191 Pre-analytic variables and multiplex immunofluorescence assays

Pre-analytic variables in formalin-fixed paraffin embedded (FFPE) specimens are a large barrier to assay reliability, and the characterization and potential correction of such variables represent a large unmet need in dermatopathology. Multiplex immunofluorescence (mIF) is an emerging assay modality, which is tissue-sparing and also allows for the assessment of marker co-expression and intensity. The purpose of this study was two-fold: 1) characterize FFPE block age as a pre-analytic variable, and 2) determine whether a novel pan-membrane (PM) marker could be used for normalization. We used tissue microarrays (TMAs) constructed from archival FFPE specimens from 125 patients with melanoma collected over a 20-year period. The TMAs were stained with a mIF panel (FoxP3, CD8, PDL1, PD1, Tumor, CD163), a novel PM cocktail, and DAPI for nuclei, imaged using Vectra Polaris, and analyzed in HALO with the Multi-IF module to evaluate the impact of pre-analytic variables on cell density and marker intensity. Cell densities did not vary significantly with block age for 5/6 of the markers, but FoxP3+ cell density decreased with block age (-1.5%/yr, p<0.001). Marker intensities showed several small shifts with block age: CD163 (+0.5%/yr), tumor (+1.3%/yr), and PM (+0.82%/yr) increased (p<0.001), while PD-L1 decreased (-0.26%/yr, p<0.01). Overall, these data suggest that for most markers, there are small changes with respect to block age; however, for more rare cell types such as FoxP3, changes may become noticeable with block age. We next considered pre-analytic variables beyond block age and evaluated whether a novel PM stain might serve as a proxy for block immunoreactivity. Linear regressions were performed to evaluate PM stain intensity vs marker intensity. All markers except PD-L1 showed positive regression coefficients (p<0.001). This suggests our novel PM stain may be a useful surrogate of block immunoreactivity and allow for normalization of marker intensity between individual specimens, correcting for pre-analytic variation.