Hyperhydration using different hydration agents does not affect the haematological markers of the athlete biological passport in euhydrated volunteers.

Athlete Biological Passport (ABP) is an indirect approach, implemented by WADA, aimed at detecting blood manipulation based on abnormal changes in haematological markers. Cases report the use of hyperhydration as masking method during anti-doping urine sample collection which could potentially mask suspicious fluctuations on ABP profiles. This study investigated the hyperhydration effect on haemoglobin concentration, reticulocyte percentage and OFF-hr score (an algorithm based on haemoglobin concentration and reticulocyte percentage), with and without recombinant human erythropoietin (rHuEPO) administration. A five-week clinical study performed; Baseline and rHuEPO Phase. Water and a sports drink were used as hyperhydration agents. To examine the hyperhydration effect on the normal ABP profile per volunteer, hyperhydration was implemented at 0, 24 and 48 hours during the baseline. During the rHuEPO phase, volunteers received Epoetin beta (3000 IU) with hyperhydration to be implemented at 0, 24 and 48 hours after drug administration. Blood and urine samples were collected and analysed according to WADA guidelines. No significant effect on ABP markers was observed due to hyperhydration at any time during the study. Pre- and post-hyperhydration data were not statistically different compared to individual baseline data. In conclusion, hyperhydration does not affect the ABP haematological markers under the examined conditions.

Hyperhydration-Induced Decrease in Urinary Luteinizing Hormone Concentrations of Male Athletes in Doping Control Analysis.

Low urinary luteinizing hormone (LH) values have been discussed as a marker to detect steroid abuse. However, suppressed LH concentrations related to highly diluted urine samples could be a misleading indication of anabolic steroid abuse. One aim of the present study was to examine the effect of hyperhydration on the interpretation of LH findings during doping control analysis and to investigate different possibilities to correct volume-related changes in urinary LH concentrations. Seven healthy, physically active, nonsmoking White males were examined for a 72-hr period, using water and a commercial sports drink as hyperhydration agents (20 ml/kg body weight). Urine samples were collected and analyzed according to the World Anti-Doping Agency's technical documents. Baseline urinary LH concentrations, expressed as the mean ± SD for each individual, were within the acceptable physiological range (7.11 ± 5.42 IU/L). A comparison of the measured LH values for both hyperhydration phases (Phase A: 4.24 ± 5.60 IU/L and Phase B: 4.74 ± 4.72 IU/L) with the baseline ("normal") values showed significant differences (Phase A: p < .001 and Phase B: p < .001), suggesting the clear effect of urine dilution due to hyperhydration. However, an adjustment of urinary LH concentrations by specific gravity based on a reference value of 1.020 seems to adequately correct the hyperhydration-induced decrease on the LH levels.

Identification of human in vitro metabolites of the haemoglobin S polymerization inhibitor voxelotor for doping control purposes.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

QuEChERS as alternative extraction procedure in doping analyses.

QuEChERS is a dispersive solid phase extraction commonly applied in food analysis for residues, such as pesticides or mycotoxins for more than 20 years. Due to the quick and easy sample preparation procedure, a QuEChERS method based on ammonium acetate combined with formic acid in acetonitrile was tested for the preparation of urine samples for doping control purposes. Testing urine samples with different pH and specific gravity, using the combination of 10 M ammonium acetate with 3% formic acid in acetonitrile, 312 out of 342 tested compounds could be extracted at their respective minimum required performance levels according to the World Anti-Doping Agency (WADA) technical documents. For nine selected analytes representing six categories of WADA's Prohibited List, we validated the QuEChERS extraction method fulfilling WADA's requirements for a confirmation procedure of the nonthreshold substances investigated. Especially for the intact stanozolol-glucuronides analyzed by high-resolution mass spectrometry, the described extraction method might be an alternative for confirmation procedures as it is time- and cost-saving compared with the commonly applied solid phase extraction.

Individual responses to short-term heat acclimatisation as predictors of football performance in a hot, dry environment.

OBJECTIVES: To identify the relationship between field performance in a hot environment and individual heat acclimatisation responses in football players. METHODS: Nineteen semiprofessional football players completed a match in 21°C followed by 6 days of acclimatisation in dry heat (38-43°C, 12-30% relative humidity) and a match in ~43°C. A heat-response test (30 min walk+30 min seated; 44°C) was performed at the beginning and end of the acclimatisation period. RESULTS: The acclimatisation period increased sweat rate by 34% during a standard heat-exposure test and reduced sweat sodium concentration by 18% (both p≤0.005). Plasma volume changes showed large interindividual differences (-10 to +20%). Match-running performance was impaired in hot ambient condition and demonstrated marked interindividual differences (total distance -6.0±5.8%, high-intensity running -16.4±21.5%, both p≤0.002). Only haematological markers investigated during the heat-response test correlated with the ability of the player to cope with heat stress in a competitive situation; that is, changes in haematocrit between the heat-response tests were correlated to changes in total running during the game, r=-0.75; 90%CI [-0.88 to -0.51]. CONCLUSIONS: Heat acclimatisation responses and in turn, match-running performance in the heat, are highly individual. The players displaying the largest haematological adaptations were able to maintain the same activity when playing in the heat as when playing in temperate conditions. As such, team doctors might use acclimatisation indicators obtained from a heat-response test to predict the ability of individual players to cope with heat in competitive situations and individualise their preparation accordingly.

Physiological strain associated with high-intensity hypoxic intervals in highly trained young runners.

To examine the physiological strain associated with hypoxic high intensity interval training (HHIT), 8 highly trained young runners (age, 18.6 ± 5.3 years) randomly performed, 5 × 3-minute intervals in either normoxic (N, 90% of the velocity associated with VO(2max), vVO(2max)) or hypoxic (H, simulated 2,400-m altitude, 84% of νVO(2max)) conditions. Cardiorespiratory (ventilation [V(E)], oxygen consumption [V(O2)], heart rate [HR], oxygen saturation [SpO(2)]), rating of central perceived exertion (RPE(C)) responses, changes in neutrophils, erythropoietin (EPO), blood lactate ([La]) and, bicarbonate ([HCO(-)(3)]), vagal-related indices of HR variability (natural logarithm of the square root of the mean of the sum of the squares of differences [Ln rMSSD]) and maximal sprint and jump performances were compared after each session. Compared with N, H was associated with similar V(E) (Cohen's d ± 90% confidence limits, 0.0 ± 0.4, with % chances of higher/similar/lower values of 15/61/24) but at least lower VO(2) (-0.8 ± 0.4, 0/0/100), HR (-0.4 ± 0.4, 1/21/78), and SpO(2) (-1.8 ± 0.4, 0/0/100). Rating of perceived exertion was very likely higher (+0.5 ± 0.4, 92/8/0). Changes in [HCO(3)] (-0.6 ± 0.8, 5/13/83), [La] (+0.2 ± 0.4, 52/42/5), and EPO (+0.2 ± 0.4, 55/40/5) were at least possibly greater after H compared with those after N, whereas changes in neutrophils were likely lower (-0.5 ± 0.7, 4/15/81). Changes in 20-m sprint time (+0.20 ± 0.23, 49/50/1) were possibly lower after H. There was no clear difference in the changes in Ln rMSSD (+0.2 ± 1.7, 48/18/34) and jump (+0.3 ± 0.9, 60/25/15). In conclusion, although perceived as harder, HHIT is not associated with an exaggerated physiological stress in highly trained young athletes. The present results also confirm that HHIT may not be optimal for training both the cardiorespiratory and neuromuscular determinants of running performance in this population.

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

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.

Untargeted Metabolomics Identifies a Novel Panel of Markers for Autologous Blood Transfusion.

Untargeted metabolomics was used to analyze serum and urine samples for biomarkers of autologous blood transfusion (ABT). Red blood cell concentrates from donated blood were stored for 35−36 days prior to reinfusion into the donors. Participants were sampled at different time points post-donation and up to 7 days post-transfusion. Metabolomic profiling was performed using ACQUITY ultra performance liquid chromatography (UPLC), Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The markers of ABT were determined by principal component analysis and metabolites that had p < 0.05 and met ≥ 2-fold change from baseline were selected. A total of 11 serum and eight urinary metabolites, including two urinary plasticizer metabolites, were altered during the study. By the seventh day post-transfusion, the plasticizers had returned to baseline, while changes in nine other metabolites (seven serum and two urinary) remained. Five of these metabolites (serum inosine, guanosine and sphinganine and urinary isocitrate and erythronate) were upregulated, while serum glycourdeoxycholate, S-allylcysteine, 17-alphahydroxypregnenalone 3 and Glutamine conjugate of C6H10O2 (2)* were downregulated. This is the first study to identify a panel of metabolites, from serum and urine, as markers of ABT. Once independently validated, it could be universally adopted to detect ABT.

Integrating Whole Blood Transcriptomic Collection Procedures Into the Current Anti-Doping Testing System, Including Long-Term Storage and Re-Testing of Anti-Doping Samples.

Introduction: Recombinant human erythropoietin (rHuEPO) administration studies involving transcriptomic approaches have demonstrated a gene expression signature that could aid blood doping detection. However, current anti-doping testing does not involve collecting whole blood into tubes with RNA preservative. This study investigated if whole blood in long-term storage and whole blood left over from standard hematological testing in short-term storage could be used for transcriptomic analysis despite lacking RNA preservation. Methods: Whole blood samples were collected from twelve and fourteen healthy nonathletic males, for long-term and short-term storage experiments. Long-term storage involved whole blood collected into Tempus™ tubes and K2EDTA tubes and subjected to long-term (i.e., ‒80°C) storage and RNA extracted. Short-term storage involved whole blood collected into K2EDTA tubes and stored at 4°C for 6‒48 h and then incubated at room temperature for 1 and 2 h prior to addition of RNA preservative. RNA quantity, purity, and integrity were analyzed in addition to RNA-Seq using the MGI DNBSEQ-G400 on RNA from both the short- and long-term storage studies. Genes presenting a fold change (FC) of >1.1 or < ‒1.1 with p ≤ 0.05 for each comparison were considered differentially expressed. Microarray analysis using the Affymetrix GeneChip® Human Transcriptome 2.0 Array was additionally conducted on RNA from the short-term study with a false discovery ratio (FDR) of ≤0.05 and an FC of >1.1 or < ‒1.1 applied to identify differentially expressed genes. Results: RNA quantity, purity, and integrity from whole blood subjected to short- and long-term storage were sufficient for gene expression analysis. Long-term storage: when comparing blood tubes with and without RNA preservation 4,058 transcripts (6% of coding and non-coding transcripts) were differentially expressed using microarray and 658 genes (3.4% of mapped genes) were differentially expressed using RNA-Seq. Short-term storage: mean RNA integrity and yield were not significantly different at any of the time points. RNA-Seq analysis revealed a very small number of differentially expressed genes (70 or 1.37% of mapped genes) when comparing samples stored between 6 and 48 h without RNA preservative. None of the genes previously identified in rHuEPO administration studies were differently expressed in either long- or short-term storage experiments. Conclusion: RNA quantity, purity, and integrity were not significantly compromised from short- or long-term storage in blood storage tubes lacking RNA stabilization, indicating that transcriptomic analysis could be conducted using anti-doping samples collected or biobanked without RNA preservation.

Horseradish-peroxidase-conjugated anti-erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept.

Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish-peroxidase (HRP)-conjugated anti-EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non-specific cross-reactivity with secondary antibodies. An in-house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti-human EPO-HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in-house anti-EPO-HRP conjugate was performed as well as the current two-step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in-house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti-EPO-HRP conjugate showed comparable specificity but had lower sensitivity to both the in-house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP-conjugated anti-EPO antibody to reduce analysis time for EPO in doping control.

Biosafety Level 2 cabinet UV-C light exposure of sports antidoping human urine samples does not affect the stability of selected prohibited substances.

The current study examined the stability of several antidoping prohibited substances analytes in urine after 15-min exposure to UV-C light in a Biosafety Level 2 cabinet. The urine matrices were exposed within the original antidoping bottles with the aim to destroy DNA/RNA and possible SARS CoV-2. The analytes small molecules Phase I and Phase II metabolites and peptides, in total 444, endogenous, internal standards, and prohibited substances, pH, and specific gravity in urine were studied. The accredited analytical methods were used by Anti-Doping Laboratory Qatar for the comparison of data of the same urine samples analyzed with and without UV-C exposure. In the study conditions, no problems of stability were detected in the substances spiked in the urine samples exposed in the UV-C irradiation.

Total hemoglobin mass, iron status, and endurance capacity in elite field hockey players.

The aims of this study were as follows: To evaluate total hemoglobin mass (tHbmass) in international field hockey players; to examine the correlation between tHbmass and maximum oxygen uptake (VO2max); and to assess influences of iron status on tHbmass and on VO2max. The players of the German women's (N = 17, aged 24.8 +/- 3.0 [21-31] years) and men's (N = 17, aged 24.2 +/- 2.9 [19-32] years) national field hockey team were investigated. tHbmass was measured by an optimized carbon monoxide rebreathing method. The following parameters were measured in venous blood: Hemoglobin concentration (Hbconc), hematocrit (Hct), number and percentage of reticulocytes, reticulocyte hemoglobin content, serum iron, serum ferritin, serum transferrin, unsaturated iron-binding capacity, and serum soluble transferrin receptor concentration. VO2max was determined in a treadmill test. tHbmass (women: 10.6 +/- 1.1 g/kg; men: 12.5 +/- 0.9 g/kg) correlated to VO2max (women: 46.6 +/- 2.9 mL/min/kg; men: 55.8 +/- 4.0 mL/min/kg) in women (r = 0.56, p < 0.05) and in men (r = 0.57, p < 0.05), whereas Hbconc and Hct did not. The investigated parameters of iron status showed no association to tHbmass or to VO2max. In conclusion, tHbmass can be used as an indicator for endurance capacity in elite field hockey players, whereas Hbconc may not. tHbmass or VO2max were not influenced by the actual iron status of the investigated athletes.

Erythroferrone as a sensitive biomarker to detect stimulation of erythropoiesis.

Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to erythropoietin stimulation. ERFE suppresses the hepatic synthesis of the master iron-regulatory hormone, hepcidin. The impact of erythropoiesis stimulation on ERFE secretion in humans is poorly understood. This paucity of information is due in part to the lack of available means for ERFE quantification in serum samples. The present study tested a new sensitive sandwich immunoassay for human ERFE. This assay was used to demonstrate that injection of various erythropoiesis stimulating agents (ESAs) increased the blood ERFE levels in healthy volunteers. After exogenous stimulation of erythropoiesis, ERFE increased up to 8-fold with a detection window of 13 days. The impact of one unit of blood withdrawal on erythropoiesis stimulation of ERFE was also tested. ERFE significantly increased after blood withdrawal in subjects injected with both iron and saline solution, suggesting that iron supplementation did not mask the ERFE increase after blood withdrawal. The effects of exercise-induced muscle damage on ERFE was assessed by comparing ERFE levels with creatine kinase levels in samples from subjects with heavy exercise loads, and determined that this was not a confounder. The ERFE assay is a sensitive means to investigate the connection between iron metabolism and erythropoiesis in humans, and to detect ESA abuse in the antidoping field.

A novel mixed living high training low intervention and the hematological module of the athlete biological passport.

Exposure to either natural or simulated hypoxia induces hematological adaptations that may affect the parameters of the Athlete Biological Passport (ABP). The aim of the present study was to examine the effect of a novel, mixed hypoxic dose protocol on the likelihood of producing an atypical ABP finding. Ten well-trained middle-distance runners participated in a "live high, train low and high" (LHTLH) altitude training camp for 14 days. The participants spent ˜6 hr.d-1 at 3000-5400 m during waking hours and ˜10 h.d-1 overnight at 2400-3000 m simulated altitude. Venous blood samples were collected before (B0), and after 1 (D1), 4 (D4), 7 (D7), and 14 (D14) days of hypoxic exposure, and again 14 days post exposure (P14). Samples were analyzed for key parameters of the ABP including reticulocyte percentage (Ret%), hemoglobin concentration ([Hb]), and the OFF-score. The ABP adaptive model was administered at a specificity of 99% to test for atypical findings. We found significant changes in [Hb] and Ret% during the hypoxic intervention. Consequently, this led to ABP threshold deviations at 99% specificity in three participants. Only one of these was flagged as an "atypical passport finding" (ATPF) due to deviation of the OFF-score. When this sample was evaluated by ABP experts it was considered "normal". In conclusion, it is highly unlikely that the present hypoxic exposure protocol would have led to a citation for a doping violation according to WADA guidelines.

Hyperhydration Effect on Pharmacokinetic Parameters and Detection Sensitivity of Recombinant Human Erythropoietin in Urine and Serum Doping Control Analysis of Males.

Excessive fluid intake, that is, hyperhydration, may be adopted by athletes as a masking method during antidoping sample collection to influence the excretion patterns of doping agents and, therefore, manipulate their detection. The aim of this exploratory study was to assess the hyperhydration effect on the detection sensitivity of recombinant human erythropoietin (rHuEPO) by sodium N-lauroyl sarcosinate ("sarkosyl") polyacrylamide gel electrophoresis analysis. The influence of hyperhydration on the serum and urinary pharmacokinetic (PK) profiles of rHuEPO was also investigated. Seven healthy physically active nonsmoking Caucasian males participated in a 31-day clinical study comprising a baseline (days 0, 1-3, and 8-10) and a drug phase (days 15-17, 22-24, and 29-31). Epoetin beta was administered subcutaneously at a single dose of 3000 IU on days 15, 22, and 29. Hyperhydration was applied in the morning on 3 consecutive days (days 1-3, 8-10, 22-24, and 29-31), that is, 0, 24, and 48 h after first fluid ingestion. Water and a commercial sports drink were used as hyperhydration agents (20 mL/kg body weight). Serum and urinary concentration-time profiles were best described by a one-compartment PK model with zero-order absorption. Delayed absorption was observed after hyperhydration and, therefore, lag time was introduced in the PK model. Results showed no significant difference (p > 0.05) on serum or urinary erythropoietin concentrations under hyperhydration conditions. A trend for decreasing volume of distribution and increasing clearance after hyperhydration was observed, mainly after sports drink consumption. However, no significant differences (p > 0.05) due to hyperhydration for any of the serum PK parameters calculated by noncompartmental PK analysis were observed. Renal excretion of endogenous erythropoietin and rHuEPO, as reflected on the urinary cumulative amount, was increased approximately twice after hyperhydration and this supports the nonsignificant difference on the urinary concentrations. Analysis of serum and urine samples was able to detect rHuEPO up to 72 h after drug administration. The detection window of rHuEPO remained unaffected after water or sports drink ingestion. Hyperhydration had no effect on the detection sensitivity of EPO either in serum or urine samples.

Stability tests for hematological parameters in antidoping analyses.

The purpose of this study was to evaluate the influences of delayed sample analysis on the stability of erythrocyte parameters and reticulocyte parameters for antidoping tests performed on the ADVIA120 system. We analyzed erythrocyte count, hemoglobin, hematocrit, mean cell volume, percentage of hypochromic erythrocytes, percentage of macrocytes, absolute reticulocyte count, percentage of reticulocytes, mean cell volume of reticulocytes, cell hemoglobin of reticulocytes, percentage of high-fluorescent reticulocytes, and OFF-Score during a 48-hour storage period at 4 degrees C or 21 degrees C. Data analysis was performed by fitting linear or nonlinear mixed-effects models. We then modeled appropriate trends and tested the curve parameters' interaction with the ambient temperature. We observed that chilling of samples to 4 degrees C generally proved more advantageous than storage at room temperature. Sufficient stability during 48-hour intervals was demonstrated for erythrocyte count, hemoglobin, percentage of reticulocytes, absolute reticulocyte count, cell hemoglobin of reticulocytes, percentage of hypochromic erythrocytes, percentage of high-fluorescent reticulocytes, and OFF-Score. We concluded that for the establishment of individual blood profiles the corresponding samples should be transported and stored at 4 degrees C. Analysis should be performed not later than 48 hours after sampling.

Whole Blood Storage in CPDA1 Blood Bags Alters Erythrocyte Membrane Proteome.

Autologous blood transfusion (ABT) has been frequently abused in endurance sport and is prohibited since the mid-1980s by the International Olympic Committee. Apart from any significant performance-enhancing effects, the ABT may pose a serious health issue due to aging erythrocyte-derived "red cell storage lesions." The current study investigated the effect of blood storage in citrate phosphate dextrose adenine (CPDA1) on the red blood cell (RBC) membrane proteome. One unit of blood was collected in CPDA1 blood bags from 6 healthy female volunteers. RBC membrane protein samples were prepared on days 0, 14, and 35 of storage. Proteins were digested in gel and peptides separated by nanoliquid chromatography coupled to tandem mass spectrometry resulting in the confident identification of 33 proteins that quantitatively change during storage. Comparative proteomics suggested storage-induced translocation of cytoplasmic proteins to the membrane while redox proteomics analysis identified 14 proteins prone to storage-induced oxidation. The affected proteins are implicated in the RBC energy metabolism and membrane vesiculation and could contribute to the adverse posttransfusion outcomes. Spectrin alpha chain, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase, and ankyrin-1 were the main proteins affected by storage. Although potential biomarkers of stored RBCs were identified, the stability and lifetime of these markers posttransfusion remain unknown. In summary, the study demonstrated the importance of studying storage-induced alterations in the erythrocyte membrane proteome and the need to understand the clearance kinetics of transfused erythrocytes and identified protein markers.

Analysis of RBC-microparticles in stored whole blood bags - a promising marker to detect blood doping in sports?

Blood doping in sports is prohibited by the World Anti-Doping Agency (WADA). To find a possible biomarker for the detection of blood doping, we investigated the changes in blood stored in CPDA-1 blood bags of eight healthy subjects who donated one unit of blood. Aliquots were taken on days 0, 14, and 35. Platelet-free plasma was prepared and stored at -80°C until analysis on a flow cytometer dedicated for the analysis of microparticles (MPs). Changes in the number of red blood cell (RBC) -MPs were highly significant (p < 0.0001) with a mean of 219 (10^3/µL) on day 0 changing to 23 120 (10^3/µL) on day 14 and 29 310 (10^3/µL) on day 35. We conclude that RBC-MPs seem to be a promising biomarker for doping control but confirmation by a transfusion study is necessary.

Analytical progresses of the World Anti-Doping Agency Olympic laboratories: a 2016 update from London to Rio.

The 2016 Olympic and Paralympic Games, the biggest event in human sports, was held in Rio de Janeiro with more than 10,500 athletes from 206 countries over the world competing for the highest of sports honors, an Olympic medal. With the hope that the Olympic ideal accompanies all aspects of the XXXI Olympiad, WADA accredited antidoping laboratories use the spearhead of analytical technology as a powerful tool in the fight against doping. This review summarizes the main analytical developments applied in antidoping testing methodology combined with the main amendments on the WADA regulations regarding analytical testing starting from the 2012 London Olympics until the 2016 Olympic Games in Rio de Janeiro.

The effect of a period of intense exercise on the marker approach to detect growth hormone doping in sports.

The major objective of this study was to investigate the effects of several days of intense exercise on the growth hormone marker approach to detect doping with human growth hormone (hGH). In addition we investigated the effect of changes in plasma volume on the test. Fifteen male athletes performed a simulated nine-day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race + three days before and after). Plasma volumes were estimated by the optimized CO Rebreathing method. IGF-1 and P-III-NP were analyzed by Siemens Immulite and Cisbio Assays, respectively. All measured GH 2000 scores were far below the published decision limits for an adverse analytical finding. The period of exercise did not increase the GH-scores; however the accompanying effect of the increase in Plasma Volume yielded in essentially lower GH-scores. We could demonstrate that a period of heavy, long-term exercise with changes in plasma volume does not interfere with the decision limits for an adverse analytical finding.