Immunofluorescence and confocal microscopy for ex‐vivo diagnosis of melanocytic and non‐melanocytic skin tumors: A pilot study

Background Ex‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of t...

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Vydáno v:	Journal of biophotonics Ročník 11; číslo 3
Hlavní autoři:	Hartmann, Daniela, Krammer, Sebastian, Vural, Secil, Bachmann, Mario Raphael, Ruini, Cristel, Sárdy, Miklós, Ruzicka, Thomas, Berking, Carola, von Braunmühl, Tanja
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	Weinheim WILEY‐VCH Verlag GmbH & Co. KGaA 01.03.2018 Wiley Subscription Services, Inc
Témata:	Antibodies basal cell carcinoma Confocal microscopy Diagnosis diagnostics in dermatology Dilution Fluorescence fluorescence confocal microscopy Histopathology Immunofluorescence Immunohistochemistry Lasers melanoma metastasis Microscopy Protocol Reflectance Scanning microscopy Skin Skin cancer skin surgery skin tumors Tumors diagnostics in dermatology metastasis fluorescence confocal microscopy basal cell carcinoma melanoma skin surgery skin tumors immunofluorescence
ISSN:	1864-063X, 1864-0648, 1864-0648
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Abstract	Background Ex‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex‐vivo CLSM. Methods The tissue probes from various skin tumors were stained with FITC‐labeled S‐100A10, Melan‐A and anti‐Ber‐EP4 antibodies before examination with ex‐vivo CLSM in the fluorescence and reflectance modes. Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex‐vivo CLSM. Results S100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline [PBS], without blocking agent). Melan A immunostaining was positive in 66.7%, the best dilution was 1:500 and protocol B (PBS, without blocking agent) was the most suitable. Ber‐EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results. Conclusion The use of fluorescent‐labeled antibodies in ex‐vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors. Ex‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) enables intraoperative tissue diagnostics on a level equivalent to histopathology. Tissue probes from melanoma, melanocytic nevus, basal and squamous cell carcinoma were stained with FITC‐labeled S100, Melan A and Ber‐EP4 antibodies. S100 immunostaining was successful in 55.6%, Melan A in 66.7% and Ber‐EP4 in 85.7%. In combination with the application of fluorescent dyes, ex‐vivo CLSM offers unique possibilities of rapid tissue examination.
AbstractList	BackgroundEx‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex‐vivo CLSM.MethodsThe tissue probes from various skin tumors were stained with FITC‐labeled S‐100A10, Melan‐A and anti‐Ber‐EP4 antibodies before examination with ex‐vivo CLSM in the fluorescence and reflectance modes. Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex‐vivo CLSM.ResultsS100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline [PBS], without blocking agent). Melan A immunostaining was positive in 66.7%, the best dilution was 1:500 and protocol B (PBS, without blocking agent) was the most suitable. Ber‐EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results.ConclusionThe use of fluorescent‐labeled antibodies in ex‐vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors. Ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex-vivo CLSM.BACKGROUNDEx-vivo confocal laser scanning microscopy (ex-vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex-vivo CLSM.The tissue probes from various skin tumors were stained with FITC-labeled S-100A10, Melan-A and anti-Ber-EP4 antibodies before examination with ex-vivo CLSM in the fluorescence and reflectance modes. Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex-vivo CLSM.METHODSThe tissue probes from various skin tumors were stained with FITC-labeled S-100A10, Melan-A and anti-Ber-EP4 antibodies before examination with ex-vivo CLSM in the fluorescence and reflectance modes. Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex-vivo CLSM.S100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline [PBS], without blocking agent). Melan A immunostaining was positive in 66.7%, the best dilution was 1:500 and protocol B (PBS, without blocking agent) was the most suitable. Ber-EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results.RESULTSS100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline [PBS], without blocking agent). Melan A immunostaining was positive in 66.7%, the best dilution was 1:500 and protocol B (PBS, without blocking agent) was the most suitable. Ber-EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results.The use of fluorescent-labeled antibodies in ex-vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors.CONCLUSIONThe use of fluorescent-labeled antibodies in ex-vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors. Ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex-vivo CLSM. The tissue probes from various skin tumors were stained with FITC-labeled S-100A10, Melan-A and anti-Ber-EP4 antibodies before examination with ex-vivo CLSM in the fluorescence and reflectance modes. Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex-vivo CLSM. S100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline [PBS], without blocking agent). Melan A immunostaining was positive in 66.7%, the best dilution was 1:500 and protocol B (PBS, without blocking agent) was the most suitable. Ber-EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results. The use of fluorescent-labeled antibodies in ex-vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors. Background Ex‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex‐vivo CLSM. Methods The tissue probes from various skin tumors were stained with FITC‐labeled S‐100A10, Melan‐A and anti‐Ber‐EP4 antibodies before examination with ex‐vivo CLSM in the fluorescence and reflectance modes. Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex‐vivo CLSM. Results S100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline [PBS], without blocking agent). Melan A immunostaining was positive in 66.7%, the best dilution was 1:500 and protocol B (PBS, without blocking agent) was the most suitable. Ber‐EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results. Conclusion The use of fluorescent‐labeled antibodies in ex‐vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors. Ex‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) enables intraoperative tissue diagnostics on a level equivalent to histopathology. Tissue probes from melanoma, melanocytic nevus, basal and squamous cell carcinoma were stained with FITC‐labeled S100, Melan A and Ber‐EP4 antibodies. S100 immunostaining was successful in 55.6%, Melan A in 66.7% and Ber‐EP4 in 85.7%. In combination with the application of fluorescent dyes, ex‐vivo CLSM offers unique possibilities of rapid tissue examination.
Author	Vural, Secil Bachmann, Mario Raphael Ruini, Cristel Berking, Carola von Braunmühl, Tanja Ruzicka, Thomas Hartmann, Daniela Sárdy, Miklós Krammer, Sebastian
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Keywords	diagnostics in dermatology metastasis fluorescence confocal microscopy basal cell carcinoma melanoma skin surgery skin tumors immunofluorescence
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Snippet	Background Ex‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination... Ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination... BackgroundEx‐vivo confocal laser scanning microscopy (ex‐vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination...
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SubjectTerms	Antibodies basal cell carcinoma Confocal microscopy Diagnosis diagnostics in dermatology Dilution Fluorescence fluorescence confocal microscopy Histopathology Immunofluorescence Immunohistochemistry Lasers melanoma metastasis Microscopy Protocol Reflectance Scanning microscopy Skin Skin cancer skin surgery skin tumors Tumors
Title	Immunofluorescence and confocal microscopy for ex‐vivo diagnosis of melanocytic and non‐melanocytic skin tumors: A pilot study
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