NACHWEIS VON BLUT- UND EPODOPING.UNTERSUCHUNGEN ZUR VALIDITÄT DER DIREKTEN UND INDIREKTEN METHODEN.

Kurzzusammenfassung Die hier vorliegende Arbeit hat folgende Themengebiete mit Relevanz in der Dopinganalytik untersucht: Die Lagerungsstabilität von zellulären Blutparametern (1), den Einfluss unterschiedlicher Analysegeräte auf Hämoglobinmasse, Hämoglobinkonzentration und Retikulozyten (2), die Reproduzierbarkeit der Hämoglobinmassenbestimmung (3), den Einfluss einer Flugreise auf Hämoglobinkonzentration und Retikulozyten (4), den diurnalen Rhythmus der Hämoglobinkonzentration vor und während eines Etappenrennens im Radsport (5), den Einfluss von belastungsinduzierten Schwankungen im Plasmavolumen auf die Hämoglobinkonzentration (6), die Validität der Methode zur direkten Detektion der homologen Bluttransfusion (7) und den Einfluss hochintensiver Belastung auf die Nachweismethoden von rekombinantem Erythropoietin (8). Zu 1.: Die Ergebnisse unserer Untersuchungen zeigen, dass zur Harmonisierung der präanalytischen Bedingungen von Blutproben eine gekühlte Lagerung um 4°C klar gegenüber einer Lagerung um 21°C zu favorisieren ist. Bei einer Lagerungstemperatur von 21°C sollte die Analyse der Proben innerhalb von 24 Stunden erfolgen. Zu 2.: Geräteunterschiede führten bei der Hämoglobinkonzentration zu Abweichungen von durchschnittlich 0,38 g/dl und bei Retikulozytenprozent zu durchschnittlich 0,33 % (Sysmex KX21N und R500 verglichen mit Siemens ADVIA120). Bei der Bestimmung der Hämoglobinmasse mit dem Radiometer OSM3 und dem Roche Cobas 221 konnte ein Unterschied von durchschnittlich 83 g dokumentiert werden. Dies zeigt, dass im Rahmen des biologischen Passes oder in wissenschaftlichen Zeitreihenstudien, Messungen auf dem gleichen Gerätetyp eine Grundvoraussetzung darstellt. Zu 3.: Die Reproduzierbarkeit der Hämoglobinmasse am selben Analysegerät wurde durch Messungen innerhalb von zwei Tagen untersucht. Unsere Ergebnisse zeigten eine durchschnittliche Abweichung von 17,8 g (± 15,8 g). Die höchste gemessene Abweichung betrug 67,3 g, ohne dass ein Fehler in der Anwendung der Methode erkennbar war. Aus der Messunsicherheit wird deutlich, dass die Hämoglobinmassenbestimmung in Bereich des biologischen Passes nur beschränktes Potential aufweist. Die Methode ist nicht sensitiv genug, um die Verabreichung eines Erythrozytenkonzentrates das 50g Hämoglobin enthält, sicher zu detektieren. Zu 4. und 5.: Wir konnten zeigen, dass eine achtstündige Flugreise mit zwei Stunden Zeitunterschied keinen Einfluss auf die Hämoglobinkonzentration und den Retikulozytenprozentwert hat und dass der diurnale Rhythmus dieser Parameter selbst während eines Etappenrennens bestehen bleibt. Zu 6.: Belastungsinduzierte Schwankungen des Plasmavolumens weisen eine sehr hohe Relevanz bezüglich der Parameter des biologischen Passes auf. Ein Proband der in dieser Arbeit beschriebenen Studie erreichte innerhalb von 5 Tagen eine Vergrößerung seines Plasmavolumens um 1389 ml, was einen Abfall der Hämoglobinkonzentration von 15,7 g/dl auf 12,9 g/dl bedeutete. Diese Daten machen deutlich, dass der Zeitpunkt der Probennahme und der Einfluss von Belastung in der Interpretation von Blutwerten des biologischen Passes immer berücksichtigt werden müssen. Zu 7.: Zur Überprüfung der Validiät der durchflusszytometrischen Methode zur Bestimmung von homologen Bluttransfusionen wurde die Spezifität, Präzision, Linearität und Robustheit untersucht. Unsere Ergebnisse zeigten eine Spezifität von 100%. Die Präzision betrug je nach Antikörper 2,7% bis 19,8%. Antikörper des IgG Typs wiesen verglichen mit dem IgM Typ eine bessere Präzision auf. Die Linearität wurde für alle untersuchten Antigene bestätigt. Die Methode wurde als robust für qualitative, allerdings nicht für quantitative Aussagen bewertet. Die durchschnittliche Nachweisgrenze liegt unter 1% und ist für die meisten Antigene besser als 2%, was unter der Menge Fremdblut liegt, die bei einer Transfusion erwartet wird. Zu 8.: In der Untersuchung zum Einfluss von hochintensiver Belastung auf die Nachweismethoden von rekombinantem Erythropoietin konnten wir keinen signifikanten Zusammenhang zwischen VO2max, Laktatkonzentration, maximaler Wattzahl auf dem SRM und relativer Mobilität oder BAP feststellen. Alle Nachbelastungsproben zeigten höhere Werte für die BAP, jedoch wurden die WADA Kriterien zur Definition einer positiven Probe in keinem Fall erfüllt. Die relative Mobilität zeigte keine Werte über 0,559, was weit unter der Obergrenze des 99,9% Vertrauensintervalls für verdächtige Proben liegt. Die relative Mobilität war nach Belastung vermindert und es gab keine Verlagerung der Banden in den Bereich des rekombinanten EPOs. Unsere Ergebnisse demonstrieren, dass die SDS-PAGE eine Möglichkeit darstellt, um zwischen Belastungsurinen und rekombinantem humanen Erythropoietin zu unterscheiden. Abstract The investigations performed in this PhD thesis were about: The stability of cellular blood parameters (1), the effect of different analyzers on Hemoglobin (Hb) mass, Hb concentration [Hb] and Reticulocytes (2), the reproducibility of Hb mass estimations (3), the effect of air travelling on [Hb] and Reticulocytes (4), the diurnal rhythm of the [Hb] before and during a cycling stage race (5), the effect of exercise induced variations in plasma volume on the [Hb] (6), the validity of the direct method to detect homologous blood transfusions (7) and the influence of high intense exercise on the direct methods to detect recombinant erythropoietin in urine (8). To 1: The results of our investigations showed that blood samples should preferably be stored at 4°C (when compared to 21°C ) to harmonize pre-analytical conditions. When stored at 21°C samples should be analyzed within maximum 24 hours. To 2: Analyses on different instruments can result in mean differences of 0.38 g/dL for [Hb] and 0.33% for Reticulocytes (%) (Sysmex KX21N and R500 vs. Siemens ADVIA120). Hb mass estimations on the Radiometer OSM3 and the Roche Cobas 221 resulted in mean differences of 83 g. These results confirm that it is required to perform measurements for the biological passport or in scientific time series studies always on the same instrument type. To 3: The reproducibility of Hb mass estimations was performed by repeated measurements of the same subjects between two days. Our results showed a mean difference of 17.8 g (± 15.8 g). The maximum measured difference was 67.3 g, without any visible error. The observed measurement uncertainty confirms that the Hb mass method has only limited potential for the use in the biological passport. The method is not sensitive enough to safely detect the transfusion of one red blood cell concentrate (50 g Hb). To 4 and 5: We could demonstrate that 8 hours of air travelling did not influence [Hb] or Reticulocytes and that the diurnal rhythm of these parameters remained stable even during a cycling stage race. To 6: Exercise induced variations in plasma volume play a major role for parameters of the biological passport. In our study we had one subject who had within 5 days a plasma volume expansion of 1389 ml, which correlated with a decrease in [Hb] of 15.7 g/dl to 12.9 g/dl. These results highlight the importance of the time of blood sampling and the influence of exercise. To 7: For the flow-cytometric method to detect homologous blood transfusions we investigated the validity by testing specificity, precision, linearity and robustness. Our results showed a specificity of 100%. Depending on the used antibody the precision was within 2.7% to 19.8%. IgG antibodies showed a better precision when compared to IgM antibodies. Linearity was confirmed for all investigated antigens. The method was robust for qualitative but not for quantitative analyses. The mean limit of detection was below 1% and is for most antigens better than 2%. This is below the amount of homologous blood which is expected after one transfusion. To 8: We did not find any significant correlation between VO2max, lactate concentration or maximal power output (W) on a bicycle ergometer and erythropoietin (EPO) relative mobility (rmv) or EPO basic area percentage (BAP). All post exercise samples showed increased BAP values, however all samples remained negative according to WADA criteria. The rmv showed no values above 0.559, which is far below the 99.9% confidence interval for suspicious samples. The rmv was reduced in post exercise samples and no sample reached the area of recombinant EPO. Our results show that the SDS-PAGE method is a possibility to differentiate between effort urine EPO and recombinant EPO

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

Erythroferrone as a sensitive biomarker to detect stimulation of erythropoiesis.

Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to erythropoietin stimulation. ERFE suppresses the hepatic synthesis of the master iron-regulatory hormone, hepcidin. The impact of erythropoiesis stimulation on ERFE secretion in humans is poorly understood. This paucity of information is due in part to the lack of available means for ERFE quantification in serum samples. The present study tested a new sensitive sandwich immunoassay for human ERFE. This assay was used to demonstrate that injection of various erythropoiesis stimulating agents (ESAs) increased the blood ERFE levels in healthy volunteers. After exogenous stimulation of erythropoiesis, ERFE increased up to 8-fold with a detection window of 13 days. The impact of one unit of blood withdrawal on erythropoiesis stimulation of ERFE was also tested. ERFE significantly increased after blood withdrawal in subjects injected with both iron and saline solution, suggesting that iron supplementation did not mask the ERFE increase after blood withdrawal. The effects of exercise-induced muscle damage on ERFE was assessed by comparing ERFE levels with creatine kinase levels in samples from subjects with heavy exercise loads, and determined that this was not a confounder. The ERFE assay is a sensitive means to investigate the connection between iron metabolism and erythropoiesis in humans, and to detect ESA abuse in the antidoping field

A public health threat in Hungary: obesity, 2013

Background: In Hungary, the last wide-range evaluation about nutritional status of the population was completed in 1988. Since then, only limited data were available. Our aim was to collect, analyze and present updated prevalence data. Methods. Anthropometric, educational and morbidity data of persons above 18 y were registered in all geographical regions of Hungary, at primary care encounters and within community settings. Results: Data (BMI, waist circumference, educational level) of 40,331 individuals (16,544 men, 23,787 women) were analyzed. Overall prevalence for overweight was 40.4% among men, 31.3% among women, while for obesity 32.0% and 31.5%, respectively. Abdominal obesity was 37.1% in males, 60.9% in females. Among men, the prevalence of overweight-obesity was: under 35 y = 32.5%-16.2%, between 35-60 y = 40.6%-34.7%, over 60 y = 44.3%-36.7%. Among women, in the same age categories were: 17.8%-13.8%, 29.7%-29.0%, and 36.9%-39.0%. Data were presented according to age by decades as well. The highest odds ratio of overweight (OR: 1.079; 95% CI [1.026-1.135]) was registered by middle educational level, the lowest odds ratio of obesity (OR: 0.500; 95% CI [0.463-0.539]) by the highest educational level. The highest proportion of obese people lived in villages (35.4%) and in Budapest (28.9%). Distribution of overweighed persons were: Budapest (37.1%), other cities (35.8%), villages (33.8%). Registered metabolic morbidities were strongly correlated with BMIs and both were inversely related to the level of urbanization. Over the previous decades, there has been a shift in the distribution of population toward being overweight and moreover obese, it was most prominent among males, mainly in younger generation. Conclusions: Evaluation covered 0.53% of the total population over 18 y and could be very close to the proper national representativeness. The threat of obesity and related morbidities require higher public awareness and interventions

Age-related changes in global motion coherence: conflicting haemodynamic and perceptual responses

Our aim was to use both behavioural and neuroimaging data to identify indicators of perceptual decline in motion processing. We employed a global motion coherence task and functional Near Infrared Spectroscopy (fNIRS). Healthy adults (n = 72, 18-85) were recruited into the following groups: young (n = 28, mean age = 28), middle-aged (n = 22, mean age = 50), and older adults (n = 23, mean age = 70). Participants were assessed on their motion coherence thresholds at 3 different speeds using a psychophysical design. As expected, we report age group differences in motion processing as demonstrated by higher motion coherence thresholds in older adults. Crucially, we add correlational data showing that global motion perception declines linearly as a function of age. The associated fNIRS recordings provide a clear physiological correlate of global motion perception. The crux of this study lies in the robust linear correlation between age and haemodynamic response for both measures of oxygenation. We hypothesise that there is an increase in neural recruitment, necessitating an increase in metabolic need and blood flow, which presents as a higher oxygenated haemoglobin response. We report age-related changes in motion perception with poorer behavioural performance (high motion coherence thresholds) associated with an increased haemodynamic response

Abnormal motor activity during anaesthesia in a dog: a case report

Seizures or convulsions that occur during anaesthesia in veterinary patients are infrequently reported in the literature. Consequently, the incidence of such events is unknown. Several drugs commonly used in clinical veterinary anaesthesia have been shown to induce epileptiform activity in both human clinical patients and experimental candidates. The present case report describes convulsions in a four-year old male Bernese mountain dog during maintenance of anaesthesia with isoflurane after premedication with acepromazine and methadone followed by co-induction with propofol and ketamine. The dog had no history of previous convulsions. The use of several sedative and anaesthetic drugs makes it difficult to find one single causative pharmaceutical

Design of Experiments for Screening

The aim of this paper is to review methods of designing screening experiments, ranging from designs originally developed for physical experiments to those especially tailored to experiments on numerical models. The strengths and weaknesses of the various designs for screening variables in numerical models are discussed. First, classes of factorial designs for experiments to estimate main effects and interactions through a linear statistical model are described, specifically regular and nonregular fractional factorial designs, supersaturated designs and systematic fractional replicate designs. Generic issues of aliasing, bias and cancellation of factorial effects are discussed. Second, group screening experiments are considered including factorial group screening and sequential bifurcation. Third, random sampling plans are discussed including Latin hypercube sampling and sampling plans to estimate elementary effects. Fourth, a variety of modelling methods commonly employed with screening designs are briefly described. Finally, a novel study demonstrates six screening methods on two frequently-used exemplars, and their performances are compared