Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition

This protocol details image acquisition steps for RCM and RCM-OCT devices at the bedside. It is quintessential to acquire good quality images for correct diagnoses and management of the skin lesions. These devices images skin at histopathological resolution in vivo, saving unnecessary benign biopsies while detecting early skin cancers.

Diagnoses is rendered at the bedside reducing wait time and anxiety for the patients. Imaging with this microscope helps us to non-invasively guide detection and diagnosis of skin lesions and skin cancers on patients at the bedside. In addition, the imaging helps us triage skin lesions into those that may be superficial versus those that may be deeper, which helps guide treatment.

So the imaging helps us integrate or combine diagnosis and treatment into a single patient visit, what's now being called as a one-stop shop patient care paradigm. Besides using imaging to detect and diagnose skin lesions and skin cancers, we're also using the imaging to guide detection and diagnosis of oral lesions and oral cancers on patients. Demonstrating the procedure will be Ms.Ucalene Harris, a histotechnician from my laboratory.

To begin, select the WP-RCM device for lesions located on the flat surfaces of skin, such as the trunk and extremities, and the HH-RCM device for lesions on curved surfaces, like the nose, ear lobes, eyelids, and genitalia. For imaging, position the patient on a fully reclining chair or a flat examination table with support. Then clean the lesion and surrounding skin with an alcohol wipe.

Add a drop of mineral oil to the center of the plastic window and spread it carefully across the window width. Remove the paperback from the adhesive side of the window, stretch the skin gently, and attach the window. To acquire images using the WP-RCM device, position the probe perpendicular to the lesion.

Then acquire dermoscopy images by hovering the WP-RCM probe over the plastic window cap, and estimate the best angle of insertion for the probe. Align the small white arrow located on the side of the probe with the arrow on the side of the dermoscopy camera. Insert the dermoscopy camera into the plastic window cap.

Ensure that the dermatoscope image covers the entire lesion surface, and press the trigger on the camera to acquire an image. Then remove the dermatoscope. Then place a pea sized amount of ultrasound gel inside the disposable plastic window cap.

Insert the RCM probe into the plastic disposable cap until a sharp click is heard. Using the dermoscopy image acquired in the previous step, select the center of the lesion and identify the topmost or brightest layer of the skin, which is the anuclear layer of the stratum corneum. Set the imaging depth to zero at this level.

Press the stack icon to acquire a stack in the center of the lesion. From the dropdown menu, select an anatomical site, either face or body. Set stepsize to 4.5 microns and depth to 250 microns.

Identify the dermal-epidermal junction, or DEJ layer in the stack and select an 8x8 millimeter square to cover the entire lesion. Then press the mosaic icon to acquire the first mosaic at the DEJ. To acquire subsequent mosaics, click on a structure on the DEJ mosaic to view it on the live view.

Scroll down from the DEJ to acquire mosaics at the dermis, and then up from the DEJ to take mosaics in the epidermis. Capture one stack in each quadrant and one in the center of the lesion with a homogeneous pattern on dermoscopy, or acquire additional stacks for heterogeneous lesions. After completing an image session, detach the protective cone surrounding the microscope lens.

Clean the tip of the objective lens with an alcohol swab and dry the lens. Reattach the plastic cone to the microscope probe. To generate a confocal diagnostic evaluation report click on New Evaluation.

Enter the diagnosis from the pre-selected options in the dropdown menu. Select the features seen during imaging from the report checklist, and select the applicable management. Click on Finish and Sign.

Generate the report as a PDF and print it. to acquire images using the HH-RCM device, first position the patient and clean the lesion site and circle the lesion with a paper ring. Apply a pea sized amount of ultrasound gel to the objective lens of the HH-RCM and cover it with the plastic cap.

Add a generous drop of mineral oil to the side of the plastic cap that will be touching the skin. Press the probe to the lesion side on the skin with firm pressure. Use the Z-depth controls on the HH-RCM device to move up and down at various depths within the lesion.

Acquire multiple single frame images and stacks in the regions of interest. For large lesions, take continuous videos at various layers by moving the HH-RCM probe over entire lesion surface and clicking on the video capture symbol. Record the movement of blood cells within vessels.

After the imaging session is complete, press Done Imaging. Clean the lesion with an alcohol swab, remove the ultrasound gel from the objective lens of the probe, and reattach the plastic cap. Position the patient for imaging and ensure that the boundary of the lesion is free of excessive hair and topical impurities.

After preparing the probe as described in the previous section, click on Imaging Settings. Select the Line Imaging mode to acquire an RCM image. Set the stepsize to five microns and the number of steps to 40.

Acquire stacks as shown in the previous section by clicking on Grab. Then click on the Freeze button after completion. Click on Imaging Settings and select Raster Mode to acquire a correlative OCT video for the lesion architecture.

Switch to the technician tab. Once complete, click on the Grab button and immediately press the Save button. Using this protocol, high quality RCM images were acquired at relevant depths in the skin.

Artifacts such as air bubbles, surface debris, and motion artifacts which hinder diagnostic evaluation should be avoided. On RCM, various skin layers were observed such as the stratum cornea, composed of anucleated keratinocytes, and the stratum spinosum, composed of tightly packed nucleated cells with bright cytoplasm creating a typical honeycomb pattern. This was followed by the stratum basale, identified by the cobblestone pattern formed by the melanin cap of the basal keratinocytes.

And the DEJ characterized by the bright ringed pattern. The papillary dermis was composed of bright collagen fibers and blood vessels. RCM images for common skin cancers, like Basal Cell Carcinoma, showed tumor nodules with clefting and palisading.

Squamous Cell Carcinoma showed an atypical honeycomb pattern and buttonhole vessels. And melanoma showed clusters of bright, large round pagetoid cells in the epidermis. For Basal Cell Carcinoma, the RCM image was unable to show a definitive tumor nodule.

Whereas RCM-OCT images interpreted by both stacks and rasters showed a distinct gray tumor nodule connected to the epidermis, along with clefting and thickened collagen bundles. The diagnosis was confirmed by H&E Stained Biopsy. It is important to acquire diagnostic quality mosaics from five layers of the skin with the WP-RCM Imaging.

Take a smooth video and stacks using HH-RCM, and both a stack and a raster in the same area using the RCM-OCT device.