Digital droplet polymerase chain reaction to monitor ultravioletC treatment of single‐donor and buffy coat platelet units

BACKGROUNDUVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) in platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of th...

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Bibliographic Details
Published in:Transfusion (Philadelphia, Pa.) Pa.), 2020-08, Vol.60 (8), p.1821-1827
Main Authors: Voglau, Uta, Müller, Thomas H, Seltsam, Axel, Gravemann, Ute, Handke, Wiebke, Doescher, Andrea
Format: Article
Language:English
Subjects:
Agitation
Apheresis
Binding sites
Buffy coat
Data analysis
Deoxyribonucleic acid
DNA
Droplets
Leukocytes
Mitochondrial DNA
Monitoring
Nucleic acids
Nucleotides
Optimization
Outliers (statistics)
Pathogens
Platelets
Polymerase chain reaction
Primers (coatings)
Quality control
Subgroups
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Summary:BACKGROUNDUVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) in platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of this purely physical pathogen reduction technology.STUDY DESIGN AND METHODSSamples from PCs (n = 530) were tested with an established LightCycler PCR (LC PCR) for QC of the UVC procedure. RNR2 and TRNK/ATP8 genes were sequenced in the PCs (n = 21) with out‐of‐specification results in the LC PCR. A digital droplet PCR (ddPCR) was developed to minimize the outliers and cross‐validated by testing the 530 PCs. The ddPCR was further evaluated in a subgroup of 300 PCs without mtDNA extraction and in samples from systematic variations of UVC dose and agitation speed.RESULTSApheresis PCs (n = 380) resulted in 5.3% outliers in LC PCR versus only 0.7% in buffy coat pool PCs (n = 150). Sequencing of these outliers revealed single‐nucleotide polymorphisms in the primer‐ and probe‐binding sites of LC PCR. The development of a ddPCR assay with modified probe sequences reduced the outliers to 0.4%. The ddPCR analysis of PCs both with and without mtDNA extraction demonstrated low intra‐ and interassay variabilities and congruent results also compared to LC PCR. Experiments varying the UVC dose and the agitation speed demonstrated that the ddPCR results closely reflect functional effects of the UVC treatment.CONCLUSIONThe ddPCR assay offers a valid and reliable tool for QC of routine production of the UVC‐treated PCs as well as for monitoring treatment conditions during optimization of the UVC procedure.
ISSN:0041-1132
1537-2995
DOI:10.1111/trf.15904