Apheresis buffy coat collection without photoactivation has no effect on apoptosis, cell proliferation, and total viability of mononuclear cells collected using photopheresis systems

Abstract

BACKGROUND Extracorporeal photopheresis (ECP) has been approved for the treatment of advanced cutaneous T‐cell lymphoma since 1988. While the precise mechanisms resulting in clinical effects are not fully understood, the photoactivation of mononuclear cells (MNCs) using ultraviolet A (UVA) light and methoxsalen is believed to be the predominant initiating process. The effects of MNC passage through the instrument without photoactivation are unknown. The objective of this study was to evaluate the effect of cell processing through the photopheresis instruments on MNCs. STUDY DESIGN AND METHODS Fourteen healthy male subjects underwent one simulated ECP procedure without reinfusion of buffy coats (BCs) in a two‐center, open‐label, prospective trial. Baseline peripheral blood BC, apheresis‐separated untreated BC (BC1), and photoactivated BC (BC2) were evaluated in culture for viability by dye exclusion, apoptosis by annexin V binding, and cell proliferation response to phytohemagglutinin (PHA) stimulation by bromodeoxyuridine (BrdU) incorporation. RESULTS Photoactivation (BC2) resulted in 88% expression of annexin V by Day 1 of culture compared with 37 and 39% for baseline and untreated BC1. Cell viability by propidium iodide exclusion was reduced to 10% in BC2 on Day 1 versus 65 and 60% for baseline and BC1. The proliferative response to PHA stimulation was 97% inhibited in the photoactivated BC2. CONCLUSIONS These results demonstrate that the mechanical processes used for cell separation and processing of the BC in the absence of photoactivation do not induce a significant amount of apoptosis compared to the standard ECP with methoxsalen and UVA photoactivation.

CD71 surface analysis of T cells: a simple alternative for extracorporeal photopheresis quality control

“…The CFSE assay was found to be a reliable test system, in agreement with previously reported results 20. We observed a strong inhibitory effect in all tested samples of healthy donors similar to the results of Szczepiorkowski et al 30. In the context of ECP quality control, CD71 was examined for the first time in this study; therefore, no comparative values are available in the literature.…”

Pilot study of a new online extracorporeal photopheresis system in patients with steroid refractory or dependent chronic graft vs host disease

“…The impact of ECP on lymphocyte proliferation is likely less relevant to the mechanism of action, mainly confirming the direct effects of ECP on treated lymphocytes. The trends we observed for both of these parameters in Amicus ECP‐treated cells are in line with values reported for other online and offline systems 18‐20 and meet ECP verification standards proposed by an Italian consensus group 21 and the French regulatory body, ANSM 22…”

“…This may in part be due to the rapid kinetics of apoptosis in the ECP‐treated cells from the previous day's procedure. Szczepiorkowski and colleagues recently reported that a median of 92% of ECP‐treated cells are apoptotic after 24 hours of culture when using the Therakos CELLEX device, albeit in healthy subjects 18 . We observed a similarly high median of 86% apoptotic lymphocytes within the same time frame in our cGVHD patient population treated with the Amicus ECP system.…”

A paired trial comparing mononuclear cell collection in two machines for further inactivation through an inline or offline extracorporeal photopheresis procedure

“…Unanswered questions and future research ECP protocols are usually defined according to the blood volume processed to collect a variable number of MNCs. 12,13 However, the real objective should be a prefixed number of MNCs that we define as the MNC standard dose. This is a quantity of MNCs that has not been determined definitively in any study.…”