Quantitative criteria for the evaluation of gel imagesfor SAR-PAGE analysis of rEPO

Code: 24T04CR

Unfortunately, the mass difference between rEPO and uEPO/bEPO is not large enough to result in a clear separation on SAR- and SDS-PAGE. Instead, a “mixed” band containing both compounds is frequently observed. The lower the rEPO concentration becomes, the fainter the area above the endogenous band becomes (“diffuse or faint area” according to TD2024EPO, p. 17). In case of rEPO-doping, this faint area must extend beyond the cut-off line defined by the band apex of Epoetin-δ (Dynepo) or Epoetin-α.

The weaker the area becomes (e.g. due to intravenous rEPO microdosing), the more difficult the band profile is to interpret. So far, the interpretation relies solely on the experience of the analyst and second opinion provider(s) and thus has been challenged by the athletes’ attorneys in the past. The aim of this project is to define a criterion that enables an objective assessment of the diffuse area. Since all WADA labs use GASepo platform for image analysis, the criterion will be developed using this software. Two approaches will be evaluated: (1) the ratio of relative pixel volumes above the rEPO migration cut-off line (“Dynepo-line”), and (2) the differentiation of the band side plot.

The evaluation will be first done with urine samples after SAR-PAGE analysis. If successful, studies on serum/plasma and possibly DBS will be performed immediately. As part of this project, a revision of GASepo will be implemented to enable an automated cutting of bands along the “Dynepo” line. The project consists of three phases, namely (1) the intralaboratory phase phase (including a GASepo revision) performed by our lab leading to the proposal of threshold criteria for "AAF", "ATF" and "negative findings", (2) the iterlaboratory validation phase performed by 4 WADA accredited laboratories, and (3) the blinded test samples phase