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 L&#243 ; 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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lass="h4">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).

lass="h4">Design

Transversal, comparative case series.

lass="h4">Participants

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

lass="h4">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.

lass="h4">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.

lass="h4">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.

lass="h4">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.

lass="h4">Financial Disclosure(s)

Proprietary or commercial disclosure may be found after the references.