Radiographic evaluation of subcutaneously injected, water-soluble, iodinated contrast for lymphography

Sentinel lymph node (SLN) mapping is common in many types of human cancers, and is gaining utility in veterinary medicine. There are currently many different methods described in veterinary medicine for pre-operative SLN mapping, however, most of these are restricted to referral institutions due to cost and need for specialized equipment. The purpose of this prospective, pilot study was to evaluate the feasibility of radiographic evaluation of water-soluble, iodinated contrast (WIC) injected subcutaneously for lymphography in dogs. Eight dogs were injected with 1-2 milliliters of WIC into the subcutaneous tissues overlying the tarsus in 4 separate locations mimicking a circumferential, peri-tumoral injection. Radiographs were taken at select time points up to 50 minutes. Image sequences were evaluated by a single, board-certified radiologist. All 8 dogs had visible contrast-enhancing lymphatic channels. Median time to lymphatic enhancement was immediately post-injection. Seven dogs (88%) had 8 contrast enhancing lymph nodes (7 popliteal and 1 superficial inguinal). Median time to lymph node enhancement was 20 minutes. In this study, the plantar aspect of the pes drained to the superficial inguinal lymph node, and the dorsal aspect of the pes drained to the popliteal lymph node. Subcutaneously-injected WIC was readily identifiable in the lymphatic channels and draining lymph node(s). Subcutaneously injected WIC may offer a practical alternative to previously described pre-operative methods of SLN mapping. Additionally, one cannot assume that the popliteal lymph node alone, drains the distal pelvic limb.


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Sentinel lymph nodes (SLN) are defined as the first lymph node(s) receiving lymphatic 37 drainage from a tumor. Sentinel lymph node mapping and biopsy is performed to stage many 3 3 38 types of human cancers [1][2][3][4] and is gaining increasing utility in veterinary medicine. [5][6][7][8][9][10][11][12][13] 39 Sentinel lymph nodes can be presumed based on location of a primary lesion; however, a recent 40 study showed that 8 of 20 dogs (40%) with naturally occurring mast cell tumors demonstrated 41 aberrant lymphatic drainage from that expected based on anatomic location. [14] Additionally, 42 one veterinary study showed that evaluation of only the mandibular lymph nodes could result in 43 up to a 45% under-diagnosis of metastases of oral tumors. [15] Several studies have shown that 44 lymph node metastasis has prognostic implications in different types of cancer, and removal of 45 lymph nodes with confirmed metastasis improves survival in both human [3,4,16,17] and 46 veterinary medicine. [14,[18][19][20][21][22][23][24][25] Based on this information, the importance of identification and 47 sampling of the SLN is clear. 48 Described methods of pre-operative SLN mapping in veterinary medicine include 49 lymphoscintigraphy, [14] contrast-enhanced ultrasonography, [6,26] lipid-soluble iodinated 50 contrast (LIC) with radiography 12,14,15 or computed tomography, [5,7,8,27,28] and water-51 soluble iodinated contrast (WIC) with computed tomography. [13,[28][29][30] Lymphoscintigraphy 52 requires special licensure and specialized equipment that limit the availability of this diagnostic 53 technique. Similarly, the available literature only describes the use of WIC with computed 54 tomography for lymphography. This requires referral to specialty practices or academic 55 institutions in most instances, which may preclude access to this diagnostic technique. While 56 LIC can be used for radiographic evaluation and computed tomographic evaluation, LIC is 57 expensive and can be difficult to obtain. Additionally, optimal imaging evaluation does not occur 58 until 24 to 48 hours after administration [5,7] which adds additional cost and time.

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Previous studies have shown that subcutaneously administered WIC is rapidly absorbed into 60 the local lymphatic channels. [8,9,11,12,28,30,31] However, such studies only describe 4 4 61 evaluating SLN and lymphatic channels using computed tomography. Development of a more 62 economic and readily available technique for SLN mapping could be beneficial to veterinary 63 oncology patients. Water-soluble contrast is inexpensive and available in most veterinary 64 practices, including primary care veterinary facilities. Radiography is also more readily available 65 in veterinary practices than is computed tomography.

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The objective of this study was to identify the feasibility of lymphography in healthy dogs 67 using iopamidol Bracco,Milan,Italy), a WIC, using digital radiography. The 68 hypotheses of this study were that subcutaneously-injected WIC would be identifiable and 69 traceable in the lymphatics and the draining lymph node. Additionally, it was hypothesized that 70 the popliteal lymph node would be the primary lymphatic drainage of the distal pelvic limb.

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A prospective, pilot study was designed. The study protocol was approved by the 73 Institutional Animal Care and Use Committee at Auburn University (Protocol #2017-3199). All 74 dogs underwent a thorough physical examination by one of the investigators (CEL) to rule out 75 any pre-existing conditions. All dogs were re-examined immediately after subcutaneous injection 76 of the WIC, after recovery from sedation, and 24 hours post-injection of WIC to assess adverse 77 events at the injection sites.

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Eight 9-month old, intact male Beagles weighing 13 to 14 kg obtained from a licensed USDA 6 6 105 source were included in this study. All animals were deemed healthy on the basis of physical 106 examination. These dogs were obtained for a use unrelated to this study, however, no other 107 interventions were done prior to this study.

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Mediolateral radiographic images were obtained of the injected limb pre-injection and at 109 times 0, 3, 5, 10, and 20 minutes post-injection in all dogs. Based on degree of lymph node 110 enhancement in the first three dogs the study protocol was modified to include radiographs up to 111 60 minutes if necessary to achieve lymph node enhancement. Thus, mediolateral images were 112 taken at 30 and 40 minutes in 5 of the 8 dogs and at 50 minutes in 1 dog.

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One milliliter of WIC was injected into the subcutaneous tissue on the plantar aspect of the 114 pes in 1 dog, 2 milliliters of contrast was injected on the plantar aspect in 6 dogs, and 1 milliliter 115 was injected into the subcutaneous tissues of both the plantar and dorsal aspects, for a total of 2 116 milliliters, of the pes in 1 dog. The volume of WIC was increased after the first dog, as the 117 contrast material was visible within the lymphatics and approaching the popliteal lymph node 118 within the study period, however, nodal enhancement did not occur. The final dog in this study 119 was injected on both the plantar and dorsal aspects of the pes in response to identification of a 120 lymphatic pathway from the dorsal aspect of the pes to the popliteal lymph node.

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All dogs had visible enhancement of lymphatic channels. The median time to initial 122 enhancement of lymphatic channels was immediately post injection (range 0-5 minutes) and 123 depletion of lymphatic channel enhancement was not identified during the study period.   the lymph nodes was 30 minutes (range 10-50 minutes). In 3 of 7 dogs and 4/8 lymph nodes that 150 enhanced, maximum contrast enhancement occurred at the last radiograph taken. All but 1 dog 151 exhibited enhancement of the draining lymph node(s) that continued to increase or remained 152 static once maximal enhancement was reached throughout the duration of the study period. One 153 lymph node, which had initial enhancement at 5 minutes, had mild decrease of contrast 154 enhancement at 40 minutes, but the lymph node remained enhanced in comparison to pre-155 contrast images. One dog had enhancement of the lymphatic vasculature, but not the draining 156 lymph node. This was the initial dog studied injected with only 1 ml of contrast, prompting the 157 increase to 2 ml for subsequent evaluations. Enhancement of both the superficial inguinal and 158 popliteal lymph node occurred in one dog at 20 and 50 minutes, respectively (Fig 3).

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Enhancement of separate afferent lymphatics leading to each lymph node from the injection site 160 were identifiable, and there were no visible efferent lymphatics from either lymph node.

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The caudal lymphatic channel, depicted by the short, solid white arrows, is contrast enhanced  Alternatively, 1 milliliter of contrast material may not be sufficient to enhance the draining 208 lymph node in all cases, irrespective of time after injection. In a study by Grimes et al[32], 78% 209 (7/9) of dogs injected with 1 ml of contrast had SLNs identified by computed tomographic 210 evaluation in comparison to 100% (9/9) of dogs injected with 2 milliliters of contrast.

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Confounding factors theorized to be impacting detection of drainage to the SLN in that study 212 included tumor compression of lymphatics, patient positioning, and/or endotracheal tube ties 213 causing collapse or restriction of lymphatic flow. These factors were not a part of this study 214 suggesting that an additional confounder, such as time or volume of contrast material, was the 215 main factor for the negative result here. Considering these findings, the authors recommend 216 using 2 mls of WIC for lymphographic evaluation with radiographs. In this study, the achievement of maximal enhancement of the lymph nodes occurred at a 218 median of 30 minutes. However, with the exception of one dog in which lymph node 219 enhancement decreased, maximal lymph node enhancement occurred at the last taken radiograph 220 in 50% of dogs, with the remaining dogs having reached maximal enhancement at the next to last 221 radiograph. It is possible that if the radiographic studies were extended, further enhancement 222 might have occurred. Contrarily, it is also possible that a decrease of contrast enhancement might 223 have occurred in more of these cases if images were acquired at later times due to spread beyond 224 the primary lymph node. There was no observed decrease in lymphatic vasculature enhancement 225 in this study. Therefore, if using this technique for SLN mapping, the authors would recommend 226 obtaining radiographs within 30 minutes post injection to follow the lymphatic channels to the 227 sentinel lymph node and, if necessary, intermittently thereafter until a distinct sentinel lymph 228 node is identifiable.

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Limitations of this study included use of young, healthy dogs without masses or lesions 230 whose lymphatic drainage could be mapped and the lack of orthogonal radiographs. However, as 231 the goal of this study was to determine the feasibility of indirect lymphography using WIC and 232 radiographic interpretation. Additionally, the slight variations in WIC injection techniques did 233 not detract from the aforementioned goal. Further studies to assess the utility of this technique 234 for sentinel lymph node mapping in patients with a variety of cutaneous and subcutaneous 235 neoplasms are warranted.

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Based on the results of this study, subcutaneously-injected, water-soluble, iodinated contrast 237 material provides a relatively quick and effective means of tracing the lymphatic channels from 238 the pes to the draining lymph node(s). The results of this study also show that one cannot assume 239 that the distal pelvic limb will have primary lymphatic drainage only to the popliteal lymph node.