Ex Vivo Confocal Microscopy: Revolution in Fast Pathology in Dermatology

J. Malvehy; J. Pérez-Anker; A. Toll; R. Pigem; A. Garcia; L.L. Alos; S. Puig


The British Journal of Dermatology. 2020;183(6):1011-1025. 

In This Article

Clinical Application of Ex Vivo Confocal Microscopy

Different studies have reported the use of ex vivo CM in the clinical setting. This includes different indications in dermato-oncology, inflammatory diseases of the skin, infections and fast assessment of skin fillers. However, new applications for ex vivo CM have been explored in other situations in surgical pathology, and possibly a list of indications will appear soon in the literature. In Table 4 the clinical applications of ex vivo CM are summarized.

Ex Vivo Confocal Microscopy for the Evaluation of Resection Margins of Skin Cancer

At present the main clinical application of ex vivo CM in dermatology is the intraoperative control of surgical margins of cutaneous tumours, as an alternative to the traditional optical microscopy examination of frozen sections or paraffin stained with H&E. In a recent study in Mohs surgery with a new-generation scanning multimodal confocal microscope, Pérez-Anker et al. reported a mean scanning time of 7 min (range 3–15).[23] In a recent study with an FCM device with different samples from punch biopsies from Mohs specimens, Peters et al. reported a median total time to generate and evaluate a confocal laser scanning microscopy (CLSM) image of 5·17 min (range 2·05–20·17).[34]

Most of the ex vivo CM studies were conducted on BCCs. Subsequently, this technique has been used for other epithelial tumours, melanocytic tumours, dermatofibrosarcoma and Paget disease.[15,16,39–41]

Basal Cell Carcinoma. BCC is the main cancer that has been evaluated, in several studies, comparing ex vivo CM with histopathology and studying the application of the technique in clinical practice. In fluorescence mode with a contrast agent that stains nuclei, such as acridine orange, BCC islands are highly fluorescent and are distinguishable from the surrounding tissue because the endogenous autofluorescence from the dermis is relatively weak (Figures 3–5).[14–16,39] The reported criteria that enable identification of BCC islands under FCM are summarized in Table 5.[32,42]

Figure 3.

Different subtypes of basal cell carcinoma scanned with the VivaScope 2500M-G4 device, stained with acetic acid and acridine orange, flattened with the magnet device. (a) Superficial subtype: the stromal reaction is larger and laxer than in other subtypes (yellow arrowhead). (b) A 1-mm punch biopsy showing an infiltrating basal cell carcinoma with a dense, low cellular, stromal reaction around it (blue arrowhead). (c) Nodular and micronodular basal cell carcinoma (green arrowhead). The stromal reaction is often better circumscribed and surrounds the tumour. (d) Micronodular basal cell carcinoma with denser stromal reaction around it (black arrowhead).

Figure 4.

Micronodular basal cell carcinoma scanned with the VivaScope 2500M-G4, stained with acetic acid and acridine orange, flattened with the magnet device. (a) Clinical and dermatoscopic image: pale whitish area with fine short telangiectasias on the dorsum of the nose. (b) Fluorescence laser: nuclear details are better appreciated. (c) Reflectance laser: stromal details are seen that are not appreciated with the fluorescence laser. (d) Fusion confocal microscopy. (e) Digital colours of fusion confocal microscopy, showing the benefit of both lasers combined in colours similar to those of conventional haematoxylin and eosin (HE). (f) Conventional histological correlation.

Figure 5.

Correlation of in vivo and ex vivo confocal microscopy in reflectance mode in nodular basal cell carcinoma, scanned with the VivaScope 2500M-G4, stained with acetic acid and acridine orange. (a, b) Clinical and dermoscopic images of a pigmented nodular basal cell carcinoma. (c) In vivo confocal microscopy showing bright tumour islands with dendritic cells that correspond to melanocytes. (d) Ex vivo reflectance confocal microscopy revealing bright tumour islands with crowding and nuclear pleomorphism, clefting, palisading, stromal reaction and dendritic cells because of the stain protocol applied with acetic acid and acridine orange. (e) Conventional histological correlation with haematoxylin and eosin (HE).

The overall sensitivity and specificity of fluorescence-mode ex vivo CM for detecting BCC with narrow or incomplete margins are 88–96·6% and 89·2–99%, respectively.[24,34,43–47]Table 6 presents the results of diagnostic accuracy in different studies with CM on fresh tissue. Sensitivity was inferior to specificity in three studies, equal in one, and superior in three when compared with conventional histopathology with frozen or paraffin sections. The main reason for the lack of correlation in Mohs surgery is the different preparation of the specimen. For standard histological examination, the outer sections of the tissue block are discarded. This outer tissue is not discarded in fresh-tissue examination with ex vivo CM. However, it has been shown that confocal imaging of fresh tissue can avoid some problems in the identification of the tumour compared with classic methods.[34] Table 7 summarizes the reasons for lack of correlation with conventional histopathology.

Squamous Cell Carcinoma. Using the reflectance mode, SCC can be identified by the presence of keratinocytes with densely packed and irregularly distributed nuclei.[26,48,49] A study of 13 SCCs by Longo et al. demonstrated that ex vivo CM with the fluorescence mode could also be used to grade the degree of differentiation (Table 5).[50] A study by Hartmann et al. found that 30 SCCs were in situ and 72 invasive.[49] Of these, 29 invasive SCC tumours were well differentiated, 19 moderately, 15 poorly and nine undifferentiated. The authors concluded that ex vivo CM allowed rapid examination of SCC and provided useful information on invasiveness and grading of the tumour.

A few case series have reported the use of ex vivo CM for controlling the surgical margins of SCC.[26,48] Regarding the reflectance mode, an initial study by Chung et al. found a positive correlation with histopathology in only 13 of 23 SCCs using acetic acid,[26] whereas a sensitivity of 95% and a specificity of 96% for the identification of SCC were achieved in a second study on 10 lesions by Horn et al.[48] In the study by Longo C et al., ex vivo CM agreed with histopathology in 41 of 43 mosaics obtained from 34 tumour margins from 13 SCCs.[50]

Using the combination of reflectance and fluorescence modes, images of SCC similar to histopathology can be obtained in a few minutes with the new systems of ex vivo CM (Figures 6–8).

Figure 6.

Squamous cell carcinoma with an in situ area represented with moderate differentiation seen with the VivaScope® 2500M-G4 device, stained with acetic acid and acridine orange, flattened with the magnet device. Conventional histopathological diagnostic features are seen: hyperkeratosis (black triangle) with parakeratosis (yellow triangle), acanthosis with architectural disarrangement (blue triangle), cellular atypia (square) and peritumoral inflammation (grey triangle). Detail of the cellular atypia: loss of cellular cohesion (green triangle), dyskeratosis (purple triangle), nuclear pleomorphism and mitotic figures (red triangle). No keratinizing pearls can be observed.

Figure 7.

Bowen disease scanned with the VivaScope 2500M-G4, stained with acetic acid and acridine orange, flattened with the magnet device. (a–c) Clinical and dermoscopic images: glomerular vessel pattern beyond a scar from a previous excision. (d) Digital stain of ex vivo confocal microscopy. Black frame: acanthosis with cellular atypia in all the layers of the epidermis surrounded by a dense inflammatory infiltrate. (e) Conventional haematoxylin and eosin (HE) histology of the same lesion.

Figure 8.

Image of a well-differentiated invasive squamous cell carcinoma obtained with the VivaScope 2500M-G4 device, stained with acetic acid and acridine orange, flattened with the magnet device. Invasive areas are observed (yellow triangles) along with a keratin pearl (blue triangle).

Melanocytic Lesions. In a first experience using ex vivo CM at the bedside, Bennàssar et al. imaged fresh tissues from shave biopsies obtained from two whitish facial papules with equivocal dermoscopic diagnosis that were diagnosed as infiltrating BCC and intradermal naevus, respectively, within 170 s of tissue excision.[51] The authors used this information to perform electrodesiccation of the naevus and Mohs surgery of the BCC in the same procedure after ex vivo CM. This report, for the first time, demonstrated the impact in clinical management of FCM with acridine orange staining in equivocal skin tumours.

Recently, melanoma has been imaged by ex vivo CM.[35,52–54] Hartmann et al. described a proliferation of atypical melanocytes in the epidermis, consumption of the epidermis and nests of atypical melanocytes in the dermis using ex vivo CM in a study on six melanomas with similar features to those seen by in vivo RCM.[53] The same group also reported a pilot study on 10 melanomas to measure melanoma thickness, showing a good correlation with classical pathology using H&E, with the possible future benefit of being able to choose the correct size of the surgical margins before surgical excision.[52] More recently the use of immunofluorescent staining in ex vivo CM has been reported for the first time in 16 fresh or frozen melanoma metastases, SCCs and BCCs.[35] The authors tested different staining protocols with S-100, melan-A and Ber-EP4, with positive fluorescence obtained in most of the tumour cells in the cytoplasm (S-100 and melan-A) and nuclei (S-100). The authors concluded from this pilot study that the combination of rapid examination and the use of specific staining would lead to a revolutionary method for intraoperative diagnosis.

Ex vivo RCM and FCM (VivaScope 2500) were also performed on the surgical specimen of a dorsal melanoma of a male patient. Fresh tissue was incubated with a few drops of dendronized nanoparticles (NPs10@D1_ICF_Alexa647_DOTAGA Fe3+) and showed a fluorescence signal of the tumour cells at 658 nm that did not involve the surrounding tissue.[55,56] Colocalization of melanoma cells visible in the CM images acquired in the reflectance mode and with the probe-labelled dendronized nanoparticle in the fluorescence mode indicated that this staining is specific for these cells.[55,56]

Ex Vivo Confocal Microscopy in Nails. Ex vivo CM could be particularly useful for nail tumours as it allows intraoperative confirmation of the diagnosis of the biopsy specimen before proceeding to final excision without waiting for the histological examination. Notably, ex vivo CM is more suitable than the classic optical examination of frozen sections of this organ because of the reduced size of tumours in nails, and no tissue sections are wasted if subsequently they have to be used for diagnosis.

In a pilot study on six malignant epithelial tumours of the nail apparatus, Debarbieux et al.[57] showed that ex vivo CM in fluorescence mode could be a useful tool for the diagnosis of invasive SCC, invasive onycholemmal carcinoma and Bowen disease, showing marked nuclear and cytological atypia and the presence of numerous dyskeratotic cells. In particular, well-demarcated epithelial nests deeply invading the dermis, nuclear pleomorphism (variable size and shape of the nucleus) and densely packed and irregularly organized nuclei have been observed in invasive SCC and onycholemmal carcinoma.[57–59] Therefore, in these cases it would be possible to perform wide excision of the tumours just after the observation of a biopsy specimen under ex vivo CM, shortening the management time. However, in situ SCC and minimally invasive well-differentiated SCC were more difficult to diagnose with the first-generation CM device, showing only focal epithelial acanthosis and not cytological atypia.[57]

The same study by Debarbieux et al. with this device included three benign epithelial tumours (two onychomatricomas and one onychopapilloma) that were differentiated from SCC because their cellular density was similar to that of the adjacent nailbed and because the cells had small monomorphic nuclei. Ex vivo CM could also be useful for melanonychia striata because the in vivo device used directly on the nail matrix performs well at this special site.[60] Debarbieux et al. also used the in vivo device in reflectance mode for the ex vivo examination of nail biopsies of pigmented subungual melanoma in a series of eight cases.[61] However, this procedure has limitations in that the specimen is not fixed, tends to move, and is difficult to orientate when using the ex vivo device. In addition, no staining method in RCM mode was used in that study. In future, the use of immunostaining for FCM could improve the accuracy of this method in melanocytic lesions of the nail.

Dermatofibrosarcoma Protuberans. Lamberti et al. reported the examination of four cases of dermatofibrosarcoma protuberans with ex vivo CM (Figure 9).[41] They showed a strong correlation among CM and histology, which suggests that this technique could be used to assess tumour surgical margins. Further studies should be performed in order to evaluate the ex vivo sensitivity of CM to detect tumour cells of dermatofibrosarcoma protuberans and to compare ex vivo CM with micrographic Mohs surgery, which was not performed in their series.

Figure 9.

Dermatofibrosarcoma protuberans scanned with the VivaScope® 2500M-G4, stained with acetic acid and acridine orange. (a, b) Conventional haematoxylin and eosin (HE) histology showing a characteristic storiform pattern. (c) Fusion ex vivo confocal microscopy with acetic acid and acridine orange: spindle-shaped cells with a storiform pattern (upper side) infiltrating the adipose tissue in the subcutaneous tissue (lower side). Adipose tissue lobules are seen as black round spaces in the image (asterisks). (d) Fluorescence mode with acridine orange stain: bright nuclei of the tumour are seen in green. (e) Reflectance mode: fusiform cellular distribution, nuclei and stroma with collagen fibres are well recognized due to the stain with acetic acid. Images 'd' and 'e' are complementary, as seen in the fusion image.

Syringomatous Carcinoma. Ex vivo CM in the fluorescent mode with acridine orange has been performed in only two syringomatous carcinomas.[62] They appeared as highly fluorescent neoplastic cords of monomorphous cells in the dermis.

Ex Vivo Confocal Microscopy and Infections

Ex vivo CM has the great advantage over conventional microscopy in that the sample is left intact, with the subsequent possibility of localizing an infectious agent directly in the whole tissue. This could be particularly useful for the fast and precise identification of a fungus in the case of a deep fungal infection such as mucormycosis or hair dermathophytes.[63,64]

As with in vivo CM, ex vivo CM can show a viral cytopathic effect.[65,66]Ex vivo CM has also been used successfully in fluorescence mode with anti-herpes simplex virus (HSV)1 antibodies coupled with fluorescein isothiocyanate for the identification of HSV1 from the roof of six vesicles from three different patients.[65] This application is an example of how this device has the potential to identify any pathogen disposing of specific antibodies conjugated with any fluorescent agent that can be excited by a wavelength of 488 nm or 658 nm. Comparison studies with standard methods such as the Tzanck test, histopathology or polymerase chain reaction for HSV infection have not been reported.

Other Applications in Dermatology

As ex vivo CM may be used to perform rapid cutaneous histology at the bedside, multiple applications in general dermatology can be presumed. In a recent publication Cinotti et al. described the identification of skin fillers.[67]

Bertoni et al.[68] identified key features with ex vivo FCM for the differential diagnoses of several cutaneous inflammatory diseases: psoriasis, eczema, lichen planus and discoid lupus erythematosus. In that retrospective study, the authors used ex vivo FCM and histological evaluations for correlation. Ex vivo FCM enabled the distinction of the main inflammatory features in most cases, providing a substantial concordance with histopathological diagnoses. Preliminary results in this indication suggest that dermatologists may be able to interpret ex vivo FCM images satisfactorily for correct real-time diagnoses.

Ex vivo CLSM was used by Bağcı et al. in cutaneous vasculitis in 49 patients, and they compared its diagnostic accuracy with that of direct immunofluorescence microscopy.[37] Eighty-two sections with relevant direct immunofluorescence microscopy findings were examined using ex vivo CLSM following staining with fluorescent-labelled IgG, IgM, IgA, C3 and fibrinogen antibodies. Direct immunofluorescence microscopy showed immunoreactivity of vessels with IgG, IgM, IgA, C3 and fibrinogen in 2%, 50%, 12%, 59% and 45% of the patients, respectively. Ex vivo CLSM detected positive vessels with the same antibodies in 2%, 39%, 8%, 43% and 37% of the patients, respectively. The detection rate of positive superficial dermal vessels was significantly higher with direct immunofluorescence microscopy than with ex vivo CLSM (P < 0·05). However, ex vivo CLSM identified positive deep dermal vessels more frequently than standard microscopy. The authors concluded that the two methods showed comparable performance in diagnosing vasculitis. The same authors in another study reported the results of immunofluorescence staining in bullous pemphigoid using the reflectance and fluorescence modes following staining with fluorescent-labelled IgG and C3 antibodies.[36]

Ex Vivo Confocal Microscopy in Other Tumours

Ex vivo CM has the potential for incorporation into surgical pathology practice in other tumours. For the field of surgical pathology the potential applications of ex vivo CM include rapid bedside tissue qualification of specimens, such as intraoperative assessment of margins of surgical resections, endoscopic biopsies or core needle biopsies, and rapid examination of any small fragments that are sent by the surgeons for frozen-section processing. It can also be used to identify tissue representative of the lesion for biobanking sampling. Ex vivo CM has been described in other tumours. FCM has been previously applied to different surgical settings, such as breast, lymph node, thyroid, cervix, oral mucosa, lung, prostate, urinary bladder, oesophagus, stomach, liver and colon.[69–71]

In a study in 2019, Krishnamurthy et al.[71] imaged 118 tissue fragments obtained from 40 breast, 23 lung, 39 kidney and 16 liver surgical excision specimens of normal tissue and benign and malignant tumours. They acquired CFM images in 2–3 min and achieved a sensitivity of 95·5%, specificity of 97·3%, positive predictive value of 95·5% and negative predictive value of 97·3%. In the same year, a first study on prostatic tissue in clinical practice reported the application of ex vivo CFM using fusion images of RCM and FCM to unfixed tissue specimens from non-neoplastic and prostatic adenocarcinoma compared with the histopathological diagnosis.[72] In that study Puliatti et al. enrolled 13 patients and analysed 89 specimens from locally localized prostate cancer extracted from punch biopsies. They found an overall substantial diagnostic agreement between FCM and histopathological diagnoses, with 91% correct diagnosis (κ = 0·75), 83·3% sensitivity and 93·5% specificity. They concluded that this technique may potentially be used for intraoperative pathological specimen analysis.