Effects of temperature on inflammatory markers assessment during sputum processing

Abstract

Objective To evaluate the effects of temperature on cytology of inflammatory cell and detection of inflammatory markers during sputum processing.Methods Fifty-two asthmatic patients were recruited from Guangzhou Institute of Respiratory Diseases between March and August 2013.Following sputum induction,0.1％ dithiothreitol (DTT),at a ratio of 4∶1 (v/w),was added for incubation consecutively at 0 ℃ and 37 ℃,for 15 min (15 min ice-incubated group and 15 min water bath group,n=28) and 30 min (30 min ice-incubated group and 30 min water bath group,n=10).The cell pellet was re-suspended for cytology assessment followed by hematoxylin-eosin (HE) staining.Commercially available luminex kits were employed to measure the levels of interleukin (IL)-5,IL-10,IL-17,interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the sputum supernatant.Effects of different temperatures on cytology of inflammatory cell and detection of inflammatory markers during sputum processing were subsequently compared.Results Sputum induction was successful in 28 out of 52 patients (success rate 53.8％).There was no difference in sputum weight,total cell counts,cell viability and differential cell counts between the 15 min ice-incubated group and water bath group,30 min ice-incubated group and water bath group (all P＞0.05).There were no differences in the levels of IL-5,IL-10,IL-17,IFN-γ and TNF-α between 15 min ice-incubated group and 15 min water bath group (all P＞0.05).There were no differences in the levels of IL-10,IL-17 and IFN-γ between 30 min ice-incubated group and 30 min water bath group (all 9＞0.05).However,the levels of IL-5 and TNF-α in 30 min water bath group were significantly higher than those in 30 min ice-incubated group [2.55 (2.20-4.35) ng/L vs 2.03 (1.52-2.50) ng/L,19.41 (9.07-37.92) ng/L vs 10.78 (8.58-17.93) ng/L,all P＜0.05].Conclusions Levels of inflammatory mediators can be influenced by the different temperatures upon processing.Sputum processed with DTT at 0 ℃ may better reflect the true levels of inflammatory markers. Key words: Asthma; Cytokines ; Sputum induction; Temperature