Digital droplet polymerase chain reaction to monitor ultraviolet C treatment of single-donor and buffy coat platelet units. 2020

Uta Voglau, and Thomas H Müller, and Axel Seltsam, and Ute Gravemann, and Wiebke Handke, and Andrea Doescher
DRK-Blutspendedienst NSTOB, Institut Bremen-Oldenburg, Oldenburg, Germany.

UVC 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. Samples 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. Apheresis 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. The 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.

UI MeSH Term Description Entries
D010983 Plateletpheresis The preparation of platelet concentrates with the return of red cells and platelet-poor plasma to the donor. Blood Plateletpheresis,Thrombocytapheresis,Thrombocytopheresis,Blood Plateletphereses,Plateletphereses,Plateletphereses, Blood,Plateletpheresis, Blood,Thrombocytaphereses,Thrombocytophereses
D011786 Quality Control A system for verifying and maintaining a desired level of quality in a product or process by careful planning, use of proper equipment, continued inspection, and corrective action as required. (Random House Unabridged Dictionary, 2d ed) Control, Quality,Controls, Quality,Quality Controls
D001792 Blood Platelets Non-nucleated disk-shaped cells formed in the megakaryocyte and found in the blood of all mammals. They are mainly involved in blood coagulation. Platelets,Thrombocytes,Blood Platelet,Platelet,Platelet, Blood,Platelets, Blood,Thrombocyte
D004272 DNA, Mitochondrial Double-stranded DNA of MITOCHONDRIA. In eukaryotes, the mitochondrial GENOME is circular and codes for ribosomal RNAs, transfer RNAs, and about 10 proteins. Mitochondrial DNA,mtDNA
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D014466 Ultraviolet Rays That portion of the electromagnetic spectrum immediately below the visible range and extending into the x-ray frequencies. The longer wavelengths (near-UV or biotic or vital rays) are necessary for the endogenous synthesis of vitamin D and are also called antirachitic rays; the shorter, ionizing wavelengths (far-UV or abiotic or extravital rays) are viricidal, bactericidal, mutagenic, and carcinogenic and are used as disinfectants. Actinic Rays,Black Light, Ultraviolet,UV Light,UV Radiation,Ultra-Violet Rays,Ultraviolet Light,Ultraviolet Radiation,Actinic Ray,Light, UV,Light, Ultraviolet,Radiation, UV,Radiation, Ultraviolet,Ray, Actinic,Ray, Ultra-Violet,Ray, Ultraviolet,Ultra Violet Rays,Ultra-Violet Ray,Ultraviolet Black Light,Ultraviolet Black Lights,Ultraviolet Radiations,Ultraviolet Ray
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D058621 Blood Buffy Coat The fraction of a blood sample, following CENTRIFUGATION, that is distinguished as a thin light-colored layer between the RED BLOOD CELLS, underneath it, and the PLASMA, above it. It is composed mostly of WHITE BLOOD CELLS and PLATELETS. Buffy Coat, Blood,Blood Buffy Coats,Buffy Coats, Blood
D020641 Polymorphism, Single Nucleotide A single nucleotide variation in a genetic sequence that occurs at appreciable frequency in the population. SNPs,Single Nucleotide Polymorphism,Nucleotide Polymorphism, Single,Nucleotide Polymorphisms, Single,Polymorphisms, Single Nucleotide,Single Nucleotide Polymorphisms
D024101 Mitochondrial Proteins Proteins encoded by the mitochondrial genome or proteins encoded by the nuclear genome that are imported to and resident in the MITOCHONDRIA. Proteins, Mitochondrial,Mitochondrial Protein,Protein, Mitochondrial

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