Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin

Vinzent Kevin Ortner; Aditi Sahu; Miguel Cordova; Kivanc Kose; Saud Aleissa; Christi Alessi-Fox; Merete Haedersdal; Milind Rajadhyaksha; Anthony Mario Rossi

doi:10.1002/jbio.202000207

Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin

Vinzent Kevin Ortner^*, Aditi Sahu, Miguel Cordova, Kivanc Kose, Saud Aleissa, Christi Alessi-Fox, Merete Haedersdal, Milind Rajadhyaksha, Anthony Mario Rossi

^*Corresponding author for this work

Research output: Contribution to journal › Journal article › Research › peer-review

6 Citations (Scopus)

19 Downloads (Pure)

Abstract

We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.

Original language	English
Article number	e202000207
Journal	journal of biophotonics
Volume	14
Issue number	4
ISSN	1864-063X
DOIs	https://doi.org/10.1002/jbio.202000207
Publication status	Published - 2021

Bibliographical note

Funding Information:
This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC's NIH/NCI Cancer Center Support Grant P30 CA008748.

Funding Information:
National Institute of Biomedical Imaging and Bioengineering, Grant/Award Number: R01EB020029; NIH/NCI Cancer Center Support Grant, Grant/Award Number: P30 CA008748 Funding information

Funding Information:
The authors would like to thank Dr. Manu Jain, Ucalene Harris, William Philips, and Sanjee Abeytunge for their support and assistance with this project. This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC's NIH/NCI Cancer Center Support Grant P30 CA008748. IRB/IEC approval status: 08-006.

Publisher Copyright:
© 2020 Wiley-VCH GmbH

Keywords

acridine orange
confocal microscopy
DRAQ5
EVCM
Mohs
tissue fixture

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10.1002/jbio.202000207

FulltextAccepted author manuscript, 2.86 MBLicence: Other

Cite this

Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin. / Ortner, Vinzent Kevin; Sahu, Aditi; Cordova, Miguel; Kose, Kivanc; Aleissa, Saud; Alessi-Fox, Christi; Haedersdal, Merete; Rajadhyaksha, Milind; Rossi, Anthony Mario.

In: journal of biophotonics, Vol. 14, No. 4, e202000207, 2021.

Research output: Contribution to journal › Journal article › Research › peer-review

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title = "Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin",

abstract = "We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.",

keywords = "acridine orange, confocal microscopy, DRAQ5, EVCM, Mohs, tissue fixture",

author = "Ortner, {Vinzent Kevin} and Aditi Sahu and Miguel Cordova and Kivanc Kose and Saud Aleissa and Christi Alessi-Fox and Merete Haedersdal and Milind Rajadhyaksha and Rossi, {Anthony Mario}",

note = "Funding Information: This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC's NIH/NCI Cancer Center Support Grant P30 CA008748. Funding Information: National Institute of Biomedical Imaging and Bioengineering, Grant/Award Number: R01EB020029; NIH/NCI Cancer Center Support Grant, Grant/Award Number: P30 CA008748 Funding information Funding Information: The authors would like to thank Dr. Manu Jain, Ucalene Harris, William Philips, and Sanjee Abeytunge for their support and assistance with this project. This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC's NIH/NCI Cancer Center Support Grant P30 CA008748. IRB/IEC approval status: 08-006. Publisher Copyright: {\textcopyright} 2020 Wiley-VCH GmbH",

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AU - Ortner, Vinzent Kevin

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AU - Cordova, Miguel

AU - Kose, Kivanc

AU - Aleissa, Saud

AU - Alessi-Fox, Christi

AU - Haedersdal, Merete

AU - Rajadhyaksha, Milind

AU - Rossi, Anthony Mario

N1 - Funding Information: This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC's NIH/NCI Cancer Center Support Grant P30 CA008748. Funding Information: National Institute of Biomedical Imaging and Bioengineering, Grant/Award Number: R01EB020029; NIH/NCI Cancer Center Support Grant, Grant/Award Number: P30 CA008748 Funding information Funding Information: The authors would like to thank Dr. Manu Jain, Ucalene Harris, William Philips, and Sanjee Abeytunge for their support and assistance with this project. This research was funded in parts by NIH/NIBIB grant R01EB020029 and MSKCC's NIH/NCI Cancer Center Support Grant P30 CA008748. IRB/IEC approval status: 08-006. Publisher Copyright: © 2020 Wiley-VCH GmbH

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N2 - We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.

AB - We investigated the utility of the fluorescent dye Deep Red Anthraquinone 5 (DRAQ5) for digital staining of optically sectioned skin in comparison to acridine orange (AO). Eight fresh-frozen thawed Mohs discard tissue specimens were stained with AO and DRAQ5, and imaged using an ex vivo confocal microscope at three wavelengths (488 nm and 638 nm for fluorescence, 785 nm for reflectance). Images were overlaid (AO + Reflectance, DRAQ5 + Reflectance), digitally stained, and evaluated by three investigators for perceived image quality (PIQ) and histopathological feature identification. In addition to nuclear staining, AO seemed to stain dermal fibers in a subset of cases in digitally stained images, while DRAQ5 staining was more specific to nuclei. Blinded evaluation showed substantial agreement, favoring DRAQ5 for PIQ (82%, Cl 75%-90%, Gwet's AC 0.74) and for visualization of histopathological features in (81%, Cl 73%-89%, Gwet's AC 0.67), supporting its use in digital staining of multimodal confocal micrographs of skin.

KW - acridine orange

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KW - DRAQ5

KW - EVCM

KW - Mohs

KW - tissue fixture

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