Efficacy of Nano-Carbon Tracing Versus Indocyanine Green in Lymph Node Detection for Minimally Invasive Colorectal Resection

Introduction

Colorectal cancer, among the 3 most common malignancies worldwide, constitutes approximately 10% of all cancer diagnoses and represents a major contributor to cancer-related mortality [1]. Diagnosis typically involves colonoscopy with histopathological confirmation, whereas staging is guided by cross-sectional imaging – computed tomography (CT) or magnetic resonance imaging (MRI) [2]. Surgical resection with regional lymphadenectomy remains the mainstay of curative therapy, complemented by adjuvant chemotherapy or radiotherapy according to disease stage [3]. Despite therapeutic advances, recurrence rates remain high, and the 5-year overall survival (OS) for advanced-stage colorectal cancer seldom exceeds 60% [4–7]. Minimally invasive surgical approaches, including laparoscopic and robot-assisted colorectal resections, are increasingly preferred because they reduce postoperative morbidity and accelerate recovery while maintaining oncologic adequacy via standardized D3 lymphadenectomy [8].

Conventional lymph node dissection primarily relies on the surgeon’s visual inspection and tactile perception, increasing the risk of incomplete lymphadenectomy, particularly for small or anatomically concealed lymph nodes. This limitation compromises surgical radicality and contributes to higher rates of postoperative recurrence [9]. To address these challenges, various intraoperative tracers have been developed. Techniques for lymph node identification include dye-based, radioactive, and fluorescence-guided methods, among which indocyanine green (ICG) fluorescence and carbon nanoparticle tracers are most widely used [10]. ICG fluorescence allows real-time imaging but requires specialized near-infrared equipment and is limited by rapid washout. In contrast, nano-carbon particles are rapidly absorbed by lymphatic capillaries, phagocytosed by macrophages, and retained in lymph nodes for prolonged periods, producing a stable black coloration visible under standard lighting [10]. This biological mechanism provides surgeons with a clear, durable visual cue to identify small or obscured lymph nodes. The results of comparative studies suggest that nano-carbon can yield higher node retrieval rates than ICG, although direct randomized evidence in colorectal cancer remains limited [11]. Existing reports on nano-carbon use have demonstrated technical feasibility and improved nodal detection; however, most studies were retrospective or single-arm designs with limited sample sizes. Furthermore, few investigations have systematically evaluated its impact on long-term clinical outcomes such as disease-free survival (DFS), OS, or patient-reported quality of life (QoL). The potential role of nano-carbon in reducing perioperative complications by minimizing unnecessary tissue trauma also has not been fully clarified [12]. Thus, prospective comparative evidence assessing both oncologic and patient-centered outcomes of this emerging technique remains insufficient.

This prospective comparative study aimed to evaluate whether endoscopy-assisted nano-carbon labeling improves the precision and efficacy of lymph node dissection among patients undergoing minimally invasive colorectal cancer surgery. Specifically, we sought to determine whether nano-carbon tracing enhances lymph node retrieval; reduces postoperative complications; and improves DFS, OS, and QoL. To our knowledge, few investigations have comprehensively assessed both surgical and patient-centered outcomes associated with nano-carbon labeling in minimally invasive colorectal cancer surgery; by performing these assessments, the present work provides robust evidence to guide clinical practice.

Material and Methods

ETHICS STATEMENT:

This study was approved by the Ethics Committee of Qujing Second People’s Hospital, China (Approval No. 2021-001-01). All procedures complied with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants before enrollment. Patient confidentiality was strictly maintained through anonymization of data; adverse events were monitored and reported according to institutional and national research guidelines.

STUDY DESIGN AND PARTICIPANTS:

This single-center, prospective comparative study was designed to evaluate the efficacy of nano-carbon labeling combined with endoscopic assistance in improving the precision of intraoperative lymph node dissection during minimally invasive colorectal cancer surgery. The study was conducted at Qujing Second People’s Hospital between June 2022 and May 2024. Eligible patients had been diagnosed with colorectal cancer and were scheduled for minimally invasive surgery. A priori sample size calculations indicated that 90 patients per group were required to detect a 1.5-node difference in lymph node yield (standard deviation=4.5, α=0.05, power=0.80); therefore, 100 patients per group were enrolled to adjust for potential attrition. Patients were allocated to the nano-carbon group or the ICG group (ie, control group) according to the operating surgeon’s tracer preference, reflecting routine clinical practice. Surgeons were necessarily unblinded; outcome assessors (pathologists and statisticians) were blinded to group assignment. Demographic and clinical variables, including age, sex, body mass index, tumor location, tumor–node–metastasis (TNM) stage, and comorbidities (eg, diabetes, hypertension), were collected from electronic medical records and operative notes to ensure comparability between groups (Table 1). All data were independently verified by 2 investigators; discrepancies were resolved by consensus.

INCLUSION AND EXCLUSION CRITERIA:

Eligible patients were men and women aged 18 to 75 years who had pathologically confirmed colorectal adenocarcinoma, excluding other tumor types. The upper age limit (≤75 years) was selected because older patients have higher perioperative risks and comorbidities that could confound outcomes [13]. All patients were clinically staged as T1 to T4 without evidence of distant metastasis on imaging; they were scheduled for laparoscopic or robot-assisted minimally invasive colorectal surgery. Eligibility was confirmed by a specialist based on histopathology and CT/MRI findings reviewed at multidisciplinary team meetings. Written informed consent to participate was obtained from all patients, with full disclosure of the study objectives, potential risks, and expected benefits. Patients were excluded if they had severe comorbidities contraindicating surgery (such as advanced cardiopulmonary, hepatic, or renal disease), a history of abdominal radiotherapy or surgeries that substantially altered anatomical structures, or documented allergies to nano-carbon components or positive allergy tests; patients were also excluded if they were pregnant or breastfeeding. Patients with suspected hypersensitivity underwent a skin-prick test with diluted nano-carbon solution before surgery to reduce risk. In total, 200 eligible patients were assigned to either the nano-carbon labeling group (experimental group, n=100) or the control group (n=100). Allocation was based on the operating surgeon’s tracer preference, as noted in the previous section.

TRACERS AND EQUIPMENT:

Purified nano-carbon particles suspended in sterile saline were used for intraoperative lymph node labeling. The standardized concentration was 25 mg/mL; a total of 2 mL was injected into each patient (0.5 mL per site at 4 quadrants). An electronic gastrointestinal endoscope (Olympus GIF-HQ190, Olympus Corp., Tokyo, Japan) was used to guide precise submucosal tracer injection and assist intraoperative localization. A high-definition laparoscopic system (Karl Storz HD, Tuttlingen, Germany), ultrasonic scalpel (Ethicon Harmonic, Johnson & Johnson, New Brunswick, NJ, USA), grasping forceps, scissors, and an electrocautery hook were used for tumor resection and lymph node dissection. All instruments were subjected to routine calibration and maintenance before use. For the control group, ICG was prepared at 0.25 mg/mL; a total of 4 mL per patient (1 mL per site at 4 quadrants) was injected into the submucosal layer under endoscopic guidance, 10 to 30 min before surgery. Near-infrared fluorescence imaging (1688 AIM, Stryker, Kalamazoo, MI, USA) was used for intraoperative visualization of ICG-labeled lymphatics. All reagents and devices were obtained from certified hospital suppliers, and batch numbers were recorded to ensure procedural reproducibility.

SURGICAL AND LABELING PROCEDURES:

All patients underwent standard preoperative bowel preparation with polyethylene glycol electrolyte solution. Abdominal CT and MRI were performed to determine tumor location and assess regional lymph node involvement [14]. A prophylactic intravenous dose of 2 g ceftriaxone was administered within 60 min before incision; anesthesia was induced with propofol (2 mg/kg), remifentanil (0.2–0.5 μg/kg/min continuous infusion), and rocuronium bromide (0.6 mg/kg). An experienced anesthesiology team ensured uniform anesthetic depth and intraoperative stability across all cases [15]. In the nano-carbon group, 2 mL of tracer solution (0.5 mL per site) were injected into the submucosal layer at 4 quadrants within 3 cm of the tumor margin under endoscopic guidance. Adequate submucosal bleb formation and uniform dispersion confirmed proper lymphatic deposition. This technique facilitated labeling of the primary lymphatic drainage basin before resection [16]. Surgery was performed using a standard 5-port laparoscopic approach, beginning with pneumoperitoneum at the umbilicus (pressure 12 mmHg). A high-definition laparoscopic system (Karl Storz HD) and ultrasonic scalpel (Ethicon Harmonic, Johnson & Johnson) were used for dissection, with ligation of major vessels along the mesenteric root. In the control group, lymph node identification was guided by ICG fluorescence visualized with a near-infrared system (1688 AIM, Stryker). All operations were conducted by the same experienced surgical team, using standardized D3 lymphadenectomy protocols to minimize operator bias. Operative times were recorded by the circulating nurse from anesthesia records. “Total operation time” was defined as the interval from skin incision to closure, and “lymphadenectomy time” was regarded as the period between vascular ligation and completion of nodal dissection (Figure 1).

EVALUATION OF LABELING:

Intraoperative visualization of labeled lymph nodes in the experimental group was scored from 1 (poor) to 5 (excellent) by the operating surgeon. The scoring scale was defined as: 1=barely visible, 2=faint rim, 3=uniform gray staining, 4=distinct black node, and 5=high-contrast jet-black with visible lymphatic channel. Two independent raters recorded scores; inter-rater agreement (κ=0.82) indicated good reliability. Visibility, labeling rate, and staining intensity were recorded. Total numbers of dissected and labeled lymph nodes were confirmed by 2 gastrointestinal pathologists blinded to tracer type, using standardized fat-clearing and microscopic verification of black-stained nodes. All dissected nodes were sent for histopathology to verify labeling accuracy [17].

SURGICAL DATA:

Surgical data included the operation time measured from skin incision to closure (as described in a previous section), with separate recording of pneumoperitoneum establishment, tumor resection, and lymphadenectomy times (in minutes). Lymph node–related parameters comprised the total number of nodes dissected, the numbers of labeled and unlabeled nodes, and the staining intensity score using the predefined scale (1–5). Additionally, the number of pathologically confirmed metastatic nodes was documented, and positive node detection rates were compared between groups to evaluate oncologic adequacy.

POSTOPERATIVE COMPLICATIONS:

Postoperative complications were documented and graded according to the Clavien-Dindo classification. Complications such as wound infection, bleeding, anastomotic leakage, and deep vein thrombosis (DVT) occurring within 30 days were recorded. All adverse events were monitored daily during hospitalization and reassessed at 30-day follow-up visits. Each complication was reviewed and validated by an independent surgical investigator blinded to group allocation.

LONG-TERM OUTCOMES:

Long-term oncologic and patient-reported outcomes were systematically evaluated after surgery. Contrast-enhanced CT or MRI examinations of the abdomen and pelvis were performed at 6-month intervals to detect local recurrence or distant metastasis. Additional imaging or colonoscopy was performed whenever symptoms or tumor markers suggested recurrence. DFS was defined as the interval from the date of surgery to the first documented recurrence, metastasis, or death from any cause. OS was defined as the interval from surgery to death from any cause. Patients alive and disease-free at last contact were censored at their most recent follow-up date. Survival status was verified through hospital records, outpatient visits, and telephone interviews (every 6 months) with patients or family members.

Patient-reported QoL was assessed using the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 (version 3.0) questionnaire, administered at 6 and 12 months postoperatively by trained study nurses who were blinded to group allocation. The validated Chinese version of the instrument was used. The questionnaire comprises 30 items evaluating 5 functional domains (physical, role, emotional, cognitive, and social), 3 symptom scales (fatigue, pain, nausea/vomiting), 6 single-item symptoms (dyspnea, insomnia, appetite loss, constipation, diarrhea, and financial difficulties), and 1 global health/QoL scale. Each item was rated on a 4-point Likert scale (1=not at all, 4=very much), except for the global health items, which used a 7-point scale (1=very poor, 7=excellent). Responses were transformed to scores of 0 to 100 according to the EORTC scoring manual, where higher functional or global health scores indicate better QoL and higher symptom scores indicate greater symptom severity. Cronbach’s α values exceeding 0.80 confirmed internal consistency. Questionnaires with more than 10% missing responses were excluded, and domain-level mean substitution was utilized for items with missing response rates of 10% or below. Group comparisons were performed using independent-sample t-tests, and a 5-point difference was considered clinically meaningful.

STATISTICAL ANALYSIS:

Statistical analysis was performed using SPSS version 27.0 (IBM Corp., Armonk, NY, USA). Continuous variables were tested for normality using the Shapiro-Wilk test, expressed as mean±standard deviation, and compared using independent-sample t-tests; non-normally distributed data were analyzed using the Mann-Whitney U test. Categorical variables were analyzed using the chi-square test or Fisher’s exact test, as appropriate. Kaplan-Meier survival curves were generated to estimate DFS and OS, and differences between groups were evaluated using the log-rank test. A multivariate Cox proportional hazards regression model was utilized to identify independent prognostic factors, adjusting for age, sex, tumor stage, comorbidities, and operation time. Proportional hazards assumptions were verified using Schoenfeld residuals. Secondary endpoints were treated as exploratory, and multiple comparisons were corrected using the Benjamini-Hochberg false discovery rate (q=0.10). Primary analyses were based on complete cases; multiple imputation was performed when the missing data rate exceeded 5% (prespecified contingency; not triggered). All tests were 2-tailed, and P-values <0.05 were considered statistically significant.

Results

BASELINE PATIENT CHARACTERISTICS:

There were no statistically significant differences between the nano-carbon labeling group and the control group in terms of age, sex, body mass index, tumor location, TNM stage, or comorbidities (all P>0.05). Preoperative imaging similarly revealed no significant differences in tumor size or location between the 2 groups (P=0.445). All 200 patients completed baseline assessments, and no data were missing. These findings confirmed that baseline characteristics were well balanced, ensuring comparability for subsequent outcome analyses (Table 1).

SURGERY-RELATED OUTCOMES:

Comparative analysis of surgical times showed that both total operation time and lymph node dissection time were significantly shorter in the nano-carbon labeling group than in the control group (mean difference in total operation time=−12.4 min, 95% confidence interval [CI] −23.0 to −1.8, P=0.027; mean difference in dissection time=−8.7 min, 95% CI −15.2 to −2.3, P=0.012). However, there were no significant differences in the time required to establish pneumoperitoneum (P=0.142) or perform tumor resection (P=0.061), indicating that nano-carbon labeling specifically improves steps related to lymph node dissection (Figure 1). The nano-carbon group also demonstrated higher lymph node labeling rates (P=0.015) and greater staining intensity scores (P=0.032) than the control group, with excellent inter-rater reliability (κ=0.82), indicating clearer visualization and enhanced intraoperative precision (Figure 2A, 2B).

LYMPH NODE DISSECTION EFFICACY:

The total number of lymph nodes retrieved was significantly higher in the nano-carbon labeling group than in the control group (mean difference=+1.65 nodes, 95% CI 0.04–3.26; P=0.045), and the number of unlabeled lymph nodes was significantly lower (P<0.001). The labeling success rate was 76.35%±9.87% in the nano-carbon group (Table 2). Additionally, the positive lymph node detection rate was greater in the nano-carbon group (risk difference=+8%, 95% CI 1–15%; P=0.021), suggesting that this technique improves intraoperative identification of metastatic nodes (Figure 3).

POSTOPERATIVE COMPLICATIONS:

Within 30 days after surgery, the incidences of postoperative infection and DVT were significantly lower in the nano-carbon labeling group than in the control group (infection: 6% vs 10%, risk difference −4%, number needed to treat=25; DVT: 4% vs 7%, risk difference −3%, number needed to treat=33; P=0.045 and P=0.031, respectively). No significant differences were observed between groups in terms of postoperative bleeding or anastomotic leakage (both P>0.05). All patients completed the 30-day follow-up, and no complication data were missing. These findings support a role for nano-carbon labeling in reducing specific postoperative complications (Figure 4).

SURVIVAL AND LONG-TERM PROGNOSIS:

Kaplan-Meier survival analysis showed a significantly higher DFS rate at 24 months in the nano-carbon group (76.0%) than in the control group (65.0%) (log-rank P=0.023). The divergence in DFS became more apparent between 12 and 24 months (Figure 5). Similarly, OS at 24 months was higher in the nano-carbon group (85.5%) than in the control group (76.2%) (log-rank P=0.037); the survival advantage increased after 18 months (Figure 6). Multivariate Cox regression identified nano-carbon labeling as an independent protective factor for recurrence or death (hazard ratio=0.67, 95% CI 0.48–0.93, P=0.018). In contrast, higher tumor stage (T3–T4) and increased age were associated with worse prognosis (hazard ratio=1.45, 95% CI 1.12–1.89, P=0.008; hazard ratio=1.25 per 10-year increase, 95% CI 1.03–1.50, P=0.029). Sex, comorbidities, and operation time were not significantly associated with survival outcomes (all P>0.05) (Table 3).

POSTOPERATIVE QOL:

Postoperative QoL, assessed using the EORTC QLQ-C30, was significantly higher in the nano-carbon group. At both 6 and 12 months, the nano-carbon group exhibited better physical and role functioning and higher overall QoL scores (mean difference in global QoL at 12 months=+5.95 points, 95% CI 1.2–10.7; P=0.015). Fatigue and pain scores were significantly lower in this group (both P<0.05). Although emotional functioning did not significantly differ at 6 months (P=0.057), it substantially improved at 12 months (P=0.049), suggesting a delayed but sustained emotional benefit. Completion rates were 94% at 6 months and 91% at 12 months; missing data were handled using complete-case analysis (Table 4).

Discussion

In this prospective comparative study of 200 patients undergoing minimally invasive colorectal resection, endoscopy-assisted nano-carbon labeling significantly improved lymph node retrieval, procedural efficiency, and short-term oncologic outcomes compared with ICG guidance. These findings are consistent with and extend those of previous reports, including Xie et al [18] and Liu et al [9], which demonstrated enhanced nodal detection with nano-carbon tracers during colorectal surgery. This study also contributes evidence by showing improvements in postoperative complications, survival, and patient-reported QoL – outcomes rarely examined in prior trials.

The present results suggest that nano-carbon labeling improves the quality of lymph node dissection in minimally invasive colorectal cancer surgery, particularly with respect to labeling success and dissection completeness. Unlike conventional intraoperative methods that rely on visual and tactile identification, nano-carbon labeling provides enhanced visualization, enabling clearer identification of lymph nodes and facilitating comprehensive dissection of critical lymphatic drainage regions surrounding the tumor [19]. The significantly greater number of lymph nodes dissected and the substantially lower proportion of unlabeled nodes in the nano-carbon group highlight the superiority of this technique for precise lymph node localization and intraoperative guidance [20]. Complete lymph node dissection is a cornerstone of curative colorectal cancer surgery because inadequate resection can lead to residual disease and increased recurrence risk. Our findings indicate that nano-carbon labeling supports more thorough dissections by accurately marking nodes near the tumor, including those at higher risk of metastasis [21]. This advantage is particularly important in identifying small or anatomically concealed lymph nodes that may be overlooked during conventional dissection. By providing clear intraoperative delineation of lymphatic structures, the nano-carbon tracer reduces the likelihood of missed nodes and contributes to more radical resection [22].

In contrast to ICG, nano-carbon provides durable black pigmentation visible under white light, eliminating the need for specialized near-infrared equipment. Although ICG offers real-time dynamic imaging, its rapid tissue washout can limit detection sensitivity for small nodes. Prior comparative studies have demonstrated mixed findings: some showed higher nodal yields using nano-carbon, whereas others favored ICG for sentinel node identification [23,24]. The present results extend these findings by demonstrating not only improved nodal harvest with nano-carbon but also associated gains in long-term survival and QoL – outcomes less frequently evaluated in earlier studies. Moreover, nano-carbon technology optimized procedural efficiency. Real-time endoscopic guidance enabled intuitive identification of labeled regions, minimizing redundant exploration and reducing intraoperative errors. This efficiency translated into shorter dissection times and reduced tissue trauma, ultimately promoting less invasive procedures and enhanced postoperative recovery [25,26]. The observed reductions of approximately 12 min in total operation time and approximately 9 min in lymphadenectomy time are clinically meaningful, particularly in complex colorectal resections where prolonged operation time increases perioperative risk. Overall, the integration of nano-carbon labeling into laparoscopic protocols provides technical refinement that enhances the precision of lymph node dissection, contributing to reduced recurrence rates and improved surgical outcomes.

Survival analysis further supports the clinical value of nano-carbon labeling. Kaplan-Meier estimates showed significantly higher DFS and OS in the nano-carbon group at the 24-month follow-up, with divergence most evident between 12 and 24 months – a critical window for recurrence risk [27]. Multivariate Cox regression confirmed that nano-carbon labeling is independently associated with improved long-term prognosis. Regardless of adjustments for age and tumor stage, patients in the nano-carbon group had considerably lower hazard ratios for recurrence and mortality, underscoring the oncologic benefit of enhanced lymph node clearance [28]. The absolute improvements in DFS (11%) and OS (9%) at 24 months suggest clinically meaningful advantages, supporting routine consideration of this technique. Given the high recurrence potential of colorectal cancer, particularly in patients with micrometastatic disease, the improved dissection accuracy facilitated by nano-carbon tracers provides additional assurance during surgical intervention. These findings reinforce the value of the technique for improving curative resection efficacy and supporting long-term oncologic outcomes [29]. Additionally, the present study identified significantly lower incidences of postoperative infection and DVT in the nano-carbon group compared with the control group. The absolute reductions of 4% (number needed to treat=25) for infection and 3% (number needed to treat=33) for DVT indicate that the benefits are clinically meaningful. These improvements are likely attributable to more accurate surgical targeting and reduced iatrogenic trauma enabled by precise lymph node mapping [30]. By localizing nodes effectively, surgeons can avoid unnecessary tissue disruption, thus reducing the incidence of complications and improving surgical safety. Beyond oncologic outcomes, nano-carbon labeling demonstrated measurable benefits in postoperative recovery and patient well-being. Patients in the experimental group reported higher scores in physical and role functioning and overall health status, with correspondingly lower fatigue and pain scores. These improvements were particularly pronounced at the 12-month follow-up, indicating sustained benefits for recovery and daily functioning [31,32]. Although the mean improvement of approximately 6 points in global QoL did not exceed the minimum clinically important difference threshold across all domains, the consistent positive trend across multiple scales supports a real patient-perceived benefit.

Despite its promising results, this study has several limitations. First, the sample size, although sufficient to detect differences in nodal yield and survival, was underpowered to assess rare complications or support subgroup analyses. Second, the follow-up period was limited to 24 months, restricting conclusions regarding long-term (5- to 10-year) survival. Third, staining intensity was scored subjectively, although reliability was good (κ=0.82), and image-based quantification may provide greater objectivity. Fourth, surgeons were not blinded to group allocation; outcomes may vary according to operator expertise and institutional protocols. Fifth, although multiplicity adjustments (false discovery rate) were applied to secondary endpoints, residual type I error risk cannot be excluded. Finally, the exclusion of patients older than 75 years may limit generalizability to older populations. Larger multicenter trials with extended follow-up are needed to validate and expand these findings across more diverse patient groups. From a safety perspective, no tracer-related adverse effects were observed. However, theoretical risks of local pigment deposition, lymphatic obstruction, or inflammatory reactions have been described in animal models [33]. Future studies should incorporate standardized adverse-event monitoring specific to nano-carbon use to further define its safety profile. Additional research should also aim to optimize the formulation and stability of nano-carbon tracers to ensure uniform performance and facilitate intraoperative use. Standardized surgeon training programs and consensus protocols for tracer administration would help reduce variability in outcomes. With confirmation in larger multicenter cohorts, nano-carbon tracing may become an integral adjunct to minimally invasive colorectal cancer surgery, improving surgical precision, safety, and long-term patient outcomes.

Conclusions

Endoscopy-assisted nano-carbon labeling enhances the precision of lymph node dissection in minimally invasive colorectal cancer surgery, improving nodal yield, short-term survival, and postoperative recovery. The technique was associated with lower rates of infection and DVT, as well as higher QoL scores, reflecting both surgical and patient-centered benefits. Despite the limited sample size and 24-month follow-up period, the findings support nano-carbon labeling as a safe, effective adjunct that may be integrated into routine surgical practice to optimize oncologic outcomes.