New insights for identification of doping with recombinant human erythropoietin micro-doses after high hydration.

To minimize the chances of being caught after doping with recombinant human erythropoietins (rhEPO), athletes have turned to new practices using micro-doses and excess fluid ingestion to accelerate elimination and decrease the probability of detection. Our objective was to test the sensitivity of detection by validated methods (IEF: isoelectric focusing; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis) when such practices are used. First, after a three-week rhEPO boost period and 10 days of wash out, detection of a single 900 IU micro-dose of Eprex® was evaluated in healthy male subjects. After an injection in the evening, urine and plasma samples were collected the following morning. Half of the subjects then drank a bolus of water and new samples were collected 80 min later. Interestingly, rhEPO was detected in 100% of the samples even after water ingestion. A second similar protocol was then performed with a single injection of a micro-dose of rhEPO (500 IU or 900 IU), without a prior rhEPO boost. In addition, urine and plasma samples were also collected 15 and 20 h post rhEPO administration. Once again drinking water did not affect the rate of detection. Urine appeared a better matrix to detect micro-doses after 10 h, enabling between 92% and 100% of identification at that time. The rate of identification decreased rapidly thereafter, in particular for the 500 IU micro-dose. However IEF analysis still resulted in 71% identification of rhEPO in urine after 20 h. These results could help to define a better strategy for controlling and identifying athletes using rhEPO micro-doses. Copyright © 2016 John Wiley & Sons, Ltd.

Acute hyperhydration reduces athlete biological passport OFF-hr score.

Anecdotal evidence suggests that athletes hyperhydrate to mask prohibited substances in urine and potentially counteract suspicious fluctuations in blood parameters in the athlete biological passport (ABP). It is examined if acute hyperhydration changes parameters included in the ABP. Twenty subjects received recombinant human erythropoietin (rhEPO) for 3 weeks. After 10 days of rhEPO washout, 10 subjects ingested normal amount of water (∼ 270 mL), whereas the remaining 10 ingested a 1000 mL bolus of water. Blood variables were measured 20, 40, 60, and 80 min after ingestion. Three days later, the subjects were crossed-over with regard to water ingestion and the procedure was repeated. OFF-hr was reduced by ∼ 4%, ∼ 3%, and ∼ 2% at 40, 60, and 80 min, respectively, after drinking 1000 mL of water, compared with normal water ingestion (P < 0.05). Forty percent of the subjects were identified with atypical blood profiles (99% specificity level) before drinking 1000 mL of water, whereas 11% (n = 18), 10% and 11% (n = 18) were identified 40, 60, and 80 min, respectively, after ingestion. This was different (P < 0.05) compared with normal water intake, where 45% of the subjects were identified before ingestion, and 54% (n = 19), 45%, and 47% (n = 19) were identified 40, 60, and 80 min, respectively, after ingestion. In conclusion, acute hyperhydration reduces ABP OFF-hr and reduces ABP sensitivity.

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.

Stability of athlete blood passport parameters during air freight.

INTRODUCTION: Fluctuations in ambient temperature and pressure, as well as physical jostling, may affect the stability of whole blood samples transported by air freight. The aim of this study was to characterize the stability of key blood variables during air freight and to investigate whether vibration or reduced pressure alone affected results. METHODS: Over a 72-h interval, we evaluated the stability of full blood count indices (plus reticulocytes) in tubes that were air-freighted a total of 2, 10 and 28 h. We also examined the impact of 24 h of reduced atmospheric pressure (750 hpa or approximately 2500 m.a.s.l) and vibration (5 Hz). Samples were measured on a Sysmex XT-2000i instrument. RESULTS: The two key variables in the context of antidoping (haemoglobin concentration, reticulocytes) remained stable over a 72-h period regardless of the duration of air freight. Atmospheric pressure and vibration had no discernible effect. CONCLUSION: Whole blood samples stored in NanoCool devices can be relied upon to remain stable for at least 72 h despite interim air freight.

Stability of athlete passport parameters during extended storage.

INTRODUCTION: Extended intervals between sample collection and analyses render athlete's 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. METHODS: We evaluated stability of full blood count indices (plus reticulocytes) in individual tubes left undisturbed during 36, 48, 72, 96, 120, 144 and 168 h of storage at approximately 4, 6 and 12 °C. Samples were measured on a Sysmex XT-2000i instrument. RESULTS: The two key variables in the context of antidoping (haemoglobin concentration, reticulocytes) were stable for at least 168 h, except under 12 °C (stable 48 h only). Volume-dependent variables changed in a predictable manner that enabled a nomogram to be generated to predict original values provided storage duration and temperature were known. CONCLUSION: Key blood results can be relied upon for at least 7 days if storage temperature is kept at 4-6 °C.

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 (Hb(mass)), but the comparability of Hb(mass) from different laboratories is unknown. To optimize detection sensitivity, the analytical variability associated with Hb(mass) measurement must be minimized. The aim of this study was to investigate the efficacy of using quality controls to minimize the variation in Hb(mass) 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 Hb(mass), expressed as the coefficient of variation, and to estimate the effect of different laboratories. The analytic variation of Hb(mass) 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 Hb(mass) values using quality controls achieved a comparable analytic variation of 1.7%. The majority of between-laboratory variation in Hb(mass) originated from the difference between hemoximeters, which could be eliminated using appropriate quality controls.

Net haemoglobin increase from reinfusion of refrigerated vs. frozen red blood cells after autologous blood transfusions.

BACKGROUND AND OBJECTIVES Two main blood storage procedures can be used for storing red blood cells: refrigeration and freezing. Nevertheless, the efficiency of these procedures measured as the increase in haemoglobin after reinfusion compared with baseline has never been examined. The main objective was to examine which storage procedure yielded the largest increase in circulating haemoglobin after reinfusion compared to baseline. MATERIALS AND METHODS Equal volumes of blood from 15 men were withdrawn and stored either frozen or refrigerated as packed red blood cells. Serial measures of circulating haemoglobin by carbon monoxide rebreathing provided an opportunity to monitor recovery from anaemia, as well as the net increase in circulating haemoglobin after transfusion. RESULTS The post-thaw yield of haemoglobin in the bags was 72% after refrigerated storage compared with only 52% after freezing. Nevertheless, frozen storage allowed haemoglobin to fully recover before reinfusion, while the haemoglobin was 10% lower in the refrigerated group compared with baseline. After reinfusion, the haemoglobin levels were 11·5% higher than the baseline values in the group reinfused with frozen blood, while for the refrigerated group, haemoglobin levels were only 5·2% higher than baseline. CONCLUSION The relatively larger recovery from anaemia in the frozen group during storage more than compensated for the larger loss of haemoglobin during freezing and resulted in a larger net gain in haemoglobin. Based on the average 23 g per week recovery of haemoglobin, extending refrigerated storage to 7-8 weeks may yield sufficient time for patients to fully replenish harvested haemoglobin from three bags of blood without reliance on frozen storage of RBC.

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.

Screening for autologous blood transfusions.

The ratio between the amount of hemoglobin in the mature erythrocyte population and the reticulocytes (RBCHb:RetHb ratio) has previously been suggested as a marker to screen for EPO-abuse. We speculated that the reinfusion of blood would lead to a marked increase in this ratio, making it a valuable parameter in the screening for autologous blood doping. Three bags of blood (approximately 201+/-11 g of Hb) were withdrawn from 16 males and stored at either -80 degrees C (-80 T, n=8) or +4 degrees C (+4 T, n=8) and reinfused 10 weeks or 4 weeks later, respectively. Seven subjects served as controls. Different erythrocyte parameters were measured on a hematological analyzer serially throughout and during a 4 week wash-out period. By using RBCHb:RetHb ratio cut-off limits of 145.7 (1:100) ('suspicious') and 182.9 (1:1000) ('positive'), 35.4% (-80 T) and 19.6% (+4 T) of all samples obtained during a 4 week wash-out period were identified as 'suspicious', and 18.8% (-80 T) and 4.3% (+4 T) as 'positive'. In total, 7 out of 16 (43.8%) subjects had at least one sample exceeding 182.9. Compared to the currently used indirect parameters, the RBCHb:RetHb ratio is the best indicator of autologous blood doping after reinfusion, and the parameter could be used in a testing setting, once stability validation has been performed.

Effects of Hemopure on maximal oxygen uptake and endurance performance in healthy humans.

Haemoglobin-based oxygen carriers (HBOCs) such as Hemopure are touted as a tenable substitute for red blood cells and therefore potential doping agents, although the mechanisms of oxygen transport of HBOCs are incompletely understood. We investigated whether infusion of Hemopure increased maximal oxygen uptake (V.O 2max) and endurance performance in healthy subjects. Twelve male subjects performed two 4-minute submaximal exercise bouts equivalent to 60 % and 75 % of V.O (2max) on a cycle ergometer, followed by a ramped incremental protocol to elicit V.O (2max). A crossover design tested the effect of infusing either 30 g (6 subjects) or 45 g (6 subjects) of Hemopure versus a placebo. Under our study conditions, Hemopure did not increase V.O (2max) nor endurance performance. However, the infusion of Hemopure caused a decrease in heart rate of approximately 10 bpm (p=0.009) and an average increase in mean ( approximately 7 mmHg) and diastolic blood pressure ( approximately 8 mmHg) (p=0.046) at submaximal and maximal exercise intensities. Infusion of Hemopure did not bestow the same physiological advantages generally associated with infusion of red blood cells. It is conceivable that under exercise conditions, the hypertensive effects of Hemopure counter the performance-enhancing effect of improved blood oxygen carrying capacity.

Live high:train low increases muscle buffer capacity and submaximal cycling efficiency.

This study investigated whether hypoxic exposure increased muscle buffer capacity (beta(m)) and mechanical efficiency during exercise in male athletes. A control (CON, n=7) and a live high:train low group (LHTL, n=6) trained at near sea level (600 m), with the LHTL group sleeping for 23 nights in simulated moderate altitude (3000 m). Whole body oxygen consumption (VO2) was measured under normoxia before, during and after 23 nights of sleeping in hypoxia, during cycle ergometry comprising 4 x 4-min submaximal stages, 2-min at 5.6 +/- 0.4 W kg(-1), and 2-min 'all-out' to determine total work and VO(2peak). A vastus lateralis muscle biopsy was taken at rest and after a standardized 2-min 5.6 +/- 0.4 W kg(-1) bout, before and after LHTL, and analysed for beta(m) and metabolites. After LHTL, beta(m) was increased (18%, P < 0.05). Although work was maintained, VO(2peak) fell after LHTL (7%, P < 0.05). Submaximal VO2 was reduced (4.4%, P < 0.05) and efficiency improved (0.8%, P < 0.05) after LHTL probably because of a shift in fuel utilization. This is the first study to show that hypoxic exposure, per se, increases muscle buffer capacity. Further, reduced VO2 during normoxic exercise after LHTL suggests that improved exercise efficiency is a fundamental adaptation to LHTL.

A comparison of the physiological response to simulated altitude exposure and r-HuEpo administration.

Concerns have been raised about the morality of using simulated altitude facilities in an attempt to improve athletic performance. One assumption that has been influential in this debate is the belief that altitude houses simply mimic the physiological effects of illegal recombinant human erythropoietin (r-HuEpo) doping. To test the validity of this assumption, the haematological and physiological responses of 23 well-trained athletes exposed to a simulated altitude of 2650-3000 m for 11-23 nights were contrasted with those of healthy volunteers receiving a low dose (150 IU x kg(-1) per week) of r-HuEpo for 25 days. Serial blood samples were analysed for serum erythropoietin and percent reticulocytes; maximal oxygen uptake (VO2max) was assessed before and after r-HuEpo administration or simulated altitude exposure. The group mean increase in serum erythropoietin (422% for r-HuEpo vs 59% for simulated altitude), percent reticulocytes (89% vs 30%) and VO2max (6.6% vs -2.0%) indicated that simulated altitude did not induce the changes obtained with r-HuEpo administration. Based on the disparity of these responses, we conclude that simulated altitude facilities should not be considered unethical based solely on the tenet that they provide an alternative means of obtaining the benefits sought by illegal r-HuEpo doping.

An evaluation of the concept of living at moderate altitude and training at sea level.

Despite equivocal findings about the benefit of altitude training, current theory dictates that the best approach is to spend several weeks living at > or =2500 m but training near sea level. This paper summarizes six studies in which we used simulated altitude (normobaric hypoxia) to examine: (i) the assumption that moderate hypoxia compromises training intensity (two studies); and (ii) the nature of physiological adaptations to sleeping in moderate hypoxia (four studies). When submaximal exercise was >55% of sea level maximum oxygen uptake (VO2max), 1800 m simulated altitude significantly increased heart rate, blood lactate and perceived exertion of skiers. In addition, cyclists self-selected lower workloads during high-intensity exercise in hypoxia (2100 m) than in normoxia. Consequently, our findings partially confirm the rationale for 'living high, training low'. In the remaining four studies, serum erythropoietin increased 80% in the early stages of hypoxic exposure, but the reticulocyte response did not significantly exceed that of control subjects. There was no significant increase in haemoglobin mass (Hb(mass)) and VO2max tended to decrease. Performance in exercise tasks lasting approximately 4 min showed a non-significant trend toward improvement (1.0+/-0.4% vs. 0.1+/-0.4% for a control group; P=0.13 for group x time interaction). We conclude that sleeping in moderate hypoxia (2650-3000 m) for up to 23 days may offer practical benefit to elite athletes, but that any effect is not likely due to increased Hb(mass) or VO2max.

Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis.

BACKGROUND AND OBJECTIVES: The detection of recombinant human erythropoietin (r-HuEPO) abuse by athletes remains problematic. The main aim of this study was to demonstrate that the five indirect markers of altered erythropoiesis identified in our earlier work were reliable evidence of current or recently discontinued r-HuEPO use. A subsidiary aim was to refine weightings of the five markers in the initial model using a much larger data set than in the pilot study. A final aim was to verify that the hematologic response to r-HuEPO did not differ between Caucasian and Asiatic subjects. DESIGN AND METHODS: Recreational athletes resident in Sydney, Australia (Sydney, n = 49; 16 women, 33 men) or Beijing, China (Beijing, n=24; 12 women, 12 men) were randomly assigned to r-HuEPO or placebo groups prior to a 25 day administration phase. Injections of r-HuEPO (or saline) were administered double-blind at a dose of 50 IU/kg three times per week, with oral iron (105 mg) or placebo supplements taken daily by all subjects. Blood profiles were monitored during and for 4 weeks after drug administration for hematocrit (Hct), reticulocyte hematocrit (RetHct), percent macrocytes (%Macro), serum erythropoietin (EPO) and soluble transferrin receptor (sTfr), since we had previously shown that these five variables were indicative of r-HuEPO use. RESULTS: The changes in Hct, RetHct, %Macro, EPO and sTfr in the Sydney trial were qualitatively very similar to the changes noted in our previous administration trial involving recreational athletes of similar genetic origin. Statistical models developed from Fisher's discriminant analysis were able to categorize the user and placebo groups correctly. The same hematologic response was demonstrated in Beijing athletes also administered r-HuEPO. INTERPRETATION AND CONCLUSIONS: This paper confirms that r-HuEPO administration causes a predictable and reproducible hematologic response. These markers are disturbed both during and for several weeks following r-HuEPO administration. This work establishes an indirect blood test which offers a useful means of detecting and deterring r-HuEPO abuse. Ethnicity did not influence the markers identified as being able to detect athletes who abuse r-HuEPO.

Reticulocyte parameters as potential discriminators of recombinant human erythropoietin abuse in elite athletes.

This study investigated using reticulocyte (retic) parameters as indirect markers of human recombinant erythropoietin (r-HuEPO) abuse in elite athletes. Absolute reticulocyte count (# retic), the per cell haemoglobin content of reticulocytes (CHr), reticulocyte haemoglobin mass per litre of blood (RetHb) and red blood cell:reticulocyte haemoglobin (RBCHb:RetHb) ratio were assessed using flow cytometry. Venous blood was drawn from 155 elite athletes from six sports during regular training to establish reference ranges (95% confidence interval) for these parameters. The reference ranges were compared with those of a non-athletic population (n = 23), four groups of athletes (n = 24) before and after exposure to simulated altitudes (2,500-3,000 m for 11-23 nights), two groups of elite cyclists (n = 13) before and after four weeks of training at natural altitude (1,780 and 2,690 m), and with those of non-athletic subjects from a separate study (n =24) before and 1-2 days after they were injected with 1,200 U x kg(-1) r-HuEPO over a 9-10 day period. Generally the changes induced by r-HuEPO injection exceeded by approximately 100% the magnitude of the changes associated with natural altitude exposure. Simulated altitude exposure did not significantly alter the reticulocyte parameters. From the sample of 155 non-users and 24 r-HuEPO users, the population mean and variance, as well as the 95% confidence limits for the population mean and population variance, were estimated. Relative to arbitrarily chosen cut-off levels, the confidence limits for the rate of true positives and rate of true negatives were also calculated. Based on the lowest rate of false positives and highest rate of true positives, the best discriminator between r-HuEPO users and non-users was # retic, marginally superior to RBCHb: RetHb ratio and RetHb. At a cut-off for # retic of 221 x 10(9)x L(-1) we could be 95% sure that we would find no more than 7 false positives in every 100,000 tests. We would expect to pick up 51.8% of users, and could be 95% sure of picking up at least 38% of current or recent users. This result highlights the potential power of retic parameters for detecting r-HuEPO abuse among athletes. However, the efficacy of these cut-offs for detecting r-HuEPO abuse is unknown if an athlete is a chronic user or stops using r-HuEPO several weeks before being tested.

A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes.

BACKGROUND AND OBJECTIVE: The use of recombinant human erythropoietin (r-HuEPO) to enhance athletic performance is prohibited. Existing tests cannot readily differentiate between exogenous and endogenous EPO. Therefore the aim of our study was to investigate possible indirect detection of r-HuEPO use via blood markers of altered erythropoiesis. DESIGN AND METHODS: Twenty-seven recreational athletes were assigned to three groups prior to a 25 day drug administration phase, with the following protocols: EPO+IM group (n = 10), 50 Ukg(-1) r-HuEPO at a frequency of 3wk(-1), 100 mg intramuscular (IM) iron 1wk(-1) and a sham iron tablet daily; EPO+OR group (n = 8), 50 U.kg(-1) r-HuEPO 3wk(-1), sham iron injection 1wk(-1) and 105 mg of oral elemental iron daily; placebo group (n = 9), sham r-HuEPO injections 3wk(-1), sham iron injections 1wk(-1) and sham iron tablets daily. Each group was monitored during and for 4 weeks after drug administration. RESULTS: Models incorporating combinations of the variables reticulocyte hematocrit (RetHct), serum EPO, soluble transferrin receptor, hematocrit (Hct) and % macrocytes were analyzed by logistic regression. One model (ON-model) repeatedly identified 94-100% of r-HuEPO group members during the final 2 wk of the r-HuEPO administration phase. One false positive was recorded from a possible 189. Another model (OFF-model) incorporating RetHct, EPO and Hct was applied during the wash-out phase and, during the period of 12-21 days after the last r-HuEPO injection, it repeatedly identified 67-72% of recent users with no false positives. INTERPRETATION AND CONCLUSIONS: Multiple indirect hematologic and biochemical markers used simultaneously are potentially effective for identifying current or recent users of r-HuEPO.

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

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, VO2max = 70.2 ml x kg(-1) x 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 VO2max= 68.9 ml x kg(-1) x 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 x ml(-1)] in the HIGH group after the 1st night [11.8 (1.9) mU x ml(-1), 57%], and were also higher on the 5th night [10.7 (2.2) mU x 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.

"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.

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.

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

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 Women's 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.

Can reticulocyte parameters be of use in detecting iron deficient erythropoiesis in female athletes?

BACKGROUND: The purpose of this study was to investigate whether monitoring reticulocyte profiles, which are known to respond to iron store depletion in sedentary populations, could also be utilised with intensely training athletes. METHODS: A retrospective study of blood samples from 134 national level athletes (61 males, 73 females) at the Australian Institute of Sport were analysed, from which reference ranges were calculated. To ascertain the stability of reticulocyte profiles during periods of intense physical training, the intra-individual variation of these parameters in 12 iron-replete female athletes over a four month period of training was documented. The precision with which the analyzer measured these parameters was also determined using duplicate samples from 37 female athletes. To establish whether reticulocyte parameters were sensitive to iron deficient erythropoiesis in athletes, reticulocyte profiles of five female athletes diagnosed by medical personnel as having depleted iron stores were compared before and after iron therapy to seven controls. RESULTS: Corpuscular hemoglobin concentration mean (CHCMr) and mean corpuscular volume (MCVr) showed little variation over time in iron-replete females, with 95% of all fluctuations being within 5.8% and 4.3% of original values, respectively. Iron supplementation in athletes with depleted iron stores elicited an increase in CHCMr (p = 0.01), and a decrease in the distributions of reticulocyte volume (RDWr, p = 0.01) and cell hemoglobin concentration (HDWr, p < 0.01). The ratios of reticulocyte to mature cell MCV (p < 0.01) and CHCM (p < 0.01) also changed following iron therapy. No such changes occurred in non-supplemented controls with normal iron stores. CONCLUSIONS: These data lend support to the thesis that monitoring of reticulocyte parameters can be of use in detecting iron deficient erythropoiesis in female athletes.