Lymph Node Mapping in Primary Care Practice

In veterinary oncology, identification of metastatic disease allows for accurate patient staging and guides both treatment and prognostication. For many skin cancer types, such as squamous cell carcinoma, mast cell tumors, and melanoma, identification of metastatic lymph nodes is important in understanding the extent of disease to guide treatment recommendations (TABLE 1). In some cases, removing metastatic lymph nodes may also have survival benefit.^{3,4,7,8,10-12}

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Identifying Metastatic Lymph Nodes

The gold standard for assessing lymph nodes for metastasis is histopathology.^13,14 In most cases, the entire lymph node is removed, or extirpated, to allow full assessment for evidence of disease.¹⁵ Studies in human medicine have shown the accuracy of ultrasonography, computed tomography, and palpation in detecting lymph node metastasis to vary depending on tumor type.^16,17 Fine-needle aspiration and cytology of the lymph nodes is considered an inferior method of assessing for metastatic disease; reasons for inaccurate results include failure to reach the metastatic region of the lymph node during sampling and failure to sample the metastatic lymph node when sampling a single lymph node within a chain or cluster.^18-20

Two main challenges exist when attempting to identify and remove the lymph nodes involved with draining malignant skin tumors. The first is that the first draining lymph node, or sentinel lymph node (SLN), is not always the lymph node(s) closest to the tumor (regional lymph node). The second is identifying and surgically removing the correct lymph node(s).

Identifying the Sentinel Lymph Node

The SLN can be identified using a technique called SLN mapping, or lymphography. Preoperatively, this technique uses injection of a contrast agent and subsequent radiographs or advanced imaging to identify uptake of the agent by lymph nodes; intraoperatively, dye is used to aid in visual identification of the SLN. Each has limitations; therefore, it is recommended that clinicians use a combination of preoperative and intraoperative lymphography to guide which lymph node(s) to remove.

The equipment and supplies needed for both preoperative and intraoperative SLN mapping are shown in FIGURE 1. The syringe size (1 to 5 mL) depends on the volume to be injected; use of a ruler and skin marker depends on clinician preference. For preoperative mapping, nonsterile gloves may be used in place of sterile gloves if desired.

Figure 1. Supplies required for sentinel lymph node mapping. (A) Iodinated contrast medium or blue dye (Omnipaque 350 shown), 25-gauge needle(s), 3- to 5-mL syringe, sterile gloves, skin marker, ruler, 3-step prep, and clippers.

Figure 1B. Close-up image of Omnipaque 350.

Figure 1C. Close-up image of methylene blue.

Preoperative Indirect Radiographic Lymphography

A readily accessible preoperative technique for SLN mapping involves peritumoral injection of a radiopaque iodinated contrast agent followed by orthogonal (2-view) survey radiography of the area to visualize where the contrast drains from the injection sites to the lymph node.

Contrast Agent Choice and Volume

The choice of iodinated contrast agent depends on what is most accessible and financially feasible. Various water-soluble contrast agents are available, including iopamidol (IsoVue 200, 300, or 370) and iohexol (Omnipaque 180, 240, 300, or 350). The concentration listed on the bottle refers to the iodine concentration, with higher concentrations being more readily visible radiographically. Generally, these solutions are available in multidose bottles and have a shelf life of 1 to 3 years but need to be used within 6 to 24 hours of opening, so it is important to plan accordingly. The authors prefer to use iohexol rather than iopamidol for this technique due to accessibility and cost in their institution.

Ethiodized oil (Lipiodol) is an iodinated, poppyseed oil–based contrast agent that can be used in place of water-soluble contrast. Due to the lipid component, it is taken up into the lymphatic system more slowly than water-soluble agents. Using ethiodized oil may carry the benefit of more definitive contrast enhancement within the lymph node for a longer period of time; however, some local injection reactions have been described and this contrast can be cost prohibitive in some geographic regions.^21-24

The volume of contrast agent administered depends on the distance between the primary tumor and the predicted SLN. For water-soluble contrast, a total volume of 3 to 4 mL (approximately 1 mL per injection site) is used in most circumstances; however, a larger volume (> 5 mL) can be considered if a greater distance (e.g., > 50 cm) of travel is anticipated. If more than 5 mL of water-soluble contrast is to be used, the contrast can be diluted 1:1 with sterile saline to help decrease the agent’s viscosity, decreasing transit time. However, this step will also dilute the iodine concentration, which can make radiographic visualization more challenging. In very small patients, or in locations that cannot accommodate a large volume of fluid intradermally, the injection volumes may be decreased to 0.25 to 0.5 mL per injection site (1 to 2 mL total); dilution of water-soluble contrast is not recommended in these cases. Ethiodized oil (0.8 to 1.6 mL total) should be used full strength.^21-23

Patient Preparation

Sedation or general anesthesia is required to avoid the need for restraint as well as any movement that could limit lymphatic flow. It is important that, immediately following injection, there is no compression on any of the tissues within the lymphatic field (e.g., endotracheal tube ties around the jaw). The area around the primary tumor should be clipped and prepared with standard surgical preparation. The optimal patient positioning depends on the primary tumor’s location; however, wide-field survey radiographs of the whole region should be obtained to ensure visualization of lymphatic tracts.

Approach

1. Obtain baseline orthogonal survey radiographs of the patient prior to injection to rule out any preexisting lymphatic or tissue mineralization (FIGURE 2).
   Figure 2A. Baseline orthogonal radiographs (prior to injection) should include limited collimation to show all lymph nodes within the region. For a mast cell tumor on the thorax, both (A) lateral and (B) dorsoventral images were taken to include axillary and superficial cervical lymph nodes.

   Figure 2B. Baseline orthogonal radiographs (prior to injection) should include limited collimation to show all lymph nodes within the region. For a mast cell tumor on the thorax, both (A) lateral and (B) dorsoventral images were taken to include axillary and superficial cervical lymph nodes.

   Figure 2C. For a mast cell tumor on the pelvic limb, (C) lateral and (D) dorsoventral images were taken to include inguinal, iliosacral, and popliteal lymph nodes.

   Figure 2D. For a mast cell tumor on the pelvic limb, (C) lateral and (D) dorsoventral images were taken to include inguinal, iliosacral, and popliteal lymph nodes.
2. Inject contrast intradermally using the peritumoral 4-quadrant technique described in BOX 1 (FIGURES 3 AND 4).
   Figure 3A. A surgical marker is used to identify tumor margin, resection margin, and injection quadrant for mast cell tumors (A) in the skin fold between the ventral abdomen prepuce, (B) on the lateral thorax, and (C) on the pelvic limb.

   Figure 3B. A surgical marker is used to identify tumor margin, resection margin, and injection quadrant for mast cell tumors (A) in the skin fold between the ventral abdomen prepuce, (B) on the lateral thorax, and (C) on the pelvic limb.

   Figure 3C. A surgical marker is used to identify tumor margin, resection margin, and injection quadrant for mast cell tumors (A) in the skin fold between the ventral abdomen prepuce, (B) on the lateral thorax, and (C) on the pelvic limb.

   Figure 4. Peritumoral 4-quadrant injection technique. (A) A 25-gauge needle and 5-mL syringe are used to inject 4 mL of iodinated contrast agent intradermally in the marked quadrant (1 mL per 30-second injection) around a mast cell tumor on the left pelvic limb for indirect radiographic lymphography.

   Figure 4B. A 25-gauge needle and 1-mL syringe are used to inject diluted methylene blue (0.2 mL in 0.8 mL saline) within the marked quadrant (0.25 mL per 30-second injection) around a prepuce mast cell tumor for intraoperative visualization. Note that the bevel is oriented upward for both injections. For all intradermal injections, additional gauze should be kept close in case of spillage and when removing the needle from the skin. Following the injection, a small bubble may appear just under the skin surface and dissipate after massage or within a few hours.
3. If desired, massage the injection site for 30 seconds to encourage lymphatic flow to the SLN.
4. Take orthogonal radiographs (ideally starting with the lateral view).
   • With water-soluble agents, radiography should immediately follow massage, as these agents are rapidly cleared from the system. For diagnostic studies in dogs with mast cell tumors, a maximum time of 18 minutes was reported for visible contrast.²⁵
   • With ethiodized oil, radiographs are taken 24 hours following injection, and the contrast has been shown in healthy, purpose-bred dogs to still be visible at the level of the lymph node 1 to 2 months after injection. Lymphatic vessels and lymph nodes are consistently observed at 24 hours.²⁴
5. Assess for evidence of lymphatic tracking toward a lymph node or evidence of contrast within the lymph node compared with preoperative radiographs (FIGURE 5).
   Figure 5. Indirect radiographic lymphography. Dorsoventral radiograph of a dog with a lateral thorax mast cell tumor, immediately following peritumoral 4-quadrant injection of a total volume of 4 mL of Omnipaque 350 (blue arrow). Contrast is visible in the intradermal space and mild tracking is visible (lymphatic vessels) from the injection site extending cranially to the left axillary region where 2 ovoid structures, the left accessory axillary lymph nodes, have contrast uptake (white arrow).
6. If contrast is seen within the lymph node, no further imaging is required. If contrast is not seen within the lymph node on the first set of radiographs, repeat the radiographs at 5 minutes postinjection (for water-soluble agents).
   • If no SLN is visible 5 minutes postinjection with water-soluble contrast, repeat steps 2 through 5.
   • If no SLN is visible at 24 hours with ethiodized oil, repeat steps 2 through 5, repeating radiographs every 6 to 12 hours, if desired.
7. Following documentation of the location of the SLN (FIGURES 5 THROUGH 9), the margins of the site of interest can be clipped preemptively to accurately demarcate the surgical field.
   Figure 5. Indirect radiographic lymphography. Dorsoventral radiograph of a dog with a lateral thorax mast cell tumor, immediately following peritumoral 4-quadrant injection of a total volume of 4 mL of Omnipaque 350 (blue arrow). Contrast is visible in the intradermal space and mild tracking is visible (lymphatic vessels) from the injection site extending cranially to the left axillary region where 2 ovoid structures, the left accessory axillary lymph nodes, have contrast uptake (white arrow).

   Figure 6A. Indirect radiographic lymphography. (A) Lateral and (B) dorsoventral orthogonal radiographs of a dog with a mast cell tumor of the left proximal antebrachium following injection of a total volume of 4 mL of Omnipaque 350 using the 4-quadrant peritumoral method (white arrows). Mild to moderate tracking of contrast is seen from the left elbow through the afferent lymphatic vessels (yellow arrows) directly to 2 ovoid structures, the axillary and accessory axillary lymph nodes (blue arrows).

   

   Figure 6B. Indirect radiographic lymphography. (A) Lateral and (B) dorsoventral orthogonal radiographs of a dog with a mast cell tumor of the left proximal antebrachium following injection of a total volume of 4 mL of Omnipaque 350 using the 4-quadrant peritumoral method (white arrows). Mild to moderate tracking of contrast is seen from the left elbow through the afferent lymphatic vessels (yellow arrows) directly to 2 ovoid structures, the axillary and accessory axillary lymph nodes (blue arrows).

   Figure 7A. Indirect radiographic lymphography. (A) Lateral, (B) extended lateral, and (C) dorsoventral orthogonal radiographs of a dog with a mast cell tumor in the skin fold between the ventral abdomen and prepuce. A total volume of 4 mL of Omnipaque 350 was injected using the 4-quadrant peritumoral method (blue arrows). Mild to moderate tracking of contrast through the afferent lymphatic vessels (yellow arrows) directly to the inguinal lymph nodes (white arrows) is seen. Following identification of the inguinal lymph nodes, surgical staples were placed (circles) as landmarks to guide surgical incision.

   Figure 7B. Indirect radiographic lymphography. (A) Lateral, (B) extended lateral, and (C) dorsoventral orthogonal radiographs of a dog with a mast cell tumor in the skin fold between the ventral abdomen and prepuce. A total volume of 4 mL of Omnipaque 350 was injected using the 4-quadrant peritumoral method (blue arrows). Mild to moderate tracking of contrast through the afferent lymphatic vessels (yellow arrows) directly to the inguinal lymph nodes (white arrows) is seen. Following identification of the inguinal lymph nodes, surgical staples were placed (circles) as landmarks to guide surgical incision.

   Figure 7C. Indirect radiographic lymphography. (A) Lateral, (B) extended lateral, and (C) dorsoventral orthogonal radiographs of a dog with a mast cell tumor in the skin fold between the ventral abdomen and prepuce. A total volume of 4 mL of Omnipaque 350 was injected using the 4-quadrant peritumoral method (blue arrows). Mild to moderate tracking of contrast through the afferent lymphatic vessels (yellow arrows) directly to the inguinal lymph nodes (white arrows) is seen. Following identification of the inguinal lymph nodes, surgical staples were placed (circles) as landmarks to guide surgical incision.

   Figure 8A. Indirect radiographic lymphography. (A) Lateral and (B) dorsoventral orthogonal radiographs of a dog with a cutaneous mast cell tumor on the left flank. Ethiodized oil was injected (white arrows), and the left inguinal lymph nodes (blue arrows) can be seen. Courtesy Julius Liptak

   Figure 8B. Indirect radiographic lymphography. (A) Lateral and (B) dorsoventral orthogonal radiographs of a dog with a cutaneous mast cell tumor on the left flank. Ethiodized oil was injected (white arrows), and the left inguinal lymph nodes (blue arrows) can be seen. Courtesy Julius Liptak

   Figure 9A. Indirect radiographic lymphography. (A) Lateral and (B) dorsoventral orthogonal radiographs of a dog with a mammary osteosarcoma. Ethiodized oil was injected (white arrows), and the right axillary lymph nodes (blue arrows) can be seen. Courtesy Julius Liptak

   Figure 9B. Indirect radiographic lymphography. (A) Lateral and (B) dorsoventral orthogonal radiographs of a dog with a mammary osteosarcoma. Ethiodized oil was injected (white arrows), and the right axillary lymph nodes (blue arrows) can be seen. Courtesy Julius Liptak

Intraoperative Lymphography

The intraoperative use of blue dyes can be helpful to guide the surgeon with improved visualization of lymphatic tracts and lymph nodes. Methylene blue (MB) is the most described blue dye in the veterinary literature^26-28 and is referenced here, although others (e.g., patent blue, isosulfan blue) can be used in a similar manner. It is important to note that MB and new methylene blue are chemically different dyes and cannot be used interchangeably.

Blue Dye

MB is generally safe when injected locally but can cause tissue staining; therefore, any important surgical boundaries should be defined prior to injection. Care should be taken to avoid intravascular injection of MB in cats. Because direct visualization of blue lymphatic vessels and lymph nodes is required, this technique should be combined with preoperative radiographic mapping so that the surgeon is informed of the targeted lymph node(s) and the location for surgical planning.

The volume of blue dye administered depends on the distance between the primary tumor and the predicted SLN. A small volume (1 mL total) can be used in most circumstances; however, a larger volume (3 mL) can be considered if a further distance of travel is anticipated. To decrease the degree of staining in the local surgical field, the lead author prefers to dilute MB 1:5 or 1:10 with sterile saline (i.e., 0.1 mL MB to 0.9 mL NaCl), but it can occasionally be more challenging to visualize blue coloration within the smaller lymphatic vessels. If desired, a total volume of up to 1 mL of MB can be used, diluted as appropriate for larger volumes. MB moves quickly through the lymphatic system and in most cases can be visible before completion of the injection. Retention times are not well documented; therefore, lymphadenectomy soon after MB injection is recommended.

Patient Preparation

The area(s) around the primary tumor and the lymph node to be removed should be prepared routinely for surgery. Lymphadenectomy should be performed prior to mass excision; therefore, positioning for this procedure while allowing access to the tumor for injection is prioritized. Often, it is possible to inject around the tumor with the patient positioned for lymphadenectomy with some creativity, but in some cases, repositioning the patient to first access the tumor and then prepare and drape for lymphadenectomy is required. Depending on the location of the primary tumor and SLN, 2 surgical fields may be required. If the patient requires repositioning after lymphadenectomy, the primary tumor site does not require draping at the time of MB injection but will need to be reprepped and draped prior to removal of the mass. The equipment and supplies needed for this procedure are shown in FIGURE 1A.

Approach

1. Demarcate the surgical margins of the primary tumor using a sterile surgical marker and ruler (FIGURE 3).
2. Inject MB intradermally using the peritumoral 4-quadrant technique described in BOX 1 (FIGURE 4).
3. If desired, massage the injection site for 30 seconds to encourage lymphatic flow to the SLN.
4. Reposition the patient as necessary to perform the lymphadenectomy. Ideally the surgical site is draped in prior to peritumoral injection but if this is not possible, repositioning, prep, and draping should be performed as quickly as possible.
5. With the guidance of preoperative imaging and anatomic landmarks, make an incision over the anticipated site of the SLN. Use the blue staining of MB to identify the SLN and perform lymphadenectomy as described elsewhere (FIGURES 10 AND 11).²⁹
   Figure 10. Initial dissection of the inguinal lymph node in a dog with a mast cell tumor on the prepuce. (A) Mild blue staining can be seen in the lymphatic vessels (white arrow). The primary tumor is visible at the right of the image outlined with sterile surgical marker.

   Figure 10B. Blue staining of the inguinal lymph node.

   Figure 10C. At the primary tumor site, the underlying tissues are stained blue from the injection.

   Figure 11. Accessory axillary lymph node stained with methylene blue in a dog with a mast cell tumor on the lateral thorax. (A) The primary tumor is at the top of the image outlined with a sterile surgical marker; an additional line indicates the planned surgical margin.

   Figure 11B. Blue staining of the lymph node and lymphatic vessels.
6. Proceed with closure of the lymph node site(s) and excision of the primary tumor (FIGURE 12).
   Figure 12. Appearance of the closed lymphadenectomy and primary mast cell tumor sites in a dog.

Summary

SLN mapping is a straightforward procedure that can be performed in primary care practices that undertake curative-intent skin mass excisions. With limited additional equipment and cost, practitioners can identify the most appropriate lymph nodes to remove and improve staging and possible treatment in these patients.