Browsing by Author "Costa, Alcina"
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- Alfa-talassémia delecional e fenótipo hematológico: parâmetros associados às diferentes deleções na casuística de 2015 a 2019Publication . Gaspar, Gisela; Ramalho, Rita de Mira; Seuanes, Filomena; Feliciano, Carla; Duarte, Guida; Copeto, Sandra; Costa, Alcina; Santos, João Xavier; Miranda, ArmandinaAs talassémias são caracterizadas por um desequilíbrio quantitativo nas cadeias globinicas devido à redução ou supressão da síntese de uma das cadeias. Foram avaliados retrospetivamente os resultados de 496 casos suspeitos de α-talassémia delecional e correlacionados com os dados hematológicos. A pesquisa de deleções causadoras de α-talassémia foi efetuada por Gap e Multiplex Gap-PCR. A maioria dos casos (n=190) apresentou um genótipo normal (αα /αα), seguido de heterozigotia (-α 3 ,7 /αα) (n=148) e homozigotia (-α 3 ,7 /α 3 ,7 ) (n=141) para a deleção de 3,7kb. Detetaram-se ainda 5 casos de heterozigotia para a deleção de 4,2Kb (-α 4,2 /αα), 4 de dupla heterozigotia ( α 3 ,7 /α 4,2 ), 7 de heterozigotia α 0 (-- S E A /αα ), e 1 de Hb H (-- S E A /-α 3 ,7 ). Os resultados evidenciaram que o VGM e o HGM são excelentes índices hematológicos de rastreio e seleção dos testes moleculares, sendo o seu valor tanto mais baixo quanto maior o número de genes delecionados. Os resultados obtidos são ainda concordantes com o descrito na literatura e reforçam que o valor de cut-off de 25 pg (HGM), tem sensibilidade adequada para inferir da presença de uma deleção α 0 -talassémia. A deteção da deleção α 0 assume particular importância na prevenção da ocorrência de Hb Bart’s na descendência de um casal de portadores. O diagnóstico de α-talassémia é efetuado por métodos moleculares, no entanto os índices hematológicos são importantes marcadores preditivos do número de genes alfa delecionados e da relação fenótipo / genótipo.
- An integrated solution using bench spreadsheets to monitor Quality control indicators and performance in medicine laboratoriesPublication . Miranda, Armandina; Costa, Sandra; Costa, Alcina; Alvim, Marta; Correia, Helena; Carletto, Aline; Faria, Ana PaulaLaboratory Quality Control (LQC) in medical laboratories is a tool to monitor the procedures of pre analytical, intra analytical and post analytical phases. The data statistic analyze allow the quantification of the random errors using the variation coefficients (CV%) obtained by Internal Quality Control (IQC) and the systematic errors (bias%), from the results of External Quality Control (EQC). These results are combined to calculate the Total Error (TE) and Measurement of Uncertainty (MAU), that allows the knowledge of the precision and accuracy and follow the performance of laboratory by comparison with the quality specifications. The main objective is to present a tool as a bench spreadsheet developed in National Institute of Health Doctor Ricardo Jorge (INSA), aiming to help the national and portuguese speaking countries laboratories, to calculate the main indicators of the LQC: TE, Sigma level and MU using IQC and EQC results, in a simplified way.
- Anemia de células falciformes: avaliação da hemoglobina fetal num grupo de crianças angolanas antes e após tratamento com hidroxiureiaPublication . Almeida, Priscilla; Costa, Alcina; Seuanes, Filomena; Romão, Raquel; Brito, Miguel; Silva, Isabel Moreira da; Miranda, Armandina
- Biomarker of Chronic Alcohol Abuse – Carbohydrate-deficient Transferrin (CDT): Methodology VerificationPublication . Gomes, Filomena; Costa, Alcina; Fernandes, Lília; Silva, Ana Maria V. da; Vasconcelos, Miguel; Miranda, ArmandinaIntroduction: Transferrin is a glycoprotein synthesized in hepatocytes that can appear with different isomorphic forms in the plasma, acquiring different levels of sialization (1,2). In a healthy person, penta, tetra and trisial isoforms are detectible in plasma. However, in an alcohol abuse and/or dependence, asialo, monosialo and disialotransferrin isoforms are also present called carbohydrate-deficient transferrin (CDT) (3) . This is considered a specific biomarker of alcohol abusive and/or dependence, being useful in the diagnosis and monitoring of this pathology (4) . Aim: Verify compliance with the requirements of the manufacturer of the capillary electrophoresis method in laboratory practice and its suitability in determining the CDT. Materials and methods: The MiniCap System (Sebia) was used with calibrators traceable to the IFCC international reference procedure and normal and pathological internal control samples. Repeatability and intermediate precision tests were performed on control samples. From participation in External Quality Assessment (EQA) program (5 rounds - 2 samples each), Bias%, Deviation Index (DI) and Total Laboratory Error (TELab) were obtained. The Measurement Uncertainty was calculated by the Top Down Method (combined and expanded with a factor of 1.96), using the internal (CV%) and external (Bias%) quality control results. Results: In the repeatability tests, normal control samples (n=22, mean = 1.4%) were obtained, CV = 5.7%; for the pathological sample (n=24, mean = 5.4%), CV = 2.2%. In intermediate precision tests for the normal control sample, (n= 12, mean = 1.4%), CV = 6.7%; for the pathological sample (n=12, mean = 5.3%), CV = 4.9%. In samples from EQA program, mean Bias = -1.0% and TELab = 11.5. In the evaluation of the method by DI, 1 satisfactory, 7 good and 2 excellent results were obtained. The obtained Expanded Uncertainty (1.3% ± 0.3) is consistent with that indicated by the manufacturer. Conclusion: The TELab obtained meets Westgard`s desirable specifications (5) , being considered an appropriate methodology for use in laboratory practise for diagnosis. However, it is considered important to monitor the method with Internal Control samples, and participate in EQA programs, as well as periodic evaluation of Quality Indicators.
- Deletional alpha-thalassemia and hematological phenotype: predictive parameters of different deletionsPublication . Gaspar, Gisela; Ramalho, Rita de Mira; Seuanes, Filomena; Feliciano, Carla; Duarte, Guida; Copeto, Sandra; Costa, Alcina; Santos, João Xavier; Miranda, ArmandinaIntroduction: Thalassemias are characterized by a quantitative imbalance of the globin chains due to the reduction or suppression of the synthesis of one of the globin chains.The hematological tests usually used as indicative for the investigation of α-thalassemia are the blood count with MCV (Mean Cell Volume) < 80 fL and/or MCH (Mean Cell Hemoglobin) < 27 pg and normal Hb A2 (< 3.5%). Aim: This study aimed to correlate the different deletional α-thalassemia genetic alterations with the corresponding hematological phenotype, based on casuistry from 2015 to 2019. Methodology: Was evaluated retrospectively 496 cases suspected of deletional α-thalassemia from 2015 to 2019 and correlated them with the hematological data available. We searched for α-thalassemia deletions by Gap-PCR and Multiplex Gap-PCR. Haematological evaluation was carried out by the erythrogram, Hb isoelectric focusing and quantification of Hb A2 and Hb F (Ion exchange high performance liquid chromatography). The statistical analysis of the results was carried out through calculating the mean, standard deviation, median, and t-Student test, with a significance level of 0.05. Results and discussion: Most patients (n=190) had a normal genotype (αα/αα), followed by heterozygosity (-α3.7/αα) (n=148) and homozygosity (-α3.7/α3.7) (n=141) for the 3.7kb deletion. We also detected 5 cases of heterozygosity for the 4.2Kb deletion (-α4.2/αα), 4 of double heterozygosity (α3.7/α4.2), 7 heterozygosity α0 (--SEA /αα) and 1 of HbH (--SEA/-α3.7). The results showed that the MCV and the MCH are excellent hematological indices for screening and selection of patients for molecular testing (their value being the lower the greater the number of deleted genes). Our results are in line with those described in the literature and reinforce that the cut-off value of 25 pg (MCH) is sensitive enough to infer the presence of α0 -thalassemia deletion. The detection of the α0 deletion is very important in preventing the occurrence of Hb Bart's in the offspring of a carrier couple. The diagnosis of deletional α-thalassemia is realised by genetic testing, however hematological indices are relevant predictive markers of the number of deleted alpha genes and the phenotype /genotype correlation.
- Diagnóstico da Fibrose Quística: a prova do suor como técnica de referência para o seu diagnósticoPublication . Alvim, Marta; Costa, Alcina; Gomes, Filomena; Almeida, Suza; Silva, Conceição; Pacheco, PaulaA Fibrose Quistica (FQ) é a doença genética autossómica recessiva letal mais frequente na população caucasiana. É causada por mutações no gene que codifica a proteína CFTR (Cystic Fibrosis Transmembrane Condutance Regulator), que funciona como canal de transporte de iões Cloreto ao nível da membrana apical das células epiteliais, contribuindo para o controlo do movimento de água nos tecidos. A FQ é caracterizada pela obstrução e infecção pulmonar crónica, insuficiência pancreática, obstrução intestinal, infertilidade masculina e suor com níveis elevados de cloretos. A idade em que se começam a manifestar os sintomas e a sua gravidade estão relacionadas com o tipo de mutações no gene CFTR. O diagnóstico é baseado no fenótipo clínico associado a duas ou mais provas do suor positivas e/ou pela presença de duas mutações no gene CFTR. O presente trabalho tem como objetivo avaliar a casuística da Prova do Suor entre 2009 e Março de 2018 realizado no Departamento de Promoção da Saúde e Prevenção de Doenças não Transmissíveis do INSA, I.P Lisboa e o estudo do gene CFTR, efetuado na Unidade de Genética Molecular, Departamento de Genética Humana, INSA, I.P Lisboa. Foram analisados 594 indivíduos, com idades entre um mês e os 77 anos, sendo que a maioria dos indivíduos, 79,5%, tem até aos 12 anos de idade. Em relação ao género a distribuição da amostragem é aproximada igual, com 43,36% das amostras do sexo feminino e 56,64% do sexo masculino.
- Fibrose quística: diagnóstico laboratorial pela prova do suor num grupo populacionalPublication . Costa, Alcina; Batalha, Lídia; Almeida, Suza; Vilares, Arminda; Pacheco, Paula; Silva, Conceição; Miranda, Armandina
- Hemoglobin variants with electrophoretic mobility similar to hemoglobin SPublication . Miranda, Armandina; Faustino, Paula; Gonçalves, João; Seuanes, Filomena; Copeto, Sandra; Loureiro, Pedro; Costa, Alcina; Costa, Sandra; Seixas, Maria TeresaHemoglobinopathies are among the most common inherited diseases around the world and are one of the world’s major health problems. They are monogenic diseases of autosomal recessive transmission resulting from mutations affecting the genes responsible for the synthesis of globin chains. Abnormal hemoglobins (Hb), named Hb variants, are caused by structural defects resulting from an altered amino acid sequence in globin chains, being Hb S the more frequent and pathogenic/disease associated. The aim of this work was to identify and characterize Hb variants with mobility similar to Hb S when using common laboratorial methodologies, such as isoelectric focusing and high pressure ion exchange chromatography (HPLC). Hemoglobin analysis was performed by isoelectric focusing and HPLC. Globin chain variants were classified in alpha or beta type by reversed phase high performance chromatography. Hb S was confirmed by the solubility test. In order to identify the rare Hb variants, molecular analyses were performed in patient’s DNA. From 2010 to 2016, in the routine practice of our laboratory, 601 cases of variants of Hb were detected with mobility Hb S-like. Amongst them, 433 were confirmed as being Hb S (72.0%). Others hemoglobins also with clinical relevance, Hb D and Hb Lepore, were prevalent, 90 (15.0%) and 61 (10.2%), respectively. The remaining 17 cases were classified as rare (2.8%) and 10 of them were identified by molecular studies as: Hb Maputo (1), Hb G-Coushata (1), Hb Summer Hill (1), Hb Setif (1), Hb G Waimanalo (1), Hb D Iran (1) Hb Oleander (1), Hb Ottawa (1), Hb Etobicoque (1) and Hb Matsue-Oki (1). Hb Matsue-Oki was found in compound heterozygosity with the –α3.7kb-thalassemia deletion. We can conclude that combining the results obtained by the different biochemical methodologies allow the presumptive identification of the more prevalent variants, namely Hb S, Hb D and Hb Lepore, and direct the molecular study for the definitive identification. This study also revealed that several rare variants have similar mobility as Hb S and, consequently, some safety measures should be applied in order to achieve their accurate identification. A correct laboratorial diagnosis is essential for proper patient’s clinical management and genetic counselling.
- Hemoglobinas variantes com mobilidade eletroforética semelhante à da Hemoglobina SPublication . Miranda, Armandina; Seuanes, Filomena; Copeto, Sandra; Loureiro, Pedro; Costa, Alcina; Costa, Sandra; Seixas, Maria Teresa; Gonçalves, João; Gonçalves, João; Faustino, PaulaAs hemoglobinopatias são doenças monogénicas hereditárias, de transmissão autossómica recessiva, resultantes de mutações nos genes codificantes para as cadeias de globina da hemoglobina (Hb), ou nas suas regiões regulatórias. Encontram-se entre as doenças hereditárias mais comuns e constituem um dos principais problemas de saúde a nível mundial.
- Hemoglobinas variantes com mobilidade eletroforética semelhante à da Hemoglobina SPublication . Miranda, Armandina; Seuanes, Filomena; Copeto, Sandra; Loureiro, Pedro; Costa, Alcina; Costa, Sandra; Seixas, Maria Teresa; Gonçalves, João; Faustino, PaulaIntrodução: As hemoglobinopatias são doenças monogénicas hereditárias, de transmissão autossómica recessiva, resultantes de mutações nos genes codificantes para as cadeias de globina da hemoglobina (Hb), ou nas suas regiões regulatórias. Encontram-se entre as doenças hereditárias mais comuns e constituem um dos principais problemas de saúde a nível mundial. As variantes das hemoglobinas são causadas por defeitos estruturais resultantes de alterações na sequência de aminoácidos nas cadeias de globina, sendo a Hb S a mais frequente e associada a patologia. As hemoglobinopatias são as únicas, entre todas as doenças genéticas, em que a deteção de portadores é possível por testes hematológicos e bioquímicos. No entanto, a análise molecular do respetivo gene, deve ser realizada para a identificação definitiva de casos complexos ou quando os resultados hematológicos/bioquímicos não são conclusivos. A identificação de hemoglobinopatias é frequentemente presuntiva, com base em tempos de retenção e padrões de migração, e deve ser baseada preferencialmente no mínimo em duas metodologias com princípios de separação diferentes. A identificação definitiva requer a análise do respetivo gene, espectrometria de massa ou sequenciação de proteínas 1, 2 .Os procedimentos analíticos mais comumente utilizados devem ter capacidade de detetar as variantes de hemoglobina clinicamente mais significativas: Hb S, Hb C, Hb D Punjab, Hb Lepore , Hb E e Hb OArab. .Objetivo: Caraterizar e identificar as variantes de Hb com mobilidade eletroforética semelhante à Hb S..