**2.2 Injection techniques**

Three main areas of injection on the uterus have been described: (1) corporal, (2) endometrial and (3) cervical. Each area has been assessed with various approaches: hysteroscopic, laparoscopic and ultrasound-guided transvaginal and even with combined techniques.


In 2013, the group from the Hospital Clinic of Barcelona published their results with the transvaginal ultrasound-guided myometrial injection of radiotracer (TUMIR) technique, injecting 148 MBq of 99mTc-albumin nanocolloid (in a volume of 8 mL) in the anterior and posterior peritumoural uterine wall, with migration of 90.5% and laparoscopic identification of 74.3%. The drainage was pelvic in 87.2%, pelvic and paraaortic in 45.4% and exclusively paraaortic in 12.8% of cases. Metastatic involvement of SLN in high-risk histology appeared in 24 patients, with paraaortic extension in 30% of cases. The authors concluded that the TUMIR technique is representative of tumour drainage and can be a promising detection method in high-risk histology, although further studies on the technique are needed [7]. The TUMIR technique has shown no differences compared with hysteroscopic injections (p = 0.2) [7].

3.**Cervical**: The cervical injection is the modality most often reported in studies [23]. The technique is reproducible, because the uterine cervix is accessible and rarely distorted in patients with EC [24]. The meta-analyses by Kang et al. [25] and Bodurtha-Smith et al. [26] showed that hysteroscopic or subserosal injections were associated with lower performance (p < 0.05), while cervical administration was correlated with higher detection rates (92%; range, 62–100% [27]; p = 0.031) [4] and bilaterality (56 vs. 33%; p = 0.003) (bilaterality is defined as the detection of sentinel node on both sides of the pelvis). The disadvantage was lower detection of paraaortic drainage (95% CI 3.4–10.1%) compared with other techniques (7% cervical vs. 27% corporal; p = 0.001) [28–31].

In terms of the number of injections, there have been no differences between the injection in two or four quadrants (with detection rates of 92 vs. 88%; p = 0.38), but there is less dye dissemination if the injection is limited to two quadrants [31]. Geppert et al. [11] performed pericervical injections at 2, 4, 8 and 10 o'clock thereby obtaining 5% paraaortic drainage. The MSKCC group recommended that cervical injections in EC be bilateral (at 3 and 9 o'clock), both superficial (in the submucosa) to 1–3 mm and deep (in the stroma) to 1–2 cm. In this way, the deep injection would therefore arrive at the isthmus, ensuring parametrial drainage. Applying this technique, a threefold greater detection of lymphatic metastases is achieved (p = 0.045), without assuming an increase in the overall detection rates (86%) [32]. The systematic review by Cormier et al. [27] determined that paraaortic detection has been significantly greater with deep cervical infiltration (17 vs. 2%).

Taking into account the critical review of Frumovitz and Levenback [33] and knowing the uterine drainage pathways, the location of the tumour will determine the dissemination of the disease, and the injection technique might reflect the pattern of lymphatic mapping. However, the study by Geppert et al. [11] concluded that the cervical pathway (with submucosal and stromal injection) should be considered the standard pathway, not only due to being a safer injection technique but also because the two main pathways of lymphatic drainage of the uterus were identified regardless of the injection technique employed.

**2.3 Sentinel node identification**

*Characteristics of injection techniques.*

Surgical approach for

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

Influence on anatomical distortion or tumour size

injection:

**Table 1.**

Various tracers have been shown to be useful. The most commonly used tracers

**Characteristic Corporal Endometrial Cervical**

Hysteroscopy Transvaginal Ultrasound-guided

++ +

Transvaginal

Subserosal or transmyometrial by laparoscopy or laparotomy

Multiple injections ++ +

Increase in economic cost +/ + Technical complexity +/ + Reproducibility + ++ Detection rate/bilaterality + ++ ++ Paraaortic drainage + ++ Comfort for the patient +/ +/ *The score was assigned according to the following gradation: () = absence of the characteristic or negative evaluation, (+) = meets the characteristic or positive evaluation, (++) = complies being better; (+++) = it fulfils being superior.*

One inherent and necessary advantage of tracers is their high capacity for penetration and fixation in lymphatic tissues [30]. The objective of lymphatic mapping and preoperative marking with tracer is the ability to analyse at least one lymph

SLNB was initially performed by the colorimetric method with methylene blue (MB) or synthetic variants of blue dye. SLNB was later combined with the isotopic detection of radiocolloids, thanks to the development of endoscopic gamma-ray detection probes, becoming the standard for use. In recent years, the application of lymphatic mapping by fluorescence in the near-infrared (NIR) electromagnetic spectrum with ICG obviated the need for a nuclear medicine unit and avoiding the

The approach to studying the lymphatic pathways using nuclear medicine techniques has gained considerable momentum in recent years with the development of

A radiopharmaceutical is a radioactive compound used for the diagnosis and treatment of diseases. Radiopharmaceuticals's property of transmitting radioiso-

Radiopharmaceuticals can be classified, according to their chemical structure, into primary radionuclides and labelled compounds, the latter of which are essentially a combination of two components: a radionuclide and a particle. The particles are small molecules, colloids, proteins and cells; the most widely used are colloidal sulphur (USA) and albumin (Europe). The particle serves as a vehicle that selectively directs the radiopharmaceutical to a specific tissue in response to physiological conditions or specific patterns of gene expression [38]. The size of the particles

topes for remote detection is employed to diagnostic purposes [38].

in the lymphatic mapping of EC include 99mTc-nanocolloid, blue dyes and

indocyanine green (ICG), alone or in combination [36].

adverse effects of blue dye, providing excellent results.

*2.3.1 Preoperative marking: radiocolloid*

SLNB.

**177**

node per lymph node chain, two in the case of bilaterality [37].

In terms of the lower cervical paraaortic drainage, numerous studies have verified that the incidence rate of paraaortic metastases isolated for EC in initial stages is 5%, with approximately 50% of metastatic pelvic and paraaortic lymph nodes [34]. Ninety percent of detected SLNs are located in the pelvic area; the rest are presacral or paraaortic. The other techniques likely obtain greater paraaortic drainage, at the expense of underestimating the main pathway of pelvic drainage [29, 30].

There is no known technique that detects with absolute bilaterality, and we still do not know why a central organ, such as the uterus, has cases in which bilateral drainage is not observed [20]. Ideally, the technique is considered valid if at least one SLN is identified in each hemipelvis [11].

There is no one ideal or superior technique; all have shown good results, with advantages and disadvantages [25]. Cervical injection is effective and reproduces pelvic drainage. Corporal injection better reproduces paraaortic drainage and the hysteroscopic pathway is representative of tumour drainage, although both are less reproducible than the cervical pathway [4]. A number of authors have advocated combined methods that provide better results. Studies such as the one by Holub et al. [35] have combined cervical and subserosal injections, achieving detection rates of 80%, without observing superiority over the two methods applied individually. In other studies with cervical and fundal injection, detection rates of 72.5% were achieved, comparable results without superior paraaortic drainage (4.9 vs. 9.8%; p = 0.18) [6].

**Table 1** shows a comparison of the main characteristics of the abovementioned injection techniques.


*The score was assigned according to the following gradation: () = absence of the characteristic or negative evaluation, (+) = meets the characteristic or positive evaluation, (++) = complies being better; (+++) = it fulfils being superior.*

#### **Table 1.**

*Characteristics of injection techniques.*

### **2.3 Sentinel node identification**

Various tracers have been shown to be useful. The most commonly used tracers in the lymphatic mapping of EC include 99mTc-nanocolloid, blue dyes and indocyanine green (ICG), alone or in combination [36].

One inherent and necessary advantage of tracers is their high capacity for penetration and fixation in lymphatic tissues [30]. The objective of lymphatic mapping and preoperative marking with tracer is the ability to analyse at least one lymph node per lymph node chain, two in the case of bilaterality [37].

SLNB was initially performed by the colorimetric method with methylene blue (MB) or synthetic variants of blue dye. SLNB was later combined with the isotopic detection of radiocolloids, thanks to the development of endoscopic gamma-ray detection probes, becoming the standard for use. In recent years, the application of lymphatic mapping by fluorescence in the near-infrared (NIR) electromagnetic spectrum with ICG obviated the need for a nuclear medicine unit and avoiding the adverse effects of blue dye, providing excellent results.

#### *2.3.1 Preoperative marking: radiocolloid*

The approach to studying the lymphatic pathways using nuclear medicine techniques has gained considerable momentum in recent years with the development of SLNB.

A radiopharmaceutical is a radioactive compound used for the diagnosis and treatment of diseases. Radiopharmaceuticals's property of transmitting radioisotopes for remote detection is employed to diagnostic purposes [38].

Radiopharmaceuticals can be classified, according to their chemical structure, into primary radionuclides and labelled compounds, the latter of which are essentially a combination of two components: a radionuclide and a particle. The particles are small molecules, colloids, proteins and cells; the most widely used are colloidal sulphur (USA) and albumin (Europe). The particle serves as a vehicle that selectively directs the radiopharmaceutical to a specific tissue in response to physiological conditions or specific patterns of gene expression [38]. The size of the particles

is important (range, 2–1500 nm) because it affects the drainage speed and retention of particles in the lymph node [34].

respectively); however, preoperative lymphoscintigraphy showed no benefit. An explanation for the low resolution and correlation could be the proximity of the injection site to the drainage, which can mask the SLN in flat images, and the anatomical complexity of the pelvic area, which can cause confusion in the correct

SPECT obtained a three-dimensional image with the fusion of the scintigraphic image with that of the traditional scanner. SPECT provides image information on the intensity of the scintigraphy and the precise anatomical location of the SLN(s) by scanner [44]. In the study by Naaman et al. [45], SPECT achieved a topographical accuracy of 91%, detecting a higher number of SLNs (1.4 vs. 2.13 SLNs/patient). Buda et al. achieved a detection rate of 50% and bilaterality of 39% for lymphoscintigraphy. For SPECT, the authors achieved a detection rate of 91%, bilaterality of 53% and sensitivity and a negative predictive value of 100% [46]. Compared with lymphoscintigraphy, the high sensitivity of SPECT seems to offer significant improvement in detecting SLNs and in the anatomical location [47, 48].

The surgical SLN detection techniques can be applied to open surgery, laparoscopy and robot-assisted surgery [30], with overall detection rates of 60–100% [27]. Regarding the possibility of laparotomic detection, Mais et al. [49] used methylene blue (MB) as tracer and observed a significant difference between the laparoscopic pathway and laparotomy, with detection rates of 82 and 41%, respectively, although

The mean number of SLNs detected per patient is 2.9 (95% CI 2.5 3.3; range 1– 8) [26]. In terms of location, Abu-Rustum et al. [50] reported 89% of SLNs in the territory between the external iliac, obturator and internal iliac areas, with 4% of

The gamma emissions of the radiocolloid can be tracked intraoperatively by a portable gamma probe adapted to open surgery or laparoscopy [42]. The use of laparoscopic gamma probe increases SLN identification guided anatomically by the previous images of SPECT and lymphoscintigraphy [51]. The gamma probe emits a signal proportional to the radioactivity uptake (analogue [counts per second] and auditory), enabling the accurate localisation of the radiocolloid in the lymph node station [38]. The activity will mark this lymph node regardless of whether is it

The radiocolloid is often used along with dye to optimise the detection rate and

visual assessment [30]. Studies with only radiotracer are therefore few, with a detection rate in the range of 70–96% [6]. In the Detection Rate and Diagnostic Accuracy of Sentinel-node Biopsy in Early Stage Endometrial Cancer (SENTI-ENDO) multicentre study, the long protocol with an injection of radiocolloid increased the detection rate of lymphoscintigraphy versus the short protocol (80.3 vs. 68.2%; p = 0.02). The paraaortic detection was more frequent in the long protocol, with no intraoperative differences [40]. There was a weak correlation between preoperative and intraoperative detection (k = 0.3) [40]. Other published data indicate good detection rates of 82% [6] and bilaterality with both protocols. While some authors use to perform another lymphoscintigraphy on the morning of the surgery [43, 52], with similarly poor improvement and weak correlation [22]. Other authors have proposed injecting the radiocolloid intraoperatively after the

anatomical description.

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

**2.4 Surgical detection**

*2.4.1 Radiocolloid*

**179**

normal or pathological [38].

with the same false negative rate (FNR).

SLNs in the paraaortic area and 6% in the common iliac areas.

The most widely used diagnostic radiopharmaceutical in nuclear medicine is isomerised metastable 99mTc [38], which is nontoxic and has almost ideal physical properties: a physical half-life to 99Tc (transition metal) of 6 h, a detectable emission and monochromatic gamma ray of 140 keV, complete disintegration within 24 h and low ionising radiation with negligible exposure [36, 39]. The greatest contribution of radiopharmaceuticals is the ability to use it as a marker of distinct pharmaceutical preparations, known as *cold kits*. The most used cold kit is the Nanocoll® 500 μg/vial (GE Healthcare Bio-Sciences, S.A.U., Madrid), a compound of colloidal particles of human serum albumin measuring ≤80 nm in diameter. The compound therefore has avidity for absorption through the lymphatic capillaries, and the size of its particles does not permit displacement of the lymph node.

To perform the SLNB, low doses of radioactivity are employed (40–185 MBq/ 0.2–4 mCi) because the distribution space is highly reduced. Only the drainage to the first or second lymph nodes needs to be assessed [38]. Furthermore, the injected dose of radiocolloid is calculated based on the estimated time to surgery; the longer the interval, the larger the dose to ensure an adequate signal. The 99mTc-albumin nanocolloid is injected the day of the surgery (the radiocolloid injection is performed 6 h before the operation with preoperative imaging 30 min after the injection; the dose should be 0.2–1.0 mCi), a method known as the short protocol. If the nanocolloid is injected the day before the surgery (at a dose of 2.0–4.0 mCi), the method is known as the long protocol [40]. Therefore, the radioisotope compound deposited in the interstitial space near the tumour will migrate to the lymphatic capillaries until it reaches the first lymph node encountered by the capillaries. Due to the size of the compound, the capillaries will be trapped, indicating the first sentinel node into which the administered site drains. Moreover, both the short half-life and the low injected dose leave relatively little space for flexibility in surgical scheduling, given that hours-long delays and postponing the operation to the following day due to logistics will compromise the efficacy of the lymphatic mapping [36].

The compound is contraindicated when faced with hypersensitivity to the active ingredient or any of the excipients and during pregnancy. As the radiocolloid is primarily cleared by the kidneys but also by the hepatobiliary system, dose adjustment is required for patients with renal and/or hepatic failure [39]. There have been no reported adverse effects related to the radiopharmaceutical 99mTc-nanocolloid, except the standard risks of the technical procedure: bleeding, vasovagal syncope and local infection [6].

#### *2.3.1.1 Lymphoscintigraphy and single-photon emission computed tomography*

The disadvantages of radiocolloid are the lack of visual assessment and the need for nuclear medicine procedures [30]. Lymphoscintigraphy and single-photon emission computed tomography (SPECT) can obtain preoperative images of the lymphatic drainage [38].

Lymphoscintigraphy obtains a flat static image with morphological information of the lymphatic drainage of the injected radiocolloid [8, 41]. Lymphoscintigraphy in EC offers varying results, depending on the injection method, with poorer detection rates than the overall rates and poor correlation between the preoperative findings and intraoperative detection [42, 43]. In the meta-analysis by Bodurtha-Smith and Tanner [26] of 55 articles and 4915 included patients, preoperative lymphoscintigraphy and the combined use of radiotracer with dye improved the overall SLN detection rates (86 vs. 76%; p = 0.016 and 87 vs. 78%; p = 0.008,

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

respectively); however, preoperative lymphoscintigraphy showed no benefit. An explanation for the low resolution and correlation could be the proximity of the injection site to the drainage, which can mask the SLN in flat images, and the anatomical complexity of the pelvic area, which can cause confusion in the correct anatomical description.

SPECT obtained a three-dimensional image with the fusion of the scintigraphic image with that of the traditional scanner. SPECT provides image information on the intensity of the scintigraphy and the precise anatomical location of the SLN(s) by scanner [44]. In the study by Naaman et al. [45], SPECT achieved a topographical accuracy of 91%, detecting a higher number of SLNs (1.4 vs. 2.13 SLNs/patient). Buda et al. achieved a detection rate of 50% and bilaterality of 39% for lymphoscintigraphy. For SPECT, the authors achieved a detection rate of 91%, bilaterality of 53% and sensitivity and a negative predictive value of 100% [46]. Compared with lymphoscintigraphy, the high sensitivity of SPECT seems to offer significant improvement in detecting SLNs and in the anatomical location [47, 48].

#### **2.4 Surgical detection**

The surgical SLN detection techniques can be applied to open surgery, laparoscopy and robot-assisted surgery [30], with overall detection rates of 60–100% [27]. Regarding the possibility of laparotomic detection, Mais et al. [49] used methylene blue (MB) as tracer and observed a significant difference between the laparoscopic pathway and laparotomy, with detection rates of 82 and 41%, respectively, although with the same false negative rate (FNR).

The mean number of SLNs detected per patient is 2.9 (95% CI 2.5 3.3; range 1– 8) [26]. In terms of location, Abu-Rustum et al. [50] reported 89% of SLNs in the territory between the external iliac, obturator and internal iliac areas, with 4% of SLNs in the paraaortic area and 6% in the common iliac areas.

#### *2.4.1 Radiocolloid*

The gamma emissions of the radiocolloid can be tracked intraoperatively by a portable gamma probe adapted to open surgery or laparoscopy [42]. The use of laparoscopic gamma probe increases SLN identification guided anatomically by the previous images of SPECT and lymphoscintigraphy [51]. The gamma probe emits a signal proportional to the radioactivity uptake (analogue [counts per second] and auditory), enabling the accurate localisation of the radiocolloid in the lymph node station [38]. The activity will mark this lymph node regardless of whether is it normal or pathological [38].

The radiocolloid is often used along with dye to optimise the detection rate and visual assessment [30]. Studies with only radiotracer are therefore few, with a detection rate in the range of 70–96% [6]. In the Detection Rate and Diagnostic Accuracy of Sentinel-node Biopsy in Early Stage Endometrial Cancer (SENTI-ENDO) multicentre study, the long protocol with an injection of radiocolloid increased the detection rate of lymphoscintigraphy versus the short protocol (80.3 vs. 68.2%; p = 0.02). The paraaortic detection was more frequent in the long protocol, with no intraoperative differences [40]. There was a weak correlation between preoperative and intraoperative detection (k = 0.3) [40]. Other published data indicate good detection rates of 82% [6] and bilaterality with both protocols. While some authors use to perform another lymphoscintigraphy on the morning of the surgery [43, 52], with similarly poor improvement and weak correlation [22]. Other authors have proposed injecting the radiocolloid intraoperatively after the

induction of anaesthesia, omitting the preoperative imaging and identifying the SLN directly with the gamma probe 30 min after the injection [36].

exposed to the chemical). The dye is not recommended for use during pregnancy or

Since the inclusion of fluorescence, the authors such as Holloway et al. [31] have replaced the radiocolloid and achieved equally good results. ICG is a tricarbocyanine dye with a short half-life of 3–4 min and hepatic excretion. ICG was developed by Kodak in the 1950s for use in photography and was approved by the US Food and Drug Administration in 1956 for IV administration [36]. The dye emits an intense blue colour detectable in real time when excited by laser in the near-infrared wavelength (range 750–900 nm, with an absorption peak at 800 nm) [6]. The high avidity by lymphatic tissue enables a high detection rate and accuracy compared

ICG has been used for years in laparoscopic and open surgery and in numerous other specialties (cardiology, general surgery, ophthalmology, vascular surgery, urology, etc.). The dye has recently been included in gynaecology. Lymph node mapping by fluorescence with ICG was first described by Furukawa et al. in 2010 in patients with cervical cancer. In 2012, Rossi et al. [58] applied ICG to 20 patients with cervical cancer or EC who underwent robot-assisted laparoscopic detection,

The supposed benefits of this technology include high tissue penetration with low autofluorescence [59]. ICG has been shown to be superior to blue dyes, particularly in patients with obesity [30]. Following an interstitial injection, ICG is absorbed to the lymphatic system and travels quickly to the lymph nodes. ICG binds to plasma proteins and is excreted by the liver [36]. The dye lacks significant adverse effects. A single case of a severe allergic reaction after IV injection has been reported (incidence rate of 0.05%) [60]. However, the dye is contraindicated for use in patients with allergies to iodine [23, 56]. Nevertheless, given that iodine is a chemical element and an essential component of the human body, a number of authors have suggested the safety of using ICG in patients with iodine allergies, given that a type I allergic reaction (antibody-mediated and responsible for anaphylactic shock) will not occur [36]. The use of ICG in pregnant patients has been

To date, there has been no standard in the concentration and volume to be injected. Rossi et al. [58], Jewell et al. [62] and Holloway et al. [63] established the optimal ICG dose for detecting SLN at 1, 1.25 or 2.5 mg/mL in 4 mL [23]. Unlike 99mTc, ICG does not remain confined to a lymph node but rather diffuses rapidly through the lymphatic tissue towards the second step lymph nodes without losing intensity. It is therefore crucial to quickly start the search for the SLNs after the administration of ICG, without delaying the start of detection by more than 10 min [23], thereby reducing the risk of sampling too many lymph nodes [36], which

Erikson et al. [65] compared the isolated use of ICG and MB in 472 patients, obtaining a higher detection rate (95 vs. 81%; p < 0.001) and bilaterality (85 vs. 54%; p < 0.001) with ICG. In 2015, How et al. [66] compared ICG, MB and 99mTc-

bilaterality (65 vs. 43%; p = 0.002) with ICG than with MB and a similar rate (87 vs. 88%; p = 0.83) and bilaterality (65 vs. 71%; p = 0.36) to 99mTc. In 2016, Papadia et al. [67] published the results of a retrospective, multicentre comparative study between detection with the combined technique using radiocolloid and blue dye versus radiocolloid and ICG. The overall detection rate was 97.3% for 99mTc-blue and 96.9% for ICG (p = 0.547). The bilaterality was 84.1% with ICG and 73.5% with

nanocolloid, obtaining a higher detection rate (86 vs. 71%; p = 0.005) and

breastfeeding because the dye's safety has not been established.

*Role of Sentinel Node Biopsy in Endometrial Cancer DOI: http://dx.doi.org/10.5772/intechopen.89949*

achieving a detection rate of 85% and bilaterality of 60%.

limits the technique beyond 25–30 min of the injection [64].

*2.5.2 Indocyanine green*

with common tracers [4, 56, 57].

reported as safe [61].

**181**

The disadvantages of this procedure include the high financial and staff costs, the risk of radioactive exposure and patient discomfort during the preoperative preparation. The advantages include the longer duration of the marking and fixation in the lymph node [15]. The technical limitations are related to the massive and diffuse uptake by the tissue, the interference caused in the gamma probe (which hinders the proper differentiation of the lymphatic tissue, especially in the parametrium and obturator area) and the uptake by the reticuloendothelial system (liver, spleen and bone marrow) [6, 27].

#### **2.5 Dyes**

#### *2.5.1 Blue dye*

The dye enables the visual identification of the SLN marked or not with the radioisotope. Several dyes have been used: 1% isosulfan blue, 1% MB and 2.5% patent blue. Isosulfan blue is a blue-green hygroscopic powder that is prepared by diluting it in sterile water. MB was created in 1876 and is the first fully synthetic drug used in medicine. Patent blue is a dark blue synthetic dye used as food colouring, and its intensity fades quickly when exposed to sunlight [36].

The detection rates with only blue dye are lower (57–62%), with a bilaterality of 56.1% (50–60%) [17, 53]. MB has lower diffusion but acceptable detection rates (57.5%, range 45–92%) and bilaterality (42.4%, range 50–60%) [54]. Nevertheless, when SLNB is applied to other cancers, such as breast cancer, the various dyes have shown accuracy and equivalence in SLN marking [36]. MB is preferred due to the extensive experience with this dye and its better toxicity profile. Due to the lower detection and given that the combination with 99mTc or ICG results in higher detection rates, MB is not recommended for use in isolation [21, 27].

After the blue dye is injected interstitially, it binds to the serum proteins and is filtered to the lymphatic vessels passing to the SLN, which will be stained in blue in approximately 5–10 min. This simple method requires no specific logistics, but the SLN must be identified at the start of the procedure before the dye vanishes or is displaced further along the lymphatic system and identifies a second nonsentinel step [36].

MB is more economical and safer than isosulfan blue. The half-life is 5–6 h, with mainly urinary and, to a lesser extent, biliary excretion. The recommendation is to use 2–4 ml of a 1% solution and to not take more than 10–20 min detecting the SLN due to the diffusion of the dye through the lymphatic tissue. A number of authors have estimated the mean procedure time, with an interval of 13.4 6.2 min between the injection and visualisation and a SLN excision time of 17.4 11.2 min [31].

Adverse effects related to the blue dye occur in 2% of cases [36]. The following effects have been reported: severe allergic reactions and anaphylactic crises (0.7– 1.9%), thrombophlebitis at the injection site, tissue necrosis, haemolysis or paradoxical methemoglobinemia and serotonin syndrome in patients undergoing treatment with serotonin reuptake inhibitors [30, 55]. Patients and health professionals should be advised of transient colouring of the urine and the possibility of interference with pulse oximetry readings [36]. The use of the dye is contraindicated in cases of hypersensitivity (MD induced allergic reaction and methemoglobinemia), severe renal failure (dosage adjustments may be necessary as serum concentrations are increased in patients with impaired renal function) and glucose-6-phosphatedehydrogenase deficiency (due to haemolytic anaemia when red blood cells are

exposed to the chemical). The dye is not recommended for use during pregnancy or breastfeeding because the dye's safety has not been established.
