A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue

Abstract

Removal of excessive melanin from heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues is essential for histomorphological and molecular diagnostic assessments. Although there have been efforts to address this issue, current methodologies remain complex and time-consuming, and are not suitable for multiple molecular applications. Herein, we have developed a robust and rapid melanin-bleaching methodology for FFPE tissue specimens. Our approach is based on quick bleaching (15 min) at high temperature (80°C) with 0.5% diluted hydrogen peroxide (H 2 O 2 ) in Tris-HCl, PBS, or Tris/Tricine/SDS buffer. Immunostaining for Ki-67 and HMB45 was enhanced by bleaching with 0.5% H 2 O 2 in Tris/Tricine/SDS and Tris-HCl, respectively. In addition to histopathological applications, our approach also facilitates recovery of protein and nucleic acid from archival melanin-rich FFPE tissue sections. Protein extracted from bleached FFPE tissues was compatible with western blotting using anti-human GAPDH and AKT antibodies. Our bleaching condition significantly improved RNA quality compared with unbleached tissues without compromising the yield. Notably, the RNA/DNA obtained from bleached tissues was suitable for end point PCR and real-time quantitative RT-PCR. In conclusion, this improved melanin-bleaching method enhances and simplifies immunostaining procedures, and facilitates the use of melanin-rich FFPE tissues for histomorphological and PCR amplification-based molecular assays.

Melanin bleaching with potassium permanganate for melanocytic lesions diagnosis in veterinary medicine

“…The high occurrence of pigmented lesions found in this study shows the importance of adopting routine MB techniques in veterinary histopathology, like in human medicine (Momose et al, 2011;Liu et al, 2013;Chung et al, 2016). Furthermore, the disagreement on the diagnoses after re-evaluation using MB confirms the importance of using this method.…”

“…However, melanotic pigments may obscure the cellular morphological characteristics; therefore, histological depigmentation techniques have been widely used in human medicine to evaluate the systematic criteria for diagnosis of these lesions (Veronese et al, 2007;Liu et al, 2013;Chung et al, 2016). On the other hand, in veterinary medicine, melanin bleaching methods have rarely been employed.…”

Suitability of melanoma FFPE samples for NGS libraries: time and quality thresholds for downstream molecular tests

“…On the other hand, differences were found in QC between tumor samples and wide local excision samples, although they were not relevant for functionality in old samples. In recent samples, wide local excision samples worked better than tumors in terms of functionality, which might be explained by the role of melanin in tumor samples, which can lead to inhibition of PCR due to the association of remnants of pigment melanin with genomic DNA [14,15]. …”

Histomorphological and Molecular Assessments of the Fixation Times Comparing Formalin and Ethanol-Based Fixatives

“…We also measured RNA integrity with the paraffin-embedded RNA metric (PERM) number, which is a metric for FFPE (formalin-fixed paraffinembedded)-derived RNA quality. 32,33 The extracted RNA was treated with 2-μl TURBO DNase buffer, 4 units of TURBO DNase (Invitrogen), and 40 units of RNase inhibitor (Promega, Madison, WI) in a 100-μl reaction volume to remove possible contaminating genomic DNA. The mixture was incubated at 37C for 30 min and was purified by phenol/ chloroform extraction.…”

“…DNA extraction yield was assessed using the NanoDrop ND-1000 UV spectrophotometer (NanoDrop Technologies), and DNA quality was evaluated by amplified DNA amounts using a Bioscore Screening and Amplification kit (Enzo Life Sciences, Farmingdale, NY), as previously reported. 33,34…”