics analysis of 9 samples with rare RhD variants / 中国输血杂志

【Objective】 To study the molecular mechanism of 9 samples with rare RhD variants and their RhD epitopes and protein structure. 【Methods】 The 9 blood samples with rare RhD variants were collected from 210 644 blood donors of Shenzhen Blood Center. Regular serological assaying was used for determination of Rh type for the 9 samples. Indirect anti-human globulin test (IAT) was used to confirm the RhD antigen and to screen the antibodies. D-screen reagent was sued to analyze the RhD epitopes of the samples. RHD zygosity testing of the samples was detected by PCR-SSP. The nucleotide sequences of all 9 exons and adjacent flanking intron regions of RHD gene were sequenced. The prediction of the effects of mutations on RhD protein function were analyzed using PROVEAN, SIFT, PolyPhen-2 and MutationTaster software. Robetta and Swiss-PdbViewer 4.1.0 were used for modeling the tertiary structures of RhD. 【Results】 A total of 9 individuals with rare RhD variants were identified as follows: RHD*weak D type 25, RHD*weak D type 50, RHD*weak D type 95, RHD*weak D type 12, RHD*weak D type 128 and four novel RHD alleles. The prediction of the tertiary structures showed that the RhD protein conformation was disrupted in the 9 rare RhD variants samples. 【Conclusion】 Five rare and four novel RHD alleles have been identified. Their phenotypic and genotypic descriptions enrich the database of reported RHD alleles. Bioinformatics analysis provided evidences for further study of the structure and functions of RhD protein.

Genetic background of RhD negative blood donors / 中国输血杂志

【Objective】 To analyze the genetic background of RhD-negative blood donors by detecting RHD and RHCE genes of those donors. 【Methods】 From March 2021 to May 2022, the blood samples of RhD-negative blood donors, who had been screened out by RhD primary screening and confirmatory experiments in the Yaan Blood Center, were firstly identified whether the RHD allele was completely deleted, then whether there were deletions in 10 exons of non-RHD allele complete deletion samples, finally, the remaining samples without RHD alleles and exon deletions were further analyzed by DNA sequencing. RHCE gene was detected by SSP-PCR method. 【Results】 Among the RHD gene test results of 104 RhD-negative samples, 65 cases were completely deleted (d/d), 33 were RHD partially deleted (one allele deletion), and 6 were without RHD gene deletion. The RHD alleles of 33 samples with partial deletion were detected by 10 exons, 13 had partial exon deletion, with genotype as RHD*D-CE(3-9)-D/d and phenotype as RhD negativity, and the remaining 20 samples had no exon deletion. The exon sequencing results of the non-deletion samples showed RHD*1227A/RHD*1227A in 6 samples, RHD*1227A/d in 19, RHD*3A/d in 1; both of the last two were considered Del by ISBT. The RHCE gene test results showed that all cc genotype blood donors were RhD true negative, while Del blood donors had no cc genotype. 【Conclusion】 Through the genetic background study of RhD negative blood donors, it is found that there is a high proportion of Del with weak expression of RhD antigen, whether this blood type affects clinical blood safety needs further researches.

Prevención de enfermedad hemolítica perinatal severa con inmunoglobulina intravenosa en paciente altamente sensibilizada / Prevention of severe perinatal hemolytic disease with intravenous immunoglobulin in a highly sensitized patient

RESUMEN La enfermedad hemolítica perinatal es infrecuente hoy por la prevención que de ella se hace. Sin embargo, existen casos de madres altamente sensibilizadas que desean tener un hijo, lo que obliga a que ese embarazo deseado sea controlado de manera especial y sometido a procedimientos invasivos no exentos de morbimortalidad fetal. El uso prenatal de inmunoglobulina humana en la madre puede representar una alternativa terapéutica. Se presenta un caso en que su uso impidió el desarrollo de enfermedad intrauterina y favoreció la buena evolución neonatal a pesar de que el pronóstico inicial era muy adverso.

ABSTRACT Perinatal Hemolytic Disease is uncommon today due to its prevention. However, there are cases of highly sensitized mothers who wish to have a child, that forces this desired pregnancy to be controlled in a special way and be subjected to invasive procedures not exempt from fetal morbidity and mortality. Prenatal use of human inmunoglobulin in the mother may represent a therapeutic alternative. We present a case in which its use prevented the development of intrauterine disease and favored a good neonatal evolution despite the fact that the initial prognosis was very adverse.

Safety of RhD alloimmunization / 中国输血杂志

【Objective】 To explore the safety of RhD-positive red blood cells (RBCs) immunization schedules in RhD-negative volunteers, so as to facilitate the development of domestic anti-D immunoglobulin. 【Methods】 From January 2018 to April 2020, 23 RhD negative volunteers with informed consent were enrolled and divided into initial immunization group and booster immunization group. The initial immunization included first immunization, second immunization and third immunization. Four groups, i. e. 3 cases of 20 mL, 8 of 30 mL, 6 of 40 mL, and 6 of 50 mL, were involved in initial immunization. After the initial immunization response, booster immunizations were performed every 3 months. According to the anti-D titer before each immunization, the booster immunization doses were set to 0.5, 1 and 2 mL. Whole blood samples of 5mL/ person (time) were collected 24 h and 1 week after each infusion, and the blood routine, liver, kidney and blood coagulation function and anti-D titer were detected. The differences of detection (index) values at 24 h and 1 week after the first immunization and booster immunization in each (dose) group were compared. 【Results】 No statistically significant differences were observed in hemolysis index values (all within the range of medical reference values) 24 h or 1 week after initial immunization among RhD positive RBCs of 20, 30, 40 and 50mL(P>0.05). The differences between the hemolysis index values and the basic values before the immune response (all within the range of medical reference values) after 0.5 or 1 mL booster immunizations were also not statistically different (P>0.05). However, the differences (μmol/L)between total bilirubin levels and the basic values before the immune response (1.55±1.87, 6.29±2.66) were significantly different after 2 mL booster immunization (P<0.05). 【Conclusion】 No risks affecting the safety of RhD negative volunteers was found in the immunization schedule proposed in this study.

Frequency analysis of RHD*1227A allele among Rh negative Han population and random population in Liaoning / 中国输血杂志

【Objective】 To investigate the frequency of RHD*1227A allele in Rh negative Han polulation and random population in Liaoning. 【Methods】 Del phenotype was screened by absorption-elution test, the RHD*1227A allele was screened by PCR-SSP and confirmed by nucleotide sequence analysis of RHD full coding region, and the heterozygosity of RHD gene was detected by hybridization technique. 【Results】 24 case of Del phenotype were detected by the absorption-elution test among 117 Rh negative individuals. 23 RHD*1227A allele carriers were detected by PCR-SSP and sequencing-based typing (SBT). Genotype of 19 individuals was RHD*1227A/RHD*01N.01, while the other 4 was homozygous RHD*1227A/ RHD*1227A.11 individuals were detected as RHD*1227A allele among 1 045 random blood donors, among which 9 were RHD*1227A/RHD*01 and 2 were RHD*1227A/RHD*01N.01. 【Conclusion】 The frequency of RHD*1227A allele is 0.115 4 in Rh negative Han population and 0.005 3 in random population in Liaoning..

Genetic polymorphism and antibody screening of RhD negative Chinese Han population in Jiayuguan / 中国输血杂志

【Objective】 To investigate and analyze the polymorphism of RHD gene in RhD-negative population in Jiayuguan using molecular biological technique, so as to accurately identify RhD-negative individuals, and formulate individualized transfusion strategies. 【Methods】 The RhD negative voluntary blood donors and patients (mainly pregnant women) were recruited. After informed consent, history of blood transfusion and pregnancy of them were investigated, and samples were collected for negative D confirmation, gene sequencing as well as antibody screening and identification. 【Results】 Among the 96 samples, 73 cases were RHD gene deletion, 18 RHD*01EL.01(17 RHD1227A homozygous type and 1 RHD1227A heterozygous type), 2 weak RHD*15 type (845G/A), 1 partial D type, i. e. RHD-CE(7) -D heterozygous allele, and 2 RHD*01N.16 variant. Antibody was detected out in 4 cases, among which 2 were positive for anti-D, 1 anti-D plus anti-E, and 1 anti-Dia. 【Conclusion】 The proportion of DEL gene in RhD negative Chinese Han population in Jiayuguan is slightly lower than that in general Chinese Han population. No anti-D or RHD-HDN was observed in DEL type women due to multiple pregnancy or delivery of D positive newborns.

Identification of two novel weak D alleles by sequence-based typing / 中国输血杂志

【Objective】 To study the molecular basis of D variant and explore the molecular genetic mechanism of novel weak D alleles. 【Methods】 Blood samples were screened for D variants by serological method. The nucleotide sequences of coding region were amplified by PCR and sequenced directly, and RHD gene heterozygosity was detected. 【Results】 Weak D phenotype was confirmed by serological test, and two novel alleles were detected by DNA sequencing. The first was novel weak D 1102A allele, 1102G>A mutation in exon 8, resulting in a 368Glu>Arg substitution in two samples. The second was novel weak D 399C allele, carried a 399G>C mutation in exon 3, which led to a 133Lys>Asn substitution. 【Conclusion】 In this study, D variants were detected by sequence-based typing, and two new alleles were identified.