Sentinel Lymph Node Biopsy for Squamous Cell Carcinoma Technique

Updated: Sep 26, 2016
  • Author: Keith M Baldwin, DO; Chief Editor: Arlen D Meyers, MD, MBA  more...
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Preoperative Assessment

Contrast-enhanced CT scanning is helpful for determining the extent of tumor infiltration. Detection of grossly positive nodal disease, particularly when central necrosis is present, is increased with contrast-enhanced CT scans. The accuracy for detecting nodal metastases is reportedly improved from 70% to 93% when a physical examination is combined with CT scans, but occult disease can still be missed.

Fluorodeoxyglucose positron emission tomography (FDG-PET) is performed before removal of the primary tumor and/or dissection of the neck, and results are compared with those from histopathology studies. FDG-PET confirms the clinically identified location of the primary tumor site. FDG-PET shows promise in the initial staging of cancer of the head and neck. It also provides additional accuracy to the conventional staging process using CT scans.

MRI is beneficial for staging the size of the suggestive lymph nodes, for the evaluation of central lucency reflecting necrosis, for the assessment of irregular nodes with rim enhancement, for the evaluation of indistinct nodal margins, and for obliteration of fat or tissue planes.

The imaging studies are performed to detect any lymph node metastases. If the imaging studies fail to positively show lymph node metastases, an SLN biopsy is indicated for oral and oropharyngeal SCCs.

Step-sectioning SNLs

The current histopathologic routine is to step-section the SLNs at multiple levels and to perform immunohistochemical staining with S-100 protein and homatropine methylbromide to identify micrometastatic disease. If any question remains about abnormal cells after the first sections are taken, additional sections are obtained. Immunohistochemistry results identify an additional 10-20% of patients with positive SLNs, in whom micrometastases are not seen on routine sections stained with permanent hematoxylin and eosin (H&E). At least some of the increased rate of detection of micrometastatic disease is attributable to step-sectioning at multiple levels.

Sampling approach

The approach for sampling involves bivalving the lymph node, fixing the 2 halves face down, and subsequently sectioning each half into 10 sections, which are alternately used for H&E staining, immunohistochemistry testing, and molecular staging. The sensitivity of intraoperative frozen section examination of the SLN is disappointingly low (< 50%), although false-positive results are almost never reported. Because of concerns about tissue loss during the frozen section procedure, most centers eschew frozen sections and rely on permanent sections, except to confirm grossly suggestive metastatic disease.

Patients undergo lymphoscintigraphy up to 1 day prior to surgery. A maximum of 40 MBq technetium-99m (99m Tc)-labeled human serum albumin (ie, AlbuRES, Nanocoll) is injected throughout the normal mucosa surrounding the tumor edge and the submucosa on the deep aspect of the tumor in a volume of approximately 0.5-1 mL. A syringe with a permanently secured needle is used for injection, thereby preventing inadvertent spillage of colloid into the mouth. Colloid is injected at as many points as necessary in an attempt to completely surround the tumor. A mouthwash is used immediately following injection to prevent pooling or swallowing of residual radioactive material by the patient.

Static lymphoscintigraphy is performed at 15 minutes, 30 minutes, and 1 hour postinjection in 2 planes or until the appearance of radioactive nodes. Hot spots are usually seen 15 minutes postinjection. If nodes are still absent 1 hour after injection, the lymph nodes are too close to the injection site or radiocolloid has leaked out of the injection site.

Either a cobalt-57 (57 Co) marker is used to trace the patient outline or a flood source of57 Co or99m Tc is placed behind the patient to produce a silhouette of the patient outline. In light of the radiation dose, the marker pen is preferable. A gamma camera fitted with a low-energy, general-purpose collimator is used to acquire images of the patient. A 20% window centered on the 140 keV photopeak is selected, and the camera is interfaced to a suitable computing system. The locations of radioactive lymph nodes are marked on the patient's skin, the position of a57 Co solid-source pen is observed on the camera's persistence display, and the pen is moved until its position overlies a radioactive node. This position is then marked on the patient's skin using indelible ink. During the skin marking, a lead plate of an appropriate thickness (eg, 3 mm) is used to shield the injection site.

Following image acquisition, a software mask is applied to all images to eliminate radioactivity from the injection site. A region of interest, drawn around the image of the site of injection, is used as the basis for the mask applied.

Two colloids are commonly used for lymphoscintigraphy: AlbuRES and Nanocoll. AlbuRES has a mean particle size of 500 nm and is a slower-moving particle that remains in first-echelon (sentinel) nodes but requires a high density of terminal lymphatic vessels at the injection site. For these reasons, AlbuRES is the colloid of choice on the tongue and on the floor of the mouth. Nanocolloid has a mean particle size of 50 nm and is a faster-moving colloid, which finds lymphatic vessels despite injection into tissues with low densities of terminal lymphatics. Nanocolloid also moves readily from sentinel nodes to subsequent-echelon nodes. For these reasons, nanocolloid is the colloid of choice for primary tumors that are not located on the floor of the mouth or on the tongue. Record the choice of colloid.

The overall success rate of a SLN biopsy by blue dye is 82%, by radioactive mapping is 94%, and by a combination of both is 98%.

Lymph node triangles of the neck

Lymph node groups of the neck region are divided into 2 triangles:

  • The anterior triangle
  • The posterior triangle

The anterior triangle of the neck is bounded anteriorly by the median plane, posteriorly by the sternocleidomastoid muscle, and superiorly by the base of the mandible. In addition, a line joins the angle of the mandible to the mastoid process. The apex of the triangle lies above the manubrium sterni.

The anterior triangle is subdivided by the digastric muscle and the superior belly of the omohyoid into the following:

  • The submental triangle
  • The digastric triangle
  • The carotid triangle
  • The muscular triangle

The submental triangle is the median triangle. On each side, the boundaries are the anterior belly of the corresponding digastric muscles. Its base is formed by the body of the hyoid bone. Its apex lies at the chin. The floor of the triangle is formed by the right and left mylohyoid muscles, with the median raphe uniting them.

The digastric triangle boundaries are, anteroinferiorly, the anterior belly of the digastric; posteroinferiorly, the posterior belly of the digastric and the stylohyoid; and superiorly (base), the base of the mandible and a line joining the angle of the mandible to the mastoid process. The roof boundary is the skin, superficial fascia, and deep fascia. The floor is formed by the mylohyoid muscle anteriorly and by the hyoglossus posteriorly and anteroinferiorly.

The carotid triangle boundaries are, superiorly, the posterior belly of the digastric muscle and the stylohyoid; anteroinferiorly, the superior belly of the omohyoid; and posteriorly, the anterior border of the sternocleidomastoid muscle. The roof boundary is skin, superficial fascia, and an investing layer of deep fascia. The floor is formed by parts of the thyrohyoid muscle, the hyoglossus, and the middle and inferior constrictors of the pharynx. Most of the SLNs are found in the carotid or the digastric triangle.

The muscular triangle includes the superficial structures in the infrahyoid region. The boundaries are, anteriorly, the anterior median line of the neck from the hyoid bone to the sternum; posterosuperiorly, the superior belly of the omohyoid muscle; and posteroinferiorly, the anterior border of the sternocleidal mastoid muscle.

The posterior triangle is a space on the side of the neck situated behind the sternocleidomastoid muscle. The boundaries of the posterior triangle are, anteriorly, the posterior border of the sternocleidomastoid muscle; posteriorly, the anterior body of the trapezius; and inferiorly (base), the middle third of the clavicle. The apex lies on the superior nuchal line where the trapezius and sternocleidomastoid muscle meet.

The posterior triangle is subdivided by the inferior belly of the omohyoid into the following:

  • The omohyoid triangle
  • The supraclavicular triangle

Lymph node dissection in both the omohyoid and the supraclavicular triangles in the posterior compartment is considered to be the same level.


Overview of SNL Biopsy

At operation, 1-2 mL of Patent Blue V dye may be used. To approximate the same injection sites as for radiocolloid, ensure that the same person performs all the injections. A suitable incision is made in the neck in such a position as to facilitate excision of the incision scar if a subsequent neck dissection is necessary.

The handheld gamma probe is used to identify radioactive sentinel nodes, including those marked preoperatively during lymphoscintigraphy. To reduce detection of radiation from the injection site, a series of malleable, sterilized lead plates may be used to mask the injection site, thus aiding in vivo identification of radioactive nodes. Radioactive nodes are excised, and radioactivity within the node is confirmed ex vivo.

If the blue dye is used, stained lymphatics, if seen, are followed to the first draining lymph node, which is then harvested. Sentinel nodes are labeled according to their color and radioactivity. The anatomic neck level of the sentinel nodes is noted. Although sentinel nodes are usually harvested prior to treatment of the primary tumor, the proximity of the sentinel node to the injection site may require a further search for sentinel nodes following excision of the primary tumor. If sentinel nodes are sought after excision of the injection site, the nodes are not likely to be stained blue.

Because of the relatively high radioactivity present in the injection sites and the proximity to the sentinel node, detection of scattered radiation must be avoided as far as possible. In addition to the use of the lead plates described above, the gamma probe must have a well-collimated detector, which excludes gamma radiation except over a small angle in front of it. Set the pulse-height analysis window to only include the99m Tc photopeak with a cutoff on the low-energy side at approximately 130 keV. Check the calibration at regular intervals of not more than 1 month (depending on the make and model of the instrument). Devise a quick check of calibration, and perform this quick check before each use. Calling on appropriate scientific or technical assistance may be necessary to ensure that the gamma probe is at its optimal settings and to make an estimate of its sensitivity at these settings.

Sentinel nodes are fixed in 10% neutral, buffered formalin, and, after fixation, they are bisected through the hilum (if identifiable) or through the long axis of the node. If the halves are thicker than 2 mm, the slices are further trimmed to provide additional blocks of 2 mm. If sentinel nodes are found to be free from tumor after the initial histologic examination, step-serial sections are prepared at an additional 6 levels in the block at intervals of approximately 150 µm. One H&E-stained section is prepared at each level. If the nodes still appear negative after histologic examination, an adjacent section from each level is examined by immunocytochemistry using the multicytokeratin antibody AE1/AE3.


Pathology Code Description

The interpretation of the histopathology and immunocytochemistry results for the SLN is categorized as stages 1 through 5.

Stage 1 tumor

In stage 1, the sample is positive for tumor upon first examination using H&E stain.

Stage 2 tumor

In stage 2, the sample initially appears negative for tumor, but it is noted to be positive for tumor upon examination of the H&E stain of step-serial sections.

Stage 3 tumor

In stage 3, the sample is negative for tumor at stages 1 and 2 but positive for tumor based on immunohistochemistry results. To be categorized as positive for tumor, cells must be present that are positive based on immunocytochemistry results and are cytologically observed to be nucleated cells with the characteristics of viable epithelial cells in both the immunocytochemical preparation and the serial H&E-stained sections. Cytokeratin positivity lacking the cytological features of viable tumor cells is categorized as stage 4.

Stage 4 tumor

In stage 4, cytokeratin positivity does not show the features of viable tumor cells. This positivity likely represents either dying tumor cells (possibly apoptotic cells), characterized by eosinophilic bodies lacking normal nuclei, or macrophages with phagocytosed tumor products. Usually, these cells are single and not in small, cohesive groups. The decision to allocate nodes to this category requires careful comparison of the serial H&E-stained section and the immunocytochemical preparation.

Stage 5 tumor

The sample is negative for tumor at all stages.


Approach After Staging

If any lymph node contains a viable tumor (ie, stages 1-3 according to the pathology code description) either based on routine histology studies or based on immunohistochemistry and multiple sectioning, the patient undergoes a radical or modified radical dissection of the neck. Perform the dissection of the neck within 4 weeks of the SLN biopsy, and begin any adjuvant radiotherapy within 6 weeks of the dissection. Do not administer radiotherapy prior to a neck dissection.

For tumors in stage 4 or 5 according to the pathology code description, no further treatment to the neck is required and no prophylactic neck radiotherapy or additional surgery is necessary.

The current treatment for patients with squamous cell carcinoma includes a proper diagnosis based on a high level of consideration and examination, with subsequent staging and development of a treatment paradigm. A treatment program of surgery, radiation, or chemotherapy is best developed by an oncology team that includes surgeons, radiation and medical oncologists, and rehabilitative specialists who all have significant experience in the care of patients with cancer involving the head and the neck. By using this approach, new protocols and surgical options can be appropriately offered to those patients with advanced cancers.


Long-Term Monitoring

Patients are seen every 3 months for the first year, every 4 months for the following 2 years, and every 6 months until 5 years following SLN biopsy. At any stage, if nodal disease is detected, patients can elect to undergo surgical treatment of the neck.



Sentinel lymph node (SLN) biopsy for head and neck squamous cell carcinomas (SCCs) is assuming a growing role in the management of early-stage patients. Particularly, those with T1 and T2 tumors may benefit from the procedure since the rate of occult lymph node metastases in this group is much lower than more advanced tumors. The accuracy of identification and false-negative rates of the procedure are good, but if there is concern by the surgeon performing the procedure that the node is difficult to locate (ie, lymphoscintigraphy findings are unclear or it is difficult to identify in the operating room), a formal neck dissection should be performed. SLN biopsy will probably play an increasing role in the management of early-stage head and neck SCC in the future.