Frequency of Wra antigen and anti-Wra in Brazilian blood donors

AbstractBackgroundWra is a low-incidence antigen, which is antithetical to the high prevalence red blood cell antigen, Wrb. Anti-Wra is a naturally occurring antibody that is found in approximately 1–2% of blood donors. The aim of this study was to determine the frequency of Wra and anti-Wra in Brazilian blood donors.MethodsA total of 1662 Brazilian blood donors were molecularly analyzed using the SNaPshot methodology to determine the WR*A/B alleles and to predict the frequency of the Wra antigen. To detect the anti-Wra, samples from 1049 blood donors were analyzed using a gel test with Wr(a+) red blood cells. The serum was treated with dithiothreitol (DTT) to determine the immunoglobulin classes. Immunoglobulin (Ig)-G isotype classification was performed in a gel test using the IgG1/IgG3 card. A monocyte monolayer assay was employed to predict the clinical significance of IgG anti-Wra.ResultsOf the 1662 donors, only one sample had the DI*02.03 allele in heterozygous predicting the Wr(a+b+) phenotype. Anti-Wra was detected in 34 (3.24%) samples, 64.7% in females and 35.3% in males. Regarding the immunoglobulin class, eight (23.5%) cases of anti-Wra were classified as IgG and 26 (76.5%) as IgM. Of the eight cases of IgG anti-Wra, four were IgG1, two were IgG3 and three anti-Wra were not IgG3 or IgG1, and thus probably IgG2 or IgG4. The results of the monocyte monolayer assay showed that IgG anti-Wra might be of clinical significance.ConclusionThis study shows a very low frequency (0.06%) of the Wra antigen in Brazilian blood donors. Additionally, it shows that the frequency of anti-Wra in this population is higher than previously reported

A New Strategy To Identify Rare Blood Donors: Single Polymerase Chain Reaction Multiplex Snapshot Reaction For Detection Of 16 Blood Group Alleles.

As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood.12 Suppl 1s256-6

Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries

Conventional karyotyping detects anomalies in 3-15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cutoff for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams-Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome. (C) 2011 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CEPID (Centro de Pesquisa, Inovacao e Difusao)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Ctr Estudos Genoma Humano, BR-05508900 São Paulo, BrazilUniv São Paulo, Fac Med, Dept Oncol, BR-05508 São Paulo, BrazilUniv Fed Campina Grande, Campina Grande, PB, BrazilUniversidade Federal de São Paulo, Dept Ginecol, Lab Ginecol Mol, São Paulo, BrazilAssoc Beneficente Coleta Sangue, São Paulo, BrazilUniv São Paulo, Fac Med, Inst Crianca, BR-05508 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ginecol, Lab Ginecol Mol, São Paulo, BrazilWeb of Scienc

Increasing rate of anti-SARS-CoV-2 antibodies between the first and second waves of COVID-19 in São Paulo, Brazil: A cross-sectional blood donors-based study

Background: SARS-CoV-2 infections rapidly spread along with Brazilian territory with heterogeneous transmission and mortality rates, mostly depending on region and period. Investigation of SARS-CoV-2 antibodies is an important tool to understand virus circulation. Given that blood donors are a representative casuistic of a healthy population, the authors evaluated the seroprevalence of IgG and IgM COVID-19 antibodies in 2,806 blood donors from a blood bank located in São Paulo, Brazil. Methods: Aiming to evaluate viral behavior over time, the authors selected samples from blood donors who donated in June and October 2020, and February 2021. To determine whether socio-demographic features affected the seroprevalence, the authors analyzed samples from three different regions from São Paulo (capital, metropolitan and countryside regions) and evaluated predictors as gender, age, educational level, race, and use of public transportation. Results: As expected, the authors observed that seroprevalence increased over time. Seroprevalence was greater in São Paulo city compared to metropolitan and countryside regions, being smallest in the countryside. Characteristics associated with a lower percentage of antibodies were age above 50 years, higher educational level, self-declared Caucasian, and use of individual transportation. Conclusion: In conclusion, blood donors' samples proved to accurately reflect virus circulation in the healthy population

An easy and efficient strategy for KEL genotyping in a multiethnic population

BACKGROUND: The Kell blood group system expresses high and low frequency antigens with the most important in relation to transfusion including the antithetic KEL1 and KEL2; KEL3 and KEL4; KEL6 and KEL7 antigens. Kell is a clinically relevant system, as it is highly immunogenic and anti-KEL antibodies are associated with hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Although required in some situations, Kell antigen phenotyping is restricted due to technical limitations. In these cases, molecular approaches maybe a solution. This study proposes three polymerase chain reaction genotyping protocols to analyze the single nucleotide polymorphisms responsible for six Kell antithetic antigens expressed in a Brazilian population. METHODS: DNA was extracted from 800 blood donor samples and three polymerase chain reaction-restriction fragment length polymorphism protocols were used to genotype the KEL*1/KEL*2, KEL*3/KEL*4 and KEL*6/KEL*7 alleles. KEL*3/KEL*4 and KEL*6/KEL*7 genotyping was standardized using the NlaIII and MnlI restriction enzymes and validated using sequencing. KEL*1/KEL*2 genotyping was performed using a previously reported assay. RESULTS: KEL genotyping was successfully implemented in the service; the following distribution of KEL alleles was obtained for a population from southeastern Brazil: KEL*1 (2.2%), KEL*2 (97.8%), KEL*3 (0.69%), KEL*4 (99.31%), KEL*6 (2.69%) and KEL*7 (97.31%). Additionally, two individuals with rare genotypes, KEL*1/KEL*1 and KEL*3/KEL*3, were identified. CONCLUSION: KEL allele genotyping using these methods proved to be reliable and applicable to predict Kell antigen expressions in a Brazilian cohort. This easy and efficient strategy can be employed to provide safer transfusions and to help in rare donor screening.9910

Antibody indexes in COVID-19 convalescent plasma donors: Unanswered questions

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by high contagiousness, as well as variable clinical manifestations and immune responses. The antibody response to SARS-CoV-2 is directly related to viral clearance and the antibodies’ ability to neutralize the virus and confer long-term immunity. Nevertheless, the response can also be associated with disease severity and evolution. This study correlated the clinical characteristics of convalescent COVID-19 patients with immunoglobulin A (IgA) and IgG anti-SARS-CoV-2 antibodies. METHODS: This study included 51 COVID-19 health care professionals who were candidates for convalescent plasma donation from April to June 2020. The subjects had symptomatic COVID-19 with a polymerase chain reaction-confirmed diagnosis. We measured anti-SARS-CoV-2 IgA and IgG antibodies after symptom recovery, and the subjects were classified as having mild, moderate, or severe symptoms. RESULTS: Anti-SARS-CoV-2 antibodies were positive in most patients (90.2%). The antibody indexes for IgA and IgG did not differ significantly between patients presenting with mild or moderate symptoms. However, they were significantly higher in patients with severe symptoms. CONCLUSIONS: Our study showed an association between higher antibody indexes and severe COVID-19 cases, and several hypotheses regarding the association of the antibody dynamics and severity of the disease in SARSCoV-2 infection have been raised, although many questions remain unanswered

Altered of apoptotic markers of both extrinsic and intrinsic pathways induced by hepatitis C virus infection in peripheral blood mononuclear cells

Background: Chronic hepatitis C (CHC) has emerged as a leading cause of cirrhosis in the U. S. and across the world. To understand the role of apoptotic pathways in hepatitis C virus (HCV) infection, we studied the mRNA and protein expression patterns of apoptosis-related genes in peripheral blood mononuclear cells (PBMC) obtained from patients with HCV infection.Methods: the present study included 50 subjects which plasma samples were positive for HCV, but negative for human immunodeficiency virus (HIV) or hepatitis B virus (HBV). These cases were divided into four groups according to METAVIR, a score-based analysis which helps to interpret a liver biopsy according to the degree of inflammation and fibrosis. mRNA expression of the studied genes were analyzed by reverse transcription of quantitative polymerase chain reaction (RT-qPCR) and protein levels, analyzed by ELISA, was also conducted. HCV genotyping was also determined.Results: HCV infection increased mRNA expression and protein synthesis of caspase 8 in group 1 by 3 fold and 4 fold, respectively (p < 0.05). in group 4 HCV infection increased mRNA expression and protein synthesis of caspase 9 by 2 fold and 1,5 fold, respectively (p < 0.05). Also, caspase 3 mRNA expression and protein synthesis had level augumented by HCV infection in group 1 by 4 fold and 5 fold, respectively, and in group 4 by 6 fold and 7 fold, respectively (p < 0.05).Conclusions: HCV induces alteration at both genomic and protein levels of apoptosis markers involved with extrinsic and intrinsic pathways.Associacao Beneficente de Coleta de Sangue (COLSAN)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Financiadora de Estudos e Projetos (FINEP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Colsan Assoc Beneficente Coleta Sangue, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Gynecol, São Paulo, BrazilURDIP, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Nephrol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Gynecol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Nephrol, São Paulo, BrazilWeb of Scienc

Genes ABI3 (GLA Member Interactor-3) and ABI3BP (ABI3-Binding Protein): investigation of expression in thyroid lesions and functional analysis

We have recently demonstrated that loss of expression of the ABI3BP (ABI3-binding protein) gene is common to all thyroid carcinomas, and that ABI3BP re-expression altered tumor growth by increasing senescence and inhibiting invasion and migration. ABI3 is a potential binding partner of ABI3BP and it has been shown that ABI3 forced expression reduces motility and metastatic dissemination of malignant cells. In this study we investigated ABI3 expression in thyroid lesions. We observed that ABI3 expression is reduced or lost in most carcinomas and that there is a positive correlation between ABI3 and ABI3BP expression. The role of ABI3 was further explored by stable transfection of two carcinoma cell lines with full-length ABI3 cDNA. We found that ABI3 decreases transforming activity, cell proliferation and cell cycle progression while increasing cell senescence correlated with increased p21 expression and reduced ERK phosphorylation and E2F1 expression. Moreover, ectopic expression reduces cell viability, cell invasion and migration. In rodent models, ABI3 expression was sufficient to reduce tumor growth. Here, we suggest that ABI3 loss of expression, similar to ABI3BP loss of expression, is an important event in thyroid carcinoma. This work establishes a functional role for ABI3 in thyroid cancers and suggests that targeting ABI3 or its pathway might be a strategy for the treatment of these cancers.Utilizando a técnica de SAGE (do inglês, Serial Analysis of Gene Expression), verificamos que o transcrito do gene ABI3BP (ABI3-Binding Protein) encontra-se presente nas bibliotecas SAGE geradas a partir de tiróide normal e de adenoma folicular da tireóide e ausente na biblioteca derivada de carcinoma folicular da tiróide. Estes achados sugerem que a perda ou diminuição da expressão do gene ABI3BP pode estar associada à patogênese dos carcinomas foliculares da tiróide. Recentemente, utilizando a técnica de microarray, um grupo descreveu a perda da expressão de ABI3BP em diferentes subtipos de carcinoma, incluindo tiróide e, portanto, corroborando nossos dados iniciais. Sendo incógnito o papel de ABI3BP na patogênese dos diferentes subtipos tumorais, tivemos como objetivo inicial à caracterização da função deste gene. Para isso, o cDNA completo de ABI3BP foi clonado em vetor de expressão e transfectado de forma permanente em linhagens de carcinoma da tiróide onde este gene não se expressa. Paralelamente utilizamos um vetor de expressão vazio, como controle negativo. Os clones selecionados, expressando o gene alvo e controle, foram posteriormente avaliados quanto à capacidade de crescimento in vitro e in vivo, invasividade, migração, apoptose, senescência e progressão no ciclo celular. A re-expressão de ABI3BP foi capaz de inibir o crescimento in vivo e in vitro, diminuir a viabilidade celular, induzir apoptose e principalmente senescência, induzir uma parada no ciclo celular em G1, de modular a expressão de genes associados ao processo de senescência e ao ciclo celular e atenuar a invasividade e a migração. Além disso, demonstramos que a perda de ABI3BP não é restrita ao carcinoma folicular, sendo observada também nos outros subtipos de carcinoma da tiróide. Visto que ABI3BP originalmente foi identificado, por meio da metodologia de duplo híbrido em levedura, como uma proteína que interage com o domínio SH3 de ABI3, nossa hipótese é de que ABI3 estaria, semelhante a ABI3BP, envolvido na patogênese dos carcinomas da tiróide. Inicialmente investigamos a expressão de ABI3 nos diferentes tumores da tiróide. Verificamos que a expressão de ABI3 está diminuída nos carcinomas, quando comparados com lesões benignas e tecido de tiróide normal. Além disso, observamos uma correlação entre a perda da expressão de ABI3 e ABI3BP. A caracterização funcional de ABI3, utilizando o modelo e as metodologias descritas para ABI3BP, demonstrou que a re-expressão de ABI3 foi capaz de inibir o crescimento in vivo e in vitro, diminuir a viabilidade celular, aumentar a senescência, atenuar a invasividade e a migração, induzir uma parada no ciclo celular em G1 e de modular a expressão de genes associados ao ciclo celular. Nossos dados sugerem que os dois genes estão envolvidos na patogênese do câncer e que podem atuar em uma via comum, bem como, sugerem que podem servir como futuros alvos para a terapêutica.TEDEBV UNIFESP: Teses e dissertaçõe

Prevalence of IFNL3 gene polymorphism among blood donors and its relation to genomic profile of ancestry in Brazil

The recent development of interferon-free regimens based on direct-acting antivirals for the treatment of chronic hepatitis C virus infection has benefited many but not all patients. Some patients still experience treatment failure, possibly attributed to unknown host and viral factors, such as IFNL3 gene polymorphism. The present study assessed the prevalence of rs12979860-CC, rs12979860-CT, and rs12979860-TT genotypes of the IFNL3 gene, and its relationship with ancestry informative markers in 949 adult Brazilian healthy blood donors. Race was analyzed using ancestry informative markers as a surrogate for ancestry. IFNL3 gene was genotyped using the ABI TaqMan single nucleotide polymorphisms genotyping assays. The overall frequency of rs12979860-CC genotype was 36.9%. The contribution of African ancestry was significantly higher among donors from the northeast region in relation to southeast donors, whereas the influence of European ancestry was significantly higher in southeast donors. Donors with rs12979860-CC and rs12979860-CT genotypes had similar ancestry background. The contribution of African ancestry was higher among rs12979860-TT genotype donors in comparison to both rs12979860-CC and rs12979860-CT genotypes. The prevalence of rs12979860-CC genotype is similar to that found in the US, despite the Brazilian ancestry informative markers admixture. However, in terms of ancestry, rs12979860-CT genotype was much closer to rs12979860-CC individuals than to rs12979860-TT. Keywords: Ancestry informative markers, Chronic hepatitis C virus infection, Direct-acting antivirals, Single nucleotide polymorphism