## 2 Materials and Methods
### Methods for Analysing Accuracy
#### 2.2.3 Quantitative Accuracy  
To analyse the quantitative accuracy of the next-generation Tissue Microarray (ngTMA)\({}^{\text{\tiny{\textregistered}}}\) method, two different approaches were used:  
1. The distance between a distinct structure and the annotation;  
Figure 6: Comparison of the endothelial length. (**A**) Correlation between the sectioning depth (S1, S2, S3) and the accuracy of the ngTMA\({}^{\text{\tiny{\textregistered}}}\) method. An exemplary target structure in a paraffin block may deviate due to sectioning depth. (**B**) The figure shows the H&E section of the donor block (DB) on the left-hand side, followed to the right side by three slides of descending sectional depth (S1, S2, S3) of the respective TMA block. The endothelial surface is marked in blue. Listed underneath each picture is: 1. the sectional plane (S1, S2, S3) and the corresponding depth in \(\upmu\)m, 2. the measured length in \(\upmu\)m and 3. the deviation percentage comparing the length of the TMA slide with the annotated H&E slide. (**C**) Distribution of the endothelial length depending on the sectional plane DB: Donor-Block (\(n=90\)), sectional plane: S1: \(n=43\); S2: \(n=89\); S3: \(n=48\). The Rhombus-Symbol (\(\bullet\)) marks the arithmetic mean. There is a strong significance (\(p<0.001\)), indicated with \(\ast\), when comparing the DB with the sectional plane S1, but no statistically significance when comparing DB to sectional planes S2 and S3. 3D sketches with kind permission of [PERSON].  
2. The length of the endothelium inside the aorta. The results were also compared with the sectioning depth. 1. Distance between structure and annotation: The distance was measured between an easily recognisable structure and the inner circumference of the annotation in 446 cases (S1: \(n\) = 117, S2 \(n\) = 197, S3 \(n\) = 132). This distance was then compared on the corresponding TMA H&E slide (Figure 7). The tissues aorta, A&V and tongue with their precisely defined punching targets were used for this purpose.  
Figure 7: Quantitative Accuracy. (**A**) Example for the determination of the quantitative accuracy. The distance between a target structure on the H&E slide of a donor block and the inner margin of the set annotation was measured and compared to the length with the respective measures in the TMA-Slide. Shown here is an example of outstanding accuracy, with a difference of less than 100 \(\upmu\)m. (**B**) The graph shows the deviation between the digitally set annotation and the TMA Slide. The number of punch cores analysed was plotted against the deviation of these cores in the different sectional planes (S1, S2, S3). The coding of the deviation on the horizontal axis is as follows: 1 0–99 \(\upmu\)m deviation; 2 100–199 \(\upmu\)m deviation; etc. (**C**) The table shows the distribution of the accuracy in the different sectional planes (S1, S2, S3).  
2. Length of the endothelium in the aorta:  
For a different approach, in 90 cases, a comparison was made of the endothelial length of the aorta in the original H&E and the TMA H&E slides in different sectional depths (S1, S2, S3) was carried out (Figure 6).