Article
A new diagnostic method for limbal stem cell deficiency syndrome scoring in patients after surgical corneal reconstruction (K)
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Authors
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Anna Popova
- IRTC Eye Microsurgery Ekaterinburg Center, Laboratory, Ekaterinburg, Russia
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Oleg Shilovskih
- IRTC Eye Microsurgery Ekaterinburg Center, Laboratory, Ekaterinburg, Russia
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Alexey Ulyanov
- IRTC Eye Microsurgery Ekaterinburg Center, Laboratory, Ekaterinburg, Russia
| Published: | October 18, 2013 |
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Outline
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Purpose: Limbal stem cell deficiency syndrome (LSCDS) is a disease caused by burns, Steven-Johnson syndrome, eye injuries etc., that results in a deficiency of limbal stem cells, corneal degeneration and conjunctivisation. Conjunctivisation scoring after surgical corneal reconstruction is essential for outcome prediction. Immunocytoflurescence when applied instead of other diagnostic approaches allows precise LSCD scoring. Until now there is no common opinion what set of antibodies should be used for these purposes.
The aim of the study was to define what cytokeratins are specific for corneal and conjunctival epithelium and to establish LSCDS scoring approach in patients underwent surgical corneal reconstruction.
Methods: On the first step we analyzed imprints of 17 biomicrosicaly intact cadaver corneas. Indirect immunocytofluorescence with primary antibodies to CK 12, 3, 19 and secondary antibodies labeled with fluorescein isothiocyanate (FITC) was used. In 9 samples CK 7 expression was also evaluated. Diagnostic sensitivity and specificity were calculated. On the second step we assessed 11 patients (16 eyes) with a LSCDS after surgical corneal reconstruction, using a pair of cytokeratines – CK 12/19. For the multicolour staining we used secondary antibodies conjugated with AlexaFluor 488 and AlexaFluor 568, while nuclei counterstaining was performed with DAPI.
Results: CK12 displayed the best sensitivity for corneal epithelium detection (100%) while for conjunctival cells CK19 was the most sensitive marker (94%). CK12 and CK3 specificity for corneal epithelium was equal (64%). Specificity of CK19 for conjunctival epithelium was 94%. Thus, CK12/19 pair was chosen for the further multicolor staining. By this technology application in patients after surgical corneal reconstruction I LSCDS stage was defined in 2 eyes, II stage – in 8 eyes and III stage – in 6 eyes.
Conclusions: CK12 and CK19 were selected for corneal and cojunctival epithelium discrimination respectively. These markers expression evaluation allows precise LSCDS staging in patients underwent surgical corneal reconstruction.
