(P-TS-11) Amotosalen or Photo-Induced By-Products in Pathogen-Reduced Blood Products Do Not Induce Non-Specific in-vitro Activation or Degranulation of Basophil from Healthy Volunteers

Background/Case Studies: Platelet concentrates (PCs) and fresh frozen plasmas (FFPs) are the leading cause of hypersensitivity transfusion reactions (HTRs). PCs and FFPs can be treated with amotosalen and UV-A (INTERCEPT™ Blood System – IBS, Cerus) for pathogen reduction. The imputability of amotosalen or by-products in HTRs remains elusive but not supported by epidemiology. Our aim is to assess effects of free amotosalen or photo-induced by-products in IBS-treated PCs/FFPs on non-specific in-vitro activation of basophils from healthy volunteers using basophil activation test (BAT).

Study

Design/Methods: Free amotosalen (0.0003 to 30 µM) or supernatants derived from IBS-FFPs and IBS-PCs (1:10 and 1:20) were added to citrated whole blood for 30 min at 37°C. Samples were analysed by flow cytometry to identify basophils (IgE⁺/CD203c⁺). Activation status was assessed by the percentage of CD63⁺ cells (correlated to histamine release) and the stimulation index (SI) based on CD203c upregulation (MFI of stimulated divided by resting basophils) with a threshold at 1.6. Positive controls were a mouse anti-human IgE monoclonal antibody (clone G7-18) and fMLP (an IgE-independent activating peptide). Statistical comparisons were done by mixed model or two-way ANOVA followed by Tukey’s post-hoc test (n=5).

Results/Findings: Free amotosalen had no effect on CD203c-SI or CD63 exposure at any concentrations (3 µM = 10 FFPs or 2-3 PCs) as FFPs (1:10 = 10 FFPs) tested before and after treatment, then after CAD and 48 hours after storage liquid at +4°C. When PCs were stored at +22°C, CD63 exposure remained at basal levels, while CD203c-SI tended to increase up to day 3 only at 1:10 (equivalent to 2-3 PCs) without reaching statistical significance and remained stable thereafter up to day 7. Interestingly, untreated (non-IBS) PC supernatants also displayed a non-significant increase in CD203c-SI over time at 1:10, suggesting that amotosalen or photo-induced by-products were not responsible for this effect. When IBS-PCs were stored at +4°C (“cold-stored platelets”) up to day 21, the same tendency was observed at 1:10 for CD203c-SI as early as day 3 and then remained stable until day 21, while CD63 was not exposed. Of note, PCs stored up to day 21 at +22°C exhibited a similar tendency but reaching statistical significance only at day 21 at 1:10 (p< 0.05). These data suggest storage lesions as the main mechanism for non-specific basophil activation (without CD63 exposure).

Conclusions: These results indicate that a wide range of concentrations of free amotosalen is unable to activate blood basophils from healthy volunteers in-vitro. Moreover, PC supernatants and FFPs, including residual free amotosalen and photo-induced by-products, are unable to induce non-specific basophil activation or granule release.