Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight.

The effects of immediate versus delayed cell separation, storage temperature, presence of serum, and type of anticoagulation on the natural killer (NK) cytotoxicity of human mononuclear cells were assessed. The NK cytotoxicity of Ficoll-Hypaque-separated peripheral blood mononuclear cells (PBMC) was tested in a 3-h chromium-51 release assay with K562 cells at various effector/target cell ratios. The NK activities of PBMC from blood anticoagulated with either heparin or EDTA and then immediately separated and assayed were not different (42.9 +/- 2.5% for heparin and 40.3 +/- 4.6% for EDTA). When these separated cells were cultured in medium with 10% fetal calf serum and stored at 4,25, or 37 degrees C for 18 h before the assay, there was a significant increase in cytotoxicity. PBMC from blood stored in heparin or EDTA for 18 h before separation had reduced NK cytotoxicity, particularly if they were kept at 37 degrees C. When separated PBMC were cultured in medium with 10% human AB serum, however, samples held at 25 and 37 degrees C decreased in cytotoxicity but samples held at 4 degrees C maintained the cytotoxicity demonstrated at the baseline level with fresh cells. We recommend that heparinized blood be used for NK assays and that the PBMC be isolated immediately and held overnight at 4 degrees C in medium with 10% AB serum if the assay must be delayed. The NK cytotoxicity under these storage conditions most closely matches the results obtained when the PBMC are isolated and tested on the same day. IF PBMC isolation must also be postponed, it is best to store the blood in heparinized tubes at 25 degrees C to prevent loss of cytotoxic function.