• View in gallery
    Figure 1—

    Photograph of a dog's right mandibular lymphocentrum that has been palpated and brought to the skin surface. The dog was used in a study to evaluate the usefulness of ICG injection with NIR fluorescence imaging for transcutaneous detection of SLNs and their associated lymphatic vessels in the oral mucosa of healthy dogs. One milliliter of ICG solution was injected into the gingival mucosa dorsal to the right maxillary canine tooth. Subsequently, NIR fluorescence imaging was used to transcutaneously detect the lymphatic vessels and SLNs. The distance between the injection site and the SLN (arrow) was measured.

  • View in gallery
    Figure 2—

    Photograph to illustrate the submucosal injection of 1.0 mL of ICG solution (concentration, 0.5 mg/mL) dorsal to the right maxillary canine tooth (Triadan tooth No. 104) in the dog of Figure 1 following sedation.

  • View in gallery
    Figure 3—

    Representative photograph of the fluorescence detected under an NIR light source in the lymphatic tract and mandibular SLN at 7 minutes after a submucosal injection of ICG solution dorsal to the right maxillary canine tooth in 1 of 6 dogs. See Figure 1 for remainder of key.

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Feasibility of near-infrared fluorescence imaging for sentinel lymph node evaluation of the oral cavity in healthy dogs

Katy L. TownsendDepartment of Clinical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331.

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Milan MilovancevDepartment of Clinical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331.

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Shay BrachaDepartment of Clinical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331.

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Abstract

OBJECTIVE To evaluate the usefulness of injection of indocyanine green (ICG) solution with near-infrared (NIR) fluorescence imaging for transcutaneous detection of sentinel lymph nodes (SLNs) and their associated lymphatic vessels in the oral mucosa of healthy dogs.

ANIMALS 6 adult purpose-bred research hounds.

PROCEDURES Each dog was sedated, and 1 mL of ICG solution was injected into the gingival mucosa dorsal to the right maxillary canine tooth. Subsequently, NIR fluorescence imaging was used to transcutaneously detect the lymphatic vessels and SLNs. The distance between the injection site and each SLN was measured. Time to first evidence of node fluorescence was recorded, and velocity of ICG movement was calculated. A slide preparation of a fine-needle aspiration sample of the fluorescing structure underwent cytologic examination (to confirm presence of lymphatic tissue) and NIR fluorescence imaging (to confirm presence of ICG).

RESULTS The ipsilateral mandibular lymphocentrum was the SLN in all dogs. The time to visually detectable fluorescence ranged from 4 to 15 minutes (mean ± SD, 8.8 ± 3.76 minutes). The mean velocity was 1.94 ± 0.93 cm/min. Fluorescence was not observed in the contralateral lymph nodes. Each fluorescing structure was confirmed to be lymphatic tissue, and NIR fluorescence imaging revealed that ICG was present in the sampled SLN.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that injection of ICG solution with NIR fluorescence imaging can be used to transcutaneously identify SLNs along with associated lymphatic vessels in the oral mucosa of healthy dogs. Time from injection to identification of fluorescence was rapid with prolonged retention of material within the SLN, indicating that this procedure could be performed during surgery.

Abstract

OBJECTIVE To evaluate the usefulness of injection of indocyanine green (ICG) solution with near-infrared (NIR) fluorescence imaging for transcutaneous detection of sentinel lymph nodes (SLNs) and their associated lymphatic vessels in the oral mucosa of healthy dogs.

ANIMALS 6 adult purpose-bred research hounds.

PROCEDURES Each dog was sedated, and 1 mL of ICG solution was injected into the gingival mucosa dorsal to the right maxillary canine tooth. Subsequently, NIR fluorescence imaging was used to transcutaneously detect the lymphatic vessels and SLNs. The distance between the injection site and each SLN was measured. Time to first evidence of node fluorescence was recorded, and velocity of ICG movement was calculated. A slide preparation of a fine-needle aspiration sample of the fluorescing structure underwent cytologic examination (to confirm presence of lymphatic tissue) and NIR fluorescence imaging (to confirm presence of ICG).

RESULTS The ipsilateral mandibular lymphocentrum was the SLN in all dogs. The time to visually detectable fluorescence ranged from 4 to 15 minutes (mean ± SD, 8.8 ± 3.76 minutes). The mean velocity was 1.94 ± 0.93 cm/min. Fluorescence was not observed in the contralateral lymph nodes. Each fluorescing structure was confirmed to be lymphatic tissue, and NIR fluorescence imaging revealed that ICG was present in the sampled SLN.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that injection of ICG solution with NIR fluorescence imaging can be used to transcutaneously identify SLNs along with associated lymphatic vessels in the oral mucosa of healthy dogs. Time from injection to identification of fluorescence was rapid with prolonged retention of material within the SLN, indicating that this procedure could be performed during surgery.

Sentinel lymph node mapping is a precise method for identification of lymph nodes that may be affected by tumor metastasis.1 In patients with cancer, assessment of lymph node status is critical for determination of disease stage and prognosis. Accurate staging of patients can be challenging, and lymph node drainage cannot be predicted by anatomic location alone.2–6 An SLN is defined as the first lymph node (or nodes) within a lymphatic drainage basin to which a primary tumor reliably drains.7 Histologic examination of tissue samples from an SLN yields the most accurate patient staging information that can be used to guide treatment options and determine prognosis.8

Lymphoscintigraphy performed with a peritumoral injection of a radionucleotide and intraoperative injection of blue dye is considered the gold standard for SLN detection in humans.9 In a case series of client-owned dogs with mast cell tumors,5 8 of 19 dogs had an SLN that was not the expected anatomically regional lymph node. Disadvantages of lymphoscintigraphy are the need for radionucleotide-capable facilities and appropriately trained staff, adherence to safety aspects of radionucleotide exposure, and lack of real-time intraoperative percutaneous visual information.10 The disadvantages of the use of blue dye injection include limited depth penetration and blue staining of the surgical field. Given the limitations associated with this type of lymphoscintigraphy, the use of NIR fluorescence imaging has been developed to identify SLNs in people.11

Near-infrared light has a wavelength of 700 to 900 nm and achieves high tissue penetration to depths of millimeters to centimeters.11 The agent used, NIR machine, and tissue composition all impact the depth of fluorescence visualization.12 The human eye is insensitive to NIR wavelengths, so this does not alter the surgeon's perception of the surgical field as occurs with blue dye SLN mapping. Near-infrared fluorescence imaging that uses injection of ICG solution has been successfully applied in humans for SLN mapping in cases of melanoma and breast, cervical, gastric, skin, or vulvar cancer.13–16 Near-infrared fluorescence imaging has the potential to improve SLN mapping by allowing real-time transcutaneous visualization of the lymphatic channels and subsequent detection of the SLN during surgery.11

Indocyanine green is an FDA-approved fluorescent contrast agent that has an emission peak of 822 nm. After IV injection of the solution, ICG has a short half-life of 150 to 180 seconds, and it is cleared exclusively by the liver. Safety data indicate that ICG solution is very safe to use; at a concentration of 2.5 mg/mL, the FDA-approved IV doses are up to 25 mg in adults, up to 12.5 mg in children, and up to 6.25 mg in infants.11 Doses of ICG in solution for SLN mapping reported in the human medical literature vary considerably, ranging from as little as 10 μg17 to as much as 25 mg.18

The use of ICG solution and NIR fluorescence imaging has identified SLNs associated with various cancers in humans, and the technique has been shown to perform as well as lymphoscintigraphy with the gold-standard combination of radiocolloid and blue dye administration.11,19–21 In humans with head or neck cancer, injection of ICG solution has been used successfully to identify the SLN.22–24 To our knowledge, there have been no studies performed in dogs to assess the potential for clinical use of ICG solution and NIR fluorescence imaging for SLN mapping. To date, it is not known whether there are species differences between humans and dogs regarding use of this novel imaging technique.

The objective of the study reported here was to assess the usefulness of injection of ICG solution with NIR fluorescence imaging for transcutaneous detection of SLNs and their correlated lymphatic vessels in the oral mucosa of healthy dogs. As part of this assessment, we were interested in determining the velocity of ICG movement within the lymphatics (on the basis of the interval between injection and highlighting of the draining lymph node) of the oral mucosa in a group of healthy hounds to predict clinical timing of SLN identification. The hypothesis was that injected ICG would generate transcutaneously detectable fluorescence in the SLN and associated lymphatic vessels within a clinically appropriate time.

Materials and Methods

Six purpose-bred houndsa (weight range, 19 to 23 kg) were used in the study. There were 3 sexually intact males and 3 sexually intact females. All procedures were approved by the Institutional Animal Care and Use Committee of Oregon State University. Food was withheld from the dogs overnight (approx 12 hours), and then the dogs were sedated with dexmedetomidine hyrdrochloride (5 μg/kg, IV, once) and butorphanol tartrate (0.1 mg/kg, IV, once).

The right and left mandibular lymphocentra were each palpated and brought to the surface of the skin manually (Figure 1). The distance between the injection site and the mandibular lymphocentrum was recorded. For injection, 25 mg of ICGb was suspended in 10 mL of sterile water to yield a 2.5-mg/mL (3.2mM) stock solution; this solution was then further diluted with 5% dextrose to provide a 0.5-mg/mL (0.64mM) solution. The 0.5-mg/mL solution was used within 6 hours of preparation as specified on the manufacturer's packaging.b The ICG solution was injected as a bolus (total volume, 1.0 mL) into the buccal submucosa, dorsal to the right maxillary canine tooth (Triadan tooth No. 104), with a 22-gauge needle (Figure 2). The ICG solution was not massaged into the region after injection. Dogs were positioned in left lateral, right lateral, and dorsal recumbency for image capture without head elevation. The neck was extended when the dogs were placed in dorsal recumbency. The dogs’ positioning was changed between these 3 postures for image capture every minute, so that images for all 3 positions were captured every minute up until 10 minutes. Near-infrared imagingc was used to capture images of the lateral and ventral cervical regions in all 3 positions, focusing on the left and right mandibular lymphocentrum and retropharyngeal lymphocentrum, before the ICG injection, every minute after injection up to 10 minutes or the time of lymph node fluorescence detection, and then at 15, 30, 45, and 60 minutes after injection. Images in all 3 positions focusing on the mandibular and retropharyngeal lymphocentra were also obtained at 6 hours and 24 hours after injection. For the 6- and 24-hour imaging, the dogs were not sedated, and images were captured with the dog in a sitting position with its neck extended. The NIR imaging machine was set at image capture of 500 milliseconds and positioned 15 cm above the skin surface. The velocity of ICG movement along the lymphatics was assessed by dividing the distance between the injection site and the mandibular lymph node by the time from injection to detection of fluorescence.

Figure 1—
Figure 1—

Photograph of a dog's right mandibular lymphocentrum that has been palpated and brought to the skin surface. The dog was used in a study to evaluate the usefulness of ICG injection with NIR fluorescence imaging for transcutaneous detection of SLNs and their associated lymphatic vessels in the oral mucosa of healthy dogs. One milliliter of ICG solution was injected into the gingival mucosa dorsal to the right maxillary canine tooth. Subsequently, NIR fluorescence imaging was used to transcutaneously detect the lymphatic vessels and SLNs. The distance between the injection site and the SLN (arrow) was measured.

Citation: American Journal of Veterinary Research 79, 9; 10.2460/ajvr.79.9.995

Figure 2—
Figure 2—

Photograph to illustrate the submucosal injection of 1.0 mL of ICG solution (concentration, 0.5 mg/mL) dorsal to the right maxillary canine tooth (Triadan tooth No. 104) in the dog of Figure 1 following sedation.

Citation: American Journal of Veterinary Research 79, 9; 10.2460/ajvr.79.9.995

The left and right mandibular lymphocentra were aspirated with a 22-gauge needle at 24 hours after injection of the ICG solution. For the left side with no fluorescence present, palpation alone was used to identify the mandibular lymphocentrum. Because aspirates were performed by palpation or with percutaneous fluorescence assistance, we cannot be certain which lymph node from the mandibular lymphocentrum was aspirated. A slide preparation of each fine-needle aspirate sample underwent NIR fluorescence imaging to assess whether the aspirated material had residual fluorescence. Cytologic examination of the lymph node aspirates was performed by a board-certified veterinary clinical pathologist to confirm the presence of lymph node tissue. The injection site was monitored daily for any inflammation, and dogs were assessed for any systemic adverse effects such as vomiting, diarrhea, and lethargy over a period of 5 days. The hounds were adopted at the conclusion of the study.

Data were tested for normality with a Shapiro-Wilk test. Variables of interest are reported as mean and SD or median and range for normally distributed and non-normally distributed data, respectively. Commercially available softwared was used for the analyses.

Results

The median weight of the 6 dogs was 22 kg (range, 19 to 23 kg). For all dogs, the ipsilateral mandibular lymphocentrum was the SLN. The median distance between the injection site and the mandibular lymphocentrum was 15 cm (range, 13 to 15 cm). The time for fluorescence to be identified in the mandibular lymphocentrum following injection of ICG solution ranged from 4 to 15 minutes (mean interval, 8.8 ± 3.76 minutes). The mean velocity was 1.94 ± 0.93 cm/min. Fluorescence of the associated lymphatic tracts was also visible under the NIR light source (Figure 3). At no time was any fluorescence observed in the contralateral mandibular lymphocentrum in any dog. All lymphocentra that initially fluoresced still had detectable fluorescence at 24 hours after injection.

Figure 3—
Figure 3—

Representative photograph of the fluorescence detected under an NIR light source in the lymphatic tract and mandibular SLN at 7 minutes after a submucosal injection of ICG solution dorsal to the right maxillary canine tooth in 1 of 6 dogs. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 79, 9; 10.2460/ajvr.79.9.995

The slide preparation of the aspirate specimen obtained from each fluorescent structure confirmed that it was lymphatic tissue. All slides of the aspirated material appeared fluorescent when examined under the NIR source. Examination of slides prepared from the contralateral mandibular lymphocentrum confirmed that it was lymphatic tissue. No aspirate specimens obtained from the contralateral mandibular lymph nodes appeared fluorescent when examined under the NIR source.

For each dog, the ICG solution injection site did not develop any signs of inflammation during the 5-day postinjection period. No clinically detectable adverse reactions were identified.

Discussion

Results of the present study indicated that transcutaneous NIR fluorescence imaging following oral mucosal injection of 1.0 mL of ICG solution (concentration, 0.5 mg/mL) can be used to detect lymphatic vessels and superficial lymph nodes in healthy dogs. The movement of the ICG along the lymphatics was rapid (mean velocity, 1.94 cm/min), which provides a basis for development of this method for SLN mapping in dogs with oral tumors. Given the short interval between injection of the ICG solution and detection of fluorescence of the SLN in the study dogs, we suggest that NIR fluorescence imaging for SLN detection can be used as an intraoperative imaging technique.

Fluorescence of the SLNs was detected as early as 4 minutes after injection in the present study. In humans with breast cancer, fluorescence detection occurs at 1 to 10 minutes after injection of ICG solution (velocity, 3 to 5 cm/min).19 This rate of ICG movement is similar to what was observed in the dogs of the present study. In pigs, ICG transit time is much faster at 0.5 cm/s.25 Because there is minimal clinical information regarding the use of ICG for SLN detection in dogs, an aim of the present study was to assess the clinical timing of SLN identification following injection of ICG solution. Other SLN mapping methods report injecting substances such as iodized oil 24 hours prior to surgery or imaging.26 Other aspects that were not evaluated in the present study but that could affect lymphatic velocity of ICG included positioning, injection volume and rate, and postinjection massage of the injection site. In dogs, it has been shown that positioning can dramatically affect the flow of aqueous contrast agents during SLN mapping.27 Therefore, clinicians should be aware of the potential effect of patient positioning affecting SLN uptake when applying transcutaneous NIR fluorescence imaging after injection of ICG solution in clinical cases. In the present study, lymph nodes had residual fluorescence at 24 hours after ICG solution injection. In a recent porcine study25 that used injections of ICG solution to identify SLNs in mammary glands, fluorescence of the lymph nodes was still apparent 20 days after injection. The duration of ICG-associated fluorescence in canine lymph nodes is not known. Given the rapid movement of ICG in the lymphatic system in the dogs of the present study, second-tier lymph nodes may easily take up the ICG. Second-tier lymph nodes are defined as lymph nodes that have lymphatic drainage from a tumor in series after the SLN.28 Studies24 of ICG injections in humans have shown second-tier lymph nodes to fluoresce at variable intervals following administration, further highlighting the need for intraoperative timing for the injection to specifically target the SLN instead of a second-tier lymph node.

One of the benefits of NIR fluorescence imaging is the transcutaneous identification of lymphatic vessels. This allows a surgeon to more precisely focus the approach for lymph node resection, which can decrease surgical time and postoperative morbidity. By use of ICG and NIR fluorescence imaging, the location of lymphatic channels was very obvious in the dogs of the present study. The lymphatic channels were detected almost immediately after injection of ICG solution and before fluorescence in the node was evident. The lymphatic channels identified were likely superficial because of the ease with which they were imaged. The type of NIR fluorescence imaging machine used in the present study has a known tissue penetration depth of 2.5 cm (as determined in a study25 in pigs) and produces a decreasing signal intensity with increasing depth. Hair and skin have been shown to decrease signal intensity, whereas melanin did not affect intensity in a studye performed in canine cadavers. In the dogs of the present study, the mandibular lymphocentrum was not surgically approached to differentiate the lateral mandibular lymph node from the medial mandibular lymph node. Palpation and results of fine-needle aspiration are not sufficient to definitively determine whether samples were collected from the lateral or medial mandibular lymph node. Compared with use of methylene blue, the benefit of ICG is its fluorescent properties; however, it has the potential to be seen within lymphatic vessels and lymph nodes with the naked eye intraoperatively because of its green pigment.

In a human SLN mapping study22 involving injection of ICG solution and NIR fluorescence imaging, elevation of the platysma was often necessary to evaluate the SLN, because depth of fluorescence visualization does not exceed 5 mm in some imaging systems. Although we were able to transcutaneously identify lymphatic tracts in the dogs of the present study, a surgical approach may be necessary to evaluate deeper lymphatic vessels and lymph nodes in clinical patients. Results of a recent study29 of humans with malignant melanoma indicated that the success rate of transcutaneous identification of SLNs with NIR fluorescence imaging can be as low as 21% without a surgical approach.

Currently, ICG is the only commercially available and FDA-approved NIR fluorescence lymphatic tracer. The concentration of the ICG solution used in the present study was extrapolated from other studies involving SLN mapping in humans.11 The best vehicle for ICG dilution has been investigated. Human serum albumin has been used to increase the fluorescence intensity of ICG.30 Given that ICG binds serum proteins, the theory for use of human serum albumin premixing is to increase the hydrodynamic diameter of ICG molecules to that of the bound proteins, resulting in better ICG retention in the SLN.31 However, it has been shown that there is no notable advantage of dilution of ICG with human serum albumin,21 and in fact the procedure has potential negative aspects such as increased cost along with the potential for adverse effects. Results of a recent meta-analysis10 indicated that use of a lower concentration of ICG solution (ie, < 5 mg/mL) and an injection volume of > 2 mL may improve SLN detection. In a study30 of SLN mapping in humans with cutaneous melanoma that used ICG premixed with human serum albumin, a range of ICG injection doses (600 to 1,200μM) was evaluated, and an injection concentration of 600μM provided a strong, clinically relevant signal-to-background noise ratio in the identified SLNs. Higher concentrations of ICG can have lower signal-to-background noise ratios with a decrease in fluorescence explained by fluorescence quenching.32 Because of the challenging calculation needed to determine this concentration, we chose to use 0.5 mg of ICG/mL, which is equivalent to 0.64mM. We selected standard sterile water to make the initial dilution and then used 5% dextrose to obtain the final concentration of the ICG solution. Dextrose was chosen because it was used in a previous porcine study25 undertaken with the same imaging system, and as an attempt to increase the osmolarity of the ICG, thereby promoting its retention in the lymphatics for a longer period. It is unknown what dose and concentration of ICG solution would be optimal for use in dogs; however, in the present study, we have shown that administration of 1.0 mL of ICG solution at a concentration of 0.5 mg/mL (achieved by dilution with sterile water and 5% dextrose) is an effective dose for assessments of lymphatic in canine heads and necks performed with this NIR fluorescence imaging device.

There were several limitations to the present study. First, only 6 healthy dogs without evidence of oral cancer were used. The lymphatic system's role in metastatic spread is complex, with multiple molecular mechanisms (and no doubt many still to be identified) that facilitate interactions between tumor cells and lymphatic tissues during tumor progression.33 The present study was intended to provide more information regarding the timing and efficacy of this novel imaging technique, and not to further elucidate the complicated lymphatic drainage of primary tumors. Lymph nodes were not surgically approached to confirm that the fluorescing structure was lymphoid tissue; instead, confirmation was achieved through cytologic examination of fine-needle aspirate specimens obtained from the fluorescing structures, which did reveal lymphocytes. The mandibular nodes in dogs form a group of 2, 3, or, rarely, as many as 5 nodes that are located ventral to the angle of the mandible.34 It is not known which of these nodes within the mandibular lymphocentrum became fluorescent after injection of ICG solution or whether deeper lymph nodes, such as the medial retropharyngeal node, became fluorescent because we did not surgically approach the deeper nodes and did not obtain fine-needle aspirate specimens from them even though the dogs were repositioned multiple times during image acquisition. For clinical cases, if deeper lymph nodes are suspected of being SLNs before surgery either by anatomic location or results of other SLN mapping procedures, such as scintigraphy or CT indirect lymphangiography,35 a surgical approach could be performed prior to injection of ICG solution. A surgical approach prior to injection must be balanced against the local anatomic features and potential risk of disrupting the draining lymphatic pathways that might cause a false-positive identification of an inappropriate lymph node as an SNL.

For clinical cases in dogs with naturally occurring tumors, SLN mapping by means of NIR fluorescence imaging after injection of ICG solution could be used alone or with adjunctive preoperative SLN mapping, such as CT indirect lymphangiography or nuclear scintigraphy. The decision to proceed with preoperative SLN mapping would likely be based on the anatomic location of the primary tumor. If there is the potential that deeper lymph nodes could be the SLN for a primary tumor, then we recommend that preoperative mapping with complementary imaging, such as scintigraphy or CT indirect lymphangiography, should be performed. Patient positioning and surgical site preparation should be thoroughly evaluated before injection of ICG solution because there is the potential for patient positioning to impede lymphatic flow and a possibility that the SLN may be located in an area that is not prepared for surgery. As each NIR imaging system is associated with slightly different depths of fluorescence identification, clinicians using this type of imaging device should have prior knowledge of the specific fluorophore used in their system.11 It should also be noted that ICG is easily transferred on gloves following injection. The authors recommend double-gloving to inject the ICG solution and then removing those gloves or having an assistant perform the injection so as not to transfer ICG into the surgical field.

Results of the present study indicated that NIR fluorescence imaging after injection of ICG solution is a feasible method for transcutaneous identification of lymph nodes when the ICG solution is injected submucosally into the oral cavity of healthy dogs. Because of the relatively rapid spread of ICG along lymphatic pathways, the authors recommend that the injection be performed at the time of surgery when the patient's hair and skin are clipped and aseptically prepared and not during any prediagnostic procedures. Given the potential of deeper lymph nodes being the SLN and the inability to transcutaneously detect fluorescence in those nodes, preoperative SLN mapping with either scintigraphy or CT indirect lymphangiography may be warranted. If deeper lymph nodes are suspected from SLN mapping or anatomic location of the primary tumor (eg, SLNs for anal sac tumors), then a surgical approach to the nodes may be necessary before injection of ICG solution. The fact that ICG fluorescence was still present at 24 hours after injection in the dogs of the present study suggested there is potential for second-tier lymph nodes to fluoresce if the ICG solution is injected too early in relation to the time of the surgery. Overall, the SLN mapping method evaluated in this study appears to warrant further assessment in clinical trials.

Acknowledgments

Presented in part at the American College of Veterinary Surgeons Surgery Summit, Seattle, October 2016.

The authors have no conflicts of interest to declare.

The authors thank Dr. Duncan Russell for cytologic assessment of the lymph node slides.

ABBREVIATIONS

ICG

Indocyanine green

NIR

Near-infrared

SLN

Sentinel lymph node

Footnotes

a.

Marshall BioResources, North Rose, NY.

b.

IC-Green Akorn Inc, Lake Forest, Ill.

c.

Fluobeam 800, Fluoptics, Grenoble, France.

d.

GraphPad Prism, version 7.00 for Windows, GraphPad Software, La Jolla, Calif.

e.

Townsend KL, Sandwisch JM. Optimization of indocyanine green use in near infrared fluoresence imaging in canine cadavers for direct lymphangiography (oral presentation). Am Coll Vet Surg Surg Summit, Seattle, October 2016.

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Contributor Notes

Address correspondence to Dr. Townsend (katy.townsend@oregonstate.edu).