Assessment of sensitivity of low-cost fluorescein as an alternative to radiocolloid for sentinel lymph node biopsy in breast carcinoma

Abstract

Background

Sentinel lymph node biopsy (SLNB) is important in the treatment and staging of early-stage breast carcinoma. Traditional methods, including blue dyes and radiocolloids, are limited by high cost, radiation safety concerns, and reliance on nuclear medicine centers. Low-cost fluorescent dye, fluorescein, has been suggested as a potential alternative due to its safety, ease of visualization, and low cost. This study aimed to find the sensitivity of fluorescein as an alternative to radiocolloid in SLNB for breast carcinoma.

Materials and methods

A prospective observational study was done at a tertiary care teaching hospital in 50 female patients of early-stage breast carcinoma. Intraoperative injection of fluorescein and visualization with ultraviolet (UV) light were done. Sentinel nodes were detected with a hand-held UV light source.

Results

Fluorescein detected sentinel nodes in 96% of patients. Fluorescein had a sensitivity of 96%, specificity of 98%, and 4.2% false-negative rate (FNR). Negative predictive value (NPV) was 98.5%. No significant adverse effects were noted.

Conclusions

Fluorescein is a safe, cost-effective, and efficient replacement for radiocolloid in SLNB in breast cancer. It provides outstanding identification rates, sensitivity, and specificity and can be utilized to the best in low-resource settings. Multicenter trials are required to confirm these findings and promote more widespread adoption of fluorescein-based SLNB in clinical care.

Introduction

Sentinel lymph node biopsy (SLNB) is a significant procedure in the surgical management of breast carcinoma, which allows for the detection of the first lymph node(s) draining the area of the primary tumor. It minimizes the need for radical axillary lymph node dissection (ALND), which has high complication risks such as lymphedema, nerve injury, and loss of shoulder mobility. Traditionally, SLNB has been achieved with blue dyes and radiocolloids that are very powerful in sentinel node localization. Radiocolloid uses a radioactive tracer to tag sentinel nodes and, when blended with blue dye, it couples visual and radioactive verification of drainage lymphatic. While these methods are very effective, they are also faced with challenges, such as patient and health workers’ radiation exposure, regulatory considerations, and logistical challenges in managing radioactive substances. Additionally, radiocolloid costs and the need for advanced nuclear medicine facilities limit their use, particularly in impoverished environments [1, 2].

Due to these limitations, there has been increasing interest in other, safer, less expensive, and less complicated alternatives to SLNB. One such alternative is fluorescein, a synthetic organic chemical that emits a bright green fluorescence when exposed to ultraviolet light. Fluorescein, widely used in ophthalmology, has an established safety profile and is extremely well tolerated by patients with little risk of reaction. In contrast to radiocolloids, fluorescein does not need to be stored or handled in a special manner, obviating the need for radiation safety procedures. This is especially valuable in low-resource settings where nuclear medicine facilities are not present. Additionally, fluorescein allows for real-time visualization of lymphatic drainage, allowing for instant feedback to the surgeon and enhancing the precision of sentinel node identification [2, 3].

Several studies have found fluorescein to be a suitable alternative for radiocolloids in SLNB with similar sensitivity and specificity for sentinel node mapping. Research shows that fluorescein has excellent detection rates with minimal side effects, thereby making it a highly viable and cost-effective choice. In addition, the capability of fluorescein to give instant feedback allows an individual to make real-time changes during surgery, thereby improving the success rate of sentinel node identification. However, problems remain, that is, with the penetration of fluorescein fluorescence, which may affect the detection of sentinel nodes in more deeply located or large tumors. The scattering and absorption by tissue may also impair the fluorescence signal, and optimization of imaging modalities may be needed to provide reproducible results [4, 5].

Its addition to routine SLNB practice will require multicenter, large-scale clinical trials to determine its safety and efficacy. Such studies will help to create guidelines for fluorescein injection, imaging, and preparation, as well as determine long-term safety compared with radiocolloids [6]. Training programs will be required to allow for the safe and effective use of fluorescein in clinical practice. Furthermore, elimination of radiation exposure in the SLNB procedure should also increase patient satisfaction and reduce anxiety, particularly in those concerned about the long-term health hazards of radiation [7, 8].

Fluorescein dye

The fundamental principle of fluorescence is that certain substances absorb light at one wavelength (usually ultraviolet or blue light) and re-emit it at a longer wavelength, typically in the visible spectrum. This emitted light is what enables imaging systems to visualize the targeted tissue or structure. Fluorescein is usually indicated in diagnostic fluorescein angiography or angioscopy of the fundus and of iris vasculature.

Advantages

1. It has the ability to passively diffuse into the lymphatic system and remain within the lymphatic vessels for a sufficient amount of time to allow detection — a key factor in its effectiveness.

2. Real-time visualization of the lymphatic system and sentinel nodes during surgery.

3. Ability to be detected at low concentrations due to its high fluorescence quantum yield.

4. Does not require a complex imaging system or expensive equipment.

5. Its safety and cost-effectiveness.

6. Only transient skin and urine discoloration, resolved within 12–24 hours.

7. No adverse side-effects.

Aim

The current research aims to determine the sensitivity of fluorescein as an alternative to radiocolloids in SLNB for breast cancer.

Materials and methods

This was a prospective observational study for evaluating the sensitivity of low-cost fluorescein sodium as an alternative to radiocolloid for SLNB in breast carcinoma patients. This was conducted at a tertiary care teaching hospital with advanced departments of surgical oncology and pathology, the study spanned from April 2023 to October 2024. Participants were selected based on strict inclusion and exclusion criteria to ensure a homogeneous study population. Patients aged over 18, early-stage breast carcinoma (cT1–T3), clinically and radiologically node-negative axilla (cN0), and who signed an informed consent for participating in the study, were included in the study while pregnant women, women who had prior history of breast surgery, chemotherapy or radiotherapy, or those who experienced a hypersensitivity reaction to any of the tracers were excluded.

Consecutive sampling was employed, and the sample size was determined to be 50 patients based on power calculations for statistical significance.

The study followed a standard protocol. Preoperative workup included clinical examination and imaging studies. Sub-areolar and peritumor injections of fluorescein sodium (4%) were given, with massaging done afterward to facilitate dye diffusion. Intraoperative UV light of 480 nm wavelength illuminated fluorescent lymphatics for the detection of sentinel lymph nodes (SLNs). Resected SLNs were sent for frozen section and histopathology to corroborate findings. Data were collected via structured case records, documenting demographic, clinical, procedural, and histopathological data, and stored on a secure password-protected database to ensure patient confidentiality.

Informed consent was obtained from the patients and their guardian. Institutional ethical clearance was obtained prior to publication, vide letter no. SNMC/IEC/2023/220.

Statistical analysis

Statistical tests like Chi-square or Fisher’s exact test for categorical variables and Student’s t-test for continuous variables were used to analyze the data via IBM^® SPSS^® software platform. p-value < 0.05 was considered statistically significant. Primary focus was on the sentinel lymph node identification rate (SLN-IR), false negative rate (FNR), and concordance between intraoperative (frozen section) and final histopathological findings. Data quality was maintained by regular checking for completeness and accuracy.

Results

The concordance of intraoperative SLN identification and eventual histopathologic findings was investigated. Of 48 intraoperative SLNs detected, 46 (95.8%) were concordant with histopathology, whereas 2 (4.2%) were discordant. Such high concordance was strong evidence for the efficacy of fluorescein-guided SLNB in accurately predicting the absence or presence of metastasis. Such a high correlation between intraoperative and histopathological findings proved diagnostic validity of the technique. These findings were essential to verify the validity of the procedure in order that intraoperative decisions made with fluorescein visualization could be correlated with definitive pathological correlation (Tab. 1).

Table 1.

Concordance between intraoperative findings and histopathology

Concordance parameter	Value
Total cases evaluated	48
Concordant results	46 (95.8%)
Discordant results	2 (4.2%)

Figure 1.

The ultraviolet (UV) light and the fluorescein dye

Tumor staging revealed that 40% of the patients belonged to T1 class, 44% belonged to T2 class, and 16% belonged to T3 class. Additionally, tumor grading revealed that 20% were Grade I tumors, 56% were Grade II, and 24% were Grade III tumors. These observations were important to correlate the tumor burden with the performance of the SLNB procedure. The majority of the tumors were intermediate grade and early stage (T1 and T2), as would be expected in patients being considered for breast-conserving surgery and SLNB (Tab. 2).

Table 2.

Clinical and tumor characteristics

Parameter	n (%)
Tumor stage
T1	20 (40%)
T2	22 (44%)
T3	8 (16%)

Figure 2.

The fluorescein sentinel lymph node seen under ultraviolet (UV) light

Intraoperative mapping procedure was also standardized for consistency. The solution of fluorescein sodium 4% was utilized, and 5 mL each was injected both at sub-areolar and peri-tumoral sites. A five-minute massage was given after each injection for the purpose of dye dispersion. Then breast-conserving surgery (BCS) or modified radical mastectomy (MRM) was done followed by axillary lymph node dissection. The mean time for lymphatic visualization was 29 ± 8.5 minutes. All these parameters confirmed that the method was rapid as well as reproducible. The brief time frame between injection and observation allowed efficient surgical workflow. These mapping data were essential to ensure that the fluorescein method was viable and did not add significantly to surgery delays, thereby supporting its clinical use (Tab. 3).

Table 3.

Intraoperative mapping: injection parameters and timing

Parameter	Value
Fluorescein concentration	4%
Volume injected per site	5 mL (sub-areolar and peri-tumoral)
Massage duration	5 minutes
Mean time to visualization	29 ± 8.5 minutes

The fluorescein-guided sentinel lymph node biopsy (SLNB) procedure was very effective, with successful identification of SLN in 96% of the patients (48/50), demonstrating a great identification rate and an acceptable learning curve sufficient for various patient populations. The majority of the patients had one or two SLNs resection (mean 1.8 ± 0.7), demonstrating procedural efficiency and conformity with standard SLNB procedures without undue lymph node resection. Histopathological analysis of 86 resected nodes revealed 24% positivity for metastasis, validating the diagnostic effectiveness and correlation of the method with clinical staging (Tab. 4).

Table 4.

Sentinel lymph node (SLN) biopsy outcomes, retrieval patterns, and histopathological findings using fluorescein method

Parameters		Value	Percentage
Outcome	SLN identified	48	96%
	SLN not identified	2	4%
Number of nodes retrieved	1	25	50%
	2	15	30%
	≥ 3	10	20%
	Mean (± SD)	1.8 ± 0.7	–
Histopathological finding	SLN positive for metastasis	12	24%
	SLN negative for metastasis	38	76%

SD standard deviation

The diagnostic efficacy of the fluorescein-guided sentinel lymph node biopsy (SLNB) technique was extremely high, with sensitivity of 95.8% and specificity of 98.0%, indicating its high ability to accurately identify true positive and true negative cases, respectively. The positive predictive value (95.0%) and negative predictive value (98.5%) also indicate the accuracy of the method in predicting the presence or absence of metastasis. With 96% identification rate of SLN and low false negative rate of 4.2% based on merely two false negative cases, data evidence shows that the fluorescein method is effective and efficient in intraoperative SLN detection and metastatic assessment (Tab. 5).

Table 5.

Diagnostic performance: sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and false-negative rate (FNR)

Diagnostic metric	Value
Sensitivity	95.8%
Specificity	98.0%
PPV	95.0%
NPV	98.5%
SLN identification rate	96%
False negative cases	2
Calculated FNR	4.2%

SLN — sentinel lymph node

Fluorescein-guided SLNB was not beset by significant complications, but its safety profile was well demonstrated. Transient difficulty with node visualization in 4%. Postoperative complications were also few with 4% wound infection, seroma collection in 4%, lymphorrhea in only 2% of the patients and the remaining 90% having an uneventful postoperative course. Such a result indicates that the method is not only effective but even extremely well tolerated without much scope for a complication to develop from the technique (Tab. 6).

Table 6.

Complications

Complication		n (%)
Intraoperative	Transient difficulty in node visualization	2 (4%)
	No complications	48 (96%)
Postoperative	Wound infection	2 (4%)
	Seroma formation	2 (4%)
	Lymphorrhea	1 (2%)
	No complications	45 (90%)

Adverse reactions of the fluorescein dye were monitored both during and after the procedure. Two patients (4%) experienced transient skin discoloration, and 1 patient (2%) had a mild allergic reaction (pruritus). None of the other 47 patients (94%) had any adverse reactions to the dye (Tab. 7).

Table 7.

Adverse reactions to fluorescein

Adverse reaction	n (%)
Transient skin discoloration	2 (4%)
Mild allergic reaction (pruritus)	1 (2%)
No adverse reaction	47 (94%)

Table 8.

Lymphatic mapping

	Radiocolloid + methylene blue	Fluorescein dye + methylene blue
Cost	High	Low
Facilities	Institution level	Can be done in resource limited setting
Radiation exposure	Present	Absent
Sensitivity	97%	95.8%
Specificity	96%	98.0%
NPV	97.6%	98.5%
Nuclear medicine facilities	Required	Not required
		Hence, a cheap alternative to LN mapping with widespread application

NPV — negative predictive value; LN — lymph node

Table 9.

Types and characteristics of studies

Author/Year	Study type	Study findings	Strengths	Limitations
Ren et al. (2016) [9]	Animal study	Fluorescein sodium had higher detection rate and longer fading time than isosulfan blue in SLNB in rabbits	First preclinical demonstration of fluorescein’s efficacy; well-controlled setting	Animal model; human applicability needs validation
Cardoso-Coelho et al. (2017) [10]	Clinical diagnostic comparison study	US-guided FNA had 45% sensitivity, 100% specificity, and 73% NPV; not sufficient to replace SLNB	Simple, non-invasive, highly specific	Low sensitivity limits its standalone utility in staging
Srivastava et al. (2018) [11]	Randomized controlled trial	Fluorescein + methylene blue (FWMB) was non-inferior to technetium + methylene blue (TWMB); equal detection rate (84.6%)	Large sample size; rigorous design; supports low-cost alternative	Limited to non-radioactive comparison; generalizability in diverse settings not fully explored
Chang et al. (2019) [12]	Experimental clinical study	Fluorescein with blue LED achieved clear SLN visualization, comparable to radioisotope methods	Avoids radiation; cost-effective; high sensitivity and specificity	Requires specific lighting setup; results not compared to technetium quantitatively
Agarwal et al. (2020) [13]	Prospective comparative validation study	Fluorescein-guided SLNB closely matched detection rate of technetium-99m method; reduced delays, safe	Practical and economical; ideal for low-resource settings	No long-term outcome data; single-center study
Salmon and Benna (2020) [14]	Feasibility study	Fluorescein identified SLNs with high accuracy; low cost and low toxicity; suitable for LMICs	Highlights accessibility and safety in LMICs; supports resource optimization	Lacks quantitative performance metrics; mostly descriptive findings
Ivković-Kapicl et al. (2020) [15]	Diagnostic accuracy study	Imprint cytology comparable to frozen section in sensitivity and specificity; faster and less costly	Efficient, enables intraoperative decisions; ideal where frozen section not available	Not focused on tracer type; limited to SLN pathology assessment
Li et al. (2021) [16]	Clinical study	Fluorescein + methylene blue improved SLN visibility and reduced false-negatives; safe with no major adverse effects	Combines affordability with enhanced detection; well-tolerated	Needs larger multicenter trials for widespread adoption
Khadka et al. (2022) [17]	Randomized comparative study	Fluorescein-guided SLNB had similar detection rates to technetium + methylene blue; safe, cost-effective	Supports shift to non-radioactive alternatives; practical for underserved hospitals	Single-center; long-term oncological outcomes not assessed
Jha et al. (2024) [18]	Clinical validation study	Fluorescein sodium + methylene blue improved SLN accuracy and reduced false-negatives; affordable	Good intraoperative visibility; easy implementation	Limited by sample size and potential surgeon learning curve
Manjunath et al. (2024) [19]	Clinical feasibility study	Fluorescein sodium showed excellent visibility, minimal adverse effects, and better safety profile than methylene blue	Emphasizes patient safety and effectiveness; ideal for high-risk patients	Comparative statistical significance data not elaborated

SLNB — sentinel lymph node biopsy; FNA — fine needle aspiration; US — ultrasound; NPV — negative predictive value; LED — light-emitting diode; SLN — sentinel lymph node; LMICs — low- and middle-income countries

Discussion

The intraoperative to histopathological concordance rate was 95.8%, indicating that the fluorescein-guided technique accurately identified sentinel lymph nodes (SLNs) that were both non-metastatic and metastatic. These results are consistent with studies like those conducted by Jha et al. (2024) [18] and Li et al. (2021) [16], which add to the credibility of the effectiveness and reliability of fluorescein-guided SLNB. Such high concordance supports the use of fluorescein sodium as an effective tracer for SLNB.

The aim of this research was to evaluate the safety, effectiveness, and diagnostic efficacy of fluorescein sodium as a non-radioactive alternative for radiocolloid-based sentinel lymph node biopsy (SLNB) in breast cancer patients. Specifically, this research was intended to assess the viability of fluorescein sodium to label sentinel lymph nodes (SLNs), to measure the diagnostic accuracy (sensitivity, specificity, false negative rate), and to establish the impact on operating time and complication rate of the procedure. The research also aimed to explore the potential advantages of fluorescein sodium in the resource-constrained setting, where radioactive tracers and nuclear medicine facilities may be restrictive.

The present work highlights the utility of fluorescein-guided sentinel lymph node biopsy (SLNB) in patients with breast cancer who report early, whose high success rate of 96% mirrors the experience of Srivastava et al. (2018) [11] and Agarwal et al. (2020) [13]. T1/T2 and grade-II tumors predominated among most of the patients, as per the general selection criterion of SLNB, where accurate staging of nodes goes a long way in mapping treatment plans.

Intraoperative fluorescein mapping was rapid (mean time 29 minutes) with good visualization and minimal complications, the same as supported by Jha et al. (2024) [18] and Salmon and Benna (2020) [14], that emphasize fluorescein’s utility benefit. The two-site injection technique also enhanced reproducibility, supporting the feasibility and diagnostic value of the technique in various clinical settings.

Studies by Li et al. (2021) [16] and Jha et al. (2024) [18] also emphasized the combination of fluorescein with methylene blue for enhanced identification of SLNs and minimizing false negatives, pointing towards fluorescein’s use in accurate axillary staging without radioactive isotopes.

In addition, the mean number of SLNs retrieved (1.8 ± 0.7) in this study conforms to findings of Salmon and Benna (2020) [14] and Khadka et al. (2022) [17], which confirm that fluorescein-guided SLNB enables effective node identification without unnecessary resection. Histopathological evaluation established 24% SLN positivity for metastasis in concordance with existing literature and maintaining the accuracy of fluorescein in diagnosis. The strong correlation between intraoperative findings and histopathology, reported by Agarwal et al. (2020) [13] and Srivasatava et al. (2018) [11], supports the effectiveness of fluorescein-guided SLNB in early breast cancer, with accurate staging and optimal treatment planning.

Fluorescein-guided SLNB technique was very reliable in this research with low false negative rate (FNR) of 4.2%, as also shown by Jha et al. (2024) [18] and Li et al. (2021) [16]. Diagnostic efficacy was maximal, with 95.8% sensitivity, 98.0% specificity, 95.0% PPV, and 98.5% NPV based on results from studies by Agarwal et al. (2020) [13] and Khadka et al. (2022) [17]. These results affirm that fluorescein, particularly with the addition of methylene blue, is extremely accurate, affordable and safe in comparison to conventional radiocolloid techniques in sentinel lymph node detection in breast cancer, worth its while in resource-limited environments.

Intraoperative and postoperative complications were minimal, again confirming the safety of fluorescein-guided SLNB. There was a minor intraoperative complication of transient visualization problems (4%). Postoperatively, 90% of patients experienced no complications, with minimal wound infection (4%), seroma (4%), and lymphorrhea (2%) — all within expectations for standard SLNB procedures. These findings are in line with existing studies, e.g., by Khadka et al. (2022) [17] and Li et al. (2021) [16], to further consolidate the safety and feasibility of fluorescein.

The adverse effects of fluorescein sodium in our study were minimal, with 4% of the patients experiencing transient skin discoloration and 2% experiencing mild pruritus. These effects were self-limiting and required no additional treatment, emphasizing fluorescein’s spotless safety profile as a tracer for sentinel lymph node biopsy (SLNB). These findings are in agreement with those of previous studies, such as Salmon and Benna (2020) [13], in supporting the low incidence of side effects.

There are certain limitations of this study, including relatively small size of 50 patients, which can have an effect on the generalizability of the results, and that it was conducted in one institution, which may cause site-specific biases. To obtain a more robust and widely applicable result, future research should design a muti-centric study [20]. At present, the study is being conducted in AIIMS Deogarh.

Conclusions

This study demonstrates that fluorescein sodium is an effective and safe non-radioactive alternative to the standard radiocolloid method for SLNB in breast cancer patients, with a high SLN identification rate of 96%, a low false negative rate of 4.2%, and minimal complications. The method, having a mean operative time of 29 minutes, is not time-consuming and can be integrated into routine surgical practice. Despite the limited number of patients in the study and the lack of long-term follow-up, the study constitutes good evidence for the safety and efficacy of fluorescein-guided SLNB. Additional research should comprise bigger, multi-center studies to validate these findings and examine its use in other cancers.