Impact of low-volume blood withdrawal on hematological biomarkers for the athlete biological passport.

This study investigated the impact of low-volume blood withdrawal on the hematological biomarkers currently considered for anti-doping purposes. After baseline measurement (D - 7), a 140 mL blood withdrawal was completed (D + 0) on 12 healthy volunteers, followed by weekly monitoring for 21 days (D + 7 - 21). Each visit consisted of a full blood count (Sysmex XN-1000) and duplicate blood volume measurements by CO-rebreathing. A significant decrease in total hemoglobin mass (Hbmass) (-2.3%, p = 0.007) and red blood cell volume (RBCV) (-2.8%, p = 0.028) was reported at D + 7. Despite no atypical passport finding (ATPF) when considering the athlete biological passport adaptive longitudinal model, hemoglobin concentration ([Hb]) increased significantly at D + 21 (+3.8%, p = 0.031). Besides, ferritin (FERR) was significantly downregulated at all points following blood withdrawal, with the largest decrease occurring at D + 7 (-26.6%, p < 0.001). Regardless of the presumable effect of blood reinfusion on ABP biomarkers, these results illustrate the challenge of monitoring hematological variables for the detection of low-volume blood withdrawal. Finally, this study outlines the sensitivity of FERR to altered erythropoiesis to support the implementation of iron markers as complementary variables for the longitudinal monitoring of blood doping, despite the potential influence of confounding factors (e.g., iron supplementations).

The effects of iron injection on blood doping biomarkers in dried blood spots.

Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in antidoping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte-related-mRNAs, such as 5'-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte-related-mRNAs analyses were performed by RT-qPCR. Ferritin level in DBS was measured with enzyme-linked immunosorbent assay (ELISA) method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte-related mRNAs in DBSs as biomarkers of blood doping to target in antidoping analyses.

EPO and the athlete biological passport: Hematological results from a placebo-controlled, boosting and microdose EPO administration in male recreational athletes.

Hematological results in the context of the Athlete Biological Passport (ABP) from a placebo-controlled EPO administration study are provided here. Twelve participants administered eight subcutaneous boosting doses of epoetin alfa (at 40 IU/kg) over the course of 20 days. After a 10-day washout period, the same volunteers administered six microdoses (900 IU), intravenously, over 13 days. A blinded placebo cohort followed the same dosing pattern, administering saline instead of EPO. All participants supplemented with oral iron, daily, throughout the entirety of the study. In the EPO cohort, as expected, significant changes from baseline were identified in IRF, RET#, RET%, RDW, HCT, HGB, and RBC. No meaningful changes were identified in the placebo cohort population. From the ABP perspective, atypical passport findings (ATPF) were identified in 49% of the samples collected during the boosting and initial washout phases, and 24% of the samples during the microdosing and final washout phases. ATPFs from this cohort were flagged as late as Day 70, the final day of the study. Only a single ATPF was identified from all samples collected from the placebo cohort. ABPs from all volunteers in the study are provided as an avenue to visually convey differences in magnitude and timing of the hematological changes caused by EPO on the individual level. These data are expected to provide important content for Athlete Passport Management Units and ABP expert panels alike.

Simultaneous detection of 93 anabolic androgenic steroids in dietary supplements using gas chromatography tandem mass spectrometry.

In recent years, anabolic androgenic steroids (AASs) have been frequently detected as undeclared ingredients in dietary supplements, where the adverse analytical findings (AAFs) were obtained from analysis of athletes' urine samples after ingestion. In our present study, a GC-MS/MS method for simultaneous detection of 93 anabolic steroids was developed. The chromatographic and mass spectrometric conditions were optimized, and selective reaction monitoring (SRM) mode was adopted to obtain the necessary sensitivity. The whole sample analysis process was completed within 23 min, and the limit of detection (LOD) was 0.5-4 ng.g-1 for solid samples and 0.1-0.8 ng.mL-1 for liquid samples. This method was verified according to World Anti-Doping Agency (WADA) regulations. In addition, the method was found to be specific, accurate. The developed method was then applied to a routine analysis of more than 300 liquid and solid dietary supplements, and one testosterone-positive sample was found. Three suspected drugs, (4-hydroxyandrostenedione, DHEA, and 6-Br androstenedione) were found in three dietary supplements obtained from the Internet through the pretreatment method of this study. This study provides a high-throughput method for screening and monitoring the ingredients of supplements and their subsequent harm to public health.

Education Interventions to Improve Knowledge, Beliefs, Intentions and Practices with Respect to Dietary Supplements and Doping Substances: A Narrative Review.

The misuse of dietary supplements and doping substances is commonly associated with toxicity, nutritional imbalances, and health and psychological consequences. This is alarming especially in light of the increasing prevalence of the use of dietary supplements and doping, particularly among young adults including athletes. There is evidence that education interventions can lead to improved knowledge, intentions, and practices. However, no review has summarized and evaluated the effectiveness of such interventions. The aim of this article is to review the characteristics, contents and effects of education interventions that were designed and implemented to improve knowledge, attitudes, beliefs and intentions with respect to the use of dietary supplements and doping agents in different populations. PubMed, Scopus, CINAHL, PsycInfo and Google Scholar were searched for English-language education interventions targeting dietary supplements and doping substances. A total of 20 articles were identified and have generally provided consistent findings. Most interventions reported a significant improvement in knowledge on dietary supplements and doping agents. Unfortunately, the heavy reliance on self-reported assessment tools limits the validity of these interventions, with almost all articles targeting athletes and adolescents.

An Untargeted Urine Metabolomics Approach for Autologous Blood Transfusion Detection.

PURPOSE: Autologous blood transfusion is performance enhancing and prohibited in sport but remains difficult to detect. This study explored the hypothesis that an untargeted urine metabolomics analysis can reveal one or more novel metabolites with high sensitivity and specificity for detection of autologous blood transfusion. METHODS: In a randomized, double-blinded, placebo-controlled, crossover design, exercise-trained men (n = 12) donated 900 mL blood or were sham phlebotomized. After 4 wk, red blood cells or saline were reinfused. Urine samples were collected before phlebotomy and 2 h and 1, 2, 3, 5, and 10 d after reinfusion and analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. Models of unique metabolites reflecting autologous blood transfusion were attained by partial least-squares discriminant analysis. RESULTS: The strongest model was obtained 2 h after reinfusion with a misclassification error of 6.3% and 98.8% specificity. However, combining only a few of the strongest metabolites selected by this model provided a sensitivity of 100% at days 1 and 2 and 66% at day 3 with 100% specificity. Metabolite identification revealed the presence of secondary di-2-ethylhexyl phtalate metabolites and putatively identified the presence of (iso)caproic acid glucuronide as the strongest candidate biomarker. CONCLUSIONS: Untargeted urine metabolomics revealed several plasticizers as the strongest metabolic pattern for detection of autologous blood transfusion for up to 3 d. Importantly, no other metabolites in urine seem of value for antidoping purposes.

Analysis of cobalt for human sports drug testing purposes using ICP- and LC-ICP-MS.

Due to the current demands in the fight against manipulation of blood and blood components, commonly referred to as "blood doping" in sports drug testing, specific and sensitive detection methods enabling the detection of prohibited substances and methods of doping are required. Similar to illicit blood transfusions, erythropoiesis stimulating agents have been shown to be misused in sport, aiming at improving an athlete's aerobic capacity and endurance performance. Amongst other strategies, the administration of ionic cobalt (Co2+ ) can increase the number of erythrocytes by stimulating the endogenous erythropoietin (EPO) biosynthesis. Conversely, several organic Co-containing compounds such as cyanocobalamin (vitamin B12) are not prohibited in sports, and thus, an analytical differentiation of permitted and banned contributions to urinary Co-concentrations is desirable. An excretion study with daily applications of either 1 mg of CoCl2 or 1 mg of cyanocobalamin was conducted with 20 volunteers over a period of 14 consecutive days. Urine, plasma, and concentrated red blood cells were analyzed for their cobalt content. The samples were collected starting 7 days before the administration until 7 days after. Total Co concentrations were analyzed by using inductively coupled plasma mass spectrometry (ICP-MS), which yielded significantly elevated levels exclusively after inorganic cobalt intake. Furthermore, a liquid chromatography (LC)-ICP-MS approach was established and employed for the simultaneous determination of organically bound and inorganic cobalt by chromatographic separation within one single run. The analytical approach offers the option to further develop detection methods of illegal Co2+ supplementation in sport.

Time Trial Performance Is Sensitive to Low-Volume Autologous Blood Transfusion.

PURPOSE: This study tested the hypothesis that autologous blood transfusion (ABT) of ~50% of the red blood cells (RBC) from a standard 450-mL phlebotomy would increase mean power in a cycling time trial. In addition, the study investigated whether further ABT of RBC obtained from another 450-mL phlebotomy would increase repeated cycling sprint ability. METHODS: In a randomized, double-blind, placebo-controlled crossover design (3-month wash-out), nine highly trained male subjects donated two 450-mL blood bags each (BT trial) or were sham phlebotomized (PLA trial). Four weeks later, a 650-kcal time trial (n = 7) was performed 3 d before and 2 h after receiving either ~50% (135 mL) of the RBC or a sham transfusion. On the following day, transfusion of RBC (235 mL) from the second donation or sham transfusion was completed. A 4 × 30-s all-out cycling sprint interspersed by 4 min of recovery was performed 6 d before and 3 d after the second ABT (n = 9). RESULTS: The mean power was increased in time trials from before to after transfusion (P < 0.05) in BT (213 ± 35 vs 223 ± 38 W; mean ± SD) but not in PLA (223 ± 42 vs 224 ± 46 W). In contrast, the mean power output across the four 30-s sprint bouts remained similar in BT (639 ± 35 vs 644 ± 26 W) and PLA (638 ± 43 vs 639 ± 25 W). CONCLUSIONS: ABT of only ~135 mL of RBC is sufficient to increase mean power in a 650-kcal cycling time trial by ~5% in highly trained men. In contrast, a combined high-volume transfusion of ~135 and ~235 mL of RBC does not alter 4 × 30-s all-out cycling performance interspersed with 4 min of recovery.

Detecting Autologous Blood Transfusion in Doping Control: Biomarkers of Blood Aging and Storage Measured by Flow Cytofluorimetry.

BACKGROUND: Blood transfusions are banned by the World Anti-Doping Agency as a form of "blood doping". A method of detection of homologous blood transfusion (HBT) has been implemented by the accredited anti-doping laboratories worldwide; however, no internationally recognized method has been finalized so far for the direct detection of autologous blood transfusions, which can at present be revealed only by targeted longitudinal profiling of key blood parameters. METHODS: The present article reports the results of an investigation aimed to pre-select potential biomarkers of blood aging and storage that can be measured to identify the presence in the sample of reinfused blood. Microparticles from platelets and erythrocytes, erythrocytes size and density, annexin V (as a marker of phosphatidylserine externalization), and the membrane surface antigens CD 55 and CD 59, were specifically considered as potential biomarkers and measured by flow cytofluorimetric techniques. RESULTS AND CONCLUSION: Our results indicate that the parameters more strongly affected by the ex vivo storage of whole blood are erythrocytes size and density, annexin V and microparticles. Although the real diagnostic value of the proposed biomarkers shall obviously be confirmed by further studies carried out on blood samples collected after an actual autologous blood transfusion, these results appear very encouraging towards the development of a direct method for detecting autologous blood transfusion in sport doping.

Changes in hemoglobin profile reflect autologous blood transfusion misuse in sports.

The changes in hemoglobin (Hb) profile following autologous blood transfusion (ABT) for the first time were studied for anti-doping purposes. Twenty-four healthy, trained male subjects (aged 18‒40) were enrolled and randomized into either the transfusion (T) or control (C) groups. Blood samples were taken from the T subjects at baseline, after withdrawal and reinfusion of 450 ml of refrigerated or cryopreserved blood, and from C subjects at the same time points. Hematological variables (Complete blood count, Reticulocytes, Immature Reticulocytes Fraction, Red-cell Distribution Width, OFF-hr score) were measured. The Hb types were analyzed by high-performance liquid chromatography and the Hemoglobin Profile Index (HbPI) arbitrarily calculated. Between-group differences were observed for red blood cells and reticulocytes. Unlike C, the T group, after withdrawal and reinfusion, showed a significant trend analysis for both hematological variables (Hemoglobin concentration, reticulocytes, OFF-hr score) and Hb types (glycated hemoglobin-HbA1c, HbPI). The control charts highlighted samples with abnormal values (> 3-SD above/below the population mean) after reinfusion for hematological variables in one subject versus five subjects for HbA1c and HbPI. A significant ROC-curve analysis (area = 0.649, p = 0.015) identified a HbA1c cut-off value ≤ 2.7% associated to 100% specificity of blood reinfusion (sensitivity 25%). Hemoglobin profile changed in trained subjects after ABT, with abnormal values of HbA1c and HbPI in 42% of subjects after reinfusion. Future studies will confirm the usefulness of these biomarkers in the anti-doping field.

Transcriptomic biomarkers of altered erythropoiesis to detect autologous blood transfusion.

Autologous blood transfusion is a powerful means of improving performance and remains one of the most challenging methods to detect. Recent investigations have identified 3 candidate reticulocytes genes whose expression was significantly influenced by blood transfusion. Using quantitative reverse transcription polymerase chain reaction as an alternative quantitative method, the present study supports that delta-aminolevulinate synthase 2 (ALAS2), carbonic anhydrase (CA1), and solute carrier family 4 member 1 (SLC4A1) genes are down-regulated post-transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. Moreover, the repression of reticulocytes' gene expression was more pronounced than the diminution of immature reticulocyte fraction and reticulocyte percentage following blood transfusion. It suggests that the 3 candidate genes are reliable predictors of bone marrow's response to blood transfusion and that they represent potential biomarkers for the detection of this method prohibited in sports.

[Nutritional supplements in sports - sense, nonsense or hazard?] / Nahrungsergänzungsmittel im Sport ­ Sinn, Unsinn oder Gefahr?

The excessive sale of dietary supplements (DSs) has become a global multi-billion market as more and more people turn to DSs for a healthy lifestyle or for aesthetic reasons. DSs are also increasingly popular among athletes; 50-85% of recreational and 35-100% of competitive athletes report taking DSs, the latter more regularly. Unless pathological deficiencies are detected, the intake of DSs for recreational athletes is not recommended. While it may be advisable for competitive athletes to supplement their diet with certain macronutrients (proteins and carbohydrates), many micronutrients (vitamins, minerals) as well as allegedly performance enhancing DSs may only show minimal impact under specific conditions and for certain sports. However, most products lack proof of their effectiveness. In some cases, DSs may even have negative effects and reduce performance. Furthermore, competitive athletes should be aware of the fact that DSs may lead to positive doping tests, as they bear the risk of being contaminated with banned substances, or components may be banned substances themselves. Every single case of taking DSs should therefore be critically assessed and discussed with experts prior to use. DSs cannot replace a balanced diet and hard practice.

Understanding alterations on blood and biochemical parameters in athletes that use dietary supplements, steroids and illicit drugs.

In recent years it was verified there are an alarming growing number of teenagers and young adults using a combination of dietary supplements (DS) anabolic androgenic steroids (AAS) and drugs of abuse. This practice is used to improve physical fitness and appearance, may cause serious side effects. This article shows the alterations in the hematological and renal function parameters associate with these substances in 40 athletes. This research involved three steps: 1-the administration of a self-completion questionnaire ; 2-the assessment of hematological and biochemical parameters of renal function and; 3-toxicological urinalysis. Hematological and biochemical tests were conducted in an accredited laboratory and the toxicological urinalysis was validated in our laboratory using liquid-liquid extraction (LLE) and gas chromatography-mass spectrometry (GC-MS). The testosterone levels in the participants who consumed steroids increased 20-60% and alterations in serum creatinine, urea and uric reached values of up to 1.9; 60.6 and 7.5mg/dL, respectively. The toxicological urinalysis supports self-reports confirming the use of AAS and recreational drugs, putting at risk the health of those athletes increasing the chances of kidney diseases.

Efectos de la suplementación con testosterona sobre el rendimiento en resistencia / Efeitos da suplementação com testosterona sobre o desempenho da resistência / Effects of doping with testosterone on endurance performance

El dopaje en el deporte tiene su origen en la Grecia Clásica. Sin embargo, a lo largo del siglo pasado y hasta la actualidad, la utilización de este tipo de prácticas fraudulentas en el deporte ha ido en aumento. Entre las sustancias dopantes más utilizadas destacan la testosterona y sus derivados sintéticos, los anabolizantes sintéticos. A pesar de que estas sustancias prohibidas se han utilizado para la mejora del rendimiento en pruebas de fuerza y potencia, frecuentemente se detectan positivos en deportistas de resistencia. Los objetivos del presente estudio han sido informar acerca de los efectos ergogénicos de la suplementación con testosterona y anabolizantes sintéticos sobre el rendimiento en resistencia, a través de cambios sobre parámetros sanguíneos, así como los efectos secundarios que tienen sobre la salud. Para ello, se ha realizado una revisión en bases de datos como Elsevier, Medline, Pubmed y Web of Science incluyendo términos como testosterone, anemia, doping, endurance, erythropoietin, hepcidin e iron. La hepcidina se ha propuesto la principal reguladora de las reservas corporales de hierro y la suplementación con testosterona puede afectar a la síntesis de dicha hormona. Los efectos de la testosterona sobre la hepcidina podrían hacer mejorar tanto la capacidad de transporte como de difusión de oxígeno. De este modo, el dopaje con testosterona podría tener un potencial efecto ergogénico en modalidades de resistencia. Sin embargo, dichas mejoras pueden tener efectos negativos sobre el estado de salud del deportista, entre los que se encuentran trastornos metabólicos, orgánicos, psicológicos e inmunosupresión (AU)

Doping in sport has its origins in Ancient Greece. However, over the last century to the present, the use of such dishonest practices has increased. Among the most widely used performance enhancing drugs is the use of testosterone and its synthetics anabolics. Although these prohibited substances have been used to increase performance in test of strength and power, due to the ability to cause hypertrophy, very frequent it's detecting positive test doping by in endurance athletes by testosterone or synthetics anabolics. The aim of this study was to report the ergogenic effects of testosterone supplementation and synthetics anabolics on endurance performance, through changes on blood parameters. To this end, it has conducted a review in different databases such as Elsevier, Medline, Pubmed and Web of Science where terms such as testosterone, anemia, doping, endurance, erythropoietin, hepcidin and iron were included. Hepcidin has been proposed main regulator of body iron stores and testosterone supplementation may affect the synthesis of the hormone. The effects of testosterone on hepcidin could improve both transport capacity and oxygen diffusion. Thus, doping with testosterone could have a potential effect on ergogenic resistance patterns. However, such improvements can have negative effects on the health of the athlete like metabolic, organic, psychological disorders and immunosuppression (AU)

O doping no esporte tem suas origens na Grécia Antiga. No entanto, do último século para o presente, a utilização de tais práticas desonestas aumentou. Entre as drogas que melhoram o desempenho mais amplamente utilizadas são o uso de testosterona e seus anabolizantes sintéticos. Embora estas substâncias proibidas têm sido usados para aumentar o desempenho em testes de força e potência, devido à capacidade de causar hipertrofia, é muito frequentes detecção de doping por testosterona ou anabolizantes sintéticos no teste de atletas de endurance. O objetivo deste estudo foi relatar os efeitos ergogênicos da suplementação de testosterona e anabolizantes sintéticos sobre o desempenho de resistência, através de mudanças nos parâmetros sanguíneos. Para este efeito, procedeu a uma revisão em diferentes bancos de dados, tais como Elsevier, Medline, Pubmed e Web of Science, onde termos como a testosterona, anemia, doping, resistência, eritropoietina, hepcidina e ferro foram incluídos. Hepcidina foi proposto como principal regulador das reservas de ferro do corpo e a suplementação de testosterona pode afetar a síntese do hormonio. Os efeitos da testosterona sobre hepcidina poderia melhorar tanto a capacidade de transporte e difusão de oxigênio. Assim, a dopagem com testosterona pode ter um efeito potencial sobre padrões de resistência ergogênicos. No entanto, essas melhorias podem ter efeitos negativos sobre a saúde do atleta como metabolismo, distúrbios psicológicos orgânicos e imunossupressão (AU)

Autologous Doping with Cryopreserved Red Blood Cells - Effects on Physical Performance and Detection by Multivariate Statistics.

The discovery of erythropoietin (EPO) simplified blood doping in sports, but improved detection methods, for EPO has forced cheating athletes to return to blood transfusion. Autologous blood transfusion with cryopreserved red blood cells (RBCs) is the method of choice, because no valid method exists to accurately detect such event. In endurance sports, it can be estimated that elite athletes improve performance by up to 3% with blood doping, regardless of method. Valid detection methods for autologous blood doping is important to maintain credibility of athletic performances. Recreational male (N = 27) and female (N = 11) athletes served as Transfusion (N = 28) and Control (N = 10) subjects in two different transfusion settings. Hematological variables and physical performance were measured before donation of 450 or 900 mL whole blood, and until four weeks after re-infusion of the cryopreserved RBC fraction. Blood was analyzed for transferrin, iron, Hb, EVF, MCV, MCHC, reticulocytes, leucocytes and EPO. Repeated measures multivariate analysis of variance (MANOVA) and pattern recognition using Principal Component Analysis (PCA) and Orthogonal Projections of Latent Structures (OPLS) discriminant analysis (DA) investigated differences between Control and Transfusion groups over time. Significant increase in performance (15 ± 8%) and VO2max (17 ± 10%) (mean ± SD) could be measured 48 h after RBC re-infusion, and remained increased for up to four weeks in some subjects. In total, 533 blood samples were included in the study (Clean = 220, Transfused = 313). In response to blood transfusion, the largest change in hematological variables occurred 48 h after blood donation, when Control and Transfused groups could be separated with OPLS-DA (R2 = 0.76/Q2 = 0.59). RBC re-infusion resulted in the best model (R2 = 0.40/Q2 = 0.10) at the first sampling point (48 h), predicting one false positive and one false negative. Over all, a 25% and 86% false positives ratio was achieved in two separate trials. In conclusions, autologous re-infusion of RBCs increased VO2max and performance as hypothesized, but hematological profiling by multivariate statistics could not reach the WADA stipulated false positive ratio of <0.001% at any time point investigated. A majority of samples remained within limits of normal individual variation at all times.

Rapid assessment of the illegal presence of 1,3-dimethylamylamine (DMAA) in sports nutrition and dietary supplements using 1H NMR spectroscopy.

1,3-Dimethylamylamine (DMAA) is a stimulant that can be found in pre-workout sports nutrition and dietary supplements. This practice is illegal because DMAA is not a safe food ingredient but rather an unapproved medicinal compound due to its pharmacological action. In order to determine the DMAA content in such products, a nuclear magnetic resonance (NMR) spectroscopic method was developed and validated (DMAA was quantified as DMAA-HCl). For quantification, the collective integral from two of the methyl groups of the molecule in the range δ 0.92-0.84 ppm was used. The method was linear over the examined range of 1-21 g/kg (R(2) = 0.9937). The recoveries from spiked concentrations (0.1-6 g/kg) ranged between 85% and 105% (96% on average), with a relative standard deviation (RSD) of 1% for an authentic sample. The detection limit was 0.03 g/kg and the quantification limit was 0.08 g/kg (calculated for 75 mg sample weight). The actual DMAA-HCl content in the sample was quantified using calibration curves (external standardization) or 3,5-dinitrobenzoic acid as single-point internal standard. The developed NMR methodology was applied for the analysis of 16 products, from which 9 samples were found positive (the DMAA-HCl concentration varied between 3.1 g/kg and 415 g/kg). The method can be recommended for routine use in food testing, customs or doping control laboratories.

Detecting doping use: more than an analytical problem.

The recent Armstrong case, where more than 250 negative doping tests are confronted with the athlete's confession of erythropoietin use, blood doping, steroid, and growth hormone abuse, illustrates the limitations of current laboratory tests in detecting doping in sport. Despite numerous doping controls and simultaneous indications of common doping abuse among professional athletes in the last two decades, the number of positive urine tests for recombinant human erythropoietin (rHuEPO) remains remarkably low. Athletes are using various masking strategies, among them protease inhibitors, intravenous injections of rHuEPO and alternative erythropoiesis stimulating agents. As one of the countermeasures, the Athlete's Biological Passport has been introduced. The sensitivity of the Athlete's Biological Passport is limited if the effect of a low-dose doping remains within the intra-individual reference range. A possible solution could be the use of a novel Epo test (MAIIA Diagnostics). Another performance-enhancing strategy is the return to 'old' doping techniques, such as autologous blood transfusions. Several indirect methods to detect autologous blood transfusions have been proposed with the majority relying on changes in erythropoiesis-sensitive blood markers. Currently, an algorithm based on the haemoglobin (Hb) level concentration and the percentage of reticulocytes (OFF-hr model; Hb(g/l)-60·âˆš%ret) is approved by the World Anti-Doping Agency. Genetic factors have been identified which may interfere with test interpretation. A large inter- and intra-ethnic variation in testosterone glucuronidation and excretion has been described. Consideration of genetic variation should improve performance of the testosterone doping test. Taking into account the pre-analytical care and better tailoring of the threshold values could increase test sensitivity. Anti-doping laboratories should routinely adjust for multiple testing as failure of doping control to detect cheaters could lead to more frequent controls. Finally, despite the huge technological progress, there is a need for increased collaboration between physiologists, analytical chemists, biostatisticians, and ethicists to reduce doping in sport.

Towards the identification of autologous blood transfusions through capillary electrophoresis.

The use of autologous blood transfusions by endurance athletes has remained one of the most difficult doping practices to detect. The implementation of the Athlete's Biological Passport by some sporting bodies has proved to be effective; however, the analysis relies on the long-term monitoring of numerous biological markers, looking for abnormal variations in a number of biological markers to indicate doping. This work introduces an approach to identify autologous blood transfusions by examining the red blood cells (RBCs) directly. By using high-speed capillary electrophoretic separations, the relative distribution of the sizes of the RBCs in a sample can be established in under 3 min, following the preparation of the cells. As RBCs that have been stored for transfusions undergo vesiculation, the relative size of the transfused cells differs from the native cells. The capillary electrophoretic separation allows for a rapid examination of this distribution and the changes that are seen when transfused RBCs are mixed with native cells. In this work, the effectiveness of this approach is demonstrated in the identification of simulated (in vitro) autologous blood transfusions performed with blood samples from three highly trained cyclists; it was possible to rapidly identify when as little as 5 % of the RBCs in the sample were from a simulated autologous transfusion.

A DNA-based method for detecting homologous blood doping.

Homologous (or allogeneic) blood doping, in which blood is transferred from a donor into a recipient athlete, is the easiest, cheapest, and fastest way to increase red cell mass (hematocrit) and therefore the oxygen-carrying capacity of the blood. Although thought to have been rendered obsolete as a doping strategy by the increased use of rhEPO to increased hematocrits, there is evidence that athletes are still using this potentially dangerous method to improve endurance performance. Current testing for homologous blood doping is based on identification of mixed populations of red blood cells by flow cytometry. This paper proposes that homologous blood doping could also be tested for by high-resolution qPCR-based genotyping and demonstrates that assays could be developed that would detect second populations of cells even if the "donor" blood was depleted of 99% of the DNA-containing leukocytes. Issues of test specificity and sensitivity are discussed as well as some of the ethical considerations that would have to be addressed if athletes' genotypes were to be used by the anti-doping authorities to prevent, or detect, the use of prohibited ergogenic practices.

Blood doping: the flip side of transfusion and transfusion alternatives.

Blood doping in sports has been a hot topic of present. Longitudinal follow up of hematological parameters in different endurance sports, during the 1990s and early 2000s, has provided considerable suspicions about extensive blood manipulation, with performance enhancing effects. Recent doping revelations in the media also prove that blood doping is not an anticipated myth but it is, in fact, real. Erythropoiesis stimulating agents and autologous blood transfusions are used in synergy with substantial effect on the maximum oxygen uptake and delivery to muscles. Whilst both methods of blood manipulation represent a potential health hazard, in the context of an elevated hematocrit, nevertheless despite a number of suspicious deaths amongst athletes, this has not yet been fully documented. A reliable test for detection of recombinant human erythropoietin was implemented in 2000, but this is probably circumvented by microdose regimens. The Athlete's Biological Passport represents the progeny of the idea of an indirect approach based on long term monitoring of hematological parameters, thus making it possible to detect autologous blood doping and erythropoietin use after the substance is excreted. Nevertheless with advances in anti-doping measures it is possible that the levels of excretion of substances used can be masked. Clearly more sensitive and specific diagnostic tools and research/development in these areas of major concern are warranted, which, combined with changes in the athlete's attitude, will help in reaching the vision of fair play.