**2. Materials and Methods**

### *2.1. Patients*

The patients included within this study were picked randomly and independently of their medical history as part of the preoperative assessment ahead of surgery. All patients had a modified Allen's test and a TIVITA™ (Diaspective Vision, Pepelow, Germany) scan as part of the assessment. The study was approved by the local ethic committee of Rhineland-Palatinate (registration number: 2020-15022\_1) and was conducted in accordance with the protocol and in compliance with moral, ethical and scientific principles governing clinical research as set out in the Declaration of Helsinki of 1975 as revised in 1983.

#### *2.2. Methods*

All tests were performed by the same surgeon to avoid interobserver variability.

#### 2.2.1. Allen's Test

Modified Allen's test (MAT) was carried out as previously described by Habib et al. and Abdullakutty et al. (the patient makes a fist for 30 s, while pressure is applied on the ulnar and radial artery to occlude them. The patient opens the fist, and the ulnar artery is selectively released) [7,22]. An 8 s cut-off point was used to discriminate between positive test and results without pathological findings [7,23,24]. A longer period to arterial refill indicated a vascular anomaly potentially resulting in ischemia after radial artery harvest.

A time period of 9 s and above until reperfusion was evaluated as positive and therefore pathological; tests with reperfusion times of 0–8 s were considered negative and therefore non-pathological.

### 2.2.2. HSI Imaging

The TIVITA™ Tissue System (Diaspective Vision, Pepelow, Germany) is an HSI system consisting of a hyperspectral camera, a lens, an illumination unit, a medical cart, a box-computer and the integrated TIVITA™ Suite basic software. The light source is arranged around the camera lens and consists of six halogen spots (20 W each). The standard measurement distance is 50 cm, represented by two indicator light points in an overlapped position. The patient was asked to place the hand flat on a table with the palm up. The central point for all measurements was the middle of the palm. After HSI images are recorded over 10 s, additional 8 s are needed to compute a RGD (red, green and blue) true colour image and additional four pseudo-colour images, representing the physiologic parameters. The camera-specific software package (TIVITA™ Suite) was used to quantify the generated information [20]. Three circular shaped regions of interest (ROI) that contain the mean value of the spectral and spatial information per pixel were manually positioned. The ROI were: in the middle of the palm (40 pixel), the proximal phalanx of the thump (15 pixel) and the proximal phalanx of the index finger (15 pixel). The software automatically calculated

the average values for each perfusion parameter (StO2, THI, NPI and TWI). Afterwards, a mean of the three measurements was calculated (Figure 2). thump (15 pixel) and the proximal phalanx of the index finger (15 pixel). The software automatically calculated the average values for each perfusion parameter (StO2, THI, NPI and TWI). Afterwards, a mean of the three measurements was calculated (Figure 2).

**Figure 2.** (**A**) Course of the experiment. First, a baseline measurement was taken (time point I, both arteries open), then both, the radial and the ulnar artery were occluded (time point II) and at time point III, the ulnar artery was released. Hyperspectral images were taken at all three time points throughout the course of the experiment as part of the hyperspectral accompanied MAT. In (**B**) the baseline measurement is displayed showing the NIR perfusion index, oxygenation and tissue hemoglobin index. Three circular shaped regions of interest (ROI) were manually positioned. (**C**) After occlusion of the ulnar and radial artery, the hand obviously turns pale. The values for NIR perfusion index decrease from 57 to 49 in the middle of the palm indicated by a less red and more green color of the hand. The same applies for the tissue oxygenation showing many red spots before occlusion and a green and blue color after occlusion of the vessels (StO2 decrease from 63% to 37%). Changes in THI are most obvious indicated by a color change from green to dark blue (THI decrease from 38 to 5). (**D**) shows the measurements after release of the ulnar artery in a healthy participant. The return to perfusion is indicated by the red and green colors in all pictures. After reperfusion, there is a slight hyperemia of the hand, which is evidenced by the increase in the measured values (NIR1: **Figure 2.** (**A**) Course of the experiment. First, a baseline measurement was taken (time point I, both arteries open), then both, the radial and the ulnar artery were occluded (time point II) and at time point III, the ulnar artery was released. Hyperspectral images were taken at all three time points throughout the course of the experiment as part of the hyperspectral accompanied MAT. In (**B**) the baseline measurement is displayed showing the NIR perfusion index, oxygenation and tissue hemoglobin index. Three circular shaped regions of interest (ROI) were manually positioned. (**C**) After occlusion of the ulnar and radial artery, the hand obviously turns pale. The values for NIR perfusion index decrease from 57 to 49 in the middle of the palm indicated by a less red and more green color of the hand. The same applies for the tissue oxygenation showing many red spots before occlusion and a green and blue color after occlusion of the vessels (StO<sup>2</sup> decrease from 63% to 37%). Changes in THI are most obvious indicated by a color change from green to dark blue (THI decrease from 38 to 5). (**D**) shows the measurements after release of the ulnar artery in a healthy participant. The return to perfusion is indicated by the red and green colors in all pictures. After reperfusion, there is a slight hyperemia of the hand, which is evidenced by the increase in the measured values (NIR1: 57 and NIR3:61; StO21: 63% and StO23: 68%; THI1: 38 and THI3:50).

57 and NIR3:61; StO21:63% and StO23: 68%; THI1: 38 and THI3:50). The measurement was performed in a dark room with constant temperature. (I) First, a basis recording was conducted in order to show the individual hands' perfusion (perfusion). (II) Then, MAT was carried out by occluding both the radial and the ulnar artery (occlusion). This was followed by a HSI measurement. (III) To verify the hand perfusion by the ulnar artery only, the radial artery stayed occluded while releasing the ulnar artery (reperfusion). Here, HSI-image acquisition for reperfusion started 1 s after release of the ulnar artery. For each time point the mean values of the following parameters were recorded: (StO2 (0–100%)), tissue hemoglobin index (THI (0–100)), near infrared perfusion The measurement was performed in a dark room with constant temperature. (I) First, a basis recording was conducted in order to show the individual hands' perfusion (perfusion). (II) Then, MAT was carried out by occluding both the radial and the ulnar artery (occlusion). This was followed by a HSI measurement. (III) To verify the hand perfusion by the ulnar artery only, the radial artery stayed occluded while releasing the ulnar artery (reperfusion). Here, HSI-image acquisition for reperfusion started 1 s after release of the ulnar artery. For each time point the mean values of the following parameters were recorded: (StO<sup>2</sup> (0–100%)), tissue hemoglobin index (THI (0–100)), near infrared perfusion index/deep perfusion (NIR (0–100)) and tissue water index (TWI (0–100)).

#### index/deep perfusion (NIR (0–100)) and tissue water index (TWI (0–100)). *2.3. Statistics*

number of analyzed hands was on average 104. Case number calculation (according to [25]):

*2.3. Statistics*  In their review concerning the reliability and validity of the modified Allen's test, Romeu-Bordas and Ballesteros-Pena listed a total of 9 studies that were included in the In their review concerning the reliability and validity of the modified Allen's test, Romeu-Bordas and Ballesteros-Pena listed a total of 9 studies that were included in the review. While the number of analyzed patients ranged from 42 to 150 (mean 81.88), the number of analyzed hands was on average 104.

review. While the number of analyzed patients ranged from 42 to 150 (mean 81.88), the

Case number calculation (according to [25]):

$$m = K \ast \frac{\left[\left(\mathbb{R} + 1\right) - p\_2 \ast \left(\mathbb{R}^2 + 1\right)\right]}{\left[p\_2 + \left(1 - \mathbb{R}\right)^2\right]}\tag{1}$$

The calculation is based on the study by Grambow et al. [26] and the measured differences in tissue oxygen content, which were found to be significantly different.

$$n = 7.85 \ast \frac{\left[\left(0.757 + 1\right) - 0.7 \ast \left(0.757^2 + 1\right)\right]}{\left[0.7 + \left(1 - 0.757\right)^2\right]} = 124.578\tag{2}$$

Considering the previous studies and the sample size calculation, the numbers of cases were averaged, resulting in a total necessary volume of 114 patients.

In order to test the assumption that the measured values follow a normal distribution, a Kolmogorov–Smirnov and a Shapiro–Wilk test were previously performed. Correlation analyses were performed using Pearson and Spearman test. Furthermore, Wilcoxon tests were used in order to assess whether the population mean ranks differ between related samples (non-parametric statistical hypothesis test). To measure the strength of the relationship between two variables in the statistical population, the effect size was calculated according to the following formula:

$$r = \left| \frac{Z}{\sqrt{n}} \right| \tag{3}$$

The following definitions were used:

0.1 ≤ *r* < 0.3 ∼ weak effect 0.3 ≤ *r* < 0.5 ∼ medium effect *r* ≥ 0.5 ∼ strong effect

Values are displayed as mean and standard deviation; where appropriate confidency intervals are given. Moreover, in addition to the measured values, a fictive value was calculated to reflect the return-to-normal perfusion. This was done as follwos:

$$RTP = 100 - \left(\frac{TP3}{TP1} \ast 100\right) \tag{4}$$

with *RTP* and *TP* denoting return to perfusion time point, respectively. Statistical analyses were performed using SPSS version 24 for Windows (IBM, Armonk, NY, USA); A *p*-value ≤ 0.05 was termed significant.

#### **3. Results**

A total of 114 patients were included within this study, all of whom had a modified Allen's test and an HSI-scan as part of the assessment. The results were categorized into two groups according to the results of the MAT. Non-pathological results were those with a time to reperfusion of less than 9 s; if it took 9 or more seconds, the MAT was considered pathological.
