Why certain dyes are useful for localizing the sentinel lymph node

Abstract

Location of the sentinel lymph node in malignant melanoma and early breast cancer patients requires separate injections of radiocolloid and blue dye. These agents are administered at alternative times because of their different rates of transit. This study has elucidated why particular dyes are absorbed by the lymphatic system from an investigation of dye molecular structure as a function of protein binding ability.

Methods: A dye-protein binding assay was developed using size-exclusion chromatography and ultraviolet spectrophotometry and applied to a series of 20 sulfonic acid group-containing dyes. Radiochemical analyses were also used with 3 99mTc-labeled dyes to rationalize which functional groups are involved in the protein binding reaction.

Results: Methylene blue resulted in no protein affinity at 37 degrees C, whereas disulfonate dyes separated by 1 atom such as Patent blue or Indigo carmine gave <30% binding. Optimum protein binding (84%-100%) was achieved with those dyes containing at least 2 sulfonic acid groups separated by 2-6 atoms in their chemical structure. Seven symmetric tetrasulfonic acid azo dyes were examined, including Evans blue, to result in 59%-71% binding.

Conclusion: Ionizable groups (sulfonic acids) that are present in the structure of dyes are directly involved in dye-protein binding. At the molecular level, there is a sulfonation reaction between sulfonic acid dyes and amino groups on the protein surface to form sulfonamide complexes. This reaction shows how the soluble dyes Evans blue and Patent blue are trapped in lymph after subdermal injection during the sentinel node biopsy procedure.