A multi-parametric approach to remove the influence of plasma volume on the athlete biological passport during a Union Cycliste Internationale cycling stage race.

Fluctuations in plasma volume (PV) present potential confounders within the concentration-based markers of the haematological athlete biological passport (ABP). Here, a multi-parametric approach involving a simple blood test is applied to the current ABP adaptive model in an attempt to remove the influence of PV expansion, induced by a cycling stage race. Blood samples were obtained from 29 professional cyclists (14 male, 15 female) before, during and after 4-5 consecutive days of racing. Whole blood was analysed in accordance with the World Anti-Doping Agency ABP guidelines for haemoglobin ([Hb]) concentration and platelets. Serum and plasma were analysed for transferrin, albumin, calcium, creatinine, total protein and low-density lipoprotein. PV variation (Z-scores) was estimated using a multi-parametric model (consisting of the biomarkers mentioned earlier) and compared against calculated variations in PV (measured via CO-rebreathing). Significant reductions in [Hb] and the OFF-score were observed in female cyclists after 3 and 4 days of racing, with accompanying increases in PV, which returned to baseline values 4 days post competition. Similarly, a significant increase in PV was observed in male cyclists after 3 and 5 days of racing. When individual estimations of PV variance were applied to the adaptive model, the upper and lower reference predictions for [Hb] and the OFF-score were refined such that all outliers consistent with racing-induced PV changes were removed. The PV model appears capable of reducing the influence of PV on concentration-dependent markers during competition. This is an important step towards the inclusion of the PV correction in the ABP haematological module.

Validation of a blood marker for plasma volume in endurance athletes during a live-high train-low altitude training camp.

Altitude is a confounding factor within the Athlete Biological Passport (ABP) due, in part, to the plasma volume (PV) response to hypoxia. Here, a newly developed PV blood test is applied to assess the possible efficacy of reducing the influence of PV on the volumetric ABP markers; haemoglobin concentration ([Hb]) and the OFF-score. Endurance athletes (n=34) completed a 21-night simulated live-high train-low (LHTL) protocol (14 h.d-1 at 3000 m). Bloods were collected twice pre-altitude; at days 3, 8, and 15 at altitude; and 1, 7, 21, and 42 days post-altitude. A full blood count was performed on the whole blood sample. Serum was analysed for transferrin, albumin, calcium, creatinine, total protein, and low-density lipoprotein. The PV blood test (consisting of the serum markers, [Hb] and platelets) was applied to the ABP adaptive model and new reference predictions were calculated for [Hb] and the OFF-score, thereby reducing the PV variance component. The PV correction refined the ABP reference predictions. The number of atypical passport findings (ATPFs) for [Hb] was reduced from 7 of 5 subjects to 6 of 3 subjects. The OFF-score ATPFs increased with the PV correction (from 9 to 13, 99% specificity); most likely the result of more specific reference limit predictions combined with the altitude-induced increase in red cell production. Importantly, all abnormal biomarker values were identified by a low confidence value. Although the multifaceted, individual physiological response to altitude confounded some results, the PV model appears capable of reducing the impact of PV fluctuations on [Hb].

A step towards removing plasma volume variance from the Athlete's Biological Passport: The use of biomarkers to describe vascular volumes from a simple blood test.

The haematological module of the Athlete's Biological Passport (ABP) has significantly impacted the prevalence of blood manipulations in elite sports. However, the ABP relies on a number of concentration-based markers of erythropoiesis, such as haemoglobin concentration ([Hb]), which are influenced by shifts in plasma volume (PV). Fluctuations in PV contribute to the majority of biological variance associated with volumetric ABP markers. Our laboratory recently identified a panel of common chemistry markers (from a simple blood test) capable of describing ca 67% of PV variance, presenting an applicable method to account for volume shifts within anti-doping practices. Here, this novel PV marker was included into the ABP adaptive model. Over a six-month period (one test per month), 33 healthy, active males provided blood samples and performed the CO-rebreathing method to record PV (control). In the final month participants performed a single maximal exercise effort to promote a PV shift (mean PV decrease -17%, 95% CI -9.75 to -18.13%). Applying the ABP adaptive model, individualized reference limits for [Hb] and the OFF-score were created, with and without the PV correction. With the PV correction, an average of 66% of [Hb] within-subject variance is explained, narrowing the predicted reference limits, and reducing the number of atypical ABP findings post-exercise. Despite an increase in sensitivity there was no observed loss of specificity with the addition of the PV correction. The novel PV marker presented here has the potential to improve the ABP's rate of correct doping detection by removing the confounding effects of PV variance.

The athlete's hematological response to hypoxia: A meta-analysis on the influence of altitude exposure on key biomarkers of erythropoiesis.

Altitude training is associated with changes in blood markers, which can confound results of the Athlete?s Biological Passport (ABP). This meta-analysis aims to describe the fluctuations during- and post-altitude in key ABP variables; hemoglobin concentration ([Hb]), square-root transformed reticulocyte percentage (sqrt(retic%)) and the OFF-score. Individual de-identified raw data were provided from 17 studies. Separate linear mixed effects analyses were performed for delta values from baseline for [Hb], sqrt(retic%) and OFF-score, by altitude phase (during and post). Mixed models were fitted with the hierarchical structure: study and subject within study as random effects. Delta values as response variables and altitude dose (in kilometer hours; km.hr = altitude (m) / 1000 x hours), sex, age, protocol and baseline values as fixed effects. Allowances were made for potential autocorrelation. Within two days at natural altitude [Hb] rapidly increased. Subsequent delta [Hb] values increased with altitude dose, reaching a plateau of 0.94 g/dL [95%CI (0.69, 1.20)] at ~1000 km.hr. Delta sqrt(retic%) and OFF-score were the first to identify an erythrocyte response, with respective increases and decreases observed within 100 to 200 km.hr. Post-altitude, [Hb] remained elevated for two weeks. Delta sqrt(retic%) declined below baseline, the magnitude of change was dependent on altitude dose. Baseline values were a significant covariate (p<0.05). The response to altitude is complex resulting in a wide range of individual responses, influenced primarily by altitude dose and baseline values. Improved knowledge of the plausible hematological variations during- and post-altitude provides fundamental information for both the ABP expert and sports physician.

Within-subject haemoglobin variation in elite athletes: a longitudinal investigation of 13 887 haemoglobin concentration readings.

The Athlete Biological Passport (ABP) estimates individualized reference ranges for key blood markers, such as haemoglobin concentration ([Hb]), using predetermined population mean, between- and within-subject variances. Here, we aim to reassess previously published estimates for within-subject [Hb] variance and determine whether sex-, analyzer-, sport-, or season-specific values are required. Our reference population contains 7723 male (mean ± SD, 22.3 ± 4.6 years of age) and 6164 female (21.6 ± 4.3) athlete observations from 49 sports. [Hb] was calculated using one of three cytometers; Bayer-H3 (1997-1999, n = 4554), ADVIA-120 (1999-2010, n = 8636) and Sysmex XT-2000i (2010-2012, n = 697). The final model was a linear mixed model for [Hb] with analyzer (H3, ADVIA, Sysmex), sex (male, female), sport (power-endurance, endurance, skill, team, disabled and non-athletes), season (summer, winter), and the interaction between sex and sport as fixed effects and athlete as a random effect. The model included an exponential correlation structure to allow for within-subject autocorrelation, and allowed different within-subject variances for each sport. Within-subject [Hb] variance (g(2) /L(2) ) was significantly less for power endurance (35.09, 95% CI 33.50 to 36.76), disabled (25.82, 95% CI 21.71 to 35.28) and non-athletes (34.30, 95% CI 28.53 to 35.87) than for endurance (40.35, 95% CI 39.62 to 47.22) and team sports (38.70, 95% CI 37.68 to 39.76) athletes. No new evidence was found to justify adjusting the current within-subject [Hb] variance estimate.

High altitude, prolonged exercise, and the athlete biological passport.

The Athlete Biological Passport (ABP) detects blood doping in athletes through longitudinal monitoring of erythropoietic markers. Mathematical algorithms are used to define individual reference ranges for these markers for each athlete. It is unclear if altitude and exercise can affect the variables included in these calculations in a way that the changes might be mistaken for blood manipulation. The aim of this study was to investigate the influence of the simultaneous strenuous exercise and low to high altitude exposure on the calculation algorithms of the ABP. 14 sea level (SL) and 11 altitude native (ALT) highly trained athletes participated in a 14-day cycling stage race taking place at an average altitude of 2496 m above sea level (min. 1014 m, max. 4120 m), race distances ranged between 96 and 227 km per day. ABP blood measures were taken on days -1,3,6,10,14 (SL) and -1,9,15 (ALT) of the race. Four results from three samples of two different SL athletes exceeded the individual limits at the 99% specificity threshold and one value at 99.9%. In ALT, three results from three samples of three different athletes were beyond the individual limits at 99%, one at 99.9%. The variations could be explained by the expected physiological reaction to exercise and altitude. In summary, the abnormalities observed in the haematological ABP´s of well-trained athletes during extensive exercise at altitude are limited and in line with expected physiological changes.

Intravenous iron supplementation in distance runners with low or suboptimal ferritin.

PURPOSE: Iron deficiency is prevalent in distance runners and may impair endurance performance. The current practice of oral supplementation is slow and often not well tolerated. The aim of this study was to assess the efficacy of intravenous (IV) iron supplementation (ferric carboxymaltose) compared with oral supplementation (ferrous sulfate) on iron status, hemoglobin mass (Hbmass), and physiological indices of running performance in distance runners. METHODS: Twenty-seven highly trained distance runners with low (LOW) (ferritin <35 µg·L(-1) and transferrin saturation <20%, or ferritin <15 µg·L(-1)) or suboptimal (SUB) iron status (ferritin <65 µg·L(-1)) were supplemented with either IV iron (Ferinject®) or oral (ORAL) supplements (Ferrogradumet) for 6 wk. Iron status and Hbmass were assessed before supplementation and at 1, 2, 4, 6, and 8 wk in the four groups (IV LOW, IV SUB, ORAL LOW, and ORAL SUB). In addition, athletes completed a treadmill running test for running economy, lactate threshold, and V˙O2max before and after supplementation. RESULTS: Both forms of supplementation substantially increased ferritin levels in all four groups. IV supplementation resulted in higher ferritin in both IV groups compared with both ORAL groups from week 1 onward. Hemoglobin concentration did not change substantially in any group. Hbmass increased in IV LOW (mean = +4.9%, 90% confidence interval [CI] = 1.1%-8.9%) and was accompanied by an increase in V˙O2max (mean = +3.3%, 90% CI = 0.4%-6.3%) and run time to exhaustion (mean = +9.3%, 90% CI = 0.9%-18.3%. CONCLUSIONS: IV supplementation can effectively increase iron stores in iron-deficient runners within 6 wk and, if Hbmass is compromised, may enhance endurance capacity by facilitating erythropoiesis. Hbmass appears a more sensitive tool for measuring changes in whole body hemoglobin than hemoglobin concentration and may be useful in the diagnosis and follow-up for iron deficiency.