**3. Results**

## *3.1. IDIF Predicts AIF*

In the first cohort of five subjects, arterial blood sampling was performed to measure the AIF. We then compared the distribution volume calculated with the AIF (VT,AIF) and IDIF (VT,IDIF) to determine whether IDIF is an appropriate surrogate for measurement of 18F-DPA-714 quantification. The extraction process of the IDIF in one subject of the blood-sampling cohort is demonstrated in Figure 1 for the three steps: segmentation, signal decomposition, and scale optimization. Figure 1a shows the summed PET image over the first 60 s post tracer injection as a maximum intensity projection. Figure 1b,c show one axial slice within the neck area and the segmented contour for the carotid arteries. The extracted tissue and blood components are shown in Figure 1d, and the IDIF after the scale adjustment is plotted in Figure 1e with the measured arterial input function (AIF). The IDIF curve demonstrated a satisfactory agreemen<sup>t</sup> with the AIF curve. *<sup>ω</sup>i*,*AIF sAIF* averaged 0.28 ± 0.30, and *<sup>ω</sup>i*,*<sup>T</sup> ISSUE sT ISSUE* averaged 0.55 ± 0.36 across the five subjects. The comparison of IDIF and AIF for the other four subjects in the blood-sampling cohort is shown in Figure S1.

**Figure 1.** *Cont*.

**Figure 1.** Demonstration of the IDIF extraction. (**a**) The coronal maximum intensity projection (MIP) of the summed early frames of the DPA-714 scan of one subject. (**b**) One axial slice of the neck area at the early frame. (**c**) The segmented regions (green contour) for carotid artery over the same slice. (**d**) The MBMF-extracted time-activity curves for the blood and tissue components. Note both curves are in the normalized and dimensionless form. (**e**) The resultant IDIF (blue circle) after the magnitude is re-scaled by the proposed method. The activities measured with arterial blood sampling are plotted as red stars, showing a satisfactory agreemen<sup>t</sup> between AIF and IDIF.

Table 1 summarizes the VT derived from the AIF and IDIF for the nine target regions in the blood-sampling cohort. The overall error for VT,IDIF was −5.8 ± 7.8% against the reference VT,AIF. In all regions, the mean error of VT was less than 10% across all subjects. The amount of error for VT in the healthy controls is similar in the PD patients. Figure 2 shows the scatter plot and the Bland–Altman plot for VT,IDIF and VT,AIF of all the target regions and demonstrates a satisfactory agreemen<sup>t</sup> between them. In the linear mixed effect model analysis, the overall VT,IDIF and VT,AIF were highly correlated with each other (*p* < 0.001), adjusting for the effect of regions.

**Figure 2.** (**a**) The scatter plot for VT,IDIF plotted against VT,AIF. The dashed line is the unity line. The red line is the fitted regression trend line. The scatter plot shows a strong correlation between VT,IDIF plotted against VT,AIF. The statistical test also showed a strong correlation through the mixed effect model analysis. (**b**) The Bland–Altman plot for VT,IDIF and VT,AIF.


**Table 1.** Comparison between the VT,AIF and VT,IDIF in the blood-sampling cohort. Overall error is −5.8 ± 7.8% for VT,IDIF when compared to VT,AIF. The error does not appear to be dependent on the target region.

#### *3.2. IDIF Method Distinguishes High-Affinity Binders from Mixed-Affinity Binders*

To validate our IDIF quantification methodology, we next tested whether IDIF could distinguish control subjects who were MAB from those who were HAB as determined by TSPO SNP genotyping. In the SUV measurements, all nine brain regions showed significantly higher uptake of 18F-DPA-714 in the HAB group than in the MAB group (*p* < 0.05). The HAB uptake averaged 31 ± 3% higher than the MAB in SUV (Figure 3). The distribution volume as determined by IDIF also showed significantly higher uptake in HAB vs. MAB overall (*p* < 0.05). Region-wise, mean VT,IDIF was statistically higher in the HAB group in all nine brain regions (Figure 4). VT,IDIF was 37 ± 3% higher in the HAB group overall. On the other hand, the distribution volume determined by using the cerebellum as a reference region showed only a mildly increased VT,REF of 3 ± 3% in the HAB group compared to the MAB group (Figure 5). Only one out of the nine brain regions revealed a statistically significant increase in VT,REF in HAB vs. MAB subjects with the reference region approach. The mixed effect model showed a significant difference in the SUV (*p* = 0.010) and VT,IDIF (*p* = 0.010) but not in the VT,REF (*p* = 0.069) after adjusting for the effects of regions.

**Figure 3.** The box plot of the SUV measured for target brain regions for high- and mixed-affinity binders in the nonblood-sampling cohort. \* denotes significant differences under two-sample *t*-test (*p* < 0.05), while \*\* denotes *p* < 0.01. All nine target regions showed significant differences between the HAB and MAB subjects. SUV is 31 ± 3% higher in HAB than in MAB.

**Figure 4.** The box plot of the VT,IDIF. VT,IDIF is 37 ± 3% higher in HAB than in MAB. All nine regions were found with significant difference (\*: *p* < 0.05, \*\*: *p* < 0.01) between the HAB and MAB subjects. The overall increase pattern in the HAB is similar as the SUV pattern of increase.

**Figure 5.** The box plot of the VT,REF using the cerebellum as the reference region. The difference between HAB and MAB is 3 ± 3% and only reaches significance (\*: *p* < 0.05) in just one of nine regions.
