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Novel Molecular Diagnostic System of Limbal Stem Cell Deficiency Based on MUC5AC Transcript Detection in Corneal Epithelium by PCR-Reverse Dot Blot Iker Garc´ıa,1 Jaime Etxebarria,2 Jes´ us Merayo-Lloves,3 Josep Torras,4 Ana Boto-de-los-Bueis,5 David D´ıaz-Valle,6 Rosal´ıa Méndez-Fernández,6 Arantxa Acera,1 and Tatiana Suárez-Cortés1 1Bioftalmik,

Derio, Vizcaya, Spain Department of Ophthalmology, Cruces Hospital, Baracaldo, Vizcaya, Spain 3 Universidad de Oviedo and Fundaci´ on de Investigaci´ on Oftalmológica, Oviedo, Spain 4 Hospital Clinic de Barcelona, Barcelona, Spain 5Hospital La Paz, idiPaz, Madrid, Spain 6 Hospital Cl´ınico San Carlos, Madrid, Spain 2

Correspondence: Tatiana SuárezCortés, Bioftalmik, Parque Tecnol´ ogico de Vizcaya, Ed. 800, 2nd Floor, E-48160 Derio, Vizcaya, Spain; [email protected]. IG and JE contributed equally to the work presented here and should therefore be regarded as equivalent authors. Submitted: February 26, 2013 Accepted: July 10, 2013 Citation: Garc´ıa I, Etxebarria J, MerayoLloves J, et al. Novel molecular diagnostic system of limbal stem cell deficiency based on MUC5AC transcript detection in corneal epithelium by PCR-reverse dot blot. Invest Ophthalmol Vis Sci. 2013;54:5643–5652. DOI:10.1167/iovs.13-11933

PURPOSE. To evaluate the sensitivity and specificity of a PCR-strip system based on reverse dot blot for detection of MUC5AC mRNA in corneal epithelium samples from patients with limbal stem cell deficiency (LSCD), and to determine the correlation with clinical diagnosis. METHODS. We obtained 87 corneal impression cytology (IC) samples from 55 subjects (37 patients clinically diagnosed with LSCD and 18 control subjects). Total RNA was extracted from each IC sample and retrotranscribed to cDNA. MUC5AC mRNA transcript was amplified by a customized RT-PCR assay and detected in PCR strips based on reverse dot blot hybridization and in agarose gels. Conjunctival IC samples were used as positive controls. RESULTS. Forty-four of 45 corneal IC samples obtained from patients clinically diagnosed with LSCD were positive for MUC5AC, whereas 34 of 42 corneal ICs from healthy subjects were negative for MUC5AC. Four healthy corneas were found MUC5AC positive, and four rendered inconclusive results. A correlation of 91.4% (P < 0.001) between molecular testing and clinical diagnosis was found. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR-strip system were 98%, 89%, 92%, and 97%, respectively. CONCLUSIONS. Corneal epithelium MUC5AC transcript detection by reverse dot blot PCR-strip is a highly sensitive technique for LSCD diagnosis. The test results strongly correlate with clinical diagnosis of characterized LSCD cases. The PCR-strip system may be used for early detection, and for the detection of mild cases of LSCD, and constitutes an objective clinical tool for the monitoring of treatments and surgical decisions. Keywords: cornea, limbal stem cell deficiency, MUC5AC, impression cytology, PCR-strip, reverse dot blot

imbal stem cell deficiency (LSCD) is a pathological eye condition in which the stem cells located at the limbus malfunction or are either partially or totally depleted, resulting in various degrees of stem cell deficiency and abnormalities at the corneal surface. Limbal stem cells act as a ‘‘barrier’’ to conjunctival epithelial cells preventing them from migrating onto the corneal surface.1,2 LSCD can have many causes and its severity can range from mild, as seen in contact lens overwear, to severe, as in chemical burns and ocular cicatricial pemphigoid (OCP). Causes of LSCD mainly include aniridia, ectodermal dysplasia, toxicity from topical medications, chemical or thermal injury, radiation, Stevens-Johnson Syndrome (SJS), OCP, cryotherapy, multiple surgery, and contact lens wear.3 Epithelial cells (ECs) of the ocular surface are characterized by their expression of the keratin antigens of intermediate filaments, which are highly tissue-specific molecular markers. The expression of K3/12 is currently thought to be a hallmark

of corneal ECs, and has been used for LSCD diagnosis.4–10 However, the corneal specificity of K3 has recently been brought into question, with the consequence that the expression patterns of K3/12 in the ocular surface epithelium are not as clear-cut as believed.11–18 The diagnosis of LSCD has long relied on the detection of goblet cells in the IC of the corneal epithelium, either by periodic acid–Schiff staining12,19 or by more specific techniques, such as mucin 5AC staining.20,21 Since the MUC5AC transcript is considered to be a molecular marker for goblet cells, its detection in the corneal epithelium is indicative of LSCD. We have recently reported the molecular detection of the MUC5AC transcript in the cornea as a tool for diagnosing LSCD.22 In that study, we evaluated the efficiency and reproducibility of the molecular technique in comparison with IC followed by PAS-hematoxylin staining. Having demonstrated the advantages of the RT-PCR method for LSCD diagnosis, a system for MUC5AC transcript detection in corneal epithelium

Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783

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IOVS j August 2013 j Vol. 54 j No. 8 j 5644

LSCD Diagnostic System via MUC5AC Transcript Detection was then developed to allow the diagnostic procedure to be performed in situ in hospital laboratories. This novel method is based on reverse dot-blot technology, and detection of the amplified fragment used for diagnosis is performed by visualizing a colorimetric band on a membrane. This type of PCR-strip–based diagnostic system is widely employed for the clinical diagnosis of several other several diseases, such as celiac disease,23 human papillomavirus,24 oligospermia,25 hemochromatosis,26 among others. The PCR-strip–based diagnostic system is easy to perform, does not require highly experienced personnel, and is routinely used in clinical diagnostic laboratories worldwide. The main objective of this study was to determine the sensitivity and specificity of the PCR-strip system for LSCD diagnosis by MUC5AC transcript amplification in the corneal epithelium and to correlate these results with clinical diagnosis.

RNA Isolation, Quality Assessment, and Reverse Transcription Total RNA was isolated using a commercial RNA kit (RNeasy plus micro kit; Qiagen). Quantification and quality assessment of total RNA was performed using a microfluidics-based platform (2100 Bioanalyzer; Agilent Technologies, Inc., Santa Clara, CA) and RNA pico kits, using 1 lL of each sample (2 replicates for each sample). Reverse transcription of 10 lL of total RNA to cDNA was carried out using a cDNA kit (Transcriptor First Strand cDNA Synthesis Kit; Roche Diagnostics Deutschland GmbH, Mannheim, Germany) as previously described.22 Negative PCR controls consisted on the inclusion of RNAse-free water in RT and PCR instead of a template. No limitation on sample RNA concentration or integrity (RIN) was established for inclusion in the study.

Polymerase Chain Reaction and Detection

METHODS Patients A transversal study was carried out in five hospitals: Cruces Hospital (Baracaldo, Vizcaya), Hospital Cl´ınico (Barcelona), Instituto Fernández Vega (Oviedo), La Paz Hospital (Madrid), and Cl´ınico San Carlos Hospital (Madrid). The work was conducted in two consecutive studies, analyzing a total of 87 eyes from 55 subjects (Table 1), including 37 patients clinically diagnosed with LSCD by means of slit-lamp examination (15 men, 22 women, with a mean age of 46.6 6 13.3 years) and 18 healthy volunteers (including 7 men, 11 women, with a mean age of 32.1 6 6.3 years). Patients were clinically diagnosed with LSCD due to a variety of etiologies including chemical burn, multiple glaucoma surgery, OCP, aniridia, StevensJohnson Syndrome, contact lens wear, ocular surface melanoma surgery and others (Table 2). IC samples were obtained after patients had signed an informed consent form in accordance with the principles of the Declaration of Helsinki on Biomedical Research Involving Human Subjects.

Impression Cytology Sample Collection A representative illustration of impression cytology (IC) sample collection and sample processing is shown in Figure 1. IC samples were obtained using 8-mm diameter cellulose acetate discs (HAWP304; Millipore, Bedford, MA). Samples were always obtained in the following order: first from the cornea and then from the conjunctiva (when sampled), after having applied topical anesthesia to the eye surface, using a mixture of oxybuprocaine hydrochloride, tetracaine hydrochloride, and chlorobutanol (Colircusi double anesthetic; Alcon Cus´ı, Barcelona, Spain). The presence of goblet cells in the conjunctival samples was used as a positive control when available. Additionally, the specificity of MUC5AC transcript detection was verified in corneal samples from healthy volunteers (MUC5AC negative controls). The procedure was performed as previously described by Garcia et al.22 Both sides of each disc were placed in contact with the epithelium using sterile tweezers to obtain the highest possible number of cells. One disc was placed on the corneal epithelium and a separate disc was placed on the bulbar conjunctival epithelium when sampled. Slight pressure was applied to the discs for a few seconds to improve sampling efficacy. The discs were immediately placed in an RNA protecting buffer (RNAprotect Cell Reagent; Qiagen, Valencia, CA) and stored at 48C until use.

Ten lL cDNA were used as a template for all PCR reactions. PCR and detection reactions were performed by using the kit for the detection of MUC5AC expression in corneal cDNA (Limbokit; Operon S.A., Zaragoza, Spain), following the procedure described by the manufacturer. This kit includes all the reagents for PCR reactions (PCRmix, custom primers for amplification of MUC5AC and GADPH), and GoTaq Flexi DNA Polymerase, as well as reagents for the detection of amplification bands on the reverse-blot PCR strips. Briefly, 7 lL PCRpremix, 2 lL primers, 1 lL Taq, and 10 lL cDNA were mixed per reaction. The amplification conditions were: 968C, 3 min/ [968C, 15 seconds; 608C, 15 seconds; 728C, 15 seconds] 403/ 48C on hold. The resulting amplicons were detected using the reverse-blot PCR-strips as described by the manufacturer (Operon S.A.) and an automated dispensing system (Autolipa; Innogenetics NV, Gent, Belgium). The system automatically performed all the steps for the detection with PCR-strips including: denaturation, hybridization, washing, and developing. All steps were performed at 428C. The PCR products were also visualized by gel electrophoresis on 2.5% agarose-TAE gels. The results of amplicon detection by PCR strips versus 2.5% TAE-agarose gels were compared and the sensitivity of the reverse-blot PCR-strips was tested.

Statistical Analysis Statistical analysis was performed using statistical software (SPSS 19.0; IBM SPSS Statistics, Chicago, IL). The correlation between the results obtained with the PCR-strip for detection of the MUC5AC transcript in corneal epithelium and clinical diagnosis was evaluated using the Spearman correlation test. The level of statistical significance was considered to be P < 0.01. The sensitivity and specificity values of the technique were analyzed. The positive predictive value (PPV) was calculated by analyzing the number of true positives/(number of true positives þ number of false positives) and the negative predictive value (NPV) was defined as the number of true negatives/(number of true negatives þ number of false negatives).

RESULTS To facilitate the interpretation of the results, an example of results using a TAE-agarose gel and a PCR-strip is illustrated (Fig. 2), with a positive and a negative sample for MUC5AC detection.

LSCD Diagnostic System via MUC5AC Transcript Detection TABLE 1. Descriptive Summary of the Samples Employed in the Study Subjects

Corneal IC Samples

Group

LSCD

Healthy

LSCD

Healthy

Study 1 Study 2 P ¼ Total

15 22 37

7 11 18

19 26 45

15* 27† 42

55

87

Conjunctival IC Samples 2 16 18 18

* Fourteen samples from seven healthy patients (both eyes), and one from the healthy eye of a patient diagnosed with LSCD in the contralateral eye. † Twenty-two corneal IC samples from 11 healthy volunteers (both eyes) and five from the contralateral healthy eye of patients diagnosed with LSCD.

Molecular Diagnosis of LSCD by the PCR-Strip System Versus Clinical Diagnosis An evaluation of the PCR-strip system for amplification of the MUC5AC transcript and detection of bands by reverse dot blot strips was performed in two studies analyzing 87 corneal epithelium samples collected by IC. Assessment of its value for diagnosis of LSCD was tested using 45 clinically diagnosed samples, and the specificity of the system was evaluated using 42 clinically healthy corneal samples. In the first study, a total of 34 corneas (19 LSCD and 15 healthy), and two conjunctivas from 22 patients were tested. The results of this assay are presented in Table 3. The PCR-strip system showed 20 positives (LSCD), 19 from LSCD patients and one from a control subject; 13 negatives (healthy corneas); and one inconclusive sample. Both conjunctivas analyzed and used as positive controls were positive for MUC5AC gene transcript. The correlation between the PCR-strip system and clinical diagnosis was 94.4% (P < 0.001). The results obtained by the PCR-strip system were confirmed using TAE agarose gels. This comparison indicated that all 36 samples tested showed exactly the same results in strip and gel (100% correlation). Although a weakly amplified band could be observed in the gel from one of the samples (sample A4), this band is longer in size than the MUC5AC band and was not observed in the strip, suggesting nonspecific amplification. A representative illustration of the results obtained is presented in Figure 3. In this study, an additional comparison of results obtained with the PCR-strip system and the standard PCR technique as reported by Garcia et al.22 was performed using the same corneal epithelium cDNA samples under exactly the same experimental conditions. The results indicated a correlation of TABLE 2. Causes of LSCD Condition/Cause of LSCD

Individuals, n

Chemical burn Pemphigoid Aniridia Stevens-Johnson Syndrome Contact lens wear Ocular surface melanoma/carcinoma Glaucoma surgery Severe infections of ocular surface Severe inflammations (of different origin) Transplant failure due to herpes Idiopathic Others Total

9 2 1 1 1 2 1 3 3 1 5 8 37

IOVS j August 2013 j Vol. 54 j No. 8 j 5645 97.2% (P < 0.001) between the two techniques (35 from 36 samples with the same result), and revealed that the PCR-strip system was slightly more sensitive than the standard PCR technique (data not shown). In the second study, a total of 53 cornea samples (26 LSCD and 27 healthy) and 16 conjunctivas from 33 subjects were tested using the PCR-strip system procedure only. The results confirmed that 25 of 26 samples clinically diagnosed as LSCD were positive for MUC5AC, and one sample was negative for MUC5AC. Similarly, 21 of the 27 samples clinically classified as being healthy (no LSCD) were found to be negative for MUC5AC, three were found to be positive, and three were inconclusive because insufficient sample was collected by IC. All 16 conjunctivas were found to be positive for MUC5AC. These data indicate a correlation of 89.9% between the PCR-strip system and clinical diagnosis (Table 3). Finally, the results obtained by PCR strip were confirmed in TAE agarose gels (Fig. 4). This comparison indicated that, in general, no divergent results are observed in gel versus strips. However, clear differences in sensitivity and specificity were observed in 10 samples. In all those samples, better results were always obtained on the strips. Two samples showed a MUC5AC band, but did not present a GAPDH band in the agarose gel. However, a clear signal band for both MUC5AC and GAPDH was observed in the corresponding strips (samples indicated by dotted arrows in Fig. 4). In 3 cases, the GAPDH band was clear, but the MUC5AC band signal was very weak in the gel; however, clear bands were observed in strips (indicated by single asterisks in Fig. 4). Additionally, weak band signals for both GAPDH and MUC5AC were observed in gel for three samples, but clear bands were seen on the corresponding strips (double asterisks, Fig. 4). These samples represent the cases in which the increased sensitivity provided by the strip is crucial, because MUC5AC amplification is hardly detectable in gel. It was also observed that one sample (094 CJ-OD) did not present GAPDH in the gel nor in the strip; this sample showed very weak MUC5AC amplification band in the gel, while it showed clear MUC5AC amplification in the strip (indicated triangle in Fig. 4). Given that MUC5AC expression was detected in this sample, it was considered positive regardless of GAPDH amplification. Finally, one healthy sample (154 C-OD) seemed to show weak MUC5AC amplification in the gel, while no band was observed in the strip (indicated by a bold arrow in Fig. 4). This points to nonspecific amplification of an amplicon that is similar in length to the MUC5AC amplicon. This could lead to a false positive if only gel results were considered. All these observations indicated that PCR-strips enhanced the visualization of results, and thus represent a more sensitive technical format than agarose gels for viewing and evaluating results. Finally, a joint analysis of the results obtained in studies 1 and 2 indicated a global correlation between the PCR-strip system and clinical diagnosis of 91.4% (Table 4), since 44 of 45 samples clinically diagnosed for LSCD were positive for MUC5AC amplification, and only one was negative. In the healthy corneal samples included as negative controls, 34 of the 42 samples analyzed were negative for MUC5AC amplification, four healthy corneas were found to be positive for MUC5AC, and four samples were inconclusive because of insufficient sample in the corneal IC. All 18 conjunctivas used as positive controls were MUC5AC positive, ensuring the reliability of the results. Negative controls of PCR reactions, including the use of H2O instead of cDNA, were processed under exactly the same experimental conditions. There was no amplification of either MUC5AC or GAPDH in these samples, thus confirming the absence of any contamination and the specificity of the reactions. The amplification of the GAPDH housekeeping gene in all healthy corneal epithelium samples confirmed the integrity

LSCD Diagnostic System via MUC5AC Transcript Detection


FIGURE 1. Schematic representation of sample collection and processing. Corneal IC samples were collected using sterile tweezers without touching the conjunctiva. The membrane carrying the IC sample was immediately transferred to a RNase-free tube and store at 48C until analysis. Processing involved the extraction of total RNA extraction and its retrotranscription to cDNA with PCR being carried out in a final volume of 20 lL. PCR and detection were performed using the MUC5AC detection kit following the manufacturer’s instructions. The PCR products were visualized on PCR strips and in agarose gels.

of the samples, the appropriateness of PCR conditions, and the reliability of the negative MUC5AC results. According to the results obtained using the PCR-strip system compared with clinical diagnosis, the sensitivity of the PCR-strip for MUC5AC detection as an indication of LSCD in corneal epithelium was 98% and its specificity was 89%. The calculated positive predictive value (PPV) was 92% and negative predictive value (NPV) was 97% (Table 4).

One of the samples in study 2 was used for a final study of the clinical potential of this PCR-strip system; we evaluated the possibility of an autologous limbal transplantation in an 18year-old patient with LSCD in the left eye. LSCD was due to eye caustication, and this eye had previously been treated with amniotic membrane. The right eye was proposed as a limbal donor candidate for autologous transplantation, and MUC5AC expression was tested in both eyes prior to surgery (Fig. 5).

FIGURE 2. Representation of MUC5AC amplification results obtained using gel (A) and PCR strip (B). Amplification of the MUC5AC transcript in the presence or absence of GAPDH is an indication of positive diagnosis for LSCD. The absence of MUC5AC with the presence of GAPDH is an indication of negative diagnosis for LSCD. The absence of both MUC5AC and GAPDH is considered a nonvalid result and is denoted as ‘‘inconclusive.’’ (A) 2.5% TAE agarose gel. Lane 1: Molecular weight markers. Lanes 2 and 3: positive results for MUC5AC; LSCD in cornea. Lane 4: amplification of GAPDH band only (i.e., a negative result [healthy cornea]). Lane 5: no amplification of either MUC5AC or GAPDH bands; negative control (dH2O). (B) PCR-strip results. Lane 1: nondeveloped strip; only control line positions are observed. Lanes 2 and 3: positive results for MUC5AC. Lane 4: presence of GAPDH and control developing bands only; negative result for MUC5AC. Lane 5: appearance of control developing band only (i.e., inconclusive result). The developing control band is only visible after a correct developing of the strip. (C) Representation of the PCR-strip principle. The multiplex reaction mixture is subjected to PCR, and the amplification products including MUC5AC and GAPDH biotinlabeled amplicons are hybridized to their respectively probes that are anchored to the strip. After washing, a conjugate buffer is added and incubated. Finally the substrate is added and incubated until the appearance of bands. The reaction is stopped by washing with distilled water.


LSCD Diagnostic System via MUC5AC Transcript Detection TABLE 3. MUC5AC PCR-Strip Test Detection Versus Clinical Diagnosis of LSCD Clinical Diagnosis of LSCD MUC5AC PCR Strip

þ

19 0 0 19 19/19

1 13 1 15 13/15

20 13 1 34 32/34 94.4%

25 1 0 26 25/26

3 21 3 27 21/27

28 16 22 0 3 0 53 16 46/53 16/16 89.9%

Total

CJ

Study 1 þ NC Total Correlation Overall correlation

2 0 0 2 2/2

Study 2 þ NC Total Correlation Overall correlation NC, not conclusive.

Impression cytologies were taken from the cornea and conjunctiva of both eyes and processed for a reverse dot blotting PCR-strip. The test confirmed the presence of MUC5AC transcript in the damaged (left) eye cornea, and the absence of this transcript in the donor (right) eye. Conjunctival samples were used as positive controls for MUC5AC amplification. Based on these results, the candidate donor eye was confirmed to be normal, thus verifying the absence of involvement in the original injury, and further corroborating the viability of autologous limbal transplantation in this patient.

DISCUSSION LSCD has traditionally been diagnosed on the basis of patient history coupled with clinical observation of corneal conjunctivalization associated with persistent epithelial defects, which strongly hint at limbal stem cell deficiency.27 PAS-hematoxylin

staining of corneal epithelium samples for the detection of goblet cells also facilitates the diagnosis. However, the fact that the latter provides limited sensitivity for diagnostic purposes, together with the unavailability of objective diagnostic tests, points to the necessity to develop more sensitive diagnostic methods. In an earlier study, we reported that the conventional PAS hematoxylin technique had a sensitivity of 37%. This contrasted dramatically with the 97% sensitivity estimated for the molecular diagnosis of LSCD involving direct MUC5AC transcript detection in IC samples. Other highly specific techniques for the diagnosis of LSCD, such as the immunohistochemical and biochemical characterization of proteins, including MUC5AC and several keratins and proteins (e.g., DNp63), have been reported.4,11,13,14,16 However, these techniques are likely to be less sensitive than molecular detection based on PCR amplification, particularly in samples in which tissue is scarce, such as in IC samples. Highly sensitive techniques combined with IC are required to accurately diagnose LSCD, because erroneous diagnosis can lead to the patient undergoing unnecessary surgery.28 Confocal microscopy can be used for LSCD diagnosis,29–33 but the equipment required is rather expensive (not feasible for all centers) and a highly qualified professional is needed to correctly interpret the resulting images, thereby limiting its use. The present study evaluated the use of a novel technique for diagnosing LSCD that involves the detection of the MUC5AC transcript in corneal epithelium based on a reverse-blot PCRstrip. This system enables detection and visualization of the amplified MUC5AC fragment as a colorimetric band on a strip. We report here an overall correlation between the reverse-blot PCR-strip system and clinical diagnosis of 91.4%, demonstrating the high value of the system for MUC5AC transcript detection in corneal epithelium for diagnostic purposes. Discrepancies between molecular and clinical diagnoses were found in five samples. In study 1, the difference between clinical and molecular diagnosis was observed in one healthy cornea which expressed MUC5AC. The clinical history of this patient was consulted and confirmed the healthy condition. It is thus most likely that this false positive result may be due to imprecise sample collection. When the membrane for the IC was placed on the cornea, it could have touched the

FIGURE 3. Representative example of results obtained from samples tested for MUC5AC amplification in study 1. (A) Visualization of amplification bands on the PCR strips. (B) Visualization of same amplification products in a TAE 2.5% agarose gel. The improvement in the PCR-strip results is illustrated by sample A4. The arrow in (B) indicates the weak band that could be observed in the gel at a higher position than that corresponding to the MUC5AC band, whereas in (A), the arrow indicates the hypothetical position on the strip. The hybridization process and thorough washing of the strip rules out the presence of unspecific PCR product.

LSCD Diagnostic System via MUC5AC Transcript Detection


FIGURE 4. Results obtained in study 2 for MUC5AC amplification. Bands obtained in the PCR strips (top) and in agarose gels (bottom) are shown. For ease of interpretation of results in the PCR strips, a pattern of bands is provided. In agarose gels, the MUC5AC and/or GAPDH bands are presented when appropriate. Dotted arrows indicate those samples in which only the MUC5AC band was visualized in agarose gel, whereas in the PCR strips, both MUC5AC and GAPDH could be seen in the same samples. Single asterisks indicate those samples in which the GAPDH band was clearly apparent, but MUC5AC was very weak in the gel; however, they were clearly positive in PCR strips. The bold arrow indicates a sample (154C-OD) that was clearly negative in the PCR-strip, while weak and nonspecific amplification was observed in the agarose gel. This finding highlights the importance of the specific detection of the PCR strips, provided by the specific probe anchored to the strip membrane. Sample 094-CJ-OD (indicated by a triangle) illustrates a case in which MUC5AC amplification can be observed in the absence of the GADPH band; the result is considered MUC5AC positive. This sample also showed a very weak MUC5AC amplification band in the gel, while the PCR-strip indicated a clear MUC5AC band. Squares highlight the comparable areas in both gel and strips. C-OD, cornea-right eye; CJ-OD, conjunctiva-right eye; C-OS, cornealeft eye; CJ-OS, conjunctiva-left eye; CL, control line position; DC, developing control band; U, inconclusive result; N, PCR negative control consisting of the addition of H2O instead of cDNA template in PCR.

conjunctiva and been contaminated by goblet cells, thus producing a false positive. Since the reverse blot PCR-strip system involves transcript amplification based on polymerase chain reaction, it is a highly sensitive procedure in which a minimum quantity of goblet cells can lead to a positive result. This is a key advantage of this method, particularly when working with small samples of corneal epithelium. However, it is precisely this feature which makes correct sample collection a crucial step in the procedure. In study 2, differences between clinical and molecular diagnoses were found in four corneal samples. Three of them were clinically healthy corneas in which MUC5AC transcript was detected. All three samples were collected in the same hospital center, and a review of the corresponding clinical

records confirmed their normal condition. Two of these samples were taken from the same volunteer diagnosed with dry eye, treated with artificial tears, and not submitted to any surgery. The third sample was taken from the left eye of a 58year-old patient with LSCD in the right eye. This patient had a history of chronic inflammation in the left eye since childhood, but no clinical evidence of LSCD was apparent. Thus, this may be a case of subclinical LSCD with clinical signs that have not yet become apparent. Thus, the molecular method reported herein may be useful for the early detection of LSCD. The fourth incongruous sample was a corneal sample clinically diagnosed with LSCD; however, no MUC5AC amplification was evident. Detection of GAPDH in the sample confirmed the correct processing of the sample. This patient


LSCD Diagnostic System via MUC5AC Transcript Detection TABLE 4. Global Correlation, Sensitivity, and Specificity of the MUC5AC PCR-Strip Detection System Versus Clinical Diagnosis of LSCD Clinical Diagnosis of LSCD MUC5AC PCR Strip

þ

þ NC Total Correlation Overall correlation Sen ¼ TP/(TP þ FN) Spe ¼ TN/(TN þ FP) PPV ¼ TP/(TP þ FP) NPV ¼ TN/(TN þ FN)

44 1 0 45 44/45

4 34 4 42 34/42

Total

CJ

48 18 35 0 4 0 87 18 78/87 18/18 91.4% 98% 89% 92% 97%

FN, false negatives; FP, false positives; Sen, sensitivity; Spe, specificity; TN, total negatives; TP, total positives.

was resampled and retested, but MUC5AC expression continued to be absent. This patient presented peripheral corneal pannus only, which was thought to be due to LSCD with stable corneal epithelium in the central area. Thus, if the corneal IC has been collected from a more central area of the cornea, affected tissue would not have been obtained in the sample, thus explaining the absence of MUC5AC in this LSCD positive case. Alternatively, this case may involve a peripheral corneal pannus induced by causes other than LSCD. Four samples provided an inconclusive result (no MUC5AC or GAPDH amplification) likely due to insufficient sampling by IC. All inconclusive samples were from healthy corneas; one was found in study 1 and three in study 2. The RNA

concentration and RIN value of the sample tested in study 1 were 215 pg/lL and 7.15 respectively, which are satisfactory values for amplification purposes. RNA degradation after quantification could therefore account for these results. The other three inconclusive PCR samples showed very low RNA concentrations (