Evaluation of DNA mixture detection in Dried Blood Spots as a detection method for homologous transfusion

Code: ISF19E03AM

Blood doping and in particular transfusions were used by athletes for decades, as an easy way to increase red blood cells, oxygen transportation to muscles and endurance. Homologous transfusion (blood from a compatible donor) was the simplest method used.

However, in 2004 a method based on flow cytometry was implemented in anti-doping laboratory to identify homologous transfusion. While being efficient to identify around 2% stranger blood in a sample, it requires the use of mnay antibodies and still could lead to false negative results. Nowadays, only few anti-doping laboratories are still running flow cytometry and athletes could feel free to come back to homologous transfusion.

With the development of forensic science, DNA has proven to be a reliable source of identification of the presence of two different DNA in a single blood sample. The power of amplification of DNA based techniques is sufficient to start from very small volumes and Dried Blood Spots (DBS) is an interesting matrix for the future of doping controls. The aim of the project is to evaluate the interest of implementing a new test to detect homologous transfusion by using a forensic DNA-based protocol on DBS for anti-doping purpose.

The objectives of the project are:

1. To validate the conditions to propose a robust protocol for detection of DNA mixture from Dried Blood Spots.

2. To estimate the window of detection after transfusion of healthy volunteers by analyzing DBS spotted from venous blood and to compare these results with those of the current flow cytometry method for detection of homologous blood transfusion.

Main findings

The protocol was successfully developed and validated. With 100μL of dried blood collected on a card treated to protect nucleic acids. A mix of two DNA was robustly identified when the minor blood proportion was 2% or higher in vitro when two whole bloods were mixed. Following one month of storage of the cards, at room temperature, the sensitivity of detection was not altered.

A blinded in vitro study with samples containing mixed compatible, bloods in different proportions as well as negative samples (one DNA) was conducted. For DNA analysis of DBS the limit of detection was confirmed at 2% donor blood. while the flow cytometry method performed on fresh blood was slightly more sensitive and could sometimes detect 1% donor blood in a sample.

However, the in vivo evaluation of this method with transfusion of 150 mL of a red blood cells concentrate (RBCC) using the forensic DNA approach gave disappointing results as no analyzed DBS showed any presence of a second DNA even one day following transfusion.

This result is most likely due to a lack of a sufficient number of white cells (the only blood cells containing DNA) from the donor in the RBCC and consequently in the DBS. On the contrary the flow cytometry method was very efficient and the transfusion could still be detected for more than 40 days. The classic flow cytometry method was performed with a tube by tube analysis of 11 antigens (C, c, E, e, Jka, Jkb, S, K, Fya, Fyb and s). A new simplified protocol for sample analysis by flow cytometry was also developed and evaluated using 96 well plates with 9 antigens detected (C, c,E, Jka, Jkb, S, Fya, Fyb and s). In each well two antibodies against two different RBC antigens can be mixed if one is an Immunoglobulin subtype G (IgG) and the other an immunoglobulin subtype M (IgM). Two secondary antibodies with two different fluorophores are added in each well to identify presence/absence or mixed expression of each antigen. Both flow cytometry methods detected well the double populations for several antigens after transfusion and were sufficiently sensitive to confirm at least two antigens with a double population at day 42 and day 50 post-transfusion.

In conclusion, analysis of DBS with the validated forensic DNA method to detect DNA mixes did not demonstrate enough performance to be considered as an alternative to the flow cytometry method for detection of HBT. In particular, it completely failed to detect the transfusion of the RBCC while the flow cytometry method was very efficient with a window of detection of 1.5 months. To simplify the laboratory work needed to analyse blood by flow cytometry, a technical evolution of the method was evaluated with success and the new protocol will soon be fully validated for a future application to doping control samples.