Validation and signal amplification of a flow cytometric method to detect homologous blood transfusion

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In endurance sport the maximum oxygen capacity is an important factor influencing athletic performance. An improvement of this factor is possible by increasing haemoglobin amounts with blood transfusion. Since 2004 homologous blood transfusion is commonly determined in routine doping control analysis using a procedure described by Nelson and Ashenden. Due to the fact that there has no validation data been published for the use of IgM antibodies so far and the fluorescence of the fluorophore FITC (Fluorescinisothiocyanate) is less intense compared to PE (Phycoerythrin) we aimed to improve the previously described method by using primary IgM antibodies in combination with secondary PE-conjugated antibodies. A panel of eight different primary antibodies and two different Phycoerythrin (PE) conjugated secondary antibodies were tested in this investigation. The flow-cytometer used in this study was the BD FACSArray instrument. Mixed red blood cell populations were prepared from phenotype known donors. Linearity, specificity, recovery, precision, robustness (concentration of the primary antibody ± 15%) and interday-precision were controlled for every primary antibody which is used in the procedure. Linearity of the method was verified by the Mandel test and was confirmed for every primary antibody. Another objective of this project was to identify the fluorescence of antigen expressing red blood cells with weak or heterozygous expression to improve and facilitate the interpretation of histograms. A technique to amplify the fluorescence signal by additional experimental steps was developed based on the use of a biotin-conjugated antibody directed against the fluorescent dye of the secondary antibody, In addition, streptavidin, conjugated to the same dye like the secondary antibody reacted with biotin-conjugated antibody. Aus dem Text (geändert)
TitelRecent advances in doping analysis (14) : Proceedings of the Manfred Donike Workshop ; 24th Cologne Workshop on Dope Analysis 4th to 9th June 2006
Redakteure/-innenWilhelm Schänzer, Hans Geyer, A. Gotzmann, Ute Mareck
Herausgeber/inSport & Buch Strauß
PublikationsstatusVeröffentlicht - 2006
VeranstaltungCologne Workshop on Dope Analysis - Köln, Deutschland
Dauer: 04.06.200609.06.2006
Konferenznummer: 24

ID: 194285

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