Consecutive Immunohistochemical Staining on a Single Slide is Effective in Confirming Melanocytic Derivation.

BACKGROUND

Metastatic melanoma in sentinel lymph nodes is often elusive to detect with morphology alone. Per American Joint Committee on Cancer staging guidelines, a single atypical melanocyte in lymph node qualifies as metastasis, whether identified by morphology or immunohistochemistry, but single cell staining must be convincing. We propose that the use of a second immunohistochemical run performed on a single slide will allow for more confident diagnosis of micrometastases.

MATERIALS AND

METHODS

We designed a technical study to determine whether a second antibody application on previously stained slides can successfully detect the same population of cells. Melanocytic neoplasms were stained with SOX-10 using Ventana Benchmark Ultra stainers, coverslipped, and examined, followed by coverslip removal and application of MART-1 (Ventana A103). The order of antibody application and chromagen detection kit (AP-RED vs. DAB) was reversed to establish reliability and robustness of the protocol.

RESULTS

All melanocytes marked with SOX-10 and MART-1, and produced a range of staining quality that varied based on order of stain application and chromagen kit were used. The optimal combination was red MART-1 applied first followed by brown SOX-10 applied second.

CONCLUSIONS

Consecutive staining of melanocytes with SOX-10 and MART-1 may improve diagnostic confidence of melanocyte identification, particularly in detection of single cell, micrometastases in sentinel lymph nodes or in situations where dual immunohistochemical stains may be unavailable.