Purpose: We investigated whether immature reticulocyte fraction (IRF) and the immature reticulocytes to red blood cells ratio (IR/RBC) are sensitive and specific biomarkers for microdose recombinant human erythropoietin (rHuEPO) and whether the inclusion of reticulocyte percentage (RET%) and the algorithm "abnormal blood profile score (ABPS)" increased the athlete biological passport (ABP) sensitivity compared to hemoglobin concentration ([Hb]) and the OFF-hr score ([Hb]-60 × √RET%).

Methods: Forty-eight (♀ = 24, ♂ = 24) participants completed a 2-wk baseline period followed by a 4-wk intervention period with three weekly intravenous injections of 9 IU × kg bw^-1 epoetin β (♀ = 12, ♂ = 12) or saline (0.9% NaCl, ♀ = 12, ♂ = 12) and a 10-d follow-up. Blood samples were collected weekly during baseline and intervention as well as 3, 5, and 10 d after treatment.

Results: The rHuEPO treatment increased [Hb] (time×treatment, P < 0.001), RET% (time×treatment, P < 0.001), IRF (time×treatment, P < 0.001) and IR/RBC (time×treatment, P < 0.001). IRF and IR/RBC were up to ~58% (P < 0.001) and ~ 141% (P < 0.001) higher compared to placebo, and calculated thresholds provided a peak sensitivity across timepoints of 58% and 54% with ~98% specificity, respectively. To achieve >99% specificity for IRF and IR/RBC, sensitivity was reduced to 46% and 50%, respectively. Across all timepoints, the addition of RET% and ABPS to the ABP increased sensitivity from 29% to 46%. Identification of true-positive outliers obtained via the ABP and IRF and IR/RBC increased sensitivity across all timepoints to 79%.

Conclusions: In summary, IRF, IR/RBC, RET% and ABPS are sensitive and specific biomarkers for microdose rHuEPO in both males and females and complement the ABP.