Open Veterinary Journal, (2026), Vol. 16(6): 3464-3469

Research Article

10.5455/OVJ.2026.v16.i6.17

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Laparoscopic lymphadenectomy guided by intradermal lymphography with methylene blue and hemosiderin in female dogs with mammary neoplasms

João Pedro Scussel Feranti^1,2*, Roberta do Nascimento Libardoni², Bernardo Nascimento Antunes³,
Vanessa Milech³, Jéssica Tomio² and Maurício Veloso Brun²

¹Department of Veterinary Medicine, Federal University of Pampa (UNIPAMPA), Uruguaiana, Brazil

²Department of Veterinary Medicine, Federal University of Santa Maria (UFSM), Santa Maria, Brazil

³Department of Veterinary Medicine, Federal University of Piauí, Bom Jesus, Brazil

*Corresponding Author: João Pedro Scussel Feranti. Department of Veterinary Medicine, Federal University of Pampa (UNIPAMPA), Uruguaiana, Brazil. Email: joaoferanti [at] unipampa.edu.br

Submitted: 13/11/2025 Revised: 05/05/2026 Accepted: 15/05/2026 Published: 05/06/2026

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Abstract

Background: Mammary tumors are the most common neoplasms in intact female dogs and have significant metastatic potential. Accurate oncologic staging often requires the assessment of regional and deep lymph nodes, including the medial iliac lymph node (MILN), which can be difficult to identify intraoperatively. Lymphatic mapping techniques may facilitate minimally invasive lymphadenectomy.

Aim: This study aimed to evaluate the effectiveness of intradermal lymphatic marking using methylene blue and hemosiderin for laparoscopic identification of the MILN in female dogs with mammary tumors.

Methods: Thirty-two female dogs with mammary neoplasms were prospectively allocated into four groups (n=8 per group) according to dye type (methylene blue or hemosiderin) and injection site (peritumoral at the fifth mammary gland or dorsal torso). No formal sample size calculation was performed, and the study was considered exploratory. All dogs underwent laparoscopic medial iliac lymphadenectomy followed by ovariohysterectomy and unilateral radical mastectomy. The primary outcome was the intraoperative identification of MILN, defined as visible lymphatic marking before removal. The secondary outcomes included time to lymph node visualization, staining intensity, and perioperative complications.

Results: Methylene blue enabled the intraoperative identification of MILN in all cases (100%), with rapid lymphatic visualization typically within 1 minute and strong staining contrast that facilitated laparoscopic dissection. In contrast, hemosiderin resulted in less consistent intraoperative identification (62.5%) and weaker staining, with lymph node visualization occurring only after adipolymphatic tissue removal. The identification time differed significantly between the groups (p=0.016), with faster detection observed in dogs receiving methylene blue. No intraoperative complications or adverse reactions related to dye administration were observed. Histopathological evaluation was performed per lesion (n=36), revealing carcinomas (75%), adenomas (16.7%), and lobular mammary hyperplasia (8.3%). No metastases were detected in the MILNs.

Conclusion: Intradermal methylene blue is a practical, safe, and effective dye for the laparoscopic identification of the MILN in dogs with mammary tumors. Although hemosiderin is a low-cost alternative, its lower staining contrast limits its intraoperative applicability. These findings demonstrate the feasibility of lymphatic mapping to assist minimally invasive lymphadenectomy; however, no conclusions regarding oncologic outcomes or staging accuracy have been drawn.

Keywords: Canine, Lymphatic mapping, Mammary neoplasms, Veterinary oncology, Vital dyes.

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Introduction

Mammary tumors represent the most frequent neoplasm in intact female dogs, accounting for approximately 50% of all neoplasms in female dogs. They are notable for their metastatic potential and impact on prognosis and patient survival (Cassali et al., 2020). Lymphatic dissemination initially occurs to regional lymph nodes, such as the axillary and superficial inguinal nodes, and may reach deeper nodes, such as the medial iliac, especially in advanced stages of the disease (Feranti et al., 2024).

The medial iliac lymph node (MILN) has been recognized as a potential metastatic target in female dogs with malignant mammary neoplasm, and its lymphadenectomy is used for accurate oncologic staging and individualized therapeutic planning, even in the absence of evident macroscopic metastasis (Feranti et al., 2018; Feranti et al., 2024). Laparoscopic lymphadenectomy has emerged as a minimally invasive alternative to conventional access, allowing the resection of deeper lymph nodes with lower morbidity and good diagnostic accuracy (Steffey et al., 2015).

Different methods of lymphatic mapping have been used to facilitate the intraoperative localization of sentinel lymph nodes, including the application of vital dyes such as methylene blue and low-cost alternatives, such as hemosiderin. However, direct comparisons of their efficacy and clinical applicability are still needed (Maués et al., 2016; Antunes et al., 2025). Therefore, this study aimed to evaluate the effectiveness of intradermal lymphatic marking using methylene blue and hemosiderin for laparoscopic lymphadenectomy of the MILN in female dogs with mammary neoplasms, considering identification time, staining intensity, clinical applicability, and procedural safety.

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Materials and Methods

Dog population

This study included 32 female dogs who underwent laparoscopic lymphadenectomy, ovariohysterectomy (OVH), and mastectomy for mammary neoplasm treatment. The mean age of the patients was 10.5 ± 2.7 years (range: 4–15 years), and the breeds observed were mixed breed (n=24), Yorkshire Terrier (n=3), Lhasa Apso (n=2), Pinscher (n=2), and Dachshund (n=1). Tumor size ranged from 1.0 to 12.0 cm (mean: 4.6 ± 2.8 cm), with 9 of 32 (28.1%) patients presenting with ulceration on physical examination. Most females (n=25; 78.1%) had not previously received progestogens.

All animals underwent complete clinical evaluation, laboratory testing, thoracic radiography, abdominal ultrasonography, and fine-needle aspiration cytology of the mammary tumors for preoperative staging.

Lymphatic mapping was performed using methylene blue (n=16) or hemosiderin (n=16), administered intradermally according to group allocation, prior to laparoscopic lymphadenectomy of the MILN ipsilateral to the primary mammary tumor, followed by laparoscopic OVH and unilateral radical mastectomy.

Experimental design

The female dogs were prospectively allocated into four groups of eight animals each, according to the lymphatic dye/marker used and the site of administration: GAI (Intradermal Methylene Blue Group), 1% methylene blue administered intradermally around the M5 mammary gland; GAD (Dorsal Methylene Blue Group), 1% methylene blue administered intradermally in the lumbar region, near the deep circumflex iliac artery/vein; GHI (Intradermal Hemosiderin Group), hemosiderin administered intradermally around the M5 mammary gland; and GHD (Dorsal Hemosiderin Group), hemosiderin administered intradermally in the lumbar region (near the deep circumflex iliac artery/vein).

Allocation was performed sequentially according to case admission, maintaining equal group sizes. No randomization or stratification based on tumor characteristics or body weight was performed.

Intradermal marking in the groups with torso application was performed as described by Antunes et al. (2025) ensuring standardization of the application site and reproducibility of the method. The methylene blue dose was 0.5 mg/kg, and the volume of hemosiderin used was 1 ml. In all groups, lymphatic mapping was followed by laparoscopic lymphadenectomy of the MILN, laparoscopic OVH, and unilateral radical mastectomy.

No formal sample size or statistical power calculation was performed prior to the study; therefore, the study should be interpreted as exploratory.

Dyes and marker preparation

Methylene blue was used as a lymphatic dye because of its well-established affinity for lymphatic vessels and widespread clinical use.

Hemosiderin was used as an alternative lymphatic marker. The hemosiderin-based preparation was obtained from the autologous blood collected from each patient. Whole blood (10 ml) was collected and subjected to controlled processing to induce erythrocyte degradation and subsequent hemosiderin formation, following a previously described protocol (De Aguiar et al., 2017). After processing, approximately 1 ml of a hemosiderin-containing suspension suitable for intradermal administration was obtained and used for lymphatic marking. The remaining material was discarded. The preparation was performed on the same day.

The use of autologous materials aims to ensure biocompatibility and reduce the risk of adverse reactions.

Anesthetic and analgesic protocol

The female dogs were previously medicated with methadone (0.3 mg/kg, intramuscular), followed by intravenous infusion of lactated Ringer’s solution (5 ml/kg/h). Anesthetic induction was performed with propofol (4 mg/kg, intravenous), and maintenance was achieved with inhaled isoflurane in 100% oxygen, using a nonrebreathing circuit. For hemodynamic monitoring, the dorsal artery of the pelvic limb was catheterized with a 24G catheter connected to a pressure transducer. Systolic arterial pressure, mean arterial pressure, and diastolic arterial pressure were recorded every 10 minutes. When necessary, fentanyl (5 µg/kg, IV) was administered as rescue analgesia.

At the end of the surgery, local infiltration with bupivacaine (4 mg/kg) was performed around the surgical wound. In the postoperative period, meloxicam (0.1 mg/kg, SID for 4 days), tramadol (5 mg/kg, QID for 5 days), and dipyrone (25 mg/kg, TID for 3 days) were administered intravenously and subsequently orally.

Surgical procedures

A single surgical team trained in laparoscopic techniques performed the surgeries. The animals were positioned in dorsal recumbency with a slight head-down tilt (15°). Three abdominal ports were placed along the midline: the first, a 10-mm port, was introduced using an open technique between the umbilicus and the pubis, with CO₂ insufflation (1.5 l/min) up to a pressure of 8 mmHg; a 5-mm port was placed at the umbilical scar; and a 10-mm port was positioned at the level of the inguinal ring.

Lymphatic marking was performed immediately after the introduction of the three access ports and visualization of the lymph node dissection site to ensure adequate initial inspection of the cavity and target area. Marking was observed a few seconds after the application in most cases, allowing visualization of the lymphatic pathway and the lymph node up to 1 minute after administration, particularly with methylene blue. However, in some cases, especially with hemosiderin, the staining was faint, and intraoperative visualization was limited, with the coloration becoming evident only after removal of the lymph node and adjacent adipose tissue outside the abdominal cavity. Laparoscopic MILN lymphadenectomy was performed by blunt dissection using Kelly forceps, with removal of adipolymphatic tissues and the lymph node through the 10-mm port.

Subsequently, laparoscopic OVH was performed using bipolar cautery for ovarian and uterine vessel hemostasis. After hemostasis verification, the cavity was desufflated, and the ports were sutured. The procedure concluded with unilateral total mastectomy, including excision of the regional axillary and inguinal lymph nodes.

Evaluation and analysis of data

The primary outcome variable assessed was the effectiveness of lymphatic marking in the intraoperative identification of the MILN via laparoscopy, defined as visible staining of the lymphatic pathway or of the lymph node itself prior to its removal. A positive result was considered when such staining allowed the intraoperative identification of MILN.

Secondary variables included the time elapsed between dye administration and lymph node visualization, staining intensity, and lymph node dissection ease. Additionally, intraoperative events, dye use-related complications, and anesthetic-surgical adverse effects were documented.

After lymphadenectomy, the quality of lymph node staining was subjectively classified outside the abdominal cavity, allowing complete removal of adjacent adipose tissue and adequate evaluation of the isolated lymph node. Staining was categorized as follows: absent (no macroscopic staining), mild (up to one-third of the lymph node stained), moderate (up to one-half of the lymph node stained), and intense (more than one-half of the lymph node stained), corresponding to an ordinal scale from 0 to 3. This classification was based on the evaluation performed by the operating surgeon.

Owing to the evident visual differences between the dyes used, blinding of the evaluator was not feasible, which represents a limitation of the study.

Furthermore, the time required for lymph node identification after dye administration was categorized as follows: 1=≤10 seconds; 2=11–30 seconds; 3=31–60 seconds; 4=>60 seconds or not identified intraoperatively. These categories allowed standardization of comparisons among the experimental groups.

The Kruskal–Wallis test was used for statistical analysis to compare the four experimental groups. When significant differences were detected, a value of p < 0.05 was considered statistically significant. Data are expressed as median and interquartile range (IQR).

Considering the factorial structure of the study design (dye/marker × injection site), results were interpreted considering the potential influence of both variables; however, no formal factorial analysis was performed due to sample size limitations.

Ethical approval

The Ethics Committee on Animal Use of UFSM approved this study (Protocol n° 5437070419). The animal guardians signed an informed consent form after receiving a full explanation of the procedures, risks, and potential consequences.

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Results

Lymphatic staining was successfully achieved in all animals treated with MB, with visible lymphatic vessel impregnation occurring within 60 seconds after administration. Intraoperative identification of the MILN was achieved in 16 of 16 (100%) dogs in the MB groups. Identification occurred more rapidly in the dorsal torso (median category=1.5; IQR=1.0) than in the peritumoral (M5) region (median=2.5; IQR=1.25).

In dogs treated with hemosiderin, intraoperative MILN identification was less consistent and more time-consuming. Visible lymphatic marking during laparoscopy was achieved in 10 of 16 (62.5%) cases, while in the remaining animals (6 of 16; 37.5%), identification was only possible after removal of the lymph node and surrounding adipolymphatic tissue. The hemosiderin M5 group had the longest identification time (median=4.0; IQR=0.25), whereas the torso group had intermediate results (median=3.5; IQR=1.0).

Statistical analysis using the Kruskal–Wallis test demonstrated a significant difference among groups regarding lymph node identification time (χ²=10.27; p=0.016), with faster identification observed in the methylene blue groups.

Regarding staining intensity, methylene blue administered in the dorsal torso resulted in intense staining in 7/8 (87.5%) cases and moderate staining in 1/8 (12.5%). In the peritumoral methylene blue group, staining was classified as intense in 3/8 (37.5%), moderate in 2/8 (25%), and mild in 3/8 (37.5%) patients. In contrast, hemosiderin administered around M5 resulted predominantly in mild staining (6/8; 75%), whereas staining intensity was moderate in 4/8 (50%), mild in 2/8 (25%), and intense in 2/8 (25%) animals in the torso group. No statistically significant difference in staining intensity was observed between the groups (χ²=5.75; p=0.124). No intraoperative complications, dye-related adverse reactions, or conversions to open surgery were observed in any procedure.

All mammary lesions removed during unilateral radical mastectomy were histopathologically evaluated. As some dogs presented with more than one lesion within the surgical specimen, results were reported per lesion rather than per animal, totaling 36 evaluated lesions. The diagnoses included carcinomas (n=27/36; 75%), adenomas (n=6/36; 16.7%), and lobular mammary hyperplasia (n=3/36; 8.3%).

The following histological subtypes were identified among carcinomas: carcinoma and malignant myoepithelioma (n=13), simple tubulopapillary carcinoma (n=4), complex carcinoma (n=3), mixed-type carcinoma (n=2), simple tubular carcinoma (n=2), carcinoma in mixed tumor (grade I) (n=1), solid carcinoma with metastasis to the inguinal lymph node (n=1), and neoplastic emboli in lymphatic vessels associated with carcinoma and malignant myoepithelioma (grade II) (n=1).

Preoperative cytological evaluation revealed mixed mammary neoplasia (n=5), epithelial mammary neoplasia (n=4), and malignant epithelial neoplasia (n=1), as well as occasional mixed lesions associated with inflammatory or cystic processes. Histopathological examination confirmed a malignant tumor in all cases previously classified as suspicious for malignant neoplasia. Cytological diagnoses suggestive of mixed or epithelial tumors corresponded to confirmed neoplasms, including mixed tumor, simple tubulopapillary, and mixed-type carcinomas. Lesions interpreted as benign or cystic were diagnosed as adenomas or lobular mammary hyperplasia. Overall, cytology demonstrated good agreement as a screening method, although definitive classification required histopathological analysis.

No metastases were identified in any of the MILNs evaluated in this study.

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Discussion

This study evaluated the feasibility and intraoperative applicability of intradermal lymphatic mapping using methylene blue and hemosiderin to assist in the laparoscopic identification of the MILN in female dogs with mammary neoplasms. The results demonstrate that both dyes are capable of promoting lymphatic marking; however, compared with hemosiderin, methylene blue provided faster and more consistent intraoperative identification, with superior staining contrast.

Methylene blue allowed rapid visualization of lymphatic pathways and MILN, typically within 1 minute, facilitating laparoscopic identification in all cases. In contrast, hemosiderin showed lower intraoperative visibility, with successful identification during laparoscopy in only a few cases, and in a substantial proportion, visualization occurred only after lymph node removal. These findings highlight the importance of dye selection in minimally invasive procedures, where visual contrast is critical for efficient dissection. Previous studies evaluating lymphatic mapping in veterinary oncology reported similar advantages of methylene blue over other dyes (Maués et al., 2016; Antunes et al., 2025).

The injection site’s influence on lymphatic mapping was also observed. Although both peritumoral and dorsal torso applications resulted in lymphatic marking, administration in the dorsal torso tended to allow faster identification, particularly when combined with MB. Although a formal factorial analysis was not performed due to the limited sample size, these findings suggest that both dye type and injection site may influence lymphatic drainage patterns and visualization efficiency, and their potential interaction should be further explored in future studies.

Despite the differences in staining intensity among the groups, no statistically significant difference was observed for this variable. This result may be partially explained by the limited sample size and the subjective nature of staining assessment, which may have reduced the statistical power to detect differences in ordinal outcomes. In addition, staining intensity may be less clinically relevant than the ability to achieve reliable intraoperative identification, which is the primary objective of minimally invasive lymphadenectomy.

No intraoperative complications, adverse reactions related to dye administration, or conversions to open surgery were observed, reinforcing the safety and feasibility of laparoscopic lymphadenectomy combined with lymphatic mapping. These findings are consistent with previous reports highlighting laparoscopy as a minimally invasive approach associated with reduced morbidity and favorable recovery profiles in veterinary patients (Steffey et al., 2015; Lim et al., 2017).

In the present study, no metastases were detected in the MILNs. Therefore, the findings should not be interpreted as evidence of oncologic benefit or staging accuracy improvement. Instead, the results primarily demonstrate the technical feasibility of lymphatic mapping to facilitate intraoperative MILN identification. The clinical relevance of routinely including the MILN in oncologic staging protocols remains dependent on further studies evaluating metastatic rates and prognostic impact on larger populations.

The absence of metastasis in this cohort may be related to tumor characteristics, disease stage, or sample size and does not preclude the potential involvement of the MILN in more advanced or biologically aggressive cases. Previous studies have reported the metastatic involvement of this lymph node in dogs with malignant mammary tumors, supporting its consideration as a potential staging target (Feranti et al., 2018; Feranti et al., 2024).

This study has several limitations that should be acknowledged. First, the sample size was relatively small, and no formal power calculation was performed, characterizing the study as exploratory. Second, the group allocation was not randomized, which may introduce selection bias. Third, the staining intensity was subjectively assessed by the operating surgeon, and blinding was not possible due to the evident color differences between dyes, potentially introducing observational bias. These limitations highlight the need for future studies with larger sample sizes and more objective assessment methods.

In recent years, fluorescence-guided techniques using indocyanine green and near-infrared imaging have demonstrated high detection rates and improved sentinel lymph node mapping visualization. However, these technologies’ availability and cost still limit their routine use in many veterinary settings. In this context, methylene blue represents a practical and accessible alternative that can be readily implemented in clinical practice. Future studies may explore hybrid approaches combining vital dyes and fluorescence techniques to optimize lymphatic mapping while maintaining cost-effectiveness.

Further investigation is warranted to better define the role of the MILN in oncologic staging of canine mammary tumors and to evaluate the impact of lymphatic mapping–guided lymphadenectomy on clinical outcomes such as recurrence, survival, and treatment planning. In addition, comparative studies involving different dyes, imaging modalities, and application techniques may contribute to the development of standardized protocols for minimally invasive lymph node staging in veterinary oncology.

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Conclusion

Intradermal lymphatic mapping is a feasible and safe technique for the laparoscopic identification of the MILN in female dogs with mammary tumors. Among the dyes evaluated, MB provided faster and more consistent intraoperative visualization, with superior staining contrast compared to hemosiderin, making it more suitable for minimally invasive lymphadenectomy.

Although hemosiderin is a low-cost alternative, its low intraoperative visibility limits its practical applicability. The findings of this study should be interpreted within the context of an exploratory design and do not allow conclusions regarding oncologic outcomes or staging accuracy to be drawn.

Further studies with larger sample sizes and robust designs are warranted to better define the role of lymphatic mapping–guided lymphadenectomy in canine mammary tumor staging.

Conflict of interest

The authors have no conflicts of interest to declare.

Funding

This study was financially supported by the National Council for Scientific and Technological Development (CNPq) – process no. 153411/2018-0.

Authors' contributions

Dr. João Pedro Scussel Feranti conducted the experiment, collected and analyzed the data, and drafted the manuscript. Dr. Roberta do Nascimento Libardoni, Dr. Bernardo Nascimento Antunes, Dr. Vanessa Milech, and Dr. Jéssica Tomio assisted in conducting the experiment. Dr. Maurício Veloso Brun supervised the study, critically reviewed the manuscript for intellectual content, and approved the definitive version for publication. All authors approved the definitive version of the manuscript.

Data availability

All data were provided in the manuscript.

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