Download Comparative Study of Limbal Stem Cell Deficiency Diagnosis Methods

Comparative Study of Limbal Stem Cell
Deficiency Diagnosis Methods: Detection of
MUC5AC mRNA and Goblet Cells in
Corneal Epithelium
Iker Garcia, DSC,1 Jaime Etxebarria, MD,2 Ana Boto-de-Los-Bueis, MD,3 David Díaz-Valle, PhD, MD,4
Luis Rivas, PhD,5 Itziar Martínez-Soroa, MD,6 Nerea Saenz, MD,7 Carlos López, MD,8
Almudena Del-Hierro-Zarzuelo, MD,3 Rosa Méndez, MD,4 Javier Soria, PhD,1 Nerea González, DSC,1
Tatiana Suárez, PhD,1 Arantxa Acera, PhD1
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/␮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.
Ophthalmology 2012;xx:xxx © 2012 by the American Academy of Ophthalmology.
Limbal stem cells reside in the sclerocorneal limbus at the
level of the palisades of Vogt. It is believed that these cells
are responsible for the regenerative function that permits the
maintenance of the corneal epithelium and for the so-called
barrier function that prevents the migration of conjunctival
cells over the cornea.1 Limbal stem cell deficiency (LSCD)
involves the loss of these functions, which may be a consequence of the direct destruction of this cell population or
its stromal microenvironment, or in the majority of cases,
due to exogenous factors that destroy the limbal stem cells,
including chemical or thermal burns, ultraviolet or ionizing
radiation, Stevens–Johnson syndrome, ocular cicatricial
pemphigoid, surgery, cryotherapy, contact lens use, or microbial infection.2 The partial or total destruction of the
© 2012 by the American Academy of Ophthalmology
Published by Elsevier Inc.
limbal epithelium leads to abnormal wound healing of the
corneal epithelium. This gives rise to a series of clinical
signs, including the invasion of the conjunctival epithelium
in the area of the cornea, vascularization, and chronic inflammation of the corneal stroma.3–5
Corneal conjunctivalization involves the loss of limbal
epithelium, which normally acts as a barrier between the
corneal and the conjunctival epithelia. Limbal stem cell
deficiency is characterized at a histopathologic level by the
presence of goblet cells on the cornea, vascularization,
destruction of the corneal basal membrane, and chronic
inflammation.2
The conjunctiva is responsible for the production of a
variety of mucins, which are essential for tear stability and
ISSN 0161-6420/12/$–see front matter
doi:10.1016/j.ophtha.2011.10.031
1
Ophthalmology Volume xx, Number x, Month 2012
corneal transparency. Epithelial mucins are a heterogeneous
group of large proteins (⬎200 kDa) and are components of
all the mucous secretions present in the epithelia. They are
highly glycosylated, so much so that a high percentage
(⬎50%) of their molecular mass is made up of sugars, thus
hindering their biochemical analysis.6 Epithelial mucins can
be classified as being transmembrane or secreted. There
are currently 11 known types of transmembrane mucins
(MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13,
MUC15, MUC16, MUC17, MUC20, and MUC21) and 7
types of secreted mucins, which can in turn be subdivided
into soluble (MUC7 and MUC9) and gel-forming (MUC2,
MUC5AC, MUC5B, MUC6, and MUC19) molecules.6 –13
Goblet cells are intercalated between non-secretory epithelial cells in the conjunctival epithelium. Their function is
to synthesize and secrete the gel-forming MUC5AC mucin
into the tear film.13 Thus, the MUC5AC mucin that is only
found in these cells has been postulated to be a specific
marker of goblet cells.
At present, the most popular technique for LSCD diagnosis is impression cytology (IC) followed by periodic
acid-Schiff (PAS)-hematoxylin staining because of the simplicity of the method, which is noninvasive and permits an
analysis of the morphology and status of the ocular surface.2
However, this technique, which is useful for evaluating the
anatomopathologic status of the conjunctival epithelium, is
less sensitive for determining corneal conjunctivalization by
means of evaluating the presence of goblet cells in the
corneal epithelium. Another technique for detecting LSCD
is the use of immunohistochemistry with monoclonal antibodies to evaluate the presence of CK19 in epithelial conjunctival cells14 –16 and the presence of mucin 5AC in goblet
cells in the cornea.
A number of studies have reported the detection of the
MUC5AC transcript in the conjunctival epithelium in
healthy individuals with the use of reverse transcriptionpolymerase chain reaction (RT-PCR),17,18 in different pathologies such as dry eye, and in contact lens users.19
However, none of these studies used the detection of the
MUC5AC transcript in the cornea as a diagnostic tool or
evaluated the efficiency and reproducibility of this molecular technique in comparison with conventional IC followed
by PAS-hematoxylin staining.
The objective of the present study was to compare the
efficacy of 2 techniques to detect the presence of goblet
cells in the cornea of patients with clinical signs of LSCD:
IC coupled with PAS-hematoxylin staining and the specific
detection of MUC5AC mRNA by RT-PCR.
Materials and Methods
Patients
A transversal study was carried out in 6 hospitals pertaining to the
Spanish National Health Service: Hospital de Cruces (Baracaldo,
Vizcaya), Hospital de Donostia (San Sebastian, Guipúzcoa), Hospital La Paz (Madrid), Hospital Clínico San Carlos (Madrid),
Hospital 12 de Octubre (Madrid), and Hospital de GaldakaoUsansolo (Galdakao, Vizcaya). A total of 43 patients diagnosed
with LSCD by means of slit-lamp examination were enrolled
2
between February 2009 and February 2010. Impression cytology
samples were obtained after patients had signed informed consent,
following the Principles of the Declaration of Helsinki on Biomedical Research Involving Human Subjects.
Independent IC samples were obtained on different days to
comparatively analyze the 2 detection techniques by using cellulose acetate discs (HAWP304, Millipore, Bedford, MA). Samples
were always obtained in the following order: first from the cornea
and next from the conjunctiva, having applied topical anesthesia to
the ocular surface using a mixture of oxybuprocaine chlorhydrate,
tetracaine hydrochloride, and chlorobutanol (Colircusi double anesthetic, Alcon Cusí, Barcelona, Spain). The presence of goblet
cells in the conjunctival samples of the same patient was evaluated
as a positive control. In addition, samples from healthy control
individuals were also tested as negative controls in the first stages
of the study. The specificity of detection of the MUC5AC transcript
was verified by using mRNA samples from the corneas of healthy
individuals (MUC5AC negative). RNAse-free water was also used
in RT-PCR instead of template as a negative control to demonstrate the absence of false positives by contamination.
Periodic Acid-Schiff–Hematoxylin Staining
Impression cytology samples for PAS-hematoxylin staining were
obtained on 5⫻5-mm strips of cellulose acetate, immediately fixed
in 96% ethanol, and subsequently stained with PAS-hematoxylin
according to the Locquin and Langeron protocol, modified by
Rivas et al.20 The samples were later examined under light microscopy. The presence of goblet cells in the cornea was considered to be indicative of LSCD. In addition, we measured the areas
of the cytoplasm and nucleus of non-secretory cells, cytoplasmic
alterations and staining, nuclear alterations, and ratio of the nuclear
and cytoplasmic areas.
RNA Isolation, Quality Assessment, and Reverse
Transcription
For RT-PCR assays, two 8-mm diameter cellulose acetate discs
were used. Both sides of each disc were placed in contact with the
epithelium using a sterile tweezers to obtain the highest possible
number of cells. One disc was placed on the corneal epithelium,
and subsequently the other disc was placed on the epithelium of
the superior bulbar conjunctiva. Slight pressure was applied to the
discs for a few seconds to improve the efficacy of the sample. The
discs were immediately placed in an RNA-conserving buffer
(RNAprotect Cell Reagent, Qiagen, Valencia, CA) and stored at
4°C until use.
Total RNA was isolated using the RNeasy plus micro kit
(Qiagen). The membrane containing epithelial cells was transferred to a clean 1.5-ml RNAse-free Eppendorf tube (Hamburg,
Germany) and briefly centrifuged to completely eliminate the
RNA-conserving buffer with a micropipette. Next, 350 ␮l of lysis
buffer were added to the tube containing the membrane, and after
vortexing for 1 minute at maximum speed, the resulting lysate was
transferred to the gDNA eliminator column. The rest of the steps
were performed as described by the manufacturer, using 16 ␮l of
diethyl pyrocarbonate-treated H2O for final RNA elution. All steps
were performed at room temperature.
Quantification and quality assessment of total RNA were performed using a 2100 Bioanalyzer (Agilent Technologies, Inc.,
Santa Clara, CA) and RNA Pico kits (Agilent Technologies, Inc.),
using 1 ␮l of each sample. Only those samples with an RNA
Integrity Number ⬎7 were included in the study. Although no
limitation for RNA concentration was established for positive
results, we set a cutoff of 1.2 ng/␮l for a reliable negative result,
Garcia et al 䡠 LSCD Diagnosis by Detection of MUC5AC mRNA
Table 1. Demographic Data
Purification and Sequencing of Amplicons
Variable
Value (n)
Eyes
Patients
Gender
Male
Female
Mean age (yrs) ⫾ SD
59
43
24
19
59.2⫾14.6
SD ⫽ standard deviation.
below which the result was considered to be inconclusive or
unreliable.
Reverse transcription of total RNA to cDNA was carried out
using a Transcriptor First Strand cDNA Synthesis Kit (Roche,
Mannheim, Germany), with a mixture of Oligo(dT) and random
hexamers as primers. The reaction conditions included a denaturation step (60°C for 10 minutes and then immediately to 4°C) for
the RNA and primer mixture, followed by high-temperature RT
after adding the remaining reagents (25°C, 10 minutes; 55°C, 30
minutes; 85°C, 5 minutes; 4°C).
Primer Design
Because of the repetitive and palindromic structure of the
MUC5AC gene and its transcript, together with its high homology
with MUC5B, several analyses were performed to identify suitable
regions for primer design (data not shown), including Nucleic Acid
Dot Plots (available at: http://www.vivo.colostate.edu/molkit/
dnadot/, accessed February 5– 8, 2009), RNAfold from Vienna
RNA Suite (available at: http://rna.tbi.univie.ac.at/cgi-bin/RNA
fold.cgi, accessed February 10, 2009), Primer BLAST (available
at: http://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed February 12, 2009), BLAST (available at: http://blast.ncbi.nlm.nih.
gov/Blast.cgi, accessed February 13, 2009), and BLAT (available
at: http://genome.ucsc.edu/cgi-bin/hgBlat?command⫽start, accessed February 13, 2009). Primers were tailor designed on the basis
of theoretically optimal candidate regions that ensured specificity
(low homology with other family transcripts) and efficacy (e.g.,
absence of RNA secondary structure domains). The corresponding
amplicons were theoretically reevaluated again by means of
Primer BLAST (March 5, 2009), BLAST (March 5, 2009), BLAT
(March 5, 2009) for specificity, and RNAfold (March 6, 2009) for
palindromic structure. A primer pair was thus designed and denominated MUC5AC-F (CCTGCAAGCCTCCAGGTAG) and
MUC5AC-R (CTGCTCCACTGGCTTTGG).
To confirm that PCR products represented the amplified gene
transcript, amplicons obtained during initial assays were excised
from agarose gels, purified using Illustra GFX PCR DNA and Gel
Band Purification kit (GE-Healthcare, Little Chalfont, UK), and
sequenced on a 3100 genetic analyzer using Big Dye 3.1 terminator chemistry (both analyzer and software from Applied Biosystems, Foster City, CA).
Results
A total of 59 eyes from 43 patients were included in this study
from the outpatient clinics of the ophthalmology departments of
each of the participating hospitals. The mean age of patients was
59.2⫾14.6 years (24 men, 19 women), as indicated in Table 1.
Patients were clinically diagnosed with LSCD due to a variety of
causes, including chemical burn, pemphigoid, aniridia, Stevens–
Johnson syndrome, trachoma, contact lenses, rosacea, idiopathic
disease, and others (Table 2). In Figures 1, 2, and 3, corresponding
to patients 9, 15A, and 4, respectively, some representative cases
of LSCD are shown, each from a different hospital.
Of the 59 eyes included in this study, 47 IC samples were also
available for PAS-hematoxylin staining. The results indicated that
of these, 17 (36.2%) had conjunctival goblet cells in the cornea (as
illustrated in Fig 4A, corresponding to patient 9), thus confirming
LSCD, whereas 27 (57.5%) did not (Fig 4B, corresponding to
patient 11). Although the sample was negative in this patient
according to PAS-hematoxylin, the sample was found to be positive by PCR). Moreover, 3 samples (6.4%) showed inconclusive
results because of the scarce number of cells in the corneal epithelium samples. The results are summarized in Table 3. In addition, the conjunctival epithelium from all patients exhibited squamous metaplasia; most of these were cases of grade 2 to 4, which
correlated with clinical signs. Conjunctival epithelial cells presented a nuclear-cytoplasmic ratio between 1:10 and 1:15, with
small and pyknotic nuclei, and a cell shape that was large and
polygonal (Fig 4C). Figure 4C shows an example of a grade 3
squamous metaplasia.
To verify the specificity of the RT-PCR assay, purified PCR
amplicons were sequenced. The sequencing chromatograms and
alignment of the resulting sequences with the MUC5AC cDNA
reference sequence confirmed the specificity of the amplified fragment (data not shown).
To ensure the specificity of MUC5AC transcript amplification,
the primers were also tested with RNA from corneal epithelium
samples from healthy volunteers, under exactly the same experimental conditions as the rest of samples analyzed in the study, to
further demonstrate that no amplification was detectable in healthy
Polymerase Chain Reaction
BioTaq DNA polymerase, reaction buffer, magnesium chloride,
and dNTPs (Bioline Inc., Tauton, MA) were used to perform the
amplification reaction. Custom primers (synthesized by MWGBiotech, Inc., High Point, NC) were used to specifically amplify a
103– base-pair (bp) fragment of the reversely transcribed
MUC5AC transcript (cDNA). Detection of amplicons was performed using 2.5% agarose–Tris-acetate-EDTA gels. Primers for
the ACTB and GAPDH housekeeping genes (Applied Biosystems,
Warrington, UK) were used as internal controls for the quality and
quantity of the reversely transcribed RNA (cDNA). The amplification conditions were 95°C, 3 minutes/(95°C, 15 seconds; 60°C,
15 seconds; 72°C, 15 seconds) ⫻40/72°C, 5 minutes/15°C on
hold.
Table 2. Causes of Limbal Stem Cell Deficiency
Pathology
Eyes (n)
Chemical burn
Pemphigoid
Aniridia
Stevens–Johnson syndrome
Trachoma
Contact lenses
Rosacea
Idiopathic disease
Others
Total
8
7
6
5
3
2
2
8
18
59
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Ophthalmology Volume xx, Number x, Month 2012
Figure 1. Eye of a 68-year-old man (patient 9) with Stevens–Johnson
syndrome. Note 360-degree corneal vascularization.
Figure 3. Eye of a 20-year-old man (patient 4). Corneal opacity due to
chemical burn.
corneas. For this purpose, 2 healthy volunteers were tested, and no
amplification of MUC5AC transcript was detected in the cornea
sample; nevertheless, amplification of the GAPDH and ACTB
housekeeping genes in the corneal epithelium samples and adequate RNA concentration (⬎1.2 ng/␮l) confirmed the reliability of
the MUC5AC negative result (Fig 5A, B, available at http://
aaojournal.org). In these healthy samples, MUC5AC transcript
amplification was also tested in conjunctival epithelium as a positive control, as was carried out in all samples of the study.
Of the 59 eyes analyzed by RT-PCR, 56 (94.9%) were
MUC5AC ⫹ according to PCR, therefore confirming LSCD,
whereas 3 (5.1%) were MUC5AC – (Table 3). The PCR products
were resolved using 2.5% Tris-acetate-EDTA–agarose gel electrophoresis. The samples shown in Figure 5C and D (available at
http://aaojournal.org) correspond to 2 eyes clinically diagnosed
with LSCD. Three different bands are shown in each of these gels:
MUC5AC (103 bp, lanes 1 and 3), ACTB (171 bp, multiplexed with
MUC5AC, lanes 1 and 3), and GAPDH (122 bp, individual reaction, lanes 2 and 4). Two separate reactions were performed to
detect 3 amplicons in each tissue because of the difficulty of
multiplexing more than 2 PCRs and the close vicinity of the 3
amplicons that could lead to confusing results. Lanes 1 and 2
correspond to the corneal samples, whereas lanes 3 and 4 correspond to the conjunctival samples used as a positive control for the
presence of the MUC5AC transcript. Lanes 5 and 6 correspond to
negative controls (lane 5, omission of reverse transcriptase in the
RT reaction; lane 6, omission of cDNA in the PCR). Amplification
of ACTB and GAPDH served as quality and quantity controls of
the cDNA of each tissue. Examples of 2 cases diagnosed with
LSCD are presented. Figure 5C (available at http://aaojournal.org)
shows the case of a patient with LSCD (patient 9) for whom
MUC5AC mRNA was detected as a 103-bp amplicon in both
cornea (lane 1) and conjunctiva (lane 3). In contrast, Figure 5D
(available at http://aaojournal.org) shows the case of a patient with
LSCD (15A) with no MUC5AC mRNA being detected in the
cornea. Amplification of ACTB (171 bp, multiplexed with
MUC5AC, lane 1) and GAPDH (122 bp, individual reaction, lane
2) in the corneal mRNA sample indicates that RNA quantity and
quality were satisfactory, thus confirming the negative result for
MUC5AC.
Comparative analysis of the 2 techniques indicated that 36.2%
of analyzed samples (17/47) were positive according to both
techniques. However, 57.5% of the total samples (27/47) were
negative according to the PAS-hematoxylin technique. Finally,
6.4% corresponding to 3 samples were inconclusive according to
PAS-hematoxylin because of the scarcity of the sample (Table 3).
Discussion
Figure 2. Right eye of a 68-year-old woman (patient 15A) with a history
of deep myopic right amblyopia and childhood keratitis in both eyes. Right
eye corneal biomicroscopy revealed micropannus (arrow), central corneal
opacity, and ghost vessels. The periodic acid-Schiff– hematoxylin staining
and reverse transcription-polymerase chain reaction test results were negative. This eye was the donor for autologous expanded limbal stem cell
transplantation to the severely damaged left eye.
4
In the present work, we present a method for the diagnosis
of LSCD based on the highly specific detection of MUC5AC
mRNA in the cornea. The sensitivity of this molecular
technique was compared with that of conventional PAShematoxylin staining of IC samples and clinical diagnosis.
Numerous cases arise in clinical practice in which despite the presence of a clinical suspicion of LSCD, there is
Garcia et al 䡠 LSCD Diagnosis by Detection of MUC5AC mRNA
Figure 4. A, Microphotograph of corneal impression cytology (IC) with periodic acid-Schiff– hematoxylin stain from patient 9. Large epithelial cells that
have lost their rounded morphology can be observed. Intercellular junctions are often lost, and spaces appear between the cells. Goblet cells can be seen
in this sample (arrow), confirming the diagnosis of limbal stem cell deficiency. Magnification, 40⫻. B, In this corneal epithelium sample from patient 11,
no goblet cells were observed (20⫻). C, Representative IC of conjunctiva. In this sample, the conjunctival epithelium exhibited grade 3 squamous
metaplasia. Conjunctival epithelial cells presented a nuclear-cytoplasmic ratio of 1:15, with small and pyknotic nuclei, and a large and polygonal cell shape
(20⫻).
no cytologic evidence of this pathology according to conventional IC. This discrepancy may be due to a number of
reasons: Damage of limbal corneal cells may not be sufficiently severe to destroy the entire cell population, LSCD
may still be subclinical and worsen later, IC might not be
sufficiently sensitive, and some patients simply may not
manifest signs of LSCD.21 For this reason, a more sensitive
and specific method is needed to diagnose LSCD, particularly in a patient who has only mild LSCD, and to more
rigorously classify or select patients with corneal pathology
who could undergo keratoplasty with a reasonable probability of success to minimize the risk of failure of this
surgical technique. In addition, this molecular method can
be used together with conventional IC to verify the restoration of the corneal phenotype and the regression of goblet
cells.22,23 The current study has shown that RT-PCR– based
analysis is more sensitive and specific than PAS-hematoxylin staining because RT-PCR detected more positive cases:
56 of 59 eyes clinically diagnosed with LSCD, equivalent to
a diagnostic efficacy of approximately 95% (Table 3).
The specificity of the RT-PCR assay was ensured by
means of the custom design of primers for the amplification
of the MUC5AC transcript. During the first stages of our
study, several published primer pairs for this gene were
bioinformatically evaluated.24 –27 However, our analyses
showed that most of these primers were designed on the
basis of non-validated sequences available at that time in
public databases, such as predicted sequences and partial
clones, or expected to simultaneously amplify MUC5B
Table 3. Summary of Results for the Different Diagnostic
Methods
Clinical
ⴙ
—
Inconclusive
Total
PAS
RT-PCR
n
%
n
%
n
%
59
0
0
59
100
—
—
17
27
3
47
36.2
57.5
6.4
56
3
0
59
94.9
5.1
—
PAS ⫽ periodic acid Schiff; PASS ⫽ PAS-hematoxylin staining; RTPCR⫽reverese transcription-polymerase chain reaction.
(primers tested by means of the National Center for Biotechnology Information’s PrimerBlast). This is possible because of the high homology between MUC5AC and
MUC5B, both of which are derived from the same genomic
locus with different transcription initiation sites. The
possibility of obtaining nonspecific results discarded
the use of these published primer sequences as a suitable
method for diagnosis and encouraged us to design the
primers ourselves.
Thus, multiple bioinformatic analyses were performed to
identify the most suitable regions to be targeted by specific
primers. After selecting a candidate primer pair, it was
bioinformatically tested for specificity. Subsequently, the
specificity of PCR detection was demonstrated by sequencing the PCR products obtained in our first assays; all the
sequenced fragments (obtained from 3 different samples)
were found to be part of the targeted MUC5AC transcript
and showed no significant nonspecific targets throughout
the human transcriptome or genome. Of note, no homology
with MUC5B was observed.
According to our results, of the 59 eyes analyzed by
RT-PCR, 56 were found to present detectable levels of
MUC5AC, confirming the LSCD diagnosis, whereas 3 did
not. Negative results were found for patients 15A, 16B, and
17. The reliability of the negative results was evaluated with
strict RNA quantity and quality controls using both the
Bioanalyzer and housekeeping genes. On the other hand,
positive PCR controls consisting of RNA from conjunctival
epithelium were also simultaneously performed to evaluate
whether the absence of amplification was due to RT or PCR
inhibitors.
Revision of the clinical history of patients who were
LSCD negative by RT-PCR revealed that patient 16B had
been clinically diagnosed with left eye superior limbic keratoconjunctivitis and LSCD on the basis of positive IC in
2005 and 2008; patient 16B’s condition improved with
treatment, and she was included in this study to analyze the
status of her limbus 1 year later. The fact that the MUC5AC
transcript was not detected in her cornea suggests that the
treatment had been effective and that LSCD had been
corrected.
5
Ophthalmology Volume xx, Number x, Month 2012
Patient 15A reported a history of childhood keratitis. Her
right eye exhibited micropannus, central corneal opacity,
and ghost vessels (Fig 2). She presented with severe painful
LSCD, whose cause was associated with the protracted use
of glaucoma eyedrops plus corneal erosions. Both PAS and
RT-PCR test results of her right eye were negative, and
therefore this eye was used as a donor for the autologous
transplantation to the severely damaged left eye of limbal
stem cells expanded in vitro.
Patient 17 presented corneal conjunctivalization that was
restricted to the superior zone. It is conceivable that the disc
used to obtain the sample during IC might not have been
placed in contact with the affected zone, thereby explaining
the absence of goblet cells in the cornea and the negative
RT-PCR result for the MUC5AC transcript.
The method we have presented represents a good tool for
clinical practice because it is highly specific, sensitive,
rapid, and noninvasive. In addition, it facilitates the selection of patients who can undergo keratoplasty with a higher
probability of success, with a view to reducing the risk of
failure of the said technique. This method can also facilitate
the selection or rejection of a cornea as an adequate source
of stem cells for autologous limbal transplantation.
The large number of cases that were RT-PCR positive
and PAS negative highlights the significantly improved
sensitivity afforded by the exquisitely sensitive PCR technique. Another clear advantage is that the RT-PCR– based
method produces objective results, which are not subject to
subjective interpretation.
Nevertheless, there are admittedly a number of inconveniences associated with this molecular technique, such as
the obtaining of the sample. Thus, it is critically important
to obtain an optimal number of corneal epithelial cells,
because few cells lead to lower performance and falsenegative results. For this reason, we suggest a minimal RNA
concentration of 1.2 ng/␮L, below which negative results
should be considered as unreliable, and the inclusion of
housekeeping genes as an additional quantity and quality
control. More important, it is vital that samples are taken
exclusively from the cornea; false-positive results can
arise if the sampling disc touches part of the conjunctival
epithelium, because the PCR technique is extremely sensitive. In addition, given that this method is based on
RNA isolation and expression analysis, the lability of
RNA must be considered, and working under RNAse-free
conditions becomes essential. Finally, this technique
does not provide spatial information about the distribution of goblet cells in the cornea, making full versus
partial LSCD indistinguishable.
Several reports about the use of confocal microscopy for
LSCD diagnosis have been published in recent years.28 –30
This technique could circumvent some of the limitations of
the PCR-based technique, especially when trying to distinguish between full and partial LSCD. Although the use of
this technology is promising, it has its own drawbacks, such
as the high costs of the equipment, the limited number of
laboratories with a confocal microscope, and the need for
specialized expertise when manipulating the apparatus and
interpreting the data. In contrast, PCR-based methods are
widespread now and can be performed in any laboratory at
6
a low cost by routine technicians. The PCR technique also
produces objective and easily interpretable results. Moreover, the exquisite sensitivity of RT-PCR facilitates the
early detection of LSCD, which can be more difficult to
detect with confocal microscopy. In this regard, the possible
absence of cytologic evidence of LSCD during the initial
stages of the pathology can easily produce false-negative
results with confocal microscopy, a limitation that can be
significantly reduced when using PCR-based technologies.
In conclusion, the method described constitutes a robust
system for the early detection of mild cases of LSCD and
the corroboration of uncertain clinical cases. Moreover, this
methodology has multiple potential applications beyond
diagnosis, including the monitoring of treated patients, the
evaluation of the evolution of the corneal epithelium after
keratoplasty or amniotic membrane transplant, and the examination of the limbal condition of donor eyes before
autologous stem cell transplantation. Finally, the use of this
technique in clinical practice can have important economic
repercussions, because it can advise against a corneal transplant in some patients diagnosed with LSCD.
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Footnotes and Financial Disclosures
Originally received: May 19, 2011.
Final revision: October 24, 2011.
Accepted: October 24, 2011.
Available online: ●●●.
Manuscript no. 2011-755.
1
Bioftalmik, Parque Tecnológico de Vizcaya, Vizcaya, Spain.
2
Hospital de Cruces, Vizcaya, Spain.
3
Hospital La Paz, Madrid, Spain.
4
Hospital Clínico San Carlos, Madrid, Spain.
5
Hospital Ramón y Cajal, Madrid, Spain.
6
Hospital de Donostia, Guipúzcoa, Spain.
7
Hospital 12 de Octubre, Madrid, Spain.
8
Hospital de Galdakao-Usansolo, Vizcaya, Spain.
Financial Disclosure(s):
The author(s) have made the following disclosure(s): The authors declare
that the results reported in this article, including the patented MUC5AC
primer sequences, may be of commercial interest to Bioftalmik, Vizcaya,
Spain.
Financial Support: The Centre for the Development of Industrial Technology partially supported this work through its NEOTEC Program, Grant
IDI-20080118.
Correspondence:
Arantxa Acera, PhD, Bioftalmik, Parque Tecnológico de Vizcaya, Ed. 800,
2a. Planta, E-48160 Derio, Vizcaya, Spain. E-mail: arantxa.acera@
bioftalmik.com.
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