Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion.
OBJECTIVE
Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).


METHODS
Activity and antigen of plasma components were determined following RF-PRT in the presence or absence of dissolved molecular oxygen.


RESULTS
Employing ADAMTS13 as a sentinel molecule in plasma, our data show that its activity and antigen are reduced by 23 ± 8% and 29 ± 9% (n = 24), respectively, which corroborates with a mean decrease of 25% observed for other coagulation factors. Western blotting of ADAMTS13 shows decreased molecular integrity, with no obvious indication of additional proteolysis nor is riboflavin able to directly inhibit the enzyme. However, physical removal of dissolved oxygen prior to RF-PRT protects ADAMTS13 as well as FVIII and fibrinogen from damage, indicating a direct role for reactive oxygen species. Redox dye measurements indicate that superoxide anions are specifically generated during RF-PRT. Protein carbonyl content as a marker of disseminated irreversible biomolecular damage was significantly increased (3·1 ± 0·8 vs. 1·6 ± 0·5 nmol/mg protein) following RF-PRT, but not in the absence of dissolved molecular oxygen (1·8 ± 0·4 nmol/mg).


CONCLUSIONS
RF-PRT of single plasma units generates reactive oxygen species that adversely affect biomolecular integrity of relevant plasma constituents, a side-effect, which can be bypassed by applying hypoxic conditions during the pathogen inactivation process.
Transfusion Research Center, Belgian Red Cross-Flanders, Ghent, Belgium.
H B
HB
Feys
B
B
Van Aelst
K
K
Devreese
R
R
Devloo
J
J
Coene
P
P
Vandekerckhove
V
V
Compernolle
D051722:ADAM Proteins / Q000097:blood / Q000737:chemistry; D000071120:ADAMTS13 Protein; D001779:Blood Coagulation Factors / Q000032:analysis; D016913:Blood Component Transfusion; D001798:Blood Proteins / Q000737:chemistry; D058731:Blood Safety / Q000379:methods*; D017848:Blood-Borne Pathogens / Q000187:drug effects / Q000528:radiation effects; D004203:Disinfection; D006801:Humans; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D017319:Photosensitizing Agents / Q000494:pharmacology*; D010949:Plasma / Q000187:drug effects / Q000528:radiation effects; D050050:Protein Carbonylation; D012256:Riboflavin / Q000494:pharmacology*; D013481:Superoxides / Q000737:chemistry; D014466:Ultraviolet Rays
D001779:Blood Coagulation Factors; D001798:Blood Proteins; D017319:Photosensitizing Agents; D013481:Superoxides; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D010100:Oxygen; D012256:Riboflavin
pathogen inactivation; plasma; reactive oxygen species