Evaluation of the detection of the homologous transfusion of a red blood cell concentrate in vivo for antidoping.

Two doping cases of homologous blood transfusion (HBT) during Tokyo 2020 Summer Olympics have shown that more controls are needed. The method of detection using flow cytometry to evaluate the expression of minor blood group antigens from red blood cells (RBCs) and identify different RBC populations is efficient but still complex to perform with multiple antigens detection. Recently, the interest of using forensic DNA analysis was also highlighted as a potential new method to detect HBT, with possibility to start from dried blood spots (DBS) instead of fresh blood. After a first phase of development, a protocol was validated for HBT detection using DNA analysis after extraction from DBS. Presence of a second DNA was clear down to 2% of donor blood in vitro. A flow cytometry protocol was also developed with preparation and analysis in 96-well plates and detection of two different antigens per well using two secondary antibodies with distinct fluorophores. The objective of the project was to evaluate the window of detection of an HBT performed in vivo with 150 mL of RBC concentrate. Blood samples obtained over 7 weeks post-transfusion were analyzed. DNA profiling from DBS was not sensitive enough to detect the presence of a second DNA even 1 day after transfusion. On the contrary, the flow cytometry protocol was very efficient and allowed identification of several double populations of RBC (expressing/non-expressing several antigens) until day 50 post-transfusion. This protocol can be fully validated for a future application to doping control samples.

Adaptation of Elecsys® anti-severe acute respiratory syndrome coronavirus-2 immunoassay to dried blood spots: proof of concept.

Aim: Several automated immunoassays have been validated on serum/plasma to evaluate the presence of significant levels of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies, signs of a present or past infection, but the use of dried blood spots (DBS) would facilitate sampling, shipping and storage. Objective: The aim of this project was to give proof of concept of the possibility to use of the automatized Elecsys® anti-SARS-CoV-2 immunoassay with a volumetric DBS device. Results: Linearity and correlation were satisfactory between volumetric DBS and plasma. A cut-off value was suggested and should be validated with more samples. Conclusion: this study strongly support the possibility to work with volumetric DBS instead of serum/plasma to test for anti-SARS-CoV-2 antibodies.

EPO transgene detection in dried blood spots for antidoping application.

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-µl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-µl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-µl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.

Use of Quantitative Dried Blood Spots to Evaluate the Post-Vaccination Level of Neutralizing Antibodies against SARS-CoV-2.

To combat the COVID-19 pandemic, vaccines against SARS-CoV-2 are now given to protect populations worldwide. The level of neutralizing antibodies following the vaccination will evolve with time and vary between individuals. Immunoassays quantifying immunoglobulins against the viral spike (S) protein in serum/plasma have been developed, but the need for venous blood samples could limit the frequency and scale of control in populations. The use of a quantitative dried blood spot (DBS) that can be self-collected would simplify this monitoring. The objective of this study was to determine whether a quantitative DBS device (Capitainer qDBS 10 µL) could be used in combination with an Elecsys anti-SARS-CoV-2 S immunoassay from Roche to follow the development and persistence of anti-S antibodies. This objective was carried out through two clinical studies. The first study investigated 14 volunteers who received two doses of the Comirnaty (Pfizer) vaccine. The levels of anti-S antibodies and the progression over time post-vaccination were studied for three months. The level of produced antibodies varied between subjects, but a similar trend was observed. The anti-S antibodies were highly stimulated by the second dose (×100) and peaked two weeks later. The antibody levels subsequently decreased and three months later were down to 65%. DBS proved to be sufficiently sensitive for use in evaluating the immune status against SARS-CoV-2 over a prolonged time. The second cohort was composed of 200 random patients from a clinical chemistry department in Stockholm. In this cohort, we had no information on previous COVID-19 infections or vaccination. Nevertheless, 87% of the subjects had anti-S immunoglobulins over 0.8 U/mL, and the bias between plasma and DBS proved to be variable, as was also seen in the first vaccination study.

Use of capillary dried blood for quantification of intact IGF-I by LC-HRMS for antidoping analysis.

Background: IGF-I is used as a biomarker to detect Growth Hormone doping in athletes' blood samples. Objective: Our aim was to develop and validate a fast, high-throughput and accurate quantification of intact IGF-I from volumetric absorptive microsampling (VAMS) dried blood using LC coupled to high resolution mass spectrometry (LC-HRMS). Methodology & results: IGF-I was extracted from the VAMS, released from its binding proteins, concentrated using microelution SPE and analyzed by LC-HRMS. The method was successfully validated in accordance with the World Anti-Doping Agency's requirements. Subsequently, IGF-I measurements from capillary dried blood and serum were compared. Conclusion: The combination of VAMS, microelution SPE and LC-HRMS is a promising strategy applicable to IGF-I quantification in athletes' samples.

Detection of Hypoxia-Regulated MicroRNAs in Blood as Potential Biomarkers of HIF Stabilizer Molidustat.

BACKGROUND: The recent development of drugs that stabilize HIFalpha, called HIF stabilizers, offers a new strategy for treating anemia. Although these drugs are still in clinical trials, misuse for doping has already begun. Identifying the biomarkers of HIF stabilizers would therefore help in detecting this drug misuse by athletes. OBJECTIVE: Our aim was twofold: to determine whether hypoxamiRs, the microRNAs associated with the cellular response to hypoxia, are potential biomarkers of HIF stabilizers in blood and whether the response to treatment with an HIF stabilizer differs from the response to a hypoxic environment. METHOD: Rats were treated for 6 days with either a placebo or 2mg/kg of Molidustat, an HIF stabilizer, or they were put under hypoxia (10% oxygen) for the same length of time. Plasma samples were analyzed before, during and 48 hours after the treatments. RESULTS: EPO concentration increased significantly in plasma during hypoxia and Molidustat treatment and showed a negative retro-control 2 days after the end of the treatments. On the contrary, circulating levels of VEGF were not modified. Among the hypoxamiRs tested, miR-130a and miR-21 were significantly increased during Molidustat treatment and miR-21 was still increased 48 hours after treatment end. CONCLUSION: Although using these microRNAs as biomarkers seems unlikely due to other possible factors of regulation, this study provides the first identification of a specific effect of HIF stabilizers on microRNAs. Further investigations are needed to better understand the possible consequences of such regulation.

Combined administration of microdoses of growth hormone and erythropoietin: Effects on performance and evaluation of GH detection capability using anti-doping methods.

The combination of growth hormone (GH) and recombinant erythropoietin (rEPO) is thought to be used particularly in endurance sports. Our objective was to reproduce a 2-week administration of rEPO microdoses alone or in combination with GH microdoses (three times a week) on healthy and athletic male subjects and to evaluate if GH had any additional effects compared to EPO treatment alone. The effects of the treatments on hematological parameters and VO2max were studied as well as the detection of GH in serum. While the rEPO microdose regimen was associated with a significant increase in reticulocytes, no clear elevation in hemoglobin concentration (HGB) was observed. Using a correction by plasma volume did not reveal more effects of EPO on HGB. Our results did not show any additional effect when the GH microdoses were co-administered. In addition, no clear increase in VO2max was observed after treatment, with an elevation in only half the subjects in both groups (EPO and EPO+GH). A clear effect of GH on insulin-like growth factor I (IGF-I) was seen but it was lower on procollagen III amino-terminal propeptide (P-III-NP). GH detection using the direct isoform test identified only one subject 24 hours after receiving GH. The GH biomarker test combining IGF-I and P-III-NP was not able to detect the GH administration. However, a longitudinal follow-up of the intraindividual variations showed a significant increase in IGF-I 24 and 48 hours after GH administration in most subjects, while the effect of GH microdoses on P-III-NP was less straightforward.