Detection of EPO injections using a rapid lateral flow isoform test

Abstract

Misuse of recombinant human erythropoietin (rhEPO) is a major concern in competitive sports, and the implementation of tests allowing for higher detection rates than what current tests are capable of is required. In this study, a novel lateral flow EPO isoform test kit, EPO WGA MAIIA, is evaluated on the basis of plasma and urine samples obtained from eight healthy males in connection with a 28-day rhEPO injection period. rhEPO was injected every other day during the first 14 days of the study, and the method proved to be 100 % effective in detecting rhEPO in the concomitantly obtained samples. Seven days after the last injection, three positive (>99.99 % confidence limit (CL)) subjects were found. When using 99 % CL as the cut-off limit, six of the eight subjects (75 %) were found to be suspected of doping. Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO. A previous study using indirect methods to determine EPO doping on the same samples indicated only that two of the subjects had suspicious values 7-21 days after the last injection. We propose implementing the easy to-use EPO WGA MAIIA test as an initial screening procedure in anti-doping work to (1) increase the detection rate of potential rhEPO doping athletes and (2) allow for a 10-to 20-fold higher analytical rate than what is possible today. days rhEPO was injected every second day during the first 14 days of the study and the method proved to be very effective (xx out of xx) in detecting thisdoping infor the concomitantly obtained samples collected prior to injection. Samples obtained 14 and 21 days after the last injection showed no detectable rhEPO. Seven days after the last injection three positive (>99.99% CL) subjects were found with the test kit. When using 99% CL as cut-off limit, six of the eight subjects (75%) were suspectedfound to be suspicious for doping.Samples obtained 14 and 21 days after the last injection showed no detectable trace of rhEPO.A previous study using indirect methods to determinefor detecting EPO doping on the same samples indicated only that two of the subjects hadwere suspicious valuespositive during 7-21 days after the last injection.We proposesuggest implementing the easy to-use EPO WGA MAIIA rapid lateral flow test as an initial screening procedure in anti-doping work to 1) increase the detection rate of potential rhEPO doping athletes and 2) allow for analysis of a ten to twenty folds higher analytical rate than what is possible todaymore samples than what is possible today.

Kidney-synthesized erythropoietin is the main source for the hypoxia-induced increase in plasma erythropoietin in adult humans

“…Samples containing different EPO glycoforms (e.g. recombinant and endogenous EPO) have PMI values that are intermittent of the "pure glycoforms" (Lönnberg et al 2012a(Lönnberg et al , b, 2008Lönnberg and Lundby 2013) and therefore a potential contribution of non-renal-derived EPO to the circulating EPO The PMI values are directly related to the glycoform composition of EPO in a given sample. Hence, for the HA samples an increase in PMI value from SL towards UC would be a consequence of a sample containing both renal and hepatic EPO.…”

“…The glycoform heterogeneity of the samples was analyzed by an EPO WGA MAIIA kit (MAIIA Diagnostics, Uppsala, Sweden) that previously have been used for detection of recombinant EPO in humans and horses (Lönnberg et al 2012b;Lönnberg and Lundby 2013;Mørkeberg et al 2013). In the present study we included umbilical cord (UC) plasma, which is known to contain mainly liver-derived EPO (Dame et al 1998;Ohls 2002), as positive control for non-renal-derived EPO.…”

Recombinant Erythropoietin in Humans Has a Prolonged Effect on Circulating Erythropoietin Isoform Distribution

“…In contrast, PMI in a solution of Epoetin beta, a form of rhEPO synthesized in Chinese hamster ovary cells, is around 30 % [12] . In samples from subjects that were injected with Epoetin beta (5,000 IU) every second day for 14 days and then again at days 21 and 28, PMI decreased to a nadir of around 30 % at day 16 [13] . Seven days after the last injection, when the plasma concentration of EPO had returned to normal, PMI was still decreased compared with baseline values [13] .…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…Increasing the initial testing frequency by competitively sensitive but potentially cheaper and faster approaches was the subject of studies employing the so‐called EPO WGA MAIIA test kit. Lönnberg and Lundby conducted an administration study using 65 IU of NeoRecormon/kg of bodyweight, injected subcutaneously every other day over a period of 14 days, and plasma as well as urine were sampled up to 21 days after the last injection . In a different setting, Dehnes et al .…”

“…Lönnberg and Lundby conducted an administration study using 65 IU of NeoRecormon/kg of bodyweight, injected subcutaneously every other day over a period of 14 days, and plasma as well as urine were sampled up to 21 days after the last injection. [85] In a different setting, Dehnes et al used intravenously administered microdoses of 7.5 IU of NeoRecormon/kg of bodyweight, injected twice per week over a period of 21 days. Also here, blood and urine was collected for analysis.…”