Influence of blood sample storage and different types of anticoagulants on results of NK cytotoxicity tests

Introduction:
Natural killer (NK) cells are important players in the human immune response. Impaired NK function may lead to serious, life-threatening conditions. Defects may be consequences of genetic mutations or results of secondary factors such as infections, malignancies and autoimmune diseases. The cytotoxicity test is very useful, but its accessibility is limited to special immunological laboratories. Blood samples are often transported to remote centers, which takes time and requires special conditions. The aim of this study was to compare cytotoxicity assay results between samples preserved with three different anticoagulants to standardize the diagnostic procedure.

Material and methods:
Peripheral blood from healthy donors was taken with three anticoagulants: heparin, K2EDTA and citrate. Peripheral blood mononuclear cells (PBMC) were isolated and tested directly after blood drawing and after 24-hour storage. Cytotoxic abilities of NK cells were tested in 4 h co-culture with K562. NK cytotoxicity was measured by flow cytometry.

Results:
In most cases of analyzed healthy donors, cytotoxicity results were similar regardless of type of anticoagulant. However, the highest mean values were obtained in samples with citrate. There was a significant decrease in cytotoxicity after 24 hours of storage of the whole blood at ambient temperature. The mean drop in cytotoxicity results was substantial for all anticoagulants: 76% for heparin, 67% for citrate and 70% for EDTA.

Conclusions:
Results of spontaneous NK cytotoxicity seem to be affected by the anticoagulants used for blood protection. Commercial instant cytotoxicity testing and delayed analysis after blood storage gave the highest results in blood with sodium citrate.