Evaluation of blood parameters by linear discriminant models for the detection of testosterone administration.

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites to identify samples suspicious for the use of synthetic forms of Endogenous Anabolic Androgenic Steroids (EAAS). Multiple recent studies have suggested that monitoring of blood parameters may provide enhanced detectability of exogenous testosterone administration. Transdermal and intramuscular testosterone administration studies were carried out in 15 subjects, and the effect on blood steroidal levels, hematological parameters, and gonadotropins was evaluated. Serum testosterone and dihydrotestosterone levels increased while gonadotropin levels were suppressed after administration. A modest increase in reticulocytes was also observed. The blood parameters that were responsive to the administrations were combined into several linear discriminant models targeting both administration (on) and washout (off) phases. The models were effective in detecting the large dose intramuscular administration but were less successful in the detection of the lower dose transdermal application. The blood profiling models may provide complementary value but do not appear to be substantially more advantageous than longitudinal urinary profiling.

Core Temperature Responses to Cold-Water Immersion Recovery: A Pooled-Data Analysis.

PURPOSE: To examine the effect of postexercise cold-water immersion (CWI) protocols, compared with control (CON), on the magnitude and time course of core temperature (Tc) responses. METHODS: Pooled-data analyses were used to examine the Tc responses of 157 subjects from previous postexercise CWI trials in the authors' laboratories. CWI protocols varied with different combinations of temperature, duration, immersion depth, and mode (continuous vs intermittent). Tc was examined as a double difference (ΔΔTc), calculated as the change in Tc in CWI condition minus the corresponding change in CON. The effect of CWI on ΔΔTc was assessed using separate linear mixed models across 2 time components (component 1, immersion; component 2, postintervention). RESULTS: Intermittent CWI resulted in a mean decrease in ΔΔTc that was 0.25°C (0.10°C) (estimate [SE]) greater than continuous CWI during the immersion component (P = .02). There was a significant effect of CWI temperature during the immersion component (P = .05), where reductions in water temperature of 1°C resulted in decreases in ΔΔTc of 0.03°C (0.01°C). Similarly, the effect of CWI duration was significant during the immersion component (P = .01), where every 1 min of immersion resulted in a decrease in ΔΔTc of 0.02°C (0.01°C). The peak difference in Tc between the CWI and CON interventions during the postimmersion component occurred at 60 min postintervention. CONCLUSIONS: Variations in CWI mode, duration, and temperature may have a significant effect on the extent of change in Tc. Careful consideration should be given to determine the optimal amount of core cooling before deciding which combination of protocol factors to prescribe.

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.

Temporal changes in physiology and haematology in response to high- and micro-doses of recombinant human erythropoietin.

There is evidence to suggest athletes have adopted recombinant human erythropoietin (rHuEPO) dosing regimens that diminish the likelihood of being caught by direct detection techniques. However, the temporal response in physiology, performance, and Athlete Biological Passport (ABP) parameters to such regimens is not clearly understood. Participants were assigned to a high-dose only group (HIGH, n = 8, six rHuEPO doses of 250 IU/kg over two weeks), a combined high micro-dose group (COMB, n = 8, high-dose plus nine rHuEPO micro-doses over a further three weeks), or one of two placebo control groups who received saline in the same pattern as the HIGH (HIGH-PLACEBO, n = 4) or COMB (COMB-PLACEBO, n = 4) groups. Temporal changes in physiology and performance were tracked by graded exercise test (GXT) and haemoglobin mass assessment at baseline, after high dose, after micro-dose (COMB and COMB-PLACEBO only) and after a four-week washout. Venous blood samples were collected throughout the baseline, rHuEPO administration, and washout periods to determine the haematological and ABP response to each dosing regimen. Physiological adaptations induced by a two-week rHuEPO high-dose were maintained by rHuEPO micro-dosing for at least three weeks. However, all participants administered rHuEPO registered at least one suspicious ABP value during the administration or washout periods. These results indicate there is sufficient sensitivity in the ABP to detect use of high rHuEPO doping regimens in athletic populations and they provide important empirical examples for use by anti-doping experts. Copyright © 2017 John Wiley & Sons, Ltd.

Reliability and accuracy of six hand-held blood lactate analysers.

The reliability and accuracy of five portable blood lactate (BLa) analysers (Lactate Pro, Lactate Pro2, Lactate Scout+, Xpress™, and Edge) and one handheld point-of-care analyser (i-STAT) were compared to a criterion (Radiometer ABL90). Two devices of each brand of analyser were assessed using 22 x 6 mL blood samples taken from five subjects at rest and during exercise who generated lactate ranging ~1-23 mM. Each sample was measured simultaneously ~6 times on each device. Reliability was assessed as the within-sample standard deviation (wsSD) of the six replicates; accuracy as the bias compared with the ABL90; and overall error (the root mean squared error (√MSE)) was calculated as the square root of (wsSD(2) and bias(2)). The √MSE indicated that both the Edge and Xpress had low total error (~0-2 mM) for lactate concentrations <15 mM, whereas the Edge and Lactate Pro2 were the better of the portable analysers for concentrations >15 mM. In all cases, bias (negative) was the major contribution to the √MSE. In conclusion, in a clinical setting where BLa is generally <15 mM the Edge and Xpress devices are relevant, but for athlete testing where peak BLa is important for training prescription the Edge and Lactate Pro2 are preferred. Key pointsThe reliability of five common portable blood lactate analysers were generally <0.5 mM for concentrations in the range of ~1.0-10 mM.For all five portable analysers, the analytical error within a brand was much smaller than the biological variation in blood lactate (BLa).Compared with a criterion blood lactate analyser, there was a tendency for all portable analysers to under-read (i.e. a negative bias), which was particularly evident at the highest concentrations (BLa ~15-23 mM).The practical application of these negative biases would overestimate the ability of the athlete and prescribe a training intensity that would be too high.

Prediction of Crohn's disease aggression through NOD2/CARD15 gene sequencing in an Australian cohort.

AIM: To investigate the association between mutations in oligomerisation domain 2/caspase recruitment domains 15 (NOD2/CARD15) and the natural history of Crohn's disease (CD) to identify patients who would benefit from early aggressive medical intervention. METHODS: We recruited thirty consecutive unrelated CD patients with a history of ileo-caecal or small bowel resection during the period 1980-2000; Fifteen patients of these had post-operative relapse that required further surgery and fifteen did not. Full sequencing of the NOD2/CARD15 gene using dHPLC for exons 3, 5, 7, 10 and 12 and direct sequencing for exons 2, 4, 6, 8, 9 and 11 was conducted. CD patients categorized as carrying variants were anyone with at least 1 variant of the NOD2/CARD15 gene. RESULTS: About 13.3% of the cohort (four patients) carried at least one mutant allele of 3020insC of the NOD2/CARD15 gene. There were 20 males and 10 females with a mean age of 43.3 years (range 25-69 years). The mean follow up was 199.6 mo and a median of 189.5 mo. Sixteen sequence variations within the NOD2/CARD15 gene were identified, with 9 of them occurring with an allele frequency of greater than 10 %. In this study, there was a trend to suggest that patients with the 3020insC mutation have a higher frequency of operations compared to those without the mutation. Patients with the 3020insC mutation had a significantly shorter time between the diagnosis of CD and initial surgery. This study included Australian patients of ethnically heterogenous background unlike previous studies conducted in different countries. CONCLUSION: These findings suggest that patients carrying NOD2/CARD15 mutations follow a rapid and more aggressive form of Crohn's disease showing a trend for multiple surgical interventions and significantly shorter time to early surgery.

A population study of urine glycerol concentrations in elite athletes competing in North America.

Glycerol is an endogenous substance that is on the World Anti-Doping Agency's list of prohibited threshold substances due to its potential use as a plasma volume expansion agent. The WADA has set the threshold for urine glycerol, including measurement uncertainty, at 1.3 mg/mL. Glycerol in circulation largely comes from metabolism of triglycerides in order to meet energy requirements and when the renal threshold is eclipsed, glycerol is excreted into urine. In part due to ethnic differences in postprandial triglyceride concentrations, we investigated urine glycerol concentrations in a population of elite athletes competing in North America and compared the results to those of athletes competing in Europe. 959 urine samples from elite athletes competing in North America collected for anti-doping purposes were analyzed for urine glycerol concentrations by a gas chromatography mass-spectrometry method. Samples were divided into groups according to: Timing (in- or out-of-competition), Class (strength, game, or endurance sports) and Gender. 333 (34.7%) samples had undetectable amounts of glycerol (<1 µg/mL). 861 (89.8%) of the samples had glycerol concentrations ≤20 µg/mL. The highest glycerol concentration observed was 652 µg/mL. Analysis of the data finds the effects of each category to be statistically significant. The largest estimate of the 99.9(th) percentile, from the in-competition, female, strength athlete samples, was 1813 µg/mL with a 95% confidence range from 774 to 4251 µg/mL. This suggests a conservative threshold of 4.3 mg/mL, which would result in a reasonable detection window for urine samples collected in-competition for all genders and sport classes.

Altitude training and haemoglobin mass from the optimised carbon monoxide rebreathing method determined by a meta-analysis.

OBJECTIVE: To characterise the time course of changes in haemoglobin mass (Hbmass) in response to altitude exposure. METHODS: This meta-analysis uses raw data from 17 studies that used carbon monoxide rebreathing to determine Hbmass prealtitude, during altitude and postaltitude. Seven studies were classic altitude training, eight were live high train low (LHTL) and two mixed classic and LHTL. Separate linear-mixed models were fitted to the data from the 17 studies and the resultant estimates of the effects of altitude used in a random effects meta-analysis to obtain an overall estimate of the effect of altitude, with separate analyses during altitude and postaltitude. In addition, within-subject differences from the prealtitude phase for altitude participant and all the data on control participants were used to estimate the analytical SD. The 'true' between-subject response to altitude was estimated from the within-subject differences on altitude participants, between the prealtitude and during-altitude phases, together with the estimated analytical SD. RESULTS: During-altitude Hbmass was estimated to increase by ∼1.1%/100 h for LHTL and classic altitude. Postaltitude Hbmass was estimated to be 3.3% higher than prealtitude values for up to 20 days. The within-subject SD was constant at ∼2% for up to 7 days between observations, indicative of analytical error. A 95% prediction interval for the 'true' response of an athlete exposed to 300 h of altitude was estimated to be 1.1-6%. CONCLUSIONS: Camps as short as 2 weeks of classic and LHTL altitude will quite likely increase Hbmass and most athletes can expect benefit.

Improved alignment of reticulocyte counts between Sysmex XT-2000i instruments.

BACKGROUND: Reticulocytes are the most sensitive index available to authorities who seek to sanction athletes for blood doping based on deviations beyond individual reference ranges. Because such data comprise longitudinal results that are generated by different laboratories, the comparability of reticulocyte counts from different instruments is of crucial importance. AIMS: To enhance between-instrument comparability of reticulocyte counts reported by the Sysmex XT-2000i automated haematology analyser. METHODS: We optimised recalibration of instruments towards assigned values of control material (e-CHECK) in tandem with fresh blood verification. RESULTS: In terms of reticulocyte counts reported as a percentage of all cells in a fresh blood sample, it was possible to recalibrate all three test instruments so that the mean of 10 samples was within 0.1% of the comparative instrument's mean value. CONCLUSIONS: This approach provides a straightforward means of reducing between-instrument differences in reticulocyte counts generated by the Sysmex XT-2000i.

Pilot study of remote telemonitoring in COPD.

BACKGROUND: Remote in-home monitoring (RM) of symptoms and physiological variables may allow early detection and treatment of exacerbations of chronic obstructive pulmonary disease (COPD). It is unclear whether RM improves patient outcomes or healthcare resource utilization. This study determined whether RM is feasible in patients with COPD and if RM reduces hospital admissions or length of stay (LOS) or improves health-related quality of life (HRQOL). SUBJECTS AND METHODS: Forty-four patients were randomized to standard best practice care (SBP) (n=22) or SBP+RM (n=22). RM involved daily recording of physiological variables, symptoms, and medication usage. RESULTS: There were no differences (mean±SD, SBP versus SBP+RM) in age (68±8 versus 70±9 years), gender (male:female 10:12 in both groups), or previous computer familiarity (59% versus 50%) between groups. The SBP group had a lower forced expiratory volume in 1 s (0.66±0.24 versus 0.91±0.34 L, p<0.01) and more current smokers (six versus none, p<0.05). There were no differences in number of COPD-related admissions/year (1.5±1.8 versus 1.3±1.7, p=0.76), COPD-related LOS days/year (15.6±19.4 versus 11.4±19.6, p=0.66), total admissions/year (2.2±2.1 versus 2.0±2.3, p=0.86), total LOS days/year (22.1±29.9 versus 21.6±30.4, p=0.88), or HRQOL between the two groups. CONCLUSIONS: The addition of RM to SBP was feasible but did not reduce healthcare utilization or improve quality of life in this group of patients already receiving comprehensive respiratory care.

Evaluation of the MAIIA dipstick test to detect recombinant human erythropoietin in plasma.

Recently a novel technology, referred to as the 'EPO WGA MAIIA' test, has been developed by Swedish researchers to discriminate between endogenous and recombinant human erythropoietin. In contrast to existing electrophoretic methods that are used by antidoping laboratories, this dipstick-based technique is simple and fast. Moreover it can be applied to either blood or urine specimens. These characteristics could prove advantageous if the test were adopted by antidoping authorities to determine blood doping in sport. We evaluated the sensitivity of EPO WGA MAIIA to detect the presence of recombinant human erythropoietin (rhEPO) in some archived plasma specimens which had been collected from healthy, active subjects either 72 h or 96 h after a 'microdose' intravenous injection of rhEPO. Under these conditions the test had modest sensitivity to discriminate rhEPO, with only two of nine subjects exceeding an arbitrary cut-off 3.09 SDs beyond the expected population mean. Sensitivity was improved to five out of six subjects if positivity was assessed according to the subject's own previous values rather than a population-based threshold. We conclude that, with further refinement, the dipstick test may supplement existing antidoping tests.

Preanalytical mixing of whole-blood specimens in the context of the Athlete Passport.

BACKGROUND: Full blood counts are now used as evidence that athletes have used banned blood doping. This has led to legal scrutiny of the efficacy of preanalytical procedures such as specimen homogenisation. AIMS: To characterise the impact of different mixing strategies on whole-blood homogeneity. METHODS: Manual inversion, mechanical mixing and automatic mixing performed by the Sysmex XT-2000i were evaluated. RESULTS: Automated mixing by the instrument, or 1 min of mechanical mixing, thoroughly homogenised specimens even for tubes that had been refrigerated and left undisturbed for 36 h. Manual inversions were almost as effective, provided that specimens were first allowed to equilibrate to ambient temperatures. CONCLUSIONS: Current sport guidelines that mandate at least 15 min of mechanical mixing are excessive. Except where specimens are presented to the instrument manually, mechanical mixing is redundant in the context of full blood counts.

Within-subject variation in hemoglobin mass in elite athletes.

UNLABELLED: Illicit autologous blood transfusion to improve performance in elite sport is currently undetectable, but the stability of longitudinal profiles of an athlete's hemoglobin mass (Hbmass) might be used to detect such practices. PURPOSE: Our aim was to quantify within-subject variation of Hbmass in elite athletes, and the effects of potentially confounding factors such as reduced training or altitude exposure. METHODS: A total of 130 athletes (43 females and 87 males) were measured for Hbmass an average of six times during a period of approximately 1 yr using carbon monoxide rebreathing. Linear mixed models were used to quantify within-subject variation of Hbmass and its associated analytical and biological components for males and females, as well as the effects of reduced training and moderate altitude exposure in certain athletes. RESULTS: The maximum within-subject coefficient of variation (CV) for Hbmass was 3.4% for males and 4.0% for females. The analytical CV was ~2.0% for both males and females, and the long-term biological CV, after allowing for analytical variation, was 2.8% for males and 3.5% for females. On average, self-reported reduced training resulted in a 2.8% decrease in Hbmass and altitude exposure increased Hbmass by 1.5% to 2.9%, depending on the duration and type of exposure. CONCLUSIONS: The within-subject CV for Hbmass of ~4% indicates that athletes may experience changes up to ~20% with a 1-in-1000 probability. Changes of this magnitude for measures taken a few months apart suggest that Hbmass has a limited capacity to detect autologous blood doping. However, changes in Hbmass may be a useful indicator when combined with other measures of blood manipulation.

Current markers of the Athlete Blood Passport do not flag microdose EPO doping.

The Athlete Blood Passport is the most recent tool adopted by anti-doping authorities to detect athletes using performance-enhancing drugs such as recombinant human erythropoietin (rhEPO). This strategy relies on detecting abnormal variations in haematological variables caused by doping, against a background of biological and analytical variability. Ten subjects were given twice weekly intravenous injections of rhEPO for up to 12 weeks. Full blood counts were measured using a Sysmex XE-2100 automated haematology analyser, and total haemoglobin mass via a carbon monoxide rebreathing test. The sensitivity of the passport to flag abnormal deviations in blood values was evaluated using dedicated Athlete Blood Passport software. Our treatment regimen elicited a 10% increase in total haemoglobin mass equivalent to approximately two bags of reinfused blood. The passport software did not flag any subjects as being suspicious of doping whilst they were receiving rhEPO. We conclude that it is possible for athletes to use rhEPO without eliciting abnormal changes in the blood variables currently monitored by the Athlete Blood Passport.

A randomised trial of domiciliary, ambulatory oxygen in patients with COPD and dyspnoea but without resting hypoxaemia.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) who are not severely hypoxaemic at rest may experience significant breathlessness on exertion, and ambulatory oxygen is often prescribed in this circumstance despite a lack of conclusive evidence for benefit. This study aimed to determine whether such patients benefit from domiciliary ambulatory oxygen and, if so, which factors may be associated with benefit. METHODS: This was a 12 week, parallel, double-blinded, randomised, placebo-controlled trial of cylinder air versus cylinder oxygen, provided at 6 l/min intranasally, for use during any activity provoking breathlessness. Patients underwent baseline measurements of arterial blood gases and lung function. Outcome measures assessed dyspnoea, health-related quality of life, mood disturbance, functional status and cylinder utilisation. Data were analysed on an intention-to-treat basis, p≤0.05. RESULTS: 143 subjects (44 female), mean±SD age 71.8±9.8 years, forced expiratory volume in 1 s (FEV(1))1.16±0.51 litres, Pao(2) 9.5±1.1 kPa (71.4±8.5 mm Hg) were randomised, including 50 patients with exertional desaturation to ≤88%. No significant differences in any outcome were found between groups receiving air or oxygen. Statistically significant but clinically small improvements in dyspnoea and depression were observed in the whole study group over the 12 weeks of the study. CONCLUSION: In breathless patients with COPD who do not have severe resting hypoxaemia, domiciliary ambulatory oxygen confers no benefits in terms of dyspnoea, quality of life or function. Exertional desaturation is not predictive of outcome. Intranasal gas (either air or oxygen) may provide a placebo benefit. CLINICAL TRIAL NUMBER: ACTRN12605000457640.

Low testosterone and anaemia in men with type 2 diabetes.

OBJECTIVE: Anaemia is frequently found in patients with diabetes, in whom it is associated with increased morbidity and mortality. Low testosterone levels are also common in men with type 2 diabetes. We hypothesized that low testosterone levels are also associated with anaemia in men with type 2 diabetes, over the effects of chronic kidney disease. DESIGN: Cross-sectional cohort study, performed in 2005 in a tertiary diabetes clinic. Patients 464 men with type 2 diabetes. MAIN OUTCOME MEASURE: Anaemia (haemoglobin (Hb) < 13.7 g/dl in men aged < 60, or < 13.2 g/dl in men aged 60 and older). RESULTS: About 24% of study participants had anaemia, which was associated with the presence and severity of chronic kidney disease, systemic inflammation, increased age, and reduced iron availability. In addition, testosterone levels were independently associated with reduced Hb levels, determining between 6 and 8% of the total variability in raw Hb levels in this population after adjusting for these other factors. Individuals with total testosterone level < 10 nmol/l (43% of the cohort) were more likely to have anaemia (adjusted odds ratio 1.7; 95% CI 1.1-2.8). Similarly, anaemia was twice as common in individuals with a calculated free testosterone of < 0.23 nmol/l (adjusted odds ratio 2.0, 95% CI 1.2-3.1). CONCLUSIONS: These findings suggest that testosterone deficiency may contribute to the increased frequency of anaemia in men with type 2 diabetes. However, the appropriate clinical response to testosterone deficiency in anaemic patients remains to be established by prospective clinical trials.

Low testosterone levels are common and associated with insulin resistance in men with diabetes.

CONTEXT: Low testosterone levels are common in men with type 2 diabetes and may be associated with insulin resistance. OBJECTIVE: We investigated prevalence of testosterone deficiency and the relationship between testosterone and insulin resistance in a large cohort of men with type 2 and type 1 diabetes. DESIGN: The study was a cross-sectional survey of 580 men with type 2 diabetes and 69 men with type 1 diabetes. A subgroup of 262 men with type 2 diabetes was then reassessed after a median of 6 months. RESULTS: Forty-three percent of men with type 2 diabetes had a reduced total testosterone, and 57% had a reduced calculated free testosterone. Only 7% of men with type 1 diabetes had low total testosterone. By contrast, 20.3% of men with type 1 diabetes had low calculated free testosterone, similar to that observed in type 2 diabetes (age-body mass index adjusted odds ratio = 1.4; 95% confidence interval = 0.7-2.9). Low testosterone levels were independently associated with insulin resistance in men with type 1 diabetes as well as type 2 diabetes. Serial measurements also revealed an inverse relationship between changes in testosterone levels and insulin resistance. CONCLUSIONS: Testosterone deficiency is common in men with diabetes, regardless of the type. Testosterone levels are partly influenced by insulin resistance, which may represent an important avenue for intervention, whereas the utility of testosterone replacement remains to be established in prospective trials.