Comparative Study of Limbal Stem Cell Deficiency Diagnosis Methods: Detection of MUC5AC mRNA and Goblet Cells in Corneal Epithelium

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• 作者：Iker Garcia ; DSC¹ ; Jaime Etxebarria ; MD² ; Ana Boto-de-Los-Bueis ; MD³ ; David Dí ; az-Valle ; PhD ; MD⁴ ; Luis Rivas ; PhD⁵ ; Itziar Martí ; nez-Soroa ; MD⁶ ; Nerea Saenz ; MD⁷ ; Carlos Ló ; pez ; MD⁸ ; Almudena Del-Hierro-Zarzuelo ; MD³ ; Rosa Mé ; ndez ; MD⁴ ; Javier Soria ; PhD¹ ; Nerea Gonzá ; lez ; DSC¹ ; Tatiana Suá ; rez ; PhD¹ ; Arantxa Acera ; PhD¹ ; ^{arantxa.acera@bioftalmik.com}
• 刊名：Ophthalmology
• 出版年：2012
• 期刊代码：211_01616420
• 类别：med
• 出版时间：May, 2012
• 卷：119
• 期：5
• 页码：923-929
• 文件大小：1189 K

摘要

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Purpose

To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription-polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC).

Design

Transversal, comparative case series.

Participants

We studied 59 eyes from 43 patients clinically diagnosed with LSCD.

Methods

Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair.

Main Outcome Measures

Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels.

Results

Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results.

Conclusions

The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/渭l is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure.

Financial Disclosure(s)

Proprietary or commercial disclosure may be found after the references.