Recombinant erythropoietin found in seized blood bags from sportsmen

Abstract

Recombinant erythropoietin found in seized blood bags from sportsmenDuring an anti-doping investigation, the Spanish Guardia Civil confiscated blood bags from elite sportsmen. A novel immuno-purification method demonstrated that plasma samples with elevated erythropoietin (EPO) contained recombinant material (rEPO). This shows that rEPO is used before autologous blood transfusions and that rEPO analysis in plasma can be reliably addressed. Haematologica 2008 Feb; 93:(2) 313-314. DOI: 10.3324/haematol.12059 A network of blood transfusion for doping purposes was detected in Spain in 2006. The so-called Operation Puerto (OP) led to the seizure of samples of whole blood, plasma, and red blood cell concentrates for autologous blood transfusion, allegedly belonging to elite sportsmen from different countries. The juridical proceedings were dismissed according to the Spanish law applicable at that time. For the moment, in Spain, the indictment has been shelved.High (supra-physiological) concentrations of erythropoietin (EPO) were found in some plasma samples. EPO is available as a recombinant pharmaceutical product (rEPO) and included as a doping agent, together with its analogue NESP (darbepoetin alfa), in the prohibited list of the World Anti-Doping Agency (WADA). A method based on isoelectric focusing (IEF) can detect rEPO in urine 1 but its direct application to plasma is not feasible due to substantial matrix effects.We investigated whether the OP plasma samples with elevated EPO concentrations contained rEPO to trigger red blood cell production before storing blood for future transfusion, as clinically used.2 To disclose the origin of the elevated EPO, a new method allowing its purification was applied.Bags of human plasma from Operation Puerto (OP) were received together with an official juridical request for their analysis. In addition, urine and plasma samples from healthy control subjects were used as control.EPO concentrations in plasma were determined using a chemiluminescence immunometric assay (Immulite 1000 EPO; Diagnostic Products Corporation (DPC), USA) and confirmed by an ELISA assay (Quantikine IVD Erythropoietin; R&D Systems Inc, USA).An immuno-purification method using microtiter plates (Corning Incorporated, USA) was developed. A 10 µg/mL solution of a monoclonal anti-human erythropoietin antibody (clone 9C21D11; R&D Systems, USA) in 50 mM NH4HCO3 (100 µL/well) was applied for 1 hr. at 37ºC. Plates were then washed three times with 0.01M Phosphate-Buffered Saline (PBS), pH 7.4 and blocked with 1% polyvinylpirrolidone in PBS overnight at 4ºC with shaking. After aspiration, plasma samples (4-8 mL) were applied (100 µL/well) and incubated overnight at 4ºC with shaking. Wells were washed twice with 0.01M PBS. EPO was eluted with 100 µL/well of 0.7% acetic acid, the eluates were frozen in liquid nitrogen and lyophilized. Finally, samples were re-suspended in 18 µl of H2O and applied to the IEF gel as previously described 3 with additional modifications. 4Eight of the samples from the OP (Figure 1, l...

Evaluation of the MAIIA dipstick test to detect recombinant human erythropoietin in plasma

“…Electrophoresis is especially effective in urine samples, where presence of rhEPO can be identified several days after the last injection. By comparison, detection of rhEPO in serum or plasma, whilst still possible [2] is more challenging due to the lower resolution attainable between the two populations in the more acidic urine matrix [3] as well as the different pI range of endogenous isoforms in blood and urine.…”

Applications and Biomonitoring Issues of Recombinant Erythropoietins for Doping Control

“…30 Nevertheless, on September 28, 2007, the French sports newspaper L'Equipe reported that the French antidoping laboratory had detected Dynepo (Shire Pharmaceuticals, Hampshire, UK) (epoetin delta) in the urine of Michael Rasmussen during the 2007 Tour de France. As shown by various researchers, 30,73 the IEF profile of epoetin delta, in which more acidic isoforms can be observed with respect to Chinese hamster ovary-derived marketed rhEPOs, 7,74,75 differs from that of endogenous EPO (from either urine or plasma). With the rigorous structural analysis of epoetin delta completed, it can be concluded that when compared with other recombinant EPOs, the more acidic bands present in IEF are the result of a higher degree of sialylation.…”

Purification of erythropoietin from human plasma samples using an immunoaffinity well plate

“…The three suspicious samples with elevated plasma hEPO concentrations resulted in profiles compatible with the presence of rhEPO. When these samples were immunopurified using the basic and acidic conditions, the profiles obtained were identical, since all bands present are in the basic area for which acidic conditions showed good recoveries [20].…”