A simple method for removing low-density granulocytes to purify T lymphocytes from peripheral blood mononuclear cells.

Abstract

Low-density granulocytes (LDGs) can form neutrophil extracellular traps (NETs) spontaneously and excessively. When peripheral blood mononuclear cells (PBMCs) are used for studying T lymphocytes, LDGs contained in the PBMCs may decrease the threshold of activating T lymphocytes by forming NETs. This study focused on the profiles of LDGs in common autoimmune diseases and methods for removing LDGs from PBMCs. The percentages of LDGs in PBMCs from 55 patients with dermatomyositis (DM), 15 with polymyositis (PM), 42 with rheumatoid arthritis (RA), 25 with systemic lupus erythematosus (SLE), and 19 healthy controls were determined by flow cytometry. Three methods of removing LDGs were explored and compared. After removal, PBMCs from six patients with positive T-SPOT.TB were tested again to find out if LDGs contained in the PBMCs could influence T lymphocyte reactions. Significantly higher LDG percentages were found in PBMCs from patients with DM ((8.41±10.87)%, P<0.0001), PM ((8.41±10.39)%, P<0.0001), RA ((4.05±6.97)%, P=0.0249), and SLE ((7.53±11.52)%, P=0.0006), compared with the controls ((1.28±0.73)%). The T-SPOT.TB values significantly decreased after LDGs were removed. Increasing relative centrifugal force (RCF) within a limited range can decrease the LDG percentage from an initial high level, but not markedly increase the LDG clearance rate. Compared with the whole blood sediment method, the PBMC adherence method can significantly remove LDGs yet scarcely influence the T lymphocyte percentage in PBMCs. The LDG percentage in PBMCs is significantly increased in patients with SLE, DM, PM, and RA. The influence of LDGs on T lymphocytes cannot be ignored in PBMC cultures. The adherence method is a simple and easy-to-use method for removing LDGs and purifying T lymphocytes from PBMCs.