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Retinal thickness measurement obtained with spectral domain optical coherence tomography assisted optical biopsy accurately correlates with ex vivo histology.

Ferguson LR, Grover S, Dominguez JM, Balaiya S, Chalam KV - PLoS ONE (2014)

Bottom Line: The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17.All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida, United States of America.

ABSTRACT

Background: This study determines 'correlation constants' between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice.

Methods: Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as 'correlation constants'.

Results: Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (± 1.09) and 219.58 µm (± 2.67), respectively. The mean 'correlation constant' for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.

Conclusion: This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

Show MeSH
C57BL/6 mouse retina showing the reference points for SD-OCT and histological measurements.Dotted circle – optic nerve head (ONH); black solid circle – 1 mm diameter central area surrounding the ONH; inferior, middle and superior reference points, each 200 µm apart, both to the left and the right of the ONH.
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pone-0111203-g001: C57BL/6 mouse retina showing the reference points for SD-OCT and histological measurements.Dotted circle – optic nerve head (ONH); black solid circle – 1 mm diameter central area surrounding the ONH; inferior, middle and superior reference points, each 200 µm apart, both to the left and the right of the ONH.

Mentions: SD-OCT images were obtained with the InVivoVue Clinic software (Bioptigen, Inc., Durham, NC). Briefly, a 3×3 mm perimeter scanning protocol was used to obtain an imaging sequence comprising of 100 B-scans, with each B-scan consisting of 1000 A-scans, through a 50-degree field of view from the mouse lens. Once the ONH was centered within the InVivoVue Clinic imaging application, three scanning sequences were acquired. Measurements were restricted to retinal regions outside a radius of 500 µm from the center of the ONH. Retinal layer measurements were performed via the automated segmentation software provided by the instrument manufacturer (Bioptigen, Inc., Durham, NC). All layers were measured except for the retinal pigment epithelial layer because of the extensive artifactual changes associated with histological preparation and the limited depth penetrance associated with the SD-OCT 4.5 µm axial resolution. Total retinal thickness (TRT) represented the summation of all retinal layers spanning from the retinal nerve fiber layer (RNFL) to the outer segment of photoreceptors/inner segment of photoreceptors/external limiting membrane (OS/IS/ELM) region. Retinal sublayer measurements consisted of the OS/IS/ELM, outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer/ganglion cell (IPL/GC), and the RNFL. In order to standardize retinal thickness measurements from SD-OCT scans and histology sections, three arbitrary points were selected. The “inferior point” was the reference point that passed through the inferior margin of the ONH. The “middle point” was designated as the B-scan 200 µm above the “inferior point”. Lastly, the “superior point” was comprised of the B-scan that measured 400 µm above the “inferior point”. To determine the overall total retinal and sublayer thickness for each study eye, measurements were made at each of these reference points to both the left and right of the ONH (Fig 1). These six measurements were then averaged together to acquire final retinal thickness values.


Retinal thickness measurement obtained with spectral domain optical coherence tomography assisted optical biopsy accurately correlates with ex vivo histology.

Ferguson LR, Grover S, Dominguez JM, Balaiya S, Chalam KV - PLoS ONE (2014)

C57BL/6 mouse retina showing the reference points for SD-OCT and histological measurements.Dotted circle – optic nerve head (ONH); black solid circle – 1 mm diameter central area surrounding the ONH; inferior, middle and superior reference points, each 200 µm apart, both to the left and the right of the ONH.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216007&req=5

pone-0111203-g001: C57BL/6 mouse retina showing the reference points for SD-OCT and histological measurements.Dotted circle – optic nerve head (ONH); black solid circle – 1 mm diameter central area surrounding the ONH; inferior, middle and superior reference points, each 200 µm apart, both to the left and the right of the ONH.
Mentions: SD-OCT images were obtained with the InVivoVue Clinic software (Bioptigen, Inc., Durham, NC). Briefly, a 3×3 mm perimeter scanning protocol was used to obtain an imaging sequence comprising of 100 B-scans, with each B-scan consisting of 1000 A-scans, through a 50-degree field of view from the mouse lens. Once the ONH was centered within the InVivoVue Clinic imaging application, three scanning sequences were acquired. Measurements were restricted to retinal regions outside a radius of 500 µm from the center of the ONH. Retinal layer measurements were performed via the automated segmentation software provided by the instrument manufacturer (Bioptigen, Inc., Durham, NC). All layers were measured except for the retinal pigment epithelial layer because of the extensive artifactual changes associated with histological preparation and the limited depth penetrance associated with the SD-OCT 4.5 µm axial resolution. Total retinal thickness (TRT) represented the summation of all retinal layers spanning from the retinal nerve fiber layer (RNFL) to the outer segment of photoreceptors/inner segment of photoreceptors/external limiting membrane (OS/IS/ELM) region. Retinal sublayer measurements consisted of the OS/IS/ELM, outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer/ganglion cell (IPL/GC), and the RNFL. In order to standardize retinal thickness measurements from SD-OCT scans and histology sections, three arbitrary points were selected. The “inferior point” was the reference point that passed through the inferior margin of the ONH. The “middle point” was designated as the B-scan 200 µm above the “inferior point”. Lastly, the “superior point” was comprised of the B-scan that measured 400 µm above the “inferior point”. To determine the overall total retinal and sublayer thickness for each study eye, measurements were made at each of these reference points to both the left and right of the ONH (Fig 1). These six measurements were then averaged together to acquire final retinal thickness values.

Bottom Line: The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17.All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida, United States of America.

ABSTRACT

Background: This study determines 'correlation constants' between the gold standard histological measurement of retinal thickness and the newer spectral-domain optical coherence tomography (SD-OCT) technology in adult C57BL/6 mice.

Methods: Forty-eight eyes from adult mice underwent SD-OCT imaging and then were histologically prepared for frozen sectioning with H&E staining. Retinal thickness was measured via 10x light microscopy. SD-OCT images and histological sections were standardized to three anatomical sites relative to the optic nerve head (ONH) location. The ratios between SD-OCT to histological thickness for total retinal thickness (TRT) and six sublayers were defined as 'correlation constants'.

Results: Mean (± SE) TRT for SD-OCT and histological sections was 210.95 µm (± 1.09) and 219.58 µm (± 2.67), respectively. The mean 'correlation constant' for TRT between the SD-OCT and histological sections was 0.96. The retinal thickness for all sublayers measured by SD-OCT vs. histology were also similar, the 'correlation constant' values ranged from 0.70 to 1.17. All SD-OCT and histological measurements demonstrated highly significant (p<0.01) strong positive correlations.

Conclusion: This study establishes conversion factors for the translation of ex vivo data into in vivo information; thus enhancing the applicability of SD-OCT in translational research.

Show MeSH