Magnetic bead technology for viral RNA extraction from serum in blood bank screening

Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. OBJECTIVE: Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. METHODS: Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. RESULTS: Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. CONCLUSION: The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Associação Beneficente de Coleta de SangueUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

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

Magnetic bead technology for viral RNA extraction from serum in blood bank screening

Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. OBJECTIVE: Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. METHODS: Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. RESULTS: Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. CONCLUSION: The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products