Evaluation of natural killer cell assay performance on shipped blood specimens

Documenting the importance of NK cell function as a biomarker for diseases and physiologic conditions including myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), will require assays amenable to clinical implementation and standardization. Research studies typically perform NK functional assays on the day of sample collection. This pilot study was conducted to compare assay formats and specimen processing to identify those that are most tolerant of conditions required for shipping and amenable to standardization as shown by inter-assay and inter-laboratory correlation of results. We compared performance within and between assays that measure NK cell function using direct cytotoxicity [chromium-51 release (CRCA) or fluorescence (Flow Cytometry Cytotoxicity Assay, FCCA)] or an indirect surrogate marker (CD107a surface expression)]. Additional variables for within/between assay comparisons included time of testing (same day as specimen collection or next day within 24 h), specimen types [whole blood or isolated peripheral blood mononuclear cells (PBMCs)], and processing method (fresh or cryopreserved). Statistical measures included number of samples tested in assay conditions (n), medians (x͂), interquartile range (IQR), Pearson correlation coefficient (R²), and correlation p-value (p). Samples came from 3 clinics and included 31 participants. Same day testing was only available for the subset of participants enrolled from the site of the laboratory performing CRCA. Results from same day CRCA testing of whole blood were considered the gold standard [n = 10, x͂=10.0%, IQR = 7.2%], and correlated well with PBMCs isolated next day [n = 26, x͂= 15.6%, IQR = 13.1%] [R² = 0.59, p = 0.03]. Next day CRCA results were compromised using whole blood or frozen PBMCs. Next day FCCA cytotoxicity in PBMC [n = 30, x͂=34.1%, IQR = 15.5%] correlated with same day CRCA PMBC [R² = 0.8, p = 0.001] and next day CRCA PMBC [R² = 0.5, p < 0.0001]. CD107a expression after induction by PMA and ionomycin did not correlate with other cytotoxicity measures. NK function can be measured in PBMCs isolated after overnight shipping/storage at ambient temperature and CRCA and FCCA results on this sample type are well correlated.