Detection of Autologous Blood Transfusions by flow cytofluorimetry: a multiparametric approach

Code: 12B8FD 

Blood doping is banned by the World Anti-Doping Agency in all sports due to its effects on sport performance, especially in endurance disciplines. WADA accredited laboratories have developed testing methods for the detection of blood doping by eryhtropoietins, synthetic hemoglobins, RSR13, and homologous blood transfusions, while no direct, internationally recognized method is yet available for the detection of autologous blood transfusions. We propose a flow cytofluorimetric approach based on the recognitions of markers of storage in red blood cells. More specifically, markers of apoptosis (namely, phosphatidylserine) and markers to detect reduction of antigen expression on red blood cells membrane (primarily among them CD55 and CD59 proteins) have already been evaluated by our laboratory as potential diagnostic parameters to detect the infusion of previously stored blood, with very promising results. Preliminary data obtained on several different blood samples, tested at different times after collection and in different storage conditions, showed that the selected parameters are significantly modified by red blood cells storage. We are planning to implement the number of markers considered for this study, possibly broadening the panel of diagnostic markers to be monitored to effectively detect the recourse to autologous blood transfusions. Once the most suitable diagnostic markers will be identified and selected, the effectiveness of the approach will be verified on subjects undergoing preoperative autologous donation in the framework of pre and post- surgery practices. 
We strongly believe that the proposed approach could effectively complement the analysis presently under evaluation for the detection of autologous blood transfusions, representing a significant advancement towards the development of a robust and reliable direct method to detect autologous blood transfusion.

Main Findings:

One  of  the  current  challenges  for  the  Antidoping  Laboratories  worldwide  is  the  detection  of  Autologous Blood Transfusion (ABT). At present, ABT can be detected only by indirect methods, which require the longitudinal evaluation of the stability of selected hematological parameters, as in the hematological module of the athlete biological passport.  
This  project  aimed  to  study  and  to  explore  a  multi---parametric  strategy,  based  on  flow  cytofluorimetry, targeting specific morphological/biochemical changes of red blood cells, as well as  the  alteration  of  other  hematological  parameters  (e.g.  the  increase  of circulating  microparticles), consequent to the storage period of the withdrawn blood prior to the reinfusion in  the  receiver  person.  This  approach  is  in  fact  a  “direct”  detection  strategy,  since  it  recognizes  specific changes on the “exogenous” (this meaning the transfused) red blood cells (RBCs).  
We have conducted a series of experiments on human whole blood samples that were focused on the  identification  of  diagnostic  signs  of  red  blood  cells  aging  also  matching  all  the  following  conditions: (i) to be easily detected by flow cytofluorimetry; (ii) to be stable after addition of the stored sample to fresh blood; and (iii) to show a sufficiently pronounced variation, allowing their identification also in mixed blood samples generated after a transfusion practice.   
From the data we obtained two main conclusions can be drawn: i) Flow  cytofluorimetry  is  able  to  identify  multiple  indicators  of erythrocytes  aging  that  are generated after a storage time in the fridge and blood banks condition.
ii) The  signals  of  RBC  aging  that  have  been  detected  in  our experimental  conditions  (I.e.,  by considering a period of storage up to 40 days) are the following:a. reduction of the expression of surface proteins on RBC;
b. moderate increase in the concentration of glycated HB (HbA1c);
c. reduction of RBC main size, that generates a population of smaller and more dense erythrocytes;
d. the  consequent  formation  of  a  population  of  microparticles,  that is  a  direct consequence of red blood cells microvesiculation process. Unluckily,  it  was  not  possible  to  verify  in  vivo  the  observation  recorded  ex  vivo,  as  well  as  to  evaluate  the  potential  effectiveness  of “markers  of  reinfusion”,  due  to  the  lack  of  samples collected from auto---transfused subjects. Experiments in this direction are still in progress, thanks to newly activated, ongoing cooperation with other research groups and clinical laboratories.  
In  spite  of  the  above  limitation,  the  data  we  collected  at  this  stage  are  very  promising  in  the  development of a universal, direct method for detecting both autologous and homologous blood transfusions: based on our results, a detection strategy based on the counting of the number of microvesicles and the counting of red blood cells of the more dense fraction (smaller in size) seem to present a higher diagnostic value than that based on the variation over time of the expression of specific proteins on the red blood cell membrane.  
Finally, it has to be stressed out that our proposed approach needs a solid standardization: indeed, the  same  counting,  when  performed  on  different  flow  cytofluorimetry  instruments,  that  use  different  detectors settings,  can  lead  to  different  measurements,  inevitably  resulting  in  an increase of the inter---laboratory variability of the results.