Biological quality control for extracorporeal photochemotherapy: Assessing mononuclear cell apoptosis levels in ECP bags of chronic GvHD patients

Abstract

Extracorporeal photochemotherapy (ECP) is a treatment approved by the FDA for cutaneous T-cell lymphoma, and it is currently used off-label for graft-versus-host disease (GvHD) and other conditions. In agreement with good practices for the therapeutic use of human cells, quality control has to be performed to validate the ECP procedure with the off-line technique. Since no gold-standard biological test is available, we assessed the apoptosis generated in the ECP bag using a flow cytometric analysis. Thirty-one ECP procedures performed on 13 patients with chronic GvHD were studied by monitoring the induction of mononuclear cell (MNC) apoptosis using annexin V/propidium iodide double staining; residual lymphocyte proliferation to standard mitogens was also measured in 17 of the procedures. The kinetics of apoptosis was analyzed at different times in MNCs untreated or treated with 8-methoxy-psoralen plus ultraviolet A; the variation (ΔAPOPTOSIS ) after 24 h revealed the efficacy of the treatment. In 88.6% of the 31 ECP procedures, ΔAPOPTOSIS was >15% (the "alerting" threshold for ΔAPOPTOSIS was set at 15% on the basis of our data); in the remainder (19.4%), the increment in apoptosis was lower. In four procedures, the proliferation assay was useful for assessing the effect of ECP on the apheretic bag. In conclusion, both flow cytometric assays enabled a biologically significant result to be obtained. In our opinion, the apoptosis test-being faster and easier than the proliferation test-could be a reliable way to validate ECP procedures.

Does Offline Beat Inline Treatment: Investigation into Extracorporeal Photopheresis

“…Although apoptosis is an ongoing process, the time point 24 h after irradiation treatment provides the largest increase in apoptosis [26] and was therefore used as a representative marker of apoptosis. In general, apoptosis levels were consistent with findings published by other groups [27]. Apoptosis induction was significantly higher in the inline system ( p < 0.001), which also had a higher “background” apo­ptosis of untreated cells after 24 h compared to the offline procedure.…”

Evaluation of lymphocytes inactivation by extracorporeal photopheresis using tetrazolium salt based-assay

“…Validation criteria however have not been yet established. The Italian Society of Hemapheresis and Cell Manipulation and Italian Group for Bone Marrow Transplantation proposed that the percentage of CD3+/7-AAD+ lymphocytes after 72-96 hours of culture should be not less 50% [8], and Taverna et al [7] proposed to set a Δapoptosis "alerting" threshold, defined as the difference between the % of annexin V positive cells in the pre and post ECP, of at least 15%.…”

“…In 2014, Taverna et al [7] performed the ECP quality control in flow cytometry by evaluating the induction of …”

Cutaneous manifestations of chronic graft-versus-host disease

“…Для контроля эффективности лечения кожной формы РТПХ методом экстракорпоральной фото-химиотерапии используются цитологические тесты (оцениваются апоптоз мононуклеарных клеток и пролиферация лимфоцитов) [12]. Имеется единич-ное описание использования ультразвукового мето-да исследования (20 MHz) с акцентом на плотность и толщину очагов поражения [13].…”

“…Так, в исследовании F. Taverna и соавт. [12] показано, что при проведении фототерапии отмечается общая тенденция снижения интенсивности кожных про-явлений РТПХ, благодаря чему может быть ограни-чено использование системных ГКС. Местное лече-ние подразумевает применение топических ГКС, нестероидных топических иммуномодуляторов, то-пических ингибиторов кальциневрина [3].…”