The Warthin–Starry stain for detection of cutaneous melanin: more than a historical curiosity

Abstract

Cutaneous melanin plays an important role in human health and disease; therefore, accurate detection of melanin in skin is essential for reliable investigations on the mechanisms underlying physiological and aberrant pigmentation in skin. Histological visualisation of melanin has most commonly been performed using the Fontana-Masson procedure, originally developed over 100 years ago. By contrast, the Warthin-Starry stain, developed in 1920 for the identification of spirochaetes, was reported in 1980 to be more sensitive and specific for detecting cutaneous melanin than the Fontana-Masson proce- ). The role of melanin pigmentation in protection against ultraviolet radiation (UVR) through its photoabsorptive, antioxidant and free radical scavenger properties is obvious from varying skin cancer incidence across pigmentary phenotypes. Moreover, as most fair-skinned individuals will agree (possibly with envy), the role of melanin in protection against sunburn is valuable, especially in regions with high ambient UVR. Melanins are also present in non-cutaneous organs such as the eyes, heart and brain, and also play a role in the biology of many organisms through protection against thermal/chemical stresses and as part of innate immunity in insects, birds and mammals. 7 Melanins can also produce a wide spectrum of colours through their high refractive indices, which may be beneficial for camouflage and sexual selection. 8In humans, the protective role against UVR-induced carcinogenesis and the cosmetic effects of cutaneous pigmentation have become more relevant in modern times. As skin cancer is the most common type of neoplasia worldwide, understanding the role of melanin and other mechanisms of cutaneous photoprotection is highly relevant. Skin pigmentary phenotypes as well as polymorphisms relating topigmentary genes such as MC1R are known to influence skin cancer susceptibility.

Visible light and human skin pigmentation: The importance of skin phototype

“…Moreover, the ratio of these two pigment types correlates with high‐performance liquid chromatography (HPLC) quantification performed by others 28,29 as discussed by Ito and Wakamatsu 30 . These histochemical data strengthen our preliminary observations and showcases the WTS stain's potential to study melanin subtypes in human epidermis, offering advantages versus other perhaps less‐specific melanin staining techniques (eg Fontana‐Masson, Haematoxylin‐eosin, Von Kossa) 21,22 …”

“…To complement the above macroscopic observations, we analysed alteration in melanin levels in both skin phototypes at a histological level by WTS 12,21,22 . While a histologically detectable increase in melanin was present in the tanned epidermis of UVR‐irradiated SPT‐II and SPT‐III histocultures as predicted, SPT‐I epidermis responded unexpectedly to blue and green light irradiation, with an increase in WTS‐detectable melanin not observed post‐UVR (Figure 1C, D, G, H, K and L).…”

“…30 These histochemical data strengthen our preliminary observations and showcases the WTS stain's potential to study melanin subtypes in human epidermis, offering advantages versus other perhaps less-specific melanin staining techniques (eg Fontana-Masson, Haematoxylin-eosin, Von Kossa). 21,22 Our results show for the first time in fair SPT-I skin that visible light can induce melanin production, which may have a beneficial effect in protecting these individuals from the harmful effects of UVR. This finding may concur with our previously reported observation that cultured melanocytes from red-haired individuals can indeed produce significant melanin if stimulated in the absence of their keratinocyte partner.…”

“…This allowed us to distinguish two differentially stained pigment types, which included an intensely black pigment product (termed WTS-black) and a lighter reddish-brown pigment product (termed WTS-reddish/ brown). 12,21,22 To distinguish these variable WTS-staining pigment characteristics optimally for image capture, we optimised the saturation and gamma levels on microscope acquisition software to more faithfully represent what was being observed via the microscope eyepiece (Leica D2500 microscope with a DFC7000 T camera). Using ImageJ software, we applied colour thresholds to disambiguate the WTS-black or WTS-reddish/brown pigment products on the skin sections.…”

Plant poisoning leads to alpha-synucleinopathy and neuromelanopathy in kangaroos

“…WS stain relies on the ability of certain pigments, including melanin to bind silver ions from solution. Of importance, we elected to use WS to aid in the identification of the granules revealed by H&E stain due to its superior binding to melanin and neuromelanin compared to other suboptimal reactions, such as Fontana-Masson and Schmorl’s stains 33,34 . WS stain displayed intense dark granular staining in Phalaris-affected EGK sometimes leading to complete obliteration of the cell cytoplasm while retaining a clear interface with the neuropil (Fig.…”