Journal of Dermatology for Physician Assistants

The official journal of the Society of Dermatology Physician Assistants

Reflectance Confocal Microscopy: An Introduction

By Megan Dauscher, MS, PA-C, and Rachel Manci, BS

Megan Dauscher, MS, PA-C, is a physician assistant for the Dermatology Service at Memorial Sloan Kettering Cancer Center in Hauppauge, New York, and a guest lecturer and Master’s Thesis Advisor of the Hofstra University Physician Assistant Studies Program in Hempstead, New York.
Rachel Manci, BS, is a Medical Student Fellow in Dermatology at Memorial Sloan Kettering Cancer Center in Hauppauge, New York.

Acknowledgment: The authors wish to thank Drs. Ashfaq Marghoob and Miguel Cordova from Dermatology Service, Department of Medicine at Memorial Sloan Kettering Cancer Center in Hauppauge, New York, for their continued commitment to education and their ability to provide exceptional patient care with novel technology.

Disclosures: The authors have disclosed no potential conflicts of interest, financial or otherwise, relating to the content of this article.

Reflectance confocal microscopy (RCM) is a noninvasive, in-vivo, imaging modality used to diagnose and manage skin cancers, benign skin neoplasms, and inflammatory dermatoses. Although previously considered an academic tool, the increasing number of available RCM resources make it necessary for dermatology physician assistants to expand their knowledge base within this field.

There have been many modern advances made in the field of dermatology, particularly in evaluating the skin lesions for malignancies. Initially, the basic morphologies of skin lesions were evaluated by the naked eye, then occasionally with the assistance of a magnifying glass. Patients and providers alike have followed guidance from the American Academy of Dermatology (AAD) and other medical organizations to look for the “ABCDEs of melanoma.” This well-known mnemonic for assessing characteristics of melanoma, including Asymmetry, Border irregularity, Color variation (both intralesional color variation as well as a color that is different from the patient’s other nevi), Diameter greater than 6mm, and Evolving (a new or changing lesion).1 Dermoscopy, a noninvasive technique that involves using a hand-held light magnifier (usually 10-fold magnification) to visualize a skin lesion, allows clinicians to observe the surface and subsurface structures that are invisible to the naked eye.2

Although dermoscopy has been shown to increase the accuracy of diagnosing cutaneous malignancy, the challenge for clinicians to accurately recognize malignancy while simultaneously minimizing unnecessary surgical procedures, remains.3,4 One technique that has proven successful in meeting this clinical challenge is reflectance confocal microscopy (RCM).

An understanding of the basic mechanics of RCM is essential to its application and interpretation. A diode laser is the source of coherent, monochromatic light. The emitted light beams are projected into the skin after passing through a beam splitter, a scanning and focusing optical lens, and a probe (Figure 1).5 Each light beam is projected into the skin, which causes illumination of a small point within it. Light is then reflected from this focal point back through the lens via a small pinhole onto a photodetector; the pinhole selectively allows light from the focal point to pass through, hence confocal, and inhibits light from other tissue points or planes. The generation of RCM images relies on differing reflectivity and back scattering of light from chemical and molecular structures due to their differing refractive indices. Structures with higher refractive indexes result in brighter images. For example, melanosomes and melanin have a high refractive index, resulting in white structures in a confocal image.

The 830 nm diode laser used in RCM allows for the visualization of the papillary and upper reticular dermis, depending on the anatomic location; it does not cause any tissue injury (including injury to the eye) due to this limited imaging depth. Increasing the intensity of the laser would result in deeper imaging capacity, but at the cost of increased morbidity to operator and patient.

By utilizing the VivaScan system, the user can obtain sets of images that have en-face orientation at varying depths (z), calculated as micrometers below the skin’s surface. The types of imaging functionalities available through the VivaScan system include the capture of single images, blocks, stacks, cubes, and movies, all of which can be used to analyze lesions of up to 8mm x 8mm. To obtain images representative of a single depth, the user can obtain single images and blocks. A single, basic image measures 500μm x 500μm and can be viewed onscreen in real time. A block is a large mosaic of up to 256 basic images of identical depth that are sequentially stitched together to create an en-face field of view much larger than that of a single, basic image (Figure 7). To optimize lesion analysis and diagnostic accuracy, mosaic images should be obtained at the suprabasal epidermis, the DEJ and the papillary dermis.6 This field of view ranges in size from 1mm x 1mm to 8mm x 8mm. Additionally, a user can obtain images that provide vertical depth information by obtaining stacks and cubes. A stack is a series of basic 500μm x 500μm en face images taken at set depth increments, commonly obtained by starting at the level of the stratum corneum and ending in the dermis (Figure 8). Cubes are similar to stacks, but instead of a series of basic 500μm x 500μm images, cubes are formed when multiple blocks are obtained at set depth increments. Finally, to obtain a real-time evaluation of the native tissue, movies or short videos can be recorded in the form of AVI files up to two minutes in length for the real-time showcase of specific features such as blood flow and other vascular structures.

Two commercially available devices for RCM in the clinical setting are the VivaScope 1500 –wide probe RCM and the VivaScope 3000 – handheld RCM; both devices offering similar image resolution quality (Figures 2 and 3).6

To utilize the VivaScope 1500 device, a drop of oil is placed on the target site, and then a polymer window must be directly adhered to the patient’s skin atop the drop of oil. The VivaScope dermoscopic camera is first placed inside the polymer window, and a dermoscopic image of the lesion is generated (Figure 4). After applying ultrasound jelly to the inside of the polymer window, the Vivascope 1500 imaging probe is then connected to the window, and RCM imaging can begin (Figure 6). The dermoscopic image allows for targeted RCM imaging of areas of interest (Figures 5 and 7). Although the VivaScope 1500’s required polymer window provides for more controlled imaging of skin lesions, it does limit the utility of the device for smaller anatomic areas or areas with a large amount of contouring, such as the nose, ears, perioral, and orbital areas.

VivaScope 3000 handheld probe provides for increased operator dexterity, but at the cost of a smaller field of view (1000 x 1000 μm2) and inability to generate mosaic images; the device can only obtain movies, stacks, and single images. It is unable to correlate RCM images with the dermoscopic image.

In order to recognize abnormalities utilizing RCM, it is essential to first understand how normal skin appears. Starting with the most superficial layer, the stratum corneum appears as a very bright – highly refractive- surface surrounded by dark furrows, which represent skin folds or dermatoglyphs.5,6 Here the operator will visualize large (10-30 μm) polygonal-shaped corneocytes that lack a visible nucleus. This layer is typically located about 0 to 20 μm below the outermost surface of the skin (Figure 8a).

Located 15-20 μm below the outermost surface of the skin, the stratum granulosum is the first layer of epidermis with retained nuclei, which appear sparse and large on confocal imaging (Figure 8b). The keratinocytes in this layer have well-demarcated outlines, forming a honeycombed pattern. The keratohyalin granules and organelles give a white, grainy appearance to the cytoplasm that surrounds the round or oval shaped, dark central nuclei.5,7

Penetrating deeper into the epidermis, the stratum spinosum exists at depths 20-100 μm below the skin’s surface. Here, the keratinocytes are smaller in comparison to those of the granular layer, measuring about 15-25 μm in diameter. These cells are also polygonal shaped, containing thin, white cytoplasm, which surrounds oval, dark nuceli forming a honeycomb pattern (Figure 7c).

At an average depth of 50-100 μm below the skin’s surface lays the stratum basalis: a single layer of germinative cells residing just above the dermis. These cells are smaller than spinous keratinocytes, measuring about 7-12 μm, but appear brighter due to the presence of melanin caps on top of their nuclei. Pigmented keratinocytes and melanocytes have a high refractive index due to the presence of melanin, and appear as solitary round or oval structures. These two cell types are virtually indistinguishable from one another.

Skin phototypes affect the appearance of basal keratinocytes in RCM. Due to the lack of pigment in skin phototype 1, basal keratinocytes are difficult to delineate due to their low refractive index. In phototypes II-IV, the increased melanin in keratinocytes yields a higher refractility resulting in a cobblestone pattern.

At the dermo-epidermal junction (DEJ), melanocytes and basal keratinocytes form bright rings surrounding dark colored dermal papillae, an arrangement known as “edged papilla” (Figure 8d).5 Within the dermal papillae, arterioles and blood flow can be observed with real-time RCM examination which aids in identification of the DEJ (Figure 8d arrow).

The papillary dermis can be found about 100-150 μm below the surface of the skin followed by the reticular dermis which lies >150 μm below the skin’s surface which contain a network of fibers and bundles. Collagen fibers appear as elongated, bright, acellular, anucleated, fibrillar structures situated side by side throughout the dermis (Figure 8e). Blood vessels in this layer appear as dark tubular structures and blood flow can be observed during real-time examination (Figure 8d arrow). Collagen fibers that surround these blood vessels are usually distributed as rings or coils in the papillary dermis but appear as parallel bundles gathered into large fascicles in the reticular dermis.

RCM imaging alone has been shown to significantly improve the early detection of and diagnostic accuracy of melanocytic and nonmelanocytic skin cancers when compared with dermoscopic and clinical examination alone.6

A 2016 meta-analysis of 21 studies and 3602 lesions showed that the combined results for sensitivity and specificity of all malignant tumors were 93.6 percent (95% CI: 0.92-0.95) and 82.7 percent (95% CI: 0.81-0.84) respectively.8 Subgroup analysis for detection of cutaneous melanomas amongst these lesions showed a sensitivity of 92.7 percent (95% CI: 0.90-0.95) and a specificity of 78.3 percent (95% CI: 0.76-0.81). A sensitivity of 91.7 percent (95% CI: 0.87-0.95) and specificity of 91.3 percent ( 95% CI: 0.94-0.96) was discerned for detecting basal cell carcinoma with RCM.8

Regarding specific melanoma subtypes, the reported sensitivities and specificities vary, but all are still greater than those seen with dermoscopic and clinical exam alone. A 2020 meta-analysis of 7 studies and 1111 lesions demonstrated that the sensitivity and specificity of RCM for the diagnosis of amelanotic/hypomelanotic melanomas were 67 percent (95% CI: 0.51-0.81) and 89% (95% CI: 0.86-0.92), respectively.9 Another literature review article stated that the sensitivity and specificity of RCM for the diagnosis of lentigo maligna ranges between 85 and 93 percent and 76 to 82 percent, respectively.10

Because RCM is a valid method of accurately diagnosing malignant skin tumors, it has been used for evaluation of equivocal lesions to determine the best course of action: biopsy, wide local excision, clinical monitoring etc.3 The addition of RCM examination to dermoscopic evaluation and/or digital follow-up has decreased the number of biopsies performed on benign lesions.11 Identifying known RCM features can help distinguish benign nevi from malignant melanoma lesions, thus driving down the number of biopsies on lesions eventually proven to be benign.11 Some of the RCM features associated with melanoma include round pagetoid cells and large atypical bright cells in the epidermis, cellular atypia at the basilar layer, non-edged papillae at the DEJ, and atypical nucleated cells in the dermis.11 In contrast, benign nevi demonstrate well conserved honeycomb and cobblestone patterns in the epidermis, and edged papillae at the DEJ.11

RCM is also useful for monitoring equivocal lesions at routine intervals to determine if the lesion displays any changes, similar to short term mole monitoring with dermoscopy. RCM can be utilized to determine response in lesions treated with imiquimod, photodynamic therapy, cryotherapy as well as other treatment modalities.1 RCM can also be used in conjunction with laser ablation to optimize treatment by using a targeted histologic approach for fast and minimally invasive removal of nodular and superficial basal cell carcinomas.13

RCM may also be used in the surgical setting to optimize patient care. Handheld RCM can be used utilized in conjunction with Wood’s lamp and dermoscopy to formulate a map for dermatologic surgeons to follow during staged excisions of lentigo maligna (LM) and lentingo maligna melanoma (LMM). These maps can result in improved patient counseling based on the increased accuracy of the anticipated defect size as well as sparing of healthy tissue by reducing the number of biopsies that are necessary for clinically ambiguous areas.14 In theory, this can also result in more efficient surgeries by decreasing the number of stages required and therefore minimizing the time that the patient needs to be in office. Similarly, real time RCM and video-mosaicking have been used in Moh’s surgery for intraoperative evaluation of surgical wounds to detect the presence of residual tumor cells as an alternative to the labor intensive, time consuming process of frozen sections.15

Although RCM proves to be very promising, there are some limitations to this imaging method. The process of image acquisition can be time consuming, ranging from several minutes to up to an hour, with the average lesion taking about 20-30 minutes.16 Image interpretation is additionally time consuming and therefore should be performed by experienced operators for maximal efficiency.6 To this end, operators should be adequately trained prior to attempting image acquisition and interpretation. This process has been estimated to take about 4 to 6 months as well as a fundamental understanding of histology and dermoscopy, and the evaluation of thousands of cases prior to obtaining an acceptable level of diagnostic expertise.6 However, there are many, emerging, training resources available for clinicians today: online courses (Confocal 101), textbooks (Reflectance Confocal Microscopy for Skin Diseases) and conferences (MSK’s Annual Basic Course in RCM: Non-Invasive Diagnosis of Skin Cancer). Sufficient training is paramount, and it is recommended that dedicated long-term staff acquire images due to the lengthy learning curve.17

As previously mentioned, the maximum depth of RCM is about 150 μm, after which the resolution significantly decreases, which limits the clinician’s ability to detect the tumor depth, posing challenges for evaluation of nodular, hyperkeratotic, or ulcerated lesions.6 A proposed method to overcoming this barrier is to couple RCM with Optical Coherence Tomography (OCT) as it has the ability to penetrate deeper into the skin but lacks the clarity and precision of RCM. By combining these two imaging modalities, practitioners are able to improve their diagnostic capabilities at the bedside as well as broaden the scope of care that they can provide for patients.18

Another limitation is availability and affordability of these commercial devices. Because these devices can be expensive to obtain, it is rare to find them in an outpatient, nonacademic setting. However, these devices are now available to lease and since the procedure can be reimbursed through insurance, there is increased incentive for clinicians to introduce these devices into their practices.

It is evident that RCM can be utilized in a variety of ways in clinical practice, and it is now recognized as a billable procedure. Unfortunately, the VivaScope 1500 is the only device with approved CPT codes including acquisition of images (96932), image interpretation and production of a report (96933), amongst others.6 To ensure reimbursement, one must obtain 3 to 5 mosaics at varying depths and stacks at points of interest or concern. The reimbursement for obtaining and interpreting images of a single lesion (96931) is approximately $175, and then $100 for each additional lesion (96934).19


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