Expression of Platelet (PLT) P-Selectin and PLT-Neutrophil (PMN) Interaction After Treatment of Whole Blood with Mirasol® PRT.
Authors: Ambruso Daniel R. , Seewald Ryan , Marschner Susanne , Goodrich Raymond P. 
Published: October 2019

Abstract
Abstract 2288

Introduction
Platelet concentrates develop biologically active compounds during storage which may play a role in adverse events of transfusion. Although many studies have focused on release of soluble pro-inflammatory compounds, changes in cells such as platelets may contribute to the response or pro-inflammatory effects of transfusion products. In previous studies we have shown that pathogen reduction with Mirasol PRT of apheresis PLT concentrates induced changes in PLTs which increased during storage including expression of P-selectin and the capacity to form PLT-PMN aggregates. These changes were also associated with increased adherence of stored PLTs in a microfluidic chamber. In the current study we evaluated P-selectin and PLT-PMN aggregate formation in whole blood after treatment with Mirasol PRT.

Methods
Ten units of whole blood from healthy donors were drawn into CPD under an IRB approved protocol. Five units where treated with Mirasol PRT and five units remained untreated. Aliquots were taken before and after treatment (or at same times for untreated units) on Day 0 and 24–27 hours after draw on Day 1 after storage at 22–24°C. Platelet rich plasma (PRP) was produced by standard technique and PLTs isolated on a Sepharose 2B column. PLTs were incubated with APC-CD62P and FITC-CD41a for comparison with P-selectin expressed as percent positive increase over isotype control. PMNs were isolated from 50 ml of heparinized peripheral blood drawn from healthy, ABO-matched donors. After a 5 min pre-incubation at 37°C in a shaking water bath, untreated, treated PLTs, and PMNs were incubated separately or together for 3 min then quenched at 4°C. FITC-CD41a and FITC-IgG1 isotype for platelet-PMN aggregate quantitation or PE-CD11b for CD11b expression on neutrophils were added. Platelet-PMN aggregates was expressed as percent of all PMN events above CD41a isotype control. CD11b expression was determined for PMNs after exposure to PLTs by flow cytometry.

Results
P-selectin positive cells did not change significantly over time in samples from untreated products (Table 1). After Mirasol PRT treatment, cells positive for P-selectin increased on Day 0 and 1 (p<0.005, two-tailed t-test). Treated products, compared to untreated products demonstrated significantly increased P-selectin expression on Day 1.
Results for PMN-PLT aggregates parallel expression of P-selectin (Table 2). No increase in aggregate formation was seen for untreated samples; however, treated samples showed increased aggregates at Day 0 and 1 (p<0.005). Aggregates with PMNs were increased in treated samples in comparison to untreated samples on Day 1.

Conclusion
Treatment of whole blood with Mirasol PRT results in increased expression of P-selectin on PLTs and capability of forming aggregates with PMNs. The changes occur soon after initiation of treatment, and the implications for clinical response to of platelet infusion and adverse events of transfusion need to be established.

Disclosures:
Ambruso: Terumo BCT: Research Funding. Seewald:Terumo BCT: Research Funding. Marschner:Terumo BCT: Employment. Goodrich:Terumo BCT: Employment.