The combination of growth hormone (GH) and recombinant erythropoietin (rEPO) is thought to be used particularly in endurance sports. Our objective was to reproduce a 2‐week administration of rEPO microdoses alone or in combination with GH microdoses (three times a week) on healthy and athletic male subjects and to evaluate if GH had any additional effects compared to EPO treatment alone. The effects of the treatments on hematological parameters and VO2max were studied as well as the detection of GH in serum. While the rEPO microdose regimen was associated with a significant increase in reticulocytes, no clear elevation in hemoglobin concentration (HGB) was observed. Using a correction by plasma volume did not reveal more effects of EPO on HGB. Our results did not show any additional effect when the GH microdoses were co‐administered. In addition, no clear increase in VO2max was observed after treatment, with an elevation in only half the subjects in both groups (EPO and EPO+GH). A clear effect of GH on insulin‐like growth factor I (IGF‐I) was seen but it was lower on procollagen III amino‐terminal propeptide (P‐III‐NP). GH detection using the direct isoform test identified only one subject 24 hours after receiving GH. The GH biomarker test combining IGF‐I and P‐III‐NP was not able to detect the GH administration. However, a longitudinal follow‐up of the intraindividual variations showed a significant increase in IGF‐I 24 and 48 hours after GH administration in most subjects, while the effect of GH microdoses on P‐III‐NP was less straightforward.

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A direct detection method for anti-doping control of recombinant human erythropoietin (rHuEPO) abuse in racehorses is proposed. This method involves screening of plasma (or serum) by an enzyme-linked immunosorbent assay specific for human EPO and confirmation in urine samples by characterization of the urinary EPO isoelectric profile. This method was tested on horses that were administered epoetin alpha (rHuEPO) and the hyper-glycosylated form of this drug (darbepoetin alpha).

Anti-doping controls in non-major events are relatively infrequent and athletes that compete only in these events are less likely to be controlled. The French Anti-Doping Agency carried out an anti-doping operation during a regional cycling competition in Guadeloupe. The urine and serum samples were analysed by the French anti-doping laboratory. Out of 42 athletes, 7 were positive for one or more substances prohibited by the World Anti-Doping Agency. Four serum samples contained continuous erythropoietin receptor activator (CERA) and one a recombinant erythropoietin. However, no traces were found in the corresponding urines. One of the athletes positive for CERA was also positive for growth hormone (GH), identified using the GH isoform test. The same serum was negative with the GH biomarkers test, probably because of the brief interval between injection and control (less than a day). The stimulants heptaminol and dimethylbutylamine, as well as the glucocorticoid prednisone and its metabolite prednisolone, were also found. Strikingly, 16.6% of the controlled athletes were using one or more prohibited drugs. These findings indicate that doping is widespread in athletes competing regionally and that CERA is still a popular drug for endurance sports. They underline the need for more controls, particularly blood sampling during non-major competitions.

by lymphocytes and it is expected that its levels steadily increase together with the progressive expansion of the leukemic clone suggesting a close correlation between stage (which measures tumor burden) and β2-m levels. Although a correlation with disease stage likely exists, there was a substantial proportion of patients with high β2-m levels already at Binet A stage (low tumor burden). Possibly, CLL cells from these patients are more activated in vivo and shed more abundant β2-m. Taken all the above into consideration, the data indicate that the role of β2-m as a prognostic tool should be re-evaluated possibly in prospective studies involving large patient cohorts.

To reproduce a potential doping scenario, a 2 week administration of recombinant erythropoietin (rEPO) microdoses alone or in combination with growth hormone (GH) microdoses (three times a week) was performed on healthy and athletic male subjects. The aim of this study was to evaluate the identification capability of rEPO in samples obtained during and post treatment. Detection was tested in urine and blood using the antidoping techniques for rEPO detection (iso-electric focusing (IEF)-, sodium-dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and for some urine samples the sarcosyl (SAR)-PAGE method) with some improvements: for blood samples, instead of a simple concentration step, immuno-extraction of EPO was performed for all urines to limit protein contamination that can affect migration. In addition, elution buffer modifications also improved the quality of migration. The use of a recently validated biotinylated anti-EPO antibody simplified the protocols, allowing a single transfer step instead of a double-blot even by IEF with a lowered background. The criteria for suspicious blood and urine samples by IEF were also reevaluated. While endogenous EPO was not decreased over the course of the study, EPO microdoses were detectable in blood and urine between 24 h and 72 h after an administration. Detection in urine in combination with SDS-PAGE was the most sensitive combination for prolonged detection (100% identification after 48 h, 91% after 72 h), slightly better than IEF. Urine samples also tested by SAR-PAGE indicated a similar sensitivity of detection to SDS-PAGE. GH co-administration had no impact on rEPO elimination/detection.

Recombinant erythropoietins (rEPOs) are still among the substances endurance athletes use for doping. Detection methods are based on an electrophoretic separation of the proteins followed by a western blot and immunodetection with specific anti‐EPO antibodies. In addition to IEF‐PAGE, the SDS‐PAGE method has been used to differentiate endogenous EPO from rEPOs by their molecular weight (MW). However, to adapt to new generations of rEPOs exhibiting higher MW, which were not well detected after SDS‐PAGE, sodium lauroyl sarcosinate (SAR) is now used instead of sodium dodecyl sulfate (SDS) for the initial EPO testing procedure on doping control samples. The SAR‐PAGE method is nevertheless expensive as it requires frequent buffer preparations using highly purified sarkosyl powder. In addition, this reagent needs to be handled with care due to acute toxicity by inhalation. The aim of this work was to improve the SDS‐PAGE method by increasing its sensitivity and transfer of high‐MW rEPOs. First, using a biotinylated primary anti‐EPO antibody and avoiding the use of a secondary antibody increased the general sensitivity of both SDS‐PAGE and SAR‐PAGE to all rEPOs about four‐fold. Then, by changing the buffer system during the protein transfer, with a CAPS buffer and a discontinuous buffer transfer system, high‐MW rEPOs, EPO‐Fc and CERA were transferred with higher efficiency and detected with high sensitivity. This optimized SDS‐PAGE protocol could be adopted by anti‐doping laboratories as an alternative to SAR‐PAGE.

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