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.

Prevalence of Rh and K phenotypes among blood donors from different ethnicities in Samtah (Southwestern Region) Saudi Arabia.

INTRODUCTION: Rh and Kell blood group systems are amongst the most important blood group systems; being highly immunogenic after ABO system. The aim of this study was to evaluate the frequencies of Rh antigens, haplotypes and K antigen among blood donors belonging to various ethnicities in Samtah, Jazan, Saudi Arabia. METHODS: This study was conducted during January 2019 and August 2020 at Samtah General Hospital, Samtah. Records of all blood donors recruited during this period were included for data acquisition. A total of 4977 blood donors' records were reviewed and data were analysed. A total of 3863 donors' results were considered in the final analysis. RESULTS: In comparison to Saudi blood donors, C antigen was less frequent in Sudanese donors (69.7% and 34.0%), the c antigen was less frequent in Indian (79.2% and 59.3%) and Philippine (79.2% and 40.0%) donors and more frequent in Sudanese (79.2% and 97.9%) donors, the E antigen was less frequent in Yemini (27.0% and 19.5%) and the e antigen was more frequent in Yemini (96.7% and 99.2%) donors. The DcE haplotype was less frequent (3.1% and 0.7%) and the ce haplotype was more frequent (4.3% and 7.6%) in Yemini donors. The K antigen was less frequent in Pakistani (11.9% and 4.1%; p = .041) and Indian (11.9% and 1.9%; p = .023) donors. CONCLUSION: Rh and K antigens showed marked variations in their frequencies among blood donors of different ethnicities. Utilization of blood from various ethnicities warrant extended phenotyping of Rh and K antigens to avoid the risk of alloimmunization in multiply transfused patients.

Análise do perfil de Rh e Kell em doadores de sangue em Porto Alegre, Rio Grande do Sul / Phenotypic profile analysis of Rh and Kell blood group systems among donors in Porto Alegre, Rio Grande do Sul

Introdução: A hemoterapia é uma prática terapêutica pelo meio de transfusão sanguínea. Devido ao baixo estoque de bolsas de sangue e o aumento de pacientes crônicos e emergenciais, se faz necessária a realização de testes imuno-hematológicos para minimizar os riscos de reações transfusionais e aloimunizações em doadores e receptores de sangue. Deste modo, no estudo foi avaliada a prevalência dos antígenos dos sistemas Rh e Kell em doadores de sangue de Porto Alegre ­ RS.Métodos: Estudo quantitativo, transversal e retrospectivo que foi realizado através da análise das informações dos doadores de sangue contidas no banco de dados do Hemocentro do Estado do Rio Grande do Sul, nos anos de 2018 e 2019.Resultados: Das 6.479 amostras fenotipadas, quanto ao sistema Rh, 44,6% são Rh positivo e 55,4% são Rh negativo. As frequências dos antígenos encontradas foram de, CC 10,1%, Cc 27%, cc 62,9%, EE 1,2%, Ee 13,9%, ee 84,9%. E, para o sistema Kell, K1 positivo 7,1% e K1 negativo 92,9%.Conclusões: Antígenos do sistema Rh e Kell exibem um grande nível de imunogenicidade e uma forte ligação com a Doença Hemolítica do Recém-nascido, podendo ocorrer a sensibilização em pacientes caso não haja a compatibilidade sanguínea. Este estudo ressalta a importância da implementação da fenotipagem eritrocitária em doadores de sangue, sugere-se mais estudos com períodos distintos para a pesquisa de resultados satisfatórios.

Introduction: Hemotherapy is a therapeutic practice consisting of blood transfusion. Low blood supply and an increase in chronic and emergency patients have made it necessary to conduct immunohematology tests to minimize the risks of adverse reactions and alloimmunization in donors and recipients. Therefore, this study aimed to assess the prevalence of Rh and Kell blood group antigens among blood donors in Porto Alegre, Rio Grande do Sul, Brazil.Methods: We conducted a quantitative, cross-sectional, retrospective study. Information from blood donors included in the Rio Grande do Sul's Blood Center database from 2018 to 2019 were analyzed.Results: A total of 6,479 samples were phenotyped, of which 44.6% were Rh-positive and 55.4% were Rh-negative. Antigen prevalence was CC (10.1%), Cc (27%), cc (62.9%), EE (1.2%), Ee (13.9%), and ee (84.9%). As for the Kell group, 7.1% were K1-positive and 92.9% were K1-negative.Conclusions: The Rh and Kell antigens are highly immunogenic and have a strong link with the hemolytic disease of the newborn. Sensitization may occur in patients if there is no blood compatibility. This study highlights the importance of implementing erythrocyte phenotyping in blood donors. Further studies should be conducted in different time frames to achieve satisfactory results.

Anti-D alloimmunization in Rh(D) negative adults with severe traumatic injury.

INTRODUCTION: Widely varying rates of alloimmunization associated with transfusing uncrossmatched RBC products to trauma patients as part of hemostatic resuscitation have been reported. We characterized the rates of RBC alloimmunization in our severely injured Rh(D) negative trauma population who received uncrossmatched Rh(D) positive RBC products. METHODS: In a 10-year retrospective analysis to assess Rh(D) alloimmunization risks, Rh(D) negative adult trauma patients initially requiring uncrossmatched group O Rh(D) positive RBC products with either RBC units or low titer group O whole blood as part of massive transfusion protocol (MTP) activation were identified. Only those Rh(D) negative patients whose initial antibody screenings were negative were included. Duration of serologic follow-up from date of MTP activation to either date of anti-D detection or most recent negative antibody screening was calculated. RESULTS: There were 129 eligible Rh(D) negative trauma patients identified. Median injury severity score was 25. Anti-D was detected in 10 (7.8%) patients after a median of 161.5 days; the median duration of serologic follow-up in those who did not have anti-D detected was 220 days. Patients who had anti-D detected were less severely injured and received fewer Rh(D) positive RBC products versus those who did not. DISCUSSION: In our severely injured adult trauma patients with MTP activation requiring uncrossmatched group O Rh(D) positive RBC products, the rate of anti-D detection was low. Additional studies are necessary to determine generalizability of these findings and fully characterize alloimmunization risks in trauma patients with varying extents of injury.

Rate of RhD-alloimmunization after the transfusion of multiple RhD-positive primary red blood cell-containing products.

INTRODUCTION: Early transfusion reduces mortality in bleeding patients. In this setting, RhD-positive blood products might be transfused. This study determined the association between the RhD-alloimmunization rate and the number of RhD-positive products transfused. METHODS: RhD-negative patients between 13 and 50 years who were transfused with ≥1 RhD-positive red blood cell (RBC) or whole blood units between January 1, 2000 and December 31, 2019 in a healthcare network were identified. Study patients had to have had at least one antibody detection test performed ≥14 days after the index RhD-positive transfusion and not receive RhIg. Patients were stratified into groups that received 1, 2, 3-5, 6-10, 11-20, and >20 RhD-positive transfusions and the RhD-alloimmunization rate was determined for each group. RESULTS: There were 335 patients included; 52/335 (15.5%) were females. Overall, there were 117/335 (34.9%, CI: 29.8%-40.3%) recipients who became RhD-alloimmunized. There was no significant dosage effect in the RhD-alloimmunization rates as the exposure to RhD-positive units increased from one RhD-positive unit to more than 20 RhD-positive units (p = .270 for non-parametric trend test). In an exploratory analysis, patients who received 100% of their RhD-positive transfusions within 72 h of the index transfusion had a significantly higher rate of RhD-alloimmunization compared to those who were transfused over a longer period of time (42.3% vs. 21.4%, respectively; p = .001). CONCLUSION: These results suggest that there may not be an increased RhD-alloimmunization risk with transfusing multiple RhD-positive units after one RhD-positive unit has been transfused. These findings need confirmation in larger studies.

An unusual case of anti-D detection in two consecutive D+ patient samples: Antibody carryover on an automated gel platform.

BACKGROUND: Laboratory results can be affected by sample to sample carryover. Carryover of different analytes occurring in automated clinical chemistry, immunology, hematology, and molecular laboratories is well described. However, carryover in a transfusion service laboratory is not reported in medical literature. MATERIALS AND METHODS: Immunohematology testing results, demographic data, and clinical data were reviewed on three patients retrospectively from 2015 to 2019. RESULTS: Type and screen samples tested on automated gel platform from two D+ patients were affected by anti-D carryover from a patient sample with a very high-titer anti-D. Additional immunohematology and molecular testing confirmed that anti-D in samples of two D+ patients was due to carryover. CONCLUSION: A case of anti-D carryover caused false detection of anti-D in two D+ patients. Carryover can have implications for patient management. Transfusion laboratory staff need to be aware of it and investigate any unexpected results further.

Does blood type affect the COVID-19 infection pattern?

Among the many aspects that characterize the COVID-19 pandemic, two seem particularly challenging to understand: i) the great geographical differences in the degree of virus contagiousness and lethality that were found in the different phases of the epidemic progression, and, ii) the potential role of the infected people's blood type in both the virus infectivity and the progression of the disease. A recent hypothesis could shed some light on both aspects. Specifically, it has been proposed that, in the subject-to-subject transfer, SARS-CoV-2 conserves on its capsid the erythrocytes' antigens of the source subject. Thus these conserved antigens can potentially cause an immune reaction in a receiving subject that has previously acquired specific antibodies for the source subject antigens. This hypothesis implies a blood type-dependent infection rate. The strong geographical dependence of the blood type distribution could be, therefore, one of the factors at the origin of the observed heterogeneity in the epidemics spread. Here, we present an epidemiological deterministic model where the infection rules based on blood types are taken into account, and we compare our model outcomes with the exiting worldwide infection progression data. We found an overall good agreement, which strengthens the hypothesis that blood types do play a role in the COVID-19 infection.

An agnostic study of associations between ABO and RhD blood group and phenome-wide disease risk.

Background: There are multiple known associations between the ABO and RhD blood groups and disease. No systematic population-based studies elucidating associations between a large number of disease categories and blood group have been conducted. Methods: Using SCANDAT3-S, a comprehensive nationwide blood donation-transfusion database, we modeled outcomes for 1217 disease categories including 70 million person-years of follow-up, accruing from 5.1 million individuals. Results: We discovered 49 and 1 associations between a disease and ABO and RhD blood groups, respectively, after adjustment for multiple testing. We identified new associations such as a decreased risk of kidney stones and blood group B as compared to blood group O. We also expanded previous knowledge on other associations such as pregnancy-induced hypertension and blood groups A and AB as compared to blood group O and RhD positive as compared to negative. Conclusions: Our findings generate strong further support for previously known associations, but also indicate new interesting relations. Funding: Swedish Research Council.

The blood types that many people are familiar with, such as O-negative or AB-positive, are determined by two systems of antigens or proteins on the surface of the red blood cells: the ABO system and the RhD system. The ABO system types people's blood as A, B or AB if they have A and/or B antigens, or as type O if they have neither; while the RhD system provides the positive or negative label depending on whether or not the RhD antigen is present. Previous studies have found that some ABO blood groups are linked to increased risk and severity of a variety of conditions, including blood clots in veins, bleeding disorders and gastric ulcers. Despite the known influence that blood groups can have on disease, the connection has not been fully studied in many conditions, particularly for RhD status. Knowing the differences in risk and disease severity between different populations could help clinicians identify individuals that they need to monitor more closely and include blood group information in prediction models. To fill this gap in information, Dahlén et al. systematically looked for relationships between diseases and blood groups using records from 5.1 million people on a Swedish national blood donation-transfusion database. Examining 1,217 disease categories revealed that the vast majority did not appear to have a connection to either the ABO or RhD systems of classifying blood. However, the analysis identified 49 diseases with links to ABO blood types and one linked to RhD status. One notable finding was that people with blood group B have an decreased risk of kidney stones. The distribution of blood groups varies significantly around the world, so this relationship between disease and blood group may in part explain regional differences in disease occurrence. In the future, identifying relationships with blood groups may help to better understand the underlying biological mechanisms of diseases and lead to new avenues of research.

Conservation of Rh negative Low Titer O Whole Blood (LTOWB) and the need for a national conversation to define its use in trauma transfusion protocols.

Low-titer group O whole blood (LTOWB) use is growing steadily in the United States. Although the percentage of O negative LTOWB use by Red Cross hospitals has remained steady at ~23% over the last 2 years, this elevated use rate is twice that of O negative RBC components. Given the more restricted group O donor pool, this level of use will make it difficult to expand the use of this product. Evaluation of hospital practices regarding females of childbearing potential show significant variability with some hospitals transfusing O positive, with others choosing to restrict this population to O negative LTOWB or only O negative RBC component therapy. To ensure access of LTOWB to all patients who may benefit and to maintain sufficient supplies, we recommend developing standardized practice recommendations for its use.

Evaluating automated titre score as an alternative to continuous flow analysis for the prediction of passive anti-D in pregnancy.

OBJECTIVES: To evaluate the potential of the automated titre score (TS) as an alternative method to continuous flow analysis (CFA) for the prediction of the nature of anti-D in pregnancy. BACKGROUND: The 2016 revised British Society for Haematology (BSH) antenatal guidelines recommended a measurement of anti-D concentration by CFA to ensure the detection of potential immune anti-D. Due to high referral costs and resource pressures, uptake has been challenging for hospital laboratories. Serious Hazards of transfusion (SHOT) data have previously shown that this has contributed to missed antenatal follow ups for women with immune anti-D and neonates affected by haemolytic disease of the fetus/newborn. METHODS/MATERIALS: In this multicentre comparative study, samples referred for CFA quantification were also tested by an ORTHO VISION automated anti-D indirect antiglobulin test (IAT) serial dilution and then converted to TS. CFA results and history of anti-D prophylaxis were used to categorise samples as passive or immune, with the aim of determining a potential TS cut-off for CFA referral of at risk patients. RESULTS: Five UK National Health Service (NHS) trusts generated a total of 196 anti-D TS results, of which 128 were classified as passive and 68 as immune. Diagnostic testing of CFA and TS values indicated a TS cut-off of 35 to assist in distinguishing the nature of anti-D. Using this cut-off, 175 (89%) results were correctly assigned into the passive or immune range, giving a specificity of 92.19% and a negative predictive value of 91.47%. CONCLUSION: TS in conjunction with clinical and anti-D prophylaxis history can be used as a viable and cost-effective alternative to CFA in a hospital laboratory setting.

Assessment of Fetal Rhesus D and Gender with Cell-Free DNA and Exosomes from Maternal Blood.

The detection of fetal cell-free DNA (cfDNA) from maternal plasma has enabled the development of essential techniques in prenatal diagnosis during recent years. Extracellular vesicles including exosomes were determined to carry fetal DNA fragments. Considering the known difficulties during isolation and stability of cfDNA, exosomes might provide a new opportunity for prenatal diagnosis and screening. In this study, comparison of cfDNA and exosome DNA (exoDNA) for predicting the fetal sex and Rhesus D (RHD) genotype was performed by using real-time polymerase chain reaction with simultaneous amplification of sequences of SRY and RHD genes. Fetal sex and RHD were determined in 100 and 81 RHD-negative pregnant women with cfDNA and exoDNA, respectively. The gestation ages of pregnant women were between 9 and 40 weeks. The results were compared with the neonatal phenotype for gender and a serological test for RHD. The cfDNA revealed 95.75% sensitivity and 100% specificity in RHD positivity and 100% sensitivity and 95.45% specificity in SRY positivity. Cohen's agreement coefficient in the Kappa test ranged from 0.8 to 1.0 (P < 0.00001). Although the exoDNA failed to amplify 16 cases, the remaining 65 cases revealed a true estimate for both fetal RHD and SRY genes with 100% sensitivity and specificity. Successful application of exoDNA and cfDNA with real-time PCR for fetal genotyping enables this technique to be applied in the assessment of fetal RHD and gender during pregnancy, allowing initiation of early treatment methods and avoiding unnecessary interventions and cost.

Two new RHD alleles with deletions spanning multiple exons.

BACKGROUND: The most common large-deletion RHD allele (RHD*01N.01) includes the entire coding sequence, intervening regions and untranslated regions. The rest of large-deletion RHD alleles reported to-date consist of single-exon deletions, such as RHD*01N.67 which includes exon 1. MATERIALS AND METHODS: Samples from two donors with RhD-negative serology yielded unclear or inconclusive results when subject to confirmatory testing on RHD genotyping arrays. To determine their RHD genotypes, genomic DNA was analyzed with a combination of allele-specific PCR, long-range PCR, Sanger sequencing, and next-generation sequencing assays. RESULTS: Allele-specific PCR failed to detect products for RHD exons 1 to 3 in one sample and RHD exons 1 to 5 in the other. A quantitative next-generation sequencing assay confirmed deletion of exons 1 to 3 and 1 to 5 respectively, and detected the absence of an RHD gene in trans in both samples. Long-range PCR and Sanger sequencing enabled identification of the breakpoints for both alleles. Both deletions start within the 5' Rhesus box (upstream of the identity region for the 1-to-3 deletion, downstream of it for the 1-to-5 deletion), and end within introns. CONCLUSIONS: Resolution of unclear or inconclusive results from targeted genotyping arrays often leads to the discovery of new alleles. The 5' Rhesus box may be a hot spot for genetic recombination events, such as the large deletions described in this report.

Does ABO and RhD matching matter for platelet transfusion?

Platelets express ABO antigens and are collected in plasma, which contains ABO antibodies as would be consistent with the donor ABO group. Platelet ABO antigens that are incompatible with recipient ABO antibodies may have accelerated clearance from circulation and result in lower count increments. ABO antibodies that are passively transferred from donor plasma may result in hemolysis of recipient red blood cells. Although platelets do not express Rh antigens, they contain small numbers of intact red blood cells or fragments, which can lead to alloimmunization in the recipient. Alloimmunization to the RhD antigen may occur when platelets obtained from RhD-positive donors are transfused to RhD-negative recipients. All of these compatibility considerations must be balanced against the available supply, which may be limited due to the 5- to 7-day shelf life of platelets. This articles describes considerations for platelet ABO and RhD selection for platelet transfusions, including the impact of major ABO incompatibility on count increments, the risks of hemolysis associated with minor ABO incompatibility, and the risk of RhD alloimmunization when RhD-negative patients receive platelets obtained from RhD-positive donors.

Impact of red blood cell alloimmunization on fetal and neonatal outcomes: A single center cohort study.

BACKGROUND: Alloimmunization can impact both the fetus and neonate. STUDY OBJECTIVES: (a) calculate the incidence of clinically significant RBC isoimmunization during pregnancy, (b) review maternal management and neonatal outcomes, (c) assess the value of prenatal and postnatal serological testing in predicting neonatal outcomes. STUDY DESIGN AND METHODS: A retrospective audit of consecutive alloimmunized pregnancies was conducted. Data collected included demographics, clinical outcomes, and laboratory results. Outcomes included: incidence of alloimmunization; outcomes for neonates with and without the cognate antigen; and sensitivity and specificity of antibody titration testing in predicting hemolytic disease of the fetus and newborn (HDFN). RESULTS: Over 6 years, 128 pregnant women (0.4%) were alloimmunized with 162 alloantibodies; anti-E was the most common alloantibody (51/162; 31%). Intrauterine transfusions (IUTs) were employed in 2 (3%) of 71 mothers of cognate antigen positive (CoAg+) neonates. Of 74 CoAg+ neonates, 58% required observation alone, 23% intensive phototherapy, 9% top up transfusion, and 3% exchange transfusion; no fetal or neonatal deaths occurred. HDFN was diagnosed in 28% (21/74) of neonates; anti-D was the most common cause. The sensitivity and specificity of the critical gel titer >32 in predicting HDFN were 76% and 75%, respectively (negative predictive value 95%; positive predictive value 36%). The sensitivity and specificity of a positive direct antiglobulin test (DAT) in predicting HDFN were 90% and 58%, respectively (NPV 97%; PPV 29%). CONCLUSION: Morbidity and mortality related to HDFN was low; most alloimmunized pregnancies needed minimal intervention. Titers of >32 by gel warrant additional monitoring during pregnancy.

RHD and RHCE molecular analysis in weak D blood donors and in patients with Rh antibodies against their own corresponding Rh antigen.

BACKGROUND: The Rh system is the largest and most polymorphic blood group system. The existence of a large number of RH alleles results in variant phenotypes that often complicate blood donor phenotyping and the distinction between auto- and allo-antibodies in recipients who have anti-Rh antibodies in the presence of their own corresponding Rh antigen. Knowledge of these variants is necessary in order to make blood transfusion safer. MATERIALS AND METHODS: Samples from 48 blood donors with serological weak D and from 29 patients who had anti-Rh antibody in the presence of their own corresponding Rh antigen were evaluated molecularly for RHD and RHCE alleles using a blood-multiplex ligation-dependent probe amplification assay and Sanger sequencing. RESULTS: Rh variants were found in 45 of the 48 blood donors: 24/45 (53%) were weak D, 2/45 (4%) partial D and 19/45 (42%) were weak and partial D. The remaining three donors (6%) did not show a mutation in the RHD allele. Among the 29 patients, 13/29 had anti-e, of whom 4/13 had genotypes that predicted a partial e antigen; 11/29 had anti-D, with 6/11 being identified as partial D; 2/29 had anti-c, of whom 1/2 was predicted to express partial c antigen; 4/29 who had anti-E and 4/29 who had anti-C did not show mutations in RHCE*C or RHCE*E. DISCUSSION: It was possible to find individuals with clinically significant Rh phenotypes due to the weak reactivity of the D antigen, detected through serological tests in blood donors. In patients, when found with the anti-Rh antibody in the presence of the same Rh antigen, it is difficult to distinguish an auto-antibody from an allo-antibody by serological tests; in these cases, molecular methods (genotyping) can help us to determine whether there are changes in the RH alleles and to discover the nature of the antibody (allo or auto).

Sistema Rh-Hr y variantes del antígeno D en tres poblaciones afroecuatorianas del Valle del Chota / Rh-Hr system and D antigen variants in three Afro-Ecuadorian populations in the Chota Valley / Sistema Rh-Hr e variantes do antígeno D em três populações afro-equatorianas no vale do Chota

Resumen La identificación inequívoca del antígeno D en medicina transfusional es de vital importancia para evitar reacciones postransfusionales y la enfermedad hemolítica del recién nacido. Es común el uso de reactivos serológicos monoclonales o tarjetas de gel y su interpretación está definida por cruces, de acuerdo con la reacción serológica. El propósito de este estudio fue determinar la frecuencia del factor Rh y las variantes del antígeno D en una población afroecuatoriana. Se trató de un estudio descriptivo, transversal con muestreo aleatorio simple de 541 pobladores. Para la tipificación del factor Rh se utilizó la metodología en tubo con antisueros monoclonales y para la detección de las variantes de D se utilizaron tarjetas de gel IDCoombs Anti-IgG. Las lecturas se verificaron mediante el análisis del índice kappa. Se aplicó estadística descriptiva y el análisis de Chi cuadrado para establecer la relación de las variables y su significación. Se identificó una frecuencia del 92% de individuos Rh(D) positivo y un 8% Rh(D) negativo. El 4,80% de los individuos presentaban la variante D débil y el 79% reacciones serológicas entre 2 y 3(+) indicativas de otras variantes del antígeno D. El fenotipo más común fue el R0/R0. Estos datos demuestran la necesidad de confirmar la existencia de variantes del antígeno D en esta población para un mejor manejo de la sangre. Una limitante constituye la disponibilidad de técnicas moleculares para la genotipificación de D; sin embargo, se podría implementar la fenotipificación RHCE como estrategia pretransfusional.

Abstract The unequivocal identification of D antigen in transfusion medicine is of vital importance to avoid post-transfusion reactions and hemolytic disease of the newborn. The use of monoclonal serological reagents or gel cards is common and their interpretation is defined according to the serological reaction by crosses. The purpose of this study was to determine the frequency of Rh factor and D antigen variants in the Afro-Ecuadorian population. This was a descriptive, cross-sectional study with simple random sampling of 541 residents. Tube typing with monoclonal antisera was used to typify Rh factor and ID-Coombs Anti-IgG gel cards were used to detect D variants, and the readings were verified by analysis of the kappa index. Descriptive statistics and Chi-square analysis were applied for the relationship of the variables and their significance. A frequency of 92% of Rh(D) positive individuals and 8% Rh(D) negative individuals were identified. Almost 5% (4.80%) of the individuals presented the weak D variant and 79% serological reactions between 2-3(+) indicative of other D antigen variants, the most common phenotype being R0/R0. These data demonstrate the need to confirm the existence of D antigen variants in this population for better management and availability of blood. A limitation is the availability of molecular techniques for D genotyping, however, RHCE phenotyping could be implemented as a pretransfusion strategy.

Resumo A identificação inequívoca do antígeno D na medicina transfusional é de vital importância para evitar reações pós-transfusionais e a doença hemolítica do recém-nascido. É comum o uso de reagentes sorológicos monoclonais ou cartões de gel e sua interpretação é definida por cruzamentos de acordo com a reação sorológica. O objetivo deste estudo foi determinar a frequência do fator Rh e as variantes do antígeno D numa população afro-equatoriana. Foi um estudo descritivo, transversal, com amostragem aleatória simples de 541 residentes. Para a tipagem do fator Rh foi utilizada a metodologia em tubo com anti-soros monoclonais e para a detecção das variantes de D, os cartões de gel ID-Coombs Anti-IgG. As leituras foram verificadas por análise do índice kappa. Foi aplicada estatística descritiva e para estabelecer a relação das variáveis e sua significação se utilizou a análise do qui-quadrado. Identificando uma frequência de 92% dos indivíduos Rh (D) positivos e 8% Rh (D) negativos. 4,80% dos indivíduos apresentavam a variante D fraca e 79% reações sorológicas entre 2 e 3(+) indicativas de outras variantes do antígeno D, sendo o fenótipo mais comum o R0/R0. Esses dados demonstram a necessidade de confirmar a existência de variantes do antígeno D nessa população para melhor gerenciamento e disponibilidade de sangue. Uma limitação é a disponibilidade de técnicas moleculares para a genotipagem de D, no entanto, a fenotipagem de RHCE poderia ser implementada como uma estratégia de pré-transfusão.

Molecular characterisation of RhD variants in North Indian blood donor population.

OBJECTIVES: A molecular analysis of serologically RhD variant samples was conducted to find the incidence of various D variants in our blood donor population. BACKGROUND: Determining a blood donor's RhD phenotype and genotype is important as transfusion of units with a weak D or partial D phenotype can result in immunisation of the recipients. METHODS: Samples with discrepant D and weak D phenotypes identified on testing with at least five different monoclonal anti-D antisera were considered serological RhD variant and subjected to molecular testing (Massarray kit, Agena Bioscience, San Diego) for variant RHD gene. RESULTS: A total of 39 samples, including 19 RhD discrepant samples and 20 weak D samples, were identified as serological RhD variant from a total of 4386 samples. Thirteen (13/39) samples carried variants leading to weak D phenotype, and eight samples had variants leading to partial D categories. Seven samples (7) could not be characterised, whereas 11 samples were identified as Rh negative (RHD*01N.01) after molecular testing. Overall incidence of D variants in the study population was 0.48%. RHD*weak D type 1(5, 0.1%) and RHD*DFR1 (5, 1%) were the most common variants identified. CONCLUSIONS: Few samples with weak reaction on serological testing were found to be partial D variant and vice versa. Donor centres should develop a protocol for genotyping of samples with aberrant results on serological testing for assessing the actual RhD status of an individual as results of serological testing may be misleading.