Naranbhai, Vivek.Bartman, Pat.Ndlovu, Dudu.Ramkalawon, Pamela.Ndung'u, Peter Thumbi.Wilson, Douglas Paul Kinghurst.Altfeld, Marcus.Carr, William Henry.2013-07-052013-07-0520102010Published in final edited form as: Naranbhai, V., et al. 2011. Impact of blood processing variations on natural killer cell frequency, activation, chemokine receptor expression and function. J Immunol Methods. 366 (1-2) pp. 28–35.0022-1759http://dx.doi.org/10.1016/j.jim.2011.01.001.http://hdl.handle.net/10413/9261Understanding the role of natural killer (NK) cells in human disease pathogenesis is crucial and necessitates study of patient samples directly ex vivo. Manipulation of whole blood by density gradient centrifugation or delays in sample processing due to shipping, however, may lead to artifactual changes in immune response measures. Here, we assessed the impact of density gradient centrifugation and delayed processing of both whole blood and peripheral blood mononuclear cells (PBMC) at multiple timepoints (2–24 h) on flow cytometric measures of NK cell frequency, activation status, chemokine receptor expression, and effector functions. We found that density gradient centrifugation activated the NK cells and modified the chemokine receptor expression. Delays in processing beyond 8 h activated NK cells in PBMC but not in whole blood. Likewise, processing delays decreased chemokine receptor (CCR4 and CCR7) expression in both PBMC and whole blood. Finally, delays in processing PBMC were associated with a decreased ability of NK cells to degranulate (as measured by CD107a expression) or secrete cytokines (IFN-γ and TNF-α). In summary, our findings suggest that density gradient centrifugation and delayed processing of PBMC can alter measures of clinically relevant NK cell characteristics including effector functions; and therefore should be taken into account in designing clinical research studies.enKiller cells, Natural--Immunology.Receptors, Chemokine.Flow cytometry.Natural killer cells.Peripheral blood mononuclear cells (PBMC)Peer reviewed journal article