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10.1245/ASO.2006.04.020
Annals of Surgical Oncology 13:692-700 (2006)
© 2006 Society of Surgical Oncology
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Original Article

99mTc–Evans Blue Dye for Mapping Contiguous Lymph Node Sequences and Discriminating the Sentinel Lymph Node in an Ovine Model

Chris Tsopelas, PhD1, Elaine Bevington, MB, BS2, James Kollias, MB, BS2, Sabah Shibli, MB, BS2, Gelareh Farshid, MB, BS3, Brendon Coventry, MB, BS, PhD4 and Barry E. Chatterton, MB, BS1

1 Nuclear Medicine Department, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia
2 Department of Breast and Endocrine Surgery, Royal Adelaide Hospital, North Terrace, Adelaide, South Australia 5000, Australia
3 Tissue Pathology Section, Institute of Medical and Veterinary Sciences, Frome Road, Adelaide, South Australia 5000, Australia
4 Department of Surgery, University of Adelaide, Frome Road, Adelaide, South Australia 5000, Australia

Correspondence: Address correspondence and reprint requests to: Chris Tsopelas, PhD; E-mail: ctsopela{at}mail.rah.sa.gov.au.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Background: The aim of this study was to investigate the potential of 99mTc–Evans blue for discriminating the sentinel lymph node in multitiered lymph node sequences by using an ovine model. 99mTc–Evans blue is an agent that has both radioactive and color signals in a single dose. Previous studies in smaller animal models suggested that this agent could have advantages over the dual-injection technique of radiocolloid/blue dye.

Methods: Doses of 99mTc–Evans blue (~ 21 MBq) containing Evans blue dye (approximately 4 mg) were administered to the hind limbs or fore limbs of sheep to map the lymphatic drainage patterns, validate its ability to identify the sentinel lymph node, and examine the reproducibility of the technique. The study protocol was repeated with 99mTc–antimony tri-sulfide colloid and Patent Blue V dye. After the operative exposure, lymph nodes were identified with the gamma probe and then excised and analyzed for radioactivity (percentage of injected dose) and blue color.

Results: After the administration of 99mTc–Evans blue, all lymph nodes harvested (35 of 35) in either short chains or long basins were hot and blue. The sentinel lymph nodes concentrated more radioactivity than the second-tier nodes to the extent of 2:1 to 215:1. For radiocolloid/Patent Blue V, the ratios were lower, at 2:1 to 3:1.

Conclusions: 99mTc–Evans blue was found to better discriminate the sentinel lymph node than 99mTc–antimony trisulfide colloid/Patent Blue V in variable multitier lymph node anatomy, and it is an agent that promises to have positive clinical applications.

Key Words: Radioactive blue dye • Evans blue • Lymphoscintigraphy • Sentinel lymph node • Multitier nodes • Sheep


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Lymphatic mapping and sentinel node biopsy techniques are currently used to identify and remove the sentinel lymph node(s) or any node(s) that receive lymphatic drainage from a primary tumor. The sentinel node biopsy is rapidly replacing axillary dissection to stage the axilla in cases of early breast cancer1 and has evolved into standard practice for the surgical management of melanoma.2,3 The lymphatic mapping agents used in clinical practice are 99mTc-labeled colloids and vital dyes, which provide the radioactive signal and the color signal, respectively. When both lymphatic mapping agents are used in tandem, the false-negative rate for sentinel node biopsy can be decreased significantly.4–7 Therefore, certain investigators have recommended the use of the two lymphatic mapping agents in clinical practice.8,9 The literature has shown that concordant blue and radioactive nodes can be identified in 30%10 to 90%11 of cases.

The preference of radiocolloid or vital dye for sentinel lymph node detection differs among continents. 99mTc–antimony trisulfide colloid (ATC) is used routinely in Australia, 99mTc–tin fluoride colloid or 99mTc-phytate in Japan,12 filtered 99mTc–sulfur colloid in the United States, and 99mTc–albumin nanocolloid in Europe. Patent Blue V13 (Guerbet, Roissy, France) is used in Australia and Europe, whereas isosulfan blue and indocyanine green14 are used in the United States and Japan, respectively. Evans blue (EB) has been used in clinical practice for >80 years as a pharmaceutical product, principally for the determination of patient plasma volume. Recently, this particular dye was also used to identify the sentinel lymph node in patients with breast cancer.15,16

99mTc-EB (Fig. 1Go) has been previously described as an agent combines both radioactive and colored signals and can be administered as a single dose. It is prepared from an instant "cold" kit by efficient radioactive labeling, without the need for further purification.17,18 Earlier studies in a rabbit model19 demonstrated that 99mTc-EB behaved exactly the same as EB in vivo with respect to the lymph migration rate and the retention by lymph nodes. Furthermore, 99mTc-EB binds with plasma proteins20 to the same extent as EB. In the search for an agent that combines blue and radioactive signals, the aim of this study was to validate the potential of 99mTc-EB for detecting the sentinel lymph node in multitiered lymph node basins by using a large-animal model. The reproducibility and pattern of 99mTc-EB uptake by higher-tier lymph nodes was examined in a sheep model, and this technique was also compared with the dual-injection technique of 99mTc-ATC and Patent Blue V.


Figure 1
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  		FIG. 1. The structure of Evans Blue with the coordinated radioisotope (99mTc).

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Nonradioactive cold kits containing EB (16 mg; Pharmalab, Brookvale, New South Wales, Australia) and stannous chloride reductant were prepared and quality control–tested according to a previously described technique.18,19 EB injection (25 mg/5 ml) was used as the raw material for cold kits. Sodium 99mTc-pertechnetate was obtained from the daily elution of a 99Mo/99mTc generator (Gentech; Australian Radioisotopes, Sydney, Australia). Radiochemical species identified by instant thin layer chromatography were characterized by their Rf values, defined as distance migrated divided by distance of solvent migrated. All results are reported as mean ± SE.

Radiolabeling and Quality Control
99mTc-pertechnetate (80–140 MBq) diluted in saline (.9%; .5 mL) was added to the EB cold kit and allowed to stand for at least 5 minutes at room temperature until further use. Radiochemical purity was determined with ascending instant thin-layer chromatography by using a stationary phase of instant thin-layer chromatography silica gel–impregnated glass fiber strips (1 x 16 cm; Gelman Sciences, Ann Arbor, MI). The mobile phase of either acetone or saline was used to quantify the level of known impurities of 99mTc-pertechnetate at Rf = 1.0 or 99mTcO2 at Rf = 0, respectively. Each strip was marked from the origin every centimeter for 12 cm, spotted with sample, developed in the solvent, and then sectioned and counted in a gamma counter (Cobra II Auto-Gamma; Canberra Packard, Victoria, Australia) over a 99mTc window (90–190 KeV). The percentage of 99mTc-EB was calculated as 100 – (% 99mTc-pertechnetate activity)– (%99mTcO2 activity).

99mTc-ATC was prepared according to the manufacturer’s instructions,21 and radiochemical purity was determined by instant thin-layer chromatography, as described previously in saline (99mTc-per-technetate at Rf = 1.0). 99mTc-ATC with radiochemical purity >95% was used in the study.

Sheep Studies
Experiments performed with the sheep complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council) and according to a protocol approved by the Animal Ethics Committee of the Institute of Medical and Veterinary Science, Adelaide, South Australia.

Operative Protocol
A total of nine male Merino sheep (4–6 tooth; 37–50 kg) were used in the study, and each animal was premedicated by intravenous injection of thiopentone sodium (12 mg/kg) via the jugular vein. Each sheep was secured by adhesive tape to an operating table in the supine position, and then four limbs were shaved. Anesthesia was maintained by intubation of halothane in air (2.5 L/min) plus oxygen (4 L/min) throughout the entire procedure. For each sheep, a single dose (.4–.5 mL) containing 99mTc-EB (14–29 MBq) with EB (3.3–5.8 mg) was administered intradermally on the dorsum of the respective limb 5 cm above the hoof (of the fore limbs or hind limbs), and then the bleb at the injection site was massaged for 2 minutes to facilitate migration.

A short vertical incision was made in the skin of the popliteal fossa or axilla approximately 20 minutes after injection of 99mTc-EB, and the blue lymphatic channels were identified. Subsequently, careful dissection was performed toward the popliteal/prescapular nodes within the extensive adipose tissue. A gamma probe (SRP Mk II; Gammasonics, NSW, Australia) was used to assist in localizing the sentinel lymph node(s). The regional lymphatic anatomy was surgically displayed in vivo. The same procedure was repeated to locate the superficial/ deep inguinal nodes, higher-tier iliac nodes for the hind limbs, or prescapular nodes for the fore limbs in six of the nine sheep. At a selected time point, lymph node chains or basins (two or more nodes) were harvested simultaneously, and individual node samples were stored separately in neutral buffered formalin (4% w/v) for ≤4 hours before analysis. Upon completion, sheep were sacrificed by intra-cardiac puncture of pentobarbitone (325 mg/mL; 23 mL). In three of the nine sheep, the operative protocol was repeated except that 99mTc-ATC (30–35 MBq in .1 mL) and Patent Blue V (CAS 3536-49-0; 25 mg/mL; .5 mL) replaced 99mTc-EB on one side of the hind limbs. The radiocolloid was injected first, followed by the dye 20 to 30 seconds later as close as possible to the first injection site. Radioactive or "hot" lymph nodes were defined as >.001% injected dose (ID), whereas those with blue stain in all or part of the node were defined as colored lymph nodes.

Counting and Sample Analyses
The distance traveled by the radiotracer from the injection site to the sentinel lymph node or from one node to the next node was determined by using lengths of silk ligature and then calibrating each of those lengths with a ruler in millimeters. Harvested lymph nodes were carefully processed to remove adipose tissue and assessed for color. Each sample was then counted five times in a large-volume gamma counter (Biosentry; AEI-EKCO, Australia) linked to a multichannel analyzer (model 3100; Canberra Industries Inc., Meriden, CT) over a 99mTc window (70–210 KeV). Results were calculated on the basis of a representative 99mTc dose standard (.1 mL) and reported as the percentage of the ID. All counted samples were background-corrected. The reliability of the counting procedure was examined for counts 20 to 2,000, 2,001 to 50,000, and 50,001 to 999,999 and calculated as a percentage of the standard error divided by the mean value.

The fore limbs were used to assess the reproducibility of the anatomical lymph drainage plus the tracer administration technique. Reproducibility was calculated as a percentage of the standard error divided by the mean value of percentage ID in the prescapular lymph nodes at the selected time after injection.

Pathologic Handling of Sentinel Nodes
The lymph nodes in one case were submitted for pathologic examination. Fresh lymph nodes were sent to the laboratory. These were examined grossly and then serially sliced into 1- to 2-mm slices along the longest axis of each node. By pressing the cut section onto a glass slide, an imprint was made from each node. This imprint was stained by using the DiG-Quick (Perth Scientific, Perth, Australia) rapid-staining technique. The nodes were then fixed in 10% buffered formalin and submitted for histological analysis. One hematoxylin and eosin section of each node was examined.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
99mTc-EB was prepared with radiochemical purity exceeding 95% in all 14 kits used. The radiotracer was allowed to stand at room temperature for approximately 20 minutes before use without loss of stability. After injection of the radioactive dye, the sheep’s limbs were scanned for radioactivity with the gamma probe at different intervals. In the hind limbs, counts had accumulated at the popliteal fossa within 5 minutes of 99mTc-EB injection. The sentinel lymph node was exposed and easily identified from its visible blue color (Fig. 2Go), with blue afferent and efferent channels, as well as from detection of a high concentration of counts with the gamma probe. Where the intensity of color was similar, EB was a darker blue than Patent Blue V, and it provided excellent contrast relative to the surrounding tissue.


Figure 2
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  		FIG. 2. Surgical exposure to display 99mTc-Evans Blue retained in superficial inguinal lymph node basins in two sheep.

 
All of the sequential lymph nodes were removed simultaneously. The gamma probe detected fewer counts in the remaining tissue (<10%), versus counts of the harvested sentinel lymph node from that area. Each node was assessed for color and radioactivity, where the counting apparatus (large-volume counter) was found to be highly reproducible for 20 to 2,000, 2,001 to 50,000, and 50,001 to 999,999 counts, yielding values of 3.9% ± .6% (n = 23), .3% ± .1% (n = 19), and .1% ± .1% (n = 28), respectively.

Lymphatic Mapping Patterns in Sheep and Sentinel Lymph Node Characteristics for 99mTc-EB
The characteristics of each lymph node sequence are listed in Table 1Go for 99mTc-EB. The distance migrated by the tracer from the injection site was 462 ± 11 mm to the popliteal lymph node, 629 ± 11 mm to the first superficial inguinal node, and 565 ± 85 mm to the prescapular node (Fig. 3Go). 99mTc-EB migrated from the injection site in sheep 1 and 2 to the superficial inguinal node basins, found at 1 to 3 mm below the skin surface. Of these small to medium-sized lymph nodes, the first one in each sequence received the highest percentage of the ID and resulted in a first:second node ratio of >21:1. The smaller sentinel nodes were visible in vivo and contrasted well against the extensive adipose tissue. The gamma probe assisted with their location. In sheep 2, two contiguous prescapular lymph nodes separated by a short distance were hot (.32% first node and .02% second node at 30 min) and blue. The second node, however, was barely stained at the cortex and was visible under normal magnification only after the tissue was bisected (node depth, 13–14 mm).


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  		TABLE 1. Lymph node sequences harvested after subdermal administration of 99mTc–Evans blue agent
 

Figure 3
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  		FIG. 3. A typical prescapular lymph node of dimension 40 x 20 mm excised at 22 minutes after 99mTc-Evans Blue injection. Note the appearance of blue color is not uniform, and maps functional segments of this large node.

 
The inguinal node was absent from the lymphatic sequence in 7 of 11 lower limbs of sheep, where the popliteal node drained lymph directly to the second-tier iliac lymph node (Fig. 4Go). The more common popliteal to iliac node drainage sequences in sheep 4 to 9 (seven of seven limbs) also resulted in higher uptake of the first draining node when nodes were separated by long distances (190–273 mm). The lower ratio of first- to second-tier nodes in sheep 5 was attributed to the presence of a denser-than-average popliteal node. In sheep 7, from the injection site two drainage paths were observed: one that migrated to the popliteal fossa and a second channel that led to a pair of unrelated superficial inguinal lymph nodes (Fig. 5aGo). Of the nodal pair, the sentinel lymph node was blue and contained 99mTc-EB (.08% ID), whereas in the other node, both radioactivity and color were absent at 29 minutes.


Figure 4
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  		FIG. 4. Surgical exposure of a sheep iliac lymph node stained by 99mTc-Evans Blue.

 

Figure 5
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  		FIG. 5. Complex lymphatic drainage fields in which lymph flows via two channels (a) from the hoof in a hind limb and (b) from an iliac node to another sequence in the lower abdominal area. The shaded areas represent blue coloration; open circles are nonblue lymph nodes.

 
Lymphatic Mapping Patterns in Sheep and Sentinel Lymph Node Characteristics for 99mTc-ATC/Patent Blue V
The characteristics of each lymph node sequence for 99mTc-ATC/Patent Blue V uptake are listed in Table 2Go. Similar to 99mTc-EB, 99mTc-ATC gave popliteal node (vs. second-tier inguinal or iliac nodes) uptake ratios of ≥2:1. There was also a noticeably higher uptake of 99mTc-ATC by lymph nodes than of 99mTc-EB for nodes harvested during 24 to 33 minutes. All lymph nodes excised were blue, except for one hot iliac node in sheep 6 which was located downfield by 10 mm from the previous node. In sheep 7, the first iliac node in the chain was also (visibly) linked to a short sequence of three iliac nodes (separated by 3, 1, and 1 mm); however, none of these lymph nodes was hot or blue (Fig. 5bGo).


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  		TABLE 2. Lymphatic chains or basins harvested after subdermal administration of 99mTc-ATC and then a subsequent dose of Patent Blue V
 
Drainage to the Prescapular Lymph Node
The 99mTc-EB dose was taken up to the extent of 1.00% ± .13% ID by the right prescapular lymph node at 22.0 ± .30 minutes (n = 6) and .56% ± .08% ID by the left prescapular node at 34.5 ± .80 minutes (n = 3). This indicates that the anatomical lymph drainage and tracer administration were reproducible to the extent of ±13% and ±14%, respectively. Most of these nodes contained one main afferent vessel, in conjunction with at least two minor vessels converging locally and up to two major efferent vessels emanating from another point. In sheep 2, the 99mTc-EB agent had reached the exposed prescapular node in <7 minutes, and the migration of colored tracer was infrequently visible through the skin of the extremity. Further exploration for lymphatic communication beyond the prescapular node into the thorax (in one sheep) failed to identify radioactivity or blue color at the internal mammary and intercostal regions. Also, the gamma probe did not detect any counts in the thyroid or salivary glands.

Pathologic Findings
Three lymph nodes were received. These were large nodes with a mean for the longest axis of 40 mm. Faint blue staining was noted on some exterior surfaces of the nodes. The cut section of each node revealed that the blue-staining regions corresponded to the cortical areas of the node and extended along the cords into the medullary regions (Fig. 6Go). The imprints from the nodes and the histological sections showed normal morphological characteristics. Areas of sinus histiocytosis were noted in the medullary sinuses, but because the sections were stained with histological dyes, the microanatomy of the patterns of localization of the EB dye in specific regions of the node could not be assessed.


Figure 6
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  		FIG. 6. Gross histological lymph node samples (after fixation).

 
Lymph nodes stored for at least 12 months in formalin at room temperature were persistently blue when the agent was EB, and no color had diffused into solution. This observation is consistent with a strong retention of this agent within the nodes.19 The converse was true for Patent Blue V, for which most color was leached into solution, thus making the native color of the lymph nodes apparent.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The amount of EB at 6 mg per dose yielded a sufficiently strong visual signal during the operation in vivo, substantially less than that of Patent Blue V (12.5 mg) to achieve the same intensity, and this was due to the high staining content of EB (80%). Histological assessment of the macroanatomy of lymph nodes indicated the presence of EB in the cortical and medullary areas. Retention of this dye by lymph nodes, in the medullary regions where leukocytes reside,22 suggests that strong binding is related to these cell types. In the hind limbs of sheep, 99mTc-EB uptake by lymph nodes did vary between animals with time, resulting in .2% to 3.1% ID at 28 to 29 minutes, .2% to 2.8% ID at 32 minutes, or .7% to 1.6% at 37 to 62 minutes for the popliteal nodes examined. The reasons can be attributed to the type and location of node sequences, the variable diameter of lymphatic vessels, and the posture of sheep limbs as secured to the operating table. From the results listed in Tables 1Go and 2Go, the data best highlight a relative comparison of lymph nodes linking a chain or basin within each sheep, rather than an absolute comparison between sheep. Generally for 99mTc-EB, the first- to second-tier node ratio was above unity for either the lymphatic chains or the short basins examined at dissection times up to 1 hour after injection. The inguinal node basins of sheep 1 and 2, for example, demonstrated that the 99mTc-EB agent could clearly discriminate the first-draining hot and blue nodes linked over very short distances (4–14 mm) at approximately 22 minutes (Fig. 7Go), irrespective of the size of the lymph node (3–22 mm).


Figure 7
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  		FIG. 7. Exposure of multiple inguinal nodes.

 
The prescapular lymph nodes were larger and thicker than the other node types, and on one occasion the denser tissue disguised color. This may be a problem during a search for lymph nodes of larger dimensions during the early stages of afferent lymph flow. Although the uptake ratios of the first- to second-tier prescapular nodes exceeded 17 in 2 instances (sheep 2 and 4), the lack of further radioactive or blue lymph nodes within the time frame indicated that this lymphatic drainage system was a less than adequate multitier model. The primary aim of the study regarding these nodes was to quantify the tracer administration technique and lymphatic drainage to them. Ultimately, the reproducibility on either the left or right side was good, considering the variable distance from the injection site and the node size. From a limited exploration in the thoracic region, no hot (99mTc-EB) and/or blue nodes were identified, either because the localization time was short or because the lymphatic drainage was directed via another undefined course, perhaps traversing directly to the thoracic duct or the right lymphatic duct. The lack of any radioactivity in the thyroid and salivary glands indicated that no 99mTc-pertechnetate was formed in vivo. There were low bladder counts detected by the gamma probe in sheep 9; however, the activity did not interfere with the identification of two linked iliac nodes by both color and radioactive signals at 36 minutes after injection. The counts detected from the bladder were likely to be due to 99mTc products produced from the hepatic metabolism of 99mTc-EB.19

Of the lymph node chains containing two or more consecutive nodes (sheep 3 to 9), the first-draining node did concentrate more 99mTc-EB counts than second-tier nodes, to the extent of 2:1 to 215:1. The size of the node or the longer internodal distances (4–273 mm) did not affect the discriminating ability of 99mTc-EB for the sentinel lymph node over nonsentinel lymph nodes. From the data listed in Table 2Go, there was higher uptake of 99mTc-ATC by lymph nodes than 99mTc-EB (Table 1Go) at the respective time points. However, where the first-draining node was hotter than the second-tier node for all three sheep, the node ratios (2–3:1) were consistently lower than those with 99mTc-EB at 24 to 33 minutes after injection. This suggests that the discriminating ability of 99mTc-EB for the sentinel node is greater than that of 99mTc-ATC in a multitiered nodal basin and that it may have positive clinical implications for the sentinel node biopsy in humans. The similarity in drainage patterns for 99mTc-ATC/Patent Blue V and 99mTc-EB in the hind limbs of sheep suggests that lymphatic mapping with 99mTc-EB may also be similar to that with 99mTc-ATC/Patent Blue V in clinical practice.

After intradermal injection of Patent Blue V and 99mTc-ATC into the hind limb of sheep 6, a third-tier radioactive iliac node was harvested that was clearly linked within the chain but absent of any blue color. It is not clearly understood, but the incongruent radioactive and color signals in this one of nine nodes could be attributed to migration of Patent Blue V dye via an alternative pathway. Radiocolloid particles are handled differently than soluble dye molecules in vivo because of their different physical and chemical properties. In cases of lymphatic mapping for breast cancer, congruence of blue and hot nodes after dual injections of isotope and vital dye can range from 30% to 70%. Other explanations include differences in the location and depth of these two injected lymphatic mapping agents, thus leading to different lymphatic drainage. For 99mTc-EB, despite the variable sheep lymphatic anatomy in short basins or long chains, the harvested lymph nodes (35 of 35) were all hot and blue. This observation is consistent with the radioactive molecules having the same properties as the colored molecules in the 99mTc-EB dose.

In terms of the clinical setting, the advantages of 99mTc-EB would be as follows: (1) a single dose is administered at one location, as opposed to radio-colloid and blue dye injections at two separate locations that might result in unrelated lymphatic drainage, and (2) its rapid migration and retention by the lymphatic system could allow for an early injection such that a same-day operation23 is easily achieved, even for operating room schedules for which surgery commences in the morning. This soluble radiotracer has the potential to discriminate the sentinel node and challenge existing methodology. The results of this work and the extensive use of EB dye for plasma volume studies in healthy subjects,24 early pregnancy,25–27 or pediatric patients28,29 support a validation study planned with 99mTc-EB for sentinel node biopsy in humans in the near future.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In this study, which used a sheep model of multitiered lymphatic anatomy, 99mTc-EB clearly identified lymph nodes in short basins or long lymphatic chains by concordant color and radioactive signals. This particular agent was retained strongly by lymph nodes and was superior to the dual-injection technique employing 99mTc-ATC and Patent Blue V dye for discriminating the sentinel node in sequentially draining lymph nodes. The 99mTc-EB agent is therefore likely to have clinical utility.


   ACKNOWLEDGMENTS
 
The authors thank Glenda Summersides, Tim Kuchel, BVSc (Hons), MVS, DVA, MRCVS, and the Animal Care Facility staff at the Institute of Medical and Veterinary Sciences for their assistance in preparing sheep before surgery.

Received for publication April 19, 2005. Accepted for publication October 28, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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