Red blood cell derived extracellular vesicles during the process of autologous blood doping.

The purpose of this pilot study was to investigate the effects of the transfusion of one erythrocyte concentrate on the number of circulating red blood cell extracellular vesicles (RBC-EVs) and their clearance time. Six, healthy volunteers donated their blood and were transfused with their RBC concentrate after 35-36 days of storage. One K2 EDTA and one serum sample were collected before donation, at four timepoints after donation and at another six timepoints after transfusion. RBC-EVs were analyzed on a Cytek Aurora flow cytometer. A highly significant increase (p < 0.001) of RBC-EVs from an average of 60.1 ± 19.8 (103 /µL) at baseline to 179.3 ± 84.7 (103 /µL) in the first 1-3 h after transfusion could be observed. Individual differences in the response to transfusion became apparent with one volunteer showing no increase and another an increased concentration at one timepoint after donation due to an influenza infection. We concluded that in an individualized passport approach, increased RBC-EVs might be considered as additional evidence when interpreting suspicious Athletes Biological Passport (ABPs) but for this additional research related to sample collection and transport processes as well as method development and harmonization would be necessary.

The effect of cumulative endurance exercise on leptin and adiponectin and their role as markers to monitor training load.

Leptin and adiponectin play an essential role in energy metabolism. Leptin has also been proposed as a marker for monitoring training load. So far, no studies have investigated the variability of these hormones in athletes and how they are regulated during cumulative exercise. This study monitored leptin and adiponectin in 15 endurance athletes twice daily in the days before, during and after a 9-day simulated cycling stage race. Adiponectin significantly increased during the race (p = 0.001) and recovery periods (p = 0.002) when compared to the baseline, while leptin decreased significantly during the race (p < 0.0001) and returned to baseline levels during the recovery period. Intra-individual variability was substantially lower than inter-individual variability for both hormones (leptin 34.1 vs. 53.5%, adiponectin 19% vs. 37.2%). With regards to exercise, this study demonstrated that with sufficient, sustained energy expenditure, leptin concentrations can decrease within the first 24 hours. Under the investigated conditions there also appears to be an optimal leptin concentration which ensures stable energy homeostasis, as there was no significant decrease over the subsequent race days. In healthy endurance athletes the recovery of leptin takes 48-72 hours and may even show a supercompensation-like effect. For adiponectin, significant increases were observed within 5 days of commencing racing, with these elevated values failing to return to baseline levels after 3 days of recovery. Additionally, when using leptin and adiponectin to monitor training loads, establishing individual threshold values improves their sensitivity.

The effect of fasting during Ramadan on parameters of the haematological and steroidal modules of the athletes biological passport - a pilot study.

This study investigated the effect of Ramadan on the haematological and steroid module of the Athletes Biological Passport (ABP) of the World Anti-Doping Agency (WADA). Nine healthy physically active subjects were tested in the morning and afternoon for two days before and three days during Ramadan. Sample collection and all analyses were performed according to WADA technical documents. Although there were significant changes in the haemoglobin concentration during Ramadan, especially during the first fasting week, none of the subjects in this study exceeded the individually calculated thresholds of the ABP. No significant effects on testosterone/epitestosterone (T/E) ratio were observed but only the afternoon specific gravity (SG) of the urine was elevated. Thus, when urinary steroid concentrations are required, SG corrections need to be performed. The haematological and the steroid module of the ABP can be reliably applied during Ramadan as the observed changes are only marginal.

New parameters and reference values for monitoring iron status in Middle Eastern adolescent male athletes.

AIM: Hematological and biochemical parameters of 160 Middle Eastern adolescent male athletes (aged from 12-18 years) were tested in order to investigate their iron status and to establish reference values for this population. A focus of this study was also the investigation of Reticulocyte hemoglobin (RetHe) and soluble transferrin receptor (sTfR). METHODS: Complete blood count, reticulocyte and sera parameters were analyzed at the beginning of the training season for these adolescent athletes. As the diagnosis of iron deficiency in adolescents is extremely difficult subjects were subdivided in three age groups (12-13, 14-15, 16-18). RESULTS: For most of the parameters our results confirmed the existing reference values reported in young athletes. Exceptions were however found with lower Mean Cell Volumes (79.9±4.3 fl) in this group when compared to other age matched data. RetHe, ferritin and sTfR levels were monitored for the interpretation of the iron status in this population and reference values for these parameters were also established. CONCLUSIONS: Information to help evidence based decision making about the need for supplementation or further investigations is provided to physicians and nutritionists. RetHe with a proposed threshold value of 25 pg expands the list of parameters which can be used to monitor athletes.

Variability of serum markers of erythropoiesis during 6 days of racing in highly trained cyclists.

The athlete biological passport for the fight against doping is currently based on longitudinal monitoring for abnormal changes in cellular blood parameters. Serum parameters related to altered erythropoiesis could be considered for inclusion in the passport. The aim of this study was to quantify the changes in such parameters in athletes during a period of intense exercise.12 highly trained cyclists tapered for 3 days before 6 days of simulated intense stage racing. Morning and afternoon blood samples were taken on most days and analysed for total protein, albumin, soluble transferrin receptor and ferritin concentrations. Plasma volume was determined via total haemoglobin mass measured by carbon-monoxide rebreathing. Percent changes in means from baseline and percent standard errors of measurement (analytical error plus intra-athlete variation) on each measurement occasion were estimated with mixed linear modelling of log-transformed measures. Means of all variables changed substantially in the days following the onset of racing, ranging from -13% (haemoglobin concentration) to +27% (ferritin). After the second day, errors of measurement were generally twice those at baseline. Plasma variables were affected by heavy exercise, either because of changes in plasma volume (total protein, albumin, haemoglobin), acute phase/inflammatory reactions (ferritin) or both (soluble transferrin receptor). These effects need to be taken into consideration when integrating a plasma parameter into the biological passport model for athletes.

The effect of a period of intensive exercise on the isoform test to detect growth hormone doping in sports.

OBJECTIVE: The major objective of this study was to investigate the effects of several days of intense exercise on growth hormone (hGH) testing using the World Anti-Doping Agencies hGH isoform differential immunoassays. Additionally the effects of circadian variation and exercise type on the isoform ratios were also investigated. STUDY DESIGN: 15 male athletes performed a simulated nine day cycling stage race. Blood samples were collected twice daily over a period of 15 days (stage race+three days before and after). hGH isoforms were analysed by the official WADA immunoassays (CMZ Assay GmbH). RESULTS: All measured isoform ratios were far below the WADA decision limits for an adverse analytical finding. Changes in the isoform ratios could not be clearly connected to circadian variation, exercise duration or intensity. CONCLUSIONS: The present study demonstrates that the hGH isoform ratios are not significantly affected by exercise or circadian variation. We demonstrated that heavy, long term exercise does not interfere with the decision limits for an adverse analytical finding.

Monitoring fitness, fatigue and running performance during a pre-season training camp in elite football players.

OBJECTIVES: To examine the usefulness of selected physiological and perceptual measures to monitor fitness, fatigue and running performance during a pre-season, 2-week training camp in eighteen professional Australian Rules Football players (21.9±2.0 years). DESIGN: Observational. METHODS: Training load, perceived ratings of wellness (e.g., fatigue, sleep quality) and salivary cortisol were collected daily. Submaximal exercise heart rate (HRex) and a vagal-related heart rate variability index (LnSD1) were also collected at the start of each training session. Yo-Yo Intermittent Recovery level 2 test (Yo-YoIR2, assessed pre-, mid- and post-camp, temperate conditions) and high-speed running distance during standardized drills (HSR, >14.4 km h(-1), 4 times throughout, outdoor) were used as performance measures. RESULTS: There were significant (P<0.001 for all) day-to-day variations in training load (coefficient of variation, CV: 66%), wellness measures (6-18%), HRex (3.3%), LnSD1 (19.0%), but not cortisol (20.0%, P=0.60). While the overall wellness (+0.06, 90% CL (-0.14; 0.02) AU day(-1)) did not change substantially throughout the camp, HRex decreased (-0.51 (-0.58; -0.45)% day(-1)), and cortisol (+0.31 (0.06; 0.57) nmol L(-1)day(-1)), LnSD1 (+0.1 (0.04; 0.06) ms day(-1)), Yo-YoIR2 performance (+23.7 (20.8; 26.6) m day(-1), P<0.001), and HSR (+4.1 (1.5; 6.6) m day(-1), P<0.001) increased. Day-to-day ΔHRex (r=0.80, 90% CL (0.75; 0.85)), ΔLnSD1 (0.51 (r=0.40; 0.62)) and all wellness measures (0.28 (-0.39; -0.17)

The impact of long-haul air travel on variables of the athlete's biological passport.

INTRODUCTION: Dehydration, fluid shifts or changes in coagulation occurring during air travel can trigger distinct reactions in the haematological system. Athletes are concerned that these effects might impair sporting performance, increase the risk of thrombosis or cause abnormalities in blood values that might be mistaken for doping in the 'Athlete's biological passport' (ABP) a longitudinal monitoring of haematological variables in antidoping. The aim of the study was to investigate key variables of the ABP before and after a long-haul flight in athletes. METHODS: Fifteen endurance athletes were submitted to ABP blood samples in the morning before and after arrival of an 8 h flight. Two additional samples were obtained in the morning and the evening 3 days after the travel. Twelve nontravelling subjects served as controls. RESULTS: Haemoglobin concentration was higher before than after travel in athletes (+0.5 g/dL, P = 0.038), a similar pattern was observed 3 days after the travel. No difference was observed in the control group. Reticulocyte% did not show any significant changes in neither of the groups. CONCLUSION: The observed changes are in line with normal diurnal variations. There is no indication that travel will affect haematological variables in way that might be mistaken for blood doping.

Physiological and performance adaptations to an in-season soccer camp in the heat: associations with heart rate and heart rate variability.

The aim of the present study was to examine the associations between adaptive responses to an in-season soccer training camp in the heat and changes in submaximal exercising heart rate (HRex, 5-min run at 9 km/h), postexercise HR recovery (HRR) and HR variability (HRV). Fifteen well-trained but non-heat-acclimatized male adult players performed a training week in Qatar (34.6 ± 1.9°C wet bulb globe temperature). HRex, HRR, HRV (i.e. the standard deviation of instantaneous beat-to-beat R-R interval variability measured from Poincaré plots SD1, a vagal-related index), creatine kinase (CK) activity, plasma volume (PV) changes, and post-5-min run rate of perceived exertion (RPE) were collected at six occasions in temperate environmental conditions (22°C). Players also performed the yo-yo intermittent recovery test level 1 (Yo-Yo IR1) in the same environmental conditions (22°C), both at the beginning and at the end of the training week. Throughout the intervention, HRex and HRV showed decreasing (P < 0.001) and increasing (P < 0.001) trends, respectively, while HRR remained unaffected (P = 0.84). Changes in HRex [-0.52, 90% confidence limits (-0.64; -0.38), P < 0.001] and SD1 [0.35 (0.19; 0.49), P < 0.001] were correlated with those in PV. There was no change in RPE (P = 0.92), while CK varied according to training contents (P < 0.001), without association with HR-derived measures. Yo-Yo IR1 performance increased by 7 ± 9% (P = 0.009), which was correlated with changes in HRex [-0.64 (-0.84; -0.28), P = 0.01]. In conclusion, we found that an in-season soccer training camp in the heat can significantly improve PV and soccer-specific physical performance; both of which are associated with changes in HRex during a 5-min submaximal run.

Temperature-dependent development of the parasitoid Tachinaephagus zealandicus on five forensically important carrion fly species.

The influences of temperature and host species on the development of the forensically important parasitoid Tachinaephagus zealandicus Ashmead (Hymenoptera: Encyrtidae) were studied at six constant temperatures in the range of 15-30 degrees C. T. zealandicus completed development successfully between 15 degrees C and 27 degrees C on five species of Calliphoridae, Calliphora albifrontalis Malloch, Calliphora dubia Macquart, Lucilia sericata Meigen, Chrysomya rufifacies Macquart and Chrysomya megacephala Fabricius. No adult parasitoids emerged from any of the host species reared at 30 degrees C. Temperature and host species significantly influenced development time, emergence success and progeny size. Development was significantly longer on Ch. megacephala and Ch. rufifacies at 18-24 degrees C and significantly longer on Ch. rufifacies and C. albifrontalis at 15 degrees C and 27 degrees C. Parasitoid emergence success was greatest at 21 degrees C, declined at the temperature extremes (15 degrees C and 27 degrees C) and was significantly lower on Ch. megacephala and Ch. rufifacies than on the three other host species. Progeny numbers per host pupa were highest at 21-24 degrees C, declined on either side of this temperature range and were significantly lower on L. sericata, Ch. rufifacies and Ch. megacephala than on either C. dubia or C. albifrontalis. An effect of host species on sex ratio was only observed at 27 degrees C, at which a higher proportion of T. zealandicus females emerged from Ch. megacephala and Ch. rufifacies than from the other host species. The thermal requirements for development (developmental thresholds, thermal constant, optimum temperature) of T. zealandicus in each host species were estimated using linear and non-linear models. Upper and lower developmental thresholds ranged between 29.90 degrees C and 31.73 degrees C, and 9.73 degrees C and 10.08 degrees C, respectively. The optimum temperature for development was estimated at between 25.81 degrees C and 27.05 degrees C. Given the significant effect of host species on development time, the use of parasitoid-host-specific developmental data in forensic application is recommended.

Detection of homologous blood transfusion.

The aim of the present study was to improve and validate a flow cytometric method for the detection of homologous blood transfusion in doping control analysis. A panel of eight different primary antibodies and two different phycoerythrin-conjugated secondary antibodies was used for the detection of different blood populations. The flow cytometer used in this study was the BD FACSArray instrument. Mixed red blood cell populations were prepared from phenotype known donors. Linearity, specificity, recovery, precision, robustness and interday-precision were tested for every primary antibody used in the presented assay. The technique of signal amplification was utilized for an improved separation of antigens with weak or heterozygous expression to improve the interpretation of histograms. The resulting method allowed to clearly identify mixed red blood cell populations in homologous blood transfusion samples containing 0.3 - 2.0 % of donor blood.

Abdominoplasty combined with gynecologic surgical procedures.

Cosmetic surgery of the abdomen is requested frequently by patients and is being performed increasingly at the time of elective gynecologic surgery. However, little information is available regarding the safety of combining these procedures. In this study intraoperative and postoperative morbidity was compared in the following groups of patients: 1) abdominoplasty plus one of five common gynecologic procedures (N = 76); 2) one of the five gynecologic procedures alone, matched for age, weight, and time of operation (N = 76); and 3) abdominoplasty alone (N = 70). Patients in group 1 experienced significantly longer operative time, longer hospital stays, and greater blood loss, which required more transfusions than group 2 or 3. These problems were accentuated in patients who weighed more than 70 kg or were older than age 35. In addition, five patients (6.6%) in group 1 had a documented pulmonary embolus within 18 days of surgery, whereas no pulmonary emboli occurred in group 2 or 3. Because of the increased morbidity, careful patient selection is necessary when abdominoplasty and gynecologic procedures are performed at the same time.

Ultrasound and the pelvic mass.

We compared the findings from pelvic examination, preoperative ultrasound and surgery on 50 patients who underwent exploratory laparotomy for a pelvic mass at Tulane University Medical Center. Of the 50 cases, 34 (68%) were correctly diagnosed as a pelvic mass by both pelvic examination and ultrasonography. Ultrasound had nine false negatives (21%) and only one false positive (2%). Pelvic examination had 7 false positives (16%). There was no statistical difference by chi-square analysis in the accuracy between ultrasonography and pelvic examination in the detection or diagnosis of the masses. Ultrasound indicated pathology not associated with the suspected mass in ten patients; surgery confirmed pathology in five of them. More errors occurred on both pelvic examination and ultrasonography in obese patients than in nonobese ones: seven of the ten ultrasound errors occurred in obese patients, and five of the seven pelvic exam errors also occurred in obese patients. In the nine cases of false-negative ultrasound findings surgery was postponed, but all of them eventually underwent surgery because of a deteriorating clinical course. In our study pelvic ultrasonography was not beneficial in the management of patients with a known or suspected pelvic mass, but it was very accurate for confirmation of a mass.