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52 | Chapter 2 cell encounter, CD16 surface levels are reduced and CD107a appears on the cell surface as a consequence of the release of the content of cytotoxic granules. Binding of CD16 to IgG 1 coated targets enhances NK cell activation and subsequent cytotoxic responses which is reflected by a more pronounced reduction in CD16 surface levels and increased levels of CD107a on the cell surface; both changes have been well reported in the literature 33 and also specifically in the presence of e.g. the EGFR-binding monoclonal antibody cetuximab 34 . The role of NK cells in eliminating metastasizing cancer cells is well acknowledged in the field of cancer immunotherapy, resulting in an ever-increasing number of clinical trials exploring their therapeutic potential 35 . Over the past decade, the development of several therapeutic IgG 1 monoclonal antibodies (mAbs) targeting different types of cancer 36 , and the recent demonstration of their clinical efficacy, has enhanced the interest in better understanding their mode of action 37 . Information on NK CD16 levels is critical for clinical trials monitoring efficacy of therapeutic ADCC mAbs. Baseline and post mAb treated PBMC samples can be compared for NK CD16 levels, and this could indicate if NK cells were able to recognize and bind therapeutic mAbs, as an indirect measure of ADCC. In addition, activation status of NK cells can be monitored measuring CD25 and NKp44 levels as included in our NK function FACS panel. The analysis of CD25 and NKp44 expression is useful for instance, to check whether NK cells are pre-activated or sensitized in peripheral blood by exposure to targets or soluble NK activating ligands 38-40 . Furthermore, NKp44 levels can also be useful for follow up in patients who had adoptive transfer of pre-activated NK cells 41 . The NK cell phenotype panel offers information onmajor NK cell activating and inhibitory receptors NKG2A, NKG2C, NKG2D and KIR2D alleles, which primarily regulate NK cell activity. In different autoimmune diseases as well as in several cancer types, down regulation of NK cell receptors such as CD16, NKp46, NKp30, NKp44 or KLRB1 and a reduction in NK cell number and functional activity are quite common occurences 42-44 . Though these alterations are not necessarily restricted to NK cells, they underscore the relevance of monitoring immune cell functions in these patients. Our NK cell phenotyping panel will prove useful in monitoring NK cells receptor changes that may affect their ability to lyse tumours. The most interesting and well-known aspect of NK cells is their ability to lyse target cells via the release of cytotoxic granules, or through Fas/FasL and TRAIL receptor interactions 45,46 . The flexible design of our panels provides a unique opportunity to measure different NK killing mechanisms, e.g. IFNγ, granzyme B and perforin, by modifying drop-in channel combinations. Similarly, natural cytotoxicity receptors (NKp46, NKp44, NKp30), DNAM-1 and other inhibitory/activating receptors can be analysed, suiting the user’s needs. Wecompared freshandcryopreservedPBMC fromhealthyvolunteers inBDLSRFortessa TM and MACSQuant ® Analyzer X. Though a small decrease in NK cell percentages was observed within living CD45+ populations post cryopreservation, it was minor allowing the acquisition of sufficient events to measure NK cell immune functions. Overall cryopreservation did