Table 5.

Checklist for complete next‐generation tissue microarray – digital image analysis (ngTMA‐DIA) workflow

Aim	Suggested analysis control	Example within this study
Control of construction	Provide data highlighting the accuracy of punched regions of interest	Tumour cell numbers per core in tumour centre versus invasive front
Control of mosaic like heterogeneity	Provide data showing the distribution of dispersed positive to negative cells	Histograms with three‐tiered intensity levels for each staining were included
Control of targeted heterogeneity	Provide data between the different targeted regions of interest	The targeted differences between tumour centre versus invasive front were analysed
Control of haphazard heterogeneity	Provide data as inter‐core‐variability	ICC values for inter‐core‐variability have been generated for each staining. Additionally, standard deviations were plotted along the cohort and a case by case search of deviating cores was performed
Control of intensity and percentages interplay	Combination of exact percentages of each intensity category (0–3) considering the expected biological function	Three categories of protein loss were generated and its impact on possible patient selection visualised in a graph
Enhanced statistics’ reliability	Increased number of punches enhances the accuracy of ngTMA biomarker analysis	As different levels of heterogeneity could be excluded, the mean of all six cores per tumour was used for further statistics
Admit limitations	Put down notes on data acquisition and data processing using DIA and corresponding software	Notes on manual tumour area recognition, differences in cell number attribution between both stainings, as well as slight threshold adaptations due to staining and intensity variations from slide to slide have been implemented