The authors used ultrashort pulsed (USP) lasers in their research as this treatment is known to inactivate a spectrum of bacteria and viruses including nonenveloped viruses, a class of virus that resists inactivation. Furthermore, the laser treatment is nonionizing and does not modify proteins covalently, meaning that proteins present in blood are likely to remain active even after exposure to USP lasers. The viruses that were tested for inactivation by USP laser in human plasma were an enveloped RNA virus human immunodeficiency virus (HIV), nonenveloped RNA virus hepatitis A virus (HAV) and enveloped DNA virus murine cytomegalovirus (MCMV).
To test the effectiveness of the laser, the researchers spiked HIV, HAV and MCMV into human plasma and exposed the plasma to a 425nm USP laser for 90 minutes while the plasma was stirred. The stirring helped ensure that all the sample was exposed to the focal point of the laser. Control plasma samples with virus were not subjected to USP laser treatment. After laser irradiation, the plasma was tested for viral titer in plaque assays for HIV and HAV, and tissue culture infectious dose (TCID50) assay for MCMV. The results showed a decrease in viral titer for all three viruses when untreated and USP laser-treated samples were compared: Two orders of magnitude decrease for HIV, one order of magnitude decrease for HAV and three orders of magnitude decrease for MCMV. Of particular interest were the HAV results as the reduction exceeded that of current approved chemical treatments.
While the viral titer was reduced, were the blood proteins in the plasma affected? Human plasma was treated with the USP laser and the various protein factors tested using standard coagulation assays (e.g., prothrombin time or the time it takes plasma to coagulate after Factor III is added). The treated plasma was altered compared to the untreated plasma but five of the nine coagulation factors fell within the normal range and only Factor VII was far from the normal range. In fact Factor VII activity was enhanced, suggesting to the authors that the treatment damaged Factor VII inhibitors in the plasma.
Plasma protein function seems to be retained, but what about other considerations? Analysis by both native and denaturing polyacrylamide gel electrophoresis did not show any difference in detergent-resistant aggregation between treated and untreated plasma, suggesting there was no induction of protein aggregation. Detergent-resistant aggregation was associated with viral inactivation based on previous work by these researchers. Fibrinogen was one of the coagulation factors reduced by USP laser treatment so Tsen et al. performed further analysis using purified fibrinogen to see if there were any changes to the protein. Purified fibrinogen was treated with USP laser and compared protein structure and aggregation state with untreated fibrinogen. Based on light-scattering tests, the amount of fibrinogen monomers were identical between USP laser treatment and untreated purified protein, and the circular dichroism spectrum of control and treated purified fibrinogen showed no difference in the protein structure.
The authors demonstrated using a USP laser at 425nm was sufficient to reduce viral titer of viruses in human plasma without altering structure and function of coagulation factors. With additional testing and fine tuning of the laser, this method offers a nonchemical alternative for reducing blood-borne pathogens in plasma and increasing the safety of the blood supply.
Tsen, S.W.D., Kibler, K., Jacobs, B., Sizemore, S., Vaiana, S.M., Anderson, J., Tsen, K.T, and Achilefu, S. (2014) Pathogen reduction in human plasma using an Ultrashort Pulsed Laser. PLOS ONE, 9 (11) e111673. DOI: http://dx.doi.org/10.1371/journal.pone.0111673
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