Adherence of Stored Apheresis Platelets (PLTs) with and without Pathogen Reduction Treatment (PRT) to Collagen in a Microfluidic Flow Chamber

Previous studies by our laboratory have demonstrated changes in apheresis collected, PLT concentrates during storage that are enhanced by Mirasol^®PRT treatment. These include increased expression of P-selectin on PLTs and increased formation of neutrophil (PMN)/PLT aggregates. To explore the effect of these changes in a microvascular environment, we measured PLT adherence in a microfluidic chamber coated with collagen.

Three, double apheresis platelet products were collected from healthy volunteers with TRIMA system under an IRB approved protocol. One product of each pair was treated with Mirasol PRT and both were stored under standard conditions. Aliquots were sterilely removed from products during storage. To 1000 μl of fresh, ABO type specific, citrate anticoagulated whole blood was added 60 μl of apheresis platelet concentrate with or without PRT treatment on day 1 or 7 of storage and incubated for 5 min at 37C. The sample was recalcified with 20 mM CaCl₂ and then pulled through the inlet of a microfluidic chamber at a flow sheer rate of 100 s⁻¹. The flow chamber was composed of two parts, a glass slide patterned with collagen and a series of four polydimethylsiloxane microfluidic chambers per inlet. Stored PLTs were labeled with fluorescently labeled anti-CD41 to distinguish from PLTs of the whole blood. The chamber was mounted on an inverted fluorescence microscope, and video microscopic images sampled on observation field every 7 sec over 7 min. The number of fluorescent cells (stored PLTs) per field and the total area covered by all PLTs were determined at 7 min.

The area, expressed as % of total field, covered by all of the PLTs in the sample was not different for whole blood alone (29 ± 3); Day 1 untreated (23 ± 9); Day 1 PRT treated (26 ± 8); Day 7 untreated (21 ± 5); and Day 7 treated (25 ± 9) PLTs. In contrast, there was an increase in the number of treated apheresis PLTs at Day 1 compared to untreated apheresis platelets binding to the chamber (2.26 ± 0.61 untreated, vs. 5.45 ± 1.45 treated, numbers are mean ± SEM for numbers of cells adhered normalized to the PLT count in the product, significant, p=0.0229). There was also an increase in binding of treated compared to untreated PLTs at Day 7 (2.43 ± 0.47 untreated, vs. 4.42 ± 1.35 treated). However, the results for untreated PLTs on Day 1 and 7 and treated PLTs on study days were not different. The increased binding of Mirasol PRT treated PLTs parallels the small decrease in recovery seen in clinical studies of patients receiving treated PLT concentrates compared to untreated products.

PRT treatment increases adherence of PLTs early in storage but does not appear to progress further. The changes may have implications for clinical responses to platelet transfusions. Evaluation of adherence in the microfluidic chamber provide a model which simulate the microvascular environment.

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