Michael J. Ashenden

''Live high, train low'' does not change the total haemoglobin mass of male endurance athletes sleeping at a simulated altitude of 3000 m for 23 nights

Abstract The purpose of this study was to document the effect of 23 days of “live high, train low” on the haemoglobin mass of endurance athletes. Thirteen male subjects from either cycling, triathlon or cross-country skiing backgrounds participated in the study. Six subjects (HIGH) spent 8−10 h per night in a “nitrogen house” at a simulated altitude of 3000 m in normobaric hypoxia, whilst control subjects slept at near sea level (CONTROL, n = 7). Athletes logged their daily training sessions, which were conducted at 600 m. Total haemoglobin mass (as measured using the CO-rebreathing technique) did not change when measured before (D1 or D2) and after (D28) 23 nights of hypoxic exposure [HIGH 990 (127) vs 972 (97) g and CONTROL 1042 (133) vs 1033 (138) g, before and after simulated altitude exposure, respectively]. Nor was there any difference in the substantial array of reticulocyte parameters measured using automated flow cytometry prior to commencing the study (D1), after 6 (D10) and 15 (D19) nights of simulated altitude, or 1 day after leaving the nitrogen house (D28) when HIGH and CONTROL groups were compared. We conclude that red blood cell production is not stimulated in male endurance athletes who spend 23 nights at a simulated altitude of 3000 m.

Simulated moderate altitude elevates serum erythropoietin but does not increase reticulocyte production in well-trained runners

Abstract The purpose of this study was to investigate whether the modest increases in serum erythropoietin (sEpo) experienced after brief sojourns at simulated altitude are sufficient to stimulate reticulocyte production. Six well-trained middle-distance runners (HIGH, mean maximum oxygen uptake, V˙O2max = 70.2 ml · kg−1 · min−1) spent 8–11 h per night for 5 nights in a nitrogen house that simulated an altitude of 2650 m. Five squad members (CONTROL, mean V˙O2max = 68.9 ml · kg−1 · min−1) undertook the same training, which was conducted under near-sea-level conditions (600 m altitude), and slept in dormitory-style accommodation also at 600 m altitude. For both groups, this 5-night protocol was undertaken on three occasions, with a 3-night interim between successive exposures. Venous blood samples were measured for sEpo after 1 and 5 nights of hypoxia on each occasion. The percentage of reticulocytes was measured, along with a range of reticulocyte parameters that are sensitive to changes in erythropoiesis. Mean serum erythropoietin levels increased significantly (P < 0.01) above baseline values [mean (SD) 7.9 (2.4) mU · ml−1] in the HIGH group after the 1st night [11.8 (1.9) mU · ml−1, 57%], and were also higher on the 5th night [10.7 (2.2) mU · ml−1, 42%] compared with the CONTROL group, whose erythropoietin levels did not change. After athletes spent 3 nights at near sea level, the change in sEpo during subsequent hypoxic exposures was markedly attenuated (13% and −4% change during the second exposure; 26% and 14% change during the third exposure; 1st and 5th nights of each block, respectively). The increase in sEpo was insufficient to stimulate reticulocyte production at any time point. We conclude that when daily training loads are controlled, the modest increases in sEpo known to occur following brief exposure to a simulated altitude of 2650 m are insufficient to stimulate reticulocyte production.

Effects of a 12-day live high, train low camp on reticulocyte production and haemoglobin mass in elite female road cyclists

Abstract The aim of this study was to document the effect of “living high, training low” on the red blood cell production of elite female cyclists. Six members of the Australian National Womens road cycling squad slept for 12 nights at a simulated altitude of 2650 m in normobaric hypoxia (HIGH), while 6 team-mates slept at an altitude of 600 m (CONTROL). HIGH and CONTROL subjects trained and raced as a group throughout the 70-day study. Baseline levels of reticulocyte parameters sensitive to changes in erythropoeisis were measured 21 days and 1 day prior to sleeping in hypoxia (D1 and D20, respectively). These measures were repeated after 7 nights (D27) and 12 nights (D34) of simulated altitude exposure, and again 15 days (D48) and 33 days (D67) after leaving the altitude house. There was no increase in reticulocyte production, nor any change in reticulocyte parameters in either the HIGH or CONTROL groups. This lack of haematological response was substantiated by total haemoglobin mass measures (CO-rebreathing), which did not change when measured on D1, D20, D34 or D67. We conclude that in elite female road cyclists, 12 nights of exposure to normobaric hypoxia (2650 m) is not sufficient to either stimulate reticulocyte production or increase haemoglobin mass.

Detecting autologous blood transfusions: a comparison of three passport approaches and four blood markers.

Blood passport has been suggested as an indirect tool to detect various kinds of blood manipulations. Autologous blood transfusions are currently undetectable, and the objective of this study was to examine the sensitivities of different blood markers and blood passport approaches in order to determine the best approach to detect autologous blood transfusions. Twenty‐nine subjects were transfused with either one (n=8) or three (n=21) bags of autologous blood. Hemoglobin concentration ([Hb]), percentage of reticulocytes (%ret) and hemoglobin mass (Hbmass) were measured 1 day before reinfusion and six times after reinfusion. The sensitivity and specificity of a novel marker, Hbmr (based on Hbmass and %ret), was evaluated together with [Hb], Hbmass and OFF‐hr by different passport methods. Our novel Hbmr marker showed superior sensitivity in detecting the highest dosage of transfused blood, with OFF‐hr showing equal or superior sensitivities at lower dosages. Hbmr and OFF‐hr showed superior but equal sensitivities from 1 to 4 weeks after transfusion compared with [Hb] and Hbmass, with Hbmass being the only tenable prospect to detect acute transfusions. Because autologous blood transfusions can be an acute practice with blood withdrawal and reinfusion within a few days, Hbmass seems to be the only option for revealing this practice.

Standardization of reticulocyte values in an antidoping context.

The lack of standardization of reticulocyte results hinders the ability of sports authorities to recognize the telltale fluctuations over time that are typical for athletes using illegal blood doping to improve their performance. Therefore, the aim of the present study was to devise a tenable approach for antidoping authorities to quantify, instrument bias. We evaluated reticulocyte data derived during a 42-week period from 210 hospital patient blood samples measured in duplicate simultaneously on up to 11 hematology analyzers located in a single laboratory. We found that square root transformation of reticulocyte values enabled quantification of interinstrument bias by using the mean reticulocyte value of a cohort of approximately 54 subjects as a de facto calibration agent. We also demonstrated that measurement precision associated with low reticulocyte values was not inferior to that associated with higher values.

Doping with Artificial Oxygen Carriers An Update

There is a long history of science seeking to develop artificial substitutes for body parts damaged by disease or trauma. While defective teeth and limbs are commonly replaced by imitations without major loss of functionality, the development of a substitute for red blood cells has proved elusive.There is a permanent shortage of donor blood in western societies. Nevertheless, despite whole blood transfusions carrying measurable risks due to immunogenicity and the transmission of blood-borne infectious diseases, red blood cells are still relatively inexpensive, well tolerated and widely available. Researchers seeking to develop products that are able to meet and perhaps exceed these criteria have responded to this difficult challenge by adopting many different approaches. Work has focussed on two classes of substances: modified haemoglobin solutions and perfluorocarbon emulsions. Other approaches include the creation of artificial red cells, where haemoglobin and supporting enzyme systems are encapsulated into liposomes.Haemoglobin is ideally suited to oxygen transport when encased by the red cell membrane; however, once removed, it rapidly dissociates into dimers and is cleared by the kidney. Therefore, it must be stabilised before it can be safely re-infused into humans. Modifications concomitantly alter the vascular half-life, oxygen affinity and hypertensive characteristics of raw haemoglobin, which can be sourced from outdated blood stores, genetically-engineered Escherichia coli or even bovine herds. In contrast, perfluorocarbons are entirely synthetic molecules that are capable of dissolving oxygen but biologically inert. Since they dissolve rather than bind oxygen, their capacity to serve as a blood substitute is determined principally by the oxygen pressure gradients in the lung and at the target tissue.Blood substitutes have important potential areas of clinical application including red cell replacement during surgery, emergency resuscitation of traumatic blood loss, oxygen therapeutic applications in radiography (oxygenation of tumour cells is beneficial to the effect of certain chemotherapeutic agents), other medical applications such as organ preservation, and finally to meet the requirements of patients who cannot receive donor blood because of religious beliefs.Given the elite athlete’s historical propensity to experiment with novel doping strategies, it is likely that the burgeoning field of artificial oxygen carriers has already attracted their attention. Scientific data concerning the performance benefits associated with blood substitutes are virtually nonexistent; however, international sporting federations have been commendably proactive in adding this category to their banned substance lists. The current situation is vulnerable to exploitation by immoral athletes since there is still no accepted methodology to test for the presence of artificial oxygen carriers.

Quality control technique to reduce the variability of longitudinal measurement of hemoglobin mass

The sensitivity of the athlete blood passport to detect blood doping may be improved by the inclusion of total hemoglobin mass (Hbmass), but the comparability of Hbmass from different laboratories is unknown. To optimize detection sensitivity, the analytical variability associated with Hbmass measurement must be minimized. The aim of this study was to investigate the efficacy of using quality controls to minimize the variation in Hbmass between laboratories. Three simulated laboratories were set up in one location. Nine participants completed three carbon monoxide (CO) re‐breathing tests in each laboratory. One participant completed two CO re‐breathing tests in each laboratory. Simultaneously, quality controls containing Low (1–3%) and High (8–11%) concentrations of percent carboxyhemoglobin (%HbCO) were measured to compare hemoximeters in each laboratory. Linear mixed modeling was used to estimate the within‐subject variation in Hbmass, expressed as the coefficient of variation, and to estimate the effect of different laboratories. The analytic variation of Hbmass was 2.4% when tests were conducted in different laboratories, which reduced to 1.6% when the model accounted for between‐laboratory differences. Adjustment of Hbmass values using quality controls achieved a comparable analytic variation of 1.7%. The majority of between‐laboratory variation in Hbmass originated from the difference between hemoximeters, which could be eliminated using appropriate quality controls.

Stability of athlete passport parameters during extended storage

Extended intervals between sample collection and analyses render athletes whole‐blood specimens collected in the field for antidoping purposes susceptible to storage degradation. The aim of this study was to characterize the stability of key blood variables under different storage durations and temperatures.

Skin-prick blood samples are reliable for estimating Hb mass with the CO-dilution technique.

Abstract Investigation of the impact of environmental stimuli such as altitude exposure on hemoglobin mass currently rely on invasive techniques that require venous blood sampling. This study assessed the feasibility of lancet skin pricks as an alternative to venepuncture to estimate hemoglobin mass with the carbon monoxide (CO) dilution technique, with the intent of making the technique accessible to technicians without phlebotomy training. Sixteen healthy volunteers rebreathed CO via a small-volume rebreathing apparatus. Blood was sampled simultaneously with a glass syringe (VEN) from a superficial forearm vein and with a capillary tube from either a lanced fingertip or earlobe (CAP). As a control, VEN blood was then aliquoted into capillary tubes (CONTROL-CAP). Samples were assayed for carboxyhemoglobin (HbCO) using a diode-array spectrophotometer. Mean %HbCO was higher in CAP than VEN (bias 0.3 ± 0.2%HbCO, p < 0.01), but VEN and CONTROL-CAP were not different (p = 0.55). Compared to VEN, Hb mass derived from CAP samples was overestimated by 1.7% (15 ± 22 g Hb, p = 0.01). CAP samples to estimate Hb mass demonstrated a technical error of measurement of 2.7%, which is comparable to the 1.9% reported previously with VEN samples. We conclude that using CAP samples gives a reliable measure of %HbCO, and will make the estimation of Hb mass with the CO-technique accessible to technicians without phlebotomy training.

Detection of microdoses of rhEPO with the MAIIA test

The detection of recombinant human erythropoietin (rhEPO) is difficult and becomes more challenging when only microdoses are administered intravenously. Twenty‐three subjects were divided into two groups: EPO group (n = 7) and CONTROL group (n = 16). Seven urine and blood samples per subject were collected at least 5 days apart to determine within‐ and between‐subject standard deviations in the percentage of migrating isoforms by the MAIIA test. Six injections of 50 IU/kg bw (boosting dosage) of epoetin beta (Neorecormon, Roche Diagnostics, Hvidovre, Denmark) were performed intravenously during a 3‐week period, followed by two microinjections of only 10 IU/kg bw. Blood and urine samples were collected 2, 6, 12, and 72 h after the microinjection, as well as 72 h after the last boosting dose. Sensitivities and specificities of the MAIIA test were examined by absolute and passport thresholds. Sensitivity was 100% for at least 12 h after the microinjection, with ∼30% of plasma samples still exceeding the 99.9% passport threshold 72 h after a microinjection. The specificity was higher for the passport approach compared to the absolute approach, but there were no differences in sensitivities between approaches or between specimens (urine and plasma). We conclude that the MAIIA test shows potential for detecting very small doses of rhEPO.