Projets de recherche

Code: DBS20CT34JM

The minimally invasive dried blood spot (DBS) technique has the potential to improve the time-and-cost efficiency compared to traditional matrices in doping control. Understanding the athletes’ preferred sampling site will support the drafting of WADA Collection Guidelines/ISTI in the implementation process. Furthermore, the potential impact of the sampling site e.g. finger vs. arm on the concentration of target analytes needs to be established. Doping Control Officers (DCOs) will collect capillary blood from the finger (finger-prick) and from the upper arm (specific collection device) from 108 athletes (males and females) of various sport disciplines. The DCOs will record the time needed to collect the samples, register the number of unsuccessful attempts and evaluate the usability of the collection devices and whether they prefer the collection of DBS from the fingertip or the upper arm. The athletes will fill out a questionnaire regarding the perception and painfulness of the two DBS collections and whether the collections processes have had any impact on their sport activities afterwards. The lab staff will to fill out a questionnaire once they have received the samples to understand the suitability of the samples for analysis. The DBS samples will be analysed for the concentration of endogenous testosterone.

Main findings

Currently several DBS collection devices exists allowing collection of capillary blood from different anatomical sites. Nevertheless, the suitability for collecting DBS samples in an anti-doping context depends on the sample collection experience by the athletes, doping control officers (DCO) and the handling of the sample by the laboratory staff. Furthermore, agreement between quantitative measurements is important if more than one collection method (device and/or sampling site) is approved. In this project, a total of 108 matched DBS samples from the fingertip (HemaSpot HF; lancet device) and the upper arm (Tasso-M20; microneedle device) were collected from 49 female and 59 male national level athletes from various sports (handball, weightlifting, football, running and wheelchair rugby). Following sampling, the collection process was evaluated by athletes and DCOs. Furthermore, upon reception of the samples, the laboratory staff compared the quality and usability of the samples from the two devices. The testosterone concentration was measured in all samples and the correlation between concentrations determined.

Overall, the DBS sampling was associated with minimal sensation of pain and a high general experience by the athletes, but the perceived pain was rated lower (-0.4 ± 1.6, p < 0.05) and the general experience rated higher (+0.6 ± 2.3, p < 0.001) during upper-arm DBS collection than during DBS collection from the fingertip. Likewise, the DCOs rated the general experience with the upper-arm DBS collection higher (+1.6 ± 1.1, p < 0.01) than the fingerprick DBS collection, partly due to problems occurring more frequently during the DBS collection from the fingertip (28% of collections) than from the upper arm (6% of collections). Both procedures were equally fast, lasting only around two minutes on average, which is a great advantage compared to urine. When choosing, the great majority of DCOs and athletes, independent of gender and discipline, preferred the automated DBS collection from the upper arm over the manual collection from the fingertip, and both DBS collections over conventional sample collection methods (urine and venous blood collection).

Both devices provided easy access to the DBS sample and the overall analysis time was not affected by the choice of DBS material, however, the Tasso-M20 device was the preferred device amongst the four analysts preparing the samples due to less sample handling prior to sample preparation. Endogenous testosterone was quantified in DBS samples from both devices with good repeatability (RSD < 5%) and reproducibility (RSD < 10%). The quantitative analyses showed good correlation between samples collected from the fingertip and the upper arm from athletes (r = 0.921, p<0.0001), as well as non-significant difference of the median testosterone concentrations (1.70 ng/mL for Tasso and 1.67 ng/mL for HemaSpot, p = 0.503). A small, measured bias between Tasso and HemaSpot (-7.45%) was observed, likely due to unsatisfactory volume control in the HemaSpot compared to the Tasso device. Collectively, the results suggest that there is no physiological difference in the basal testosterone concentrations in capillary blood samples collected from the fingertip and the upper arm.

In conclusion, both an automated upper-arm DBS collection device [Tasso-M20] and a manual fingerprick DBS system [HemaSpot HF] could be used for DBS collection in an anti-doping setting. However, for subsequent quantitative analyses, the volumetric control of the Tasso-M20 spots seemed more robust than for HemaSpot HF. Both DCOs and laboratory personnel seem to prefer the upper-arm DBS collection with the Tasso-M20 device. If more than one collection method (device and/or sampling site) is approved, it appears crucial that the analytical assays are validated and calibrated on the respective DBS-devices/materials.

Code: DBS20AS7JM

The minimally invasive dried blood spot (DBS) technique has the potential to improve the time-and-cost efficiency compared to traditional matrices in doping control. The potential impact of the sampling site e.g. finger vs. arm on the concentration of target analytes needs to be established, especially when analyzing for threshold substances prohibited in-competition only. Eight healthy male volunteers will receive a single oral administration of ~20 mg (‘low dosage’) and 60 mg (‘high dosage’) of ephedrine in a randomized crossover design with one week between the interventions. Parallel DBS samples from the fingertip and upper arm will be collected at 0 (pre-administration control sample), 1, 2, 4, 6 and 8 hours post-administration. From the DBS samples the ephedrine concentration will be determined. Additionally, venous blood samples will be collected through a peripheral venous catheter on the same time points to compare the DBS concentrations of ephedrine with those in plasma.

Main Findings

Dried blood spot (DBS) testing allows for fast, easy, and minimally invasive collection of microvolumes of blood. In an anti-doping context, DBS testing has particularly relevance for substances prohibited in-competition only, as it can determine the presence of pharmacologically relevant blood concentrations during the in-competition period. A wide range of collection methods and devices exist for DBS collection allowing collection of capillary blood from different anatomical sites, but the possibility to use different devices in an anti-doping setting would rely on agreement in substance concentrations between sampling sites and between devices. Furthermore, it is of interest to evaluate the agreement between concentrations of target analytes in DBS and conventional venous plasma samples. Herein, we collected matched upper-arm DBS, finger prick DBS and venous plasma samples from 8 healthy, male subjects in an 8-hour period following oral administrations of 20 mg (‘low dose’) and 60 mg (‘high dose’) of ephedrine. We observed no consistent trend in the dependence of ephedrine concentration on blood sampling site or sampling device, and the correlations between ephedrine concentrations in finger prick DBS and upper-arm DBS were very high (Pearson’s r > 0.80) after both low and high dose administration. These results indicate that DBS originating from finger prick and automated upper-arm collection, along with conventional venous blood samples, can be used for quantification of ephedrine in doping control.

Code: DBS20AS7AT

This project will assess the introduction of cut-off limits for substances that are only prohibited in competition (stimulants, corticoids, cannabinoids, and narcotics). Data about the pharmacologically relevant levels of each substance are required to interpret the measured results correctly.

Main Findings

In competition sport drug testing represents an important aspect in doping controls. Here the analysis of blood samples owns a considerable benefit compared to urine samples due to the determination of actually relevant blood concentrations during the competition. DBS sampling represents a simple, reliable, cost-efficient and robust approach to ideally support the classical urine analysis, especially for in competition testing. In the present study potential analytical cut-off limits for stimulants, corticoids, cannabinoids, and narcotics are proposed. The values are based on literature review for pharmacokinetic data as well as already existing levels valid for driving under the influence of drugs.

Code: DBS20AS2AT

This project will investigate the possibility to measure the hematocrit value of the dried blood spot (DBS) sample by near-infrared spectroscopy (NIR). This can have an influence on the result, especially when the data are evaluated quantitatively. For this purpose, the results of the NIR measurements will be compared with reference data (Sysmex etc.) obtained simultaneously.

Main Findings

In contrast to established blood sampling strategies (yielding serum or EDTA plasma), DBS consist of whole blood. Therefore, the knowledge about the hematocrit (as percentage of red blood cells) of the DBS sample represents an important parameter especially for quantitative results interpretation. Here, the hematocrit measurement with near-infrared spectroscopy (NIR) from cellulose-based DBS paper represents a valid and reliable approach. After complete drying of the cards, the non-destructive NIR-analysis enables robust hematocrit (Hct) measurements over weeks and presumably months. The correction for the actual hematocrit of the finger prick DBS samples facilitates the accurate correlation to the resulting and comparable plasma levels of the respective drugs. With regard to in-competition DBS sampling, this possibility will enhance the result interpretation significantly. In the present project, different whole blood sampling strategies (venous, finger prick, TAP, Tasso, capillary) and the subsequent hematocrit measurement (Sysmex, NIR, centrifugation) were compared. All measurements for venous EDTA-blood and capillary finger blood (finger prick) were < 10% of relative deviation compared to the ‘true’ Sysmex value. This is also true regardless of the method of measurement. However, collection of capillary blood on heparin (here using the TAP device) and subsequent analysis by NIR led to greater Hct values and a mean relative deviation ˃10%. Transfer to other laboratory is not hindered, because NIR analysis is based on calibration-based technology, which can be adapted and transferred to any other instrument using the same technology. It was additionally shown that the repeated exposure to NIR does not have a measurable impact on the subsequent chemical analysis of the prohibited drugs.

Code: DBS20AS4DE

Recent work in the anti-doping field has clearly shown that the future of blood collections is dependent on direct capillary blood collection, moving away from traditional venous draws. In that light, this project will focus only on the detectability of endogenous (blood EPO, or bEPO) and recombinant EPO in samples collected directly with capillary collection devices and will avoid venous blood spotted onto a matrix. Blood samples, both from control volunteers and patients who have been administered rEPO, will be collected using Tasso and OneDraw devices, and finger pricks.

Extraction of blood (and EPO) from the spot will be optimized using in-house protocols. Once extracted, two methods of EPO purification will be attempted. First, a conventional magnetic bead immunopurification method utilizing anti-EPO antibodies to capture the EPO in the dried blood sample will be tested (adapted from Desharnais, 2017). Next, the commercially available MAIIA EPO purification gel kit will also be utilized for efficacy. Once purified, samples will be analyzed via SAR-PAGE and Western Blotting, using the biotinylated monoclonal anti-EPO antibody described in previous Cologne Workshops (Reichel 2018, Dehnes 2020) to provide a higher quality protein signal. Using the methods described above, the sensitivity and specificity of bEPO and rEPO in DBS will be characterized.

Main findings

This project was executed as a follow-up verification from work completed in the Barcelona laboratory in 2018 in which various ESAs were detected in dried blood spots using electrophoretic methods. The purpose of this project was to verify these results in a second laboratory and to answer the following questions:

1. What is the optimal method to extract and purify EPO from a dried capillary blood spot?

2. How does the optimal method perform in samples collected directly from capillary blood, specifically in samples from a drug administration?

Extraction and purification using StemCell ELISA and anti-EPO magnetic bead technology were studied in this project. Although MAIIA technology was also proposed as a purification method, this technology is currently under study in other laboratories and therefore was omitted from this project. Initial and follow-up studies both showed superior extraction and purification using the StemCell ELISA method, and thus this method was deemed more appropriate for ESA analysis in DBS.

Next, a clinical study was designed in which nine male subjects each received a single dose of EPOGEN (epoetin alfa) at a dose of 40 IU/kg s.c. Finger prick (spotted onto DMPK-C cards) and capillary blood from Tasso M20 devices was collected from each patient before their dose and then intermittently up to 72 hours after their dose. All samples were extracted using the StemCell ELISA method and were analyzed by SAR-PAGE and Western Blotting according to laboratory standard operating procedures. On average, rEPO was detectable in both finger prick and Tasso M20 samples up to 48 hours.

In most cases, the rEPO detected in the samples appeared on the gels as a double band, with the recombinant portion migrating on the gel similarly to the Dynepo standard and the endogenous portion migrating further. While this has been seen in urine and blood samples following controlled administrations, it differs from the conventional ‘mixed band’ or ‘smeared band’ commonly seen with rEPO-positive samples.

Finally, while rEPO was detectable in capillary blood samples, due to factors including low concentrations of EPO in the samples and low sample volume, gel quality was an issue throughout the study. In many instances, the EPO content on the gel was low enough such that the contrast between EPO and background provided interpretation issues. It is expected that using larger sample volumes should eliminate some of these concerns.

Overall, results from this study validate the data observed in the Barcelona in 2018, showing that doses of rEPO can be detected in DBS. Prior to implementing into routine doping

control, method improvement is recommended regarding increasing sample volumes (pending MAIIA purification results). Additionally, it is recommended that a technical document be drafted by the EPO Working Group outlining sample preparation proced

Code: DBS20AS5AT

This project deals with the stability of steroid esters on DBS cards, which represent a frequently misused class of prohibited compounds. As is well known, these esters are hydrolyzed by endogenous esterases in the blood (even after sampling in serum or plasma) and this can falsify the results accordingly. Whether and to what extent this cleavage also occurs when using DBS will be investigated here.

Main findings

Anabolic steroids represent one of the most frequently misused class of compounds in sports due to their performance enhancing properties. These steroids are often applied as esters with variable length of the non-polar ester side chain that have a considerable impact of the pharmacology of the drug. These steroid esters are known for their degradation due to endogenous esterases, which are present in blood also after the sampling process. The addition of esterase inhibiting agents (such as sodium fluoride) enables a slight inhibition of the degradation in the sampling device. The present study was conducted to confirm the potential of DBS sampling enabling the storage of DBS samples with subsequent analysis of intact steroid esters. Interestingly, the storage of whole blood samples as DBS (without any esterase inhibitors) will provide a useful approach to ensure better stability during long-term storage compared to classic liquid whole blood samples. Also the application of EDTA whole blood samples (e.g. derived from the athlete biological passport sampling) to DBS cards enables the storage for subsequent analysis of steroid esters. Here, the time period from sampling until spotting should be minimized (e.g. < 2 hours). This study shows also that storage at 4°C or -20°C with a desiccant in the dark provides the best results for all steroid esters. Here even after five months of storage the esters were still detectable on the DBS cards.

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Ce document n'est actuellement disponible qu'en anglais. 

Ce document n'est actuellement disponible qu'en anglais. 

Ce document n'est actuellement disponible qu'en anglais.