Enhanced liver fibrosis (ELF) score: Analytical performance and distribution range in a large cohort of blood donors.

BACKGROUND: The Enhanced Liver Fibrosis (ELF) is a serological score that includes hyaluronic acid (HA), tissue inhibitor of matrix metalloproteinases-1(TIMP-1), and aminoterminal propeptide of type III procollagen (PIIINP) and shows good performance for detecting liver fibrosis. There are few studies evaluating ELF's intra and inter-assay variation and stability of the samples. The influence of host variables, such as age, gender and body mass index (BMI) is also not well known. We determined ELF's analytical performance and possible influences of gender, age and BMI. METHODS: The study included 958 healthy blood donors evaluated for age, gender, and BMI. RESULTS: Mean ELF scores were significantly different between female (8.53±0.75) and male groups (8.76±0.76) and also according to age strata (p<0.001). For both genders, ELF significantly varied in individuals with BMI under 25 (p<0.001). Analytes remained stable after freezing/thawing cycles and intra- and inter-assay coefficients of variation were low. CONCLUSIONS: ELF has appropriate precision and is quite robust, due to the high stability of the analytes in fresh and frozen samples. ELF's results are influenced by gender, age and BMI which should be taken into account when analyzing its results.

Thrombophilic mutations and polymorphisms, alone or in combination, and recurrent spontaneous abortion.

Generally, recurrent spontaneous abortions (RSAs) have no identifiable cause; yet, vascular alterations during pregnancy may be associated with pregnancy loss. Therefore, we evaluated the association between thrombophilic mutations and RSAs. This case-control study was conducted in 112 patients who had RSAs and 98 health control women. Genomic DNA was extracted from whole blood, and polymorphism genotyping was conducted using polymerase chain reaction. The following 6 genetic variants were analyzed: factor V Leiden, prothrombin mutation, methylenetetrahydrofolate reductase C677T and A1298C, plasminogen activator inhibitor type 1 (4G>5G), and factor XIII G103T (V34L). No correlations were found in any of the investigated polymorphisms. Moreover, 35.0% of cases and 25.5% of controls had at least 2 mutations in combination, and 4.8% of cases and 5.1% of controls had 3, but these combinations were not associated with additional risk. In conclusion, we found no association between the polymorphisms studied and the occurrence of RSAs.

How do we identify RHD variants using a practical molecular approach?

Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encountered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using specific PCR-restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algorithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants.

A new strategy to identify rare blood donors: single polymerase chain reaction multiplex SNaPshot reaction for detection of 16 blood group alleles.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. DISCUSSION: 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.

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.

DARC (Duffy) and BCAM (Lutheran) reduced expression in thyroid cancer.

Duffy or DARC (Duffy Antigen Receptor for Chemokines) is a glycosylated membrane protein that selectively binds angiogenic chemokines. Previous in vivo and in vitro studies of DARC function in cancer have associated DARC over expression with better prognosis, decreased metastatic potential, and inhibition of tumor-associated neovascularization. Another carcinogenesis-associated antigen is Lutheran or BCAM (basal cell adhesion molecule), a surface glycoprotein that acts as a receptor for the extracellular matrix protein, laminin. BCAM is a protein related to tumor progression; and, its over expression is associated with skin, ovarian and pancreatic cancers. We explored DARC and BCAM functions and investigated whether or not their expressions were altered in thyroid cancer. The expression of DARC and BCAM were evaluated by quantitative real-time PCR (qPCR) in a set of 18 normal thyroid tissues (NT), 15 follicular adenomas (FTA), 17 follicular carcinomas (FTC), and 122 papillary thyroid carcinomas (PTC), including 78 classical (CVPTC) and 44 follicular variant (FVPTC). RNA was isolated, reverse transcribed to cDNA, and used in qPCR reactions containing SYBR Green. The relative expression value was calculated using ribosomal protein S8 as an internal control. When we compared benign (NT and FTA) versus malignant samples (FTC, CVPTC and FVPTC) we observed a significant decrease of DARC and BCAM relative expression in malignant cases. Additionally, we correlated clinic-pathological features (tumor size, presence of metastasis, presence of lymphocyte infiltrate) with DARC and BCAM expression. We found a diminished expression of DARC in PTC samples, which was correlated with tumor size and presence of a lymphocyte infiltrate. We, also, found a correlation between decreased BCAM expression and tumor size or presence of metastasis. DARC and BCAM expression was associated with pathogenesis of thyroid carcinoma and correlated with clinical-pathological features.

T-Cell Response to Hepatitis B Core Antigen: Identification of Prior Exposure to and Confirmatory Testing for Screening for Anti-HBc.

Background. During routine donor screening in the blood bank, it is not uncommon to find isolated reactivity for anti-HBc in the absence of detectable HBV DNA in a first donation but absence of reactivity to anti-HBc in subsequent donations, suggesting a false-positive result for anti-HBc. Study Design and Methods. The blood donor population was screened between January 2010 and October 2011. We selected 2,126 donations positive only for anti-HBc from a total of 125,068 donations. During the process, OBI donors were identified, and their HBcAg-specific T-cell response was analyzed and compared to donors with chronic (HBsAg positive) and recovered (anti-HBc only) infection. We analyzed correlations between signal levels (Co/s) in the competitive assay for anti-HBc and HBV DNA detection. Results. In the 21-month study period, 21 blood donors with anti-HBc alone were identified as OBI (1 in each 5955 donors). The relevant finding was the observation that anti-HBc only subjects with Co/s ≥ 0.1 did not have either HBcAg-specific T-cells or detectable HBV DNA and OBI subjects presented with Co/s ≤ 0.1 and HBcAg T-cell response. In the subset of 21 OBI subjects, 9 donors remained positive for HBcAg T-cell response after four collections. In all 9 samples, we observed HBV DNA fluctuation. Conclusion. Our data suggest that HBcAg-specific T-cell response could be used to confirm anti-HBc serological status, distinguishing previous exposure to Hepatitis B virus from anti-HBc false-positive results.

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.

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.

Investigation of pseudogenes RHDΨ and RHD-CE-D hybrid gene in D-negative blood donors by the real time PCR method.

INTRODUCTION: The Rh system is the most polymorphic and immunogenic of all systems of blood groups. Currently more than 49 antigens were identified with five major antigens D, C, c, E, e. Knowledge of the molecular basis of the Rh system permitted the understanding of both the mechanism of Rh phenotype on the antigen variants of RHD and RHCE In Caucasians the primary mechanism of D-negative phenotype is the complete deletion of RHD gene, while the black Africans is the presence of pseudogene and gene hybrid RHD-CE (3-7)-D. OBJECTIVE: To determine the prevalence gene pseudogene and hybrid gene and standardization of molecular techniques in method of Taqman on real-time PCR for RHD genotyping. PATIENTS AND METHODS: 203 samples of D-negative donor were used to establish and validate the effectiveness of RHD genotyping in real-time PCR using Taqman technology. The extraction was performed using a commercial kit QIAmp DNA mini kit. Samples exon 10 and 7 positive were submitted to amplification of exon 5, confirming the pseudogene RHDΨ, whereas exon 10+exon 7--for the hybrid gene (C) cdes and mutation C733G (Leu245Val) of the RHCE gene. RESULTS: Twenty-five (12.3%) samples were positive, 14 amplified for both exons 10 and 7 while in 11 only for the exon 10. When extended the screening using exon 10, 7 and 5, only 06 amplified. The pseudogene was present in 07 samples (3.5%) and the hybrid RHD-CE (3-7) in 04 (1.97%), while in 177 (87.2%) of Rh negative donors were RHD gene deletion. In 07 samples not amplified for exon 3 had mutated and the mutation C733G antigen. CONCLUSION: The prevalence of pseudogene was 3.5% and the gene hybrid RHD-CE of 1.9%. This approach for real-time PCR as a complementary tool is technically feasible and the results of this study helped develop a new strategy for RHD genotyping.

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.

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

UNLABELLED: 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.

Soluble uric acid increases intracellular calcium through an angiotensin II-dependent mechanism in immortalized human mesangial cells.

Hyperuricemia is associated with increases in cardiovascular risk and renal disease. Mesangial cells regulate glomerular filtration rates through the release of hormones and vasoactive substances. This study evaluates the signaling pathway of uric acid (UA) in immortalized human mesangial cells (ihMCs). To evaluate cell proliferation, ihMCs were exposed to UA (6-10 mg/dL) for 24-144 h. In further experiments, ihMCs were treated with UA (6-10 mg/dL) for 12 and 24 h simultaneously with losartan (10(-7) mmol/L). Angiotensin II (AII) and endothelin-1 (ET-1) were assessed using the enzyme-linked immunosorbent assay (ELISA) technique. Pre-pro-ET mRNA was evaluated by the real-time PCR technique. It was observed that soluble UA (8 and 10 mg/dL) stimulated cellular proliferation. UA (10 mg/dL) for 12 h significantly increased AII protein synthesis and ET-1 expression and protein production was increased after 24 h. Furthermore, UA increased [Ca(2+)](i), and this effect was significantly blocked when ihMCs were preincubated with losartan. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. In addition, UA can potentially affect glomerular function due to UA-induced proliferation and contraction of mesangial cells. The latter mechanism could be related to the long-term effects of UA on renal function and chronic kidney disease.

[The molecular basis of RH system and its applications in obstetrics and transfusion medicine]. / Bases moleculares do sistema Rh e suas aplicações em obstetrícia e medicina transfusional.

The Rh system is the most polymorphic and immunogenic for all blood group systems. Currently more than 49 antigens were identified with five major antigens D, C, c, E, e. Knowledge of the Rh system's molecular basis, since its first cloning 17 years ago, allowed to understand the mechanism of Rh-negative phenotype and the variants of antigens as RHD and RHCE. Deletions, gene rearrangements and insertions are the main mutations. In Caucasians the primary mechanism of Rh-negative phenotype is the complete RHD gene deletion, while in African descendants it is the presence of pseudogene and gene RHDψ hybrid RHD-CE (4-7)-D. The authors analyze the structure of the Rh complex in red cells, molecular basis of the Rh system, mechanisms of Negativity RHD and weak and incomplete expression of RHD.

Bases moleculares do sistema Rh e suas aplicações em obstetrícia e medicina transfusional / The molecular basis of RH system and its applications in obstetrics and transfusion medicine

O sistema Rh é o mais polimórfico e imunogênico de todos os sistemas de grupos sanguíneos. Atualmente mais de 49 antígenos foram identificados sendo cinco principais os antígenos D, C, c, E, e. O conhecimento das bases moleculares do sistema Rh desde a sua primeira clonagem há 17 anos possibilitou o entendimento tanto do mecanismo do fenótipo Rh negativo quanto das variantes dos antígenos RHD e RHCE. As deleções, rearranjos gênicos e as inserções são as principais mutações encontradas. Nos caucasianos, o mecanismo principal do fenótipo Rh negativo é a completa deleção do gene RHD, enquanto nos afrodescendentes é a presença do pseudogene RHDψ e do gene híbrido RHD-CE (4-7)-D. Os autores analisam a estrutura do complexo Rh nas hemácias, as bases moleculares do Sistema Rh, os mecanismos de negatividade RHD, além da Expressão fraca e parcial de D.

The Rh system is the most polymorphic and immunogenic for all blood group systems. Currently more than 49 antigens were identified with five major antigens D, C, c, E, e. Knowledge of the Rh system's molecular basis, since its first cloning 17 years ago, allowed to understand the mechanism of Rh-negative phenotype and the variants of antigens as RHD and RHCE. Deletions, gene rearrangements and insertions are the main mutations. In Caucasians the primary mechanism of Rh-negative phenotype is the complete RHD gene deletion, while in African descendants it is the presence of pseudogene and gene RHDψ hybrid RHD-CE (4-7)-D. The authors analyze the structure of the Rh complex in red cells, molecular basis of the Rh system, mechanisms of Negativity RHD and weak and incomplete expression of RHD.

Manual de hemoterapia 2010 / Hemotherapy Manual 2010

Mesmo com a aplicação de todos os recursos disponíveis para obtenção de homocomponentes de excelente qualidade, a transfusão de um componente sanguíneo nunca estará isento de riscos. Seja inerente as reações que podem ocorrer entre substâncias do doador e do receptor, desencadeando febre, reação alérgica e reações anafiláticas, ou seja por conta de doenças transmitidas por transfusão, as quais mesmo com a grande evolução dos testes de triagem, podem ainda ser transmitidas, persistindo o risco residual. A prática da medicina transfusional exige, cada vez mais, serviços de hemoterapia bem equipados, com recursos humanos altamente qualificados e com sistema de gestão de qualidade bem implementados, com uma estrutura gerencial que contemple uma boa articulação entre todos os setores da área técnica e administrativa. A indicação da utilização terapêutica de hemocomponentes deve ser criteriosa e exige amplo conhecimento teórico-prático, o qual na maioria das vezes é de amplo domínio apenas dos profissionais especializados na área de hemoterapia. Portanto é necessária a supervisão direta ou indireta destes profissionais na prática da medicina transfusional