Evaluation of the detection of the homologous transfusion of a red blood cell concentrate in vivo for antidoping.

Two doping cases of homologous blood transfusion (HBT) during Tokyo 2020 Summer Olympics have shown that more controls are needed. The method of detection using flow cytometry to evaluate the expression of minor blood group antigens from red blood cells (RBCs) and identify different RBC populations is efficient but still complex to perform with multiple antigens detection. Recently, the interest of using forensic DNA analysis was also highlighted as a potential new method to detect HBT, with possibility to start from dried blood spots (DBS) instead of fresh blood. After a first phase of development, a protocol was validated for HBT detection using DNA analysis after extraction from DBS. Presence of a second DNA was clear down to 2% of donor blood in vitro. A flow cytometry protocol was also developed with preparation and analysis in 96-well plates and detection of two different antigens per well using two secondary antibodies with distinct fluorophores. The objective of the project was to evaluate the window of detection of an HBT performed in vivo with 150 mL of RBC concentrate. Blood samples obtained over 7 weeks post-transfusion were analyzed. DNA profiling from DBS was not sensitive enough to detect the presence of a second DNA even 1 day after transfusion. On the contrary, the flow cytometry protocol was very efficient and allowed identification of several double populations of RBC (expressing/non-expressing several antigens) until day 50 post-transfusion. This protocol can be fully validated for a future application to doping control samples.

The effect of FBI CODIS Core STR Loci expansion on familial DNA database searching.

1000 profiles chosen randomly from an in-house database of 6000 profiles were searched against the database for matches with at least one shared allele per locus. The database contains profiles that have been analyzed with Identifiler Plus (15 markers) for biological relationship and DNA identification purposes and both true and false matches are expected to be obtained. 100 pairs of at least one true paternity match and one false match were selected and were initially supplemented with 5 additional STRs representing the new Core CODIS set. Study of the LR value showed that when false matches were treated as paternity matches, the expansion of the marker set severely diminished the LR values obtained compared to true matches and the false positive ratio of familial database searching. When false matches were treated as full-sibling matches, the expansion to 20 STRs also diminished the number of false matches and the corresponding LR values compared to true full-sibling cases, but the effect was less dramatic. Addition of the SE33 marker, further promoted distinction between true and false matches both in paternity and full-sibling cases. Counting the number of shared alleles presented improved distinction efficiency between true and false matches after STR expansion to20 and 21 STRs but remains a less valuable method of familial DNA database searching compared to LR.