Comparison between standard hematological parameters and blood doping biomarkers in dried blood spots within the athlete population of Swiss Sport Integrity

Abstract

Introduction: The study demonstrated the feasibility of incorporating RNA biomarkers, specifically 5-aminolevulinic acid synthase (ALAS2) and carbonic anhydrase 1 (CA1), to improve the hematological module of the Athlete Biological Passport (ABP) in routine antidoping context.

Objective: The aim was to investigate the implementation of reticulocyte (RET) related biomarkers, specifically ALAS2 and CA1, using quantitative reverse transcription polymerase chain reaction (RT-qPCR) on dried blood spots (DBS) from elite athletes. Hemoglobin changes over time in DBS samples was measured as well. Combining hemoglobin and messenger RNA (mRNA) analyses allowed to monitor alterations of the established marker, "DBS OFF-score".

Methodology: Ten athletes were selected for sampling by the Swiss national antidoping organization, Swiss Sports Integrity (SSI). Samples were collected, transported and analyzed for ABP following the World Anti-Doping Agency (WADA) procedures and spotted onto Protein Saver DBS cards.

Results: Most athletes exhibited stable biomarker levels, except for one individual involved in ski mountaineering, who demonstrated a sustained increase in ALAS2 compared to the individual baseline. This elevation could be due to blood withdrawal or other factors, such as doping with substances outside the targeted test menu.

Conclusion: In this study, RNA-biomarkers were successfully analyzed in routine blood samples, and the project demonstrated promising results for the implementation of ALAS2 and CA1 in routine analysis to complement the ABP.

Keywords: Athlete Biological Passport (ABP); RNA biomarkers; blood doping; dried blood spots (DBS); hematological module.

Figure 1

Scheme of routine experiment processing analysis of EDTA blood tubes. (1) Blood samples are analyzed using a Sysmex XN instrument, and the data is utilized for the Athlete Biological Passport (ABP). Blood from EDTA tubes is then spotted onto dried blood spot (DBS) cards. (2) Analysis of DBS: The DBS samples undergo RNA purification, followed by cDNA preparation and analysis using quantitative reverse transcription polymerase chain reaction (RT-qPCR).

Figure 2

Monitoring of three athletes from cycling. (A) ALAS2, CA1 and%RET data as a percentage of the first sample, considered as baseline value, and corresponding 100% on the y-axis. (B) Comparison of HGB-values obtained in Sysmex-analysis and as calculated on the DBS. HGB data are represented in y-axis as g/dl. (C) Calculated OFF-score for all markers based on HGB,%RET, ALAS2 and CA1 data. ALAS2 is represented in dark grey diamonds (♦), CA1 in light grey squares (▪), %RET in black triangles (▴), HGB SYSMEX in black diamonds (♦) and HGB DBS in light grey squares (▪). Athlete 1: Cycling/Track Endurance (male); Athlete 2: Cycling/Road (female); Athlete 3: Cycling/Road (male). Samples were collected in monthly intervals.

Figure 3

Monitoring of three athletes from athletics. (A) ALAS2, CA1 and%RET data as a percentage of the first sample, considered as baseline value, and corresponding 100% on the y-axis. (B) Comparison of HGB-values obtained in Sysmex-analysis and as calculated on the DBS. HGB data are represented in y-axis as g/dl. (C) Calculated OFF-score for all markers based on HGB,%RET, ALAS2 and CA1 data. ALAS2 is represented in dark grey diamonds (♦), CA1 in light grey squares (▪), %RET in black triangles (▴), HGB SYSMEX in black diamonds (♦) and HGB DBS in light grey squares (▪). Athlete 4: Athletics/Long distance (female); Athlete 5: Athletics/Middle distance (female); Athlete 6: Athletics/Long distance (female). Samples were collected in monthly intervals.

Figure 4

Monitoring of three athletes from skiing. (A) ALAS2, CA1 and%RET data as a percentage of the first sample, considered as baseline value, and corresponding 100% on the y-axis. (B) Comparison of HGB-values obtained in Sysmex-analysis and as calculated on the DBS. HGB data are represented in y-axis as g/dl. (C) Calculated OFF-score for all markers based on HGB, %RET, ALAS2 and CA1 data. ALAS2 is represented in dark grey diamonds (♦), CA1 in light grey squares (▪), %RET in black triangles (▴), HGB SYSMEX in black diamonds (♦) and HGB DBS in light grey squares (▪). Athlete 7: Skiing/Cross-country (male); Athlete 8: Skiing/Cross-country (male); Athlete 9: Skiing/Cross-country (male). Samples were collected in monthly intervals.

Figure 5

Monitoring of on athlete from ski-mountaineering. (A) ALAS2, CA1 and%RET data as a percentage of the first sample, considered as baseline value, and corresponding 100% on the y-axis. (B) Comparison of HGB-values obtained in Sysmex-analysis and as calculated on the DBS. HGB data are represented in y-axis as g/dl. (C) Calculated OFF-score for all markers based on HGB,%RET, ALAS2 and CA1 data. ALAS2 is represented in dark grey diamonds (♦), CA1 in light grey squares (▪), %RET in black triangles (▴), HGB SYSMEX in black diamonds (♦) and HGB DBS in light grey squares (▪). Samples were collected in monthly intervals.