Abstract

Blood manipulations (e.g. blood transfusion, recombinant human erythropoietin (rHuEPO) injections) are currently being used by athletes to enhance their sporting performance despite these methods being prohibited by the World Anti-doping Agency (WADA). Recent advances in “omics” technologies have provided new high-throughput techniques that may facilitate the discovery of new biomarkers for anti-doping purposes [1, 2].

This study aimed to test the application of whole transcriptome analysis to the detection of autologous blood transfusion (ABT).

15 healthy Caucasian male (20-35 yrs old, BMI ≤ 30 kg/m²) donated 500 mL of blood that was stored and then infused 36 days later. Whole blood samples were collected 4 days and 1 day before blood transfusion and at 3, 6 and 12 hours, 1,2,3,6,9 and 15 days after blood transfusion. Total RNA was extracted and 100 ng/ul of total RNA used for gene expression analysis using the GeneChip HTA 2.0 from Affymetrix UK Ltd ( > 285,000 full-length transcripts). One-way repeated measure analysis of variance (ANOVA) was used to identify differential expressed genes following ABT. Changes considered significant at ANOVA p-value < 0.05 and a fold change (FC) of 1.5.

As the study is ongoing, we present the results of 8 subjects and only 5 time points (baseline one day before, 3, 6, 9 and 15 days after ABT). Compared to baseline, 202 genes were differentially expressed 3 days after ABT, 170 genes after 6 days, 218 genes after 9 days and 51 genes after 15 days. Approximately 91 genes were differentially expressed among time points, with 23 genes overlapping and upregulated 3, 6 and 9 days after ABT; fold changes varied from 1.5 to 2.9. Of these differentially expressed genes, three overlapped with 45 previously identified rHuEPO genes. Specifically, ALAS2 was downregulated 6 days (FC = −1.77, ANOVA p = 0.003), 9 days (FC = −1.84, ANOVA p = 0.007) and 15 days after ABT (FC = −1.5, ANOVA p = 0.011). CCR7 was upregulated (FC = 1.83, ANOVA p = 0.049) 3 days after ABT and SLC4A1 was downregulated (FC = −1.65, ANOVA p = 0.023) 6 days after ABT. Although preliminary, these results are encouraging and support the detection of autologous blood transfusion using whole genome expression.