Detecting the Near Infrared Autofluorescence of the Human Parathyroid: Hype or Opportunity?

Probe-based modalities:

When Paras et al. first reported about utilizing NIRAF for identifying PGs in real-time, they utilized a portable fluorescence spectroscopy system, which consists of a NIR laser source for excitation, a spectrometer for signal collection, a laptop for data processing and a fiber optic probe to deliver and collect the light from the sample (Figure 2a)²⁴. In early iterations of this approach, measurements were performed in patients during surgery with operating room (OR) lights turned off. Each measurement provided a plot of fluorescence intensity as a function of emission wavelength (spectrum), where the peak intensity was found to be stronger in PGs compared to thyroid glands (Figure 2b). These early studies predominantly relied on NIRAF spectroscopy for PG detection.^{24-26, 30} This ‘lab-based’ system was subsequently developed into a user-friendly clinical device called PTeye (by AiBiomed, USA) that provides visual, quantitative and auditory feedback when the handheld fiber probe touches a PG, akin to a nerve-monitoring device.^{31, 32}. The aforementioned devices that rely on a fiber optic probe for NIRAF detection will hereafter be referred to as ‘probe-based’ modalities in this review.

Imaging-based modalities:

NIRAF detection based on the ‘imaging’ approach typically uses a NIR light source in conjunction with a camera and appropriate filters to visualize a tissue of interest on a display monitor (Figure 2c, 2d). With the camera held at a defined distance from the surgical field, this approach does not require tissue contact and can help a surgeon spatially localize PGs within the surgical field as demonstrated in 2014 (Figure 2e – 2g).³⁰ NIRAF imaging also requires that the OR lights be turned off during use. Easy access to commercial NIR imaging systems developed for ICG fluorescence (FDA-approved for other applications – tissue perfusion and transfer circulation in free-flaps, plastic and reconstructive surgery) such as Fluobeam-800 (Fluoptics, France), PDE Neo II (Hamamatsu, Japan) and Karl Storz cameras (Karl Storz, Germany) further popularized this method of PG identification.^33-35

FDA approval:

Within a decade following the discovery of the NIRAF in PGs, the FDA granted approval to two of the aforementioned devices - the PTeye (probe-based) and Fluobeam-800 (imaging-based) – for label-free intraoperative PG identification.^{36, 37} Despite approval, many questions and misconceptions remain about the capability of NIRAF and its application for thyroidectomy and parathyroidectomy. We therefore present a review of the published literature prior to and after FDA approval of these two NIRAF detection devices. We will also discuss how surgeons might be able to use these tools in their practice.

1). Helping the surgeon identify PG tissue intraoperatively in real time

In the operating room, identification of PG tissue has traditionally been based on a surgeon’s visual assessment with a frozen section subsequently confirming the surgeon’s impression. Using a probe-based approach, McWade et al. first relied on spectroscopy to detect NIRAF and noted a 100% PG detection rate across 45 patients.²⁵ The authors extended the scope of this technique across 137 patients, where NIRAF was analyzed to be higher in 97% (256/264) of PGs as compared to surrounding tissue.²⁶ An interesting observation made in this study was the presence of intra-glandular spatial heterogeneity in NIRAF signal with the authors recommending at least three measurements per PG in different locations. The study further found that high calcium, low 25-hydroxyvitamin D, disease state of PG and high body mass index led to varied NIRAF intensities in PGs, but ultimately did not affect the capability of NIRAF detection in identifying PGs during surgery. The authors concluded that NIRAF detected with spectroscopy using a fiber probe was capable of reliable, label-free and real-time detection of PGs, regardless of the clinical and demographic characteristics of the patients. Subsequently, Thomas et al. compared the now FDA-approved ‘PTeye’ (Figure 3a – 3d) against the ‘lab-built’ , and found that the PTeye had an accuracy of 96.1% compared to 92.5% with the lab-built system in the same patients. ³¹ They also noted that presence of blood on tissues did not affect the performance of either system in detecting NIRAF for PG identification.

The feasibility of an ‘imaging’ approach for NIRAF detection to spatially localize PGs in thyroid or parathyroid operations was first studied by McWade et al., where 100% PG detection rate was achieved.³⁰ For this modality, a modified Karl Storz camera was placed 15 cm over the surgical field and NIRAF images were captured to be displayed in real-time on a separate monitor after OR lights were turned off. Raw images were processed post hoc to quantify the ratio of NIRAF intensity of PGs to its surrounding tissues. Based on these images, the authors inferred that high levels of NIRAF from PGs could offer a unique opportunity for PG-selective imaging, without administering injectable contrast agents.

These early results ushered the era of NIRAF imaging for label-free intraoperative PG identification. A series of manuscripts that utilized imaging modalities for NIRAF detection to visualize PGs using commercially available or custom-built systems validated the original findings (Table 1).^{33, 34, 38-42} In 2016, Falco et al. were the first to utilize a commercially available NIR imaging system (Fluobeam 800) to visualize and confirm PG tissues in the OR, where the device detected 100% of PGs.⁴³ Around the same time, De Leeuw et al. also reported on the feasibility of Fluobeam 800 for PG detection with 94% sensitivity and 80% specificity in 28 ex-vivo specimens³⁸ and observed positive NIRAF in 98.8% of in-situ PGs. Kahramangil et al. later observed that NIRAF imaging could visualize PGs earlier than ICG-based fluorescence.⁴⁴ The same authors later reported the performance of NIRAF detection via imaging in the first multi-centric study across three sites that yielded a PG identification rate of 98% in 210 patients (Figure 3e – 3g).⁴⁰ In parallel, imaging of NIRAF in PGs was successfully demonstrated with another commercially available device – the PDE system (Hamamatsu, Japan) showing 100% sensitivity and 97.3% specificity³⁴, while several other studies relied on modified iterations of Karl Storz systems to obtain PG detection rates ranging from 86.4 to 92.3% (Table 1).^{35, 45-48}

Thus, the available studies strongly support that NIRAF detection can accurately identify and confirm both healthy and diseased PG tissues in real-time without injectable labels, using either probe-based or imaging-based platforms. These modalities that rely on NIRAF detection could in theory be used in lieu of frozen section to confirm PG tissue and could save operating room time by facilitating PG identification and avoiding excessive dissection. As with any new technology, surgeon will need to prospectively study the tool in his/her clinical practice and assess how it performs against the current standard of care.

2). Visualize and “Map” the location of the PG prior to or after dissection

Early detection of PGs during neck dissection could, in concept, help avoid damage to healthy PGs and guide in their localization during thyroidectomy and/or parathyroidectomy. De Leeuw et al. was the first to suggest that imaging-based approach to detect NIRAF could guide in localizing PGs prior to surgeon visualization.³⁸ The authors indicated that imaging for NIRAF aided in finding the PG in five instances before the surgeon could observe it with the naked eye. Similarly, Kahramangil et al. reported that NIRAF imaging aided in localizing 37 to 67% of all PG candidates prior to the surgeon’s eye.⁴⁰ Falco et al. noted that the mean number of PGs identified per patient with plain white light increased significantly from 2.5 to 3.7 PGs with NIRAF imaging.³³ Recently Squires et al. stated that imaging with the PDE Neo II improved the surgeon’s ability by 20%, in detecting NIRAF and identifying PGs not initially visible.⁴⁹ The need for frozen sections was obviated in 29% of PGs as the authors felt confident in having correctly identified PGs with NIRAF detection.

In contrast to commercial imaging systems, Kim et al. described a custom-built Canon camera specifically designed to identify and locate PGs.^{41, 42} The authors noted 100% NIRAF detection rate in the PGs examined, where the system was able to localize 10 PGs that were ‘veiled’ by connective tissue, fat or blood vessels and not apparently visible to the surgeon’s naked eye. While it was noted that unexposed PGs tended to have lower NIRAF intensity than in an exposed state, their location could still be detected by the described instrument. The same authors coined the term ‘parathyroid mapping’ based on a systematic three-stage NIRAF detection approach which was applied in their subsequent studies.^{41, 50} The first stage (P1) involved aiming the camera at predicted locations of PGs prior to dissection or identification with surgeon’s plain sight (Figure 4a – 4c). For PGs not visualized with NIRAF detection in P1 stage, the imaging protocol was repeated after the surgeon dissected or identified PGs with the naked eye for the second (P2) stage. For the 3rd stage (P3), ex-vivo NIRAF images were taken from excised surgical specimens looking for PG not detected in P1 or P2 stage. Using this approach, the authors were able to detect 82.7 to 92.8% of PGs within P1 stage, 96.2 to 98.6% PGs by P2 stage and 98.1 to 100% by P3 stage, indicating that NIRAF-based parathyroid mapping may guide in early PG localization.

On the other hand, Ladurner et al. reported that the authors were unable to visualize NIRAF from PGs in 8 cases when using an optimized Karl Storz camera, because the PG were embedded in adipose tissue.³⁵ Unlike the earlier described studies, the authors recommended dissecting and partially freeing potential PGs from its connective tissue sheath for NIRAF imaging, as overlying tissue would obstruct NIRAF detection. In conformity with this finding, other studies have also observed an inability to detect NIRAF when PGs were covered with fat.^{29, 34, 46, 51}

There are currently no studies that report using probe-based modalities for NIRAF detection to spatially ‘map’ PGs. However, it is plausible that the PTeye could provide comparable results to NIRAF detection with imaging in terms of PG mapping/localization. For instance, once the thyroid lobe is systematically mobilized during surgery, the fiber optic probe can be used to ‘scan’ the surgical field for PGs and could notify the surgeon with an auditory feedback, analogous to a ‘metal detector’ or ‘nerve monitor’. The surgeon could then repeat the process after further dissection, in a similar manner to the ‘parathyroid mapping’ technique described by Kim et al. (see above).

The FDA approval granted for Fluobeam 800 and PTeye explicitly state that these two devices are meant to solely ‘assist’ and ‘not replace’ experienced visual assessment by the surgeon in identifying PG tissues. Moreover, since NIR light typically has a penetration depth of 0.4 to 5 millimeters in soft tissues⁵², the ability to localize ‘hidden’ PGs would be contingent on (i) sensitivity, exposure time and related optics of the detector used, and (ii) optical properties of the tissues overlying the PG. Therefore, current FDA-approved devices – Fluobeam 800 and PTeye – may be limited in NIRAF detection beyond a 5 mm depth, due to limitations of their existing system design. As a result, these devices would be unlikely to localize deep-seated PGs (intrathyroidal or ectopic). Currently the surgeon should fully or partially expose PG candidates prior to NIRAF detection. It is however quite possible that future technical advances and iterations for technologies that can detect NIRAF, could definitively ensure spatial mapping for ‘unseen’ PGs, as described by Kim et al.^{41, 42}

3). Distinguish healthy PGs from diseased PGs

The ability of a surgeon to differentiate between normal and abnormal PGs plays a pivotal role in the success of parathyroid surgery. Preoperative ultrasound imaging, ^99mtechnetium-sestamibi scintigraphy and computed tomography (CT) can aid in localizing hyperfunctioning/abnormal PG, but have yielded subpar results in detecting multiglandular disease present in 5 to 33% patients with primary hyperparathyroidism.^{9, 53-55} With the advent of NIRAF detection for identifying PGs, researchers have attempted to identify discriminant traits in NIRAF signals between normal and abnormal PGs. McWade et al. was the first to observe that PGs of SHPT patients had relatively weaker NIRAF intensity than normal or adenomatous PGs, which was later confirmed by other studies.^{26, 29, 31} Using PTeye, Thomas et al. found no significant difference in NIRAF intensity detected between healthy and diseased PGs associated with primary hyperparathyroidism, which was in agreement with observations by others who relied on NIRAF imaging.^{31, 45, 49}

In stark contrast to these findings, Falco et al. found that adenomas showed higher NIRAF intensity than normal PGs using Fluobeam 800 during cases of primary hyperparathyroidism.³³ They hypothesized that the higher NIRAF intensity of PG adenomas could be related to increased cellularity and lower fat concentration in adenomas. Interestingly, the exact opposite findings were recorded by Kose et al., where hyperfunctioning PGs displayed lower NIRAF intensity than normo-functioning PGs upon using the same Fluobeam 800 device.⁵⁶ The authors further determined that a normalized NIRAF intensity ratio of 2.0 or above could serve as an optimal cutoff to differentiate normo-functioning from hyperfunctioning PGs. In addition, the authors observed that hyperfunctioning PGs often displayed heterogenous patterns of NIRAF as compared to the normo-functioning ones. While McWade et al. also reported about intraglandular heterogeneity of NIRAF with fiber-optic probe measurements, this study did not report if the assessed PGs were healthy or diseased.²⁶

In summary, due to inconsistent findings in the various reported studies, there is currently no clear-cut consensus on whether NIRAF can definitively distinguish between normal and abnormal PGs. Further studies are necessary to determine whether the degree of NIRAF intensity can accurately predict the presence of hyperfunctioning PGs.

4). Prevention of hypocalcemia after total thyroidectomy.

The question of whether routine use of NIRAF detection to identify and localize PGs can improve patient outcome after total thyroidectomy was recently investigated in three studies. Benmiloud et al. evaluated the effect of NIRAF imaging on patient outcome by comparing the results of such procedures by two surgeons where one surgeon used the Fluobeam 800 to evaluate the surgical field during total thyroidectomy while the other did not. ³⁹ This study revealed that NIRAF detection with imaging reduced the incidence of inadvertent PG removal, PG auto-transplantation rates and transient hypocalcemia (Calcium <8 mg/dL at post-operative day 1 or 2). However, the surgeon that did not use NIRAF detection also showed reduction in transient hypocalcemia, presumably due to the Hawthorne or observer effect. In another study, Dip et al. compared postsurgical outcomes with use of imaging (NIR light) vs. the surgeon’s naked eye (white light).⁵⁷ For the study design, patients were block randomized to two equal groups. White light and anatomical landmarks were used to localize PGs in Group 1, while imaging with Fluobeam 800 was used in Group 2. While there was no significant difference in the incidence of transient hypocalcemia (defined as serum calcium < 8.0mg/dL) between groups, the authors reported that the incidence of severe transient hypocalcemia (defined as serum calcium < 7.6mg/dL) was significantly decreased in Group 2 as compared to Group 1. In another study, DiMarco et al. agreed that NIRAF imaging may aid in detecting accidentally excised PGs and allow timely PG auto-transplantation, but failed to find a significant reduction in missed inadvertent parathyroidectomies or postsurgical hypocalcemia (transient or permanent) upon using Fluobeam 800 during thyroidectomy.⁵¹

The present data suggest that NIRAF imaging during total thyroidectomy may help visualize PGs more readily and consequently avoid their injury. However, more clarity is required to fully determine if NIRAF imaging can minimize accidental injury or removal of PGs or reduce the incidence of postoperative hypocalcemia. To our knowledge, probe-based detection of NIRAF has not been evaluated in patient outcome studies yet, although studies are currently in progress to evaluate the role of PTeye in affecting patient outcome.

1 –. Comparing probe-based vs. imaging-based platforms for PG identification

Table 2 summarizes the capabilities of the current FDA-approved devices for intraoperative PG identification using NIRAF detection. The pen-like configuration of the fiber optic probe (Figure 2b) utilized in the PTeye allows for it to be easily handheld and used in very small incisions potentially reaching into “nooks and crannies” within a surgical field. The device is compatible with ambient OR lights and gives real-time quantitative information with an immediate auditory feedback to the surgeon when the PG is detected. The PTeye however does not provide information of PG viability/perfusion or a “field view” of the operative site. Since the PTeye probe currently needs to be in contact with the PG in question, the fiber optic probe utilized at present is essentially a disposable. While the contact-based approach requires probe sterility, it was observed that probe-based NIRAF detection was more sensitive in identifying PGs (Detection rate: 97%) as compared to a non-contact based imaging approach (Detection rate: 90.9%) in a pilot study where both probe- and imaging-based approaches of NIRAF were tested concurrently.⁵⁸

The Fluobeam 800 is a reusable non-contact camera that is typically handheld by the surgeon above the neck incision. Since it is a camera-based modality, it provides a “field view” that can be very valuable for spatially “mapping” locations of PGs, as discussed above. The Fluobeam 800, along with other imaging devices such as the PDE systems or Karl Storz cameras, have the added benefit of being used for multiple applications in surgery, as these instruments were originally approved to evaluate tissue perfusion in plastic surgery and lymphatic mapping,^{59, 60} while also being able to assess PG perfusion when used in conjunction with ICG administration. Current imaging systems such as Fluobeam 800 require that the OR lights be turned off during NIRAF measurements. To achieve optimum visualization of PGs with these camera-based systems, the surgeon may need to make wider neck incisions or ensure that the camera is held at a fixed distance from the target, as NIRAF intensities can fluctuate with varying distance between target tissue and the camera held by the surgeon.²⁹ Since there is no real-time quantitative information of NIRAF intensity from PGs, the surgeon would have to subjectively judge NIRAF intensity on display monitors for presence of potential PGs.

2 –. False positives and false negatives with NIRAF detection modalities

Using Fluobeam 800, De Leeuw et al. observed 3 false positive cases which were attributed to colloid nodules in thyroid or brown fat.³⁸ The false positives typically presented as bright spots in the images with a normalized NIRAF intensity higher than that of the thyroid. The authors also noted that although brown fat exhibited strong NIRAF, its bright autofluorescence declined rapidly after resection while that of the PGs did not. Interestingly, the bright spots of brown fat could also be observed with plain white light illumination with the NIR laser turned off, unlike the PGs. The sole false negative result obtained in this study was that of an intra-thyroidal parathyroid. In comparison, McWade et al. noted that while some PGs emitted lower NIRAF, these glands could still be detected as these signals were higher relative to the thyroid. However reduced sensitivity was recorded particularly with PGs of SHPT patients, where elevated NIRAF was observed in only 54% of PGs confirmed by histology.²⁶ Thomas et al. also observed that SHPT patients were predominantly responsible for false negatives obtained with the PTeye.³¹ In addition, false negatives were observed for parathyroid cysts with PTeye, while false positives were found to occur in fibroadipose tissues, brown fat and occasionally in lymph nodes. Lowered NIRAF intensity or detection rate were also reported in PGs of SHPT patients with NIRAF imaging in other studies.^{29, 47} Using Fluobeam 800 for parathyroidectomy, DiMarco et al. observed that 9.5% of PGs lacked NIRAF signal (false negatives) with no false positives being observed.²⁹ In addition, 3 potential PGs could not be localized neither by the surgeon nor by NIRAF imaging. In another report only 50% of patients with primary hyperparathyroidism associated with multiple endocrine neoplasia type 1 (MEN1) syndrome exhibited detectable NIRAF in PGs when viewed with PDE Neo II.⁶¹

False NIRAF positives in non-parathyroid tissues – brown fat, lymph nodes – should thus be interpreted with necessary care by surgeons. Findings with NIRAF in such scenarios should be inferred in conjunction with either intraoperative PTH levels during parathyroidectomy or frozen section biopsies in total thyroidectomy for malignant thyroid disease.³¹ The former ensures that the diseased PG is removed instead of brown fat (additionally distinguished from PG using white light illumination), while the latter ensures preservation of healthy PGs and removal of metastatic lymph nodes. In comparison, surgeons should exert attention if they were to use NIRAF detection for PG localization in SHPT and MEN1 cases, because of the associated false negative rates – possibly due to the unique histopathogenesis in PGs for these diseases. It will be cogent to note that ‘non-tissues’ such as purple vicryl suture may also have strong NIRAF due to its dye, causing false positives and interference with imaging.³⁴ Similarly, surgical kittner sponges may strongly ‘glow’ under NIR cameras as a false positive, due its radiopaque dye.⁶²

3 –. Inability of NIRAF to assess parathyroid gland viability

NIRAF modalities do not provide information regarding PG blood supply or their viability. The PG’s ability to emit NIRAF remains intact even after it loses its blood supply. The NIRAF of PGs persists after excision from the body and thus can be detected by probe or imaging-based systems in an ex vivo setting. Current clinical efforts to evaluate perfusion/viability is focused on exogenously administered contrast agents like ICG to assess perfusion/viability of PGs. As a result, imaging systems such Fluobeam 800, PDE Neo or Novadaq (Stryker, US) among others have been successfully used to assess perfusion and viability of PGs or other tissues, but only after injection of ICG.^{59, 63} Recently it was demonstrated that PG perfusion can now be assessed in a label-free manner as well by using laser speckle contrast imaging with 91.5% accuracy (Figure 5a – 5e).⁶⁴ However, no study has been reported to date where probe-based NIRAF detection was applied for assessing PG viability using ICG.