The role of reflectance confocal microscopy in the diagnosis and management of pigmentary disorders: A review

Reflectance confocal microscopy (RCM) has quickly transitioned from a research tool to an adjunct diagnostic bedside tool, providing the opportunity for noninvasive evaluation of skin lesions with histologic resolution. RCM is an optical imaging technique that uses near‐infrared excitation wavelengths and safe low‐power lasers. En‐face images of different skin layers (up to the superficial dermis) are acquired in grayscale based on the reflective indices of tissue components. Melanin has the highest reflective index (contrast) and appears bright on RCM.


| INTRODUC TI ON
Reflectance confocal microscopy (RCM) is a non-invasive imaging technique that allows for the evaluation of skin lesions at the cellular level.Highly reflective tissue structures such as melanin, collagen, hemoglobin, and keratin appear bright in confocal images.Due to its ability to detect melanin, RCM has become a valuable adjunct bedside tool for the diagnosis and monitoring of pigmentary disorders.

| MATERIAL S AND ME THODS
We reviewed the medical literature (PubMed and Ovid Medline databases) from January 2000 to June 2021, using MeSH key terms: "reflectance confocal microscopy, confocal laser scanning microscopy, pigmentary disorders, treatment, melasma, vitiligo, ephelides, solar lentigo, lentigo, tattoo, nevus, nevus of ota, nevus spilus, nevus depigmentosus, halo nevus, melanoma, skin cancers, pigmented lesions, post inflammatory, melanin, photoaging" to identify studies and review articles discussing RCM and its use in the diagnosis and management of pigmentary disorders.Further papers were identified from the reference lists of the above-retrieved papers.Our search included English-language articles published between 2000 and 2021.The selection consisted of an initial screening of titles and abstracts, followed by the evaluation of full-text articles.(DEJ), and in some cases, the presence of round and polygonal refractile structures within dermal collagen bundles were also noted.The highly refractile dermal papillary rings were found in the lesional and perilesional forehead and upper lip skin, where an abrupt transition from stratum spinosum to papillary dermis was seen that corresponded to flattened rete ridges.H&E sections showed a significant increase in the density of melanin in all epidermal layers that corresponded to the cobblestone pattern on RCM.Polygonal refractile cells in the papillary dermis corresponded to dermal melanophages on H&E.Melasma was classified into "epidermal" type where increased melanin was seen only in the epidermis, observed in 71.5% of the patients and "mixed" type where increased melanin was seen in both epidermis and the dermis, in 28.5%.Figure 1 shows the RCM features of melasma.al to distinguish labial mucosal melanosis from mucosal melanoma, proving 100% sensitive and 88% specific for diagnosing mucosal melanoma. 13Dermoscopic and RCM images of the labial melanotic macule are given in Figure S1.

| Post-inflammatory hypopigmentation
Xiang et.al note that in post-inflammatory hypopigmentation, the content of melanin and dermal papillary rings "depends on the depth and site of the inflammation". 26Melanophages were occasionally observed in post-inflammatory hypopigmentation, while not in other disorders of decreased pigmentation such as vitiligo and nevus depigmentosus. 26Melanophages may or may not be visualized. 26We report a case of post-inflammatory hypopigmentation which shows a loss of melanin reflectance signal at the DEJ and the presence of a few melanophages in the superficial dermis (Figure S2).

| Vitiligo
Vitiligo is the most common pigmentary disorder, affecting 0.5%-2% of the population worldwide, and presents as multiple achromic patches due to progressive loss of melanocytes at the DEJ. 14RCM has been utilized to diagnose, monitor disease progression, and monitor treatment outcomes in vitiligo patients in the last decade.
Vitiligo is characterized by the total or partial disappearance of dermal bright papillary rings on RCM. 27digo et al described vitiligo on RCM as a complete loss of dermal papillae rings at the DEJ, whereas perilesional skin showed 'halfring' structures corresponding to a partial loss of papillary rings. 27nversely, Kang et al. observed the 'half ring' structures in normal skin, which suggests that these structures may be a physiological variation rather than a distinguishing trait of vitiligo on RCM. 2 Additional studies have visualized melanocytes within the hair follicles, which have been described as the 'follicular reservoir' in vitiliginous skin. 28,29RCM was used to monitor treatment response in vitiliginous skin after UVB treatment, with re-pigmented areas showing large, bright dendritic melanocytes at the DEJ. 30 grafting. 15Figure S3.shows RCM images of vitiliginous skin before and 3 weeks after microneedling.

| Nevus depigmentosus
Both authors concluded that HN may show features consistent with melanoma on RCM, thus additional clinical findings should be considered while managing these lesions.
An example of a halo nevus from a 16-year-old patient is given in

| CON CLUS ION
Pigmentary disorders exhibit characteristic patterns on RCM.
Without the need for a biopsy, RCM presents as a promising adjunct tool in the diagnosis and treatment monitoring of pigmentary disorders.

CO N S E NT
Patient gave consent for skin imaging and medical information to be published in print and online with the understanding that this information may be publicly available.

AUTH O R CO NTR I B UTI O N S
Banu Farabi, Marielle Jamgochian, Babar K.

4 | DISCUSS ION 4 . 1 |
Disorders of increased pigmentation4.1.1 | Postinflammatory hyperpigmentationHistologically, hyperpigmentation presents with patchy epidermal or dermal pigmentation and the presence of melanophages in the superficial dermis.RCM shows small bright and large bright cells in the epidermis and superficial dermis.

4. 1
.2 | Melasma Melasma is characterized by light brown patches on the facial prominences and forehead.Histologically, there is increased melanin deposition in the epidermis, especially the basal layer.Melanocytes are slightly enlarged and increased in number with prominent dendritic processes.An increased number of melanophages can be visualized in the papillary dermis.Kang et al. describe RCM features of melasma including hyperrefractile cobblestone pattern at the suprabasal layer (identified in all patients) that corresponded to basal keratinocyte hyperpigmentation in histopathologic sections. 2Dendritic cells that corresponded to activated melanocytes in the epidermis (identified in 6/26 patients) were identified as well as plump bright and large cells (identified in 9/26 patients) that corresponded to melanophages in the superficial dermis.Significant solar elastosis and increased blood vessels in the dermis were noted compared to the adjacent normal skin.Liu et al. evaluated a cohort of 210 Asian melasma patients with RCM. 3 In 10 patients, they defined melasma features on RCM compared with the corresponding histopathology.Then, they used these RCM features to classify melasma in the remaining 200 patients.Reported RCM features of melasma included a cobblestone pattern in the suprapapillary layer.A flattened basal cell layer was also noted.Increased epidermal pigmentation in the spinous layer, strongly visible papillary rings at the dermo-epidermal junction

1 - 6 - 12 --
Figure 2B.Corresponding dermoscopy and histopathology are also included (Figure 2A,C,).RCM can distinguish solar lentigines from other pigmented lesions arising on sun-damaged skin, such as pigmented actinic keratosis, lichen-planus-like-keratosis, and lentigo maligna.Richtig et al. treated 12 patients with solar lentigines with a Q-switched ruby laser (QSRL) and imaged the treated areas with RCM 30 minutes and 10 days after the treatment. 25Thirty minutes after treatment, RCM revealed disruption of the stratum corneum and the presence of multiple highly reflective round-to-polygonal areas, which corresponded clinically to white scaling on the lesions.The epidermal honeycomb pattern showed indistinct borders with weak intercellular connections.Dark structureless areas varying in size and shape were observed where the laser beam penetrated the skin throughout the epidermis and in the superficial dermis.Histopathology from treated areas on two patients showed epidermal and dermal edema with multiple cell-debris-filled vacuoles in different sizes and shapes at the level of DEJ and tips of the rete ridges.On day 10, RCM images showed aggregated granules, which corresponded to extracellular melanin deposits.Polycyclic papillary contours, a hallmark feature of solar lentigines, were no longer observed.

F I G U R E 3 F I G U R E 4
Dermoscopic and RCM images of café-au lait macule (CALM) from a 27-year-old female on the upper back which was present since birth.(A) Dermoscopic image of CALM shows a homogenous light brown background pigmentation along with delicate reticular network.(B) RCM image of CALM at the level of dermo-epidermal junction shows multiple enlarged regular-edged papillae and hyperreflective peri-papillary rings.Dermoscopic and RCM images of congenital melanocytic nevus of a 31-year-old female located on the left elbow.(A) Dermoscopic image shows a symmetrical pigmented lesion in homogenous color and terminal hairs within the lesion.(B) RCM image of the lesion at the level of DEJ shows junctional nests and clusters (red arrow) along with dermal nests (white arrow).4.1.10| Labial mucosal melanosis Differential diagnosis of labial pigmented macules are labial mucosal melanosis (LMM) and mucosal melanoma.RCM features of LMM are regular epidermal honeycomb pattern and homogeneously distributed round or polycyclic ringed pattern, hyperreflective basal layer, and sparse bright dendritic cells at the basal layer. 12Mucosal melanoma presents with an irregular honeycomb pattern in the epidermis, pagetoid cells, and irregularly distributed atypical dendritic cells.An RCM lip scoring system was developed by Uribe et.

F I G U R E 6
Further studies have used RCM to assess the stage of vitiligo activity, which would lead to an improved selection of patients appropriate for F I G U R E 5 Dermatoscopic and RCM images of invasive melanoma.(A) Dermatoscopy shows an irregularly pigmented lesion with gray-brown circles and angulated lines on sun-damaged skin.(B) RCM image at the level of DEJ shows epidermal disarray with hyperreflective large round and dendritic shaped cells around the follicles.Dermoscopic and RCM images of a nevus spilus in a 23-year-old female patient from the upper back.(A) Dermoscopic image of nevus spilus shows brown, sharply demarcated pigmented lesions with regular reticular network.(B) RCM image of nevus spilus at the level of DEJ shows regular enlarged ringed-edged papillae.

4. 3 |
Exogenous pigmentation4.3.1 | TattooOgos'hi et al. reported RCM features of black, blue, and green tattoo pigment in two patients with multicolored tattoos.32Black ink was observed as densely packed, hyper-reflective granules measuring 2-3 mm at the epidermis and DEJ.Blue ink showed smaller (~1 mm in size) diffuse pigmented granules in the epidermis and dermis without clustering.Green ink showed fine-pigmented granules at the lower epidermis.Pigment granules were seen around dermal papillae under the basal layer.Complications due to decorative tattooing, as well as treatment outcomes can be assessed with RCM by revealing subtle inflammatory changes and pigment granules.Guichard et.al observed inflammatory changes in RCM that led to a diagnosis of tattoo rejection which was clinically and dermoscopically subtle.33RCM showed epidermal spongiosis and an inflammatory cell infiltrate evidenced by the presence of small bright and large bright cells concentrated in the dermal papillae.Likewise, Maier et al. reported subepidermal pigmentation and granulomatous changes on RCM after permanent make-up of the lip.Granuloma formations were seen as clustered, roundish, bright structures, and pigment-loaded macrophages at the level of the superficial dermis.Moreover, 3 months after treatment with 5-FU and triamcinolone injection, follow-up RCM showed regression of the nodular lesions with scarce pigmented bright granules.34Due to the pigmentary alteration of the tattooed skin, the diagnosis of pigmented cancers can be especially challenging.18Reilly et al. investigated 19 pigmented lesions in 15 patients occurring in or near tattooed skin with RCM. 35Tattoo pigment did not hinder the diagnosis of pigmented lesions on RCM.The authors observed round tattoo pigment deposits in 89.4% of the lesions, spindle-shaped tattoo pigment deposits in 36.8% of the lesions, and tattoo pigmentengulfed macrophages in 21% of the lesions.Ink deposits were visualized within both nevi and skin adjacent to nevi.Tattoo particles were differentiated from other reflective structures on RCM such as keratinocytes, melanocytes, melanophages, and inflammatory cells based on their size and morphology.Moreover, RCM showed an excellent correlation with tattoo pigment on clinical/dermoscopic images.Examples of dermatoscopic and RCM images of tattooed skin are presented in Figure S5.

4. 3 . 2 |
Exogenous ochronosisGil et al. reported RCM features of exogenous ochronosis due to the application of hydroquinone.In this case, authors observed welldefined round-to-oval and banana-shaped, non-reflective material deposition in the dermis.Additionally, bright-plump cells throughout the upper dermis were seen that corresponded to melanophages.36

Table 1
outlines RCM features of pigmentary disorders that have been discussed in this paper.RCM images of pigmentary disorders are included with informed consent from patients for the publishing of images. 16,26No 17n-edged dermal papillae, junctional thickening, nucleated cells in the dermal papillae, and the presence of plump bright cells.HN had overlapping RCM features with other atypical nevi and melanoma.17Pogorzelska-Antkowiaket al. reported HN had an atypical honeycomb pattern, dendritic cells in the epidermis, roundish pagetoid cells, non-edged papillae, multiple dilated blood vessels located on the peripheral white area, dense nests in the periphery, and nonhomogeneous nests.