Polymerase chain reaction inhibition assay documenting the amotosalen-based photochemical pathogen inactivation process of platelet concentrates.
BACKGROUND
The INTERCEPT Blood System (Baxter Healthcare Corp.) for platelets (PLTs) uses amotosalen-HCl (S-59) in conjunction with ultraviolet A (UVA) light to inactivate contaminating pathogens by modifying the nucleic acids of pathogens. The success of this photochemical treatment (PCT) process can be documented indirectly with a high-performance liquid chromatography assay measuring the photodegradation of amotosalen and measurement of the UVA light dose delivered by the illumination system.


METHODS
To develop an assay that documents the success of PCT directly on the effector molecule DNA, the effect of PCT on PLT-derived mitochondrial DNA (mtDNA) was examined. mtDNA-specific polymerase chain reaction (PCR) assays were tested with regard to their susceptibility for PCT, their reliability in terms of PCR performance, and the absence of polymorphic sites in primer hybridization loci.


RESULTS
Suitable PCR amplification targets were found in the regions of 16S rDNA, cytochrome c oxidase I, and cytochrome c oxidase III of mitochondria. Amplicon sizes between 868 and 1248 bp gave consistent signals before PCT and complete inhibition of the PCR signal after PCT. Amplicons of less than 300 bp were found to be transparent to PCT.


CONCLUSIONS
Based on PCT-mediated mtDNA modifications in PLTs, a PCR inhibition assay was established with a large amplicon documenting the success of PCT and a small amplicon serving as an internal control.
Bruchmüller
Iris
I
Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden Württemberg-Hessen, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Friedrich-Ebert-Strasse 107, 68167 Mannheim, Germany. i.bruchmueller@blutspende.de
Janetzko
Karin
K
Bugert
Peter
P
Mayaudon
Veronique
V
Corash
Laurence
L
Lin
Lily
L
Klüter
Harold
H
D000225:Adenine / Q000032:analysis; D001424:Bacterial Infections / Q000517:prevention & control*; D000094902:Blood Banking / Q000379:methods; D001793:Blood Preservation / Q000379:methods* / Q000592:standards; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000032:analysis / Q000187:drug effects / Q000528:radiation effects; D003576:Electron Transport Complex IV / Q000235:genetics; D011564:Furocoumarins; D006801:Humans; D010777:Photochemistry; D017713:Platelet Transfusion; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011786:Quality Control; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000187:drug effects / Q000528:radiation effects; D012680:Sensitivity and Specificity; D013941:Thymine / Q000032:analysis; D014466:Ultraviolet Rays
D017931:DNA Primers; D004272:DNA, Mitochondrial; D011564:Furocoumarins; D012336:RNA, Ribosomal, 16S; D003576:Electron Transport Complex IV; D000225:Adenine; C118577:amotosalen; D013941:Thymine