Tracking immature reticulocyte proteins for improved detection of recombinant human erythropoietin (rhEPO) abuse.

Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.

Measurement of Immature Reticulocytes in Dried Blood Spots by Mass Spectrometry.

BACKGROUND: Immature reticulocytes (IRC) are the first cells to respond to changes in erythropoiesis. For antidoping applications, measurement of IRC may improve detection of blood doping practices. Unfortunately, this small cell population has limited stability in liquid blood samples and is difficult to measure with optimal precision. We developed a method to measure 3 IRC membrane proteins in dried blood spots (DBS) to monitor changes in erythropoiesis. METHODS: DBS spots were washed with buffers to remove soluble proteins, membrane proteins remaining in the spot were digested with trypsin, and one peptide for each protein was measured by LC-MS/MS. IRC protein concentration was determined using a DBS single point calibrator. RESULTS: Intraassay precision for IRC proteins was between 5%-15%. IRC proteins were stable in DBS for 29 days at room temperature. In a longitudinal study of 25 volunteers, the mean intraindividual variation for 3 IRC proteins was 17%, 20%, and 24% from capillary blood DBS. In comparison, the mean longitudinal variation for IRC counts measured on an automated hematology analyzer was 38%. IRC protein concentration from capillary blood DBS correlated well with venous blood DBS protein concentrations. CONCLUSIONS: Measurement of IRC proteins in DBS samples provides a method to measure changes in erythropoiesis with improved analytical sensitivity, stability, and precision. When combined with the inherent advantages of capillary blood collection in the field, this method may substantially improve the detection of blood doping practices.

Investigating oral fluid and exhaled breath as alternative matrices for anti-doping testing: Analysis of 521 matched samples.

Current anti-doping testing is primarily conducted in urine and blood. Recently, due to confounding factors with urine and blood collections such as invasiveness, cost, and stringent shipping conditions, there has been a push for the use of alternative sample matrices to ameliorate these issues. Gaining support within the anti-doping field is the use of oral fluid, and more recently exhaled breath, as viable alternative or complementary matrices to traditional urine and blood for drug testing. Thus, we designed a first-in-field study with the purpose of investigating the utility of oral fluid and exhaled breath testing, and the preference of athlete participants, comparative to conventional anti-doping methods of urine testing. To accomplish this, 521 total matched samples, consisting of exhaled breath, oral fluid, and urine samples, were collected and analyzed, and the results compared across matrices. Participants in this study preferred the exhaled breath collection (rated 4.90 ±â€¯0.34 out of 5, mean ±â€¯SD) over the oral fluid collection procedure (4.29 ±â€¯0.85), and most preferred both over urine collections. Exhaled breath resulted in the shortest collection time (2.58 ±â€¯1.00 min, mean ±â€¯SD), followed by urine (3.08 ±â€¯1.50 min), and finally oral fluid (4.14 ±â€¯1.94 min). Prohibited substances from the drug categories of stimulants, narcotics, cannabinoids, diuretics, glucocorticoids, beta-blockers, and others, were analyzed in this study for a comparison of testing efficacy. Of the total findings 49% were detectable in only urine, 38% in urine + oral fluid, and 9% in all three matrices. Of the unique findings 3% were detectable in only oral fluid, 1% in oral fluid + breath, and 0% of unique findings were present only in exhaled breath. The findings from this study provide a strong foundation for the future use of oral fluid and exhaled breath as viable alternative or complementary matrices for in-competition anti-doping testing.