June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Multicentric study validation of a new molecular method for Limbal Stem Cell Deficiency diagnosis based on MUC5AC transcript detection in corneal epithelium by reverse dot-blot strip
Author Affiliations & Notes
  • Tatiana Suarez-Cortes
    Biomed R & D, Bioftalmik, Derio, Spain
  • Iker Garcia
    Biomed R & D, Bioftalmik, Derio, Spain
  • Jaime Etxebarria
    Department of Ophthalmology, Cruces Hospital, Plaza Cruces 12, Baracaldo, Spain
  • Jesus Merayo-Lloves
    Fundación de Investigación Oftalmológica, Oviedo, Spain
  • Josep Torras
    Department of Ophthalmology, Hospital Clinic de Barcelona, Barcelona, Spain
  • Ana Boto-de-los-Bueis
    Department of Ophthalmology, Hospital La Paz, idiPaz, Madrid, Spain
  • David Diaz-Valle
    Department of Ophthalmology, Hospital Clínico San Carlos, Madrid, Spain
  • Rosalia Mendez
    Department of Ophthalmology, Hospital Clínico San Carlos, Madrid, Spain
  • Xabier Landaluce
    Biomed R & D, Bioftalmik, Derio, Spain
  • Arantxa Acera
    Biomed R & D, Bioftalmik, Derio, Spain
  • Footnotes
    Commercial Relationships Tatiana Suarez-Cortes, Bioftalmik S.L. (E); Iker Garcia, Bioftalmik Applied Research (P); Jaime Etxebarria, bioftalmik (C); Jesus Merayo-Lloves, Ferrara & Hijos SL (I); Josep Torras, None; Ana Boto-de-los-Bueis, None; David Diaz-Valle, None; Rosalia Mendez, None; Xabier Landaluce, Bioftalmik (E); Arantxa Acera, BIOFTALMIK SL (E)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 547. doi:
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      Tatiana Suarez-Cortes, Iker Garcia, Jaime Etxebarria, Jesus Merayo-Lloves, Josep Torras, Ana Boto-de-los-Bueis, David Diaz-Valle, Rosalia Mendez, Xabier Landaluce, Arantxa Acera; Multicentric study validation of a new molecular method for Limbal Stem Cell Deficiency diagnosis based on MUC5AC transcript detection in corneal epithelium by reverse dot-blot strip. Invest. Ophthalmol. Vis. Sci. 2013;54(15):547.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To validate the efficacy, accuracy and sensitivity of a PCR-strip based on reverse dot-blot for detection of MUC5AC transcript as indicative of the presence of goblet cells in cornea of patients with Limbal Stem Cell Deficiency (LSCD), and to evaluate the correlation with clinical diagnosis.

Methods: Eighty-five corneal impression cytology (IC) samples from 54 subjects were analyzed in the study: 44 clinically diagnosed LSCD corneas and 41 healthy corneas. Sixteen conjunctival impression cytology (IC) samples were assayed as positive control. A total of 101 impression cytology samples were analyzed in the study. All the corneal impression cytologies were processed by RNA extraction, retrotranscription, and analyzed by the presence of goblet cells in the cornea by the PCR-strip based on reverse blotting system (Limbokit).

Results: The total IC samples analyzed indicated that 43 of 44 samples clinically diagnosed as LSCD were confirmed positive for MUC5AC, 33 of 41 healthy corneas were confirmed negative for MUC5AC, 4 healthy corneas were found positive, and 4 were rendered inconclusive results. All conjunctival impression cytologies used as positive control were confirmed MUC5A positive. The data indicate a global correlation of 91.1%. (p=0.0001). Confirmation of PCR results in TAE-agarose gels indicated that reverse dot-blot strip was more sensitive for bands detection and enhances visualization of results. The overall sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of Limbokit visualized on PCR-strips, were 98%, 89%, 91% and 97% respectively.

Conclusions: The PCR-strip test based on reverse blotting was found to be a sensitive technique for detection of MUC5AC transcript in corneal epithelium. The test results correlate well with clinical diagnosis of characterized LSCD cases. The overall sensitivity, specificity, and positive and negative predictive values were satisfactory for diagnostic purposes. The PCR-strip based test (Limbokit) constitutes a robust system for the detection of MUC5AC in corneal epithelium and may be used for early detection and for mild cases of Limbal Stem Cell Deficiency, and also as an objective clinical tool for monitoring of treatments and surgical decisions.

Keywords: 482 cornea: epithelium • 485 cornea: surface mucins • 467 clinical laboratory testing  
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