Olivier Salamin1, Emeric Gottardo2, Céline Schobinger2, Gemma Reverter-Branchat3, Jordi Segura3,4, Martial Saugy1, Tiia Kuuranne2, Jean-Daniel Tissot5, Bernard Favrat6, Nicolas Leuenberger7. 1. Center of Research and Expertise in Anti-Doping Sciences - REDs, University of Lausanne, Lausanne, Switzerland. 2. Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland. 3. Integrative Pharmacology and Systems Neuroscience Research Group, Neurosciences Research Program, IMIM - Hospital del Mar Medical Research Institute, Barcelona, Spain. 4. Catalonian Antidoping Laboratory, Doping Control Research Group, Neurosciences Research Program, IMIM - Hospital del Mar Medical Research Institute, Barcelona, Spain. 5. Transfusion Interrégionale CRS, site d'Epalinges, Switzerland. 6. Department of Ambulatory Care and Community Medicine, University of Lausanne, Lausanne, Switzerland. 7. Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; Nicolas.leuenberger@chuv.ch.
Abstract
BACKGROUND: Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS: The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). RESULTS: When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant human erythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. CONCLUSIONS: Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.
BACKGROUND: Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS: The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant humanerythropoietin). RESULTS: When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant humanerythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. CONCLUSIONS: Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.
Authors: Francesco Loria; Holly D Cox; Sven C Voss; Angela Rocca; Geoffrey D Miller; Nathan Townsend; Costas Georgakopoulos; Daniel Eichner; Tiia Kuuranne; Nicolas Leuenberger Journal: Drug Test Anal Date: 2021-07-07 Impact factor: 3.234