Why Certain Dyes Are Useful for Localizing the Sentinel Lymph Node

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FIGURE 1.
Molecular structures of 10 dyes used. Methylene blue with ▴ nucleophile reactive sites; Phenol red, monosulfonic acid dye; disulfonic acid dyes with atom spacers: Patent blue; Acid yellow 42; Naphthol blue black; Chrysophenine; Reactive blue 4; tetrasulfonic acid dyes: Potassium indigotetrasulfonate; Evans blue and Chicago sky blue, which both contain a 1-amino-8-hydroxynaphthalene ring system.
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FIGURE 2.
After subdermal injection of soluble blue dye such as Evans blue, there is diffusion from interstitial cells to lymphatic vessels as well as blood capillaries. Dye binds to surface of endogenous proteins by sulfonation reaction, remaining trapped inside lymphatic lumen and transported along with lymphatic flow. Dyes such as Methylene blue, which do not have sulfonic acid groups in their structure, do not bind to endogenous lymph proteins and continue migration into blood capillaries.
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FIGURE 3.
Dye—protein complex formed by chemical reaction of amino group on protein surface with sulfonic acid group of dye (R).

TABLE 1

Binding Affinity of Sulfonic Acid Dyes with Plasma Proteins

Dye	No. of -SO₃H groups	No. of atoms between -SO₃H groups	% bound to plasma proteins*
Methylene blue	0	0	0.0 ± 0.0
Phenol red	1	0	10.0 ± 1.2
Patent blue VF	2	1	4.7 ± 1.2
Sulforhodamine 101	2	1	14.2 ± 1.0
Orange G	2	1	15.7 ± 2.1
Acid yellow 42	2	2	84.2 ± 3.1
Naphthol blue black	2	3	99.7 ± 1.9
Nitrazine yellow	2	3	100.6 ± 1.3
Chrysophenine	2	4	96.2 ± 2.4
Direct yellow 27	2	5	97.9 ± 5.8
Reactive blue 4	2	6	102.8 ± 7.9
Indigo carmine	2	8	28.0 ± 2.3
Potassium indigotetrasulfonate	4	1 and 6	12.7 ± 0.8
Evans blue	4	1 and 20	68.1 ± 3.5
^99mTc-Evans blue	4	1 and 20	69.6 ± 2.5
Chicago sky blue	4	1 and 20	69.3 ± 1.5
^99mTc-Chicago sky blue	4	1 and 20	70.8 ± 0.6
Trypan blue	4	3 and 14	62.2 ± 2.0
^99mTc-Trypan blue	4	3 and 14	60.5 ± 0.7
Direct yellow	4	4 and 19	58.6 ± 2.4

TABLE 2

Before and After SEC Radiochemical Analyses of Percentage of Components

↵* ITLC analysis gave 0.4% ± 0.0% ^99mTcO₄⁻, 3.9% ± 1.0% ^99mTc-Evans blue, and 95.7% ± 1.0% ^99mTc-Evans blue bound to protein; 0.3% ± 0.1% ^99mTcO₄⁻, 4.5% ± 0.4% ^99mTc-Chicago sky blue, and 95.2% ± 0.4% ^99mTc-Chicago sky blue bound to protein; and 0.7% ± 0.5% ^99mTcO₄⁻, 3.7% ± 0.8% ^99mTc-Trypan blue, and 95.6% ± 0.9% ^99mTc-Trypan blue bound to protein.
Data are expressed as mean ± SD.