Pathogen Inactivation of Platelet Concentrates by UVC-Light: Influence on Platelet Quality, Vitality, Membrane Integrity and Mitochondrial Function.

Abstract

Introduction: Safety of blood products today is mainly based on strict donor selection, screening systems to detect markers for transfusion relevant viruses and bacterial culturing. Window period, pathogens not tested, emerging viruses and slow growing bacteria are remaining risks for the recipient and will cause a failure of all safety measures. Pathogen inactivation will overcome those problems, at least for platelet and plasma units. UVC light without additional substances has recently proven to inactivate a broad range of viruses (with the exception of HIV) and bacteria (Fokke et al., Transfusion 2008). We investigated the influence of UVC irradiation on platelet storage parameters, apoptosis rate and aggregation capacity. In addition we visualized membrane integrity, mitochondrial activity and intra-platelet calcium content in living platelets using live confocal microscopy.Method: 12 double dose platelet concentrates (6.0x1011 platelets, WBC content below 106) were prepared by means of apheresis (Trima, Gambro BCT, USA), automatically suspended in platelet additive solution (SSP+, Macopharma, BRD) in a 60:40 ratio (SSP+ vs. plasma) and divided into two single units after a two hours resting period. One unit was UVC irradiated immediately after resting at a dose of 0.3J/cm2 (group 2), one remained untreated and both were stored at 22° C for 7 days (group 1). Storage parameters (glucose, lactate, LDH, pH) were analyzed before irradiation and on day two, five and seven. Apoptosis rate was calculated as the percentage of Annexin V positive cells within CD-41 pos cells. Combining life confocal imaging with three fluorescent dyes, fluorescent wheat germ agglutinin (WGA; Molecular Probes, Eugene, OR, USA), tetramethylrhodamine methyl ester perchlorate (TMRM; Sigma-Aldrich) and Rhod-2 (Molecular Probes) we assessed platelet morphology (WGA), mitochondrial activity (TMRM) and intra-platelet calcium (Rhod-2). A students paired T-test was used for statistical analysis and a p value of < 0.05 was considered significant.Results: On day two, the only significant difference in the measured metabolic parameters could be seen in pH (7.28±0.04 vs. 7.25±0.05, group 1 vs. group2;p < 0.01) and differences remained significant over the whole observation period (7.37±0.06 vs. 7.18±0.07, group 1 vs. group2;p < 0.01). On day seven lactate values mg/dl (58.08±13.51 vs. 81.33±13.45, group 1 vs. group2; p < 0.01) and glucose consumption mg/dl (80.33±19.49 vs. 59.50±16,97, group 1 vs. group2; p < 0.01) reached statistical significance, only LDH (U/L) increase did not differ significantly between both groups even not on day seven (68.83±22.17 vs. 75.25±15.08 group 1 vs. group2; p= n.s.). Differences in aggregation capacity reach significance on day 5 (59.50%±10.45% vs. 55.92%±12.30%, group 1 vs. group2; p < 0.05) but not on day seven. Rate of apoptotic platelets was similar on day 2, reaches significance on day 5 (6.53%±3.02% vs. 8.52±3.53, group 1 vs. group2; p < 0.01) and increased on day seven (9.98%±7.34% vs. 18.89%±14.96%, group 1 vs. group2 ; p < 0.01). Image analysis of the confocal data comparing paired samples from untreated and UVC treated platelets revealed no significant differences for all time points. Neither mitochondrial activity, intra platelet calcium content, platelet morphology nor platelet membrane integrity differed between the compared samples.Conclusion: Influence of UVC irradiation of platelet concentrates as a pathogen inactivation method could be witnessed in metabolic parameters as well as in a decrease of aggregation capability and an increase in the rate of apoptotic cells after seven days in a paired comparison with untreated platelets. However differences are small and the total values of the measured parameters meet the quality requirements for platelets. Live confocal imaging of living platelets supports our conclusion but further recovery and survival studies with UVC treated platelets are necessary to compare in-vitro data with in-vivo data.