Serologic profiling of D variants in donor routine: unveiling the impact on false-negative results and alloimmunization.

The high number of D variants can lead to the unnecessary use of Rh immune globulin, overuse of D- RBC units, and anti-D allommunization. D variant prevalence varies among ethnic groups, and knowledge of the main variants present in a specific population, their behavior in serologic tests, and their impact on clinical practice is crucial to define the best serologic tests for routine use. The present study aimed to explore the serologic profile of D variants and to determine which variants are most associated with false-negative D typing results and alloimmunization. Donor samples were selected in two study periods. During the first period, D typing was performed on a semi-automated instrument in microplates, and weak D tests were conducted in tube or gel tests. In the second period, D typing was carried out using an automated instrument with microplates, and weak D tests were performed in solid phase. Samples from patients typed as D+ with anti-D were also selected. All samples were characterized by molecular testing. A total of 37 RHD variants were identified. Discrepancies and atypical reactivity without anti-D formation were observed in 83.4 percent of the samples, discrepant D typing results between donations were seen in 12.3 percent, and D+ patients with anti-D comprised 4.3 percent. DAR1.2 was the most prevalent variant. Weak D type 38 was responsible for 75 percent of discrepant samples, followed by weak D type 11, predominantly detected by solid phase. Among the D variants related to alloimmunization, DIVa was the most prevalent, which was not recognized by serologic testing; the same was true for DIIIc. The results highlight the importance of selecting tests for donor screening capable of detecting weak D types 38 and 11, especially in populations where these variants are more prevalent. In pre-transfusion testing, it is crucial that D typing reagents demonstrate weak reactivity with DAR variants; having a serologic strategy to recognize DIVa and DIIIc is also valuable.

Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

High prevalence of serological weak D phenotype and preponderance of weak D type 4.0.1. genetic variant in a Nigerian population: implications for transfusion practice in a resource-limited setting

ABSTRACT Introduction: Prevalence of RhD negative phenotype in Nigeria is low; this leads to scarcity of RhD negative red cells for transfusion. Serological and molecular genotyping of RhD negative individuals for weak D types could reduce this scarcity. The aim of this study was to determine the serological prevalence and molecular types of weak D phenotypes among blood donors and pregnant women in Kano, Nigeria. Methods: A total of 4482 blood donors and pregnant women from three hospitals in Kano were recruited. An indirect antiglobulin test was used to determine weak D phenotypes. Molecular genotyping was performed on genomic DNA from whole blood amplified by polymerase chain reaction sequence-specific primers (PCR-SSP) with agarose gel electrophoresis. Results: The mean age of the participants was 26.50 ±5.79 years. The prevalence of the RhD negative phenotype was 4.2% (189/4482). Of the 189 RhD negative phenotypes, 20 (10.6%) were weak D positive. Molecular genotyping of the 20 Weak D positive phenotypes revealed 15 (75%) weak D type 4, of which 11 were due to the RHD*09.03 and RHD*DAR3 (T201R, F223V) polymorphisms and 4, due to RHD* 08.01 and RHD* DFV polymorphisms; 2 (10%) were due to the 602 C>G polymorphism, while the remaining 3 (15%) constituted partial D or other rare weak D types. Conclusion: The prevalence of weak D positive phenotypes is high in this study; weak D type 4 is the most common RhD genetic variant. Routine serologic weak D testing of RhD negative blood and molecular genotyping should be encouraged in resource-limited settings.

High prevalence of serological weak D phenotype and preponderance of weak D type 4.0.1. genetic variant in a Nigerian population: implications for transfusion practice in a resource-limited setting.

INTRODUCTION: Prevalence of RhD negative phenotype in Nigeria is low; this leads to scarcity of RhD negative red cells for transfusion. Serological and molecular genotyping of RhD negative individuals for weak D types could reduce this scarcity. The aim of this study was to determine the serological prevalence and molecular types of weak D phenotypes among blood donors and pregnant women in Kano, Nigeria. METHODS: A total of 4482 blood donors and pregnant women from three hospitals in Kano were recruited. An indirect antiglobulin test was used to determine weak D phenotypes. Molecular genotyping was performed on genomic DNA from whole blood amplified by polymerase chain reaction sequence-specific primers (PCR-SSP) with agarose gel electrophoresis. RESULTS: The mean age of the participants was 26.50 ±â€¯5.79 years. The prevalence of the RhD negative phenotype was 4.2% (189/4482). Of the 189 RhD negative phenotypes, 20 (10.6%) were weak D positive. Molecular genotyping of the 20 Weak D positive phenotypes revealed 15 (75%) weak D type 4, of which 11 were due to the RHD*09.03 and RHD*DAR3 (T201R, F223V) polymorphisms and 4, due to RHD* 08.01 and RHD* DFV polymorphisms; 2 (10%) were due to the 602 C>G polymorphism, while the remaining 3 (15%) constituted partial D or other rare weak D types. CONCLUSION: The prevalence of weak D positive phenotypes is high in this study; weak D type 4 is the most common RhD genetic variant. Routine serologic weak D testing of RhD negative blood and molecular genotyping should be encouraged in resource-limited settings.

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

BACKGROUND: The 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 METHODS: Samples 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. RESULTS: RHD 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. DISCUSSION: The 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.

Frequency and characterization of RHD variant alleles in a population of blood donors from southeastern Brazil: Comparison with other populations.

BACKGROUND: The correct determination of D antigen could help to avoid alloimmunization in pregnant women and patients receiving blood transfusions. However, there are limitations in the identification of D variants as the partial and weak D phenotypes make the determination of D antigen a great challenge in the transfusion routine.' STUDY DESIGN AND METHODS: The molecular characterization of D variants was performed on blood donors from southeastern Brazil with atypical D typing. Furthermore, the serological profile of all RHD variant alleles identified was analyzed using different Anti-D clones. The prevalence of RHD alleles and genotypes found was compared with those described in other countries and in other regions from Brazil. RESULTS: Atypical serologic D typing occurred in 0.79 % of blood donors. The majority of RHD variant alleles (88 %) were first characterized by multiplex PCR and PCR-SSP as RHD*weak partial 4 (47 %), followed by RHD*weak D type 3 (29.9 %), RHD*weak D type 2 (3.9 %) and RHD*weak D type 1 (3.1 %). Genomic DNA sequencing characterized the RHD*weak partial 4 variants found in RHD*DAR1.2 (weak 4.2.2) (22 %), RHD*DAR3 (weak 4.0.1) (2.4 %), RHD*DAR3.1 (weak 4.0) (22 %) and RHD*DAR4 (weak 4.1) (0.8 %). RHD variant alleles associated with partial D, such as, RHD*DAU-4 (1.6 %), RHD*DAU-5 (2.4 %), RHD*DAU-6 (1.6 %), RHD* DIII type 8 (1.6 %), RHD*DVII (3.9 %) and RHD* DMH (0.8 %) were also observed. CONCLUSION: The prevalence of RHD variant alleles observed in this cohort differ from those found in other populations, including Brazilians from other regions. RHD allele distribution in specific regions should be considered for implementation of algorithms and genotyping strategies aiming at a more effective and safe transfusion.

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.

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.

Prevalencia del antígeno D débil y su clasificación fenotípica en donantes voluntarios de sangre / Prevalence of "weak" D antigen and its phenotypic classification in voluntary blood donours / Prevalência do antígeno D "fraco" e sua classificação fenotípica em doadores voluntários de sangue

En el campo de la medicina transfusional la correcta identificación de los fenotipos del sistema Rh y en especial del antígeno D debe ser de manera inequívoca por su relevancia clínica. El antígeno D tiene variantes denominadas D parcial, D débil y DEL, las que se producen por mutaciones de los alelos RHD/RHCE o por una supresión en la expresión fenotípica. Se trató de un estudio descriptivo, retrospectivo de corte transversal en el que se realizó una revisión de registros primarios durante el período 2011-2014 validados de acuerdo con el protocolo de Hernández-Sampieri R. Se utilizó estadística descriptiva mediante la aplicación del software informático SPSS versión 22.0 y se estableció la relación entre variables independientes a través del análisis estadístico de Chi-cuadrado. Se determinó una prevalencia de donantes RhD negativos de 1,8 a 2,5% y RhD débil de 1,79 a 2,28%. La fenotipificación serológica permitió identificar que los tipos 2 y 5 eran los más frecuentes. También se estableció la existencia de aloinmunización por anti-D, anti-C y anti-E. Se estableció de esta manera la existencia de D débil y una importante aloinmunización en la población de donantes de sangre tipificados como D negativo y D débil, por lo que se recomienda implementar un algoritmo de identificación del antígeno D en servicios de medicina transfusional.

In the field of transfusion medicine, the correct identification of the phenotypes of the Rh system and especially of the D antigen must be unequivocal for clinical relevance. The D antigen has variants called partial D, weak D and DEL. These are produced by mutations of the RHD/RHCE alleles or a suppression in phenotypic expression. The objective of this study was to establish the frequency of weak D antigen in the population of blood donours from 17 Ecuadorian states and their phenotypic combinations. It was a descriptive, retrospective cross-sectional study performed during the 2011-2014 period and validated with primary records in accordance with the Hernández-Sampieri R protocol. A descriptive statistics through the application of SPSS computer software version 22.0 was used and the relationship between independent variables through the Chi-square statistic method was established. A prevalence of RhD negative donours from 1.8 to 2.5% and weak D 1.79 to 2.28% was observed The serological phenotyping made it possible to identify that type 2 and 5 were the most frequent. The presence of alloimmunization by anti-D, anti-C and anti-E was also established. Besides, the presence of weak D types and significant alloimmunization in the donour population of blood typed as D negative and weak was established, so it is recommended to implement an algorithm for the identification of D antigen in transfusional medicine services.

No campo da medicina transfusional, a correta identificação dos fenótipos do sistema Rh e especialmente do antígeno D deve ser inequívoca devido a sua relevância clínica. O antígeno D tem variantes chamadas de D parcial, D fraca e DEL, as quais são produzidos por mutações dos alelos RHD/RHCE ou por uma supressão na expressão fenotípica. O objetivo deste estudo foi estabelecer a frequência do antígeno D fraco em uma população de doadores de sangue de 17 províncias equatorianas e suas combinações fenotípicas. Foi uma estudo descritivo, retrospectivo de corte transversal em que se realizou uma revisão dos registros primários validados de acordo com o Protocolo Hernández-Sampieri R durante o período 2011-2014. Utilizou-se estatísticas descritivas através da aplicação do software informático SPSS versão 22.0 e a relação entre variáveis independentes através da análise estatística de qui-quadrado. Foi determinada uma prevalência de doadores RhD negativos de 1,8 a 2,5% e RhD fraco de 1,79 a 2,28%. A genotipagem serológica permitiu identificar que os tipos 2 e 5 são os mais frequente. A existência de alo imunização por anti-D, anti-C e anti-E também foi estabelecida. A existência de D fraco e uma alo imunização significativa na população de doadores de sangue tipificados como D negativo e fraco, por isso é recomendado implementar um algoritmo de identificação do antígeno D em serviços de medicina transfusional.

Serologic strategy in detecting RHD altered alleles in Brazilian blood donors.

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.

Frecuencia de fenotipos del sistema Rh en donantes voluntarios de sangre / Frequency of Rh system phenotypes in voluntary blood donors / Frequência de fenótipos do sistema Rh em doadores de sangue voluntários

El sistema Rh es altamente polimórfico y está relacionado con la producción de aloanticuerpos y la enfermedad hemolítica del recién nacido. Los antígenos codificados por los genes RHD y RHCE forman el fenotipo Rh que es característico en cada población. Las variantes RHCE no han sido identificadas en la población ecuatoriana y así constituyen un riesgo de aloinmunización durante el embarazo o en transfusiones de componentes sanguíneos incompatibles. Prueba de ello es el estudio realizado en Ecuador que determinó una aloinmunización del 0,27%. Los anticuerpos con mayor frecuencia pertenecían al sistema Rh, resultados que motivaron la realización del presente estudio. Se analizaron un total de 1.298 muestras de donantes de sangre provenientes de 22 provincias ecuatorianas. Para la fenotipificación se utilizaron antisueros comerciales de la casa BIORAD y células de fenotipo conocido para el control de calidad interno, y se identificaron 20 fenotipos del sistema Rh distribuidos de forma heterogénea en las 22 provincias; el más frecuente fue Rz/R0. En donantes con fenotipo D débil el más común fue el R2/r, mientras que en los donantes Rh(D) negativo fue el fenotipo r/r. Estos datos demuestran la variedad de fenotipos en la población ecuatoriana y por ende la necesidad de su detección oportuna.

The Rh system is highly polymorphic. It is related to the production of alloantibodies and the hemolytic disease of the newborn. The antigens encoded by the RHD and RHCE genes form the Rh that is characteristic for each population. The RHCE variants have not been identified in the Ecuadorian population, constituting a risk of alloimmunization during pregnancy or in transfusions of incompatible blood components. Proof of this is the study carried out in Ecuador that determined an alloimmunization of 0.27% and the antibodies, more frequently belonged to the Rh system, results that motivated the realization of the present study. A total of 1298 samples from blood donors from 22 Ecuadorian provinces were analyzed. For the phenotyping, commercial antisera from the BIORAD house were used and cells of known phenotype for internal quality control. Identifying 20 phenotypes of the Rh system distributed heterogeneously in the 22 provinces, the most frequent was Rz/R0. In donors with weak D phenotype the most common was R2/r; whereas in Rh(D) negative donors was the r/r phenotype, these data demonstrate the variety of phenotypes in the Ecuadorian population and therefore the need for their timely detection.

O sistema Rh é altamente polimórfico e está relacionado com a produção de aloanticorpos e a doença hemolítica do recém-nascido. Os antígenos codificados pelos genes RHD e RHCE formam o fenótipo de Rh, que é característico para cada população. As variantes de RHCE não foram identificados na população equatoriana constituindo um risco de aloimunização durante a gravidez ou em transfusões de componentes sanguíneos incompatíveis. Prova disso é o estudo realizado no Equador que determinou aloimunização de 0,27%. Os anticorpos com maior frequência pertenciam ao sistema Rh, resultados que motivaram a realização do presente estudo. Um total de 1298 amostras de doadores de sangue de 22 estados equatorianos foram analisadas. Utilizou-se para a fenotipificação anti-soros comerciais BIORAD e células de fenótipo conhecido para controle de qualidade interno, identificando-se 20 fenótipos do sistema Rh heterogeneamente distribuídos nos 22 estados. O mais frequente foi Rz/R0. Em doadores com fenótipo D fraco, o mais comum foi o R2/r; ao passo que nos doadores Rh (D) negativo foi o fenótipo r/r. Esses dados demonstram a variedade de fenótipos na população do Equador, e portanto a necessidade da detecção precoce dos mesmos.

High frequency of variant RHD genotypes among donors and patients of mixed origin with serologic weak-D phenotype.

BACKGROUND: The current transfusion policy recommended for individuals with serologic weak-D phenotype is based on data derived from European-descent populations. Data referring to the distribution of RH alleles underlying weak-D phenotype among people of mixed origin are yet incomplete, and the applicability of European-based transfusion guidelines to this specific population is questionable. GOAL: To evaluate the distribution of RHD variant genotype among individuals with serologic weak-D phenotype of both African and European descent. METHODS: Donors and patients of mixed origin and with serologic weak-D phenotype were selected for the study. They were investigated using conventional RHD-PCR assays and RHD whole-coding region direct sequencing. RESULTS: One hundred and six donors and 58 patients were included. There were 47 donors and 29 patients with partial-D genotype (47/106, 44.3%, and 29/58, 50%, respectively). RHD*DAR and RHD*weak D type 38 represented the most common altered RHD alleles among donors (joint frequency of 39.6%), while weak D types 1-3 accounted for 10.4% of the total D variant samples. RHD*DAR was the most common allele identified in the patient group (frequency of 31%), and weak D types 1-3 represented 29.3% of the total. CONCLUSION: The frequency of partial D among mixed individuals with serologic weak-D phenotype is high. They should be managed as D-negative patients until molecular tests are complete.

RHD alleles among pregnant women with serologic discrepant weak D phenotypes from a multiethnic population and risk of alloimmunization.

BACKGROUND: A considerable number of RHD alleles responsible for weak and partial D phenotypes have been identified. Serologic determination of these phenotypes is often doubtful and makes genetic analysis of RHD gene highly desirable in transfusion recipients and pregnant women. We analyzed the RHD gene in a cohort of pregnant women with doubtful D phenotypes. METHODS: RHD genotyping was performed on 104 cases with D typing discrepancies or with history of serologic weak D phenotype. Laboratory-developed DNA tests, RHD BeadChip (Bioarray Solutions, Immucor), and sequencing were used to identify the RHD alleles. RESULTS: Molecular analyses showed 23 of 104 (22%) pregnant women were RHD*weak D types 1, 2, or 3 and not at risk for anti-D. Fifty-one (49%) were RHD*weak partial 4.0, 6 RHD*weak D type 38 (6%), 1 RHD*weak D type 45 (1%), 1 RHD*weak D type 67 (1%), and potentially at risk for being alloimmunized and making anti-D. Partial D was identified in 22 of 104 (21%) patients and definitively at risk for anti-D. DISCUSSION: Appropriate classification of RhD phenotypes is recommended for correct indication of RhIG in pregnant women. However, the serologic distinction between RhD-negative and RhD-positive phenotypes is a difficult task in the case of D variants due to the variations in serologic testing. Our results show a great variability in RHD variant alleles in pregnant women from this population of high admixture. According to these results, 78% of these obstetric patients are at risk for anti-D and candidates for RhIG.

Variant RHD Types in Brazilians With Discrepancies in RhD Typing.

BACKGROUND: The knowledge of D variants in patients and donors is important because anti-D alloimmunization can occur in some but not all individuals who express a variant RHD allele. Serologic distinction of RhD discrepancies is not always straightforward, which makes molecular analysis highly desirable. METHODS: A group of 223 subjects, 129 patients, and 94 blood donors was identified and analyzed on the basis of a D typing discrepancy. The D antigen expression was evaluated by tube and gel hemagglutination with four anti-D reagents. PCR-single specific primer (SSP), multiplex PCR, RHD BeadChip (Immucor), or sequencing were used for molecular analysis. RESULTS: In total, 168/223 (75%) weak D and 55/223 (25%) partial D variants were identified. Hemagglutination results varied in methods and anti-D reagents used in this process. There was no standard serologic reactivity identified, which could predict what type of D variant would be identified. Among weak D samples, types 1-3 were the most common, while DAR and DVI were most prevalent among partial D samples. CONCLUSION: Our results show that discrepancies found in the serologic typing should be investigated by molecular methods in order to determine the D variant involved and also to distinguish between weak D and partial D. The knowledge of the distribution of weak D types and partial D among populations is important for D- patients and pregnant women management.