As prohibited blood manipulations remains a challenge for anti-doping authorities, it is important to continuously investigate and evaluate new potential detection methods. A current need is to evaluate the potential of iron biomarkers and improve the sensitivity of current detection methodologies following micro-dosing regimes in a sex-specific context as well as evaluate the impact of well-known confounding factors. In addition, specific and sensitive biomarkers relevant in an antidoping context may also have merit in a blood donation context, where a rapid recovery after blood donation is a priority. Finally, it is important to determine the physiological effects of the applied regimes to establish the lower limit of relevance for anti-doping authorities.

Specifically, the present thesis investigated to which extent iron regulatory markers was affected by alterations in blood volume following a standard whole blood donation of 450 mL and a subsequent small-volume autologous blood transfusion of half a blood bag (~130 mL red blood cells). It was hypothesized that hepcidin and erythroferrone in conjunction represented clinical and anti-doping relevant markers for altered iron homeostasis. In addition, the sensitivity and specificity of the Athlete Biological Passport was investigated in males and females following both blood donation and reinfusion as well as a micro-dosing strategy of recombinant human erythropoietin (rHuEPO), where it was hypothesized that the inclusion of reticulocyte percentage and the algorithm Abnormal Blood Profile Score (ABPS) would increase sensitivity. It was also hypothesized that both the small-volume blood transfusion and a micro-dosing strategy of recombinant human erythropoietin would improve cycling time trial performance. Furthermore, it was hypothesized that altitude exposure concurrent with rHuEPO treatment would results in reduced specificity of the Athlete Biological Passport although the sensitivity would be increased due to an additive erythropoietic response. Finally, it was hypothesized that the iron regulatory hormones was sensitive to even small manipulations in erythropoetic homoestasis induced by both altitude and rHuEPO treatment.

Paper I and II confirmed our hypothesis that hepcidin are sensitive for alterations in erythropoietic homeostasis exemplified by both blood donation and a small-volume reinfusion. Interestingly, hepcidin remained attenuated after donation despite reverted hemoglobin and ferritin levels, although with large variation, indicating that the iron stores were yet to fully recover. Thus, hepcidin may be a valuable marker for blood banks to assess donors iron stores. Hepcidin was increased immediately following reinfusion and may be a valuable supplementary marker for anti-doping authorities but large intraand inter-individual variation may preclude it for being a standalone marker. In contrary to our hypothesis, the inclusion of reticulocyte percentage did not improve sensitivity following blood reinfusion, while the ABPS did although with reduced specificity. No differences were found between males and females concerning sensitivity and specificity of the passport approach. In contrast, the sensitivity following small injections of rHuEPO was additionally improved with the inclusion of reticolucyte percentage as evident from paper IV and an ongoing rHuEPO study. In accordance with our hypothesis, we observed a ~5% increase in cycling time trial performance following both autologous blood transfusion and rHuEPO injections. In paper IV, an additively erythropoietic response was evident following concurrent rHuEPO treatment and altitude exposure, resulting in an increased sensitivity when compared to sea-level, although concurrent with a reduced specificity. In paper III, we demonstrated that both hepcidin and erythroferrone are sensitive to even small erythropoietic manipulations, and that a combination of moderate altitude exposure and rHuEPO treatment additively increase the iron regulatory response. These findings demonstrate that iron regulatory hormones indeed are rapid biomakers of changes in iron demands compared to routine iron markers, which can be of high relevance in a clinical setting.

In conclusion, the present thesis demonstrate that iron regulatory hormones can be of high value both in a clinical setting such as blood banks as well as in an anti-doping context. However, large fluctuations in hepcidin caused by normal variation should be taken into account and limit hepcidin’s applicability in anti-doping, whereas erythroferrone shows promising results. Future studies should continue the efforts of developing a sensitive detection method for autologous blood transfusion and rHuEPO doping as both strategies are performance enhancing even in small doses. In addition, the current methodologies are challenged by such micro-dosing strategies evident by either a low sensitivity as well as a
compromised specificity in altitude, which is a common situation for elite athletes.