2-PRELIMINARY NOTE Models , Biological Hemosiderin . A new marker for sentinel lymph node identification

Purpose: To evaluate and present our initial results of a new marker (hemosiderin) for mammary sentinel lymph node identification in an experimental model. Methods: Skins mapped like a lymphatic duct draining to the axilla in patients submitted to breast biopsy, in our mastology service, stimulated us to try it in an animal model (female dogs). Our theory was that some blood derivate (hemosiderin) was captured by macrophages and accessed the lymphatic ducts in direction to the axilla. Six female dogs of no defined race were studied. We injected 0,2 ml of technetium on both superior mammary glands. After ten minutes, a 2,5 ml solution of hemolized blood (hemosiderin) from the own animal was injected in the subareolar lymphatic plexus on the left superior mammary gland and 2,5 ml of patent blue concomitantly and equally on the contralateral gland. Ten minutes after, incisions on both axilas were made to search, through the lymphatic mapping and a gamma probe, the sentinel lymph nodes. Results: Seven brown sentinel lymph nodes were indentified and also radiomarked on the left axilla. Six blue sentinel lymph nodes were identified and also radiomarked on the right axilla. Conclusion: Preliminary studies of a potential new dye for sentinel lymph node identification are presented. It may be the change of the current use of the blue dyes and their severe side-effects on patients submitted to sentinel lymph node biopsies.


Introduction
The incidence of female breast cancer has increased 0.5% since the year 2000, as reported by the International Agency Research on Cancer.This number is greater in developing countries due to the increase in life expectancy and change in behavior resulting in increased exposure to risk factors.
Lymph node staging is an early event carried out during initial patient evaluation in developed countries.Some 20% to 30% of all cases are diagnosed quite early (in situ lesions) with negative axillary nodes in 79% of the patients at this stage 1 .These values are somewhat different in developing countries where more than 50% of the cases are diagnosed at advanced stages of the disease.Late stages (III and IV) of breast cancer are seen in 60% of women who seek medical attention at the Maternidade-Escola (Federal University of Ceara) Breast Unit.Lack of adequate surveillance programs, the reduced number of specialists and the small number of educational programs may account for these numbers.
Increased survival rates have been reported lately.According to Parkin 2 , the overall survival rate is 91% after the first year and 65% after five years in Europe.These values increase to 96.8% after the first year in the United States.As primary prevention of breast cancer is not available as yet, early detection and treatment in the initial phase of the disease are, therefore, the most important measures for its control 3 .
Available diagnostic methods include mammography, breast ultrassonography, sentinel lymph node biopsy, and fine-needle aspiration biopsy among others 4,5 .With the definition of the sentinel lymph node as the first one which receives the drainage of the tumoral area 6 , it was possible to ensure means for the proper staging of the illness and the therapeutic approach establishing less invasive surgery techniques.
Currently, it is admitted that the presence of metastatic lymph nodes is the main predictor factor for prognosis of the breast malignant neoplasia course and subsequent therapeutic program.Lymph nodes are also valuable for staging breast cancer.In the recent past dissection of the axillary lymph nodes was required for this matter, resulting in a series of additional complications 7 .
The principles of the sentinel lymph node were first used by Cabanas (1973) in penis cancer.Morton et al. 8 used this principles on the identification of melanoma metastasis.He used Patent blue dye.Giuliano et al. 9 described its use for the breast cancer, also using the vital blue dye for lymphatic mapping and then, Krag et al. 10 additionally used a radioguided model with a gamma probe increasing the accuracy on the identification of the sentinel lymph node.
The results of the researches on breast sentinel lymph node are encouraging, although there are some methods which are controversial 11 .One of these is the vital blue dye use for the lymphatic mapping which may cause severe hypersensitivity reactions on patients 12 .Other dyes are used, as methylene blue which may also cause serious reactions, including skin and fat necrosis in the site of injection 13 .
During the management of two patients submitted to breast biopsy in our service, we observed their skin mapped as the lymphatic ducts draining to axillary lymph nodes.This observation stimulated us to try an animal model which was described for our group.Our theory was that some blood derivate (hemosiderin) was captured by macrophages and accessed the lymphatic duct in direction to the axilla (Figure 1).For these reasons, the purpose of this paper is to present our initial results of a new marker for sentinel lymph node identification.

Methods
Prospective experimental surgical study approved by the Ethic Commission in Animal Research of our university (UFC).It took place in the experimental surgery laboratory Prof. Saul Goldenberg in order to evaluate the use of a blood derivate (hemosiderin) on mammary sentinel lymph node mapping in female dogs.
Six female dogs of no defined race, with 10kg as mean weight, were given by the zoonoses control center of Fortaleza to be studied.These animals were part of a group programmed for sacrifice as they may be infected by diseases which affect men.The animals, after proper immobilization, were anesthetized with an intramuscular application of ketamine (05-10 mg/kg) immediately before the experiment.
For the hemoglobin use (hemosiderin precursor), 10 ml of blood were obtained from the left internal jugular vein of the animal to be studied.From that blood, 8 ml were stored in 2 (two) tubes with an anticoagulant solution (EDTA), 4 ml in each tube.
These tubes were placed symmetrically in a centrifuge for 5 (five) minutes at 3800 rpm.Then, in each tube, an interface was observed; the upper phase is the plasma which was extracted with a pipette.To the rest of the volume of each tube, equal volume in mL of NaCl 0,9% was added, in order to double the final volume.After manual homogenization, the tubes were replaced in the centrifuge for more 5 (five) minutes at 3800 RPM.A new interface was noted and the upper phase (a serum solution) was once again extracted.With these steps erythrocyte concentrates were obtained in each tube.
Once again, to the rest of the volume of each tube (erythrocyte concentrates), equal volume of bidistilled water was added, in order to double the final volume.Vigorous homogenization was performed manually, causing hemolysis by the osmolarity difference.The tubes were replaced in the centrifuge for more 3 (three) minutes to confirm the hemolysis.If there was no interface after this last centrifuging, the hemolysis was confirmed.The volume of 2,5 ml of one tube (hemosiderin) was taken to be used on the lymph node research.
The radioactive tracer (technetium 99m), the processed blood and the patent blue (Guerbert V 2,5%) were injected in the mammary subareolar lymphatic plexus.Following with the surgical procedure, the two first mammary pairs (superior) of the female dog were defined where 0,2 ml of the radioactive tracer were injected in each mammary gland.After 10 (ten) minutes, 2,5 ml of patent blue were injected in the right mammary gland and 2,5 ml of the processed blood were injected in the left mammary gland, at the same time.After more 10 (ten) minutes, 5 (five) cm incisions were made in both axillas for the sentinel lymph node identification by its color and with the guide of a gamma-detecting probe.

Preliminary results
Six experiments were performed according to the methodology described and showed the following results.In all animals, at least one mammary sentinel lymph node, each one colored and radiomarked, was identified from each axilla.In the total, 13 (thirteen) mammary sentinel lymph nodes colored and radiomarked were identified from all axillas (Table 1).All the sentinel lymph nodes found in the right axillas were radiomarked and had a blue color.All the sentinel lymph nodes found in the left axillas were radiomarked and had a brown color (Figure 2).We observed that both colors were different from those of control lymph nodes (Figure 3).There were no problems or difficulties on finding mammary sentinel lymph nodes in the axillas of the female dogs with these techniques.

Discussion
Sleth 14 , while discussing, in his work, the annual rise on the incidence of adverse reactions to blue dyes during sentinel lymph node biopsies, the clearly quantifiable allergic risks and the continuous evolution of the sentinel lymph node study in other tumors, asked if it was not the time to change the dye.
Nowadays, reactions to vital dyes are being more frequently reported due to its increased use in lymphatic mappings and in sentinel lymph node biopsies in patients with breast cancer and other malignancies 12,15 .Cases and meta-analysis of anaphylactic reactions are described due to the use of blue dyes in sentinel lymph node identification.The incidence of anaphylactic reactions to isosulfan blue or patent blue vary from 0,6% to 2,7% in patients submitted to sentinel lymph node biopsy and must be known by the surgeon and the anesthesiologist 16 .
Mertes et al. 15 reported 14 clinical cases of anaphylaxis induced by dyes during the years 2004 and 2006 in four different allergoanesthesia french centers.All patients suffered hypersensitive reactions to patent blue V during sentinel lymph node biopsy due to breast cancer.In this study, 43% of patients suffered life-threatening reactions (cardiovascular collapse, tachycardia or bradycardia, arrhythmias and severe bronchospasm) and 64% of these patients required prolonged continuous epinephrine infusion and transference to an intensive care unit.The authors recommended the use of dermatologic tests for these markers before surgery.
Wohrl et al. 17 described a near-fatal case of anaphylaxis due to the patent blue V dye in sentinel lymph node identification.A patient with melanoma suffered cardiac arrest for 10 minutes and required mechanic cardio-pulmonary reanimation measures after the intradermal injection of this dye.
The preliminary results of our experimental work showed that the blood derivate (hemosiderin) was capable of being used along with the radiomarker ( t e c h n e t i u m 9 9 m ) a n d c o l o r i n g wonderfully the sentinel lymph node of female dogs.The use of the blood derivate (hemosiderin) with the radiomarker also showed equal results comparing the use of the patent blue with the radiomarker in identifying the mammary sentinel lymph nodes of the female dog because they were always found in both axillas.
The hemosiderin, a blood-derived marker, theoretically does not cause side effects as allergic reactions.Besides, it may decrease personal and healthcare spendings, reduce the morbidity of the procedure and the emotional suffering, along with other indirect vantages.

Conclusion
Preliminary studies of a potential new dye for sentinel lymph node identification are presented.It may be the change of the current use of the blue dyes and their severe side-effects on patients submitted to sentinel lymph node biopsies.

FIGURE 1 -
FIGURE 1 -Skin mapped as a lymphatic duct in direction to the axilla

FIGURE 3 -
FIGURE 3 -Sentinel lymph nodes marked with hemosiderin at left (bold arrows) and with patent blue at right (thin arrows).NSL = Non-sentinel lymph node.

FIGURE 2 -
FIGURE 2 -Sentinel lymph node marked with hemosiderin in vivo

TABLE 1 -
Number of mammary sentinel lymph nodes colored and radiomarked found in each axilla by animal