Evaluation of Epithelial Changes in Limbal Stem Cell Deficiency

Using in Vivo Confocal Microscopy

ERIC CHAN, Luxia Chen, Sophie X. Deng

Cornea and Uveitis Division, Jules Stein Eye Institute, UCLA

The authors have no financial interests to disclose.

PURPOSEBackgroundLimbal Stem Cell Deficiency (LSCD) commonly occurs with destruction of the limbal stem cells (LSCs) through either acute or chronic injury. Common causes include changes to the stem cell niche environment, chemical injuries, severe dry eyes, contact lens wear, Stevens-Johnson syndrome, multiple surgeries, and widespread infection. Diagnosis of LSCD is important as a limbal graft with corneal transplant can be curative.

The gold standard of diagnosis of LSCD is through fluorescein staining for stippling or vortex staining of the cornea followed by impression cytology to determine the presence of goblet cells on the cornea. Goblet cells are normally found in conjunctival epithelium and their presence on the cornea indicates conjunctivalization, invasion of the conjunctiva tissue into the corneal layer. However, goblet cell deficiency can be concurrent with LSCD and will lead to a false-negative result as in the case of Stevens-Johnson and glaucoma drop usage.

Recent work in our lab analyzed in vivo confocal microscopy (ICM) images of both healthy and LSCD eyes. We have identified significant microstructural changes in the corneal and limbus epithelium particularly in the basal cell layer as potential diagnostic parameters.

ObjectiveTo investigate the changes in epithelium thickness (ET) in different stages of limbal stem cell deficiency (LSCD) using in vivo confocal microscopy (ICM).

METHODSLSCD ClassificationA total of 44 LSCD eyes from 31 patients were classified as early, intermediate, and late stage LSCD and then selected for impression cytology and ICM. The early stage was characterized by stippling or late fluorescein staining, the intermediate stage was characterized by persistent late fluorescein staining in a vortex pattern, and end stage was characterized by the same vortex staining with history of epithelial defect. Affected limbal and corneal areas were identified by the location of fluorescein staining by slit lamp examination. Affected areas were then stratified into superior, nasal, inferior, and temporal limbal sections. Unaffected areas were determined as the limbal sections outside of the affected sections. Ten eyes from 8 subjects with normal presentation on slit lamp examination were selected as the control group. Representative images are shown below.

Confocal Microscopy AnalysisLSCD patients were examined with the Heidelberg Retina Tomograph III Rostock Corneal Module Confococal Microscope (Heidelberg Engineering GmBH, Dossenheim, Germany). Z-scan images of the central cornea as well as the superior, nasal, inferior, and temporal limbus were collected. Epithelial layer thickness was measured by manually counting the focus positions of the initial image of the superficial epithelium to the final image of the basal cell layer. Each image plane advanced 2 µm in depth. Statistical analyses were performed with SAS software.

In Vivo Confocal Images

Cornea

Limbus

Normal Early Intermediate Late

Deng et al, 2012

LSCD PATIENT DEMOGRAPHICS

29.5%

29.5%

18.2%

22.7%

Contact lens wear (13)Multiple surgeries (13)Dry eye syndrome (8)Other (10)*

Etiology of LSCD

LSCD PATIENT DEMOGRAPHICS

41%

48%

11% Early (18)

Intermediate (21)

Late (5)

Staging of LSCD

LSCD PATIENT DEMOGRAPHICS

89%

11%

Sectoral LSCD (39)

Total LSCD (5)

Sectoral vs. Total LSCD

Progressive Decrease in Epithelium Thickness in LSCD Eyes in the Cornea and Limbus

Normal

LSCD Early In-ter-me-diate

Late05

1015202530354045

Ep

ith

elia

l T

hic

kn

ess

(µm

)

Normal

LSCD Early In-ter-me-diate

Late0

5

10

15

20

25

Ep

ithe

lial T

hic

kn

ess (

µm

)

Limbus

Cornea

Compared to normal cornea, ET was statistically significantly decreased in LSCD, intermediate, and late stage groups. The cornea ET in LSCD eyes decreased by 13.1% compared to normal.

Compared to normal limbus, ET was statistically significantly decreased in LSCD, early, intermediate, and late stage groups. The limbus ET in LSCD eyes decreased by 33.0% compared to normal.

*

*

**

* **

*

*p<0.005

Normal LSCD Early Inter-mediate

Late0

5

10

15

20

25

30

Epith

elia

l Thic

kness (

µm

) Temporal Limbus

Normal LSCD Early Inter-mediate

Late05

10152025303540

Epi

thelia

l Thi

ckness (

µm

)

Nasal Limbus

Normal

LSCD Early In-ter-me-diate

Late0

5

10

15

20

25

30

Ep

ith

elia

l

Th

ickn

ess (

µm

) Superior Limbus

Normal LSCD Early Inter-mediate

Late0

5

10

15

20

25

30

Epith

elia

l Thic

kness (

µm

) Inferior Limbus

* * **

**

*

**

** * **

Epithelium Thickness in LSCD Eyes Decreases in Clinically Unaffected Limbal Regions

Compared to normal limbus, ET in clincially unaffected sections in most stages demonstrated a statistically significant difference

All unaffected regions of LSCD eyes for each limbal location were decreased significantly compared to normal (all p<0.01)

*p<0.005

CONCLUSIONS

• Epithelial thickness was decreased in both the cornea and limbus of LSCD eyes compared to normal

• Clinically unaffected limbal sections also showed decreased epithelial thickness compared to normal suggesting subclinical disease

• Epithelial thickness can be a reliable diagnostic measure of LSCD

This project was supported by Fight for Sight (EHC) and Research to Prevent Blindness (SXD).

ACKNOWLEDGEMENTS