Automation of RNA-Based Biomarker Extraction From Dried Blood Spots for the Detection of Blood Doping

Loria, Francesco; Manfredi, Michelania; Reverter-Branchat, Gemma; Segura, Jordi; Kuuranne, Tiia; Leuenberger, Nicolas

doi:10.4155/bio-2020-0041

Cited by 22 publications

(30 citation statements)

References 27 publications

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“…17 Moreover, previous studies done by our group on RNAbased biomarkers for indirect detection of rhEPO use demonstrate that ALAS2 LC, ALAS2 L, CA1, and SLC4A1 mRNAs can be detected using DBSs. 5,6 The present study confirms that RNA-based biomarkers could offer a viable solution for increasing the sensitivity and specificity of the ABP. Furthermore, the method reported here provides a solution for distinguishing between variations due to rhEPO use and variations due to hypoxia exposure using DBSs as the matrix.…”

Section: Introductionsupporting

confidence: 79%

“…mRNA was extracted manually from DBSs and analyzed as described by Loria et al 6 using the miRNeasy Mini Kit (Qiagen, Germany), following the manufacturer's instructions with minor modifications. 5,6 After being cut from the collection card, the whole DBS was transferred into a 2-ml conical polypropylene microcentrifuge tube (Eppendorf, Switzerland). The cells were lysed by adding 1-ml QIAzol lysis reagent (Qiagen) to the tube and agitated (450 rpm) at 37 C for 15 min.…”

Section: Rna Extraction and Analysismentioning

confidence: 99%

“…In earlier studies, Salamin et al and Loria et al demonstrated that the expression of these mRNAs increased following the administration of therapeutic doses of rhEPO. [5][6][7] Both the linear and the linear + circular forms of ALAS2 mRNA (ALAS2 L and ALAS2 LC, respectively) may be useful as potential biomarkers, as both were found to increase after therapeutic injection of rhEPO. 5 Circular mRNAs have a head and tail joined at the splice site, rendering them more stable than linear mRNAs.…”

Section: Introductionmentioning

confidence: 99%

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The use of RNA‐based 5'‐aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses

Loria

Cox²,

Voss

et al. 2021

Drug Testing and Analysis

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The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with microdoses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'-aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO microdosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO microdosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO microdosing and hypoxia, whereas SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO microdosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti-doping analyses.

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Section: Introductionsupporting

confidence: 79%

Section: Rna Extraction and Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The use of RNA‐based 5'‐aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses

Loria

Cox²,

Voss

et al. 2021

Drug Testing and Analysis

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“…31,81 Recent advancements in instrumentation have achieved improved accuracy and sensitivity which has led to magnified attention in the use of microsamples. 42,46,[80][81][82] The range of applications include therapeutic drugs, [83][84][85][86] biomarkers, 87,88 proteins and peptides, 89 drugs of abuse, 90,91 hormones and steroids, 92,93 DNA/RNA 56,87 and cytokines. 27,53,94 Few established applications discussed below focus on the use of microsampling techniques in the determination of drug concentration using a validated bioanalytical method.…”

Section: Applications Of Microsampling In the Development Of Treatmentsmentioning

confidence: 99%

Microsampling: A role to play in Covid‐19 diagnosis, surveillance, treatment and clinical trials

Rajadhyaksha

Londhe

2021

Drug Testing and Analysis

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The outbreak of the new coronavirus disease changed the world upside down. Every day, millions of people were subjected to diagnostic testing for Covid‐19, all over the world. Molecular tests helped in the diagnosis of current infection by detecting the presence of viral genome whereas serological tests helped in detecting the presence of antibody in blood as well as contributed to vaccine development. This testing helped in understanding the immunogenicity, community prevalence, geographical spread and conditions post‐infection. However, with the contagious nature of the virus, biological specimen sampling involved the risk of transmission and spread of infection. Clinic or pathology visit was the most concerning part. Trained personnel and resources was another barrier. In this scenario, microsampling played an important role due to its most important advantage of remote, contactless, small volume and self‐sampling. Minimum requirements for sample storage and ease of shipment added value in this situation. The highly sensitive instruments and validated assay formats assured the accuracy of results and stability of samples. Microsampling techniques are contributing effectively to the Covid‐19 pandemic by reducing the demand for clinical staff in population‐level testing. The validated and established applications supported the use of microsampling in diagnosis, therapeutic drug monitoring, development of treatment or vaccines and clinical trials for Covid‐19.

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“…Leuenberger and colleagues have shown that an automated method of analysis they used for transcriptomic biomarkers from reticulocytes in dried blood spots was as good as or better than manual sample handling. They applied their methodology to examine changes in the expression levels of a number of factors in samples from elite athletes and volunteers after recombinant erythropoietin injection [9].…”

mentioning

confidence: 99%

Antidoping Analysis: A Special Focus

Cowan

2020

Bioanalysis

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in antidoping • gene doping • metabolomics in antidoping • peptide quantification • VAMS in antidopingThis issue of Bioanalysis has the theme of antidoping analysis and was originally planned to be published to coincide with the run-up to the 2020 Olympic and Paralympic Games in Tokyo, Japan. It was intended to give the interested reader an indication of the latest approaches used by the scientists working in the antidoping field that would likely be used during the Games in the Tokyo World Anti-Doping Agency (WADA) accredited antidoping laboratory. The COVID-19 pandemic has affected all walks of life internationally and sport is no exception; hence the games have been postponed for a year until 2021 by the International Olympic Committee, for the first time ever. The ability, under WADA and International Olympic Committee regulations, to store samples for up to 10 years means that even if some of the approaches described in this issue are not yet ready for use to take action against a cheating athlete, retrospective analysis could enable this to occur at some time in the years ahead.I have found the articles in this issue always interesting to read and informative, being written by and with the opinions of experts in the field of antidoping science. The conclusions or opinions documented should provide fertile ground for further research. Here is a summary of what you will find.Aikin and colleagues from WADA provide a well-informed perspective about the current and future use of biomarkers in antidoping testing with a special focus on athlete biological passports pioneered by their organization. They outline what is required and make it clear that discovering new biomarkers and implementing their use is no easy task, but worth the effort in making antidoping testing more effective [1].Botré and colleagues provide a commentary on the need for a metabolomics approach to deal with some of the more complex analytical challenges in antidoping, and the potential of the approach for use in the field. However, they point out numerous challenges and conclude that "stand-alone screening + confirmation techniques based on metabolomics approaches still seem premature in antidoping," but indicate that explorative metabolomics are of value even now to help identify putative biomarkers [2].An update on the state of play of peptide quantification in antidoping and how it often links with the requirements of clinical science is provided in an editorial by Moncrieffe. The need for traceable reference standards is stressed and the importance of LC-MS to provide the 'gold-standard' approach contrasting with some of the problems encountered with the use of immuno-procedures. She stresses the benefits of and need for good interlaboratory studies to show the reliability and robustness of the methods [3].Fedoruk of the United States Anti-Doping Agency (USADA) presents in his editorial how USADA has modified its approach to antidoping testing as a result of the COVID-19 pandemic [4]. He explains how athletes attempt to beat the testers ...

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Automation of RNA-Based Biomarker Extraction From Dried Blood Spots for the Detection of Blood Doping

Cited by 22 publications

References 27 publications

The use of RNA‐based 5'‐aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses

The use of RNA‐based 5'‐aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses

Microsampling: A role to play in Covid‐19 diagnosis, surveillance, treatment and clinical trials

Antidoping Analysis: A Special Focus

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