Combined Application of Technetium-99m Sulfur Colloid and Blue Dye for Sentinel Lymph Node Detection in Early-stage Breast Cancer

Summary

This article describes in detail the protocol of sentinel lymph node (SLN) mapping with combined application of technetium-99m sulfur colloid (^99mTc-SC) and methylene blue, including the preparation of ^99mTc-SC, drug injection, image acquisition and reconstruction, methylene blue mapping, and intraoperative localization of SLNs.

Abstract

Sentinel lymph node biopsy (SLNB) is the standard procedure for axillary lymph node staging in breast cancer patients, and its application has prevented unnecessary axillary lymph node dissection (ALND) in early-stage breast cancer in the past decades. Accurate sentinel lymph nodes (SLNs) localization helps improve the success rate of SLNB. The dual-tracer technique (radiocolloid plus blue dye) for SLN mapping significantly improves the identification rate compared to single tracer methods. The most widely adopted radiocolloid is ^99mTc-SC, and methylene blue is widely used in visual mapping. However, previous studies have found that the identification rate of SLN imaging varies significantly, which may be closely related to the preparation process and the operator's experience.Standardization of the SLN mapping protocol is crucial for maximizing the identification rate and minimizing false-negative results. This article provides a detailed process using ^99mTc - SC combined with methylene blue for SLN mapping in early-stage breast cancer, including procedures for the preparation of ^99mTc-SC, injection, SLN image acquisition, methylene blue mapping and intraoperative localization.

Introduction

In early-stage breast cancer, only a minority of cases present with axillary lymph node metastasis. For these patients, axillary lymph node dissection (ALND) does not provide additional survival benefits and may lead to severe complications such as upper limb lymphedema^1,2. The sentinel lymph node (SLN) is defined as the "first-station" lymph node receiving lymphatic drainage from the breast tumor³. If the SLN shows no metastasis, the probability of metastasis in downstream lymph nodes is extremely low⁴. According to the 2021 guidelines titled Management of the Axilla in Early-Stage Breast Cancer: Ontario Health (Cancer Care Ontario) and ASCO Guideline¹, patients with negative sentinel lymph node biopsy (SLNB) results can be spared ALND, while those with 1-2 metastatic SLNs may avoid ALND under specific conditions. Studies have demonstrated that SLNB spares approximately 70% of early-stage breast cancer patients from unnecessary ALND^5,6,7. Therefore, SLN detection plays a crucial role in determining the necessity of ALND, significantly improving postoperative quality of life¹.

Different studies have explored ultrasound-guided lymph node biopsy to determine nodal metastasis in breast cancer patients, but this method yields a high false-negative rate (FNR) of 6.4%-40.8%⁴. Moreover, requiring the presence of an experienced sonographer during the operation poses significant challenges for widespread clinical implementation. A variety of tracers are available for SLN mapping in breast cancer^8,9,10, radiocolloid and/or blue dyes, fluorescein, indocyanine green (ICG), and mitoxantrone hydrochloride injection. Tausch et al. reported an identification rate (IR) of 82% with blue dye alone, 85% with radiocolloid alone, and 94% when dual tracers (radiocolloid plus blue dye) were used¹¹. A meta-analysis of 13 studies revealed comparable IR for radiocolloid (96%) and blue dye (96%), while the combination achieved 97%¹². Combined tracer administration significantly reduces false-negative rates (FNR), with radiocolloid-only approaches exhibiting FNRs of 16%-20.3% versus 5.2%-10.8% for dual-tracer^{13,14,15,16,17}. Fluorescein is non-inferior to ^99mTc-SC and is more cost-effective. However, its poor tissue penetration limits its application in detecting deeper SLNs¹⁸. ICG demonstrates diagnostic efficacy comparable to ^99mTc- SC, making it particularly suitable for institutions lacking nuclear medicine resources^19,20. Nevertheless, its efficacy is low in patients with BMI > 30 or centrally located tumors²¹. Some studies have confirmed that the diagnostic performance of mitoxantrone hydrochloride injection is similar to ^99mTc- SC for SLN mapping^8,10. However, the limited number of studies necessitates further clinical validation.

Although there are many imaging agents available for detecting SLNs in recent decades, the guidelines still recommend the combination of radiocolloid and blue dye for SLN localization in breast cancer, owing to its high diagnostic efficacy, substantial evidence-based medical support, and minimal severe adverse reactions^1,20,22. Alternative agents are fluorescein or ICG without support from a nuclear medicine department. ^99mTc-SC is the most commonly used radiocolloid. Compared with other blue dyes, methylene blue is more commonly used in most centers in Asia²³, because it demonstrates similar SLN localization efficacy to isosulfan blue or patent blue, but is morecost-effective.

Reported IR of SLN with either radiocolloid or blue dye alone differ significantly (from 69.8% to 90.8%) across studies and are highly dependent on procedural protocols and operator experience^9,12,13,24. Therefore, standardized operational procedures are crucial for ensuring consistent IR. Several different protocols are currently available, which complicates standardization efforts. First is the heating time during the preparation of ^99mTc-SC. Studies have shown that reduced 3 min heating can improve the synthesis efficiency of ^99mTc-SC²⁵. ^99mTc-SC prepared by a reduced 3 min heating had comparable radiochemical purity (RCP) to the standard 5 min-heating (both >92%) at 0 h and 6 h²⁶. Furthermore, ^99mTc-SC of 3 min heating had greater lymph node retention at 24 h²⁷ . Therefore, we consider the 3 min heating protocol is superior. Second, periareolar and peritumoral injections are recommended for ^99mTc-SC administration. Studies demonstrate that the periareolar injection had a higher IR for axillary SLN compared to the peritumoral injection (99.3% vs. 91.1%, P < .001)²⁸. The periareolar technique is technically simpler and shows particular advantages for non-palpable tumors or lesions in the upper outer quadrant, mainly by preventing the "shine-through" phenomenon²⁹. Additionally, periareolar injection naturally covers lymphatic drainage from all breast quadrants, potentially reducing the missed detection rate of SLNs and proving particularly beneficial in multifocal tumors^30,31. Hence, we consider the periareolar injection to be more advantageous.

In this article, we adopted a dual-tracer protocol of preoperative ^99mTc-SC (periareolar injection) combined with intraoperative methylene blue (periareolar injection). ^99mTc-SC was prepared using a reduced 3 min heating protocol.

Protocol

This study was approved by the Clinical Research Approval Committee of the First Affiliated Hospital, Zhejiang University School of Medicine [2025B] IIT Ethics Approval No.0125. Written informed consent was obtained from all subjects. The entire synthesis procedure must be conducted inside a fume hood. The lead glass panel of the hood provides shielding against radiation, while its negative pressure airflow design prevents the dispersion of radioactive aerosols or volatiles, thereby avoiding inhalation by personnel. Dispose of vials, syringes, and needles in a ^99mTc radioactive waste container in accordance with relevant regulations. Other materials should be handled as regular medical waste. Maintain radiation protection and patient privacy throughout the process.

1. Patient selection

NOTE: According to the 2021 guidelines titled Management of the Axilla in Early-Stage Breast Cancer: Ontario Health (Cancer Care Ontario) and ASCO Guidelines, early-stage breast cancer patients requiring SLN localization are identified by the surgeon¹. SLN mapping of ^99mTc-SC was performed 3-18 h prior to surgery.

1. Set the following inclusion criteria: (1) Early-stage breast cancer with clinical axillary lymph nodes (ALNs) negative (cN0). cN0 is defined as negative results in clinical examination and imaging, or ALN was pathologically negative by ultrasound-guided needle biopsy. (2) Negative ALN after neoadjuvant therapy in cN0 patients. (3) ALN metastasis confirmed by biopsy in cN1 patients, and clinically negative after neoadjuvant therapy.
2. Set the following exclusion criteria: (1) Inflammatory breast cancer. (2) Patients with metastatic ALN confirmed by biopsy without neoadjuvant therapy. (3) Patients with positive ALN metastasis and still positive after neoadjuvant therapy.

2. Preparation of ^99mTc- SC

1. Preparations required prior to the labeling of ^99mTc-SC
   1. Pre-order the kit of the prodrug comprising three vials: Vial A containing 2.0 mL of 0.15 mol/L hydrochloric acid (HCl); Vial B containing 2.0 mL of a buffer solution composed of 49.2 mg of sodium dihydrogen phosphate (NaH₂PO₄) and 15.8 mg of sodium hydroxide (NaOH); Vial C, a lyophilized product containing a white, water-soluble powder composed of 2.0 mg of sodium thiosulfate (Na₂S₂O₃), 2.3 mg of edetate disodium (Na₂EDTA), and 18.1 mg of gelatin. Store the kit in a 4 °C refrigerator.
   2. Thawing the kit: Remove it 1 h before drug preparation to reach room temperature.
   3. Ordering technetium-99m on the day of imaging: Ensure that the technetium-99m injection is eluted from a Mo-Tc generator within 24 h and is free of oxidizing agents.
   4. Preparation of the water bath: Fill the water bath with sufficient water and heat it to 100 °C, maintaining a boiling state.
      NOTE: The ordered sodium pertechnetate [^99mTc] requires sufficient activity to account for synthesis loss, and an activity of 185 MBq per patient is recommended for ordering, with a total volume of 0.5-1 mL. When preparing for multiple patients, the total dosage of ^99mTc should be increased in accordance with the number of patients, with a total volume not exceeding 1 mL and a radioactive concentration not exceeding 18.5 GBq/mL.
2. Labeling process
   1. Prepare the ^99mTc-SC injection under radiation protection conditions, typically in a fume hood. Under aseptic conditions, inject sodium pertechnetate [^99mTc] injection into Vial C. Shake thoroughly to dissolve the lyophilized powder and let it stand for 5 min.
      ​NOTE: Perform all operations under aseptic conditions and disinfect before each withdrawal of the solution. If disinfectant swabs are contaminated with radionuclides, dispose of them as radioactive waste. Ensure that air is strictly expelled when injecting ^99mTc into the kit vials. Maintain negative pressure in the vials until the final step of solution withdrawal.
   2. Draw 1.5 mL of solution from Vial A and inject it into the reaction vial. Mix well and let it stand for another 5 min to obtain mixture D.
   3. Boiling water bath: Place the vial containing mixture D into the water bath and heat for 3 min (Figure 1A).
      NOTE: The heating time should not exceed 3 min, as prolonged heating significantly may reduce the percentage of particles < 0.2 µm ^99mTc-SC^25,26,27. Use forceps to handle the vials after boiling to prevent burns.
   4. Prepare mixture E: Remove the vial containing mixture D and allow it to cool for 2-5 min. Then, draw 1.5 mL of solution from Vial B and inject it into the vial containing mixture D. Mix thoroughly to obtain mixture E. Use a 5 mL syringe to draw the entire mixture E.
   5. Filtration: Attach a dedicated filter to the syringe. Replace the needle and insert it into a vacuum bottle. Slowly filter mixture E without applying pressure to obtain the ^99mTc-SC solution (Figure 1B).
3. Preparation of the final injection:
   1. Use a 2 mL syringe to draw the ^99mTc-SC solution. Ensure that each patient receives 37-55.5 MBq in a total volume of 0.4-1.0 mL, preferably close to 0.1 mL per site to minimize injection discomfort. After drawing the solution, replace the needle with a 4.5-gauge (26 G) needle for administration.
      NOTE: A total injected dose of 37 MBq is generally considered sufficient³².

3. Quality control of ^99mTc-SC

NOTE: A small aliquot of the radiopharmaceutical is aspirated into a syringe for quality control testing prior to use.

1. Physicochemical properties
   1. Confirm that the prepared injectate is a gelatin-stabilized, sterile, and pyrogen-free colloidal suspension suitable for intravenous administration. According to the requirements of the United States Pharmacopeia (USP)³³, ensure that the pH ranges between 4.5 and 7.5.
      NOTE: The pH value of ^99mTc-SC synthesized in this study was ~6.0.
2. Radiochemical purity (RCP)
   1. Perform the determination of radiochemical purity (RCP) of 0 h and 6 h samples from both the 3 min and 5 min heating protocols in accordance with the requirements of USP³³.
      1. Withdraw 0.1 mL ^99mTc-SC injection of 3 min heating and 5 min heating at 0 h and 6 h after preparation, then appropriately dilute it (approximately 1:10).
      2. Spot the diluted injections using a 10 µL capillary tube onto a chromatographic paper strip (25 mm × 300 mm), at a point approximately 20 mm from one end, and then allow to dry.
      3. Using 85% methanol as the mobile phase, develop the chromatogram by the ascending chromatography technique over a suitable period and dry.
      4. Determine the radioactive distribution on the chromatogram by scanning with a radioactive scanner. Calculate the RCP using the formula: RCP = 100% - (% free pertechnetate).
         NOTE: An RCP value greater than 92% was considered acceptable (Figure 2).If the RCP is less than 92% or any other quality control parameter fails to comply with standards, the injection must be unconditionally reprepared and subjected to requalification testing until all specifications are met.

4. Patient preparation and administration of ^99mTc-SC

1. Confirm patient identity, medical record number, diagnosis, and preoperative breast tumor localization (typically marked by the surgeon).
2. Inform the patient about the sentinel lymph node (SLN) mapping procedure, including potential injection pain, injection sites, and imaging timeline.
3. Measure the radioactive dose of ^99mTc-SC before injection to each patient (Figure 3A) to ensure a reasonable dose, so as to achieve excellent imaging and as little radiation damage to the patient as possible. Ensure each patient receives a total injected dose of 37 MBq in a total volume of 0.4 mL³². Disinfect the skin with iodinated swabs, then administer ^99mTc-SC via subcutaneous injection at the periareolar region (3, 6, 9, and 12 o'clock positions) of the affected breast (Figure 3B).
4. Apply pressure to the injection site with a sterile cotton swab for 5 min. After hemostasis, instruct the patient to perform a gentle breast massage to enhance lymphatic drainage and wait in the holding area for subsequent imaging³².
   NOTE: During the injection of ^99mTc-SC, use lead shields to reduce radiation exposure.

5. Image acquisition and reconstruction

1. For image acquisition³², initiate imaging ~30 min after the injection (usually between 20 and 40 min) with SPECT/CT.
   1. Ask the patient to adopt a supine and lateral decubitus (injected side adjacent to the detector) position, with arms abducted and hands positioned near the head.
   2. Use the following planar imaging parameters: H-mode, low-energy high-resolution (LEHR) collimator, 140 keV photopeak with ±10% window, 256 × 256 matrix, ZOOM 1.0, 300K counts, field of view spanning from the supraclavicular region to the diaphragm.
   3. Perform Tomo fusion imaging immediately if SLNs are visualized on planar imaging. If not, perform delayed imaging at 1 h post injection using the following Tomo fusion imaging Parameters: LEHR collimator, 140 keV photopeak, ±10% window, 128 × 128 matrix, ZOOM 1.0, 60 projections (6° intervals, 16 s/frame). CT Parameters: 120 kV, 150 mA, 2.5 mm slice thickness.
2. For image reconstruction, process images on a workstation to generate planar and fused SPECT/CT datasets. Localize SLNs on fused images, and measure nodal long-axis dimensions.
3. If SLNs are not visualized on ^99mTc-SC imaging, first rule out correctable factors³².
   1. Reconfirm with nursing staff that the injection was administered subcutaneously in the periareolar region rather than deep within the glandular tissue (if incorrect, inject ^99mTc-SC again).
   2. Check for any local compression at the injection site, which may impair lymphatic drainage. If present, release compression immediately, and instruct patients to gently massage the breast to promote lymphatic flow.
   3. In some patients with slow lymphatic drainage, extend the imaging time to 2 h, and if negative, perform an additional preoperative imaging 2 h before the surgery, within 24 h after the injection^34,35. If SLN is still negative after these steps, use methylene blue³⁶.

6. Intraoperative methylene blue mapping and SLN identification

1. On the day of surgery (within 24 h of radiopharmaceutical administration): inject 1 mL of methylene blue into the subcutaneous tissueat the 3,6,9,12 o'clock periareolar position^20,29.
2. After 10-15 min, trace blue-stained lymphatic channels to locate SLNs.
3. Confirm SLNs using a Gamma Detection System (positive signal: the hottest node and any other hot nodes with greater than 10% of counts of the hottest node).
4. Excise SLNs for frozen section analysis and ensure that the final surgical management is guided by the SLN pathologic results.
   NOTE : If the dual-tracer method cannot localize SLNs, a third tracer, such as ICG, can be attempted. However, in most cases, the cause of imaging failure may be factors such as lymph node metastasis, lymphatic obstruction, or disruption of lymphatic drainage due to previous surgery^14,37. Therefore, additional SLN tracing methods may still fail. Consequently, in most cases, current guidelines recommend proceeding with an ALND^20,38, the ultimate responsibility for this surgical decision lies with the operating surgeon.

Results

We successfully synthesized ^99mTc-SC with RCP > 92%, the curve of RCP is shown in Figure 2. We obtained high-quality SLN mapping of ^99mTc-SC to detect SLNs in early-stage breast cancer patients (Figure 4). Methylene blue mapping was helpful for the rapid localization of SLNs during operation (Figure 5). The SLNs were finally identified by a γ-ray detector, avoiding the omission of SLNs in the axillary region...

Discussion

In this study, the combined use of preoperative ^99mTc-SC and intraoperative methylene blue achieved a 99.8% identification rate of SLN, while radiocolloid alone demonstrated a similarly high rate of 99.1%. Thus, surgeons can achieve near-optimal SLN detection rate using radiocolloid alone. However, in cases that radiocolloid mapping failed, methylene blue helped the SLN detection, which aligns with previous SLN mapping studies^9,18,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% saline	China Otsuka Pharmaceutical Co.,Ltd.	24a75B3	Dilution solution
Acrodisc Syringe Filter	PALL	PN4612	0.2 μm Supor Membrane
chromatography paper	Cytiva	09927854	For radiochemical purity analysis
Fume hood	Qingdao Qingdun Medical Technology Co., Ltd.	20150710	Radiation protection in drug synthesis
Glass Spotting Capillary	Sinopharm Chemical Reagent Co.,LtD	20250510	Sample the injection for radiochemical purity analysis
Lead shield	Suzhou Hengkang Medical Devices Co.,Ltd	5mm Pb	Radiation protection in injection
Medical disposable sterilization rubber gloves	Sri Trang Gloves (Thailand) Public Company Limited	F840	Protects hands from contamination.
Methanol	Huzhou Shuanglin Chemical Technology Co.,LtD	20251021	For radiochemical purity analysis
Methylene Blue	Jumpcan Pharmaceutical Co.,Ltd.	240812	Intraoperative Methylene Blue mapping and SLN identification
MiniGita radioactive TLC thin layer scanner	Sepate Technology Co., LtD		TLC for radiochemical purity
needle	Jiangsu Kangbao Medical Equipment Co., Ltd.	20240722	4.5-gauge (26 G)
Neoprobe Gamma Detection System (GDS)	Mammotome	2331609	Intraoperative Sentinel lymph nodes detection
PH test paper	Sinopharm Chemical Regent  Co.,LtD	72002361	range:1-14
SPECT/CT	GE Health Care	Discovery NM/CT 670	Image Acquisition
syringe	Zhejiang Longde Pharmaceutical Co., Ltd.	202404004, 202412014	1 mL, 5 mL
technetium-99m	Beijing Atom High-Tech Co.,Ltd.	241020-111	It should be eluted from a Mo-Tc generator within 24 h, and it must be free of oxidizing agents.
Vial A	Beijing Shihong Pharmaceutical Co., Ltd.	241020	2.0 mL of 0.15 mol/L hydrochloric acid (HCl)
Vial B	Beijing Shihong Pharmaceutical Co., Ltd.	241020	2.0 mL of a buffer solution composed of 49.2 mg of sodium dihydrogen phosphate (NaH2PO4) and 15.8 mg of sodium hydroxide (NaOH).
Vial C	Beijing Shihong Pharmaceutical Co., Ltd.	241020	lyophilized product containing a white, water-soluble powder composed of 2.0 mg of sodium thiosulfate (Na2S2O3), 2.3 mg of disodium edetate (Na2EDTA), and 18.1 mg of gelatin
Xeleris workstation	GE Health Care	Imaging software	Process images to generate planar and fused SPECT/CT datasets, Localize SLNs