Frequency of the DI*A, DI*B and Band 3 Memphis polymorphism among distinct groups in Brazil

ABSTRACT Introduction: The Band 3 is a red blood cell protein that carries the Dia and Dib antigens from the Diego blood system. The SLC4A1 gene encodes Band 3; Band 3 Memphis is a polymorphism of normal Band 3 and has two variants, but only the variant II carries the Dia antigen. Objectives: Describe the frequencies of the DI*A and DI*B alleles and the Band 3 Memphis among blood donors, sickle cell disease (SCD) patients and Amazonian Indians. Methods: A total of 427 blood samples were collected and separated into three groups: 206 unrelated blood donors, 90 patients with SCD and 131 Amazonian Indians. We performed DI*A/B, normal Band 3 and Band 3 Memphis genotyping, using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP). Results: The frequency of the DI*A/DI*A genotype was 0.5% in blood donors and it was not found in other groups. The frequency of the DI*A/DI*B was higher in Amazonian Indians (33.6%) and the frequency of the DI*B/DI*B was highest in blood donors (92.2%). All 105 individuals tested were positive for the presence of normal Band 3 and of these individuals, only 5/105 (4.8%) presented the Band 3 Memphis mutation. Conclusion: We observed a higher frequency of the DI*B allele in blood donors and a low frequency of the DI*A/DI*A genotype in all groups studied. The Band 3 Memphis was found in a higher frequency in the blood donor group. Our findings highlight the importance of analyzing different population groups to gain a better understanding of the genetic association of blood group antigens.

Frequency of the DI*A, DI*B and Band 3 Memphis polymorphism among distinct groups in Brazil.

INTRODUCTION: The Band 3 is a red blood cell protein that carries the Dia and Dib antigens from the Diego blood system. The SLC4A1 gene encodes Band 3; Band 3 Memphis is a polymorphism of normal Band 3 and has two variants, but only the variant II carries the Dia antigen. OBJECTIVES: Describe the frequencies of the DI*A and DI*B alleles and the Band 3 Memphis among blood donors, sickle cell disease (SCD) patients and Amazonian Indians. METHODS: A total of 427 blood samples were collected and separated into three groups: 206 unrelated blood donors, 90 patients with SCD and 131 Amazonian Indians. We performed DI*A/B, normal Band 3 and Band 3 Memphis genotyping, using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP). RESULTS: The frequency of the DI*A/DI*A genotype was 0.5% in blood donors and it was not found in other groups. The frequency of the DI*A/DI*B was higher in Amazonian Indians (33.6%) and the frequency of the DI*B/DI*B was highest in blood donors (92.2%). All 105 individuals tested were positive for the presence of normal Band 3 and of these individuals, only 5/105 (4.8%) presented the Band 3 Memphis mutation. CONCLUSION: We observed a higher frequency of the DI*B allele in blood donors and a low frequency of the DI*A/DI*A genotype in all groups studied. The Band 3 Memphis was found in a higher frequency in the blood donor group. Our findings highlight the importance of analyzing different population groups to gain a better understanding of the genetic association of blood group antigens.

Application of blood group genotyping in complex cases of immunohematology.

BACKGROUND: Red blood cell (RBC) group systems are depicted by antigens on the surface of RBCs, which when transfused to a recipient that lacks them, can result in alloimmunization. Thus, transfusion of matched RBC components to the recipient is recommended, especially for the more immunogenic blood group antigens, such as Rh (E, e, C, and c), Kell, Kidd, Duffy, and MNS. AIMS: The aim of this study was to perform the blood group genotyping from blood samples of 12 polytransfused patients whose phenotyping was inconclusive or incomplete. METHODS: The amplicons were amplified by polymerase chain reaction-sequence-specific primers for the following alleles: RHCE (RHCE * C, RHCE * c, RHCE * E, and RHCE * e), KEL (KEL * 01 and KEL * 02), FY (FY * 01 and FY * 02), and KID (JK * 01 and JK * 02), in addition to the GATA1-mutated gene (FY * 02N.01). RESULTS: Discrepancies were found in the Rh (E) and Kidd systems, in addition to cases of Fyb antigen silencing attributed to the GATA mutation, which was present in all individuals with Fy (a-b-) phenotype. The technique also solved the inconclusive phenotyping caused by mixed-field agglutination. CONCLUSION: The results show the contribution of blood group genotyping in complex immunohematology cases, optimizing the delivery of RBC components suitable for transfusion safety, and expanding the number of compatible donors for patients with the Fy (a-b) phenotype related to the FY (02N.01) allele.

Molecular matching for patients with haematological diseases expressing altered RHD-RHCE genotypes.

BACKGROUND AND OBJECTIVES: The high homology and the inverted orientation of RHD and RHCE may give rise to non-functional and aberrant RH alleles. RH genotyping is used to screen RH matched donors to African descent patients. This study aimed to define a strategy for testing RHD and RHCE variants in blood donors to provide compatible units for transfusion of patients with haematological diseases. MATERIALS AND METHODS: Samples from 132 patients [101 Sickle cell disease (SCD), 14 myelodysplastic syndrome (MDS), 17 acute myelogenous leukaemia (AML)] and 198 Brazilian donors were studied. Major blood group alleles, RHD, RHCE alleles and RHD zygosity were determined by the blood-MLPA assay. Sequencing was performed to determine RHD and RHCE variant subtypes. A match was an RH genotype that did not encode Rh antigens absent in the patient, along with matching for ABO, MNS, KEL, FY, JK and DI antigens. RESULTS: Overall, 7·6% of blood donors and 17.4% of patients presented RH genotypes that predict expression of partial Rh antigens or lack of high prevalence Rh antigens. From 23 patients with clinically relevant RH genotypes, 15 had available matched donors. CONCLUSION: We report the presence of clinically relevant RH genotypes in SCD and in non-SCD patients. In our admixed population, many patients carry variant RHCE alleles in heterozygosity with normal RHCE alleles. Thus, our results suggest that donors could be selected based on the normal RH allele.

Influência do tempo de centrifugação em testes de coagulação: tempo de protrombina e tempo de tromboplastina parcial / Influence of the centrifuge time in coagulation tests: prothrombin time and partial thromboplastin time

Objetivo: Analisar os testes de coagulação: tempo de protrombina (TP) e tempo de tromboplastina parcial (TTP) em diferentes tempos de centrifugação da amostra da biológica, com relação ao protocolo padrão do Clinical Laboratory Standards Institute (CLSI). Métodos: As amostras foram divididas em cinco alíquotas de 1 mL. Foi realizada a centrifugação em 15, 10, 5, 2 e 1 minuto, com velocidade de 1500 g. O TP e TTP foram imediatamente analisados em aparelho automatizado. Os plasmas foram analisados para presença de elementos residuais: eritrócitos, leucócitos e plaquetas. Resultados: Observou-se alteração dos valores do TP nos tempos de centrifugação 10, 5, 2 e 1 minuto e do TTP em 5, 2 e 1 minuto, com relação ao protocolo padrão. Na interpretação de Bland Altman, observou-se um viés significativo do limite clínico aceitável para o TP e para o TTP em todos os tempos de centrifugação, com relação ao protocolo padrão. Apenas no tempo de centrifugação de 15 minutos não foram encontradas células residuais nas amostras analisadas. Conclusão: O tempo de centrifugação de 15 minutos é o ideal para remoção completa das células sanguíneas residuais e para garantia da confiabilidade dos resultados dos testes de coagulação TP e TTP.

Objective: To analyze the coagulation tests: prothrombin test (PT) and partial thromboplastin time (PTT) in different centrifugation times of the sample, in relation to the standard protocol of the Clinical Laboratory Standards Institute (CLSI). Methods: The selected samples were splitted up into five aliquots of 1 mL. Centrifugation of these aliquots was carried out at 15, 10, 5, 2 and 1 minute at 1500 g. The PT and PTT were analyzed in an automated apparatus. The plasmas were analyzed for presence of residual elements: erythrocytes, leukocytes and platelets. Results: The results showed a change in the values of PT at the 10, 5, 2 and 1 minute centrifugation times and the PTT at 5, 2 and 1 minutes, relative to the standard protocol. In the interpretation of Bland Altman, a significant bias of the acceptable clinical limit for TP and TTP at all centrifugation times was observed, relative to the standard protocol. Only in the 15 minute centrifugation time no residual cells were found in the analyzed samples. Conclusion: The present study demonstrated that the 15-minute centrifugation time is ideal for complete removal of residual blood cells and to ensure the reliability of the results of the PT and PTT coagulation

Análise comparativa de contagens de plaquetas entre metodologias de impedância e óptica em amostras de sangue de indivíduos hospitalizados / Comparative analysis of platelet count between electrical impedance and optical platelet count methods in blood samples from hospitalized individuals

Objetivo: Comparar resultados de contagens plaquetárias de indivíduos hospitalizados realizadas por impedância (PLT-I) e metodologia óptica fluorescente (PLT-O). Métodos: Em estudo retrospectivo, foram avaliados dados sequenciais arquivados de contagens plaquetárias de trezentos indivíduos adultos hospitalizados, incluindo casos de anemias microcíticas e hemolíticas, neoplasias hematológicas, entre outras doenças. Todos os casos continham contagens de plaquetas PLT-I e PLT-O realizadas no equipamento Sysmex XE-5000. Resultados: Não houve diferença significativa entre os valores de contagens plaquetárias entre a PLT-I e PLT-O (p=0,614). Quando avaliamos os valores de plaquetas entre diferentes grupos em relação às metodologias, não houve diferença entre as contagens plaquetárias naqueles com VCM abaixo de 80 fL (p=0,936), VCM abaixo de 70 (p=0,821), plaquetas abaixo de 100×109/L (p=0,369) e plaquetas abaixo de 50×109/L (p=0,314). Além disso, a correlação entre PLT-I e PLT-O foi forte. Conclusão: Os valores de contagens plaquetárias, provenientes de pacientes não saudáveis, realizadas no analisador XE-5000 pelos métodos óptico e impedância, mostraram forte correlação e boa concordância.