Abstract
Background
Sentinel lymph node biopsy (SLNB) is crucial for staging melanoma and determining metastasis. Indocyanine green (ICG) has emerged as a promising near-infrared fluorescence mapping technique. Recent advancements in near-infrared fluorescence imaging systems, with color-segmented fluorescence (termed “red capping”), may enhance the detection of positive sentinel lymph nodes.
Methods
This study retrospectively compared node positivity rates between a control group using standard ICG alone and a study group utilizing the newer ICG combined with red capping technology in melanoma patients undergoing SLNB. A total of 70 patients met the study criteria, with 47 in the ICG-alone control group and 23 in the ICG with red capping study group. Both groups achieved a 100% sentinel lymph node detection rate, confirmed by radiotracer.
Results
The red capping group demonstrated a node positivity rate of 26.1% compared to 19.1% in the ICG-alone group. While this represents a higher proportion of positive nodes, it is important to note that the red capping group also contained a higher percentage of aggressive tumor features, including a 30.4% rate of ulceration (vs 21.3%) and a higher proportion of tumors with >7 mitoses/mm2 (21.7% vs 6.4%). The observed node positivity likely reflects these underlying biological differences between the cohorts rather than the imaging modality itself. Both imaging modes achieved a 100% detection rate when verified by radiotracer, indicating that red capping is an effective and highly visual alternative for intraoperative nodal mapping.
Discussion
Red capping provides an effective visualization tool for intraoperative lymphatic mapping and demonstrates comparable sentinel node identification to standard ICG fluorescence.
Keywords: Fluorescence, indocyanine green, sentinel node biopsy
Sentinel lymph node biopsy (SLNB) is a crucial component in the staging and management of melanoma, providing valuable information about potential metastasis and guiding treatment decisions.1,2 The presence or absence of malignant cells in the sentinel lymph node (SLN) is the single most powerful predictor of melanoma’s biological behavior.3,4 Traditional SLNB techniques have a false-negative rate ranging from 5% to 21%.4 Factors such as age, the presence of ulceration, and tumor thickness may correlate with higher rates of false-negative SLNs.5,6 The accuracy and sensitivity of SLNB techniques are critical for achieving optimal outcomes in melanoma care.7,8
The current standard method for performing SLNB uses a combination of blue dye and radiolabeled colloids to map lymphatic pathways and identify the sentinel nodes, which are the first lymph nodes where cancer cells are most likely to spread from the primary tumor.9 However, the use of indocyanine green (ICG) near-infrared fluorescence (NIF) has gained increased attention in recent years.5 ICG-based NIF imaging has demonstrated high detection rates and ease of use in SLNB procedures.10 A meta-analysis by Wölffer et al further supports its noninferior performance compared to technetium-based lymphoscintigraphy, emphasizing its growing role in intraoperative SLN identification.11
The ongoing pursuit of improved sensitivity and accuracy has driven advancements in NIF imaging systems, such as the development of color-segmented fluorescence. This technology utilizes a color mapping algorithm to differentiate fluorescence intensity, which likely reflects vascular flow or lymph volume. By highlighting the area of highest dye concentration, this method is designed to facilitate the precise identification of the sentinel node. A handheld near infrared imaging device (Stryker iSpye system, Stryker, Kalamazoo, MI) employs color-segmented fluorescence and a proprietary algorithm to produce a heat map visualization of fluorescent dye concentration.12 Color-segmented fluorescence functions as an overlay visualization mode that assigns colors to varying fluorescence intensities. This allows surgeons to identify areas of maximal ICG concentration more easily compared with conventional grayscale fluorescence imaging. When using this system, a distinct red fluorescence pattern localized to areas of maximal ICG concentrations within SLNs was observed, which we termed “red capping.” Red capping effectively highlights regions of increased ICG uptake, providing enhanced contrast and clearer differentiation of SLNs from nearby structures.
This technological advancement has the potential to refine SLNB by improving sensitivity and positive node detection rates. While red capping is promising, limited data exist comparing it to standard ICG fluorescence in melanoma SLNB. This study evaluated whether the red capping mode of ICG imaging improves the identification of positive SLNs compared to standard ICG imaging. We hypothesized that incorporating red capping would enhance visualization of SLNs during intraoperative mapping and provide comparable SLN identification when compared with standard ICG fluorescence imaging. We found that red capping with ICG yielded a higher positive node detection rate (26.1%) compared to ICG alone (19.1%), while maintaining a 100% overall SLN detection rate as verified by radiotracer.
METHODS
This was a retrospective review of a prospectively maintained database of melanoma patients who underwent SLNB at Baylor University Medical Center between July 2018 and September 2024. The study was approved by the Baylor Scott & White Research Institute institutional review board. These cases were performed by two surgeons in one practice in one institution. A total of 70 patients were included in this study, divided into two groups: a control group using ICG alone (n = 47) and a study group using ICG with color-segmented fluorescence, or “red capping” (n = 23).
All patients underwent standard-of-care SLNB using a dual-tracer method. Technetium-99 lymphoscintigraphy was used preoperatively, and intraoperative gamma probe measurements were used to confirm SLN identification and ensure complete basin evaluation. Intraoperatively, “red capping” or standard ICG fluorescence was used to provide real-time visualization for the identification of SLNs. The radiotracer (technetium-99) and a handheld gamma probe were utilized in vivo to confirm that the ICG-avid nodes were indeed the sentinel nodes and to ensure that all nodes with a gamma count reduction of >90% were successfully removed.13 Clinical parameters collected included age, sex, and anatomical location of the tumor. Pathological parameters analyzed included tumor characteristics (Breslow thickness, ulceration, mitoses count, Clark level), pathological features (lymphovascular invasion, microsatellite status, perineural invasion), and nodal status. The primary outcome measure was the node positivity rate, defined as the proportion of patients with one or more pathologically positive SLN.
Data were analyzed using Microsoft Excel. Descriptive statistics (means, medians, and percentages) were calculated for all variables. Fisher’s exact test was used for categorical comparisons, and Student’s t test was employed for continuous variables. A P value of <0.05 was considered statistically significant. Given the small sample size, the study is acknowledged to be largely descriptive with limited statistical power.
RESULTS
The two study groups were comparable with respect to demographic characteristics (Table 1), with no statistically significant differences in mean age (P = 0.85), sex distribution (P = 0.80), or SLNB side (P = 0.87) (Table 1). The overall cohort’s mean age was 61.5 ± 15.9 years (mean ± standard deviation [SD]), with 51.4% women and 48.6% men (Table 1). The ICG-alone group had a mean age of 61.8 ± 15.5 years, and the red capping + ICG group had a mean age of 61.0 ± 16.9 years (Table 1). The most common SLNB site overall was the axillary region (57.1%), and there was no statistically significant difference in SLNB site between groups (P = 0.36) (Table 1). Tumor site category was also statistically similar between groups (P = 0.34) (Table 1). The ICG group’s two most common tumor sites were the trunk (40.4%) and arms (25.5%). For the red capping + ICG group, the most common sites were equally split between the trunk and the legs (30.4% for each).
Table 1.
Demographic and clinical characteristics of the ICG-alone group and the red capping + ICG group
| Variable | Total (N = 70) | ICG (N = 47) |
RedCap + ICG (N = 23) |
P value |
|---|---|---|---|---|
| Age (years): Mean ± SD | 61.5 ± 15.9 | 61.8 ± 15.5 | 61.0 ± 16.9 | 0.851 |
| Sex | 0.802 | |||
| Female | 36 (51.4%) | 25 (53.2%) | 11 (47.8%) | |
| Male | 34 (48.6%) | 22 (46.8%) | 12 (52.2%) | |
| SLN biopsy side | 0.872 | |||
| Bilateral | 1 (1.4%) | 1 (2.1%) | 0 (0.0%) | |
| Left | 37 (52.9%) | 24 (51.1%) | 13 (56.5%) | |
| Right | 32 (45.7%) | 22 (46.8%) | 10 (43.5%) | |
| SLN biopsy site | 0.362 | |||
| Axillary | 40 (57.1%) | 28 (59.6%) | 12 (52.2%) | |
| Cervical | 12 (17.1%) | 9 (19.1%) | 3 (13.0%) | |
| Inguinal | 16 (22.9%) | 8 (17.0%) | 8 (34.8%) | |
| Parotid | 2 (2.9%) | 2 (4.3%) | 0 (0.0%) | |
| Tumor site category | 0.342 | |||
| Arms | 18 (25.7%) | 12 (25.5%) | 6 (26.1%) | |
| Head and neck | 13 (18.6%) | 10 (21.3%) | 3 (13.0%) | |
| Leg | 13 (18.6%) | 6 (12.8%) | 7 (30.4%) | |
| Trunk | 26 (37.1%) | 19 (40.4%) | 7 (30.4%) | |
| Mitoses count (mm2) | 0.102 | |||
| 0 | 3 (4.3%) | 3 (6.4%) | 0 (0.0%) | |
| ≥1 | 49 (70.0%) | 33 (70.2%) | 16 (69.6%) | |
| >7 | 8 (11.4%) | 3 (6.4%) | 5 (21.7%) | |
| Cannot be determined | 10 (14.3) | 8 (17.0%) | 2 (8.7%) | |
| Lymphovascular invasion present | 5 (7.1%) | 2 (4.3%) | 3 (13.0%) | 0.322 |
| Ulceration present | 17 (24.3%) | 10 (21.3%) | 7 (30.4%) | 0.552 |
| SLN positive | 15 (21.4%) | 9 (19.1%) | 6 (26.1%) | 0.542 |
“RedCap” indicates color-segmented fluorescence; ICG, indocyanine green; SLN, sentinel lymph node.
P value based on: 1Student’s t test; 2Fisher’s exact test for count data.
In terms of tumor characteristics (Tables 1 and 2), ulceration was observed in 17 patients (24.3%) overall, with no statistically significant difference between the red capping + ICG group (30.4%) and the ICG-alone group (21.3%; P = 0.55) (Table 1). Overall, 60% of tumors had a Breslow thickness of 1 to 4 mm, with comparable distributions between the ICG-alone group (63.8%) and the red capping + ICG group (52.2%) (Table 2). The median tumor thickness for the red capping + ICG group was 1.4 mm versus 1.3 mm for the ICG-alone group. Mitoses counts were generally similar across groups; however, a higher proportion of tumors with a mitoses count >7 mitoses/mm2 was noted in the red capping + ICG group (21.7%) compared to the ICG-alone group (6.4%; P = 0.10) (Table 1). Clark level IV predominated overall (57.1%), with a higher percentage in the ICG-alone group (61.7%) than in the red capping + ICG group (47.8%). Lymphovascular invasion was present in 7.1% of cases overall, with a nonsignificant higher prevalence in the red capping + ICG group (13.0%) compared to the ICG-alone group (4.3%; P = 0.32) (Table 1). Perineural invasion was identified in only one patient (4.3%) in the red capping + ICG group (Table 2).
Table 2.
Tumor-specific pathological features for the ICG-alone group and the red capping + ICG group
| Characteristics | All (N = 70) |
ICG alone (N = 47) |
RedCap + ICG (N = 23) |
|---|---|---|---|
| Breslow thickness (mm) | |||
| <1 mm | 14 (20.0%) | 8 (17.0%) | 6 (26.1%) |
| 1–4 | 42 (60.0%) | 30 (63.8%) | 12 (52.2%) |
| ≥4 | 13 (18.6%) | 8 (17.0%) | 5 (21.7%) |
| Cannot be determined | 1 (1.4%) | 1 (2.1%) | 0 (0.0%) |
| Clark level | |||
| I | 1 (1.4%) | 0 (0.0%) | 1 (4.3%) |
| II | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) |
| III | 9 (12.9%) | 6 (12.8%) | 3 (13.0%) |
| IV | 40 (57.1%) | 29 (61.7%) | 11 (47.8%) |
| V | 9 (12.9%) | 5 (10.6%) | 4 (17.4%) |
| Cannot be determined | 11 (15.7%) | 7 (14.9%) | 4 (17.4%) |
| Microsatellite | |||
| Present | 1 (1.4%) | 1 (2.1%) | 0 (0.0%) |
| Not identified | 69 (98.6%) | 46 (97.9%) | 23 (100%) |
| Perineural invasion | |||
| Present | 1 (1.4%) | 0 (0.0%) | 1 (4.3%) |
| Not identified | 69 (98.6%) | 47 (100%) | 22 (95.7%) |
| SLN status | |||
| Positive | 15 (21.4%) | 9 (19.1%) | 6 (26.1%) |
| Negative | 55 (78.6%) | 38 (80.9%) | 17 (73.9%) |
“RedCap” indicates color-segmented fluorescence; ICG, indocyanine green; SLN, sentinel lymph node.
Both the ICG-alone and red capping + ICG groups achieved a 100% SLN detection rate, as confirmed by radiotracer mapping and verified by a 90% reduction in gamma counts (Figure 1). No nodal basin recurrences were observed among patients with initially negative SLNs during the available follow-up period (median follow-up: 24 months). However, given the modest sample size and limited follow-up duration, no conclusions regarding false-negative rates can be drawn. Figure 2 illustrates the visual differences between traditional NIF imaging and the color-segmented red capping technique.
Figure 1.
Scatterplot showing the percent reduction in background gamma counts after sentinel lymph node removal in the indocyanine green (ICG)-alone group and the color-segmented fluorescence (“RedCap”) + ICG group.
Figure 2.
(a) Traditional black and white image showing sentinel lymph node visualization using standard near-infrared fluorescence imaging. (b) Color-segmented red capping image showing enhanced sentinel lymph node visualization, with red indicating the areas of highest indocyanine green concentration.
DISCUSSION
Our study suggests that color-segmented fluorescence imaging (“red capping”) with ICG enhances the detection of positive SLNs in melanoma patients compared to ICG alone. This work builds upon a previous study from our institution that demonstrated that ICG and radiotracer identified SLNs in 52 of 52 patients (100% identification rate), validating the fundamental utility of ICG as a lymphatic mapping agent. Furthermore, a recent meta-analysis by Wölffer et al corroborates that ICG is comparable to radiotracer in identifying metastatic SLNs.11
In our study, although both the ICG-alone and red capping + ICG techniques achieved a 100% SLN detection rate (as confirmed by radiotracer), the red capping + ICG group identified a higher proportion of positive nodes (26.1% vs 19.1%). The positive node detection rate observed in the ICG-alone group aligns with current established benchmarks for ICG-based SLNB, typically reported at 15% to 20%.5,11,14 The 7% absolute increase in positive node detection, while not statistically significant (P = 0.54), could still be clinically meaningful if validated in a larger, adequately powered cohort. A modest improvement in detecting positive SLNs may lead to improved staging accuracy and help guide more appropriate treatment decisions.
Patient demographics, tumor characteristics, and anatomical distributions were mostly comparable between the groups, suggesting that the observed difference in node positivity should be interpreted cautiously and may reflect underlying tumor biology rather than the imaging modality itself. Specifically, the ICG-alone group had a mean age of 61.8 years and 46.8% men, compared to the red capping + ICG group with a mean age of 61.0 years and 52.2% men (Table 1). The median tumor thickness was 1.3 mm for ICG-alone versus 1.4 mm for red capping + ICG. The red capping + ICG group did, however, show a nonsignificant higher proportion of tumors with aggressive features, such as a mitoses count >7 mitoses/mm2 (21.7% vs 6.4%) and lymphovascular invasion (13.0% vs 4.3%) (Table 1). The similar SLN detection rates between groups demonstrate that color-segmented fluorescence performs comparably to standard ICG imaging while offering an alternative visualization strategy for intraoperative lymphatic mapping.
Red capping technology offers several practical advantages in clinical practice. By using color-segmented fluorescence, surgeons can more easily differentiate SLNs from adjacent tissues, potentially reducing operative time and increasing surgical precision.10 Furthermore, red capping is easy to incorporate into standard surgical workflows, building on the established benefits of ICG fluorescence without requiring extensive protocol changes. Dependence on radiotracers for SLNB poses logistical challenges and increases costs for healthcare facilities.5 The development of color-segmented fluorescence imaging, despite requiring the upfront capital expense of the handheld NIF imaging system, has the potential to simplify SLNB procedures by reducing reliance on nuclear medicine while maintaining or even enhancing node identification accuracy.14,15
Despite these promising findings, several limitations of this retrospective, exploratory study must be acknowledged. First, the modest sample size, especially the small and uneven split between groups (47 in ICG-alone vs 23 in red capping + ICG), may be insufficient to detect small but clinically meaningful differences, limiting the statistical power and generalizability of our findings. The study was not powered to confirm statistical superiority over ICG alone. Additionally, as a single-institution study, our results may not translate in broader patient populations or diverse surgical practices. We did not examine other factors that could influence fluorescence imaging, such as body mass index or surgeon experience. Finally, the financial and logistical implications, including the purchase cost of the system and associated training time and costs, warrant further evaluation to thoroughly assess red capping’s feasibility on a wider scale.
Future research should address these limitations through prospective, multicenter trials enrolling larger patient cohorts to validate the findings. Long-term clinical outcomes, such as recurrence rates and overall survival, should be examined to clarify the true impact of red capping–guided SLNB. Moreover, extending this technology to other malignancies that rely on SLNB could further broaden its clinical application. Finally, thorough cost-effectiveness analyses will be crucial to inform widespread adoption and integration into standard practice. The enhanced node positivity suggests that red capping may help address the persistent challenge of false-negative SLNB outcomes, positioning it as a promising candidate for improving the accuracy of melanoma staging.
Disclosure statement/Funding
The authors report no funding or conflicts of interest.
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