| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 2.6 MB | Adobe PDF |
Advisor(s)
Abstract(s)
As especificações para a análise de eritropoietina (EPO) e Agonistas do Recetor da Eritropoietina (ERAs) nos laboratórios de antidopagem encontram-se descritas atualmente apenas para urina e soro/plasma. A utilização de dried blood spots (DBS) para análise de substâncias dopantes tem sido uma realidade cada vez mais comum estando esta metodologia a conquistar um lugar de relevo na luta contra a dopagem. Neste sentido, o presente estudo teve como objetivo compreender se o sangue capilar em DBS é um complemento viável para a deteção de EPO e ERAs no âmbito do controlo de dopagem. O estudo envolveu 30 voluntários, aos quais foram colhidos dois spots de 60 μL de sangue capilar no dedo, amostras de sangue venoso (tubo EDTA) e ainda, a quatro voluntários, amostras de urina e sangue (tubos EDTA e seco). Todas as amostras, de DBS, foram imunopurificadas (Kit de Purificação em Gel - MAIIA) e implementados os métodos eletroforéticos sodium N-lauroysarcosinate polyacrilamide gel eletrophoresis (SAR-PAGE) e Western Blot. De modo a identificar qual a quantidade mínima do analito que pode ser detetada pelo método recorrendo a amostras DBS determinaram-se valores de limites de deteção (LODs) para as principais ERAs: BRP, NESP, EPO-Fc e CERA que se concluíram mais elevados do que os definidos para soro e urina. O estudo de estabilidade realizado com amostras de DBS, soro e urina conservadas a diferentes temperaturas (ambiente, 4ºC e -20 ºC), apresentaram resultados promissores para os DBS, ao serem a única matriz que permite a conservação de todas as ERAs ao fim de três meses a qualquer temperatura analisada. Estes resultados apoiam a utilidade dos DBS como matriz biológica complementar para a deteção de EPO e ERAs no controlo de dopagem.
ABSTRACT - Currently specifications for the analysis of Erythropoietin (EPO) and Erythropoietin Receptor Agonists (ERAs) in anti-doping laboratories are only described for urine and serum/plasma. The use of dried blood spots (DBS) for the analysis of doping substances has been increasing and this methodology gaining a prominent place in the fight against doping. For that reason, the present study aims to understand whether capillary blood in DBS is a viable complement for the detection of EPO and ERAs in relation to doping control. The study involved 30 volunteers, of whom two 60 μL spots of capillary blood on the finger and venous blood samples (EDTA tube) were collected. Urine and blood samples (EDTA and dry tubes) were also collected from 4 of the 30 volunteers. All DBS samples were immunopurified (Gel Purification Kit - MAIIA) and the electrophoretic methods of sodium N-lauroyl sarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western Blot were implemented. To determine the lowest concentration of the analyte that can be detected in DBS samples, limits of detection values (LODs) were quantified for the main ERAs: BRP, NESP, EPO-Fc, and CERA, resulting in limits of higher value than those defined for serum and urine. The stability study carried out with samples of DBS, serum, and urine, stored at different temperatures (ambient, 4ºC and -20ºC), showed promising results for DBS, as they are the only matrix that allows the conservation of all ERAs after three months at all the studied temperatures. These results support the utility of DBS as a complementary biological matrix for the detection of EPO and ERAs in doping control.
ABSTRACT - Currently specifications for the analysis of Erythropoietin (EPO) and Erythropoietin Receptor Agonists (ERAs) in anti-doping laboratories are only described for urine and serum/plasma. The use of dried blood spots (DBS) for the analysis of doping substances has been increasing and this methodology gaining a prominent place in the fight against doping. For that reason, the present study aims to understand whether capillary blood in DBS is a viable complement for the detection of EPO and ERAs in relation to doping control. The study involved 30 volunteers, of whom two 60 μL spots of capillary blood on the finger and venous blood samples (EDTA tube) were collected. Urine and blood samples (EDTA and dry tubes) were also collected from 4 of the 30 volunteers. All DBS samples were immunopurified (Gel Purification Kit - MAIIA) and the electrophoretic methods of sodium N-lauroyl sarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western Blot were implemented. To determine the lowest concentration of the analyte that can be detected in DBS samples, limits of detection values (LODs) were quantified for the main ERAs: BRP, NESP, EPO-Fc, and CERA, resulting in limits of higher value than those defined for serum and urine. The stability study carried out with samples of DBS, serum, and urine, stored at different temperatures (ambient, 4ºC and -20ºC), showed promising results for DBS, as they are the only matrix that allows the conservation of all ERAs after three months at all the studied temperatures. These results support the utility of DBS as a complementary biological matrix for the detection of EPO and ERAs in doping control.
Description
Mestrado em Tecnologias Moleculares em Saúde
Keywords
Dried Blood Spots Agonistas do recetor da eritropoietina Eritropoietina
Citation
Santos SC. Desenvolvimento e implementação de um método de pesquisa de ERAs em amostras de Dried Blood Spots (DBS) [dissertation]. Lisboa: Escola Superior de Tecnologia da Saúde de Lisboa/Instituto Politécnico de Lisboa; 2022.
Publisher
Instituto Politécnico de Lisboa, Escola Superior de Tecnologia da Saúde de Lisboa