Publications (11)11.05 Total impact
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Article: Technologies involved in molecular blood group genotyping
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ABSTRACT: Background Until the 1990s, blood screening, typing and diagnostics depended entirely on serological techniques. For over a century, agglutination has been the gold standard for red blood cell (RBC) antigen detection used in all blood services. However, haemagglutination has certain limitations, such as, the difficulty to phenotype recently and multi-transfused patients or direct antiglobulin test (DAT)-positive patients. The haemagglutination test provides only indirect indications of risk or severity of haemolytic disease of the new born. In part, to overcome these limitations, nucleic acid-based technologies have been used in immunohematology reference laboratories.Methods There are many molecular methods available for red cell genotyping: PCR, PCR-RFLP, PCR-SSP or PCR-ASP, real time PCR, DNA sequencing and pirosequencing and methods with microarrays-based systems.Other molecular techniques that are under development and may be available for red cell genotyping in the next decade include fluidic or open microarrays; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); and mini-sequencing.Conclusions Although serology may be superior for some blood group typing, genotyping assays offer a good alternative for problems encountered by serology. In many laboratories, blood group genotyping is already used at a low-throughput level for diagnostics in cases of problematic serology. Especially in case of weak expression of antigens, the presence of rare antigens or auto-antibodies or after multiple transfusions, genotyping is superior. The non-invasive determination of the foetal RHD analysis in maternal plasma by real-time PCR is well established and already offered as a clinical service in a number of countries.The recent availability of automated, high throughput, DNA-array platforms, allows to introduce into the hospital and donor centres this DNA-based typing methodology. The evolution of molecular methods combined with automation and high-quality standards will make large-scale screening a cost-effective reality.ISBT Science Series 05/2011; 6(1):1 - 6. -
Article: Weak D type 2 is the most prevalent weak D type in Portugal.
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ABSTRACT: The weak D phenotype is the most common D variant, with a frequency of 0.2-1% in Caucasian individuals. There are several weak D types, with different frequencies in European countries, which may pose serologic problems and have the potential for alloimmunization. Samples from Portuguese individuals were tested for RhD by two or three distinct monoclonal and oligoclonal antisera, in direct agglutination tests. When discrepant results were observed, samples were tested with panels of monoclonal anti-D by LISS-indirect antiglobulin test. Cases that reacted weakly with IgM but positive with IgG anti-D were analysed by PCR-sequence-specific primers and real-time PCR. Ninety-nine samples were referred after being characterized as weak D. This genotype was recognized, with a preponderance of weak D type 2 (63.6%) over type 1 (16.2%) and 3 (14.1%). The high incidence of weak D type 2 in our population is in marked contrast to studies performed in other European populations and might be due to our sample selection criteria or ethnic variation. There are advantages in genotyping serologically depressed D samples to avoid the waste of D-negative RBC units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization.Transfusion Medicine 03/2006; 16(1):63-7. · 1.14 Impact Factor -
Article: Re: Prediction of the fetal Kell blood group reduces aggressive interventions.
Australian and New Zealand Journal of Obstetrics and Gynaecology 11/2005; 45(5):464. · 1.24 Impact Factor -
Article: The clinical phenotype modulation of haemophilia by prothrombotic gene mutations.
Haemophilia 04/2003; 9(2):235-6. · 2.60 Impact Factor -
Article: Blood group antigen profile predicted by molecular biology-use of real-time polymerase chain reaction to genotype important KEL, JK,RHD, and RHCE alleles.
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ABSTRACT: The most clinically important blood group systems in transfusion medicine, excluding the ABO system, are the RH, Kell, and Kidd systems. Alloantibodies to antigens of these systems may be produced following blood transfusion or during pregnancy and can result in serious hemolytic transfusion reactions and hemolytic disease of the newborn. We developed rapid and robust techniques for RHD, RHCE, KEL, and JK genotyping with the use of a real-time polymerase chain reaction instrument. Two fluorescence-based methods for the detection of amplification products were used: for KEL1/KEL2, JK1/JK2, and RHE/RHe (exon 5) we used the hybridization probes protocol; for RHC/RHc the analysis was done in sequences of exon 1 for RHC and exon 2 for RHc; and for RHD, analysis was done in sequences of intron 4, exon 7, and exon 4 pseudogene using the SYBR Green I protocol. The genotyping tests were validated with samples from 85 Caucasian Portuguese and 15 Black European blood donors. Complete phenotype-genotype correlations were obtained. The potential use of the presented methods can be predicted in clinical transfusion medicine, allowing appropriate monitoring, early intervention, and improved care. When blood group genotyping techniques are necessary, this methodology is highly competitive for a routine laboratory.Immunohematology / American Red Cross 02/2002; 18(3):59-64. -
Article: The first case of HCV seroconversion in Portugal after the introduction of HCV NAT screening.
Transfusion 07/2001; 41(6):848-9. · 3.22 Impact Factor -
Article: Rapid genotyping of the major alleles at the Duffy (FY) blood group locus using real-time fluorescence polymerase chain reaction.
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ABSTRACT: The Duffy blood group system has clinical importance due to involvement in transfusion reactions and hemolytic disease of the newborn. Recently, the molecular basis of the two alleles, FY*A and FY*B (125G>A), and the mutation situated in the promoter region of the FY gene (-33T>C), have been elucidated. In order to develop an accurate, easy, and rapid genotyping method, we describe a procedure using the LightCycler. Samples from 53 Caucasian Portuguese blood donors and 7 black, healthy, European individuals were phenotyped with commercial antisera. DNA was extracted from blood samples and the relevant sequences were amplified with the same cycling conditions, using real-time polymerase chain reaction. The melting point of the FY*A allele was 63 degrees C and of the FY*B allele, 55 degrees C. The allele without mutation at the promoter region had a melting point at 64 degrees C and the FY*B silent allele at 58 degrees C. The results in Caucasian individuals were similar to those found in European and American populations. When FY genotyping techniques are necessary, the methodology described is preferable to conventional methods as it is reliable, high speed, and uses small volumes, providing a highly competitive technology for use by a routine laboratory.Immunohematology / American Red Cross 02/2001; 17(2):42-4. -
Article: Genetic risk factors in acute coronary disease.
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ABSTRACT: We investigate whether each of the following: HPA-1, Factor V Leiden, prothrombin gene variant and the methylene tetrahydrofolate reductase gene (MTHFR) mutation, are risk factors for acute coronary disease in Portuguese patients. 100 blood donors and 52 patients with an established diagnosis of myocardial infarction or unstable angina were evaluated for genetic risk factors, by determining HPA-1 genotype, Factor V Leiden, Prothrombin 20210 variant and MTHFR mutation. We found a prevalence of 2.0% for Factor V Leiden, 5.0% for the Prothrombin 20210 variant and 66% for the MTHFR mutation in blood donors. These values are similar to those found in the patients (1.9, 3.8 and 58%, respectively). We found that 28/100 controls had the PI(A2) polymorphism, a frequency statistically different from that in the patients (23/52). This difference was even more pronounced in patients less than 60 years old (27/96 vs. 13/24). Factor V Leiden, Prothrombin 20210 variant and MTHFR mutation do not seem to represent risk factors for acute coronary disease. However, the PI(A2) polymorphism could have a role in the pathogenesis of this disease. The presence of multiple genetic factors, more than single ones, could influence the development and outcome of myocardial infarction and unstable angina. Larger studies are needed in order to have a better insight into the pathophysiological mechanisms of this disease, along with its prevention and the development of new treatments.Haemostasis 02/1999; 29(4):212-8. -
Article: The blood bank and hepatitis G.
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ABSTRACT: HGV can be transmitted by transfusion and is capable of inducing persistent infection. Thus far it appears to have no discernable disease association. We determined the infection rate of HGV in volunteer blood donors and patient with risk factors for viral transmission through the blood. The association between HGV infection and exposure to labile blood products could be a problem for blood banks in the future.Transfusion Science 07/1998; 19(2):119-20. -
Article: Routine screening of blood donations for HCV RNA.
Vox Sanguinis 02/1998; 74(3):211. · 2.86 Impact Factor -
Article: [Parvovirus B19 infection].
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ABSTRACT: In 1975, during blood screening for hepatitis B, Cossart et al. discovered the human parvovirus B19 (B19). It is a small, single strand DNA virus of the Parvoviridae family. This virus is widespread with 40-80% of adults showing evidence of infection. It is found in the respiratory secretions of viraemic patients and direct contact has been suggested as the most likely mode of transmission. Parenteral transmission is common during treatment with clotting-factor concentrates, but rarely occurs during transfusion with single donor products. Although B19 usually causes a self-limited illness, complications of infection can be severe and at times life threatening. In pregnant women, infection can lead to spontaneous abortions and hydrops fetalis and, in patients with haemolytic anaemias or in immunocompromised individuals, can induce aplastic crisis and chronic anaemias. The diagnosis can be made by indirect (testing for B19 antibodies) or direct methods (detecting B19 viremia). There are no vaccines or specific therapy currently available. Contact isolation is recommended for hospitalized patients.Acta médica portuguesa 12(4-6):195-202.
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Institutions
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1998–1999
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Hospital de São João
Porto, Distrito do Porto, Portugal
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