2.2 Blood samples
Venous blood was collected from each of the PNS patients and each of the healthy controls and stored in ethylenediamine tetraacetic acid (EDTA)-Na2 as an anti-coagulant. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient for subsequent flow cytometric analysis . Plasma was obtained after centrifugation and stored at -80°C for analysis by cytometric bead array (CBA). CD4+CD25- T cells were immediately isolated from the patients’ peripheral blood samples, according to the manufacturer’s instructions for microbeads (11363D, Dynal; Invitrogen, USA). Cell populations were considered pure at > 97% by flow cytometry, and cell activity was investigated using the trypan blue exclusion assay (using the threshold of > 95% of the cell population as a positive indicator of significantly decreased cell activity).