## 2 Materials and Methods
### Methods for Analysing Accuracy
#### 2.2.2 Qualitative Accuracy  
The qualitative accuracy was defined as the concordance between the digitally set annotation and the corresponding core on the H&E slide of the respective TMA block. For this purpose, the actual punched TMA core was compared with the original annotation (Figure 4). Qualitative accuracy in relation to different cutting depths of the TMA-block was analysed by selecting three slides to represent the three different depths of sections of the TMA block using a maximum cutting depth of roughly 800 \(\upmu\)m (Figure 5).  
Furthermore, three groups were defined to characterise the results as target was completely hit (2), partially hit (1) or missed (0), and groups were coded accordingly.  
When studying the accuracy of the digitalised ngTMA(r) method, it is crucial to consider that the TMA block is a three-dimensional structure. The depth of the retrieved section determines the location and shape of the targeted structure (Figure 6A). To analyse the accuracy of cutting depth, the block was cut to a mean depth of 441 \(\upmu\)m from S1 so that approximately two-thirds of the block remained for further research. Three slides were selected to represent three different sectional planes.  
The following definitions were made: S1 was the 'first acceptable' section. The first cut which contained specimen and most core punches were present.  
S2 was the 'best' section. All or mostly all core punches were present but also the smallest number of defects was observed, such as overlaying, rolling up and deformation due to squashing and stretching in the water bath.  
Figure 3: Correlation of core Loss and tissue type. (**A**) H&E slide of a donor block on the left side before and on the right side after the punching procedure. Punched areas (*) and one core loss (arrow) on a renal specimen. (**B**) Core loss is shown in correlation to the different tissue types in percent. CL_pre shows the core loss before and CL_post the core loss after manual replacement of dispatched tissue cores. There is a strong significance (\(p<0.001\), Chi-Square), indicated with *, when comparing skin tissue to the other tissues and a significant difference (\(p<0.01\)), indicated with *, when comparing the Renal medulla and A&V-tissue with the renal cortex, myocard and spleen tissues. (**C**) The table contains the total number and the percentage of core loss.  
Figure 4: Example of the qualitative analysis. The left picture shows the digitalised H&E slide of the donor block with its annotation marking the carotid artery and the vagus nerve. The right image shows the corresponding core on the TMA H&E slide. (**A**) is an example of a structure ‘completely hit’, which leads to code 2. Note the different shapes of the artery and the nerve between the left and the right image, resulting from the 3-dimensional passage of the structures through the surrounding tissues. (**B**) is an example of a structure ‘partially hit’. The nerve was hit, but the aorta is only partially captured. This leads to code 1.  
Figure 5: Qualitative Accuracy regarding the Cutting plane. Colours for the S1 plane are deliberately more faint because it did not always contain all punched cores already, since the top of each core is not necessarily precisely in the same sectional plane. This circumstance limits the comparability of the S1 data. The number 2 indicates that the target was completely hit, number 1 indicates a partially hit, 0 indicates that the target was missed.