Introdução: O sistema de grupo sanguíneo Rh é altamente complexo e polimórfico sendo considerado, depois do sistema ABO, o sistema de maior significado clínico na medicina transfusional. Os anticorpos dirigidos contra os seus antígenos estão envolvidos em reações hemolíticas transfusionais, anemia hemolítica autoimune e doença hemolítica do feto e neonatos. O antígeno D é o mais imunogênico dos antígenos do sistema Rh e sua conformação na superfície das hemácias está disposta como um mosaico de diferentes epítopos. Alterações no gene RHD, que expressa a proteína RhD, podem ser detectadas pela variação na intensidade da reação sorológica, utilizando-se diferentes reagentes anti-D. As alterações ocorrem por diversos mecanismos genéticos e são responsáveis pelo aparecimento dos fenótipos D fraco e D parcial. Essas variantes podem apresentar mudanças qualitativas e/ou quantitativas na proteína RhD e estão frequentemente envolvidas em casos de aloimunização anti-D. A identificação e classificação dos fenótipos RhD variantes possuem alta relevância clínicae, portanto, requerem uma rigorosa investigação molecular, uma vez que os testes sorológicos não são capazes de distinguir entre fenótipos D fraco e D parcial. Dessa forma, esclarecer os resultados fracos ou discrepantes encontrados na sorologia e determinar as variantes RhD têm grande importância para a comunidade científica e, principalmente, para a medicina transfusional. Objetivos: Caracterizar, por métodos moleculares, antígenos RhD variantes em amostras de doadores de sangue da Fundação Hemocentro de Brasília que apresentaram fraca expressão do antígeno RhD. Métodos: Foram selecionadas para extração de DNA e investigação molecular de antígenos RhD variantes 103 de 7983 amostras de doadores de sangue que apresentaram tanto resultados negativo, inconclusivo ou fraco (menor que 2 cruzes) na fenotipagem RhD, realizada por técnica em microplacas, como resultado positivo na confirmação de D fraco. Inicialmente, as amostras foram confirmadas quanto à presença do gene RHD por PCR multiplex RHD regiões íntron4/éxon7. Na sequência, técnicas de PCR alelo específico e RFLP foram utilizadas para triar as variantes D fraco tipos 1, 2, 3 e 4 ou DAR, seguido do sequenciamento de éxons específicos do gene RHD para investigação das demais variantes. As amostras foram avaliadas quanto à zigosidade por dosagem do gene RHD, por qPCR, e quanto à presença do Pseudogene (RHD?) e da deleção do gene RHD, por PCR-SSP. Os resultados encontrados foram interpretados em conjunto com os fenótipos RhCE também analisados. Resultados: Das amostras estudadas, 68,93% foram caracterizadas como RhD parcial; 21,36% como RhD fraco; 1,94% não apresentaram polimorfismos nos éxons do gene RHD e 2,91% apresentaram ausência de éxons RHD indicando a presença de um gene híbrido RHD-CE-D não definido. Entre as amostras RhD fraco, 16,5% eram RHD*fraco tipo 3; 5,8% RHD*fraco tipo 2; 1,94% RHD*fraco tipo 1; 0,97% RHD*fraco tipo 38 e 0,97% RHD*fraco tipo 145. Entre as amostras RhD parcial, 33,01% eram RHD*DAR 3.1; 32,04% RHD*DAR 1.2; 1,94% RHD*DVII; 0,97% RHD* DOL 1; 0,97% RHD* DOL 2. Discussão e conclusão: Nossos resultados demonstraram que as reações atípicas nos testes sorológicos para o antígeno RhD são indicativas da presença de variantes RhD. Os alelos RHD*DAR subtipos foram os mais frequentemente encontrados nas amostras dos doadores de Brasília-Distrito Federal. Esses alelos estão associados à aloimunização anti-D. A caracterização de antígenos RhD variantes por meio da associação de testes sorológicos e métodos moleculares visa, principalmente, determinar os fenótipos/genótipos associados à aloimunização anti-D. Essa caracterização é muito importante para aumentar a segurança transfusional, diminuir a administração desnecessária de imunoprofilaxia anti-D em gestantes e pode, também, colaborar com a preservação dos estoques escassos de unidades de sangue RhD negativo. Além disso, o conhecimento sobre a frequência e distribuição das variantes Rh contribui para a compreensão da origem multirracial da região estudada e auxilia no desenvolvimento de futuros protocolos de genotipagem RHD que estabeleçam estratégias de buscas de unidades de sangue compatíveis para pacientes portadores de variantes RhD. / Introduction: The Rh blood group system is highly complex and polymorphic, and considered, after the ABO system, the system of greater clinical significance in transfusion medicine. Antibodies directed against its antigens are involved in hemolytic transfusion reactions, autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn. Antigen D is the most immunogenic of the Rh system and its conformation on the surface of red blood cells is a mosaic of different epitopes. Changes in the RHD gene, which expresses the RhD protein, can be detected by variations in the intensity of the serological reaction by using different anti-D reagents. The changes occur as a result of several genetic mechanisms and are responsible for the appearance of weak D and partial D phenotypes. These variants may exhibit qualitative and/or quantitative changes in RhD protein and are often involved in anti-D alloimmunization. The identification and classification of variant RhD phenotypes are of high clinical relevance. Therefore, they require a rigorous molecular investigation, since serological tests are not able to distinguish between weak D and partial D phenotypes. Thus, clarifying the weak or discrepant results found in serology and determining RhD variants are of great importance to the scientific community and especially to transfusion medicine. Objectives: To characterize, by molecular methods, variant RhD antigens in donor blood samples from the Blood Center Foundation of Brasília which showed poor expression of the RhD antigen. Methods: A total of 103 out of 7983 samples from blood donors who had negative, inconclusive or weak results (less than 2 crosses) in the microplate technique and also tested positive in the weak D confirmation test were selected for DNA extraction and molecular investigation of the RhD antigen. Initially, the presence of the RHD gene was confirmed in the samples by PCR multiplex RHD intron4/exon7 regions. Then, allele specific PCR and RFLP techniques were used to screen for weak D variant types 1, 2, 3 and 4 or DAR, followed by sequencing of specific exons of the RHD gene to investigate the other variants. Samples were evaluated for zygosity by RHD gene dosage by qPCR and for the presence of Pseudogene (RHD?) and RHD gene deletion by PCR-SSP. The results were interpreted in conjunction with the RhCE phenotypes also analyzed. Results: Of all samples studied, 68.93% were characterized as partial RhD; 21.36% as weak RhD; 1.94% had no polymorphisms in the RHD gene exons and 2.91% had no RHD exons indicating the presence of an undefined RHD-CE-D hybrid gene. Among the weak RhD samples, 16.5% were weak RHD*type 3; 5.8% RHD * weak type 2; 1.94% RHD*weak type 1; 0.97% RHD*weak type 38 and 0.97% RHD* weak type 145. Among the partial RhD samples, 33.01% were RHD*DAR 3.1; 32.04% RHD*DAR 1.2; 1.94% RHD*DVII; 0.97% RHD*DOL 1; 0.97% RHD*DOL 2. Discussion and conclusion: Our results demonstrated that atypical reactions in serological tests for the RhD antigen are indicative of the presence of RhD variants. The RHD*DAR subtype alleles were the most frequently found in donor samples from Brasília - Distrito Federal. These alleles are associated with anti-D alloimmunization. The characterization of variant RhD antigens through the association of serological tests and molecular methods mainly aims to determine the phenotypes/genotypes associated with anti-D alloimmunization. This characterization is very important to increase transfusion safety, reduce unnecessary administration of anti-D immunoprophylaxis in pregnant women and may also contribute to the preservation of scarce RhD negative blood units. In addition, knowledge about the frequency and distribution of Rh variants contributes to the understanding of the multiracial origin of the studied region, and assists in the development of future RHD genotyping protocols that establish compatible blood unit search strategies for RhD variant patients.

Monoclonal RhD antibodies (anti-RhD) may potentially be used in preventing Rh alloimmunisation of women delivering RhD-positive babies, and thereby preventing haemolytic disease of the newborn (HDN). Binding and molecular analyses were performed on a range of monoclonal anti-RhDs to understand the interaction between the antibody and antigen. A flow cytometry (FCM) assay system was developed to analyse the binding of the anti-RhD to human group O R₁R₂ red blood cells (RBC). A panel of 30 anti-RhD preparations were studied using this assay and Vmax (maximum binding) and KD (equilibrium constant) were determined for each antibody. The antibodies were categorised into 3 groups (low, medium and high binders) according to their Vmax. Furthermore, the Vmax was converted to the number of antibody molecules bound per RBC (NMBR) by using a correlation curve generated from running parallel FCM and radioiodination assays (RIA). Scatchard analysis of the RIA data indicated that the total NMBR for O R₁R₂ RBC was 27,300 antigen sites/cell. Molecular analysis involved cloning and sequencing of 11 anti-RhD. Heavy chains (HC) preferentially used gene segments from the VH3 and VH4 families, and kappa chain (κ LC) usage was restricted to DPK9. Four sets of antibodies, showed restricted D gene segments (encode part of the HCDR3) which indicated possible importance for epitope specificity. Analysis of V gene sequence indicated that common VH and VL pairings were found used by the medium binders. The high and low binders had unique VH and VL pairings, although the high binders also showed greater somatic mutations from their respective germline genes. It was concluded that the fit of the antibody to the RhD antigen is dependent on both the VH and VL usage and pairing, and that the precise epitope specificity of these antibodies may require HCDR3 interaction.

Author: Bc. Dagmar Bakerová Supervisor: MUDr. Vít Řeháček Charles University, Faculty of Pharmacy in Hradec Králové Title of diploma thesis: RhD antigen screening by molecular genetic methods This thesis deals with the genotyping of weak and partial antigens using molecular genetic methods. The main aim is to evaluate the rate of the representation of individual types of variant and weak RhD antigens in first-time blood donors, patients and pregnant women. Testing took place at the Transfusion Department of University Hospital Hradec Králové between October 2015 and February 2019. The PCR-SSP method was used for RHD genotyping using commercially supplied BAGene SSP kits from BAGene Health Care. The study includes 32 samples from first-time blood donors in the reference period to determine the specific type of RhD antigen, and 188 samples from patients and pregnant women, for whom serological methods could not be used to unequivocally identify the RhD antigen. For all pregnant women, moreover, the genotyping result was a factor in determining whether to administer anti-D immunoglobulin. This RHD genotyping for all serologically ambiguous samples has made it possible to determine a specific type of partial or weak RhD antigen. In the donor group, the weak RhD antigen was detected in 1.12 % of cases...

La prédiction de la zygotie à partir du génotype le plus probable en la comparant à la PCR-SSP, n’était pas fiable liée à la possibilité d’avoir d’autres génotypes possibles pour le même phénotype. En effet, la fréquence très proche de l’haplotype R0 et r dans la population tunisienne rend la déduction du génotype le plus probable très aléatoire. Dans un deuxième temps, l’évaluation de la méthode moléculaire la plus convenable à la détermination de la zygotie RHD a été réalisée par comparaison des trois techniques moléculaires (PCR-SSP, PCR-RFLP et Q-PCR) et par l’analyse des résultats discordants par séquençage du gène RHD et des « boîtes Rhésus ». L’analyse de 370 échantillons RH:1 à l'aide de ces trois tests moléculaires a montré que 81,9% des résultats étaient en concordance alors que 18,1% en discordance. L’analyse des cas discordants a montré que notre cohorte se compose de 193 sujets dizygotes et 145 hémizygotes et 32 dont la zygotie reste inconnue. Cette étude a révélé 19 nouveaux polymorphismes des « boîtes Rhésus » et a permis aussi de décrire trois nouveaux allèles RHD: RHD(Trp185Stop), RHD(Ala176Thr) et RHD(Ile342Ile). Cette étude a également mis en valeur l’hétérogénéité des « boîtes Rhésus » et la complexité des allèles RHD en décrivant les nouveaux polymorphismes obtenus, ce qui met en évidence les limites des approches moléculaires de la détermination de la zygotie. La Q-PCR a été la méthode la plus adaptée à la détermination de la zygotie, mais en raison des contraintes économiques locales, la PCR-RFLP pourrait être une alternative malgré l’hétérogénéité des « boîtes Rhésus » et la complexité du gène RHD. / Determination of paternal RHD zygosity can help the clinician to assess the risk of HDN. It was determined initially by both assignment of the most probable genotype and PCR-SSP. The prediction of zygosity based on the most probable genotype was not reliable due to the possibility of other genotypes for the same phenotype. In fact, the frequencies of R0 and r haplotypes in the Tunisian population are approached and make the deduction of the most probable genotype very aleatory. Secondly, the evaluation of the most convenient molecular method for RHD zygosity determination was realized by comparison of three molecular techniques (PCR-SSP, PCR-RFLP and RQ-PCR) and analysis of discordant results by sequencing of the RHD gene and Rhesus boxes. Analysis of 370 RH:1 samples by these three molecular tests showed concordant results in 81.9% and discordant results in 18.1%. Molecular investigations revealed that our cohort consists of 193 dizygous and 145 hemizygous samples and 32 which zygosity remains unknown. This study revealed 19 novel Rhesus boxes polymorphisms, and described 3 novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile). This study also underlined Rhesus boxes heterogeneity and RHD alleles complexity by describing of new polymorphisms which showed the limits of molecular approaches for RHD zygosity determination. RQ-PCR is the most convenient method for first intension paternal RHD zygosity determination in Tunisians. However taking into account local economic constraints PCR-RFLP could be an alternative despite the Rhesus boxes heterogeneity and RHD complexity.

A aplicação dos métodos de biologia molecular tem acrescentado benefícios aos métodos convencionais de diagnóstico do grupo sangüíneo Rhesus (Rh). Alguns estudos evidenciaram a superioridade prática da genotipagem RhD por reação em cadeia da polimerase (PCR) em relação às técnicas de identificação do fenótipo dos antígenos do grupo Rh obtidas pela hemaglutinação. Esta análise molecular tem sido realizada utilizando-se várias seqüências cromossômicas e diferentes tipos de técnicas. O grupo Rh contém dois genes homólogos, um codificando o antígeno D e o outro os antígenos C/c e E/e. Os objetivos deste projeto foram: (1) Detectar a presença de seqüência RhD específica dos indivíduos Rh positivo; (2) Comparar a presença/ausência destas seqüências com o grupo sanguíneo detectado pela hemaglutinação e calcular a sensibilidade e a especificidade da PCR. Para cumprir estes objetivos, foram analisadas amostras de DNA extraídas de sangue periférico de 23 indivíduos (4 homens e 19 mulheres) Rh positivo (11) e negativo (12) para amplificação de seqüências dos genes RhD e RhCE utilizando a técnica de PCR convencional. Nestas amostras, a comparação dos resultados da PCR com os da hemaglutinação demonstrou total concordância entre os testes. A sensibilidade do método foi avaliada pela realização da PCR em amostras de sangue Rh positivo diluídas em água e em sangue Rh negativo, amplificando o DNA na concentração de até 4 pg/l. Estes resultados indicaram que a técnica de PCR mostrou-se efetiva no diagnóstico da genotipagem do fator Rh, vislumbrando-se a possibilidade de sua utilização em outros tecidos de origem êmbrio-fetal para orientação de diagnóstico fetal invasivo e do uso profilático da imunoglobulina anti-D apenas nos casos de incompatibilidade Rh materno-fetal. Adicionalmente, indicaram que havendo melhora da sensibilidade desta técnica será possível detectar quantidades objetivamente menores de DNA fetal na circulação materna, fundamentando um importante passo rumo ao diagnóstico do fator Rh fetal por técnica não invasiva. / Molecular biology techniques have added some advantages to conventional diagnosis of the Rhesus (Rh) blood group. Many researches have demonstrated the practical superiority of RhD genotyping using polymerase chain reaction (PCR) over phenotypic identification tests obtained by hemagglutination. The use of different kinds of molecular analysis techniques and genetic sequences has been described. The Rh blood group contains two homologous genes, one encoding the D antigen and the other one coding C/c and E/e antigens. This study objectives were: (1) Detect the RhD sequence specific for the Rh positive individuals; (2) Compare the presence/absence of these sequences with the blood group identified by hemagglutination to calculate PCRs sensitivity and specificity rates. To accomplish these objectives, DNA extracted from venous blood of 23 individuals (4 men and 19 women), Rh positive (11) and negative (12), were analyzed using conventional PCR to amplify RhD and RhCE gene sequences. The comparison of PCR with hemagglutination results has shown total agreement. The sensitivity of this PCR method was evaluated using progressive dilutions of Rh positive samples on water and also on Rh negative samples, which demonstrated successful amplification of until 4 pg/l DNA concentration. These results have indicated that PCR was effective for the RhD genotyping, foreseeing the possibility of its utilization with other embryofetal tissues for invasive diagnosis orientation and anti-D immunoglobulin use only in cases of maternal-fetal incompatibility. Also, with an increase of this techniques sensitivity, fewer DNA amounts could be detected, which will certainly be an important step towards noninvasive fetal RhD diagnosis.

A aplicação dos métodos de biologia molecular tem acrescentado benefícios aos métodos convencionais de diagnóstico do grupo sangüíneo Rhesus (Rh). Alguns estudos evidenciaram a superioridade prática da genotipagem RhD por reação em cadeia da polimerase (PCR) em relação às técnicas de identificação do fenótipo dos antígenos do grupo Rh obtidas pela hemaglutinação. Esta análise molecular tem sido realizada utilizando-se várias seqüências cromossômicas e diferentes tipos de técnicas. O grupo Rh contém dois genes homólogos, um codificando o antígeno D e o outro os antígenos C/c e E/e. Os objetivos deste projeto foram: (1) Detectar a presença de seqüência RhD específica dos indivíduos Rh positivo; (2) Comparar a presença/ausência destas seqüências com o grupo sanguíneo detectado pela hemaglutinação e calcular a sensibilidade e a especificidade da PCR. Para cumprir estes objetivos, foram analisadas amostras de DNA extraídas de sangue periférico de 23 indivíduos (4 homens e 19 mulheres) Rh positivo (11) e negativo (12) para amplificação de seqüências dos genes RhD e RhCE utilizando a técnica de PCR convencional. Nestas amostras, a comparação dos resultados da PCR com os da hemaglutinação demonstrou total concordância entre os testes. A sensibilidade do método foi avaliada pela realização da PCR em amostras de sangue Rh positivo diluídas em água e em sangue Rh negativo, amplificando o DNA na concentração de até 4 pg/l. Estes resultados indicaram que a técnica de PCR mostrou-se efetiva no diagnóstico da genotipagem do fator Rh, vislumbrando-se a possibilidade de sua utilização em outros tecidos de origem êmbrio-fetal para orientação de diagnóstico fetal invasivo e do uso profilático da imunoglobulina anti-D apenas nos casos de incompatibilidade Rh materno-fetal. Adicionalmente, indicaram que havendo melhora da sensibilidade desta técnica será possível detectar quantidades objetivamente menores de DNA fetal na circulação materna, fundamentando um importante passo rumo ao diagnóstico do fator Rh fetal por técnica não invasiva. / Molecular biology techniques have added some advantages to conventional diagnosis of the Rhesus (Rh) blood group. Many researches have demonstrated the practical superiority of RhD genotyping using polymerase chain reaction (PCR) over phenotypic identification tests obtained by hemagglutination. The use of different kinds of molecular analysis techniques and genetic sequences has been described. The Rh blood group contains two homologous genes, one encoding the D antigen and the other one coding C/c and E/e antigens. This study objectives were: (1) Detect the RhD sequence specific for the Rh positive individuals; (2) Compare the presence/absence of these sequences with the blood group identified by hemagglutination to calculate PCRs sensitivity and specificity rates. To accomplish these objectives, DNA extracted from venous blood of 23 individuals (4 men and 19 women), Rh positive (11) and negative (12), were analyzed using conventional PCR to amplify RhD and RhCE gene sequences. The comparison of PCR with hemagglutination results has shown total agreement. The sensitivity of this PCR method was evaluated using progressive dilutions of Rh positive samples on water and also on Rh negative samples, which demonstrated successful amplification of until 4 pg/l DNA concentration. These results have indicated that PCR was effective for the RhD genotyping, foreseeing the possibility of its utilization with other embryofetal tissues for invasive diagnosis orientation and anti-D immunoglobulin use only in cases of maternal-fetal incompatibility. Also, with an increase of this techniques sensitivity, fewer DNA amounts could be detected, which will certainly be an important step towards noninvasive fetal RhD diagnosis.

Rh-blodgruppssystemet är mycket komplext med 54 blodgruppsantigen som kodas av två nära varandra belägna gener på kromosom 1 – RHD och RHCE. RHD-genen kodar för RhD-proteinet, ett membranbundet protein på erytrocyter vars antigen utgör de kliniskt viktigaste och mest immunogena efter ABOsystemets, och som kan ge upphov till transfusionskomplikationer och hemolytisk sjukdom hos foster och nyfödda. Vissa individer har varianter av RhD-protein som uttrycks svagare än normalt (”svaga D”), eller där vissa epitoper saknas (”partiella D”), och för vilka serologiska metoder inte kan ge enhetliga resultat. Detta orsakar problem vid blodtransfusion, graviditet och bloddonation, och leder ofta till användning av det redan knappa lagret av RhD-negativa blodenheter för att skydda patienten. I detta projekt har åtta prover med svaga RhD-antigenuttryck sekvenserats med avseende på RHD-genen i syfte att fastställa individernas RhDfenotyp. I sex av proverna hittades sex nukleotidpolymorfismer och två deletioner, som alla är sällsynta men dock är kända sedan tidigare. I två prover kunde inga mutationer i exon eller intilliggande intron påvisas som förklaring till de svaga uttrycken av RhD hos dessa individer. / The Rh blood group system is very complex with 54 blood group antigens encoded by two adjacent genes on chromosome 1 – RHD and RHCE. The RHD gene encodes the RhD protein, a membrane bound protein on erythrocytes whose antigens are the most clinically important and immunogenic after those of the ABO system, and which can result in transfusion complications and haemolytic disease of the fetus and newborn. Some individuals have variants of the RhD proteins that are expressed more weakly than normal (“weak D”), or have some of the epitopes missing (“partial D”), and for which serological methods cannot give a uniform result. This provides a problem in blood transfusion, pregnancy, and blood donation, and often results in the use of the already sparse supply of RhDnegative blood units for the safety of the patient. In this project, eight samples with weak RhD antigen expression have been sequenced with regard to the RHD gene in order to determine the RhD phenotype of the individuals. In six of the samples, six single nucleotide polymorphisms and two deletions were found, all of which are rare but are previously known. For two of the samples, no mutations in exons or adjacent introns could be detected to explain the weak expression of RhD in those individuals.

No description available.

There remain inconsistencies in the literature concerning the intelligence of subjects infected with latent toxoplasmosis. The main goals of this work are to find out whether, a) latent toxoplasmosis influences the intelligence of infected subjects and b) whether Rh negative and Rh positive subjects respond to the infection in different ways. In this work we used a complex test of intelligence, The Structure Intelligence Test I-S-T 2000 R. We were able to statistically control for the confounding variable the size of the place of residence in childhood. This had benefits when compared to previous studies. The differences in intelligence were measured in students of The Faculty of Science. The sample used included 46 toxoplasma-infected and 188 toxoplasma-free individuals. Using nonparametric tests we found lower numerical, fluid and general intelligence in toxoplasma-infected subjects compared to noninfected subjects. In addition, these tendencies were also observed in the results of parametric tests. Further to these components of intelligence there was also found to be a lower component of numerical knowledge in toxoplasma-infected males. By contrast, no differences between infected and noninfected individuals was found to occur in the female test subjects. No statistically significant...

Health problems of the human beings in a society are one of the main components of the social security systems due to the dimension of the financial burden it might bring on individuals, employers, insurance companies and governments. Morbidity measures, such as incidence and prevalence of a specific disease in a certain population enable researchers to estimate for individuals the probability of being diagnosed or being prone to the diseases. This information is usually not tractable because of the non-availability of the convenient data or recordings for many countries as well as Turkey. Even if it is available, it is commonly limited with largely varying characteristics about the type and coverage of the diseases. In this regard, the pattern that a population follows for an acute disease may not be the same for chronic diseases. Having those indicators determined for a group of insureds will enable underwriters to have more profitable and economical premium calculation and precision on required reserve estimation. v Based on their characteristics such as acute or chronic behaviour, the gender, and the location of residency of people, the diseases show different behaviour on their occurrences. From the insurer