title: Comparative study of limbal stem cell deficiency diagnosis methods: detection of MUC5AC mRNA and goblet cells in corneal epithelium.
abstract: OBJECTIVE
  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).
  
  
  METHODS
  Transversal, comparative case series.
  
  
  METHODS
  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.
  
  
  METHODS
  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/μl 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.
authors.affiliation: Bioftalmik, Parque Tecnológico de Vizcaya, Vizcaya, Spain.
authors.forename: Iker
authors.initials: I
authors.lastname: Garcia
authors.forename: Jaime
authors.initials: J
authors.lastname: Etxebarria
authors.forename: Ana
authors.initials: A
authors.lastname: Boto-de-Los-Bueis
authors.forename: David
authors.initials: D
authors.lastname: Díaz-Valle
authors.forename: Luis
authors.initials: L
authors.lastname: Rivas
authors.forename: Itziar
authors.initials: I
authors.lastname: Martínez-Soroa
authors.forename: Nerea
authors.initials: N
authors.lastname: Saenz
authors.forename: Carlos
authors.initials: C
authors.lastname: López
authors.forename: Almudena
authors.initials: A
authors.lastname: Del-Hierro-Zarzuelo
authors.forename: Rosa
authors.initials: R
authors.lastname: Méndez
authors.forename: Javier
authors.initials: J
authors.lastname: Soria
authors.forename: Nerea
authors.initials: N
authors.lastname: González
authors.forename: Tatiana
authors.initials: T
authors.lastname: Suárez
authors.forename: Arantxa
authors.initials: A
authors.lastname: Acera
mesh_terms: D000368:Aged; D003316:Corneal Diseases / Q000175:diagnosis* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D019573:Epithelium, Corneal / Q000473:pathology*; D005260:Female; D020397:Goblet Cells / Q000473:pathology*; D006801:Humans; D016850:Limbus Corneae / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D055271:Mucin 5AC / Q000235:genetics*; D010503:Periodic Acid-Schiff Reaction; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000473:pathology*; D055815:Young Adult
chemical_list: C524678:MUC5AC protein, human; D055271:Mucin 5AC; D012333:RNA, Messenger