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48 | Chapter 2 and 6) were significantly reduced (***p-value=0.0010) when cryopreserved samples were exposed to A431 alone. When comparing CD16 expression levels in PBNK exposed to A431 and CET, no significant differences were noted between fresh and cryopreserved samples. Further, no significant changes in NK cell expression of the activation marker NKp44 could be detected between fresh and cryopreserved nor between non-activated and cytokine activated NK cells27(Figure 6C). Upon exposure to A431 cells, freshly isolated NK cells that were stimulatedwith cytokines expressed significantly higher (*p-value=0.04) levels of CD25 when compared to NK cells from cryopreserved samples (Figure 6D and Supplementary Fig S6D and S7D online and Table 5 and 6). Figure 5: Comparison of NK cell phenotypes between fresh and cryopreserved NK cells NK cell phenotypes between freshly isolated and cryopreserved thawed PBMC samples were compared. NK cells were either activated or non-activated with cytokines (IL-2+IL-15) and target cells (A431) alone or targets coated with cetuximab (CET). Expression levels of NKG2A (A), NKG2C (B), NKG2D (C), KIR2D (D) were compared for the following conditions: i) NK only ii) NK + A431 and iii) NK + A431 + CET conditions. NK only conditions are depicted as open rectangles, followed by NK + A431 with grey shades and NK + A431 + CET conditions represented as black rectangles. Columns represent data from12 donors, from each donor the mean of triplicate values was used; with bars showing SEM. Data are from independent experiments performed in triplicates from 12 PBMC donors (6 donors: BD LSRFortessa + 6 donors: MACSQuant). Statistical analysis was performed using the Wilcoxon test.