Evaluation of Cytokine Synthesis in Human Whole Blood by Enzyme Linked Immunoassay (ELISA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Flow Cytometry

摘要

Whole blood stimulated for 2 hours with small amounts of lipopolysaccharide (LPS, l0-l000 pg/ml) was used for evaluating methods for detecting cytokine expression in leukocytes. The anticoagulants heparin, citrate, and EDTAwere examined for their influence on synthesis oftumornecrosis factor 0 (%%F-a), interleukin-l% and -l%%(IL-la and IL-l%), and interleukin-8 (IL-%). Cytokine secretion into blood was followed by enzyme liiiked immunoassay (ELISA) and rnRNA synthesis was determined by reverse transcriptase- polymerase chain reaction (RT-PCR). Leukocytes responsible for synthesis of cytokines were identified by imniunolabeling of cell surface markers and intracellular cytokines followed by detection by flow cytometry. Translation of mRNA and secretion of cytokines into the plasma were affected by the method of anticoagulation. Use of heparin resulted in higher levels of cytokines than citrate or EDTA, Heparin interfered with RT-PCR detection of mRNA if it was not removed.