Session 238 Cornea Imaging

Transcription

ARVO 2016 Annual Meeting Abstracts
238 Cornea Imaging
Monday, May 02, 2016 11:00 AM–12:45 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 1900–1940/A0175–A0215
Organizing Section: Cornea
Program Number: 1900 Poster Board Number: A0175
Presentation Time: 11:00 AM–12:45 PM
Quantitative Analysis of Depth, Distribution and Density of Cysts
in Acanthamoeba Keratitis using Confocal Microscopy
Ping Huang1, 2, Tudor Tepelus1, 2, Elmira Baghdasaryan1, 2,
Laura Vickers1, Jianyan Huang1, 2, John A. Irvine1, Hugo Y. Hsu1,
Srinivas R. Sadda1, 2, Olivia L. Lee1, 2. 1Doheny Image Reading
Center, Doheny Eye Institute, Los Angeles, CA; 2Doheny Eye
Institute, UCLA, Los Angeles, CA.
Purpose: To quantify the density, distribution and depth of invasion
of cysts in the corneas of eyes with Acanthamoeba Keratitis (AK) by
in-vivo confocal microscopy (IVCM) using a novel scanning pattern.
Methods: Patients with clinically diagnosed AK underwent IVCM
((HRT III RCM, Heildelberg, Germany) at various time points
during the clinical course. Five locations of the cornea were scanned:
the clinically affected area, as well as four standard points on the
peripheral cornea corresponding to temporal, nasal, inferior, and
superior locations. The IVCM scans were then manually graded by
trained reading center graders to quantify the maximum depth of
invasion and density of cysts (per mm2) in each of the five locations.
Results: Seventeen eyes of 14 patients with visible cysts on
IVCM were included. Cultures were positive for Acanthamoeba
in 9 eyes (52.9%). IVCM confirmed the diagnosis of AK in the
absence of positive cultures in 8 eyes (47.1%). Mean cyst density at
presentation was 215/mm2 (range, 64-484), and average cyst depth
was 169 microns (range, 70-300) in the affected area. Fourteen
eyes, with maximum cyst depth of 200 microns or less, reached
clinical resolution with medical treatment alone. The mean cyst
density became zero at the final visit in these 14 eyes. Three eyes
that ultimately underwent therapeutic PKP had cysts in all four
quadrants with deeper cyst infiltration; the average cyst depth in
these corneas was 283.3microns (range, 250-300). In one of these3
eyes, preoperatively, IVCM identified cysts extending to the limbus;
postoperatively, AK is not resolved and IVCM demonstrates cysts
within the graft host interface.
Conclusions: IVCM is clinically useful for the diagnosis of AK.
Acanthamoeba cyst density, distribution and depth of invasion
can be quantified non-invasively using IVCM, using a scanning
approach including both central and peripheral locations to detect
cysts that may otherwise be missed in routine scanning patterns.
These parameters may be employed in prognostication and treatment
decision-making in AK. A broader and deeper distribution of cysts on
IVCM appears to portend a worse prognosis.
In vivo laser confocal microscopy of Acanthamoeba Keratitis showed
double-walled cysts (arrow) and a number of highly reflective, highcontrast round-shaped particles 10–15 μm in diameter (arrow head),
suggestive of Acanthamoeba cysts.
Commercial Relationships: Ping Huang, None; Tudor Tepelus,
None; Elmira Baghdasaryan; Laura Vickers, None;
Jianyan Huang, None; John A. Irvine, None; Hugo Y. Hsu, None;
Srinivas R. Sadda, Stem Cells Inc (C), Avalanche (C), Genetech (F),
Bayer (C), Allergan (C), Optos (C), Iconic (C), Optos (F), Genetech
(C), Allergan (F), Thrombogenics (C), Regeneron (C), Novartis (C),
Carl Zeiss Meditec (F); Olivia L. Lee, Allergan (F), Allergan (C)
Corneal Endothelium and Iridocorneal Angle Biometrics in
Pediatric Glaucomatous Eyes
Grace Shih1, Dilshad Contractor2, Bibiana J. Reiser2, 1.
1
Ophthalmology, University of Southern California, Los Angeles,
CA; 2The Vision Center, Children’s Hospital Los Angeles, Los
Angeles, CA.
Purpose: Pediatric glaucoma patients are managed with a myriad of
topical eyedrops and surgical interventions. However, as glaucoma
is chronic and progressive, it is important for these young patients
to maintain relatively healthy corneas and iridocorneal angles to
maximize successful visual outcomes. Herein, we evaluated the
endothelial cell morphology and density, and iridocorneal angle
biometrics in glaucomatous and non-glaucomatous pediatric eyes.
Methods: 90 glaucomatous eyes and 39 control eyes from 80
pediatric subjects aged 0-18 years at Children’s Hospital Los Angeles
were evaluated and imaged with specular microscopy (Konan
NSP-9900, Konan Medical) and anterior segment optical coherence
tomography (AS-OCT) (iVue, Optovue, Inc.). Specular images
were manually counted to evaluate endothelial cell density and
morphology. AS-OCT images of open angles were analyzed for angle
opening distance at Schwalbe’s line (AOD-SL) and the trabecular iris
space area 500 µm posterior to Schwalbe’s line (TISA-SL).
Results: The average age of glaucoma patients and control
subjects was 9 was and 6 years, respectively. Glaucomatous eyes
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demonstrated an average cell density of 2763.84/mm2, compared to
control eyes with a cell density of 3318.46/mm2 (p=0.002). Of the
cells counted, normal eyes averaged 71.69% hexagonality, whereas
glaucomatous eyes averaged 62.89% hexagonal cells (p=0.0001). The
average pachymetry for glaucomatous eyes was 590.35µm, whereas
normal eyes averaged 587.47µm corneal thickness (p=0.044).
Average AOD-SL was 0.689 and 0.740 mm for glaucomatous and
control eyes, respectively. Average TISA-SL was 0.245 and 0.248
mm2 for glaucomatous and control eyes, respectively.
Conclusions: On average, glaucomatous pediatric eyes appeared to
exhibit lower endothelial cell density and percentage of hexagonal
cells than age-matched controls. Pachymetry measurements were
comparable between glaucomatous and control eyes. Iridocorneal
angle biometrics do not appear to be different between groups.
Although our findings suggest a disparity in the density and
morphology of corneal endothelial cells in glaucomatous eyes
compared to normal controls, further studies are required to elucidate
the etiology of these changes.
Endothelial cell loss in a pediatric glaucomatous eye.
Commercial Relationships: Grace Shih, None;
Dilshad Contractor; Bibiana J. Reiser, Allergan Sales, Inc. (F)
Normal corneal endothelium in a pediatric control eye.
Anterior Segment Spectral Domain Optical Coherence
Tomography and Histopathologic Study of Peters Anomaly
Mohamed S. Sayed, George Kontadakis, Maria Paula Fernandez,
Ta C. Chang, Kara M. Cavuoto, Sander R. Dubovy,
Mohamed Abou Shousha. Ophthalmology, Bascom Palmer Eye
Institute, Miami, FL.
Purpose: To report the results of anterior segment examination using
Spectral Domain Optical Coherence Tomography (SD-OCT) and
histopathologic study of excised corneal tissue in eyes of patients
with Peters anomaly. The study aims at studying the natural history
of corneal edema associated with Peters anomaly and determining
endothelial layer presence histologically and corneal thickness by
SD-OCT. Results of this study may guide the indications and nature
of surgical intervention in these patients, including anterior lamellar
keratoplasty.
Methods: Anterior segment SD-OCT examination was performed
on 18 eyes of 12 children with Peters anomaly using SD-OCT.
Subjective morphologic analysis of anterior segment structures
and corneal thickness measurement using the pachymetric map
display were done (Figure 1A). Corneal thickness measured as the
maximum thickness in a 6 mm zone was correlated to patient’s age.
Histopathlogic study using Periodic Acid-Schiff (PAS) stain of three
excised corneal buttons from eyes of children diagnosed with Peters
anomaly undergoing penetrating keratoplasty (PK) was performed.
Results: Corneal thickness inversely correlated with age (linear
correlation coefficient R=0.6) with lower corneal thickness values in
older children (P=0.01) (Figure 1B). Histopathologic examination
revealed presence of corneal endothelium in areas of absent or
attenuated Descemet’s membrane in two out of three eyes (Figure
2A and 2B; arrows point at areas of termination of Descemet’s
membrane; arrow heads point at endothelium present in areas with
absent or attenuated Descemet’s). The third corneal tissue (Figure 2C)
revealed the presence of a retrocorneal membrane, with underlining
attenuated and variably present endothelium (arrows).
Conclusions: The lower corneal thickness values observed in older
children with Peters anomaly can possibly be attributed to better
endothelial cell function and expansion of endothelial cells in areas of
absent or attenuated Descemet’s membrane. These results may help
optimize indications for keratoplasty and may propose deep anterior
lamellar keratoplasty as a potentially viable therapeutic option,
preserving the patient’s own endothelium and thus substantially
reducing the risk of graft rejection.
Purpose: Diabetic corneas have ultrastructural changes that can lead
to dry eye, abnormal healing after trauma. The literature is sparse
on the topic of Asian diabetics corneas with good Va and automated
refractive keratometry readings. At our retina clinic in California,
we retrospectively evaluated the keratometry readings of corneas of
Diabetic patients in an Asian population. Are there external refractive
or superficial keratometry differences in Asian Diabetic vs Asian
Control eyes?
Methods: Corneal topography was performed using TopCon KR8000PA Auto Kerato-Refractometer (Oakland, NJ) on patients as part
of the refractive screening prior to dilation for evaluation of diabetic
retinopathy. Inclusion criteria: Va = 20/15 to 20/50. Recent HbA1c/
FBS within 6 months of exam; Controls were patients without
diabetes (nl FBS). Exclusion criteria: corneal scarring, LASIK,
corneal surgery or previous macula laser.
Results: Total of 38 pts:
17 DM, 21 C; 17M, 21F; Age 43-90yrs; Avg 65.6yrs±10.8
Asians 22 pts: 11M, 11F; Age: 43-90yrs; Avg 61.4yrs±10.5
9 DM - HbA1c: Range 6.1-7.7%, Avg 6.9%±0.61; FBS: Range 95197mg/dL; Avg 136mg/dL±35
13 C - HbA1c: Range 5.5-6.0%, Avg 5.8%±0.18; FBS: Range 84119mg/dL; Avg 99mg/dL±10
Non-Whites 27 pts: 12M,15F; Age: 43-90yrs; Avg 64.4yrs ±11.2
13 DM - HbA1c: Range 6-7.7%; Avg 6.73%±0.62; FBS: Range 87197mg/dL; Avg 124.92mg/dL±33.07
14 C - HbA1c: Range 5.5-6.0%; Avg 5.84%±0.18; FBS: Range 84119mg/dL; Avg 98.08mg/dL±10.39
For OS: Asian diabetics (ADM) have a median horizontal axis (HA)
of 158° vs. Asians controls (AC) median 28° (p=0.02). Non-White
diabetics (NWDM) have a median horizontal axis of 158° vs. NonWhite controls (NWC) 28.5° (p=0.05).
Conclusions: Abnormal collagen fibrils are noted within the
cornea by ultra-structural exam in diabetic patients. The irregular
astigmatism occurring in diabetic patients may be due to the
abnormal collagen which may play a role in decreased vision for
diabetics. Asian DM eyes have an astigmatic axis closer to 150°
whereas Asian Control eyes have an astigmatic axis closer to 50°.
This has implications for refractive and cataract surgery.
Commercial Relationships: Mohamed S. Sayed, None;
George Kontadakis; Maria Paula Fernandez, None; Ta C. Chang,
None; Kara M. Cavuoto, None; Sander R. Dubovy, None;
Mohamed Abou Shousha, None
Asian Diabetic Corneas: A Study in Astigmatism
Vidhya Gunasekaran1, Jullian Nguyen2, Victor Chen3, Don B. Kim4,
Victoria Phan4, Shelby Muchamuel5, So Yeon Kim4, Gloria Wu6.
1
Aravind Eye Hospital, India, Sunnyvale, CA; 2Ross University
School of Medicine, Portsmouth, Dominica; 3UC San Diego, La Jolla,
CA; 4UC Berkeley, Berkeley, CA; 5UC Los Angeles, Los Angeles,
CA; 6Ophthalmology, UC San Francisco School of Medicine, San
Francisco, CA.
Commercial Relationships: Vidhya Gunasekaran, None;
Jullian Nguyen; Victor Chen, None; Don B. Kim, None;
Victoria Phan, None; Shelby Muchamuel, None; So Yeon Kim,
None; Gloria Wu, None
Measurements of corneal astigmatism axis with anterior
segment swept source optical coherent tomography versus auto
refractokearatometer
Shinichiro Nakano1, Masaharu Iida2, Takahiro Hiraoka2,
Tetsuro Oshika2. 1Ophthalmology, Ryugasaki Saiseikai Hospital,
Ryugasaki, Japan; 2Ophthalmology, UnIversity of Tsukuba, Tsukuba,
Japan.
Purpose: Precise preoperative measurement of corneal astigmatism
is of crucial importance to achieve good surgical results with toric
intraocular lens (T-IOL) implantation. In this study, the axis of
corneal astigmatism was assessed by two methods; anterior segment
swept source optical coherent tomography (AS-OCT) and auto
refractokearatometer before cataract surgery. The results of these
two methods were compared and the factors which influence the
difference were examined.
Methods: Two hundred twenty-six eyes of 155 patients (69 male
86 female, 74.8 ± 5.8 [mean ± SD] years) undergoing cataract
surgery and IOL implantation were included. Preoperatively,
corneal astigmatism axis was measured by two methods; auto
refractokeratometry (KR8100A, TOPCON) and AS-OCT (CASIA,
TOMEY). The difference between two methods was analyzed in
relation to various parameters including preoperative factors (age,
corrected distance visual acuity (CDVA), objective astigmatism)
and postoperative factors (3-month postoperative uncorrected visual
acuity (UCVA) and 3-month postoperative objective astigmatism). In
addition, the influence of corneal astigmatism type (against-the-rule;
ATR, with-the-rule; WTR, and oblique) was also examined.
Results: Difference in corneal astigmatism axis measured with
the two methods was within 10 degrees in 107 cases (47%), from
10 to 19 degrees in 25 cases (11%), from 20 to 29 degrees in
31 cases (14%), and 30 degrees or more in 34 cases (15%). The
difference showed no correlation with age, preoperative CDVA,
3-month postoperative UCVA, and 3-month postoperative objective
astigmatism, but exhibited a significant correlation with preoperative
corneal astigmatism power (r = 0.341, p<0.001: Pearson’s correlation
coefficient).
When compared among preoperative corneal astigmatism types, eyes
with ATR astigmatism showed significantly lower disparity in corneal
astigmatism axis between the two methods than eyes with WTR and
oblique astigmatism (p<0.001, p<0.001: Mann-Whitney U test).
Conclusions: There was considerable difference in the measurement
results of corneal astigmatism axis between AS-OCT and
refractokeratometer, and the difference was larger in eyes with greater
preoperative astigmatism as well as WTR/oblique astigmatism.
Commercial Relationships: Shinichiro Nakano, None;
Masaharu Iida, None; Takahiro Hiraoka, None; Tetsuro Oshika,
None
Novel Objective Evaluation of the Efficacy of Cyclorotation
Compensation in Topography-guided Treatments
Joyce Khandji1, George Asimellis2, Laurence Sperber1,
A. J. Kanellopoulos2, 1. 1Ophthalmology, NYU, New York, NY;
2
LaserVision.gr Clinical & Research Eye Institute, Athens, Greece.
Purpose: Topography-guided treatments have been recently
approved by the FDA for the correction of refractive error as
a custom-treatment. A parameter that may affect results is the
assumed cyclorotation (cyclotorsion) of the eye that may occur
between the imaging (upright sitting) position of the patient and
the treatment (supine) position of the patient. The purpose of this
study was to comparatively investigate the efficacy of a topographyguided treatment procedure for keratoconic eyes that incorporates
cyclorotation compensation (Group-A), as compared with the
previously applied procedure (Group-B), in which no cyclorotation
compensation was used.
Methods: In group-A (cyclo nA=110 eyes), cyclorotation
compensation was applied, while in group-B (non-cyclo nB=110),
no cyclorotation compensation was applied. Analysis was based
on digital processing of Scheimpflug-imaging derived topographic
curvature difference maps. Differences between targeted (surgical
planning) and achieved ablation patterns were objectively measured.
The vector (r, θ) corresponding to the steepest (peak topographic)
corneal point on the pre-operative surgical planning map (rp, θp)
and the curvature difference map (rd, θd) was computed, and the
difference between the peak topographic angular data: Δθ=|θp - θd|
and the weighted angular difference WΔθ=ΔθxΔr was calculated.
Results: For group-A (cyclo) average Δθ was 7.18±7.53 (range 0 to
34°) and WΔθ was 3.43±4.76 (range 0.00 to 21.41mm). For group-B
(non-cyclo), average Δθ was 14.50±12.65 (range 0 to 49°) and WΔθ
was 10.23±15.15 (range 0.00 to 80.56mm). The cyclo group-A had
on average a smaller angular difference as well as weighted angular
difference by a statistically significant margin (Δθ p=0.0058 and
WΔθ p=0.015).
Conclusions: This study introduces an objective technique for
evaluation of cyclorotation compensation in excimer laser ablation.
The data indicates that incorporation of cyclorotation compensation
in customized topography-guided treatment leads to improved
correlation between targeted and achieved changes.
Commercial Relationships: Joyce Khandji, None;
George Asimellis, None; Laurence Sperber, None;
A. J. Kanellopoulos, ISP Surgical (C), Keramed (C), i-Optics (C),
Alcon/WaveLight (C), Allergan (C), Avedro (C)
Rotating Scheimpflug Camera Curvature Decentration Indices
Nermine S. Madkour1, Maged M. Roshdy2, 1, Sherine S. Wahba1, 2.
1
Ophthalmology, Al Watany Eye Hospital, Cairo, Egypt;
2
Ophthalmology, Ain Shams University, Cairo, Egypt.
Purpose: To study the accuracy and correlation between different
curvature decentration indices; anterior corneal curvature (A) both
Inferior minus Superior value (I-S) astride horizontal meridian (h)
and I-S value astride flattest meridian (f), posterior corneal curvature
(P) I-S value astride horizontal meridian (h) and Index of Height
Decentration (IHD).
Methods: In a retrospective study; history taking, clinical
examination, and rotating Scheimpflug camera scanning (by Oculyzer
II equivalent to Pentacam HR) were done to 94 eyes; 57 normal, 20
forme frust keratoconus FFKC (apparently normal cornea whose
fellow eye with evident keratoconus(KC)) and 17 KC. All cases
of corneal scars or previous corneal surgeries were excluded. I-S
value was calculated manually from 10 points astride the horizontal
and flat meridians for anterior corneal curvature and astride the
horizontal meridian for posterior corneal curvature. IHD is calculated
automatically by the device software 1.17r119.
Results: The mean values of A I-S h in normal corneas versus (vs)
FFKC and KC eyes were 0.19 ±0.62 vs 2.19 ±3.03 (P<.001), A I-S f
0.10 ±0.60 vs 1.64 ±1.94 (P<.001), P I-S h -1.25 ±3.83 vs 0.45 ±0.50
(P=.002), and IHD 0.113±0.794 vs 0.032 ±0.041 (P=.444). The four
indices were highly correlated (P<.0001). The highest correlation
coefficient was between A I-S h and A I-S f (r 2 =.913) and the least
was between IHD and A I-S f (r 2 =.621).
Conclusions: All the three commonly used topographic decentration
indices are correlated. The three types of I-S indices are more useful
in detection of KC and FFKC than the IHD.
Commercial Relationships: Nermine S. Madkour, None;
Maged M. Roshdy, Alcon Labs (R); Sherine S. Wahba, Alcon
Labs (R)
Support: ARVO Egypt Travel Grant
Higher-order aberrations after DSEAK, “thin” DSEAK and
DMEK: comparative study about 60 cases
Naïla Houmad, Oualid Guechi, Piotr Krawczyk, Mohamed Zaidi,
Nadia OUAMARA, Alix EHRHARDT, Maxime SOT, Louis Lhuillier,
Adina Agapie, Jean-Marc Perone, Huong Duong Nguyen Viet.
Ophthalmology, CHR-Metz, Villers-les-Nancy, France.
Purpose: The aim of this study was to assess the visual acuity
after corneal graft by descemet’s stripping automated endothelial
keratoplasty (DSAEK) or Descemet’s membrane endothelial
keratoplasty (DMEK) for advanced
endothelial dysfunction. Clinical data and higher-order aberrations
(HOA) were analyzed.
Methods: A total of 60 eyes were included in our study and were
divided into 4 groups: 15 DMEK (group 1), 15 “thin” DSAEK
(group 2), 15 DSAEK (group 3) and 15 control eyes (group 4) which
received no treatment. “Thin” DSAEK was defined as a graft with
postoperative thickness inferior to 100μm. Surgeries were performed
at Regional Hospital Center of Metz-Thionville (Mercy Hospital). In
addition to the analysis of general clinical data, HOAs were measured
by Scheimpflug camera (Schwind Sirius) one year after surgery
for DSAEK and “thin” DSAEK, and six months after DMEK.
Best spectaclecorrected visual acuity (BSCVA; in logarithm of the
minimal angle of resolution [logMAR] units) was obtained. Data
were compared between the
four groups.
Results: The Mean increase of BSCVA was 0.64±0.56 logMAR for
DMEK, 0.74±0.58 logMAR for DSAEK and 0.61±0.39 logMAR
for “thin” DSAEK. There was no statistically significant difference
for the gain line average. However post-op BSCVA in DMEK group
was statistically higher than that in DSAEK group (p=0.017), but
had no statistical difference with “thin” DSAEK group (p=0.31). For
HOAs, the total and posterior root mean square (RMS) at 4 mm was
statistically different (p<0.05) between controls and others groups.
The back corneal RMS of the DMEK group is statistically lower
than that of the DSAEK group (p=0.035) and “thin” DSAEK group
(p=0.037). The difference between “thin” DSAEK and DSAEK was
not significant (p=0.24).
Conclusions: As we show in our study, DMEK provided better
visual recovery than DSAEK. Several comparative studies ended to
the same result. The explanations usually moved forward to explain
this difference include: hyperreflectivity of the interface, hyperopic
shift and HOAs. Our series tends to confirm this last point. DMEK
surgery seems to generate lower HOAs values than “thin” DSAEK
and DSAEK, but more than in control group, concerning posterior
RMS at 4 mm.
Commercial Relationships: Naïla Houmad, None;
Oualid Guechi, None; piotr krawczyk, None; Mohamed Zaidi,
None; Nadia OUAMARA, None; Alix EHRHARDT, None;
Maxime SOT, None; Louis Lhuillier, None; adina agapie, None;
Jean-Marc Perone, None; Huong Duong Nguyen Viet, None
A purely solid-state device for rapid reconstruction of 3D models
of the anterior segment of the eye with no moving parts
Pushyami Rachapudi1, Shantanu Sinha3, William S. Lee2,
Albert Redo-Sanchez3, Ramesh Raskar3. 1CEH, IIIT Hyderabad,
Hyderabad, India; 2Department of Mechanical Engineering,
Massachusetts Institute of Technology, Cambridge, MA; 3MIT Media
Lab, Massachusetts Institute of Technology, Cambridge, MA.
Purpose: Routine eye exams via slit lamp are critical in screening for
conditions like cataracts and corneal injury. The hardware complexity
of slit lamps limit their use to ophthalmic clinics by trained
professionals, rendering them impractical for resource-constrained
settings. We present purely solid-state instrumentation that employs
programmable illumination and light steering optics to simulate the
motion of a slit, thereby exhibiting functionality similar to a slit
lamp with no moving parts. Complete computational control over
illumination enables us to generate 3D models and extract meaningful
quantitative metrics to aid in disease diagnosis by untrained staff.
Methods: The prototype comprises a laser projector, an ellipsoidal
reflector and a system of cameras to image the eye. The projector and
the eye are placed at the foci of the ellipsoid so all the light emitted
by the projector converges onto the eye. The ellipsoidal reflector
serves two purposes: 1. steering the diverging beam projected into
converging slits of light. 2. multiplying the angular spread of the
beam projected to achieve a larger sweep angle. Software corrects
distortions produced to ensure projection of parallel slits illuminating
one cross-section at a time. The images captured by the cameras are
then stitched together to generate a 3D model.
Results: Our prototype exhibits functionality similar to a slit lamp
with lower hardware complexity, enabling it to be compact. Based
on the eccentricity of the ellipsoid, we are able to multiply the
angular spread of the input beam up to a factor of four. The entire
data capture process is automated to less than 2 seconds. Complete
software control over illumination allows us to precisely determine
the orientation of each cross-section. This enables us to reconstruct
a complete 3D model and extract quantitative metrics like corneal
topography and anterior chamber depth.
Conclusions:
We have proposed a design for examining the anterior segment
of the eye in resource-constrained settings. The use of solid state
instrumentation instead of mechanically moving parts allows the
device to be portable. Computational control over data capture
reduces the capture process to under 2 seconds, making it ideal
for rapid screening in remote locations. Generation of 3D models
and extraction of quantitative metrics makes the captured data
interpretable by lesser-skilled staff.
Commercial Relationships: Pushyami Rachapudi, None;
Shantanu Sinha; William S. Lee, None; Albert Redo-Sanchez,
None; Ramesh Raskar, None
Studies of collagen organisation and early stages of cell
infiltration in the primary stroma of developing chick cornea
Robert D. Young, Carlo Knupp, Elena Koudouna, Philip N. Lewis,
Keith M. Meek, Andrew J. Quantock. School of Optometry & Vision
Sciences, Cardiff University, Cardiff, United Kingdom.
Purpose: Primary stroma, the earliest rudiment of developing corneal
matrix in the chick embryo, has been implicated in organisation
of cells and lamellar structure in the mature tissue, and yet it is
absent in the immature corneas of many species, including man. We
examined early stages in chick corneal development to characterise
the 3-dimensional structure of the primary stroma to determine if
inwardly migrating neural crest cells exhibit alignment in relation to
its components.
Methods: Eyes from embryonic chicks at 3 to 7 days of development
were fixed in buffered aldehyde solution. Dissected corneas, or whole
eyes, were embedded in epoxy resin after immersion in osmium
tetroxide with potassium ferricyanide, tannic acid, osmium tetroxide
and uranyl acetate, each for 1 h, then dehydration in ethanol.
Toluidine blue-stained tissue sections were cut for orientation and the
polished block surface imaged with a backscatter electron detector in
a Zeiss Sigma VP FEG scanning electron microscope. Sequences of
up to 1000 serial images were taken, each alternating with removal
of a 100nm surface slice by an in-chamber Gatan 3View2 microtome.
3-dimensional reconstructions of tissue architecture were made
by manual or automated segmentation using Amira 5.6 or ImageJ
software.
Results: The primary stroma forms a layer of loosely distributed
collagen fibrils (mean diameter at E4, 25.5nm, S.D. 3.5, n=750),
increasing in thickness from 15 to 100µm between E3 and E7.
Fibril distribution was not homogeneous, but showed a central
condensation, approximately 5µm thick, which undulated regularly
with respect to the epithelial basal lamina between more-sparsely
dispersed fibrils distally and proximally. Immediately below the
basal lamina, fibrils showed orthogonal arrangement, but some zones
almost free of fibrils, and others with fibrils orientated at large angles
to the basal lamina were also present. Preliminary observations seem
to indicate that cells migrating into the primary stroma at E5-E6
exhibit no preferential alignment with respect to the collagen fibrils.
Conclusions: Absence of any clear alignment between presumptive
keratocytes entering the primary stroma and collagen fibrils of this
rudimentary tissue would challenge the long-held view that it serves
a role in guidance and organisation of cells and thus lamellae of the
mature stroma.
Commercial Relationships: Robert D. Young, None;
Carlo Knupp, None; Elena Koudouna, None; Philip N. Lewis;
Keith M. Meek, None; Andrew J. Quantock, None
Support: Biotechnology and Biosciences Research Council, UK:
project grant No. BB/M025349/1; Medical Research Council, UK;
programme grant 503626
Collagen Organization in Pathological Corneas Measured With
Second Harmonic Microscopy
Francisco J. Avila, Pablo Artal, Juan M. Bueno. Laboratorio de
Optica, Universidad de Murcia, Murcia, Spain.
Purpose: Since the particular distribution of collagen fibers within
the stroma is related to corneal transparency, any alteration might
seriously compromise the optical properties of the cornea. The use
of second harmonic generation (SHG) microscopy demonstrated
that healthy and pathological collagen-based tissues present
different organization. We propose to analyze corneal tissues with
SHG microscopy to quantitatively discriminate the collagen fibers
organization in pathological from healthy ones.
Methods: A custom multiphoton microscope was used for SHG
imaging of ex-vivo corneal tissues from human donors. SHG images
from corneas affected by keratoconus, edema and bullous keratopathy
were recorded. These were compared to SHG images of healthy
corneas used as control. The structure tensor was used to characterize
the collagen fiber dispersion information from SHG images. Different
quantitative information parameters were obtained from each sample:
preferential orientation (PO), structural dispersion (SD) and the
degree of isotropy (DoI) of the collagen fibers (lamellae).
Results: Control corneas showed systematically a regular lamellar
distribution with high DoI and a low SD. The PO presented a
Gaussian distribution with the maximum corresponding to the
dominant direction of the fibers. However, this pattern turned into
a non-organized arrangement in all tested pathological corneas.
The DoI was lower and consequently the SD noticeable increased.
Moreover, there was not a clear preferential orientation.
Conclusions: SHG microscopy was used to image collagen
architecture in corneal tissues under different experimental
conditions. From the images, the structure tensor approach provided
information on the fiber spatial organization in both pathologic and
normal corneal tissues based on several quantitative parameters. This
permits to differentiate between normal and pathological corneas,
as well as to establish discrimination numerical scales. Although,
currently SHG can only be applied “in vitro”, future implementations
of this techniques in clinical environments might represent a better
tool for corneal disease diagnoses.
Commercial Relationships: Francisco J. Avila; Pablo Artal, None;
Juan M. Bueno, None
Support: European Research Council Advanced Grant ERC-2013AdG-339228 (SEECAT),SEIDI, Spain (grant FIS2013-41237-R).
Sampling density and configuration of stimulator points in
determination of astigmatism, coma and higher order aberrations
of the posterior surface of the cornea using Purkinje imaging
reflection technology
Roland Piper1, Enrique Gambra1, Victor Sicam2. 1Research, Cassini
BV, Den Haag, Netherlands; 2Advanced Development, Cassini BV,
The Hague, Netherlands.
Purpose: Research on improving intraocular lens outcomes is
shifting focus onto accurate measurement of the posterior corneal
surface (Koch DD. Ophthalmology. 2015 Jun;122(6):1070-1.).
The Cassini Corneal Shape Analyzer (Cassini BV, The Hague,
Netherlands) is at the frontier of anterior and posterior surface
measurement technology (Ch. 5 of Refractive Lens Exchange, ed.
Wang MX, Slack Inc © 2016). In this paper we investigate the
sampling density required for this technique to reconstruct a Zernike
model of the posterior surface of the cornea.
Methods: Source and image points are simulated for a test eye with
an 8 mm radius spherical anterior surface and a 6.5 mm base radius
posterior surface, with corneal aberrations described by Zernike
coefficients of up to 6th order. Gaussian noise (25 μm s.d.) is added
to the image points to simulate experimental noise. Source points
are arranged in 9 configurations of concentric rings (3, 4 and 5 rings
containing 7, 14 or 21 points each) providing even angular coverage
up to a 6 mm corneal zone diameter (CZD). A novel raytracing
algorithm that considers sequential surface interactions is used to
calculate a posterior surface height map. This map is used to calculate
a Zernike fit of arbitrary order of the posterior surface. Fits of up to
3rd, 4th and 5th order are made. The precision of the resulting shape
contribution to astigmatism (2nd and 4th order), coma (3rd and 5th
order), trefoil, and quadrafoil (where possible) is compared. The
threshold of acceptable corneal height precision is 10 μm at 6 mm
CZD, relating to around 0.25 eq. Dioptre.
Results: A minimum of 37 stimulator points covering the 6 mm CZD
is found to be sufficient to achieve a precision of better than 10 μm
for astigmatism, using an order 4 Zernike fit. Third order coma is
found to have a precision of better than 5 μm with this configuration.
Attempting a fit of up to order 5 is not sufficiently stable for clinically
relevant determination of 5th order coma or higher order aberrations
with this level of experimental noise, up to and including 105 points
split between five rings.
Conclusions: The results show this algorithm reconstructs up to 4nd
order astigmatism and coma to a clinically relevant precision when
using more than 37 stimulator points as input.
Commercial Relationships: Roland Piper, Cassini BV;
Enrique Gambra, Cassini BV; Victor Sicam, Cassini BV, Cassini
BV (P)
Correlating functional and morphometric characteristics
of pterygium using high resolution anterior segment optical
coherence tomography
Bernhard Steger2, Thomas Gasser2, Vito Romano1, Stephen B. Kaye1,
Nikolaos E. Bechrakis2. 1St. Paul’s Eye Unit, University of Liverpool,
Liverpool, United Kingdom; 2Department of Ophthalmology,
Medical University of Innsbruck, Innsbruck, Austria.
Purpose: To investigate the role of high resolution anterior segment
optical coherence tomography (HR-ASOCT) in assessing the
morphology of pterygia and pingueculae and the relationship to
corneal scarring or astigmatism.
Methods: Single centre cross-sectional case series of patients with
nasal primary pterygium and or pingueculae were included. A clinical
assessment together with HR-ASOCT based morphometric analysis,
color photography and keratometry were undertaken in each patient.
Associations were tested between the parameters of the pterygium,
degree of corneal scarring, astigmatism and best corrected visual
acuity (BCVA).
Results: 29 eyes of 26 patients with pterygium and 6 patients with
pinguecula were included. Morphometric analysis of pterygia
revealed a mean horizontal corneal length of 2.8±1.3 mm,
0.5±0.2mm limbal thickness, 4.8±1.7mm limbal breadth and affected
corneal area of 9.4±6.6 mm2. Loss of integrity of the basement
membrane with anterior stromal scarring was found in 23 cases
(79.3%) reaching a mean depth of 68.8±21.7 µm (min: 33 µm,
max: 126 µm). Depth of anterior stromal scarring had a significant
inverse association with BCVA and pterygium thickness (p = 0.011
and p = 0.014). Flat bridging of the corneoscleral transition zone
was significantly associated with corneal scarring and the degree
of corneal astigmatism, while an inverse association was found for
limbal nodularity (Figure 1).
Conclusions: HR-ASOCT provides useful quantification of the
affected corneal area, pterygium thickness and stromal scarring for
planning of treatment. Morphology of the corneoscleral transition and
epithelial thickness differ between pingueculae, small and advanced
pterygia, aiding in the understanding of the pathogenic relationship
between pingueculae and pterygia.
Commercial Relationships: Bernhard Steger, None;
Thomas Gasser, None; Vito Romano, None; Stephen B. Kaye,
None; Nikolaos E. Bechrakis, None
Evaluation of corneal irregularity due to progress of primary
pterygium using Fourier harmonic analysis for multiple area
diameters
Keiichiro Minami1, Takashi Ono1, Tadatoshi Tokunaga1,
Shouta Tokuda2, Atsushi Otani3, Kazunori Miyata1, 2, Shiro Amano4.
1
Miyata Eye Hospital, Miyakonojo, Japan; 2Kagoshima Miyata Eye
Clinic, Kagoshima, Japan; 3Tomey Corporation, Nagoya, Japan;
4
Inoue Eye Hospital, Tokyo, Japan.
Purpose: Progress of pterygium increases irregularity of the corneal
surface and the increased irregularity results in degradation of
visual function. It is important to identify a central area in which
the corneal irregularity does not increase due to the progress of
pterygium for evaluating the visual functions of the eye. The purpose
of the prospective observation study was to evaluate changes in
the corneal irregularity due to the progress of pterygium using the
Fourier harmonic analysis of corneal topographic data modified to
accommodate different diameters of the analysis areas.
Methods: 609 eyes of 465 patients who had pterygium without a
history of pterygium excision surgery were enrolled. Mean age was
67.3 (SD: 11.4) years and there were 172 men and 293 women. Bestcorrected visual acuity (BCVA) and the corneal topography were
measured. Progress rates were calculated as ratios of positions of the
pterygium end with respect to the corneal diameters. The topography
data were analyzed using the Fourier series harmonic analysis within
1, 2, 3, 4, 5, and 6 mm diameters. Changes in the asymmetry and
higher-order irregularity components with the progress rates were
evaluated using segmented regression analysis for each analysis
diameter.
Results: The progress rate was ranged from 2 to 60% with a mean
of 22.2 %, and significantly correlated with the BCVA (P<0.0001,
R2=0.065). The asymmetry components showed steep increases when
the progress rate exceeded 35.9 to 36.9 % and they did not vary with
the analysis diameters. In the higher-order irregularity components,
the progress rates of 31.7, 22.5, and 14.8% for the analysis diameters
of 1, 3, and 6 mm respectively, showed steep increases at relative
starting points, according to the analysis diameters. The amplitudes
at the progress rates were within a range of 0.18 to 0.28, which were
independent of the analysis diameters.
Conclusions: The segmented regression analysis revealed that
the higher-order irregularity components for the series of analysis
diameters detected steep increases in the corneal irregularity due to
progress of pterygium. The results suggested that the progress of
pterygium and its influence on visual function could be evaluated
with multiple-area Fourier harmonic analysis.
Commercial Relationships: Keiichiro Minami, None;
Takashi Ono, None; Tadatoshi Tokunaga, None; Shouta Tokuda,
None; Atsushi Otani, Tomey Corporation; Kazunori Miyata, None;
Shiro Amano, None
High-resolution imaging of limbal structural properties using
Brillouin spectro-microscopy
Guillaume Lepert1, Ricardo M. Gouveia2, Che J. Connon2,
Carl Paterson1. 1Physics, Imperial College London, London, United
Kingdom; 2Institute of Genetic Medicine, Newcastle University,
Newcastle, United Kingdom.
Purpose: Limbal stem cell differentiation is dependent not only on
the chemical but also the mechanical environment. We previously
hypothesized that the mechanical properties of the cornea can regulate
the differentiation of limbal epithelial stem cells, and direct normal
corneal epithelium maintenance and homeostasis [1]. As such, we
proposed to characterize in fine detail the mechanical properties of the
anterior layers of the human cornea, specifically at the limbal zone.
Methods: The mechanical properties of the cornea have been
previously investigated using different methodologies. Here
we applied the non-invasive, non-destructive Brillouin spectromicroscopy technique to analyse fresh corneal tissues in vitro,
in physiological conditions. For that we used a new confocal
microscopy setup integrating a single-stage virtually imaged phased
array (VIPA) spectrometer to measure the Brillouin shift [2] with a
novel adaptive-optics interferometric filter to achieve unprecedented
rejection of elastic background signal. The Brillouin shift is directly
related to the acoustic velocity, which in turn is correlated to the
mechanical moduli. Measurements were carried out with whole
human corneas immersed in Carry-C preservation medium.
Results: This technique allowed us to quickly measure the relative
stiffness of discrete points in large areas of whole fresh human
corneas. In particular, we were able to obtain highly-detailed maps
of Brillouin shift from the centre to the corneal limbus, from the
anterior- down to the posterior-most layers of the cornea, with an
xyz sampling of up to 5×5×5 µm, (the confocal resolution or spot
size is 1x1x5 µm). Importantly, this method allowed the precise
identification of different corneal structures based on their Brillouin
shift patterns, with the anterior stroma showing higher Brillouin shifts
in central cornea compared to the limbal region.
Conclusions: Brillouin spectro-microscopy allowed us to identify
the differences in mechanical properties between the anterior layers
of the centre and limbus regions of whole fresh human corneas.
This technique thus provides a vital method to finely and precisely
characterize the stiffness of corneal tissues, and investigate further
our hypothesis that differences in compliance between the limbus and
central cornea underpin corneal epithelial homeostasis.
Commercial Relationships: Guillaume Lepert, Imperial College
London (P); Ricardo M. Gouveia, None; Che J. Connon, None;
Carl Paterson, Imperial College London (P)
Measurement of Corneal and Limbal Epithelial Thickness by
In Vivo Confocal Microscopy and Anterior Segment Optical
Coherence Tomography
Qihua Le, Yan Chen, Yujing Yang, JianJiang Xu. Ophthalmology, Eye
& ENT Hospital of Fudan University, Shanghai, China.
Purpose: To compare corneal epithelial thickness (CET) and limbal
epithelial thickness (LET) measured by anterior segment optical
coherence tomography (AS-OCT) and in vivo confocal microscope
(IVCM) in normal subjects, and evaluate the consistency between
these two techniques.
Methods: 38 normal subjects (17 men and 21 women) were enrolled
in this study. AS-OCT was performed at central cornea and the
superior, inferior, nasal and temporal limbus. Then followed by
IVCM examination performed at the same location. SPSS 13.0
software was used to compare CET and LET measured by two
techniques and analyze the correlation between them.
Results: The average CET measured by AS-OCT and IVCM was
55.9±4.0μm and 51.7±4.9μm respectively. The value measured
by IVCM was significantly lower than that measured by ASOCT (P=0.015). The average LET values tested by IVCM at
superior, inferior, nasal and temporal quadrant were 73.1±14.7μm,
71.7±15.5μm, 55.5±8.8μm and 55.9±11.4μm, which were similar
as those measured by AS-OCT at superior and inferior quadrant and
10.3% and 10.9% thinner at nasal and temporal quadrant (nasal:
P=0.019, temporal: P=0.003). In subjects older than 40 years, CET
and LET values measured by AS-OCT were significantly higher than
those by IVCM. Such differences were not found in subjects≤40
years old.
Conclusions: CET values measured by IVCM are lower than those
by AS-OCT, while LET values have good consistency between
them. These two techniques have their own advantages in measuring
epithelial thickness and are mutually complementary.
Commercial Relationships: Qihua Le, None; Yan Chen, None;
Yujing Yang, None; JianJiang Xu, None
Support: National Natural Science Foundation of China
(81020108017, 81270014)
Corneal epithelial thickness mapping in the diagnosis of ocular
surface pathologies involving the corneal epithelium
Sara Touhami, Cristina Georgeon, Nacim Bouheraoua,
Laurent Laroche, Vincent Borderie. Ophthalmology, Quinze-Vingts
Hospital, Paris, France.
Purpose: To analyze the value of corneal epithelial thickness
mapping (CETM) by Fourier-domain optical coherence tomography
(OCT) in the diagnosis and differentiation of ocular surface
pathologies involving the corneal epithelium.
Methods: Study type:Cross-sectional.
Patients:Consecutive patients with ocular surface pathology
involving the corneal epithelium who underwent CETM by Fourierdomain OCT at a tertiary center (Quinze-Vingts Hospital, Paris)
were included. For each patient, CETM parameters were compared
between the groups using Fisher-exact and one-way ANOVA tests in
order to look for distinctive features for each pathology.
Results: 171 eyes of 171 patients were included between January
2013 and September 2015. Mean age at inclusion was 46.2 years.
Patients distribution was as follows: 32.1% were healthy, 15.2%
had limbal deficiency (LD), 7% had trachoma, 15.8% had epithelial
basement membrane dystrophy (EBMD), 1.2% had herpes, 1.2%
had dry eye syndrome, 20.5% had keratoconus (KC), 3% had
Thygeson’s superficial punctuate keratitis, 1.8% had superior limbic
keratoconjunctivitis and 1.8% had in situ carcinoma. Analysis of
CETM revealed specific patterns for various pathologies. A “rayon
de roue” aspect of CETM, corresponding to a circular succession of
thickened and thinned epithelial areas, seemed specific of LD (72%
versus 4% for LD versus all others respectively, p= 4e-8). “Pyramid”
aspect of CETM involving triangularly thickened epithelium seemed
specific of EBMD (74% versus 2%, p=2e-8). “Donut” aspect of
CETM with temporally thickened epithelium, surrounding areas of
thinned epithelium seemed suggestive of KC (34.3% versus 1.4%,
p=6.4e-8). Other CETM parameters seemed distinctive of LD (LD
versus all others respectively): minimal and maximal epithelial
thicknesses (ET) (27.4 versus 44.3 microns for minimal ET and 77.4
versus 62.4 microns for maximal ET, p=0.0001), epithelium standard
deviation (15.1 versus 4.24, p=0.01) and minimal-maximal ET (-49.9
versus -18.3 microns, p= 3.6e-8).
Conclusions: CETM seems to differentiate ocular surface
pathologies in a quite reliable manner. Specific epithelial patterns
seem suggestive of LD, KC and EBMD. CETM can help characterize
ocular surface pathologies that involve the corneal epithelium beyond
what is apparent with the clinical examination, providing a noninvasive and easily available tool for diagnosis and monitoring.
Commercial Relationships: Sara Touhami, None;
Cristina Georgeon, None; Nacim Bouheraoua, None;
Laurent Laroche, None; Vincent Borderie, None
Multimodal Assessment of Corneal Thinning Using Anterior
Segment Optical Coherence Tomography, Scheimpflug Imaging,
and Ultrasound Pachymetry Compared to Clinical Measurement
Julius Oatts, Tova Mannis, Jeremy D. Keenan,
Jennifer Rose-Nussbaumer. Ophthalmology, University of California,
San Francisco, San Francisco, CA.
Purpose: Several studies have compared corneal thickness
measurements in normal eyes taken with different devices including
ultrasound pachymetry (USP), anterior segment optical coherence
tomography (ASOCT), and rotating Scheimpflug imaging
(Pentacam). Additionally, clinicians often grade and monitor corneal
thickness over time, though no studies have correlated this with
pachymetric data. We performed a prospective, cross-sectional
clinical study to assess the relationship between corneal thinning as
measured by clinicians compared to USP, ASOCT, and Pentacam.
Methods: Patients with corneal thinning were identified through
chart review and asked to return to undergo clinical examination by
two cornea specialists as well as USP, ASOCT, and Pentacam. Two
cornea specialists used standardized clinical grading to estimate
corneal thinning on slit lamp exam compared with normal cornea.
Pachymetric values were obtained directly from imaging devices.
Inter-rater agreement was assessed using the Intraclass Correlation
Coefficient (ICC) performed using Stata 13.0.
Results: Twenty-two patients with corneal thinning were available
for grading. Thinning was secondary to infectious keratitis (27%),
herpetic keratitis (18%), peripheral ulcerative keratitis (18%),
neurotrophic keratitis (18%), radiation (9%), trauma (4.5%) and
Fuchs marginal keratitis (4.5%). Mean percent stromal thinning of
grader 1 was 50% (SD 31), grader 2 was 49% (SD 33). ICC between
graders was 0.92 (95% CI 0.81 to 1.02). ASOCT showed an ICC of
0.76 (0.48 to 1.04) for percent stromal thinning with grader 1 and
0.76 (0.47 to 1.04) with grader 2. By contrast, Pentacam had an ICC
of 0.13 (0.00 to 0.93) with grader 1 and 0.00 (0.00 to 0.87) with
grader 2. USP had an ICC of 0.16 (0.00 to 0.95) for grader 1 and
0.72 (0.35 to 1.09) for grader 2. Exam corresponded to the area of
maximal thinning on ASOCT 52% of the time and Pentacam 27% of
the time.
Conclusions: Two corneal specialists had a high degree of agreement
in location and degree of corneal thinning when examining patients
in a standardized manner. ASOCT demonstrated the most agreement
with clinical grading compared with USP and Pentacam. Pentacam
underestimated degree of thinning compared to clinical grading and
ASOCT. ASOCT may be a beneficial addition to clinical grading in
patients with corneal thinning.
Commercial Relationships: Julius Oatts, None; Tova Mannis,
None; Jeremy D. Keenan, None; Jennifer Rose-Nussbaumer, None
Can we determine the true central corneal thickness? The
significance of the discrepancy between the ultrasound and
optical coherence tomography pachymetry
Magdalena Niestrata-Ortiz1, Olivia Li3, Martin Haywood2,
Nigel Davies2. 1Edinburgh University, London, United Kingdom;
2
Chelsea and Westminster Hospital, London, United Kingdom;
3
Moorfields Eye Hospital, London, United Kingdom.
Purpose: Central corneal thickness (CCT) plays an important role in
the diagnostic and therapeutic assessment of ocular pathologies. The
aim of the study was to assess the comparability of central corneal
thickness measurements by optical coherence tomography (OCT)
and ultrasound pachymetry and to determine their reliability and
interchangeability as clinical tools. Based on the universal use of
both methods in clinical practice, we hypothesised there would be no
significant difference between the measurements.
Methods: We compared measurements by Optovue RTvue-100
optical coherence tomography (OCT) and Pachmate ultrasound (US).
The data for OCT pachymetry was collected prospectively for 112
glaucoma and glaucoma suspect patients over a 5 month period.
Using Bland-Altman plots, this was compared with retrospectively
collected data on previously performed ultrasound pachymetry for the
same patients. The statistical tools used in the data analysis included
standard deviation, standard error and confidence intervals.
Results: A statistically significant difference was shown between
pachymetry results obtained by US and OCT, with the mean
difference of 26.00µm (standard error 1.72; standard deviation 18.19)
and 31.13µm (standard error 1.84; standard deviation 19.39) and the
95% confidence interval of ± 3.41µm and ± 3.65µm for the right and
left eye, respectively. The absolute measurement difference between
the two methods was 0-135µm and 0-100µm for the right and left
eye, respectively.
Conclusions: No gold standard exists for CCT measurement.
Higher US thickness may be due to off-centre readings, exacerbated
by Bell’s reflex. As CCT impacts corrected intraocular pressures
and monitoring corneal endothelial failure, we challenge the
interchangeable use of US and OCT pachymetry in clinical settings.
Commercial Relationships: Magdalena Niestrata-Ortiz, None;
Olivia Li, None; Martin Haywood, None; Nigel Davies, None
Confocal microscopy for the analysis of the sub-basal corneal
nerve plexus alternations in elderly and diabetic patients using a
new software technology
Hatim I. Batawi1, 2, Nabeel Shalabi1, 2, Madhura Joag1,
Tulay Koru-Sengul3, Jorge Rodriguez4, Parke Green4,
Mauro Campigotto5, Carol Karp1, Anat Galor1, 2. 1Ophthalmology,
Bascom Palmer Eye Institute, University of Miami, Miami, FL;
2
Ophthalmology, Miami Veterans Affairs Medical Center, Miami, FL;
3
Department of Public Health Sciences, University of Miami, Miller
School of Medicine, Miami, FL; 4Nidek, Inc, Miami, FL; 5Nidek
Technologies, SRL, Italy, Italy.
Purpose: To study corneal sub-basal nerve plexus parameters by invivo corneal confocal microscopy using a new software technology
and examine the effect of demographics and diabetes mellitus on
corneal nerve morphology.
Methods: A Confoscan4 (Nidek Technologies Srl, Padova, Italy) was
used to image the corneal sub-basal nerve plexus in 60 right eyes.
Images were analyzed using a new semi-automated nerve analysis
software program (The Corneal Nerve Analysis tool) which evaluated
9 factors including nerve fibers length (NFL), nerve fibers length
density (NFLD), number of fibers, number of branches, number of
bifurcations, number of trunks, number of beadings, beading density
and nerve tortuosity. The main outcome measure was the examination
of sub-basal corneal nerve plexus morphology by demographic
factors and co-morbidities.
Results: Inter- and intra- operator reproducibility was good for the
9 parameters studied (Intraclass Correlations 0.73 to 0.97). Image
variability between two images within the same scan was good for
all parameters (ICC 0.66 to 0.80). Overall, older individuals had
lower nerve densities with several confocal parameters negatively
correlating with age (NFL, NFLD, number of fibers and trunks, and
number of beadings), (r= -0.37 to -0.52, p<0.05 for all). In a similar
manner, diabetic patients also had lower nerve densities than nondiabetes with noted differences in NFL, NFLD, number of fibers
and trunks and number of beadings (p<0.05 for all). On the other
hand, diabetics had more tortuous nerves than their non-diabetic
counterparts (p=0.03).
Conclusions: The Corneal Nerve Analysis tool is a reproducible
diagnostic technique for analysis of the sub-basal corneal nerve
plexus. Older age and diabetes mellitus are associated with a
significant reduction in several sub-basal corneal nerve plexus
parameters.
Commercial Relationships: Hatim I. Batawi; Nabeel Shalabi,
None; Madhura Joag, None; Tulay Koru-Sengul, None;
Jorge Rodriguez, None; Parke Green, None; Mauro Campigotto,
None; Carol Karp, None; Anat Galor, None
Support: Supported by the Department of Veterans Affairs, Veterans
Health Administration, Office of Research and Development, Clinical
Sciences Research and Development’s Career Development Award
CDA-2-024-10S (Dr. Galor) Supported in part by NIH Center Core
Grant P30EY014801, Research to Prevent Blindness Unrestricted
Grant, Department of Defense (DOD- Grant#W81XWH-09-1-0675),
the Ronald and Alicia Lepke Grant, and the Lee and Claire Hager
Grant, Jimmy and Gaye Bryan Grant, The Richard Azar Family
Grant, and the Gordon Charitable Trust (all institutional grants).
Validation and quantification of neurotaxis effect in a chick
dorsal root ganglion/corneal stromal cell model
Geraint P. Williams1, 2, Gary H. Yam1, 3, Xiao-Wen Lee1,
Jodhbir S. Mehta1, 2. 1Singapore Eye Research Institute, Singapore,
Singapore; 2Singapore National Eye Centre, Singapore, Singapore;
3
ACP-EYE Duke-NUS Graduate Medical School, Singapore,
Singapore, Singapore.
Purpose: Nerve regeneration following disease or surgery could be
optimised in a microenvironment that mimics the healthy corneal
stromal. We developed an in-vitro system to study the effect of
corneal stromal cells on nerve outgrowth from chick dorsal root
ganglion (DRG). In order to reliably quantify neurite growth, we
validated a concentric circle intersection (CCI) quantification assay in
a primary DRG culture system, employing stromal cell conditioned
medium (CM) as a stimulant to nerve growth.
Methods: Primary human corneal stromal cells were obtained from
research grade cadaveric corneal stroma under full consent and
propagated to P5 in serum culture. After washing of serum factors
from culture for 24 hours using serum-free media (SFM), cells were
incubated in fresh SFM for 48 hours for medium conditioning. The
collected CM was applied to single chick DRG culture, harvested
from E10 fetuses. After 72 hours, DRG neurite explants were fixed
in situ and TuJ1-positive neurites were imaged. Using CCI assay
performed by two masked observers, the intersections of neurite
processes with concentric circles at every 100μm interval were
quantified as total count per DRG. Agreement was calculated using
Bland-Altman plots together with mean difference and level of
agreement and by correlation.
Results: Using 12 DRGs incubated with CM, quantification of
CCI showed a reduction in neurites with increasing distance from
DRG centre, with an average of 44.5 (SD 21.8) intersections at
500um through to 2.3 (SD 4.7) at 1500um. Individual intra-observer
agreement was high for both observer 1 and 2 with a mean difference
of <1.9 and <1.5 intersections, respectively, and a correlation of 0.96
to 1.0 (p<0.0001), respectively. Inter-observer agreement was also
high with a mean difference of <2.8 intersections and a correlation of
0.98 to 1.0 (p<0.0001).
Conclusions: CCI quantification assay measuring DRG neurite
extension demonstrated a predictable decline in neurite outgrowth
from DRG centre. A masked validation exercise revealed that this
means of assessing neurites intersections at defined distance from
DRG centre could offer an objective, simple and reliable method for
measuring nerve outgrowth ex vivo. This method can be applied to
study neurotaxis effect exerted by molecules, such as growth factors,
inhibitors, and in co-culture models.
Commercial Relationships: Geraint P. Williams, None;
Gary H. Yam, None; Xiao-Wen Lee, None; Jodhbir S. Mehta,
None
Support: NMRC Translational clinical research grant R1021,
Singapore.
Good inter-center reproducibility of quantification of the corneal
subbasal nerve plexus using in vivo corneal confocal microscopy
Hoang-Ton Nguyen1, Mariska Nieuwenhoff2, 4, Netty Dorrestijn3,
Frank J.P.M. Huygen4, Sjoerd P. Niehof4, Justin
L. Hay2, Annette C. Moll1, Geert Jan Groeneveld2, 5. 1Ophthalmology,
VU University Medical Center, Amsterdam, Netherlands; 2Centre
for Human Drug Research, Leiden, Netherlands; 3Rotterdam
Ophthalmic Institute, Rotterdam, Netherlands; 4Anesthesiology and
Pain Medicine, Erasmus MC University Medical Center, Rotterdam,
Netherlands; 5Neurology, VU University Medical Center, Amsterdam,
Netherlands.
Purpose: In vivo corneal confocal microscopy is a novel noninvasive imaging technique, in which corneal nerve morphology
can be imaged in great detail. Automated software to quantify these
corneal nerves is readily available, with acceptable inter-rater and
inter-operator reproducibility. Recently, studies have demonstrated
decreased corneal nerve density in various neuropathies such as
diabetic neuropathy, idiopathic small fiber neuropathy, sarcoidosis
and hereditary neuropathies. To date, there are no multi-center
studies employing in vivo corneal confocal microscopy. Inter-center
reproducibility has not been described in the literature, therefore we
aim to assess inter-center variability in corneal nerve parameters
measured by corneal confocal microscopy.
Methods: Images of 54 eyes of 27 participants (19 male, 8 female)
were obtained with corneal confocal microscopy (HRT III with
Rostock Cornea Module) in two centers by two operators, 4-10 days
apart. Both operators received the same training (prof. Malik and
colleagues, University of Manchester). Per eye, three high-quality
images of the corneal subbasal nerve plexus were selected. Images
were analyzed using ACCMetrics V2.0 (University of Manchester)
to determine corneal nerve fiber length (CNFL), corneal nerve fiber
density (CNFD) and corneal nerve branch density (CNBD). The
mean values of both eyes were averaged. Inter-center variability
was calculated using Bland-Altman plots and intraclass correlation
coefficient (ICC).
Results: Using Bland Altman plots, bias ± standard deviation was
-2.1 ± 3.9 for CNFD, -1.7 ± 2.0 for CNFL and -12.3 ± 11.5 for
CNBD. ICC was 0.82 for CNFL, 0.83 for CNFD, 0.64 for CNBD.
Conclusions: In our study, ICC was very good for CNFL and
CNFD, but not for CNBD. Even though both operators had different
experience in ophthalmic examinations (one MD/PhD student,
one ophthalmic resident) we managed to obtain high intraclass
correlation. Therefore we conclude that in vivo corneal confocal
microscopy of the corneal subbasal nerve plexus is suitable for multicenter studies when CNFL and CNFD are used as outcome measures.
Commercial Relationships: Hoang-Ton Nguyen, None;
Mariska Nieuwenhoff, None; Netty Dorrestijn, None; Frank
J.P.M. Huygen, None; Sjoerd P. Niehof, None; Justin L. Hay,
None; Annette C. Moll, None; Geert Jan Groeneveld, None
Repeatability and clinical utility of a novel method to measure
in-vivo corneal nerve migration in diabetic neuropathy
Katie Edwards1, Khaled Al Rashah1, Nicola Pritchard1,
Christopher Poole1, Cirous Dehghani1, Anthony Russell4, 5,
Rayaz A. Malik2, 3, Nathan Efron1. 1School of Optometry and
Vision Science, and Institute of Health and Biomedical Innovation,
Queensland University of Technology, Kelvin Gove, QLD, Australia;
2
Center for Endocrinology and Diabetes and Institute of Human
Development, University of Manchester, Manchester, United
Kingdom; 3Weill Cornell Medical College in Qatar, Doha, Qatar;
4
School of Medicine, University of Queensland, Brisbane, QLD,
Australia; 5Department of Diabetes and Endocrinology, Princess
Alexandra Hospital, Woolloongabba, QLD, Australia.
Purpose: We developed a novel technique to measure in-vivo corneal
nerve migration, and determined its repeatability and clinical utility
in diabetic neuropathy.
Methods: Wide-field montages of the sub-basal corneal nerve plexus
were generated at baseline and after three weeks for 14 participants
(control participants (n=4); diabetic participants without (n=5) and
with (n=5) neuropathy). Montages were manually examined side
by side to identify a referent landmark in the inferior whorl region
and 20 additional nerve landmarks throughout each montage. An
algorithm was developed to measure nerve migration by quantifying
the movement of the nerve landmarks relative to the inferior whorl
landmark over the three-week period, with migration reported in
microns per week. Intra- and inter-observer repeatabilities were
determined by measurement of nerve migration on the pair of
montaged images by one researcher on two occasions, five days
apart, and by two observers respectively. A paired t-test was used
to compare the difference between the two measurements. Bland–
Altman plots were generated to assess agreement and the intra-class
correlation coefficient (ICC) was calculated to estimate repeatability.
To illustrate it’s clinical utility, we compared the average nerve
migration between the three groups.
Results: The mean difference between observations 1 and 2 was 0.92
µm/week (p=0.10), with an ICC of 0.99 (95% confidence interval
(CI)=0.99-1.00). The mean difference between observer 1 and 2 was
3.05 µm/week (p=0.04), with an ICC of 0.97 (95% CI=0.91-0.99).
Movement of individual points was dependent on their distance from
the inferior whorl, so the method was altered to keep the distance of
the measured points at an average of 1,200±50µm from the inferior
whorl. Using this method, the mean difference between observers
was 0.38 µm/week (p=0.38), with an ICC of 0.97 (95% CI=0.991.00). The average nerve migration rate was 32.7, 53.4 and 45.3 µm/
week for the diabetic individuals with and without neuropathy and
the control participants, respectively (p=0.18).
Conclusions: This novel imaging technique allows repeatable and
non-invasive measurement of in-vivo corneal nerve migration. The
results indicate that patients with diabetic neuropathy may have a
reduced rate of nerve migration, which should be explored in future
studies.
Commercial Relationships: Katie Edwards; Khaled Al Rashah,
None; Nicola Pritchard, None; Christopher Poole, None;
Cirous Dehghani, None; Anthony Russell, None; Rayaz A. Malik,
None; Nathan Efron, None
Support: Juvenile Diabetes Research Foundation Grants 8-2008-362
and 3-2013-211
Automatic estimation of short- and long-range tortuosity of
corneal sub-basal nerves
Alfredo Ruggeri, Pedro Guimarães. University of Padua, Padua, Italy.
Purpose: To automatically grade corneal nerve images according to
two classes of nerve tortuosity (NT), representing respectively shortrange or long-range directional changes.
Methods: The clinical perception of corneal NT was recently shown
to have two distinct forms, characterized by either short-range
(high frequency, low amplitude) or long-range (low frequency, high
amplitude) directional changes (Lagali et al., IOVS Aug 2015).
Ground-truth (GT) for each NT form in 30 confocal microscopy
images was provided by 7 expert graders, who visually examined the
images and ordered them by increasing nerve tortuosity, according
to either tortuosity definition (see Fig.1 for examples). Their average
orderings were assumed as GT.
Using the same 30 images, we traced corneal nerves with a custom
computerized procedure and computed for each image more than
one hundred NT metrics, using several different mathematical
formulations, including angle-, twist- and curve-based metrics. The
ranking estimated by each formulation was then compared to the GT
(for both short- as well as long-range NT) using the Spearman Rank
Correlation (SRC) coefficient.
Results: Table 1 presents the SRC coefficients for the bestperforming NT metrics. It appears that angle-based metrics are the
best to capture short-range NT, whereas twist-based metrics are the
best for expressing long-range NT.
Conclusions: The results indicate which of the many mathematical
formulations for NT we considered is the best for describing either
the short- or the long-range NT. The next step will be to assess to
what degree the various corneal pathologies exhibit one or the other
NT class in their sub-basal nerve patterns.
Figure 1: Corneal nerves image with the highest tortuosity rank for
the short-range (top) and the long-range tortuosity (bottom).
Table 1. SRC coefficients with respect to the short-range GT or the
long-range GT for angle-based (top), curve-based (middle) and twistbased (bottom) NT metrics.
Commercial Relationships: Alfredo Ruggeri, None;
Pedro Guimarães
Quantitative alterations in nerve morphology differentiate
affected and unaffected eyes in Unilateral Keratoconus
RUSHAD SHROFF1, Rohit Shetty1, Arkasubhra Ghosh2,
Natasha Pahuja1, AARTI AGRAWAL1, HARSHA NAGARAJ1.
1
REFRACTIVE CORNEA AND CATARACT, NARAYANA
NETHRALAYA, BANGALORE, India; 2GROW RESEARCH
LABORATORY, NARAYANA NETHRALAYA, BANGALORE, India.
Purpose: Unilateral Keratoconus (KC) provides an objective model
to study corneal morphology with other risk factors remaining
constant. In vivo confocal microscopy (IVCM) has been used to
image corneal sub basal nerve plexus (SBNP) changes in KC,
however its role in unilateral KC has not been evaluated so far. The
aim of this study is to highlight changes in SBNP in unilateral KC
and gain insight regarding the role of corneal nerves in pathogenesis,
diagnosis and as a marker for disease progression in KC.
Methods: In this prospective cross-sectional study, 33 eyes of 33
patients with evidence of keratoconus in one eye (Group 3) were
compared with the other normal eye of the same patients (Group 2)
and with 30 eyes of healthy age matched controls (Group 1) for the
morphology of corneal nerves. Patients with history of contact lens
use, ocular surgery, trauma and bilateral KC were excluded from
the study. All patients underwent detailed ophthalmic examination
followed by topography with Pentacam HR (Oculus Optikgerate
GmBH, Wetzlar Germany) and in vivo confocal microscopy (IVCM)
using the Rostock Corneal Module/Heidelberg Retina Tomograph
II (Heidelberg Engineering GmbH, Dossenheim, Germany). Five
images from the center of the cornea obtained with IVCM were
analyzed using an automated CCmetrics software version 1.0
(University of Manchester, UK) for the changes in sub-basal plexus
of nerves in all eyes. Statistical analyses were performed using
Stata version 12.1 (StataCorp, College Station, TX, USA) statistical
software.
Results: Intergroup comparison showed statistically significant
reduction in the corneal nerve fiber density (CNFD) and length
(CNFL) in Group 3 as compared to Group 1 (p<0.001 and p=0.001
respectively) and Group 2 (p=0.01 and p=0.02 respectively).
Intragroup analysis in Group 3 revealed that while 18 eyes had
decentered cones, 15 had centered cones. Though all the parameters
were found to be higher in decentered cones, only the corneal nerve
branch density (CNBD) was found to be statistically significant
(p<0.01).
Conclusions: This study underscores the importance of IVCM in
early diagnosis of keratoconus. It also highlights the importance
of alterations in corneal nerve morphology that can be used as an
imaging marker for early diagnosis, monitoring of progression and
for prognostication of keratoconus.
Commercial Relationships: RUSHAD SHROFF, None;
rohit shetty, None; Arkasubhra Ghosh, None; natasha pahuja,
None; AARTI AGRAWAL, None; HARSHA NAGARAJ, None
Purpose: To investigate Color LED corneal shape measurements
of corneal aberration and irregularity indicators in healthy and
keratoconus eyes.
Methods: Cassini Color LED corneal shape analyzer (Cassini
BV, The Hague, Netherlands) measurements were done for 63
Healthy (38 OD, 25 OS) and 24 Keratoconus (9 OD and 15 OS)
eyes. Corneal aberrations (coma – RMS 3rd order, trefoil – RMS
3rd order, spherical (SA) – Z(4,0) and quadrafoil – RMS 4th order)
were characterized using Zernike convention @ 6mm diameter.
Surface Regularity Index (SRI) and Surface Asymmetry Index (SAI)
as described by Klyce and Wilson (Refract Corneal Surg 1989)
were evaluated @ 4.23 mm diameter. Mann Whitney test was used
to compare measurements between normal and keratoconus eyes.
Pearson correlation coefficient, R, of each aberration and index
compared to SRI and SAI were calculated. Sensitivity and specificity
of corneal aberrations and indices were determined with respect to its
performance in diagnosing keratoconus.
Results: Coma is strongly correlated to SAI for both healthy and
keratoconus eyes (see Table 1). For healthy eyes the correlation
to SRI/SAI gets stronger/weaker with more complexity on the
irregularity of the aberration (coma < trefoil < quadrafoil / coma >
trefoil > quadrafoil. This pattern seems to disappear with Keratoconus
eyes which may be attributed to the dominance of coma in the shape
of keratoconus eyes. SA does not correlate with either SRI and SAI
for healthy eyes, however for keratoconus eyes the correlation is
strong.
Conclusions: SAI and coma agrees well for both healthy and
keratoconus eyes indicating that coma aberration may well be an
alternative for SAI in measuring corneal asymmetry. The correlations
confirm and give insights that SRI is an indicator of local irregularity
which could be a contribution of the actual corneal shape and tear
film distribution. Correlations found for SA suggest that corneal
dystrophy in keratoconus eyes involves simultaneous increase in
SA, SRI and SAI. There is a similar statistical significant difference
between corneal aberrations and irregularity indices of healthy and
keratoconus eyes as measured by color LED corneal shape and
there is no statistical significant difference between sensitivity of
keratoconus diagnosis. However, coma aberration, SRI and SAI are
superior to trefoil, spherical and quadrafoil aberration in specificity of
keratoconus diagnosis.
Color LED corneal shape analysis of aberrations and irregularity
indicators in healthy and keratoconus eyes
Victor Sicam3, Stijn Klijn1, Maarten Huijbregtse3,
Annette J. Geerards2, Nicolaas J. Reus1. 1Rotterdam Ophthalmic
Institute, Rotterdam, Netherlands; 2Rotterdam Eye Hospital,
Rotterdam, Netherlands; 3Cassini BV, The Hague, Netherlands.
Commercial Relationships: Victor Sicam, Cassini BV, Cassini BV
(P); Stijn Klijn, None; Maarten Huijbregtse; Annette J. Geerards,
None; Nicolaas J. Reus, None
Association of fluorescein anterior corneal mosaic and corneal
K-structures by in vivo laser confocal microscopy in patients with
keratoconus
Akira Kobayashi, Hideaki Yokogawa, Toshinori Masaki,
Natsuko Mori, Kazuhisa Sugiyama. Ophthalmology & Visual
Science, Kanazawa Univ Sch of Medicine, Kanazawa, Japan.
Purpose: To report the in vivo laser confocal microscopy findings of
a cornea with keratoconus with special attention to the abnormality of
Bowman’s layer and sub-Bowman’s fibrous structures (K-structures).
Methods: Twelve kertoconic eyes in 6 keratoconus patients (2 male,
4female, mean 39.7 years old) were included in this study. Slit lamp
biomicroscopic photo with or without fluorescein staining were
taken. The existence of anterior corneal mosaic (ACM) after eyelid
rubbing under fluorescein staining were documented. In vivo laser
confocal microscopic examinations were performed for all patients in
both central cone and pheripheral cornea to examine the existence of
K-structures.
Results: ACM was observed in 5 eyes (41.7%) in cone area and 12
eyes (100%) in peripheral cornea. Also, K-structures was observed
in 5 eyes (41.7%) that had ACM in cone area and 12 eyes (100%) in
peripheral cornea. The existence and non-existence of the K-structure
was completely matched (100%) with the existence of ACM in both
central cone and pheripheral cornea.
Conclusions: The existence of ACM and K-structures in both central
cone and pheripheral cornea showed perfect accord in keratoconus
patients, indicating a strong association of ACM and K-structure
in patients with keratoconus. Further study using larger groups of
keratoconic patients is required to fully understand the significance
of ACM/K-structurse in keratoconic eyes and their association with
Bowman’s layer.
Commercial Relationships: Akira Kobayashi, None;
Hideaki Yokogawa, None; Toshinori Masaki, None;
Natsuko Mori, None; Kazuhisa Sugiyama, None
Quantification of Kayser-Fleischer ring using Scheimpflug
imaging
Niklas Telinius11, , Niklas Telinius11, , Peter Ott2, Jesper Hjortdal1.
1
Ophthalmology, Aarhus University Hospital, Aarhus, Denmark;
2
Department of Medicine (Hepatology and Gastroenterology), Aarhus
University Hospital, Aarhus, Denmark.
Purpose: In Wilson’s disease (WD) a defect in cellular copper
transport leads to accumulation of copper in tissues, which can be
seen in the eye as a Kayser-Fleischer ring (KFR). The KFR can be
used to diagnose and monitor the disease. The purpose of this study
was to assess if Scheimpflug imaging can be used to visualize KFR
and provide an objective way to quantify KFR.
Methods: The study was a retrospective study of 16 patients with
WD. All patients were examined with slit lamp and Schempflug
imaging (Pentacam, Oculus) as part of routine clinical practice.
Clinical examination was conducted by one of two cornea specialists.
Pentacam images were analyzed in ImageJ. The inferior part
of cornea was analyzed by measuring the brightness (0-256) in
approximately 30 points along a line perpendicular to the corneal
surface. 9 healthy volunteers of similar age were included as
controls. Statistical analysis included one-way ANOVA and the postBonferroni test.
Results: A KFR was identified on the inferior part of cornea on
slit lamp examination in 7 out of 16 WD patients and confirmed on
Pentacam images. The posterior part of cornea was significantly
(p<0.05) brighter in WD patients with KFR compared to WD patients
without KFR and controls; 220±19 compared to 106±14 and 92±10.
When normalized to the peak value of the anterior cornea all patients
with a KFR had a ratio over 1 (1.4±0.14) whereas all patients without
KFR and controls had a ratio below 1 (0.64±0.1 and 0.66±0.1),
p<0.05. There was no significant difference between controls and WD
patients without KFR in absolute brightness or ratio (p>0.05). Four
patients were examined 2 or 3 times over 2 years and the Pentacam
images correlated with the slit lamp examination.
Conclusions: We conclude that Scheimpflug imaging can
successfully visualize KFR and that the images can be used for
quantification. The images correlated well with the slit lamp
examination. Further analysis and follow up examinations are needed
for determining the optimal way to use Scheimpflug imaging for
monitoring KFR.
Commercial Relationships: Niklas Telinius, None; Peter Ott,
None; Jesper Hjortdal, None
Corneal In Vivo Confocal Microscopy in Ocular Graft versus
Host Disease
Kunal Suri1, Ahmad Kheirkhah1, Yureeda Qazi1,
Michael A. Arnoldner1, Pedram Hamrah2, Reza Dana1.
1
Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA;
2
Ophthalmology, Tufts Medical Center, Boston, MA.
Purpose: To compare the density of corneal immune cells and nerves
in patients with dry eye disease (DED) with and without ocular graft
versus host disease (GVHD) using in vivo confocal microscopy
(IVCM).
Methods: This retrospective study included 54 patients who had
DED with (n=33) or without (n=21) ocular GVHD. All had moderate
to severe DED, an Ocular Surface Disease Index (OSDI) score >22
and corneal fluorescein staining (CFS) score ≥4/15 (National Eye
institute; NEI scale). All had a complete ophthalmic evaluation,
which consisted of symptom questionnaires (including OSDI and
Symptom Assessment iN Dry Eye [SANDE]) and clinical assessment
including CFS, conjunctival lissamine green staining, tear breakup time (TBUT), and Schirmer’s test. All patients also had laserscanning IVCM (Heidelberg Retina Tomograph 3, Heidelberg,
Germany) of the central cornea and superior palpebral conjunctiva.
The densities of the following cells were measured in IVCM images
using ImageJ software by two masked observers: corneal subbasal
immune dendritic cells (DC), corneal subbasal nerve fibers, and
conjunctival epithelial inflammatory cells.
Results: There were no significant differences between the GVHD
and non-GVHD groups in terms of age or gender. There were no
significant differences between GVHD and non-GVHD groups
in OSDI (57.9 ± 20.5 and 53.8 ± 21.0, respectively, P=0.49) and
SANDE (63.1 ± 19.1 and 69.7 ± 16.9, respectively, P=0.36).
However, the GVHD group had significantly worse CFS (7.1±2.4
vs 5.4±1.8, P=0.01), TBUT (2.4±1.8 vs 4.3±1.9 seconds, P=0.001),
and Schirmer scores (3.9±4.3 vs 7.6±6.8 mm, P=0.01) compared
to the non-GVHD group. In IVCM images, corneal DC density,
corneal subbasal nerve fiber density, and conjunctival epithelial
inflammatory cell density were 148±135 cells/mm2, 16.3±6.1 mm/
mm2 and 670±267 cells/mm2 in the GVHD group and 122 ± 99 cells/
mm2, 18.3 ± 5.1 mm/mm2 and 572 ± 271 cells/mm2 in the non-GVHD
group. After adjusting for DED severity, none of the IVCM parameter
was significantly different between the groups (P=0.41, P=0.21 and
P=0.20 respectively).
Conclusions: After adjusting for DED severity, cell densities
assessed by IVCM are similar in patients with DED with or without
ocular GVHD. Therefore, the corneal and conjunctival IVCM
findings in ocular GVHD may solely be due to DED and not the
underlying disease.
Commercial Relationships: Kunal Suri, None;
Ahmad Kheirkhah, None; Yureeda Qazi, None;
Michael A. Arnoldner; Pedram Hamrah, None; Reza Dana, None
Support: Research to Prevent Blindness
Quantitative analysis of Central Corneal Sub-basal Inflammatory
cells/Dendritic Cells in Large-area Mosaic images obtained by In
Vivo Confocal Microscopy (IVCM)
Reza A Badian1, 2, Tor P. Utheim3, 4, Stephan Allgeier5,
Bernd Koehler6, Neil S. Lagali7. 1Faculty of Health Sciences,
Buskerud and Vestfold University College, Drammen, Norway; 2The
Norwegian Dry Eye Clinic, Oslo, Norway; 3Department of Medical
Biochemistry, Unit of Regenerative medicine, Oslo, Norway; 4Vestre
Viken Hospital Trust, Department of Ophthalmology, Drammen,
Norway; 5Karlsruhe Institute of Technology, Institute of Applied
Computer Science/ Automation, Karlsruhe, Germany; 6Karlsruhe
Institute of Technology, Institute of Applied Computer Science/
Automation, Karlsruhe, Germany; 7Department of Ophthalmology,
Faculty of Health Sciences, Linköping University, Linköping,
Sweden.
Purpose: To quantify and examine the distribution of inflammatory
dendritic cells (DCs) in large-area mosaic images of the cornea
obtained by In Vivo Confocal Microscopy (IVCM).
Methods: 82 subjects were included in the study. Using laserscanning in vivo confocal microscopy (Hidelberg HRT 3 with Rostck
corneal module), the central cornea in both eyes of study subjects
(163 eyes) was imaged. Subsequently images from the sub-basal
layer were used to assemble the best possible mosaics. Mosaics were
analyzed manually by an experienced examiner using image J. Subbasal inflammatory cells were distinguished, demarcated and grouped
into three distinct categories: Type 1) Mature dendritic cells (mDCs);
Type 2) Immature dendritic cells (imDCs); Type 3) Globular cells
(GCs); reflective, circular-shaped, with no visible dendritic processes
and variable size.
Results: Average size of mosaic images was 6.0 mm2, comprising
an average of 37 single confocal fields of view. The process of
designation, categorization and counting of inflammatory cells
for large-area mosaics took an average time of 19.38 minutes per
mosaic image. The DC counting time varied between values as high
as 147 minutes for a complex mosaic where corneal sub-basal layer
was extensively dominated with variety of inflammatory cells and
values as low as 1 minute for simple mosaic images with very few
cells in the sub-basal layer. The most prevalent inflammatory cells
in this study were type 2 immature dendritic cells (imDCs) and least
prevalent cells were type 1 mature dendritic cells (mDCs).
Complete quantitative analysis and geographic distribution patterns
are presented.
Conclusions: Inflammatory cells in the corneal sub-basal layer can
be analyzed in vivo in a large field of view of the central cornea.
The results provide information regarding the inflammatory status of
the cornea in a more complete manner than would be possible from
single image analysis, thereby possibly facilitating better diagnosis
and management of inflammatory or other pathologic conditions.
Commercial Relationships: Reza A Badian, None; Tor P. Utheim,
None; Stephan Allgeier, None; Bernd Koehler, None;
Neil S. Lagali, None
Techniques to perform Specular Microscopy: Endothelial Cell
Counting (ECC) and Endothelial Mosaic Mapping (EMM)
Fernando C. Abib1, 2. 1Clinic Hospital of Federal University of
Parana, Curitiba, Brazil; 2Cornea, Dr. Fernando Abib Eye Clinic,
Curitiba, Brazil.
Purpose: The main task is to know the endothelial behavior to plan
an objective way to perform the Corneal Endothelial Mosaic (CEM)
examination. The CEM was described according the autor, ARVO
2015, into Regular, Irregular and Chameleon Pattern. The Regular
Pattern can be examined by No Contact (NC) Corneal Specular
Microscope (CSM), Irregular and Chameleon Patterns by Contact
(C) CSM. This choice was based in how each type of CSM get the
images: NC CSM acquire CEM images when the patient sets the
target light, each model of this type of CSM has fixed positions; C
CSM can acquire CEM of any area of the endothelial surface by
sliding the objective lens on the cornea. Considering the differences
this study to presents a guideline to perform Specular Microscopy
choosing the best technique for CEM description.
Methods: All CSM were colleted in 2013 at the Dr. Fernando Abib
Eye Clinic, Curitiba, Brazil. The choose of the NC CSM (CSO,
Italy) or C CSM (BioOptics, USA) was determined by the algorithm
(Figure1A). The number of counted cells in each examination was
guided by Reliability Indexes of the Cells Analyzer USA Patent
software program (Technicall, Brazil) to obtain sample error < 5%
counting more endothelial cells than the calculated sample size
considering all endothelial cells inside of the different images as
necessary (Figure1B). The results were reported in terms of the
examinations performed for Endothelial Cell Count or Endothelial
Mosaic Mapping, respective clinical situations, and used CSM,
utilizing descriptive statistics.
Results: 879 CSM were performed in 2013; 556 (63.2%) were
performed for ECC finality and 323 (36.8%) for EMM: 91 (28.2%)
CSM with EMM finality could be performed with NC CSM or
C CSM to correct ECC and 232 (71.8%) of than with only C
CSM to correct but with focal description of the ECC. Number of
examinations: Clinical situations 21 (8.8%) for ECC and 217 (92,2%)
for EMM; Refractive situations: 370 (91.8%) for ECC and 33 (8.2%)
for EMM; Other non refractive surgical situations: 165 (69.3%) for
ECC and 73 (30.7%) for EMM.
Conclusions: The ECC was the most frequent finality of the CSM
(63.2%) and performed with NC CSM. A high number of CSM
needed the endothelial mapping (36.8%) to describe the reality of
the CEM. We strongly suggest C CSM with endothelial mapping
technique to perform CEM examination for Irregular and Chamaleon
patterns.
automated image processing application for rapid and objective
evaluation of the preprocessed donor cornea disks.
Methods: Processed donor cornea disks were stained using a
modification of Park et al. (2012). High magnification color
photographs were obtained. Images were stored on disk for further
evaluation. Image processing algorithms were developed using
MathLab software. Each image was preprocessed for brightness
and contrast and the image size was equalized for all images. Color
channels were extracted for the three main colors and analyzed. Each
channel was post processed separately. Multiple runs were performed
on several pilot images to determine the desired threshold values.
Each image analyzed by the algorithm was evaluated for adequate
feature extraction as compared to the original color photograph. The
second part of the algorithm aimed to extract the size of the corneal
disk by using image segmentation. The size of the disk was expressed
in number of pixels. Finally the software automatically calculated the
size of the extracted corneal features as a percentage of each cornea.
Results: 34 images processed and received from 2 separate Eye Bank
facilities were used to design and validate the algorithm. Satisfactory
automatic feature extraction of the corneal staining was achieved 100
%. In 5 cases additional image noise due to under- or overexposure
was manually filtered outside the area of feature extraction. The
algorithm was able to segment the cornea dimensions in 100% of the
images. Repeating the process for all images yielded 100% reliability.
The total size of the corneal features attributed to corneal alteration
varied from 8.53 - 45.83 % with median of 22.46.
Conclusions: Software image algorithm for automated quantitation
of the endothelium viability from Eye Bank processed corneas was
successfully developed and validated. It yielded 100% extraction
rate and reliability. It was found that the noise contained in the
images is a function of the photographic process which needs further
optimization.
Commercial Relationships: Brian Madow; Nicholas Sprehe,
None; Sung Lee, None; Patrick Gore, None; Stephen Kaufman,
None; Jackie Malling, None; Veronique Grimes, None;
Mitchell D. McCartney, None
Commercial Relationships: Fernando C. Abib
Automated quantitation of the endothelium alterations in Eye
Bank processed corneas
Brian Madow1, Nicholas Sprehe2, Sung Lee3, Patrick Gore2,
Stephen Kaufman3, Jackie Malling3, Veronique Grimes3,
Mitchell D. McCartney2. 1Ophthalmology, University of South
Florida, Tampa, FL; 2Lions Eye Institute, Tampa, FL; 3Minnesota
Lions Eye Bank, St Paul, MN.
Purpose: To design and validate image processing algorithm for
automated evaluation of corneal tissue processed at the Eye Bank.
The quality control of the corneal tissue supplied to the surgeons for
transplantation is of paramount importance. Currently the existing
methods are subjective and time consuming. We sought to develop
Endothelial cell recognition capability of central and paracentral
corneal areas using automatic specular microscopy counting
Richard Y. Hida1, Fábio U. Carvalho2, Ricardo Holzchuh2,
Fernando C. Abib3. 1Univ de Sao Paulo/Santa Casa de Sao Paulo, Sao
Paulo, Brazil; 2Santa Casa de São Paulo, São Paulo, Brazil; 3Federal
University of Parana, Curitiba, Brazil.
Purpose: Automatic counting of endothelial cells (EC) is subject to
errors due to the acquired image quality and to the accuracy of the
software, which can distort the specular microscopy (SM) results.
This study aims to compare the ability to recognize endothelial cells
using automatic counting of a non-contact (NC) SM between the
central area (CA) and paracentral areas (PCA) from the endothelial
mosaic.
Methods: Thirty-six right eyes of 36 patients without any ocular
abnormalities or previous surgeries were evaluated using NC SM
CEM-350 (NIDEK©). Nine endothelial images were obtained in
each examination: 1 image in CA (Group I) and 8 in PCA (Group II).
The SM software automatically counted the cells in each image. The
ability of the software to recognize endothelial cells was analyzed
by comparing the following outcome measurements: 1 – number (N)
of non-counted EC groups and total number of EC in these groups;
2 – Erroneously counted EC: Split EC (one cell counted as two or
more) and the average amount of EC created by this division, N of
cell clusters (multiple cells counted as one) and the average amount
of EC per cluster (Figure 1); 3 – Non-evaluated area of the image,
calculated as 1-[counted cells x average cell area (µm2)/image size
(µm2)]. We also compared the results of the SM parameters: counted
cells (CC), endothelial cell density (ECD), average cell area (AVG),
coefficient of variation (CV) and hexagonality percentage (HEX).
Descriptive statistics and two-tailed Student’s t test was used for
statistical analysis (p<0.05). The same examiner performed all
examinations.
Results: Results from EC recognition capability analysis (N of
non-counted EC groups, total number of EC in these groups and
erroneously counted EC) and SM parameters (CC, ECD, ACA, CV
and HEX) as well as the comparison between groups I and II are
presented in table 1.
Conclusions: EC automatic counting presented inaccuracies in both
groups, although they were more significant in group I. This study
suggests manual cell counting or individualized assessment with
correction of errors to minimize distortions in the results of specular
microscopy.
Commercial Relationships: Richard Y. Hida, None;
Fábio U. Carvalho, None; Ricardo Holzchuh, None;
Fernando C. Abib, None
Three types of cell recognition errors. A=Non-counted cells; B=Cell
cluster; C=Split cell; D=image C before automatic counting.
Diagnostic performance of the Endothelial/Descemet’s membrane
thickness versus endothelial cell density in the diagnosis of
corneal graft rejection
Georgios Kontadakis2, 1, Christopher Smith3, Daniel Kaitis3,
Rocio Bentivegna3, Jordan Winegar3, Sonia H. Yoo2, Victor L. Perez2,
Mohamed Abou Shousha2. 11st Department of Ophthalmology,
Ophthalmiatreio Eye Hospital of Athens, ATHENS, Greece; 2Bascom
Palmer Eye Institute, University of Miami, Miami, FL; 3Saint Louis
University Eye Institute, Saint Louis University, Saint Louis, MO.
Purpose: To assess the diagnostic performance of the Endothelial/
Descemet’s membrane thickness (En/DMT) as measured by the high
resolution optical coherence tomography (HD-OCT) in the diagnosis
of corneal graft rejection and compare it to that of endothelial cell
density (ECD) and central corneal thickness (CCT).
Methods: This prospective study includes 42 corneal grafts.
Assessment included detailed history, clinical examination,
automated assessment of the corneal endothelial layer with corneal
confocal microscopy, and corneal imaging with HD-OCT. Blinded
observers calculated manually CCT and EnDMT on HD-OCT
images. Eyes were divided into two groups, rejected and clear grafts.
CCT, EnDMT and ECD were compared between groups. Diagnostic
performance of EnDMT for the diagnosis of graft rejection was
evaluated using receiver operating characteristic (ROC) curves.
Results: CCT and EnDMT showed statistically significant
differences among groups. Clear grafts’ mean CCT was 525 μm (±74)
and mean EnDMT was 15μm (SD 3). Whereas, in the rejected grafts
group, mean CCT was 809μm (±251) and mean EnDMT was 48 μm
(SD 49, p<0.05 for corresponding comparisons, Mann-Whitney U
test). On the other hand, no significant difference in ECD was found
between the two groups. EnDMT showed better diagnostic accuracy
than CCT (EnDMT area under the curve, AUC was 0.93, p<0.001
and for CCT AUC was 0.81, p<0.001).
Conclusions: Endothelial/Descemet’s membrane thickness
demonstrated excellent accuracy in the diagnosis of corneal graft
rejection, which is significantly better than that of central corneal
thickness and endothelial cell density.
Commercial Relationships: Georgios Kontadakis, None;
Christopher Smith, None; Daniel Kaitis, None; Rocio Bentivegna,
None; Jordan Winegar; Sonia H. Yoo, None; Victor L. Perez,
None; Mohamed Abou Shousha, University of Miami (P)
Manual adjustment of specular images: Correcting what the
computer can’t see after DMEK
Zachary Mayko1, Beth Ann Benetz2, Christopher Stoeger1,
Harry Menegay2, Chris Donovan4, Mark A. Terry1, 3,
Jonathan H. Lass2. 1Lions VisionGift, Portland, OR; 2Department
of Ophthalmology and Visual Sciences, Case Western Reserve
University and UH Eye Institute, Cleveland, OH; 3Cornea, Devers
Eye Institute, Portland, OR; 4School of Medicine, Case Western
Reserve University, Cleveland, OH.
Purpose: To analyze a single clinical site’s method for capturing
endothelial cell density (ECD) and endothelial cell loss (ECL) by
an automated method with manual correction (AMMC) of specular
microscopy images in postop DMEK patients against the Konan
center method.
Methods: A consecutive series of fifty nine patients who underwent
DMEK surgery, and who had central donor graft as well as central 6
and 12 months post-surgery specular microscopic images available
were retrospectively identified. Specular microscopic images of
the central endothelium were first evaluated in the clinic using the
AMMC by a technician. Images were then masked and provided to
the Cornea Image Analysis Reading Center (CIARC) for analysis
using the Konan center method by certified readers using a dual
grading and adjudication process.
Results: At 6 Months postop the AMMC method was strongly
correlated with the Konan center method (r=0.92, p<0.001). We
found a mean ECD of 1939 ± 407 cells/mm2 and 1949 ± 437
cells/mm2 for the AMMC method and the Konan center method
respectively (p=0.67). At 12 Months postop the AMMC method was
strongly correlated with the Konan center method (r=0.92, p<0.001).
We found a mean ECD of 1843 ± 435 cells/mm2 and 1833 ± 470
cells/mm2 for the AMMC method and the Konan center method
respectively (p=0.63). Between 6 and 12 months, we observed an
average of 4 and 6 percent endothelial cell loss for this group when
evaluated using the AMMC and Konan center methods respectively.
No statistically significant difference was observed between the 2
methods for percent endothelial cell loss (p=0.36).
Conclusions: We found high levels of correlation between
endothelial cell densities when comparing an automated method with
manual correction to the standard Konan center method. Additionally,
each method provided equivalent amounts of percent endothelial cell
loss between 6 and 12 months. Our results suggest that as long as
a consistent method for evaluating endothelial cell density is used,
surgeons can accurately evaluate their percent endothelial cell loss
post DMEK transplant.
Commercial Relationships: Zachary Mayko, None; Beth
Ann Benetz, None; Christopher Stoeger, None; Harry Menegay;
Chris Donovan, None; Mark A. Terry, None; Jonathan H. Lass,
None
Do specular images of endothelial cell density post DMEK
preparation tell you anything new?
Christopher Stoeger1, Beth Ann Benetz2, 3, Zachary Mayko1,
Harry Menegay2, 3, Chris Donovan2, 3, Mark A. Terry4,
Jonathan H. Lass2, 3. 1Lions VisionGift, Portland, OR; 2Department
of Ophthalmology and Visual Sciences, Case Western Reserve
University, Cleveland, OH; 3UH Eye Institute, Cleveland, OH;
4
Cornea, Devers Eye Institute, Portland, OR.
Purpose: To compare a single eye bank’s measurement of endothelial
cell density (ECD) of Descemet Membrane Endothelial Keratoplasty
(DMEK) grafts before and after preparation using two separate
counting methods.
Methods: A series of sixty donor tissues were prepared for DMEK
surgery. Two to four specular microscoic images of the central
endothelium were taken both before and after preparation and ECDs
evaluated for a total of 345 unique images. An eye bank technician
analyzed each image using a center dot (CD) method and then
averaged those values. Images were then masked and provided to the
Cornea Image Analysis Reading Center (CIARC) for independent
analysis by certified readers using the HAI variable frame (VF)
method and a dual grading and adjudication process.
Results: Average ECD was 2678 ± 259 cells/mm2 before preparation
when measured by CD method. Final ECD was 2599 ± 280 cells/mm2
measured by the VF method (p<0.001, n=176). After preparation
the ECD was 2719 ± 265 cells/mm2 using the CD method and
2615 ± 344 cells/mm2 using the VF method (p<0.001, n=169). The
difference in ECD before and after DMEK preparation was not found
to be statistically significant when evaluated using the CD method
(p=0.19). The difference before and after DMEK preparation also
was not found to be statistically significant when evaluated using
the VF method (p=0.64). After DMEK preparation 80% (48/60) of
post-preparation ECD values fell within a range of ± 300 cells/mm2
using the CD method and 82%(49/60) fell within this range using the
VF method.
Conclusions: Our results show consistent measures of ECD
before and after DMEK preparation. We did not find a statistically
significant difference between ECD values before and after DMEK
preparation when comparing within each method (CD or the VF
method). While we did find statistically significant differences when
evaluating endothelial images between methods we feel they are not
clinically significant. Most eye banks when releasing donor tissue
for keratoplasty do not rely on a single image and cell count, but an
average of several images and ECDs to account for natural variation
in ECD along Descemet membrane. In this study, post-prep image
quality was diminished after DMEK preparation compared to the prepreparation images and may have contributed to the non-significant
ECD increase. More accurate results may be observed in future
studies with improved image quality.
Commercial Relationships: Christopher Stoeger, None; Beth
Ann Benetz, None; Zachary Mayko, None; Harry Menegay;
Chris Donovan, None; Mark A. Terry, None; Jonathan H. Lass, None
Development of a totally automated method for the reliable
morphometry of human corneal endothelium
Fabio Scarpa, Alfredo Ruggeri. University of Padova, Padova, Italy.
Purpose: To develop a fully automatic, reliable cell detection system
for corneal endothelium images from clinical specular and confocal
microscopy. The system should eventually allow the measurement
of the morphometric parameters that provide an objective clinical
assessment of corneal endothelium (density, pleomorphism,
polymegethism).
Methods: The proposed method detects corneal endothelial cells
contour in three main steps. 1: the centers of the endothelial cells
are automatically detected by convolving the original image with
customized two-dimensional kernels (Fig 1 - top); 2: a structure
made by connected vertices is derived from the centers using the
Euclidean distance (Fig 1 - middle); 3: the structure is fine-tuned by
means of a genetic algorithm, which combines information about the
typical regularity of endothelial cells shape with the pixels intensity
of the actual image (Fig 1 - bottom). The final structure of connected
vertices forms a set of polygons that fit the underlying cells contours.
From these contours the morphometric parameters of clinical interest
can then be easily computed.
Results: The procedure was applied to 15 images acquired with
the SP-3000P (Topcon) specular microscope and 15 images
acquired with the Confoscan4 (Nidek Technologies) confocal
microscope, from both healthy and pathological subjects. Ground
truth values for the three morphometric parameters were obtained
from manually carefully drawn cell contours. Results show that an
accurate automatic estimation is achieved: the mean absolute percent
difference between manual and automated estimation is 0.82 (range
0.00-4.38) for density, 3.13 (0.00-6.24) for pleomorphism and 3.95
(0.00-6.86) for polymegethism in images from specular microscopy,
and 1.49 (0.26-2.82) for density, 2.91 (0.00-6.49) for pleomorphism
and 5.26 (0.00-8.43) for polymegethism in images from confocal
microscopy. The procedure analyzes about 100 cells per image and
requires few minutes per image.
Conclusions: The proposed totally automatic method appears
capable of obtaining the cell contours in regions containing also
hundreds of cells and thus the clinically reliable computation of all
the important morphometric parameters used in clinical practice.
Fig 1: Representative example of the first step (top), the second step
(middle) and the final result (bottom) of the proposed automated
procedure.
Commercial Relationships: Fabio Scarpa, None; Alfredo Ruggeri
Functional Imaging of Ex Vivo Human Corneal Endothelium
Using Two-Photon Fluorescence Lifetime Imaging Microscopy
Dagny Zhu, Cosimo Arnesano, Jiun L. Do, Grace Shih, Jessica Cao,
Scott Fraser, Martin Heur. University of Southern California, Los
Angeles, CA.
Purpose: The global state of corneal endothelial health is currently
assessed with endothelial cell density and morphology using specular
microscopy. Two-photon fluorescence lifetime imaging microscopy
(2P-FLIM) is a powerful non-contact imaging modality that can
be used to characterize the metabolic activity of living tissue by
measuring the auto-fluorescence of endogenous biomolecules
essential for cellular function. Herein, we characterized the metabolic
state of ex-vivo human corneal endothelial cells by imaging free
and protein-bound nicotinamide adenine dinucleotide (phosphate)
(NAD(P)) under different stress conditions.
Methods: Human donor corneas not suitable for transplantation
were purchased from VisionShare and treated with vehicle control
(n=2) or acetazolamide (n=2). A band-pass filter of 460/80nm was
used for NAD(P) detection. Fluorescence decay time (lifetimes) of
protein-bound and free NAD(P) in endothelial cells were imaged and
analyzed at t = 0, 5, 15, and 30 min. Fluorescence lifetime images
were acquired with a Zeiss LSM-780 inverted confocal microscope
coupled to a Ti:Sapphire laser system (Coherent Chameleon Ultra
II) and an ISS A320 FastFLIM. FLIM data were acquired by the
VistaVision software from ISS and processed by SimFCS software.
Results: 2P-FLIM images of ex-vivo human corneal endothelium
showed that the ratio of protein-bound to free NAD(P) in corneas treated
with vehicle control remained unchanged over time. Treatment with
acetazolamide, a carbonic anhydrase inhibitor known to play a role in
corneal endothelial dysfunction, resulted in a shift from free to proteinbound NAD(P) over time, reflecting an overall reduction in cellular
metabolism and ATP production that is consistent with previous results
obtained with other tissues under stress conditions.
Conclusions: FLIM’s potential as a clinical optical imaging
modality has already been described for diseases of the human
retina and corneal epithelium. This study demonstrates proof of
principle of using 2P-FLIM for non-invasive functional imaging of
live human corneal endothelium. Future refinements could lead to
further research and clinical applications for evaluation of corneal
endothelial function under various pathophysiological conditions.
Commercial Relationships: Dagny Zhu, None; Cosimo Arnesano,
None; Jiun L. Do, None; Grace Shih, None; Jessica Cao, None;
Scott Fraser, None; Martin Heur, None
Support: Research to Prevent Blindness
Assessment of a standardized protocol for corneal endothelial cell
analysis and interrater reliability across educational levels using
the flex-center method
Cindy Cai1, Sina Vahedi1, Inna Stroh1, Gelareh Dastjerd2,
Hajra Channa3, Mehraz Malakooti4, Allen O. Eghrari1.
1
Ophthalmology, Johns Hopkins Hospital, Baltimore, MD;
2
University of Maryland, Baltimore County, Baltimore, MD; 3Aga
Khan University Medical College, Karachi, Pakistan; 4International
American University School of Medicine, Vieux Fort, Saint Lucia.
Purpose: The flex-center method is a semi-automated process of
measuring corneal endothelial cell density and morphology from
images obtained from specular microscopy. It is unknown whether
educational level impacts measurement reliability. We sought to
assess the reliability of a standardized training protocol for the flex-
center method of analysis across a spectrum of graders with different
educational backgrounds.
Methods: Images from 20 random healthy individuals were collected
using the Konan Specular Microscope (NSP-9900, Irvine, CA, USA).
We developed a manual to train five graders on the flex-center method.
Konan Cellchek software was used to extract measures including cell
density (CD, cells/mm2), average cell area (CA, mm2), coefficient of
variation (CV), and percent of hexagonal cells (HEX). We calculated the
intraclass correlation (ICC) based on a two-way mixed effect model to
assess inter-grader reliability of two sets of images: one set composed
of the clearer image of a participant’s two eyes, and a second set of the
more challenging image, with varying degrees of opacity and blur. We
utilized test-retest data to assess intra-grader reliability.
Results: Five reviewers, ranging from completion of secondary school
to doctorate degrees, evaluated images from 40 eyes. Among the eyes
with the clearer image, the average number of cells counted was high
across all graders (mean cells per photo: range 100 to 146.1). Inter-grader
reliability as determined by the ICC for CD (0.92, 95% CI: 0.87-0.97)
and CA (0.95, 0.91-0.98) was high. The ICC was less robust for CV
(0.46, 0.26-0.69) and HEX (0.71, 0.55-0.85). Inter-grader reliability also
remained high for CD (0.91, 0.84-0.96) and CA (0.90, 0.82-0.95) in the
set of more challenging images. Intra-grader reliability upon test-retest
again was high for CD (0.97, 0.93-0.99) and CA (0.97, 0.93-0.99) but
less so for CV (0.66, 0.32-0.85) and HEX (0.86, 0.69-0.94).
Conclusions: This standardized training protocol produces higher
inter-grader reliability measures for determination of CD and CA than
previously reported across a variety of educational backgrounds, and
remains strong in a set of challenging images. These findings provide
support for future large-scale analyses that require a diverse set of
reviewers.
Commercial Relationships: Cindy Cai, None; Sina Vahedi, None;
Inna Stroh; Gelareh Dastjerd, None; Hajra Channa, None;
Mehraz Malakooti, None; Allen O. Eghrari, None
Support: Wilmer Research Grant
Endothelial cell recognition capability of paracentral and
midperipheral corneal areas using automatic specular
microscopy counting
Fábio U. Carvalho1, Richard Y. Hida1, Ricardo Holzchuh1,
Fernando C. Abib2. 1Ophthalmology, Santa Casa de São Paulo, São
Paulo, Brazil; 2Universidade Federal do Paraná, Curitiba, Brazil.
Purpose: Despite the technological advances in capturing and
analyzing corneal endothelium images by specular microscopy (SM),
automatic counting of endothelial cells (EC) is subject to errors due
to the acquired image quality and to the accuracy of the software.
This study aims to compare the ability to recognize endothelial
cells using automatic counting of a non-contact (NC) SM between
paracentral areas (PCA) and midperipheral areas (MPA) from the
endothelial mosaic.
Methods: The same examiner performed specular microscopy in
the right eye of 36 consecutively examined patients with NC SM
CEM-350 (NIDEK©). None of the patients had any eye disease or
previous ocular surgery. Around the corneal center, 14 endothelial
images were obtained in each examination: 8 images in PCA (Group
I) and 6 in MPA (Group II). The SM software automatically counted
the cells in each image. The EC recognition capability of this
procedure was evaluated by comparing the analyzed image set by the
SM with the EC image before analysis, considering: 1 – number (N)
of non-counted EC groups and total number of EC in these groups;
2 – Erroneously counted EC: Split EC (one cell counted as two or
more) and the average amount of EC created by this division, N of
cell clusters (multiple cells counted as one) and the average amount
of EC per cluster (Figure 1); 3 – Non-evaluated area of the image,
calculated as 1-[counted cells x average cell area (µm2)/image size
(µm2)]. We also compared the results of the SM parameters: counted
cells (CC), endothelial cell density (ECD), average cell area (AVG),
coefficient of variation (CV) and hexagonality percentage (HEX).
Descriptive statistics and two-tailed Student’s t test was used for
statistical analysis (p<0.05).
Results: Results from EC recognition capability analysis and SM
parameters as well as the comparison between groups I and II are
presented in table 1.
Conclusions: Errors in identifying the EC by automatic counting
occurred both in PCA and in MPA of the endothelial mosaic, although
they were more significant in MPA. Based on these results, manual
cell counting or individualized assessment with correction of errors,
to minimize distortions in the results of specular microscopy, are
recommended.
Three types of cell recognition errors. A=Non-counted cells; B=Cell
cluster; C=Split cell; D=image C before automatic counting.
Commercial Relationships: Fábio U. Carvalho, None;
Richard Y. Hida, None; Ricardo Holzchuh, None;
Fernando C. Abib, None
Corneal Optical Properties after Swelling by Contact Lens Wear
in Fuchs Endothelial Dystrophy
Jay W. McLaren, Katrin Wacker, Katrina Kane, Sanjay V. Patel.
Ophthalmology, Mayo Clinic, Rochester, MN.
Purpose: Patients with Fuchs endothelial corneal dystrophy (FECD)
often have poor vision in the morning that improves throughout the
day. This study examined corneal high-order aberrations and scatter
as possible sources of poor vision during corneal swelling.
Methods: Twenty-one phakic eyes (21 patients) with FECD (grades
1 – 6, modified Krachmer scale) and 7 normal eyes (7 participants)
were examined by Scheimpflug photography (Pentacam HR, Oculus).
Central corneal thickness, high-order aberrations from anterior
and posterior corneal surfaces, and backscatter in standardized
scatter units (SU) from the anterior, mid, and posterior cornea were
determined from the Scheimpflug images. A low-oxygen permeable
contact lens was placed on the eye and after 2 hours of lid closure
over the lens, the lens was removed and eye was reexamined.
Scheimpflug photography was repeated at 0.5, 1, 2, and 3 hours.
After lens removal, variables were compared to those before lens
placement by using paired t-tests with Bonferroni adjustment.
Results: Immediately after removing the contact lens, corneas were
swollen by 54±13 µm (mean ± standard deviation) in FECD patients
(p<0.001) and by 48 ± 11 µm in control subjects (p<0.001). In both
groups, thickness recovered by 3 hours. Anterior corneal high-order
aberrations (root-mean-square, 6 mm-diameter optical zone) in FECD
patients (0.60±0.16 µm) decreased slightly after lens wear (0.52±0.14
µm, p<0.001) but did not change in control subjects (0.43±0.13 µm
before to 0.42 ±0.08 µm after lens wear, p>0.9). Aberrations from
the posterior corneal surface did not change in either group (p>0.19).
Backscatter from the anterior cornea increased from 1520±185
SU before lens wear to 1914±238 SU after lens removal in FECD
(p<0.001) and from 1345±80 SU to 1819±167 SU in control subjects
(p=0.001), and recovered by 2 hours in both groups. Backscatter also
increased in both groups in the mid-cornea (p≤0.02) but increased
only in the FECD group in the posterior cornea (p=0.028 FECD,
p>0.9 control).
Conclusions: Inducing corneal edema to simulate swelling after
overnight lid closure increases corneal backscatter but not high-order
aberrations. Subjective poor vision in the morning in FECD is more
likely caused by scattered light rather than by aberrations, suggesting
that these patients experience more disability glare than decreased
acuity in the morning.
Commercial Relationships: Jay W. McLaren, None;
Katrin Wacker, None; Katrina Kane, None; Sanjay V. Patel, None
Support: Research to Prevent Blindness, Mayo Foundation for
Medical Education and Research, Dr. Werner Jackstädt Foundation

Session 238 Cornea Imaging

Transcription

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