Cell Biochem Biophys DOI 10.1007/s12013-015-0625-5
ORIGINAL PAPER
Clinical Application of Confocal Laser Scanning Microscopy for Atypical Dermatoses Jie Ma • Xiaoyan Zhang • Yongjing Lv • Chenguang Zhao • Qing Li • Xueping Yang Jianbin Zhao
•
Ó Springer Science+Business Media New York 2015
Abstract Confocal laser scanning microscopy (CLSM) is a novel non-invasive imaging technique for in vivo evaluation of cutaneous lesions at near-histologic resolution. The applicability of CLSM for various neoplastic and inflammatory skin diseases has been shown. The objective of the study is to utilize the CLSM for the differential diagnosis of atypical dermatoses. Six patients with atypical clinical manifestation were detected by CLSM. In spite of non-typical clinical manifestations, CLSM can still detect their characteristic pathological changes and help differentiate them from other diseases that are liable to be confused in clinical practice. CLSM deserves wide application in clinical practice as it boasts of easy and convenient operation, broad application, no pains or traumas for patients, rapid examination reports, as well as it can relieve patient’s distress by avoiding the traumas resulting from histopathological biopsy. Keywords Confocal laser scanning microscopy Atypical Dermatoses
Introduction Confocal laser scanning microscopy (CLSM), utilizing the new generation of reflectance laser confocal scanning microscope, represents a novel computer-assisted diagnostic technique for imaging of skin lesions. Images of conventional pathological cells and sub cellular structures,
J. Ma X. Zhang Y. Lv C. Zhao Q. Li X. Yang J. Zhao (&) Department of Dermatology, The First People’s Hospital of Xuzhou, Xuzhou, Jiangsu, China e-mail: [email protected]
available for non-invasive diagnosis, differential diagnosis, and curative effect of dermatoses can be observed by CLSM in real time. Favorable results that we achieved with the application of CLSM for auxiliary diagnosis of dermatoses with atypical clinical manifestation are reported here.
Case 1 The patient was a 35-year-old female with erythema and papules on both the trunk and the limbs as well as with symptoms of itching for 10 days. She was previously diagnosed with eczema and prescribed antihistamines with poor improvement after the treatment. When she came to our hospital for the treatment, dermatologic examination revealed visible sporadic erythema and papules of maize to a rice grain size, together with excoriation (Fig. 1a), on both the trunk and the limbs. CLSM examination showed parakeratosis (red arrow in Fig. 1b), visible Munro microabscess (green arrow in Fig. 1c), psoriasiform hyperplasia in epidermis, upward dermal papillae, capillary tortuosity, extension and congestion, variable perivascular inflammatory cell infiltration (green arrow in Fig. 1d). Psoriasis was diagnosed taking both images and clinical manifestations into consideration. Clinical manifestations of this case are easy to be confused with that of allergic dermatitis, such as dermatitis and eczema, so misdiagnosis is liable to occur in clinical practice. After observation and analysis of histopathology of allergic dermatitis using CLSM technique, features of such diseases can be concluded as parakeratosis, prickle layer spongiosis, vascular dilation of shallow-layer dermis, perivascular inflammatory cell infiltration, and other nonspecific inflammatory changes. These features are clearly
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Dermatologic examination showed a circular erythema measuring about 1 cm in diameter and a strip-shaped erythema of 1 9 3 cm2 was visible on his left temporal (Fig. 2a). CLSM inspection showed epidermal hyperplasia and hypertrophy, liquefaction of basal cells (red arrow in Fig. 2b) and numerous melanophages, and inflammatory cell infiltration (red arrow in Fig. 2c) in papillary and upper layer of dermis. Therefore, combined consideration to images and clinical manifestations resulted in the diagnosis of lichen planus.
A
B
C
A
D
B
Fig. 1 a Erythema and papules on the trunk and the lower limbs. b– d CLSM images (Color figure online)
different from characteristic changes of psoriasis, including Munro microabscess, psoriasiform hyperplasia in epidermis, upward dermal papillae, capillary tortuosity, extension and congestion, and variable perivascular inflammatory cell infiltration. Previous differential diagnosis of psoriasis and such diseases were mainly based on skin histopathological examination, while poor compliance of patient often led to misdiagnosis. Utilization of CLSM, non-invasive and rapid, in differential diagnosis of psoriasis and other allergic diseases like dermatitis and eczema has significantly improved the patient compliance and diagnostic accuracy.
C
Case 2 The patient was a 15-year-old male with erythema on left temporal for 2 years. Local hospital provided him with treatment for dermatitis (specific medication unknown), no improvement happened, and the patient discontinued treatment and came to our hospital for exact diagnosis.
123
Fig. 2 a Erythema on left temporal. b, c CLSM images (Color figure online)
Cell Biochem Biophys
It was difficult to diagnose based only on the non-typical clinical manifestations of case 2 patient. However, CLSM inspection revealed epidermal hyperplasia and hypertrophy, liquefaction of basal cells and numerous melanophages, and inflammatory cell infiltration in papillary and upper layer of dermis etc. Thus, combined consideration of clinical behaviors and these precisely characteristic pathological changes in lichen planus facilitate clinical diagnosis. For clinically atypical lichen planus lesions, differential diagnosis of eczema, psoriasis, lichen striatus, lichen planus-like keratosis, and chronic cutaneous lupus erythematosus deserves attention. Distinctions between the clinical manifestations of above dermatoses are occasionally insufficient but there are still some characteristic pathological changes which can be inspected and identified by CLSM. Eczema, as illustrated in case 1, associated with typical CLSM evidence like parakeratosis, prickle layer spongiosis, even blisters can be seen, vascular dilation of shallow-layer dermis, perivascular inflammatory cell infiltration, and other nonspecific inflammatory changes. Psoriasis, as illustrated in case 1, associated with typical CLSM evidence like parakeratosis, Munro microabscess, psoriasiform hyperplasia in epidermis, upward dermal papillae, capillary tortuosity, extension and congestion, thus can be differentiated from lichen planus. The main difference between lichen striatus is the characteristic changes, such as liquefaction of basal cells in skin lesions, dermal papillae pigment cells, and mononucleate inflammatory cell infiltration observed in CLSM, can only observed in focal areas, and then combined with clinical behaviors, will facilitate diagnosis. Clinical manifestation and histopathology of Lichen planus-like keratosis and Lichen planus are very similar. But histopathologically visible parakeratosis in the CLSM of the Lichen planuslike keratosis will contribute to identification. Liquefaction of basal cells also exist in chronic cutaneous lupus erythematosus, which however is associated with expansion of follicular orifice, keratotic plugs in the openings of the hair follicles, and prickle cell layer atrophy as well. Therefore, CLSM can also facilitate differential diagnosis of chronic cutaneous lupus erythematosus and lichen planus.
Case 3 The patient was a 6-year-old female with white patches on forehead for 1 month. Her family took her to the doctor due to increasing skin lesions. Dermatologic examination showed irregularly shaped depigmentation macule with illdefined border on forehead (Fig. 3a). CLSM inspection showed clear absence of pigmented ring of basal skin layer in the white patches areas (red arrow in Fig. 3c) compared with surrounding normal skin (red arrow in Fig. 3b). So
A
B
C
Fig. 3 a White patches on forehead. b, c CLSM images (Color figure online)
vitiligo was diagnosed out of combined consideration of images and clinical manifestations. This patient suffers from hypopigmentation disorder which clinically is liable to be confused with pityriasis alba and achromic nevus. But CLSM inspection of pityriasis alba usually shows benign focal spongiosis in epidermal prickle cell layer, slight loss and uneven distribution of pigment in basal layer, sparse inflammatory cell infiltration in papillary and upper layer of dermis. CLSM inspection of achronic naevus shows pigment loss in epidermal basal cell layer, pigmented ring of basal skin layer still exists and the pigment loss is distributed relatively even. Both of these
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cases show pigment loss but not absence, obviously different from vitiligo, which shows total or local pigment absence. So this case was diagnosed as vitiligo combined with images and clinical practice.
Case 4 The patient was a 3-year-old female with papules on her forehead for 3 months, without apparent subjective symptoms in the skin lesion, and she came to our hospital for definite diagnosis. Dermatological examination illustrated two visible cuticolor moderately hard papules of maize size on her forehead (Fig. 4a, b). CLSM inspection shows skin lesion with apparent epidermal hyperplasia, stretching down to the dermis, crateriform opening on the surface
with many small moderately refractive bodies inside (Fig. 4c). Consequently, molluscum contagiosum was diagnosed out of combined consideration of images and clinical manifestations. With non-typical clinical manifestation, this case deserves differential diagnosis of verruca vulgaris and verrucae planae. CLSM inspection of verruca vulgaris shows hyperkeratosis, acanthosis, and papillomatous hyperplasia in skin lesions, and visible vacuolization cells in upper and granular cell layers. CLSM examination of verrucae planae shows characteristic arrangement of granular layer and upper prickle layer cells as in a roughly concentric circle like a bouquet of roses. CLSM evaluation of this patient shows evidence of typical molluscum bodies, while histopathologically consistent with molluscum contagiosum, distinct from the other two in spite of similar clinical manifestations.
A Case 5
B
The patient was a 33-year-old female with brown papules on her neck for 3 years. Local hospital diagnosed her with verrucae plana and prescribed medication. The treatment was poorly effective and skin lesions started to expand recently, so she came to our hospital. Dermatologic examination showed visible sporadic brown papules size of a needle tip to a rice grain on her neck (red arrow in Fig. 5a, b). CLSM inspection showed hyperkeratosis, acanthosis, increased pigmentation of the basal layer (red arrow in Fig. 5c); papillomatosis on the surface, visible keratin
A
B
C
D
C
Fig. 4 a, b Papules on forehead. c CLSM images (Color figure online)
123
Fig. 5 a, b Brown papules on the neck. c, d CLSM images (Color figure online)
Cell Biochem Biophys
pseudocyst, and visible gyriform structure under CLSM scan (red arrow in Fig. 5d). Subsequently, seborrheic keratosis was diagnosed based on combined consideration of images and clinical manifestations. Manifested by brown flat papules, it was difficult to diagnose this patient with verrucae planae or seborrheic keratosis. However, these two diseases are distinct in biological behaviors, treatment, and prognosis, so exact identification of these is of great significance. As mentioned earlier, CLSM inspection of verrucae planae shows characteristic arrangement of granular layer and upper prickle layer cells as in a roughly concentric circle like a bouquet of roses. While CLSM inspection of seborrheic keratosis shows hyperkeratosis, acanthosis, increased pigmentation of the basal layer, papillomatosis on the surface, visible keratin pseudocyst, and visible gyriform structure under CLSM scan. Differentiation of the two diseases, while clinically difficult, is easier to identify by CLSM.
A
B
C
D
E
Case 6 The patient was a 75-year-old female with black facial papules for 4 years, and she went to see a doctor as recently the skin lesions apparently started to get bigger. Dermatologic examination showed a visible black papule of a soybean size under the right medial canthus, and visible pitting on the lesion surface. CLSM inspection showed numerous pigment clumps and inflammatory cell infiltration at the dermal–epidermal interface (red arrow in Fig. 6b, c); tightly packed visible nodular structure in dermis, representing the formation of tumor mass, and monomorphic tumor cells arranged in an elongated spoke shape with poorly refractive nucleus (green arrow in Fig. 6d); rich existence of blood vessels with thickening lumen finds in skin lesions, and rapid movement of white cells along lumen and perivascular inflammatory infiltration (yellow arrow in Fig. 6e). Therefore, basal cell carcinoma was diagnosed out of combined consideration of images and clinical manifestations. Clinically manifested by black papules, this case deserves differentiation from following diseases: pigmented nevus, seborrheic keratosis, and malignant melanoma. Pigmented nevus shows the same manifestations in skin histopathological and CLSM examination—intradermal nevus cell aggregates at the dermal or dermal–epidermal interface. CLSM inspections of seborrheic keratosis show acanthosis, papillomatous hyperplasia of epidermis, visible keratin pseudocyst, and gyriform structure under CLSM scan. Typical pathological characteristics of malignant melanoma are non-typical number increase of melanocytes at the dermal–epidermal interface, invasion of oncocytes to epidermis and dermis, and diphase differentiation of oncocytes, including epithelial cells and spindled cells.
Fig. 6 a Black papule of a soybean size under the right medial canthus. b–e CLSM images (Color figure online)
Nucleus shows obvious heteromorphism and frequent spallation. CLSM inspection shows abnormality of epidermal structure; diffused distribution of relatively big and bright round or oval cells (identified histologically as Paget-like cells) in superficial epidermis; abnormal melanocytes in basal layer; disorder at the dermal–epidermal interface and rich existence of refractive particles with mononucleate cell infiltration in dermal papilla and extension and congestion of vessels. In CLSM images, both basal cell carcinoma and malignant melanoma show evidence of dense and highly refractive dendritic cells in basal layer, but the former disease shows no signs of Paget-like cell structure, and the main part of its carcinoma mass was the elongated basal layer cells. CLSM was utilized in the early diagnosis of the skin lesion in this case and the determination of excision borders.
Discussion CLSM, utilizing the new generation of reflectance laser confocal scanning microscope, represents a novel
123
Cell Biochem Biophys
computer-assisted diagnostic technique used for imaging of skin lesions [1]. At present, though serving as the golden rule for dermatoses diagnosis, histopathological examination still appears daunting to patients for the traumas resulted from biopsy. CLSM has appeared to overcome this disadvantage, as it is in situ and in vivo and offers real-time dynamic images with higher resolution of epidermis and dermal layer. CLSM has been utilized by several scholars to observe and measure the normal skin structure, diagnosis of pigmented diseases, inflammatory diseases and skin tumors, as well as to assist in treatment of malignant and premalignant skin lesions by defining borders of tumor tissue. All applications have produced promising results [2–5]. Skin lesions involved in the six cases described here cover inflammatory disease, pigmentary lesions, viral disease, and skin tumors. In spite of non-typical clinical manifestations, CLSM can still detect their characteristic pathological changes and help differentiate them from other diseases that are liable to be confusing in clinical practice. CLSM, as a novel non-invasive dermatologic examination method, deserves wide application in clinical practice as it boasts of easy and convenient operation, broad application, no pains or traumas for patients, rapid examination reports, as well as it can relieve patient’s
123
distress by avoiding the traumas resulting histopathological biopsy and saves diagnosis time.
from
Acknowledgments This study was supported by ‘‘333 program’’ of Jiangsu Province and ‘‘Six talent peaks project’’ of Jiangsu Province.
References 1. Liu, H., Zheng, Z., Ren, Q. (2006). Introduction and application of confocal laser scanning microscopy. Chinese Journal of Dermatology, 39(10), 616–619. 2. Rajadhyaksha, M., Grossman, M., Esterowitz, D., et al. (1995). In vivo confocal scanning laser microscopy of human skin: Melanin provides strong contrast. Journal of Investigative Dermatology, 104(6), 946–952. 3. Busam, K. J., Charles, C., Lee, G., et al. (2001). Morphologic features of melanocytes, pigmented keratinocytes, and melanophages by in vivo confocal scanning laser microscopy. Modern Pathology, 14(9), 862–868. 4. Gerger, A., Koller, S., Kern, T., et al. (2005). Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors. Journal of Investigative Dermatology, 124(3), 493–498. 5. Agero, A. L., Busam, K. J., Benvenuto-Andrade, C., et al. (2006). Reflectance confocal microscopy of pigmented basal cell carcinoma. Journal of American Academy of Dermatology, 54(4), 638–643.
ORIGINAL PAPER
Clinical Application of Confocal Laser Scanning Microscopy for Atypical Dermatoses Jie Ma • Xiaoyan Zhang • Yongjing Lv • Chenguang Zhao • Qing Li • Xueping Yang Jianbin Zhao
•
Ó Springer Science+Business Media New York 2015
Abstract Confocal laser scanning microscopy (CLSM) is a novel non-invasive imaging technique for in vivo evaluation of cutaneous lesions at near-histologic resolution. The applicability of CLSM for various neoplastic and inflammatory skin diseases has been shown. The objective of the study is to utilize the CLSM for the differential diagnosis of atypical dermatoses. Six patients with atypical clinical manifestation were detected by CLSM. In spite of non-typical clinical manifestations, CLSM can still detect their characteristic pathological changes and help differentiate them from other diseases that are liable to be confused in clinical practice. CLSM deserves wide application in clinical practice as it boasts of easy and convenient operation, broad application, no pains or traumas for patients, rapid examination reports, as well as it can relieve patient’s distress by avoiding the traumas resulting from histopathological biopsy. Keywords Confocal laser scanning microscopy Atypical Dermatoses
Introduction Confocal laser scanning microscopy (CLSM), utilizing the new generation of reflectance laser confocal scanning microscope, represents a novel computer-assisted diagnostic technique for imaging of skin lesions. Images of conventional pathological cells and sub cellular structures,
J. Ma X. Zhang Y. Lv C. Zhao Q. Li X. Yang J. Zhao (&) Department of Dermatology, The First People’s Hospital of Xuzhou, Xuzhou, Jiangsu, China e-mail: [email protected]
available for non-invasive diagnosis, differential diagnosis, and curative effect of dermatoses can be observed by CLSM in real time. Favorable results that we achieved with the application of CLSM for auxiliary diagnosis of dermatoses with atypical clinical manifestation are reported here.
Case 1 The patient was a 35-year-old female with erythema and papules on both the trunk and the limbs as well as with symptoms of itching for 10 days. She was previously diagnosed with eczema and prescribed antihistamines with poor improvement after the treatment. When she came to our hospital for the treatment, dermatologic examination revealed visible sporadic erythema and papules of maize to a rice grain size, together with excoriation (Fig. 1a), on both the trunk and the limbs. CLSM examination showed parakeratosis (red arrow in Fig. 1b), visible Munro microabscess (green arrow in Fig. 1c), psoriasiform hyperplasia in epidermis, upward dermal papillae, capillary tortuosity, extension and congestion, variable perivascular inflammatory cell infiltration (green arrow in Fig. 1d). Psoriasis was diagnosed taking both images and clinical manifestations into consideration. Clinical manifestations of this case are easy to be confused with that of allergic dermatitis, such as dermatitis and eczema, so misdiagnosis is liable to occur in clinical practice. After observation and analysis of histopathology of allergic dermatitis using CLSM technique, features of such diseases can be concluded as parakeratosis, prickle layer spongiosis, vascular dilation of shallow-layer dermis, perivascular inflammatory cell infiltration, and other nonspecific inflammatory changes. These features are clearly
123
Cell Biochem Biophys
Dermatologic examination showed a circular erythema measuring about 1 cm in diameter and a strip-shaped erythema of 1 9 3 cm2 was visible on his left temporal (Fig. 2a). CLSM inspection showed epidermal hyperplasia and hypertrophy, liquefaction of basal cells (red arrow in Fig. 2b) and numerous melanophages, and inflammatory cell infiltration (red arrow in Fig. 2c) in papillary and upper layer of dermis. Therefore, combined consideration to images and clinical manifestations resulted in the diagnosis of lichen planus.
A
B
C
A
D
B
Fig. 1 a Erythema and papules on the trunk and the lower limbs. b– d CLSM images (Color figure online)
different from characteristic changes of psoriasis, including Munro microabscess, psoriasiform hyperplasia in epidermis, upward dermal papillae, capillary tortuosity, extension and congestion, and variable perivascular inflammatory cell infiltration. Previous differential diagnosis of psoriasis and such diseases were mainly based on skin histopathological examination, while poor compliance of patient often led to misdiagnosis. Utilization of CLSM, non-invasive and rapid, in differential diagnosis of psoriasis and other allergic diseases like dermatitis and eczema has significantly improved the patient compliance and diagnostic accuracy.
C
Case 2 The patient was a 15-year-old male with erythema on left temporal for 2 years. Local hospital provided him with treatment for dermatitis (specific medication unknown), no improvement happened, and the patient discontinued treatment and came to our hospital for exact diagnosis.
123
Fig. 2 a Erythema on left temporal. b, c CLSM images (Color figure online)
Cell Biochem Biophys
It was difficult to diagnose based only on the non-typical clinical manifestations of case 2 patient. However, CLSM inspection revealed epidermal hyperplasia and hypertrophy, liquefaction of basal cells and numerous melanophages, and inflammatory cell infiltration in papillary and upper layer of dermis etc. Thus, combined consideration of clinical behaviors and these precisely characteristic pathological changes in lichen planus facilitate clinical diagnosis. For clinically atypical lichen planus lesions, differential diagnosis of eczema, psoriasis, lichen striatus, lichen planus-like keratosis, and chronic cutaneous lupus erythematosus deserves attention. Distinctions between the clinical manifestations of above dermatoses are occasionally insufficient but there are still some characteristic pathological changes which can be inspected and identified by CLSM. Eczema, as illustrated in case 1, associated with typical CLSM evidence like parakeratosis, prickle layer spongiosis, even blisters can be seen, vascular dilation of shallow-layer dermis, perivascular inflammatory cell infiltration, and other nonspecific inflammatory changes. Psoriasis, as illustrated in case 1, associated with typical CLSM evidence like parakeratosis, Munro microabscess, psoriasiform hyperplasia in epidermis, upward dermal papillae, capillary tortuosity, extension and congestion, thus can be differentiated from lichen planus. The main difference between lichen striatus is the characteristic changes, such as liquefaction of basal cells in skin lesions, dermal papillae pigment cells, and mononucleate inflammatory cell infiltration observed in CLSM, can only observed in focal areas, and then combined with clinical behaviors, will facilitate diagnosis. Clinical manifestation and histopathology of Lichen planus-like keratosis and Lichen planus are very similar. But histopathologically visible parakeratosis in the CLSM of the Lichen planuslike keratosis will contribute to identification. Liquefaction of basal cells also exist in chronic cutaneous lupus erythematosus, which however is associated with expansion of follicular orifice, keratotic plugs in the openings of the hair follicles, and prickle cell layer atrophy as well. Therefore, CLSM can also facilitate differential diagnosis of chronic cutaneous lupus erythematosus and lichen planus.
Case 3 The patient was a 6-year-old female with white patches on forehead for 1 month. Her family took her to the doctor due to increasing skin lesions. Dermatologic examination showed irregularly shaped depigmentation macule with illdefined border on forehead (Fig. 3a). CLSM inspection showed clear absence of pigmented ring of basal skin layer in the white patches areas (red arrow in Fig. 3c) compared with surrounding normal skin (red arrow in Fig. 3b). So
A
B
C
Fig. 3 a White patches on forehead. b, c CLSM images (Color figure online)
vitiligo was diagnosed out of combined consideration of images and clinical manifestations. This patient suffers from hypopigmentation disorder which clinically is liable to be confused with pityriasis alba and achromic nevus. But CLSM inspection of pityriasis alba usually shows benign focal spongiosis in epidermal prickle cell layer, slight loss and uneven distribution of pigment in basal layer, sparse inflammatory cell infiltration in papillary and upper layer of dermis. CLSM inspection of achronic naevus shows pigment loss in epidermal basal cell layer, pigmented ring of basal skin layer still exists and the pigment loss is distributed relatively even. Both of these
123
Cell Biochem Biophys
cases show pigment loss but not absence, obviously different from vitiligo, which shows total or local pigment absence. So this case was diagnosed as vitiligo combined with images and clinical practice.
Case 4 The patient was a 3-year-old female with papules on her forehead for 3 months, without apparent subjective symptoms in the skin lesion, and she came to our hospital for definite diagnosis. Dermatological examination illustrated two visible cuticolor moderately hard papules of maize size on her forehead (Fig. 4a, b). CLSM inspection shows skin lesion with apparent epidermal hyperplasia, stretching down to the dermis, crateriform opening on the surface
with many small moderately refractive bodies inside (Fig. 4c). Consequently, molluscum contagiosum was diagnosed out of combined consideration of images and clinical manifestations. With non-typical clinical manifestation, this case deserves differential diagnosis of verruca vulgaris and verrucae planae. CLSM inspection of verruca vulgaris shows hyperkeratosis, acanthosis, and papillomatous hyperplasia in skin lesions, and visible vacuolization cells in upper and granular cell layers. CLSM examination of verrucae planae shows characteristic arrangement of granular layer and upper prickle layer cells as in a roughly concentric circle like a bouquet of roses. CLSM evaluation of this patient shows evidence of typical molluscum bodies, while histopathologically consistent with molluscum contagiosum, distinct from the other two in spite of similar clinical manifestations.
A Case 5
B
The patient was a 33-year-old female with brown papules on her neck for 3 years. Local hospital diagnosed her with verrucae plana and prescribed medication. The treatment was poorly effective and skin lesions started to expand recently, so she came to our hospital. Dermatologic examination showed visible sporadic brown papules size of a needle tip to a rice grain on her neck (red arrow in Fig. 5a, b). CLSM inspection showed hyperkeratosis, acanthosis, increased pigmentation of the basal layer (red arrow in Fig. 5c); papillomatosis on the surface, visible keratin
A
B
C
D
C
Fig. 4 a, b Papules on forehead. c CLSM images (Color figure online)
123
Fig. 5 a, b Brown papules on the neck. c, d CLSM images (Color figure online)
Cell Biochem Biophys
pseudocyst, and visible gyriform structure under CLSM scan (red arrow in Fig. 5d). Subsequently, seborrheic keratosis was diagnosed based on combined consideration of images and clinical manifestations. Manifested by brown flat papules, it was difficult to diagnose this patient with verrucae planae or seborrheic keratosis. However, these two diseases are distinct in biological behaviors, treatment, and prognosis, so exact identification of these is of great significance. As mentioned earlier, CLSM inspection of verrucae planae shows characteristic arrangement of granular layer and upper prickle layer cells as in a roughly concentric circle like a bouquet of roses. While CLSM inspection of seborrheic keratosis shows hyperkeratosis, acanthosis, increased pigmentation of the basal layer, papillomatosis on the surface, visible keratin pseudocyst, and visible gyriform structure under CLSM scan. Differentiation of the two diseases, while clinically difficult, is easier to identify by CLSM.
A
B
C
D
E
Case 6 The patient was a 75-year-old female with black facial papules for 4 years, and she went to see a doctor as recently the skin lesions apparently started to get bigger. Dermatologic examination showed a visible black papule of a soybean size under the right medial canthus, and visible pitting on the lesion surface. CLSM inspection showed numerous pigment clumps and inflammatory cell infiltration at the dermal–epidermal interface (red arrow in Fig. 6b, c); tightly packed visible nodular structure in dermis, representing the formation of tumor mass, and monomorphic tumor cells arranged in an elongated spoke shape with poorly refractive nucleus (green arrow in Fig. 6d); rich existence of blood vessels with thickening lumen finds in skin lesions, and rapid movement of white cells along lumen and perivascular inflammatory infiltration (yellow arrow in Fig. 6e). Therefore, basal cell carcinoma was diagnosed out of combined consideration of images and clinical manifestations. Clinically manifested by black papules, this case deserves differentiation from following diseases: pigmented nevus, seborrheic keratosis, and malignant melanoma. Pigmented nevus shows the same manifestations in skin histopathological and CLSM examination—intradermal nevus cell aggregates at the dermal or dermal–epidermal interface. CLSM inspections of seborrheic keratosis show acanthosis, papillomatous hyperplasia of epidermis, visible keratin pseudocyst, and gyriform structure under CLSM scan. Typical pathological characteristics of malignant melanoma are non-typical number increase of melanocytes at the dermal–epidermal interface, invasion of oncocytes to epidermis and dermis, and diphase differentiation of oncocytes, including epithelial cells and spindled cells.
Fig. 6 a Black papule of a soybean size under the right medial canthus. b–e CLSM images (Color figure online)
Nucleus shows obvious heteromorphism and frequent spallation. CLSM inspection shows abnormality of epidermal structure; diffused distribution of relatively big and bright round or oval cells (identified histologically as Paget-like cells) in superficial epidermis; abnormal melanocytes in basal layer; disorder at the dermal–epidermal interface and rich existence of refractive particles with mononucleate cell infiltration in dermal papilla and extension and congestion of vessels. In CLSM images, both basal cell carcinoma and malignant melanoma show evidence of dense and highly refractive dendritic cells in basal layer, but the former disease shows no signs of Paget-like cell structure, and the main part of its carcinoma mass was the elongated basal layer cells. CLSM was utilized in the early diagnosis of the skin lesion in this case and the determination of excision borders.
Discussion CLSM, utilizing the new generation of reflectance laser confocal scanning microscope, represents a novel
123
Cell Biochem Biophys
computer-assisted diagnostic technique used for imaging of skin lesions [1]. At present, though serving as the golden rule for dermatoses diagnosis, histopathological examination still appears daunting to patients for the traumas resulted from biopsy. CLSM has appeared to overcome this disadvantage, as it is in situ and in vivo and offers real-time dynamic images with higher resolution of epidermis and dermal layer. CLSM has been utilized by several scholars to observe and measure the normal skin structure, diagnosis of pigmented diseases, inflammatory diseases and skin tumors, as well as to assist in treatment of malignant and premalignant skin lesions by defining borders of tumor tissue. All applications have produced promising results [2–5]. Skin lesions involved in the six cases described here cover inflammatory disease, pigmentary lesions, viral disease, and skin tumors. In spite of non-typical clinical manifestations, CLSM can still detect their characteristic pathological changes and help differentiate them from other diseases that are liable to be confusing in clinical practice. CLSM, as a novel non-invasive dermatologic examination method, deserves wide application in clinical practice as it boasts of easy and convenient operation, broad application, no pains or traumas for patients, rapid examination reports, as well as it can relieve patient’s
123
distress by avoiding the traumas resulting histopathological biopsy and saves diagnosis time.
from
Acknowledgments This study was supported by ‘‘333 program’’ of Jiangsu Province and ‘‘Six talent peaks project’’ of Jiangsu Province.
References 1. Liu, H., Zheng, Z., Ren, Q. (2006). Introduction and application of confocal laser scanning microscopy. Chinese Journal of Dermatology, 39(10), 616–619. 2. Rajadhyaksha, M., Grossman, M., Esterowitz, D., et al. (1995). In vivo confocal scanning laser microscopy of human skin: Melanin provides strong contrast. Journal of Investigative Dermatology, 104(6), 946–952. 3. Busam, K. J., Charles, C., Lee, G., et al. (2001). Morphologic features of melanocytes, pigmented keratinocytes, and melanophages by in vivo confocal scanning laser microscopy. Modern Pathology, 14(9), 862–868. 4. Gerger, A., Koller, S., Kern, T., et al. (2005). Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors. Journal of Investigative Dermatology, 124(3), 493–498. 5. Agero, A. L., Busam, K. J., Benvenuto-Andrade, C., et al. (2006). Reflectance confocal microscopy of pigmented basal cell carcinoma. Journal of American Academy of Dermatology, 54(4), 638–643.
![[Confocal laser scanning microscopy].](/assets/img/hold.png)
