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Anais Brasileiros de Dermatologia

Print version ISSN 0365-0596

An. Bras. Dermatol. vol.84 no.6 Rio de Janeiro Nov./Dec. 2009 



Reflectance confocal microscopy in the diagnosis of cutaneous melanoma*



Cintia RitoI; Juan Pineiro-MaceiraII

IMaster studies under course, Universidade Federal do Rio de Janeiro (collaboration with Universidade degli studi di Reggio-Emilia – Modena, Italy), Preceptor of Dermatoscopy, course of medical residence, Hospital Geral de Bonsucesso - Rio de Janeiro (RJ), Brazil
IIPost-Doc studies, Armed Forces Institute of Pathology, USA, Joint Professor, Universidade Federal do Rio de Janeiro - Rio de Janeiro (RJ), Brazil

Mailing Address




Skin melanoma is an international public health issue, with a considerable increase in frequency over the past few years. Early diagnosis and excision are essential for good patient prognosis. Over the past two decades dermoscopy has gained significance due to a major improvement in the accuracy of skin melanoma diagnosis in its early stage. However, there are some benign lesions of
questionable dermoscopy, which may lead to the performance of unnecessary surgery. Recently, reflectance confocal microscopy has been introduced as a promising supplementary diagnostic method. It is a noninvasive, in vivo, simple, painless and quick exam. It is the only technique capable of identifying cellular structures and to examine the epidermis and papillary dermis with a resolution similar to that
of histopathology, with a sensitivity of 97.3% and specificity of 72.3 % in the diagnosis of cutaneous melanoma. This is an important diagnostic tool, because it does not substitute post-surgical histopathological examination and allows for the rational assessment of lesions of questionable dermoscopy, thus avoiding unnecessary surgical procedures.

Keywords: diagnosis; melanoma; melanoma; microscopy, confocal




Melanoma and non-melanoma skin cancer are currently the most common types of cancer in the Caucasian population, according to some authors 1. Both tumors have shown increased incidence in the whole world. Cutaneous melanoma is a type of cancer that grows fast in the Caucasian population and its morbidity has increased dramatically in recent years 1-5.

There is strong inverted correlation between the survival rate and tumor thickness and given that there are no effective curative therapies for advanced melanoma, early diagnosis and excision are essential to reduce morbidity and increase patient survival 5-8. There are reports that this type of incidence is due mainly to the growing detection of initial melanomas (of smaller thickness). The improvement of diagnostic methods leads to increased number of low risk patient detections (that is, Breslow thickness below 0.76 mm) and this is the main reason that explains no changes to the mortality rate related with this neoplasm 2,9.

The main concern is to have early diagnosis of these lesions, which stimulates constant development of different noninvasive diagnostic techniques 10,11.

The development of dermoscopy as a diagnostic support method enabled the increase of diagnostic accuracy of melanoma in up to 90% 12,13 with greater sensitivity and specificity, compared to clinical isolated diagnosis 5,14. Currently, there is also the support of reflectance confocal microscopy 15.

Of all new noninvasive diagnostic methods, reflectance confocal microscopy is the only technique that enables examination of the epidermis and papillary dermis with resolution close to that of histopathology exam, identifying with high resolution the microanatomical structures and individual cells 16,17. Confocal images have been assessed as qualitatively and quantitatively corresponding to horizontal histological sections 11,18-24.

Reflectance confocal microscopy and dermatoscopy collect horizontal images. It facilitates the correlation between images obtained in both exams, and the correlation between some global patterns and dermatoscopic sites and their confocal microscopic characteristics has already been described 25-28.



The confocal microscope was invented by Marvin Minsky in 1957. 10,29,30 However, it required the development of an appropriate light source and computer technology to enable its use in in vivo human skin. The first report of use of reflectance confocal microscope on human skin was made in 1995 31.

Diagnostic Assessment with Reflectance Confocal Microscopy

This new diagnostic methodology presents a number of advantages, such as identification of skin structure, noninvasive procedure performed in vivo, painless procedure that does not cause tissue damage. The skin is not affected by material processing, minimizing the occurrence of artifacts; data are collected in real time, enabling quick analysis of the lesion and the test may be repeated on the skin site innumerous times. Thus, we may dynamically assess abnormalities to the lesion in response to some therapy used, and lesion borders may be assessed in vivo enabling better surgical planning 32-34. As a result of teledermatology advance, this test may be easily analyzed by different specialists and the time required to perform the test is not greater than what it takes to perform digital dermatoscopy 10,11,31,16.

One of the limitations of current reflectance confocal microscopy is the possibility of analyzing microanatomical structures only up to the depth of 350 µm, which normally means as far as the papillary dermis. For this reason, abnormalities to the reticular dermis and tumors that invade the depth cannot be properly assessed 16. However, in areas where epidermis has shallow thickness, part of the reticular epidermis may also be assessed.

Different types of devices have been manufactured and two models were recently commercialized, vivascope 1500 (Figure 1) in 2000 and vivascope 3000 in 2006, which has a pistol that facilitates the performance of the test.



Device vivascope uses diode laser with 830 nm wavelength and 30x objectives with numeric opening of 0.9, providing lateral resolution of 1-2 µm and axial resolution of 3-5 µm. The penetration of image depth ranges from 200-500 µm (on average 350 µm), which enables visualization of the epidermis and upper dermis. The laser power is below 30 mW and does not cause damage to the skin or the eyes 11,31,16.

The laser produces light that illuminates a small point inside the tissue. The light is reflected, goes through a small pinhole and an image is formed in the detector. This opening does not allow the reflected light (reflectance) to reach the detector from another tissue point. Therefore, only reflected light from the focal region (confocal) is detected. To create the image of a complete lesion, each point is scanned 31.

The device uses objective lenses for water immersion, being the refraction index of water and epidermis 1,34. It minimizes spherical aberrations. There is also a water-based gel as immersion medium, especially if the lesion is desquamative or hyperkeratotic, because the gel reduces the refraction irregularities.

The exam is easy, simple and painless. We use a metal ring that is fixed to the patient's skin with adhesive. Inside the right, there is water or gel. The ring is then fixed to the device using magnetic medium.

There are three basic differences from confocal microscopy in relation to conventional histology routine. In confocal microscopy images are obtained horizontally from the lesion, whereas in conventional histology the sections are made vertically. The images are obtained in a gray scale, similarly to what happens in radiographs 31. Moreover, we may have static and dynamic images of the skin, which may be recorded in 20-30 Hz videotape, showing events such as blood flow 31.

Images are formed by the confocal microscope using different shades of brightness. The brightness contrast mechanism in confocal microscopy is caused by different light refractions. In the gray scale of confocal microscopy, the structures are brighter with high index of refraction, compared to the structures around it. Skin components with high rate of refraction are: melanin (n=1.72), keratin (n=1.51) and collagen (n=1.43). These components seem to be bright, surrounded by epidermis (n=1.34) and dermis (n=1.41).10

Normal skin examination

To interpret the images of abnormal skin, it is necessary to be familiar with normal skin appearance. In normal skin histology, the epidermis is comprised primarily by keratinocytes and a smaller population of dendritic cells, which are the melanocytes and Langerhans cells. The dermis is comprised by blood vessels, nerves, inflammatory cells and fibroblasts involved by collagen fibers and elastin. Papillary dermis forms projections into the epidermis, named dermal papillae. The meeting point between the epidermis and the dermis is named dermal-epidermal junction. In the epidermis, the keratinocytes are differentiated to form four different layers. Reflectance confocal microscopy enables identification of these layers based on architecture and cytological characteristics, as well as on measuring depth of the section that is related to location of each layer 10,31.

Stratum korneal is the most superficial layer of the epidermis, which is placed at 0-15 µm deep and it is comprised by flat anucleated keratinocytes 10. This layer varies in depth depending on anatomical site and sun exposure. At confocal microscopy, it produces a bright image 31,17. Large polygonal anucleated korneocytes (25-50µm) can be seen. Dermatoglyphs look like dark linear valleys amidst the korneocyte groups (Figure 2) 10,31.



The granulous layer is found 10-20 µm deep and it is comprised by polygonal keratinocytes that contain kerato-hyaline granules in the cytoplasm and oval and large nucleus. At confocal microscopy, the keratinocyte measures 25-35 µm and is presented as a flat, oval dark central area corresponding to the nucleus, surrounded by bright and granulous cytoplasm. Normally inside the dark nucleus, we can see a small white central point, the nucleolus. The cytoplasm is bright owing to innumerous structures with refraction index of 0,1-1 µm, which corresponds to the organelles and to kerato-hyaline granules. Keratinocytes have well marked borders and they are arranged in a honeycomb pattern (Figure 3).10,11,31



The spinous layer is found approximately 20-100 µm deep from stratum korneal, which has approximately 5 to 10 keratinocytes in its depth, with polygonal format that is progressively flatted towards the surface. Upon examining them, they have an area of approximately 15-25 µm in size, with polygonal shape, containing a central oval or rounded dark area, which corresponds to the nucleus and the cytoplasm with bright contour. These keratinocytes have clearly demarked borders and are arranged in honeycomb shape, such as on the previous layer (Figure 4).10,31



Basal layer is found approximately 40-130 µm deep from the surface. It is comprised by only one layer of columnar keratinocytes containing melanocytes regularly distributed, in a proportion of about 1 melanocyte to each 10 keratinocytes. Melanin is the main source of contrast on the epidermis for reflectance confocal microscopy. Keratinocytes are bright cells that measure about 7-12 µm. When we obtain a deeper image, supra-papillary image of dermal-epidermal junction is shown as basal cell rings surrounding the dark dermal papilla, which frequently shows blood flow and collagen 10,31. This oval papillary standard is characteristic of benign lesions (Figure 5).



The detection of normal melanocytes is possible only in rare situations. When identified, they are oval or rounded structures in the dermal-epidermal junction with external ramifications that may correspond to the dendrites 31. From day 4 to 8 after sun exposure, denditric melanocytes may be better observed, because the melanin cover shine on the basal membrane is diminished. After day 29 post-exposure, the marked shine of melanin sheaths reappears, preventing the visualization of melanocytes 11,35.

The dermal - epidermal junction is comprised by epidermal crests and dermal papillae and it corresponds to the junction point between the epidermis and the dermis. Papillary dermis is found 50-150 µm deep and it includes the dermal papilla and a small layer under the dermal papilla that goes up to the superficial vascular plexus. It appears to be dark, with grey collagen fibers and it is surrounded by a bright keratinocyte ring. Collagen fibers may appear as a reticulated mesh of fibers that measure 1-5 µm in diameter (Figure 6) or as bright bands measuring 5-25 µm in diameter (these bands are the most characteristic of the reticular dermis). Using video, we can see the blood flow through the capillaries 10.



Reticular dermis is placed between the superficial blood plexus and the subcutaneous, deeper than 150 µm. The reflectance confocal microscopy can hardly see it, except in its upper portion, which can be seen in some specific situations. When seen, it reveals thick bands of collagen, gray colored and arranged in a fascicular pattern intertwined by dark lumens, corresponding to the blood vessels, and few bright cells that correspond to the inflammatory cells 10.

Confocal microscopic characteristics related to melanoma

In the diagnosis of cutaneous melanoma, we can reach sensitivity of 97.3% and specificity of 72.3% 36. The criteria used to make the diagnosis are 15,36-40:

- Significant cytology atypia on the basal layer (Figure 7)



- Loss of oval contour of papillae at the dermal-epidermal junction (Figure 7)

- Presence of rounded, bright cells on superficial layers (Figure 8)



- Melanocytic cells in clear pagetoid ascension throughout the whole lesion. They are pleomorphic, nucleated cells with bright cytoplasm

- Confluent cell clusters with not very bright cells, providing a characteristic cerebriform pattern inside the papillary dermis (Figure 9)



- Presence of isolated nucleated cells inside the papillary dermis, corresponding to melanocytes (Figure 10).




Confocal microscopy is a promising methodology through which we can understand many skin pathological and physiological aspects. It is capable of examining the epidermis and the dermis by assessing their tissue structures in real time, monitoring the dynamic processes. The applications include clinical research as well as diagnosis of different conditions, including cutaneous melanoma with sensitivity of 97.3% and specificity of 72.3% for the disease. Therefore, it is a significant diagnostic tool that can support dermatoscopy and histopathology.



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Mailing Address:
Cintia Rito
Rua Orcadas 447, apto 201, Jardim Carioca, Ilha
do Governador, Rio de Janeiro
Tel.:/Fax: 21 2463-2938, 7892-5693, 2463-2938

Funding: None
Conflict of interest: None



* Study carried out at Universidade Federal do Rio de Janeiro (UFRJ) – Rio de Janeiro (RJ), Brazil.

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