Segmentation and supervised classification of image objects in Epo doping-control

Abstract A software system Gel Analysis System for Epo (GASepo) has been developed within an international WADA project. As recent WADA criteria of rEpo positivity are based on identification of each relevant object (band) in Epo images, development of suitable methods of image segmentation and object classification were needed for the GASepo system. In the paper we address two particular problems: segmentation of disrupted bands and classification of the segmented objects into three or two classes. A novel band projection operator is based on convenient object merging measures and their discrimination analysis using specifically generated training set of segmented objects. A weighted ranks classification method is proposed, which is new in the field of image classification. It is based on ranks of the values of a specific criterial function. The weighted ranks classifiers proposed in our paper have been evaluated on real samples of segmented objects of Epo images and compared t

Correction of geometrical distortions in bands of chromatography images

This paper presents a methodology for correcting band distortions in Thin-LayerChromatography (TLC) images. After the segmentation of image lanes, theintensity profile of each lane column is spatially aligned with a reference profileusing a modified version of the Correlation Optimized Warping (COW)algorithm. The proposed band correction methodology was assessed using 105profiles of TLC lanes. A set of features for band characterization was extractedfrom each lane profile, before and after band distortion correction, and was usedas input for three distinct one-class classifiers aiming at band identification. In allcases, the best results of band classification were obtained for the set lanes afterband distortion correction

Validation of isoelectric focusing method for recombinant erythropoietin detection in human urine

Erythropoietin (EPO) is a substance that stimulates red blood cell production, increasing muscle oxygenation and is secreted naturally by the body and excreted in urine in low concentrations. Due to the special properties of EPO, this was quickly introduced into the world of sport and its illicit use provides advantages in sports performance. In early 2000, was developed a method for direct detection of erythropoietin (EPO) on recombinant human urine by Lasne, based on isoelectric focusing (IEF) in polyacrylamide gel, followed by double blotting, has been published and validated. In 2002, the World Anti-Doping Agency (WADA) has implemented this same method which is currently the only official method used by laboratories accredited by WADA. The starting point for this work was the need to implement and validate the reference method, for the detection of recombinant erythropoietin in human urine. The study was conducted at the Laboratory for Doping Analysis and (LAD) of the Sports Institute of Portugal (IDP), current IPDJ. Theobjective of the work focused on validation studies/investigation of different validation parameters (specificity/selectivity; ability identification, detection limit, accuracy and repeatability), according the protocol Procedimento Geral Interno of the Antidoping Laboratory of Lisbon. This method of screening and confirmation has revealed performance characteristics in accordance with the applicable requirements for what is considered valid and fit

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

Microvascular endothelial dilator function: role of COX and the effects of ecdysteroids

Cyclooxygenase (COX), which can be expressed as COX-1 or COX-2 in endothelial cells has the unique ability to regulate microvascular tone through balanced production of dilator/constrictor prostanoids. This study investigated the roles of these isoforms in microvascular endothelial dilator function and how these are affected by supplement-derived ecdysteroids. Acetylcholine or 20-hydroxyecdysone relaxation were recorded in Skeletal muscle (SKM) and mesenteric (ME) arteries from healthy sheep and omental (OM) and subcutaneous (SC) fat arteries from obese humans by wire myography in the absence and presence of inhibitors of nitric oxide synthase, cyclooxygenase (COX) isoforms 1 and 2 and endothelium-derived hyperpolarizing factors. Gene and protein expression analysis were also carried out to fully characterize the roles of COX in these arteries. Non-selective COX inhibition attenuated acetylcholine relaxation in SKM arteries but enhanced it in ME arteries. Selective inhibition of COX-1 in both SKM and ME arteries also attenuated acetylcholine relaxation. In contrast, selective inhibition of COX-2 enhanced acetylcholine relaxation in ME arteries and had no effect in SKM arteries. In OM arteries from obese patients, selective inhibition of COX-1 but not COX-2 significantly improved acetylcholine relaxation. The OM arteries also displayed enhanced responsiveness to thromboxane A2 mimetic (U46619) compared with SC arteries. 20-hydroxyecdysone caused relaxation which was attenuated by NOS inhibition compared with COX inhibition in SKM and ME arteries. COX roles in microvascular endothelial dilator function are isoform-specific and dependent on type and health of the vasculature. In healthy arteries, COX-1 promotes but COX-2 opposes vasodilation. In human obesity, COX-1 opposes while COX plays no part in OM endothelial dilator function. Although 20-hydroxyecdysone alters COX expression, its vasodilatory effect is more eNOS-dependent than COX-dependent