Comparative assessment of near-infrared
autofluorescence imaging in porcine and human
adrenalectomy specimens

Barbara Seeliger, Rupak Bose, Didier Mutter, Michel Vix

► To cite this version:

Barbara Seeliger, Rupak Bose, Didier Mutter, Michel Vix. Comparative assessment of near-infrared autofluorescence imaging in porcine and human adrenalectomy specimens. Surgery, 2025, 186, pp.109585. 10.1016/j.surg.2025.109585. hal-05212635

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Distributed under a Creative Commons Attribution 4.0 International License
adrenal surgery, near-infrared autofluorescence (NIRAF) imaging could also aid in different procedural steps.⁷ When illuminated by a filtered light source at a specific excitation wavelength, the target tissue absorbs light and emits light at a longer wavelength that is detected by a specifically designed camera. With dedicated imaging systems that use a near-infrared (NIR) light source in conjunction with a filtered camera, the autofluorescence invisible to the human eye can be displayed on a monitor.⁸ Although the molecule at the origin of adrenal autofluorescence remains unknown, its spectral profile was identified as similar to parathyroid tissues, with an emission peak in the NIR spectral range at 820 nm and a higher intensity.⁹ It is also unknown whether the NIRAF in adrenal tissue is as robust as in parathyroid tissue, where it was reported to persist ex vivo for up to 2 years.¹⁰ Whereas there are several commercially available minimally invasive and robotic surgery imaging devices for contrast-enhanced NIR fluorescence detection with an exogenous fluorophore (indocyanine green [ICG]), for contrast-free endogenous NIRAF there is only an open surgical image-based option.¹⁰

The aim of this study was to assess the NIRAF properties of healthy and neoplastic adrenal tissues in porcine and human specimens to determine their potential utility for adrenal surgery, the identification of the responsible endogenous fluorophore in adrenal tissue, and the development of targeted minimally invasive imaging systems.

## Materials and methods

### *Study design*

### *Specimens for comparative analysis*

All animals in the experimental laboratory at IHU Strasbourg were managed according to French laws for animal use and care and according to European Community Council directives (2010/63/EU) and ARRIVE Guidelines.¹¹ The animals on which the open adrenalectomies were performed on euthanasia were used in experiments approved by the French Ministry of Research (project authorization APAFIS 38189), after having been evaluated by the Ethics committee No. 35 in accordance with the National Charter on Ethics of Animal Experimentation. The AdrenalPath study was approved by the institutional board in March, 2023. Because there were no previous findings on porcine adrenal autofluorescence, a sample size of 10 adrenal glands was chosen in this exploratory study in a no-intervention/no-effect group requiring no power analysis. Left and right adrenalectomies were carried out on 5 pigs (reuse of consecutive animals in a given protocol, all female, mean weight 55 ± 0.7 kg) to be analyzed in this experimental study, and specimens preserved in formaldehyde for follow-up imaging (Q Path Formol 4% buffered prefilled containers; VWR International, France). At the University Hospitals of Strasbourg/IHU Strasbourg, robotic transabdominal left and right adrenalectomies are the standard of care. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The password-protected NIRAF imaging device (Fluobeam-LX; Fluoptics SAS, Grenoble, France) was retrospectively searched for autofluorescence imaging of resected specimens within routine clinical care, and adrenal specimen images acquired between June 2023 and September 2024 were anonymously exported and analyzed for the present study. Race and ethnicity data were not accessible in the patient records. According to the General Data Protection Regulation, the principles of data protection do not apply to personal data rendered anonymous so that the subject is not or no longer identifiable. Anonymization allows the reuse of data without infringing on people's privacy and storage of these data without limitation.

### *NIR endogenous fluorescence imaging*

Autofluorescence imaging relies on the intrinsic biomolecules that act as endogenous fluorophores, and no exogenous fluorophore (such as indocyanine green) was administered. The camera head of the open surgical system used in the present study (Fluobeam-LX; Fluoptics SAS) is intended for use as an adjunct assisting in the location of parathyroid glands (US Food and Drug Administration, K233564). At a working distance of approximately 20 cm, the camera system provides tissue illumination that can be switched from white light LEDs to a 750-nm laser (excitation wavelength) and collects fluorescence emissions of 800 nm and higher, which are displayed as video images in real time and stored as video and still images via the integrated software interface.¹²

Consequently, at a constant sensitivity setting to minimize variability between different acquisitions, the adrenal glands and tumors were examined after specimen extraction, on a back table covered with surgical drapes or gauzes. Although the imaging system is certified for use with and without ambient lights, the ambient light in the operating room had been switched off during autofluorescence imaging as per local routine. Standardized reference cards (Green Balance ICG Reference Card; Diagnostic Green GmbH, Munich, Germany) were used to enable comparability between the NIRAF image acquisitions in different operating room settings.

### *Quantitative image analysis*

For each autofluorescence image, manual annotations were performed (B.S.) where little if any overlying periadrenal tissue was present for normal and neoplastic adrenal tissue as well as in a background area. These annotations were in a layered format to enable precise spatial registration by use of a web-based photo and graphics editor that stores all files locally, and does not upload any data to a server (Photopea, https://www.photopea.com/). Ratios were calculated for the normal and neoplastic adrenal tissue NIRAF intensities relative to their background. For illustration of the quantitative NIRAF measurements, a perceptually uniform scientific color map (batlow) was chosen to prevent data distortion and visual error.¹³

The reference card (rc) image sections were converted to grayscale, and the mean intensities calculated to serve as a normalization factor between different specimens imaged on different days for comparison of specimens. A background correction was performed to obtain the absolute fluorescence intensity characteristic of the specimen and to the respective reference card. This normalization for potential image acquisition variations served to enable comparisons across different acquisitions. Statistical analyses were performed with Prism 10 for macOS (GraphPad Software, Boston, MA). For comparison between groups, tests used are parametric for data with a Gaussian distribution (Kolmogorov-Smirnov test, alpha = 0.05), and otherwise nonparametric.

To evaluate the discriminative power of key features in classifying adenomas versus non-adenomas, we employed a random forest model and assessed feature importance using the Gini impurity criterion.¹⁴ The Gini importance quantifies how frequently a feature is used to split the data, with higher values indicating greater contribution to classification accuracy. The analyzed features included the mean lesion-to-background ratio (l/b), mean lesion-to-reference card ratio (l/rc), and normalized lesion-to-reference card ratio ([l-b]/[rc-b]), with their relative importance ranked based on their ability to separate the 2 classes. Additionally, to determine an optimal diagnostic threshold, we performed 100 iterations of logistic regression on a randomized subset, calculating the mean cutoff ratio for adenoma prediction.
## Results

### Porcine specimens

All porcine adrenal glands (n = 10; 5 left and 5 right) consecutively resected for the present study were included and showed a visible autofluorescence. Figure 1 illustrates exemplary image acquisitions and separate annotations for the left and right adrenal glands of one pig at the initial time point and at 1-year follow-up.

In quantitative analysis, normal porcine adrenalectomy specimens' and background NIRAF data showed a normal distribution. The rc signal was 235 in all acquisitions (uniform absolute values).

First, ratios were calculated for the NIRAF intensities of normal adrenal parenchyma relative to the background. The mean adrenal-to-background ratio was 3.9 ± 1.5, and there was no significant difference between left- and right-sided samples (4.0 ± 1.6 vs 3.8 ± 1.5; P = .33), with a background signal of white gauzes of 24.6 ± 2.4.

Second, NIRAF intensities were calculated to compare the adrenal autofluorescence with the standardized ICG rc signal. The adrenal autofluorescence was less than half of the ICG rc NIRAF intensity, with a mean adrenal-to-rc ratio of 0.41 ± 0.17, and again

without significant difference between left- and right-sided adrenalectomy samples (0.42 ± 0.19 vs 0.40 ± 0.16; P = .58). When normalizing the ratio by background subtraction, the mean normalized adrenal-to-rc ratio was 0.34 ± 0.18.

After 12 months, the acquisitions were repeated, and the porcine adrenal glands still exhibited visible autofluorescence. The mean adrenal-to-background ratio was significantly lower than in the first acquisition (2.3 ± 0.42 vs 3.9 ± 1.5; P = .01), albeit in the context of a significantly higher background signal (61.3 ± 5.24 vs 24.6 ± 2.4; P < .0001). The mean adrenal-to-rc ratio did not show a decrease of autofluorescence intensity, as the ratio in follow-up was even higher than in the first acquisition, with the same sensitivity setting (P = .02) (Table 1).

### Human specimens

In visual assessment of the 21 adrenalectomy specimens by the surgical team, differentiated adrenocortical tissue in normal adrenal cortex (detectable in 19 of 21 specimens, 90.5%) and in all adenomas located at the surface (n = 6 of 10; 60%) exhibited autofluorescence (representative illustrations in Figure 2).

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Autofluorescence image acquisition and preparation for analysis. (A) Standard black-and-white image acquisition and (B) fluorescence intensity heatmap provided by switching modes in the Fluobeam-LX software. (C) White light photos (left), autofluorescence image (middle), and annotation of normal parenchyma in green, and background samples in blue (right), separate annotations of left (top) and right (below) adrenal gland of the same pig. (D) Corresponding image acquisitions at 12-month follow-up after specimen storage in formalin.</figcaption></figure>
<table><caption>**Table I**

Porcine adrenalectomy specimen analyses</caption><thead><tr><th></th><th>First acquisition</th><th>1-y follow-up</th><th>P value</th></tr></thead><tbody><tr><td>Mean adrenal-to-background ratio (a/b)</td><td>3.9 ± 1.5</td><td>2.3 ± 0.42</td><td>.01<br>(paired t test)</td></tr><tr><td>Mean adrenal-to-rc ratio (a/rc)</td><td>0.41 ± 0.17</td><td>0.59 ± 0.1</td><td>.02<br>(paired t test)</td></tr><tr><td>Mean normalized adrenal-to-rc ratio (a-b/rc-b)</td><td>0.34 ± 0.18</td><td>0.45 ± 0.14</td><td>ns</td></tr><tr><td>Background</td><td>24.6 ± 2.4</td><td>61.3 ± 5.24</td><td>&lt;.0001<br>(Welch t test)</td></tr></tbody></table>

Ratios for the normal adrenal parenchyma's NIRAF intensities (a) relative to the background (b) and ICG reference card (rc) are shown.

Additionally, a small adenoma that could not be distinguished from periadrenal adipose tissue on palpation and was not even visible on the cut surface in white light, however, showed subsurface contrast on NIRAF imaging (Figure 2, A). In contrast, all nonfluorescent tissues were non-adenomas, notably pheochromocytomas (n = 6; 28.6%) and adrenal medulla, metastatic lesions (n = 3; 14.3%), adrenocortical cancer (n = 1; 4.8%), hematoma (n = 1; 4.8%), and retroperitoneal adipose tissue (Table II).

NIRAF imaging after specimen removal had no impact on the operating time. NIR fluorescent light enables visualization for up to several millimeters beyond the surface. Fluorescent contrast was strongest when the fluorescent tissue was directly exposed, such as in a longitudinally incised specimen (Figure 2, A and D), or when the capsule was not covered by retroperitoneal adipose tissue. Similarly, a small margin of adrenocortical tissue exhibiting autofluorescence was observed adjacent to the nonfluorescent renal cell carcinoma metastasis to the adrenal gland but had been undistinguishable in white light illumination (Figure 2, H). As it was sometimes difficult to visually perceive the NIRAF intensity in the black-and-white view, or to assess the fluorescence intensity heatmap with the color code in the integrated software (“jet” as in Figure 3, A1–D1), basic image postprocessing served to represent the NIRAF intensity variations and gradients with a perceptually uniform scientific color map that is also compatible with color vision deficiencies (“batlow” as in Figure 3, A2–D2).¹³

In quantitative fluorescence assessment, human adrenalectomy specimens' NIRAF ratios showed a normal distribution for normal parenchyma, but not for the neoplastic regions of interest. For ease of comparability, data are represented uniformly as mean ± standard deviation. The rc signal was again 235 in all acquisitions.

First, tissue-to-background ratios were calculated for the NIRAF intensities of normal adrenal parenchyma. The mean adrenal-to-background ratio was 3.9 ± 1.8, with no significant difference between left- and right-sided samples (4.2 ± 1.7 vs 3.6 ± 2.0; P = .55, unpaired Mann-Whitney test), with a mean background signal of 32.0 ± 8.7. For the neoplastic regions of interest, the mean lesion-to-background ratio was 2.1 ± 1.4, similar between left- and right-sided neoplasia (left 2.0 ± 1.4, right 2.2 ± 1.5, P > .99).

Second, tissue-to-rc ratios were calculated, and the normal adrenal autofluorescence was about half of the ICG rc NIRAF intensity, with a mean adrenal-to-rc ratio of 0.51 ± 0.23, and a mean normalized adrenal-to-rc ratio of 0.44 ± 0.26. The mean lesion-to-rc ratio was 0.28 ± 0.20, and 0.18 ± 0.22 after background normalization.

In paired analysis of samples with both normal and neoplastic areas (n = 16), normal adrenal cortex showed significantly higher mean tissue-to-background ratios than neoplasia (3.9 ± 2.0 vs 2.2 ± 1.4, P = .02, Wilcoxon test). Correspondingly, the mean and the mean normalized tissue-to-rc ratios were also significantly higher for normal parenchyma than for neoplasia (0.50 ± 0.24 vs 0.29 ± 0.21, P = .02, and 0.42 ± 0.28 vs 0.19 ± 0.23, P = .02, respectively).

When comparing normal adrenal cortex and visible adenomas on the same specimen (n = 6), the NIRAF intensities were similar and paired analysis revealed no significant difference in quantitative NIRAF ratios (Figure 4, A). Among the visible adenomas, 4 were functional (2 Cushing, 2 Conn) and 3 nonfunctional. Functional adenomas had double the NIRAF intensities when compared with nonfunctional adenomas, although no statistically significant difference was reached in these small subgroup sizes (Figure 4, B).

When comparing all visible adenomas with all visible other neoplasias (100% of which were nonfluorescent), the quantitative analysis revealed a significantly higher mean tissue-to-background (adenomas: 3.3 ± 1.5 versus other: 1.4 ± 0.48; P < .001) and tissue-to-rc ratio (adenomas: 0.46 ± 0.21 versus other: 0.17 ± 0.05; P < .001), also after background correction (adenomas: 0.37 ± 0.25 versus other: 0.05 ± 0.06; P < .001).

Random forest feature importance to distinguish between adenomas and nonadenomas was highest for lesion-to-rc ratio (0.39), followed by normalized lesion-to-rc ratio (0.31) and lesion-to-background ratio (0.29). The logistic regression simulations revealed a mean lesion-to-rc ratio threshold of 0.36 for categorizing adenoma versus nonadenoma (Figure 5). The subclassification between non-adenomas (pheochromocytoma, hematoma, metastasis, and adrenocortical cancer) was inconclusive because of lower sample sizes in the subgroups.

### Comparative analysis of normal adrenal parenchyma between species

When comparing porcine and human normal regions of adrenalectomy specimens, the mean adrenal-to-background ratio did not show any relevant difference (porcine 3.9 ± 1.5 vs human 3.9 ± 1.8, P > .99). When using the rc as a reference, there was no significant difference either (porcine 0.41 ± 0.17 vs human 0.51 ± 0.23 mean adrenal-to-rc ratio, P = .21; porcine 0.34 ± 0.18 vs human 0.44 ± 0.26 mean normalized adrenal-to-rc ratio, P = .31).

### Discussion

In the present study, autofluorescence of porcine adrenal glands in the NIR range was investigated for the first time, and its stability during 1 year of storage in formalin was demonstrated. Although most tissues generate only a low NIR fluorescence contrast,¹⁵ this finding is similar to parathyroid tissue, where NIRAF has been shown to persist ex vivo for extended periods of up to 2 years under various conditions, including extreme temperatures, enzymatic activity, and formalin fixation.¹⁰,¹⁶⁻¹⁸ In addition, this study evaluated NIRAF in adrenal neoplasia using a series of image acquisitions that were taken on fresh surgical specimens. The examination was carried out on the back table after the specimen had been removed, without altering the intraoperative workflow. This technique did not require the administration of exogenous fluorophores such as ICG or an increase in operating time.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Autofluorescence characteristics of different adrenal tumors. (A-D) Adenomas. All adenomas located on the specimen surface exhibited autofluorescence. (A) A subsurface adenoma was only detectable with NIRAF imaging at the cut surface. (B) With a larger viewing angle, the lower autofluorescence intensity when compared with the indocyanine green (ICG) reference card is illustrated. (C) With a small adenoma in an otherwise normal and fluorescent parenchyma, the adenoma could be distinguished neither with the naked eye nor with NIRAF imaging (no specimen incision). (D) In a large lesion suspicious for malignancy, the fluorescence intensity on NIRAF imaging oriented toward diagnosis of a benign neoplasia, which was later confirmed by histopathology. (E-I) Other, nonfluorescent, neoplasias. (E, F) The pheochromocytomas (pheo) do not exhibit visible autofluorescence, and adjacent adrenocortical tissue is highlighted by its autofluorescence even under retroperitoneal adipose tissue. (G) A large adrenal hematoma. (H) A renal cell cancer (RCC) metastasis, with partly preserved autofluorescence of unaffected adrenal parenchyma. (I) Adrenocortical cancer, shown in a large field of view including the ICG reference card. NIRAF, near-infrared autofluorescence.</figcaption></figure>

Especially in bilateral disease and in many cases of unilateral disease, including patients with adrenal metastases, the enhanced intraoperative visualization of normal adrenocortical parenchyma could support cortical-sparing approaches and thus preserve postoperative functional capacity. As illustrated for the 4-cm renal cell cancer metastasis (Figure 2, H1), NIRAF can provide an enhanced demarcation of the metastasis from normal parenchyma suitable for preservation. In 766 consecutive partial

adrenalectomies in a specialized center, partial adrenalectomy for adrenal metastases was reported as optional in unilateral disease after previous contralateral adrenalectomy, dimensions <3 cm, without infiltration of adjacent structures, and in bilateral disease in individual cases, dimensions <4 cm, and no infiltration of adjacent structures.¹⁹

Fluorescence patterns varied between the different adrenal zones and tumor types, with differentiated adrenocortical
<caption>**Table II**</caption>

Human adrenalectomy specimen analyses, highlighting where the lesions (l) were visibly fluorescent on the acquired images, and corresponding quantitative measurements

<table><thead><tr><th rowspan="3">Visible fluorescence</th><th rowspan="3">Adrenal cortex</th><th colspan="3">Quantitative fluorescence</th><th colspan="3">Visible fluorescence</th><th colspan="3">Quantitative fluorescence</th><th rowspan="3">Reference card</th><th rowspan="3">Background</th></tr><tr><th>Mean adrenal-to-background ratio</th><th>Mean adrenal-to-reference card ratio</th><th>Mean normalized adrenal-to-reference card ratio</th><th rowspan="2">Lesion</th><th rowspan="2">Type</th><th rowspan="2">Diameter (cm)</th><th rowspan="2">NIRAF</th><th rowspan="2">Mean lesion-to-background ratio</th><th rowspan="2">Mean lesion-to-reference card ratio</th><th rowspan="2">Mean normalized lesion-to-reference card ratio</th></tr><tr><th>a/b</th><th>a/rc</th><th>a-b/rc-b</th><th>l/b</th><th>l/rc</th><th>l-b/rc-b</th></tr></thead><tbody><tr><td>2</td><td>Y</td><td>5.8515</td><td>0.8986</td><td>0.8821</td><td>Adenoma</td><td>0.4</td><td>Y*</td><td>4.9971</td><td>0.7674</td><td>0.7253</td><td>235.0</td><td>36.0</td></tr><tr><td>19</td><td>Y</td><td>3.7764</td><td>0.6287</td><td>0.5591</td><td>Adenoma</td><td>0.8</td><td>No lesion visible with (sub)surface NIRAF</td><td></td><td></td><td></td><td>235.0</td><td>39.1</td></tr><tr><td>18</td><td>Y</td><td>2.8116</td><td>0.4474</td><td>0.3424</td><td>Adenoma</td><td>0.9</td><td>Y</td><td>2.4397</td><td>0.3882</td><td>0.2705</td><td>234.9</td><td>37.4</td></tr><tr><td>15</td><td>Y</td><td>4.1746</td><td>0.5847</td><td>0.5232</td><td>Adenoma</td><td>1</td><td>No lesion visible with (sub)surface NIRAF</td><td></td><td></td><td></td><td>235.0</td><td>33.0</td></tr><tr><td>16</td><td>Y</td><td>4.1381</td><td>0.5342</td><td>0.4602</td><td>Adenoma</td><td>1.3</td><td>No lesion visible with (sub)surface NIRAF</td><td></td><td></td><td></td><td>235.0</td><td>30.5</td></tr><tr><td>10</td><td>Y</td><td>1.6963</td><td>0.2228</td><td>0.1053</td><td>Adenoma</td><td>3</td><td>Y</td><td>5.2145</td><td>0.6849</td><td>0.6338</td><td>234.8</td><td>30.8</td></tr><tr><td>1</td><td>Y</td><td>1.9483</td><td>0.2584</td><td>0.1461</td><td>Adenoma</td><td>5</td><td>Y</td><td>4.2550</td><td>0.5643</td><td>0.4975</td><td>235.0</td><td>31.2</td></tr><tr><td>4</td><td>Y</td><td>5.0138</td><td>0.5695</td><td>0.5063</td><td>Adenoma</td><td>5</td><td>Y</td><td>2.5090</td><td>0.2850</td><td>0.1982</td><td>235.0</td><td>26.6</td></tr><tr><td>17</td><td>Y</td><td>1.2847</td><td>0.2909</td><td>0.0835</td><td>Adenoma</td><td>5.1</td><td>Y</td><td>1.3642</td><td>0.3089</td><td>0.1079</td><td>235.0</td><td>53.2</td></tr><tr><td>20</td><td>No visible normal adrenal parenchyma</td><td></td><td></td><td></td><td>Adenoma</td><td>7</td><td>Y</td><td>2.4784</td><td>0.2152</td><td>0.1451</td><td>234.2</td><td>20.3</td></tr><tr><td>8</td><td>Y</td><td>6.5634</td><td>0.7694</td><td>0.7413</td><td>Pheo</td><td>2</td><td>N</td><td>1.2194</td><td>0.1429</td><td>0.0342</td><td>235.0</td><td>27.5</td></tr><tr><td>5</td><td>Y</td><td>4.7344</td><td>0.6824</td><td>0.6310</td><td>Pheo</td><td>3.5</td><td>N</td><td>1.3118</td><td>0.1891</td><td>0.0538</td><td>235.0</td><td>33.8</td></tr><tr><td>9</td><td>Y</td><td>5.3628</td><td>0.5557</td><td>0.5024</td><td>Pheo</td><td>4</td><td>N</td><td>1.1242</td><td>0.1165</td><td>0.0167</td><td>235.0</td><td>24.4</td></tr><tr><td>6</td><td>Y</td><td>6.5753</td><td>0.8355</td><td>0.8256</td><td>Pheo</td><td>6</td><td>N</td><td>2.0665</td><td>0.2626</td><td>0.1552</td><td>235.0</td><td>29.9</td></tr><tr><td>7</td><td>Y</td><td>2.3631</td><td>0.5251</td><td>0.3923</td><td>Pheo</td><td>6</td><td>N</td><td>0.7146</td><td>0.1588</td><td>0.0032</td><td>235.0</td><td>52.2</td></tr><tr><td>14</td><td>Y</td><td>3.2553</td><td>0.2964</td><td>0.2288</td><td>Pheo</td><td>8</td><td>N</td><td>1.6186</td><td>0.1474</td><td>0.0634</td><td>235.0</td><td>21.2</td></tr><tr><td>12</td><td>No visible normal adrenal parenchyma</td><td></td><td></td><td></td><td>Hematoma</td><td>8</td><td>N</td><td>0.9233</td><td>0.1466</td><td>0.0057</td><td>235.0</td><td>36.8</td></tr><tr><td>3</td><td>Y</td><td>7.1080</td><td>0.8436</td><td>0.8184</td><td>Metastasis</td><td>4</td><td>N</td><td>2.2547</td><td>0.2676</td><td>0.1704</td><td>235.0</td><td>27.9</td></tr><tr><td>13</td><td>Y</td><td>3.5524</td><td>0.3658</td><td>0.2982</td><td>Metastasis</td><td>4</td><td>N</td><td>1.5969</td><td>0.1644</td><td>0.0695</td><td>235.0</td><td>24.2</td></tr><tr><td>11</td><td>Y</td><td>2.0720</td><td>0.2782</td><td>0.1643</td><td>Metastasis</td><td>9</td><td>N</td><td>1.0889</td><td>0.1462</td><td>0.0161</td><td>235.0</td><td>31.6</td></tr><tr><td>21</td><td>Y</td><td>1.7886</td><td>0.1795</td><td>0.0902</td><td>ACC</td><td>9.5</td><td>N</td><td>0.9564</td><td>0.0960</td><td>0.0030</td><td>234.9</td><td>23.6</td></tr></tbody></table>

Ratios for the normal adrenal parenchyma (a) relative to the background (b) and ICG reference card (rc) near-infrared autofluorescence (NIRAF) intensities are listed. Adenomas were Conn (pink), Cushing (green), and non-secreting (blue).

ACC, adrenocortical cancer.

<footnote>* Subsurface adenoma (see Figure 2, A).</footnote>
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Regions of interest in processed images. A change of color code can highlight the regions of interest that might be less obvious in the standard black-and-white view. (A) The small subsurface adenoma is easier to see adjacent to the normal adrenal parenchyma with a strong (red, A1; and yellow, A2) cortical fluorescence surrounding the nonfluorescent medulla than in **Figure 2, A1**. B) Weak autofluorescence gets highlighted and is easier to distinguish from the background than in **Figure 2, B1**. (C, D) The margin of normal adrenocortical tissue adjacent to the pheochromocytomas is also highlighted (see **Figure 2, E** and **F**). If such image guidance were available intraoperatively, cortical-sparing adrenal surgery could be facilitated.</figcaption></figure>

**A** Normal cortex vs. Adenoma

<figure><img src="image_5.png" /></figure>

**B** Comparison between adenomas

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Quantitative NIRAF intensity comparison between normal adrenal cortex and different types of adenomas. (A) There were no significant differences in mean tissue-to-background ratio for normal cortex 3.1 ± 1.9 (a/b) vs adenomas 3.5 ± 1.6 (l/b), *P* > .99; in mean tissue-to-rc ratio 0.45 ± 0.26 (a/rc) vs 0.50 ± 0.20 (l/rc), *P* = .84; or in mean normalized tissue-to-rc ratio 0.34 ± 0.31 (a-b/rc-b) vs 0.41 ± 0.25 (l-b/rc-b), *P* = .84. (B) The NIRAF intensity differences between secreting versus nonsecreting adenomas did not reach statistical significance with a small sample size (orange: mean adenoma-to-background ratio 4.2 ± 1.3 vs 2.1 ± 0.65, *P* = .23; green: mean adenoma-to-rc ratio 0.60 ± 0.16 vs 0.27 ± 0.05, *P* = .06; blue: mean normalized adenoma-to-rc ratio 0.53 ± 0.20 vs 0.15 ± 0.05, *P* = .06). NIRAF, near-infrared autofluorescence; rc, reference card.</figcaption></figure>

parenchyma and adenomas exhibiting highest NIRAF—in agreement with the literature on endogenous adrenal fluorescence by 3 working groups7,9,12,20,21 and exogenous fluorescence after

intravenous fluorophore administration22,23—when compared to medullary tissue and other tumor types and tissues. All adenomas located on the specimen surface displayed autofluorescence. In
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Logistic regression analysis to classify adrenal neoplasia. (A) The logistic regression curve (green) from a sample simulation using the feature lesion-to-reference card ratio for NIRAF prediction to distinguish between fluorescent (NIRAF Y) and nonfluorescent (NIRAF N) adrenal neoplasia. (B) Accuracy versus threshold plot from 100 logistic regression simulations to determine the optimal classification cutoff for fluorescent adenoma versus nonfluorescent other neoplasia. The highest predictive accuracy was achieved at a lesion-to-reference card ratio threshold of 0.36. NIRAF, near-infrared autofluorescence.</figcaption></figure>

addition, because NIR light enables surgeons to see beneath the tissue surface as a result of its penetration depth of several millimeters,⁸ one small adenoma located under the cut surface could be localized with NIRAF (750 nm excitation wavelength) although it was neither visible in white light nor palpable. It is worth noting that an optimized excitation wavelength (785 nm used in a handheld probe-based system and a custom-built camera device) can result in higher NIRAF-to-background ratios than with the commercially available cameras (Fluobeam-LX with 750 nm and PDE-Neo II [Hamamatsu Photonics, Hamamatsu-shi, Shizuoka-ken, Japan] with 760 nm excitation wavelengths).⁹ ICG—with its emission peak at 830-835 nm providing a higher signal-to-background ratio and quantum yield—could also have enabled deeper tissue imaging than autofluorescence.¹⁰ The limited resolution of NIR light beyond 5 mm depth requires an incision to effectively scan larger specimens, as shown in Figure 2, A and D. Normal porcine and human adrenocortical tissue exhibit similar autofluorescence properties, which may help identify the molecule responsible for NIRAF by use of porcine samples.

Quantitative image analysis with the tissue-to-rc ratios allowed to demonstrate that normal adrenal autofluorescence is only half as intense as the standardized ICG signal as provided by the reference card, and even weaker for adrenal neoplasia. Current laparoscopic and robotic imaging systems, which are optimized for ICG fluorescence, lack the required sensitivity to detect NIRAF for adrenal surgery.²⁰ Our assessment with the latest IMAGE1 S Rubina (KARL STORZ SE & Co. KG, Tuttlingen, Germany) and Da Vinci Xi systems (Intuitive Surgical, Sunnyvale, CA, USA) in the integrated NIR imaging modes confirmed that the adrenal autofluorescence signal is too weak for effective intraoperative image guidance. Consequently, autofluorescence assessments in the present study were performed using a system designed for open surgery to analyze the specimen ex vivo. In a similar quest for convenient endoscopic NIRAF visualization of parathyroid glands, some groups have modified the filters of a commercially available minimally invasive imaging system in collaboration with the manufacturer,²⁴ or used a prototype parathyroid autofluorescence imager with a custom-designed fiberoptic light-guide to transmit visible and NIR light and display both in a split-screen view during endoscopic thyroid surgery.²⁵ Commercial availability of such advancements would facilitate adrenal and tumor margin delineation and localization, as has already been demonstrated with ICG-enhanced adrenal²,²²,²³,²⁶⁻²⁹ and paraganglioma imaging.³⁰ Future developments with a minimally invasive imaging system capable of detecting

relatively weak autofluorescence signals and thus providing depth information could enhance adrenal visualization, particularly under thin layers of retroperitoneal adipose tissue.

Despite current imaging system limitations, adrenal fluorescence imaging provides complementary insights to standard white light visualization and has potential for guiding adrenal surgery with further quantitative and automated image analyses. However, literature on adrenal tumor fluorescence “signatures” remains scarce for automatic intraoperative characterization. A limitation of this pilot investigation is the small clinical sample size. Given the variability of background fluorescence in back table NIRAF imaging, standardized reference card measurements were useful for methodologic consistency across different operating room environments.

Analyzing adrenal neoplasia is challenging because of signal inhomogeneity from overlying retroperitoneal tissue and fluorescent adrenocortical tissue at the edge of nonfluorescent lesions. Regions of interest had to be labeled manually, and we could not yet achieve automatic detection. Our random forest algorithm-based analysis to evaluate feature importance for classifying different adrenal neoplasia revealed that the lesion-to-rc ratio was more discriminative than the lesion-to-background ratio. Prioritizing invariant reference standards over dynamic background measurements may enhance classification accuracy, as background variability introduces noise into classification models.

To automatically distinguish between benign or indeterminate and malignant lesions, binary logistic regression was recently reported to have promising diagnostic accuracy in a cohort of 107 patients. Benign tumors and neoplasms with uncertain malignant potential had significantly higher median normalized NIRAF intensities than malignant tumors.²¹ However, the study did not report comparison of tissue-to-background NIRAF ratios between hormonally active (n = 53) and nonsecreting (n = 16) adrenocortical adenomas, and differences in NIRAF intensity between functional and nonfunctional adenomas require further investigation. A prior study from the same group reported higher adenoma-to-background ratios for cortisol-secreting (median 2.5, IQR 2.2) and aldosterone-secreting (median 1.9, IQR 3.0) adrenocortical adenomas than for nonsecreting ones (median 0.6, IQR not available), but no statistical comparisons.¹² Another study assessing 55 adrenal glands with NIRAF, of which 16 (29.1%) had cortisol-secreting, 6 (10.9%) aldosterone-secreting, and 3 (5.5%) nonsecreting adrenocortical adenomas, reported the strongest NIRAF levels (tissue-to-background ratios) in adenomas irrespective of whether they were
secreting or nonsecreting, consistently with a commercial image-
based system (PDE-Neo II), laboratory-built camera and
spectrometer systems, and a commercial fiber-optic probe-based
system (PTeye; Medtronic, Galway, Ireland).⁹ Another study re-
ported higher mean fluorescence levels for cortisol-producing (n =
10) than aldosterone-producing tumors (n = 3), but had no non-
secreting adenomas for comparison.⁷ Whether or not the NIRAF
intensity we observed in favor of functional adenomas correlates
with secretory status necessitates clarification through a multi-
institutional comparison of existing data.

Integrating quantitative fluorescence intensity and dynamic assessment into commercial imaging systems will facilitate pattern recognition and standardize NIRAF criteria for tissue characterization. Video-based recordings and real-time quantitative analysis should be explored for correlating intraoperative assessments with postoperative adrenal function, particularly in cortical-sparing approaches.³¹

Overall, there have been relatively few studies, despite growing
interest in adrenal autofluorescence. At this moment, there is still
insufficient evidence for a robust differentiation between all types
of benign and malignant disease based on NIRAF imaging alone.
Centralized access to image acquisitions and collaborative quanti-
tative analysis, including artificial intelligence-driven lesion clas-
sification, may lead to enhanced intraoperative image guidance in
the near future.

Funding/Support

This work was funded by the French National Agency for
Research (Agence Nationale de la Recherche [ANR]) within the
framework of the project AI-DIAL (ANR-22-CE17-0019-01). This
work was also supported by French state funds managed by the
ANR within the "Programme d'investissements d'avenir" France
2030 (reference ANR-10IAHU-02).

Conflict of Interest/Disclosures

Barbara Seeliger declares that she is the recipient of a grant from the French National Agency for Research (Agence Nationale de la Recherche [ANR]) within the framework of the project AI-DIAL—Diagnostic Imaging of Adrenal Lesions (ANR-22-CE17-0019-01 and ANR-23-IACL-0004) and has a consultant agreement with Intuitive Surgical. Rupak Bose, Didier Mutter, and Michel Vix have no conflict of interest to declare.

Acknowledgments

BS would like to thank the preclinical research team at IHU
Strasbourg, in particular Fanélie Wanert and Cindy Vincent, for
assistance during the porcine adrenalectomies, and is grateful for
fruitful discussions with Dale J. Waterhouse and Farahdiba Zarin on
certain optical/technical aspects of data extraction for quantitative
NIRAF analysis. The authors would also like to thank Michele Diana,
who conducted pioneering studies on quantitative fluorescence
imaging at IHU Strasbourg, and a device was used of which the
purchase had been funded by his grant from the ARC Foundation
for Cancer Research (www.fondation-arc.org) as part of the ELIOS
(Endoscopic Luminescent Imaging for precision Oncologic Surgery)
project.

Use of AI and Al-assisted Technologies Statement

AI has not been used in the writing process.

CRediT authorship contribution statement

**Barbara Seeliger:** Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Rupak Bose:** Writing – review & editing, Methodology, Formal analysis. **Didier Mutter:** Writing – review & editing, Validation, Supervision, Resources, Investigation. **Michel Vix:** Writing – review & editing, Validation, Resources, Investigation.

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