Related Experiment Video
Updated: Jan 12, 2026
Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples
Published on: July 11, 2025
Optimisation of a Standardised Automated Bleaching and Staining Protocol for Melanin-Rich Cytology Specimens
Chia-Hsing Liu1, Shu-Jyuan Chang1,2, Shu-Han Chang1
1Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Background:
Melanin-rich cytologic specimens, particularly those from melanocytic lesions, present diagnostic challenges due to pigment-induced obscuration of cellular details and interference with immunocytochemistry (ICC) interpretation. These limitations are especially pronounced in cell transfer preparations, which differ significantly from tissue sections in cellular distribution and density. Existing bleaching protocols are inconsistent, often incomplete in pigment removal and can compromise cellular morphology. This study aimed to develop and evaluate an automated platform incorporating optimised melanin bleaching, ICC and cytomorphologic staining to enhance diagnostic accuracy in heavily pigmented cytologic samples.
Methods:
Ten melanoma cell transfer smears were processed using an optimised protocol. Slides underwent melanin bleaching with 10% hydrogen peroxide at 60°C for 25 min, followed by automated ICC for Melan-A and SOX-10. Chromogenic detection was performed using either 3,3'-diaminobenzidine (DAB) or alkaline phosphatase (AP). In a parallel workflow, Papanicolaou (Pap) staining was performed after bleaching to assess cytomorphologic preservation.
Results:
The integrated protocol completed bleaching and staining within 2 h, with bleaching effectively removing melanin pigment while enhancing nuclear and cytoplasmic visibility without compromising morphological detail. Post-bleaching Pap staining preserved cytologic features, enabling accurate morphological interpretation. Both markers exhibited strong, specific immunoreactivity with either chromogen; however, AP yielded superior contrast and clearer antigen localisation. In contrast, residual melanin occasionally masked DAB signals, limiting interpretability.
Conclusion:
This automated protocol, combining melanin bleaching, Pap staining and ICC, improves visualisation and diagnostic interpretation of melanin-rich cytologic specimens. The bleaching step preserves cellular and antigenic integrity, while AP chromogen provides enhanced clarity in the presence of residual pigment. This reproducible and practical workflow facilitates more accurate cytopathologic evaluation of melanocytic lesions.
