Intraoperative near-infrared autofluorescence in parathyroidectomy: associations with gland morphology, body mass index, and histopathology

Introduction

As the core endocrine organ that regulates calcium and phosphorus metabolism, dysfunction of the parathyroid gland can directly cause serious metabolic disorders. Primary hyperparathyroidism (PHPT) is the third most common endocrine disorder, with a prevalence of 1 in 500 to 1 in 1,000 (1). Secondary hyperparathyroidism (sHPT) is more progressive in patients with chronic kidney disease: epidemiological data indicate that its incidence rises to 57 per 1,000 person-years when the glomerular filtration rate declines to stage G3, and increases sharply to 230 per 1,000 person-years with the onset of end-stage renal disease (stage G5) (2). Complications such as persistent hypercalcemia, osteoporosis and vascular calcification make this disease a multidisciplinary clinical problem (3).

As the primary treatment for hyperparathyroidism, surgery requires reliable intraoperative identification of parathyroid glands to support appropriate surgical decision-making. Visual identification of parathyroid glands remains effective in routine practice and experienced surgeons can reliably identify parathyroid glands using conventional visual and anatomical cues; however, adjunctive technology may be helpful in complex cases where gland morphology is altered or identification is uncertain. Residual parathyroid glands can lead to incomplete treatment of the disease and persistent or recurrent hypercalcemia levels after surgery (4). Therefore, accurate identification of the parathyroid glands during surgery is one of the important challenges faced by surgeons.

Although the emerging supportive technologies in recent years have partially alleviated this dilemma, they still face key bottlenecks: indocyanine green fluorescence imaging relies on exogenous contrast agents and is subject to hemodynamic interference (5); nano-carbon negative imaging technology may contaminate the surgical field and affect the subsequent pathological evaluation (6), while the rapid detection of parathyroid hormone (PTH) requires ex vivo tissue processing, which makes it difficult to achieve real-time dynamic monitoring (7). In this context, near-infrared autofluorescence (NIRAF) has been introduced as an adjunct tool for intraoperative identification of parathyroid glands using endogenous fluorescence signals. Parathyroid glands were first reported to exhibit intrinsic autofluorescence under near-infrared excitation (800–900 nm) by Das et al. in 2006, representing a major breakthrough in intraoperative parathyroid identification (8). Subsequently, Paras et al. demonstrated that, at a peak excitation wavelength of 822 nm, both thyroid and parathyroid tissues exhibited a single emission peak, suggesting the presence of a shared endogenous fluorophore and laying the foundation for clinical application of NIRAF (9). This technology enables identification without the need for exogenous tracers by capturing the autofluorescence signal characteristic of parathyroid tissue at 780–950 nm (10).

Of note, the latest generation of probe-based NIRAF systems, such as PTeye, offers unique advantages in complex surgical fields by optimizing spectral acquisition algorithms to improve signal resolution to the sub-millimeter level (11). It may be convenient to use during surgery, and it has been reported to be used for parathyroid protection during thyroid surgery. However, most of the existing studies focus on the recognition of normal parathyroid glands, and there is still a gap in the systematic study of fluorescence signal characteristics in pathological states. In particular, the applicability of NIRAF technology has not been clarified for specific pathological changes such as nodular hyperplasia and marked structural remodeling that are common in patients with sHPT (12).

The primary objective of this study was to identify patient subgroups within hyperparathyroidism in which NIRAF is most applicable and reliable for intraoperative parathyroid identification, rather than to establish the intrinsic diagnostic accuracy of the NIRAF device. We present this article in accordance with the STROBE reporting checklist (available at https://gs.amegroups.com/article/view/10.21037/gs-2025-420/rc).

Methods

Patients inclusion

This prospective cohort study included a patient population that underwent parathyroidectomy in the Department of General Surgery of Nanfang Hospital, Southern Medical University between June 2023 and June 2024. We consecutively enrolled a cohort of patients undergoing parathyroidectomy. The inclusion criteria included: age 18–80 years old, meeting the indications for hyperparathyroidism, and clear localization on preoperative ultrasound/technetium 99m sestamibi (99mTc-MIBI) imaging. Patients with severe cardiopulmonary insufficiency [New York Heart Association (NYHA) cardiac function class III–IV or Chronic Obstructive Pulmonary Disease Global Initiative for Chronic Obstructive Lung Disease (COPD GOLD) stage 3–4], prior neck radiation therapy or parathyroid surgery, or known allergic reaction to NIRAF detection system components were excluded. PHPT was diagnosed based on elevated serum calcium levels in combination with inappropriately elevated PTH levels and the absence of chronic kidney disease. sHPT was defined by elevated PTH levels in the setting of chronic kidney disease with normal or low serum calcium levels. The surgical approach has been clarified to specify standard open cervical exploration performed by experienced endocrine surgeons, with NIRAF used as an adjunctive technique. The study aimed to include all consecutive eligible patients during the study period, and no prior sample size calculation was performed. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University (No. NFEC-2023-308). All participants provided written informed consent before enrollment in the study.

Variables and data sources

Clinical and pathological features were considered analysis variables, including age, gender, body mass index (BMI), preoperative blood calcium, PTH level, parathyroid gland maximum diameter and volume (estimated as an elliptical sphere), and pathological type (nodular hyperplasia, diffuse hyperplasia, adenoma) confirmed by histopathology. The fluorescence intensity ratio (FR) was assessed using a handheld, probe-based NIRAF detection system probe-based NIRAF imaging system (BN-YGXX-100) with a 2-mm fiber-optic probe for intraoperative parathyroid identification. This system, previously developed and clinically applied by our group, operates at a laser power of 20 mW with a fluorescence sensitivity of 0.3 nmol/L (13). The primary outcome was the intraoperative NIRAF detection status, defined as positive (FR ≥4.0) or negative (FR <4.0). This threshold was selected empirically based on the observed distribution of fluorescence intensity ratios within the study cohort, rather than a predefined diagnostic cutoff. The measurement protocol (including dynamic baseline calibration and multiple measurements per gland) was designed to minimize measurement variability and potential bias (Figure 1).

Figure 1 Real-time intraoperative detection using NIRAF probe in different surgical approaches. (A) Intraoperative NIRAF detection of the left inferior parathyroid gland via open anterior cervical approach. (B) Intraoperative NIRAF detection of the left superior parathyroid gland via transaxillary endoscopic approach. (C) Synchronized display of endoscopic view (above, cervical surgical field) and NIRAF monitoring interface (below), showing real-time fluorescence intensity waveform (FR =7.1) with auditory feedback (continuous beep triggered when FR exceeds preset threshold of 4.0). FR, fluorescence intensity ratio; NIRAF, near-infrared autofluorescence.

Statistical analysis

SPSS 26.0 was used for data analysis. (I) Data distribution test: the normality of continuous variables is confirmed by Shapiro-Wilk test; (II) descriptive statistics: normally distributed data are expressed as mean ± standard deviation (SD), and non-normal data are described as median [interquartile range (IQR)]; Categorical variables are presented in frequency (percentage); Continuous variables are expressed as mean ± SDs, and categorical variables are described in terms of frequency (percentage). Independent-samples t-test or Mann-Whitney U test (non-normally distributed data) were used for comparison between groups, and Chi-squared test or Fisher’s exact test was used for categorical variables. A multivariate logistic regression model (stepwise regression method, inclusion criterion P<0.10) was constructed with fluorescence detection status (positive/negative) as the dependent variable, and covariates such as age, BMI, and glandular volume were included, and the adjusted odds ratio (OR) value and 95% confidence interval (CI) were calculated. The significance threshold was set to two-sided P<0.05. No data were missing for the variables included in the final analysis.

Results

Baseline data

During the 1-year study period, a total of 20 patients with PHPT and 19 patients with sHPT underwent surgery for 98 parathyroid lesions. The baseline characteristics of the 39 patients and 98 lesions. The mean age of the patients was 52.4±11.8 years, and 69.23% were female. The mean preoperative serum calcium level was 2.81±0.18 mmol/L, and the median PTH level was 356 pg/mL (range, 98–3,800 pg/mL). The average maximum diameter of the parathyroid glands was 1.5±0.4 cm, with a mean volume of 1.5±0.6 cm³. Parathyroid pathological subtypes included adenoma (21.43%), diffuse hyperplasia (41.84%), and nodular hyperplasia (36.73%). No missing data were observed for the characteristics presented in Table 1.

Analysis of clinical factors related to the positive rate of parathyroid detection

In this study, all intraoperatively excised tissues were pathologically confirmed as parathyroid tissue. Among these confirmed parathyroid glands, 60% demonstrated positive NIRAF signals intraoperatively, as defined by an FR ≥4. As shown in Table 2, univariate analysis showed that the positive rate of parathyroid detection was correlated with BMI, PTH level, maximum diameter of parathyroid gland, and pathological nature of parathyroid gland. BMI was negatively associated with intraoperative NIRAF positivity, with a significantly higher detection rate observed in patients with BMI <24 kg/m² compared with those with BMI ≥24 kg/m² (OR =0.385, 95% CI: 0.148–0.998, P=0.05).

PTH levels were inversely associated with intraoperative NIRAF positivity. The positive detection rate was significantly higher in patients with lower PTH levels [lower log₁₀ (PTH)] compared with those with higher PTH levels (OR =0.208, 95% CI: 0.075–0.578, P=0.003).

Similarly, parathyroid gland size was negatively associated with NIRAF positivity. Patients with a smaller maximum parathyroid diameter (<2 cm) demonstrated a significantly higher positive detection rate than those with larger glands (OR =0.082, 95% CI: 0.028–0.240, P<0.001).

Pathological subtype was significantly associated with intraoperative NIRAF positivity. Compared with diffuse hyperplasia and adenoma, parathyroid nodular hyperplasia demonstrated a significantly lower positive detection rate (OR =7.119, 95% CI: 2.869–17.663, P<0.001). In multivariate logistic regression analysis, maximum parathyroid gland diameter and pathological subtype remained independent predictors of a positive NIRAF signal (both P<0.001). In contrast, the association between BMI and NIRAF positivity was no longer statistically significant after adjustment for other covariates (Table 3).

In patients with sHPT (n=19), intraoperative NIRAF positive rate of nodular hyperplasia by NIRAF probe was 72%, which was significantly lower than that of the diffuse hyperplasia group (92%) (P<0.05). In addition, the NIRAF positive rate of smaller glands (<1.0 cm³) was higher (OR =2.3, 95% CI: 1.2–4.5, P=0.01), however there was no significant correlation between age and detection rate (P=0.23).

Correlation analysis between the maximum diameter of the parathyroid gland and the positive rate of the test

In this study, the intraoperative NIRAF positivity rate (defined as FR ≥4) was 38.89% (21/54) in histologically confirmed parathyroid glands with a maximum diameter ≥2 cm, and increased to 88.64% (39/44) in glands with a diameter <2 cm (P<0.001). Specificity and negative predictive value were not calculated because all evaluated tissues were pathologically confirmed parathyroid glands, and true negative samples were not available in this surgical cohort. Accordingly, positive predictive value was 100% by study design. This indicates that the lesions of the parathyroid glands with a maximum diameter of <2 cm are easily detected by near-infrared autofluorescent probes. In addition, parathyroid lesions larger than 2 cm in diameter were associated with a significantly lower NIRAF positivity rate compared with lesions <2 cm.

Discussion

This study confirms the application value of NIRAF in hyperparathyroidism. The results show that autofluorescence intensity is correlated with parathyroid weight, patient age, and nodular hyperplasia. Application of NIRAF may be particularly helpful in patients with small parathyroid glands, lower BMI (<24 kg/m²), and diffuse hyperplasia or adenoma.

This probe-based parathyroid detection system provides real-time intraoperative feedback and can assist surgeons during parathyroidectomy by supporting intraoperative decision-making. Previous studies have demonstrated the feasibility of NIRAF-based identification of parathyroid tissue using devices such as PTeye and Fluobeam, primarily in the setting of thyroid surgery (14-16). However, most clinical studies have focused on the use of NIRAF to distinguish normal parathyroid glands from surrounding tissues or to identify adenomas in patients with PHPT (17). In contrast, secondary and tertiary hyperparathyroidism, which are characterized by diffuse or nodular hyperplasia rather than discrete adenomas, have been less extensively investigated despite their distinct pathological and clinical features. Several studies have compared the structural and functional characteristics of nodular hyperplasia with parathyroid adenoma, indicating that pathological subtype may influence autofluorescence behavior (18,19). These differences highlight the need for a more systematic evaluation of NIRAF performance across hyperparathyroid disease categories and pathological patterns.

The observed association between lower BMI and higher NIRAF positivity is more likely attributable to technical and anatomical factors rather than inherent biological differences in parathyroid pathology. Increased cervical adipose tissue in patients with higher BMI may attenuate near-infrared signal transmission and reduce the signal-to-background contrast, thereby impairing intraoperative autofluorescence detection. At present, there is limited evidence to suggest that BMI is directly associated with intrinsic autofluorescence properties of parathyroid adenomas or hyperplastic glands. Therefore, the association observed in this study should be interpreted primarily as a context-related imaging effect rather than a pathological difference in parathyroid tissue itself.

Our results indicate that gland size and pathological subtype significantly influence NIRAF performance. Larger glands often exhibit increased structural heterogeneity, including variable cellular composition, fibrosis, necrosis, hemorrhage, or cystic degeneration, which may attenuate or unevenly distribute autofluorescence signals (16). In addition, increased tissue thickness may alter optical signal transmission, further affecting NIRAF detection (20). Similarly, diffuse hyperplasia showed stronger fluorescence signals than nodular hyperplasia, likely reflecting differences in mitochondrial density and stromal architecture (21).

Notably, the mechanism underlying differences in autofluorescence between hyperfunctioning and normal parathyroid tissue remains unclear (22). Calcium-sensing receptors have been proposed as potential contributors to this signal because of their abundant expression in parathyroid tissue; however, their precise role as fluorophores has not been conclusively established (23). But this view has not yet been conclusively confirmed. In this study, it was found that fluorescence correlated with the type of parathyroid hyperplasia. Decreased expression of calcium-sensitive receptors in parathyroid tissue has been reported in patients with SHPT, particularly in nodular hyperplasia parathyroid tissue (21). Further studies integrating optical characteristics with histopathological features are warranted to better elucidate the determinants of NIRAF variability and to refine its clinical application (24).

Although this study has achieved certain results in exploring the application of NIRAF technology in parathyroidectomy, there are still some limitations. First, the sample size is relatively small, which may limit the generalizability and statistical power of the findings. Second, NIRAF measurements were obtained using a single probe-based detection system, and no direct comparison was performed with commercially available NIRAF platforms or other intraoperative identification modalities. Therefore, the generalizability of the results across different devices remains uncertain. Importantly, the primary aim of this study was to identify patient subgroups in which NIRAF is most applicable, rather than to evaluate or compare the technical performance of different systems. In addition, the mechanism of action of NIRAF technology has not been fully elucidated, and further basic research is needed to further understand how it works in parathyroid tissue. Lack of technical standardization is also a problem, and the current lack of unified operating procedures and equipment specifications may affect the comparison between different studies and the reproducibility of results. Future research needs to be improved and explored in depth in these areas.

Conclusions

This study confirms the value of NIRAF in hyperparathyroidism, and the results show that autofluorescence intensity is associated with parathyroid weight, patient age, and parathyroid nodular hyperplasia, and it may be particularly helpful in identifying smaller parathyroid lesions, elderly patients, and diffuse hyperplasia in sHPT.

Acknowledgments

We would like to express our gratitude to the Department of Pathology of Nanfang Hospital of Southern Medical University for their professional support in sample processing and pathological diagnosis. In addition, we would like to thank all the patients and their families who participated in this study for their trust and cooperation.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://gs.amegroups.com/article/view/10.21037/gs-2025-420/rc

Data Sharing Statement: Available at https://gs.amegroups.com/article/view/10.21037/gs-2025-420/dss

Peer Review File: Available at https://gs.amegroups.com/article/view/10.21037/gs-2025-420/prf

Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 82373366), Science and Technology Program of Guangzhou (No. 2023A04J2393), and High-tech, Major and Unique Clinical Technology Projects of Guangzhou (No. 2023P-TS02).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://gs.amegroups.com/article/view/10.21037/gs-2025-420/coif). L.C. is employed as an engineer by Beneeon (Jiangsu) Medical Technology Co., Ltd., the manufacturer of the near-infrared autofluorescence device used in this study. The company had no involvement in the study design, data acquisition, data analysis, interpretation of results, or manuscript preparation. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Nanfang Hospital of Southern Medical University (No. NFEC-2023-308). All participants provided written informed consent before enrollment in the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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