Assessment of nucleic acid modification induced by amotosalen and ultraviolet A light treatment of platelets and plasma using real-time polymerase chain reaction amplification of variable length fragments of mitochondrial DNA.
Author(s): Bakkour S, Chafets DM, Wen L, Dupuis K, Castro G, Green JM, Stassinopoulos A, Busch MP, Lee TH
Publication: Transfusion, 2016, Vol. 56, Page 410-20
PubMed ID: 26446053 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of amotosalen and ultraviolet A (UVA) treatment on the amplification of four different length amplicons of mitochondrial DNA from platelets (Plts) and plasma and investigated the effects of leukoreduction or plasma concentration through centrifugation.
Conclusion of Paper
Cycle threshold (CT) values for mitochondrial DNA increased when white blood cell (WBC) DNA, Plts, or plasma were exposed to amotosalen and UVA illuminated, but a similar increase was not observed when WBC DNA was exposed to amotosalen or UVA illuminated alone. The magnitude of the increase was dependent on the amplicon size with larger increases observed for longer amplicons and when plasma was centrifuged rather than not. The difference in the CT values between the 73 bp and 1065 bp products was able to differentiate treated from untreated Plt or plasma specimens.
Studies
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Study Purpose
This study investigated the effects of amotosalen and UVA treatment on the amplification of four different length amplicons of mitochondrial DNA from Plts and plasma and investigated if there was any effect of leukoreduction or plasma concentration through centrifugation. Six units of individual apheresis Plts were placed in InterSol platelet additive solution (PAS). Leukoreduced and nonleukoreduced Plts were obtained using SEPACALL from eight Plt units obtained by the Plt-rich plasma preparation method. Plasma units were obtained from 10 apheresis units or from whole blood and DNA was extracted directly or after centrifugation at 21,000 x g for 15 min. All Plts and plasma specimens were stored frozen and shipped on dry ice for analysis. Specimens were sampled and treated with amotosalen and then UVA illuminated. The ability of the PCR-based assay to distinguish treated and untreated specimens was checked using 110 Plt units and 100 plasma specimens in a blind study. DNA was extracted using the QIAamp DNA blood kit and mitochondrial DNA was quantified by real-time PCR amplification.
Summary of Findings:
Real-time PCR efficiency decreased with increasing amplicon size (100% for 73 bp, 95% for 561 bp, 81% for 1065 bp, and 80% for 1409 bp). The CT of the 73 bp, 1065 bp, and 1409 bp products increased by 7.2, 12.1, and 14.1 cycles (respectively) when Plts in 35% plasma 65% InterSol were treated with amotosalen and then UVA irradiated, but the CTs were comparable to untreated specimens when WBC DNA in 35% plasma 65% PAS was treated with amotosalen or UVA alone. When plasma was treated with amotosalen and UVA irradiated, a similar reduction in the CT of all three products was observed. Interestingly, inclusion of a centrifugation step increased the difference between the CT values of the 73 bp and 1065 bp products, but leukoreduction had no effect. An assay based on the difference in the CT values of the 73 bp and 1065 bp products was able to differentiate treated from untreated specimens using 110 Plt specimens and 100 plasma specimens.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood processing method Untreated
UVA and amotosalen treated
Only amotosalen treated
Only UVA treated
Leukoreduced
Non-leukoreduced
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Real-time qPCR Specific Length of gene fragment 73 bp
561 bp
1065 bp
1409 bp
Biospecimen Aliquots and Components Blood and blood products Platelets
Plasma