BASIC INVESTIGATION
Immunocytochemical Diagnosis of Limbal Stem Cell Deficiency: Comparative Analysis of Current Corneal and Conjunctival Biomarkers Muriel Poli, MD, PhD,* Carole Burillon, MD, PhD,* Celine Auxenfans, MD,† Marie-Rose Rovere,† and Odile Damour, MD†
Purpose: To evaluate and compare corneal and conjunctival biomarkers for immunocytochemical diagnosis of limbal stem cell deficiency (LSCD).
Methods: In accordance with the current literature, we selected K12 as the corneal biomarker and K7/K13/K19/MUC5AC as the conjunctival ones. The specificity and accuracy for each biomarker were assessed and compared on 10 healthy subjects and tissues of deceased donors. Twelve eyes of 9 patients clinically suspected of LSCD were enrolled. Epithelial cells (ECs) from the central cornea were collected using impression cytology (IC) and assessed for each biomarker. The presence of conjunctival cells in the central cornea was diagnostic proof of LSCD, whereas the detection of corneal residual cells would quantify the degree of LSCD. Results: K12 and K7/K13/MUC5AC are, respectively, highly specific of corneal and conjunctival differentiation, whereas K19 is not. Normal corneal ECs are not desquamative enough to be suitable for IC. Among 12 eyes with suspected LSCD, 84% (10 of 12) of IC samples were suitable for analysis. K3/K7/K19 immunostaining was positive in 100%, MUC5AC in 40%, and K12 was never observed. Conclusions: Clinical examination can lead to misdiagnosis of LSCD. Immunocytochemical detection of K7/K13 on corneal ECs collected by IC is reproducible, noninvasive, and highly effective in this indication, but without any quantification of the degree of the disease. This time-consuming technique requires skilled technicians and laboratory facilities, reserving it for planned limbal reconstruction. Key Words: limbal stem cell deficiency, impression cytology, cytokeratin, MUC5AC (Cornea 2015;34:817–823)
Received for publication January 18, 2015; revision received March 7, 2015; accepted March 10, 2015. Published online ahead of print May 13, 2015. From the *Clinique Ophtalmologique, Pavillon C, Hôpital Edouard Herriot, Lyon, France; and †Laboratoire des Greffes et Substituts Cutanés, Pavillon I, Hôpital Edouard Herriot, Lyon, France. The authors have no funding or conflicts of interest to disclose. Reprints: Muriel Poli, MD, Clinique Ophtalmologique, Pavillon C, Hôpital Edouard Herriot, 5 place d’Arsonval, 69003 Lyon, France (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cornea Volume 34, Number 7, July 2015
T
he corneal limbus contains stem cells involved in corneal epithelial renewal. Its impairment leads to limbal stem cell deficiency (LSCD) syndrome, characterized by reduced corneal epithelial healing with persistent corneal epithelial defects, conjunctivalization of the corneal epithelium, and severe corneal scarring.1 Graft rejection is the rule without prior limbus reconstruction, and evaluating the viability of stem cells is essential to predicting its outcome. Thus, unfailing diagnosis and quantification of the disease are required in cases of planned limbus reconstruction. Biomicroscopic examination cannot be considered as a reliable technique to ensure diagnosis of LSCD because clinical signs are not specific for the disease. The first minimally invasive techniques for diagnosing LSCD were based on the detection of goblet cells collected by corneal epithelium impression cytology (IC) by means of mucin staining (periodic acid-Schiff staining or immunocytochemical staining of MUC5AC).2–4 Despite the high specificity of this conjunctival marker, goblet cell detection is not sensitive enough to be considered as a valuable tool for LSCD diagnosis because strong goblet cell density reduction can be related to the disease.5–9 Recently, many studies have suggested that the positive immunocytochemical staining against conjunctival keratin on corneal IC samples was a noninvasive and highly specific method to investigate LSCD.4,10–13 Keratin (K) is a group of proteins that form intermediate filaments in epithelial cells (ECs) with high tissue specificity, divided by Moll et al 14 into types I (small, acidic, including K9–K20) and II (large, neutral to basic, including K1–K8). Expression of the K3/K12 pair has long been considered as a specific marker of the corneatype differentiation.14–17 K13, K19, and K7 have recently been reported as markers of the conjunctival-type differentiation.12,18–20 However, the conjunctival specificity of K19 has recently been questioned,4,13,21,22 as has the corneal specificity of K3,4,11,13,20,23,24 strongly suggesting that the actual expression patterns of keratin in the ocular surface epithelium are not as clear-cut as the current dogma suggests. The aim of this study was to evaluate and compare the immunocytochemical detection of corneal (K12) and conjunctival (K7, K13, K19, MUC5AC) marker accuracy to provide a minimally invasive technique for unfailing diagnosis and quantification of LSCD. www.corneajrnl.com |
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MATERIALS AND METHODS
Impression Cytology
Human Samples This prospective and monocentric study followed the tenets of the Declaration of Helsinki and was approved by the institutional review board. All patients were cared for in the Department of Ophthalmology, Hospital Edouard Herriot, Lyon University Hospital, Lyon, France. The inclusion and exclusion criteria for healthy subjects and pathological patients have been described previously.13 Mild LSCD was defined by the absence of visual axis involvement despite peripheral neovascularization and conjunctivalization, whereas severe LSCD was considered in case of complete conjunctivalization and neovascularization, corneal scarring or thinning. Between both, LSCD was scored as moderate. Normal tissue samples were collected from tissues of deceased donors: corneoscleral buttons comprising corneal, limbal, and conjunctival epithelia, not suitable for therapeutic use, were obtained from the tissue and cell bank (Lyon, France), covered with formaldehyde fixative for 0.5 hours, and then paraffin embedded to be sectioned for histological examination. Permission to use the donated tissues for research was obtained from all donor families. Ocular surface cells were obtained after prior informed consent by cytological impression of cornea and bulbar conjunctiva of healthy volunteers (n = 10 eyes of 10 patients, 3 men and 7 women; mean age, 61.0 6 25.0 years) and of patients with a clinical diagnosis of LSCD [n = 12 eyes of 9 patients; 7 men and 5 women; mean age, 51.1 6 13.6 years, with the following etiologies: chemical burn (n = 5), Lyell syndrome (n = 2), idiopathic LSCD (n = 2), thermal burn (n = 1), infection (n = 1), and neurotrophic keratopathy (n = 1)] (Table 1). The patients were ranked by increasing order of severity of the disease.
The technique of ocular surface IC has been described previously.13 The sampling sites chosen were the central cornea and the remote temporal bulbar conjunctiva (10 mm from the limbus) for healthy subjects and areas of maximum corneal conjunctivalization for those patients clinically suspected of having LSCD.
Immunostaining Primary antibodies were against K12, specific for corneal ECs, and K7, K13, K19, and MUC5AC, specific for conjunctival ECs (Table 2). We did not use K3 because previous studies had demonstrated its lack of corneal specificity.4,11,13,20,23,24 The secondary antibodies were Alexa Fluor 488 IgG (H + L) (Invitrogen, diluted 100-fold) and Alexa Fluor 568 IgG (H + L) (Invitrogen, diluted 100-fold) (Table 3). Keratin specificity was previously labeled on healthy samples: the corneal tissue of a healthy deceased patient was the positive control for the K12 corneal marker and the negative control for the K7/K13/K19/MUC5AC conjunctival markers, whereas both bulbar conjunctival tissues of a healthy deceased patient and conjunctival ECs collected by IC from healthy subjects were the positive controls for the K7/K13/ K19/MUC5AC conjunctival markers and negative controls for the K12 corneal marker. In all immunostainings, anisotype negative control (IgG) was carried out. As in a previous study, we did not impress healthy corneas for immunostaining because of the low quantity of desquamative tissue, which partly explains why it was difficult to collect corneal ECs.13 We hypothesized that the expression of K7, K13, K19, or MUC5AC, markers of conjunctival differentiation, on corneal ECs collected by IC was diagnostic proof of LSCD. The presence of K12, a marker of corneal epithelial differentiation, would make it possible to quantify the severity of the disease.
TABLE 1. Clinical Characteristics and Qualitative Fluorescence Immunocytochemistry Analysis of Corneal Impression Cytology Specimens From Patients With Clinically Suspected Limbal Stem Cell Deficiency (n = 12) No
Sex
Age
VA Snellen
Eye
Associated Pathology
Clinical LSCL
First Treatment
1 2 3 4 5 6 7 8 9 10 11 12
F F F M M F F M M M M M
54 43 43 22 56 46 46 53 62 62 49 75
20/50 20/100 CF 20/63 20/63 CF LP LP LP HM LP HM
R R L R R L R R L R L L
Neurotrophic keratopathy Chemical burn (basic) Chemical burn (basic) Chemical burn (basic) Thermal burn Lyell Lyell Chemical burn (basic) Idiopathic Idiopathic Chemical burn (basic) Infection
Mild: PCN, CCO Mild: PCN, CCO Moderate: PCN, CCC Moderate: PCN, CCC Moderate: PCN, CCC Severe: PCN, CVA, CS Severe: PCN, CVA Severe: CC + NV(360°) Severe: CC + NV(360°) Severe: CC + NV(360°) Severe: CC + NV(360°) Severe: CC + NV(360°)
Amniotic membrane Penetrating keratoplasty None Amniotic membrane None None Amniotic membrane None Amniotic membrane Penetrating keratoplasty None Amniotic membrane
K7
+ + + + + + + + + +
K13
+ + + + + + + + + +
K19
K12
Acellular Acellular + 2 + 2 + 2 + 2 + 2 + 2 + 2 + 2 + 2 + 2
MUC5AC
+ 2 2 2 2 2 2 + + +
CC, complete conjunctivalization; CCC, clear central cornea; CCO, central corneal opacification; CF, count fingers; CS, conjunctival synechiae; CVA, conjunctivalization of the visual axis; F, Female; HM, hand motion; LP, light perception; M, male; NV, neovascularization; PCN, peripheral areas of conjunctivalization and neovascularization; VA, visual acuity.
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Cornea Volume 34, Number 7, July 2015
Immunocytochemical Diagnosis
TABLE 2. Primary Antibodies Used During the Study Antigen K7 K12 K13 validation step K13 diagnostic step K19 MUC5AC
Clone
Type
Host
RCK-105 12 (N-16) ks13.1 C-16 NCL-CK19/b170 CLH2
Monoclonal Polyclonal Monoclonal Polyclonal Monoclonal Monoclonal
Mouse Goat Mouse Goat Mouse Mouse
RESULTS Immunostaining on Impression Cytology Validation on Normal Corneal and Conjunctival Epithelia In the first stage, we validated the keratin labeling method using cells from healthy subjects. In normal conditions, IC was simple, fast, and easy to carry out for the operator and painless and atraumatic but uncomfortable for healthy subjects. The results obtained in terms of cell density were highly reproducible. Indeed, 100% of conjunctival ICs provided a high rate of return, giving carpets of many joint cells containing at least 200 cells per field, at ·200 magnification. As previously described, normal corneal ECs were difficult to sample by the IC method, only yielding a handful of isolated cells.13 The cell density decreased significantly as the filters were impressed, because this operation could not be renewed more than 3 times. As shown in Figure 1, K12 was expressed in all layers of both central and peripheral corneal epithelium (Figs. 1I, J) and in the suprabasal and superficial layers of the limbal epithelium (Fig. 1H) but absent from the conjunctiva (Figs. 1F, G), confirming as expected its corneal specificity. Both K7 and K13 were expressed in all layers of the conjunctival epithelium (Figs. 1A, B, F, G) and in the suprabasal and superficial layers of the limbal epithelium (Figs. 1C, H) but were absent in the central and peripheral corneal epithelium (Figs. 1D, E, I, J), confirming their high conjunctival specificity. K19 staining was not only positive in all layers of the conjunctival epithelium (Figs. 1P, Q) but also in the suprabasal layers of both limbal (Fig. 1R) and peripheral corneal
Immunogen Human Human Human Human Human Human
K7 K12 K13 K13 K19 K19
Manufacturer Affinity BioReagents, Golden, CO Santa Cruz Biotechnology, Santa Cruz, CA Chemicon international, Inc, Temcula, CA Santa Cruz Biotechnology, Santa Cruz, CA Visionbiosystems, Novocastra, Nanterre, France Visionbiosystems, Novocastra, Nanterre, France
Dilution 1/100 1/100 1/100 1/100 1/100 1/100
epithelia (Fig. 1S). Thus, although the central corneal epithelium did not stain for this marker (Fig. 1T), K19 was not considered as specific of the conjunctiva. MUC5AC expression was restricted to the conjunctival tissue (Fig. 1L) and conjunctival cells collected by IC (Fig. 1K) but was never observed in any of the samples of limbal and corneal ECs (Figs. 1M–O). Thus, MUC5AC was considered as a specific marker of the conjunctiva. All IgG isotype negative controls were negative.
Assessment of Patients With Clinically Suspected LSCD In the second stage, we looked for keratin expression on cells harvested from patients with a clinical diagnosis of LSCD. Carrying out IC proved painless but uncomfortable for patients with LSCD. No side effects were found to be related to sampling. It was easy to identify the areas of maximum conjunctivalization with the naked eye. In patients 1 and 2, IC samples did not harvest enough cells for analysis (Fig. 2). Therefore, for both of them, immunostaining alone was not able to provide unfailing diagnosis. Except for these 2 cases, a carpet of cells was harvested, respecting the architecture of the epithelial tissue and intercellular junctions. The number of cells decreased with the number of impressions, leaving sufficient cells on the second impressed slide for analysis. Finally, on slides impressed too strongly, the cell cytoplasm appeared disorganized and the cell nuclei were poorly individualized. Of the 10 analyzable samples (patients 3–12), the cells collected strongly expressed K7, K13, and K19 with no expression of K12, confirming the replacement of corneal
TABLE 3. Secondary Antibodies Used During the Study Antigen
Secondary Antibodies
K7 validation step K13 validation step K19 diagnostic step MUC5AC all steps K19 validation step
Alexa Fluor 568 IgG TRITC
Rabbit
Mouse
Invitrogen Corporation, Carlsbad, CA
1/100
Alexa Fluor 488 IgG FITC Alexa Fluor 488 IgG FITC
Goat
Mouse
Invitrogen Corporation, Carlsbad, CA
1/100
Donkey
Goat
Invitrogen Corporation, Carlsbad, CA
1/100
K12 diagnostic step K13 diagnostic step
Host
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Immunogen
Manufacturer
Dilution
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FIGURE 1. Cellular expression of keratin both after cytological impression of the bulbar conjunctiva and analysis of corneoscleral buttons comprising corneal, limbal, and conjunctival epithelia (corneal tissue is the positive control of the cornea and the negative control of the conjunctiva; conjunctival tissues are positive controls of the conjunctiva and negative controls of the cornea). K12 is specific to corneal epithelium because it is present in 100% of corneal cells and absent from conjunctival cells (F–J); K7, K13, and MUC5AC are specific to conjunctival epithelium because they are present in conjunctival cells and absent from corneal cells (A–O). K19 is not a specific marker of the conjunctiva because it is expressed in suprabasal layers of the peripheral cornea (S). Scale bar: 100 mm.
epithelium by conjunctival epithelium (Table 1, Fig. 2). In these cases, immunostaining alone provided unfailing diagnosis of LSCD without quantification of the degree of LSCD severity. Concerning MUC5AC, the immunostaining was positive in 4 of 10 patients clinically suspected of having LSCD related to chemical burn (patients 3 and 11), idiopathic etiology (patient 10), and infection (patient 12) (Table 1, Fig. 2). Despite pathognomonic clinical features of LSCD and positive staining for conjunctival markers, MUC5AC staining was negative for the other patients, demonstrating a major lack of sensitivity with that method.
Unfailing diagnosis of LSCD is a determinant in the management of the disease and has a great influence on the
treatment outcome. However, it is currently based on clinical examination (medical history and slit-lamp biomicroscopy) looking for nonspecific signs characterizing the disease. Our aim was to evaluate and compare new ocular surface markers for minimally invasive LSCD diagnosis to improve patient management, looking for evidence of limbal stem cell niche impairment and/or corneal conjunctivalization. The first step in this study confirmed that cytological impression of the ocular surface is a very suitable minimally invasive technique for collecting conjunctival ECs, with no side effects or contraindications compared with biopsy. Furthermore, when LSCD is present, many cells can be collected. In contrast, in healthy subjects, it retrieves few corneal ECs, probably because the corneal epithelium has little desquamative tissue. As demonstrated in a previous study, we concluded that a rich cytological impression is
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DISCUSSION
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Immunocytochemical Diagnosis
FIGURE 2. Biomicroscopic characteristics, corneal IC, and RT polymerase chain reaction detection of MUC5AC mRNA of 4 of the 12 eyes with a clinical diagnosis of LSCD. Patient 1: no cells were collected by corneal IC, suggesting a normal corneal differentiation. Patients 4, 7, and 11: Collected cells strongly expressed K7, K13, and K19 with no expression of K12, confirming the replacement of a corneal epithelium by a conjunctival epithelium even for patient 4 who had a moderate form of the disease. MUC5AC staining was rarely positive (patient 11). Scale bar: 100 mm.
therefore a characteristic that is specific to the conjunctival epithelium, and the high retrieval rate of cells by corneal IC is in itself a criterion of conjunctival differentiation and thus a diagnosis of LSCD.13 Healthy corneal and conjunctival epithelia were assessed for specific immunostaining techniques. Among the markers that we selected in accordance with the current data in the literature, strong corneal specificity was demonstrated for K12 and strong conjunctival specificity for K7, K13, and MUC5AC, as recently confirmed by many studies12,21,25 (Fig. 1). In contrast, K19-positive staining in the basal layers of the peripheral cornea confirmed the lack of conjunctival specificity of this marker.4,12,21,26,27 The second step in this study demonstrated that in LSCD conditions, IC produces strong returns with 84% of samples (10 of 12) suitable for analysis. In 2 cases, patients 1 and 2, IC did not collect enough cells. Both patients were clinically suspected of mild LSCD, and biomicroscopic examination found peripheral mild corneal neovascularization and central opacification, which are major but unspecific Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
clinical signs of LSCD. Patient 1 suffered from neurotrophic keratopathy related to facial palsy and patient 2 from bilateral chemical burn, which are common etiologies of LSCD. According to previous results obtained from healthy donors, lack of cellularity could have been the hallmark of corneal epithelium. Nevertheless, it could also have been related to a lack of sensitivity of the cell collection technique. Of the 10 analyzable samples, all were strongly positive for K7, K13, and K19 (100% of collected cells), suggesting their great sensitivity in the moderate to severe form of the disease (Table 1, Fig. 2, patients 3–5), but the immunocytochemical search for both K7 and K13 should be more efficient than K19 in diagnosing LSCD given the K19’s lack of conjunctival specificity. The negative predictive value of the test was confirmed on healthy histological tissue. In those cases, immunostaining on IC could alone be sufficient to provide certain diagnosis of LSCD, but without any quantification of the degree of the disease. Recently, levels of fluorescence expression of the different markers were www.corneajrnl.com |
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quantified through quantitative fluorescence immunohistochemistry to provide a grading of LSCD extent with promising results.4,28 MUC5AC-positive staining indicates the presence of goblet cells and thus indicates the conjunctival phenotype of the collected ECs.2–4 Of the 10 analyzable samples, MUC5AC immunostaining was negative in 6 cases of moderate to severe LSCD, whereas keratin immunostaining proved the conjunctival phenotype for all of them, suggesting its low accuracy for LSCD diagnosis by lack of sensitivity (Table 1, Fig. 2). Indeed, it has been clearly established that goblet cell density could decrease by up to 99% from the ocular surface depending on the etiology of LSCD.5–8 Recently, a new method of MUC5AC screening by means of RT polymerase chain reaction and IC has been reported with 98% of sensitivity and 89% of specificity, overcoming the variability of goblet cell density.25 Finally, K12 staining, a marker of corneal differentiation, could be used to refute an LSCD diagnosis or to quantify the severity of the disease when combined with conjunctival markers, its detection defining incomplete LSCD. In this study, K12 staining was negative for all LSCD samples. Two main hypotheses could explain this result: first, in patients 6 to 12 suspected of severe LSCD, the corneal phenotype could have been totally replaced by a conjunctival phenotype. Second, for patients 1 to 5 suspected of mild to moderate LSCD, K12 negativity could be related to how difficult it was to collect normal corneal ECs using IC related to the low quantity of desquamative tissue. Indeed, it should be remembered that a significant sampling error does exist in the frequency of K12+ cells sampled as shown in healthy subjects, where few corneal ECs were retrieved by IC.13 Therefore, K12 immunostaining is not recommended. Recently, in vivo confocal microscopy of the ocular surface has been evaluated in the diagnosis and monitoring of LSCD with promising results.29–31 This technique could quantify the severity of the disease, detecting residuals corneal ECs and/or palisades of Vogt.
interpretation, limiting its expansion in clinical practice and reserving it for planned limbal reconstruction. REFERENCES
We demonstrated that clinical examination could lead to misdiagnosis of LSCD while unfailing diagnosis of the disease and quantification of its severity are essential in cases of planned limbal reconstruction. Immunocytochemical detection of conjunctival keratin on corneal ECs collected by IC is a reproducible, noninvasive, and highly accurate method to investigate LSCD. We recommend both K7 and K13 in this indication, 2 equivalent and highly specific biomarkers of conjunctival differentiation. This labeling overcomes the variability of goblet cell density and leads to unfailing diagnosis of LSCD in 84% of cases, but without any quantification of the severity of the disease. The use of K19 should be avoided because of its lack of conjunctival specificity, whereas both MUC5AC and K12 detection are not sensitive enough to be recommended. This time-consuming technique requires skilled technicians and laboratory facilities for processing, staining, and
1. Dua HS. The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol. 1998;82:1407–1411. 2. Chen JJ, Tseng SC. Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. Invest Ophthalmology Vis Sci. 1991;32:2219–2233. 3. Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology. 1995;102:1476–1485. 4. Barbaro V, Ferrari S, Fasolo A, et al. Evaluation of ocular surface disorders: a new diagnostic tool based on impression cytology and confocal laser scanning microscopy. Br J Ophthalmol. 2010;94:926–932. 5. Nelson JD, Wright JC. Conjunctival goblet cell densities in ocular surface disease. Arch Ophthalmol. 1984;102:1049–1051. 6. Rivas L, Oroza MA, Perez-Esteban A, et al. Morphological changes in ocular surface in dry eyes and other disorders by impression cytology. Graefes Arch Clin Exp Ophthalmol. 1992;230:329–334. 7. Kinoshita S, Kiorpes TC, Friend J, et al. Goblet cell density in ocular surface disease. A better indicator than tear mucin. Arch Ophthalmol. 1983;101:1284–1287. 8. Ohji M, Ohmi G, Kiritoshi A, et al. Goblet cell density in thermal and chemical injuries. Arch Ophthalmol. 1987;105:1686–1688. 9. Sacchetti M, Lambiase A, Cortes M, et al. Clinical and cytological findings in limbal stem cell deficiency. Graefes Arch Clin Exp Ophthalmol. 2005;243:870–876. 10. Donisi PM, Rama P, Fasolo A, et al. Analysis of limbal stem cell deficiency by corneal impression cytology. Cornea. 2003;22:533–538. 11. Espana EM, Di Pascuale MA, He H, et al. Characterization of corneal pannus removed from patients with total limbal stem cell deficiency. Invest Ophthalmol Vis Sci. 2004;45:2961–2966. 12. Jirsova K, Dudakova L, Kalasova S, et al. The OV-TL 12/30 clone of anti-cytokeratin 7 antibody as a new marker of corneal conjunctivalization in patients with limbal stem cell deficiency. Invest Ophthalmol Vis Sci. 2011;52:5892–5898. 13. Poli M, Janin H, Justin V, et al. Keratin 13 immunostaining in corneal impression cytology for the diagnosis of limbal stem cell deficiency. Invest Ophthalmol Vis Sci. 2011;52:9411–9415. 14. Moll R, Franke WW, Schiller DL, et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982;31:11–24. 15. Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;103:49–62. 16. Kasper M, Moll R, Stosiek P, et al. Patterns of cytokeratin and vimentin expression in the human eye. Histochemistry. 1988;89:369–377. 17. Chaloin-Dufau C, Sun TT, Dhouailly D. Appearance of the keratin pair K3/K12 during embryonic and adult corneal epithelial differentiation in the chick and in the rabbit. Cell Differ Dev. 1990;32:97–108. 18. Krenzer KL, Freddo TF. Cytokeratin expression in normal human bulbar conjunctiva obtained by impression cytology. Invest Ophthalmol Vis Sci. 1997;38:142–152. 19. Ang LP, Tan DT, Beuerman RW, et al. Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system. Invest Ophthalmol Vis Sci. 2004;45: 1789–1795. 20. Elder MJ, Hiscott P, Dart JK. Intermediate filament expression by normal and diseased human corneal epithelium. Hum Pathol. 1997;28: 1348–1354. 21. Ramirez-Miranda A, Nakatsu MN, Zarei-Ghanavati S, et al. Keratin 13 is a more specific marker of conjunctival epithelium than keratin 19. Mol Vision. 2011;17:1652–1661. 22. Garcia I, Etxebarria J, Boto-de-Los-Bueis A, et al. Comparative study of limbal stem cell deficiency diagnosis methods: detection of MUC5AC mRNA and goblet cells in corneal epithelium. Ophthalmology. 2012;119: 923–929. 23. Kawasaki S, Tanioka H, Yamasaki K, et al. Clusters of corneal epithelial cells reside ectopically in human conjunctival epithelium. Invest Ophthalmol Vis Sci. 2006;47:1359–1367.
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24. Wei ZG, Wu RL, Lavker RM, et al. In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci. 1993;34:1814–1828. 25. Garcia I, Etxebarria J, Merayo-Lloves 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. 26. Yoshida S, Shimmura S, Kawakita T, et al. Cytokeratin 15 can be used to identify the limbal phenotype in normal and diseased ocular surfaces. Invest Ophthalmol Vis Sci. 2006;47: 4780–4786. 27. Kim HS, Jun Song X, de Paiva CS, et al. Phenotypic characterization of human corneal epithelial cells expanded ex vivo
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Immunocytochemical Diagnosis
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from limbal explant and single cell cultures. Exp Eye Res. 2004; 79:41–49. Pisella PJ, Brignole F, Debbasch C, et al. Flow cytometric analysis of conjunctival epithelium in ocular rosacea and keratoconjunctivitis sicca. Ophthalmology. 2000;107:1841–1849. Lagali N, Eden U, Utheim TP, et al. In vivo morphology of the limbal palisades of vogt correlates with progressive stem cell deficiency in aniridiarelated keratopathy. Invest Ophthalmol Vis Sci. 2013;54:5333–5342. Araujo AL, Ricardo JR, Sakai VN, et al. Impression cytology and in vivo confocal microscopy in corneas with total limbal stem cell deficiency. Arq Bras Oftalmol. 2013;76:305–308. Goldberg MF. In vivo confocal microscopy and diagnosis of limbal stem cell deficiency. Photographing the palisades of vogt and limbal stem cells. Am J Ophthalmol. 2013;156:205–206.
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Immunocytochemical Diagnosis of Limbal Stem Cell Deficiency: Comparative Analysis of Current Corneal and Conjunctival Biomarkers Muriel Poli, MD, PhD,* Carole Burillon, MD, PhD,* Celine Auxenfans, MD,† Marie-Rose Rovere,† and Odile Damour, MD†
Purpose: To evaluate and compare corneal and conjunctival biomarkers for immunocytochemical diagnosis of limbal stem cell deficiency (LSCD).
Methods: In accordance with the current literature, we selected K12 as the corneal biomarker and K7/K13/K19/MUC5AC as the conjunctival ones. The specificity and accuracy for each biomarker were assessed and compared on 10 healthy subjects and tissues of deceased donors. Twelve eyes of 9 patients clinically suspected of LSCD were enrolled. Epithelial cells (ECs) from the central cornea were collected using impression cytology (IC) and assessed for each biomarker. The presence of conjunctival cells in the central cornea was diagnostic proof of LSCD, whereas the detection of corneal residual cells would quantify the degree of LSCD. Results: K12 and K7/K13/MUC5AC are, respectively, highly specific of corneal and conjunctival differentiation, whereas K19 is not. Normal corneal ECs are not desquamative enough to be suitable for IC. Among 12 eyes with suspected LSCD, 84% (10 of 12) of IC samples were suitable for analysis. K3/K7/K19 immunostaining was positive in 100%, MUC5AC in 40%, and K12 was never observed. Conclusions: Clinical examination can lead to misdiagnosis of LSCD. Immunocytochemical detection of K7/K13 on corneal ECs collected by IC is reproducible, noninvasive, and highly effective in this indication, but without any quantification of the degree of the disease. This time-consuming technique requires skilled technicians and laboratory facilities, reserving it for planned limbal reconstruction. Key Words: limbal stem cell deficiency, impression cytology, cytokeratin, MUC5AC (Cornea 2015;34:817–823)
Received for publication January 18, 2015; revision received March 7, 2015; accepted March 10, 2015. Published online ahead of print May 13, 2015. From the *Clinique Ophtalmologique, Pavillon C, Hôpital Edouard Herriot, Lyon, France; and †Laboratoire des Greffes et Substituts Cutanés, Pavillon I, Hôpital Edouard Herriot, Lyon, France. The authors have no funding or conflicts of interest to disclose. Reprints: Muriel Poli, MD, Clinique Ophtalmologique, Pavillon C, Hôpital Edouard Herriot, 5 place d’Arsonval, 69003 Lyon, France (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
Cornea Volume 34, Number 7, July 2015
T
he corneal limbus contains stem cells involved in corneal epithelial renewal. Its impairment leads to limbal stem cell deficiency (LSCD) syndrome, characterized by reduced corneal epithelial healing with persistent corneal epithelial defects, conjunctivalization of the corneal epithelium, and severe corneal scarring.1 Graft rejection is the rule without prior limbus reconstruction, and evaluating the viability of stem cells is essential to predicting its outcome. Thus, unfailing diagnosis and quantification of the disease are required in cases of planned limbus reconstruction. Biomicroscopic examination cannot be considered as a reliable technique to ensure diagnosis of LSCD because clinical signs are not specific for the disease. The first minimally invasive techniques for diagnosing LSCD were based on the detection of goblet cells collected by corneal epithelium impression cytology (IC) by means of mucin staining (periodic acid-Schiff staining or immunocytochemical staining of MUC5AC).2–4 Despite the high specificity of this conjunctival marker, goblet cell detection is not sensitive enough to be considered as a valuable tool for LSCD diagnosis because strong goblet cell density reduction can be related to the disease.5–9 Recently, many studies have suggested that the positive immunocytochemical staining against conjunctival keratin on corneal IC samples was a noninvasive and highly specific method to investigate LSCD.4,10–13 Keratin (K) is a group of proteins that form intermediate filaments in epithelial cells (ECs) with high tissue specificity, divided by Moll et al 14 into types I (small, acidic, including K9–K20) and II (large, neutral to basic, including K1–K8). Expression of the K3/K12 pair has long been considered as a specific marker of the corneatype differentiation.14–17 K13, K19, and K7 have recently been reported as markers of the conjunctival-type differentiation.12,18–20 However, the conjunctival specificity of K19 has recently been questioned,4,13,21,22 as has the corneal specificity of K3,4,11,13,20,23,24 strongly suggesting that the actual expression patterns of keratin in the ocular surface epithelium are not as clear-cut as the current dogma suggests. The aim of this study was to evaluate and compare the immunocytochemical detection of corneal (K12) and conjunctival (K7, K13, K19, MUC5AC) marker accuracy to provide a minimally invasive technique for unfailing diagnosis and quantification of LSCD. www.corneajrnl.com |
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Cornea Volume 34, Number 7, July 2015
Poli et al
MATERIALS AND METHODS
Impression Cytology
Human Samples This prospective and monocentric study followed the tenets of the Declaration of Helsinki and was approved by the institutional review board. All patients were cared for in the Department of Ophthalmology, Hospital Edouard Herriot, Lyon University Hospital, Lyon, France. The inclusion and exclusion criteria for healthy subjects and pathological patients have been described previously.13 Mild LSCD was defined by the absence of visual axis involvement despite peripheral neovascularization and conjunctivalization, whereas severe LSCD was considered in case of complete conjunctivalization and neovascularization, corneal scarring or thinning. Between both, LSCD was scored as moderate. Normal tissue samples were collected from tissues of deceased donors: corneoscleral buttons comprising corneal, limbal, and conjunctival epithelia, not suitable for therapeutic use, were obtained from the tissue and cell bank (Lyon, France), covered with formaldehyde fixative for 0.5 hours, and then paraffin embedded to be sectioned for histological examination. Permission to use the donated tissues for research was obtained from all donor families. Ocular surface cells were obtained after prior informed consent by cytological impression of cornea and bulbar conjunctiva of healthy volunteers (n = 10 eyes of 10 patients, 3 men and 7 women; mean age, 61.0 6 25.0 years) and of patients with a clinical diagnosis of LSCD [n = 12 eyes of 9 patients; 7 men and 5 women; mean age, 51.1 6 13.6 years, with the following etiologies: chemical burn (n = 5), Lyell syndrome (n = 2), idiopathic LSCD (n = 2), thermal burn (n = 1), infection (n = 1), and neurotrophic keratopathy (n = 1)] (Table 1). The patients were ranked by increasing order of severity of the disease.
The technique of ocular surface IC has been described previously.13 The sampling sites chosen were the central cornea and the remote temporal bulbar conjunctiva (10 mm from the limbus) for healthy subjects and areas of maximum corneal conjunctivalization for those patients clinically suspected of having LSCD.
Immunostaining Primary antibodies were against K12, specific for corneal ECs, and K7, K13, K19, and MUC5AC, specific for conjunctival ECs (Table 2). We did not use K3 because previous studies had demonstrated its lack of corneal specificity.4,11,13,20,23,24 The secondary antibodies were Alexa Fluor 488 IgG (H + L) (Invitrogen, diluted 100-fold) and Alexa Fluor 568 IgG (H + L) (Invitrogen, diluted 100-fold) (Table 3). Keratin specificity was previously labeled on healthy samples: the corneal tissue of a healthy deceased patient was the positive control for the K12 corneal marker and the negative control for the K7/K13/K19/MUC5AC conjunctival markers, whereas both bulbar conjunctival tissues of a healthy deceased patient and conjunctival ECs collected by IC from healthy subjects were the positive controls for the K7/K13/ K19/MUC5AC conjunctival markers and negative controls for the K12 corneal marker. In all immunostainings, anisotype negative control (IgG) was carried out. As in a previous study, we did not impress healthy corneas for immunostaining because of the low quantity of desquamative tissue, which partly explains why it was difficult to collect corneal ECs.13 We hypothesized that the expression of K7, K13, K19, or MUC5AC, markers of conjunctival differentiation, on corneal ECs collected by IC was diagnostic proof of LSCD. The presence of K12, a marker of corneal epithelial differentiation, would make it possible to quantify the severity of the disease.
TABLE 1. Clinical Characteristics and Qualitative Fluorescence Immunocytochemistry Analysis of Corneal Impression Cytology Specimens From Patients With Clinically Suspected Limbal Stem Cell Deficiency (n = 12) No
Sex
Age
VA Snellen
Eye
Associated Pathology
Clinical LSCL
First Treatment
1 2 3 4 5 6 7 8 9 10 11 12
F F F M M F F M M M M M
54 43 43 22 56 46 46 53 62 62 49 75
20/50 20/100 CF 20/63 20/63 CF LP LP LP HM LP HM
R R L R R L R R L R L L
Neurotrophic keratopathy Chemical burn (basic) Chemical burn (basic) Chemical burn (basic) Thermal burn Lyell Lyell Chemical burn (basic) Idiopathic Idiopathic Chemical burn (basic) Infection
Mild: PCN, CCO Mild: PCN, CCO Moderate: PCN, CCC Moderate: PCN, CCC Moderate: PCN, CCC Severe: PCN, CVA, CS Severe: PCN, CVA Severe: CC + NV(360°) Severe: CC + NV(360°) Severe: CC + NV(360°) Severe: CC + NV(360°) Severe: CC + NV(360°)
Amniotic membrane Penetrating keratoplasty None Amniotic membrane None None Amniotic membrane None Amniotic membrane Penetrating keratoplasty None Amniotic membrane
K7
+ + + + + + + + + +
K13
+ + + + + + + + + +
K19
K12
Acellular Acellular + 2 + 2 + 2 + 2 + 2 + 2 + 2 + 2 + 2 + 2
MUC5AC
+ 2 2 2 2 2 2 + + +
CC, complete conjunctivalization; CCC, clear central cornea; CCO, central corneal opacification; CF, count fingers; CS, conjunctival synechiae; CVA, conjunctivalization of the visual axis; F, Female; HM, hand motion; LP, light perception; M, male; NV, neovascularization; PCN, peripheral areas of conjunctivalization and neovascularization; VA, visual acuity.
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Cornea Volume 34, Number 7, July 2015
Immunocytochemical Diagnosis
TABLE 2. Primary Antibodies Used During the Study Antigen K7 K12 K13 validation step K13 diagnostic step K19 MUC5AC
Clone
Type
Host
RCK-105 12 (N-16) ks13.1 C-16 NCL-CK19/b170 CLH2
Monoclonal Polyclonal Monoclonal Polyclonal Monoclonal Monoclonal
Mouse Goat Mouse Goat Mouse Mouse
RESULTS Immunostaining on Impression Cytology Validation on Normal Corneal and Conjunctival Epithelia In the first stage, we validated the keratin labeling method using cells from healthy subjects. In normal conditions, IC was simple, fast, and easy to carry out for the operator and painless and atraumatic but uncomfortable for healthy subjects. The results obtained in terms of cell density were highly reproducible. Indeed, 100% of conjunctival ICs provided a high rate of return, giving carpets of many joint cells containing at least 200 cells per field, at ·200 magnification. As previously described, normal corneal ECs were difficult to sample by the IC method, only yielding a handful of isolated cells.13 The cell density decreased significantly as the filters were impressed, because this operation could not be renewed more than 3 times. As shown in Figure 1, K12 was expressed in all layers of both central and peripheral corneal epithelium (Figs. 1I, J) and in the suprabasal and superficial layers of the limbal epithelium (Fig. 1H) but absent from the conjunctiva (Figs. 1F, G), confirming as expected its corneal specificity. Both K7 and K13 were expressed in all layers of the conjunctival epithelium (Figs. 1A, B, F, G) and in the suprabasal and superficial layers of the limbal epithelium (Figs. 1C, H) but were absent in the central and peripheral corneal epithelium (Figs. 1D, E, I, J), confirming their high conjunctival specificity. K19 staining was not only positive in all layers of the conjunctival epithelium (Figs. 1P, Q) but also in the suprabasal layers of both limbal (Fig. 1R) and peripheral corneal
Immunogen Human Human Human Human Human Human
K7 K12 K13 K13 K19 K19
Manufacturer Affinity BioReagents, Golden, CO Santa Cruz Biotechnology, Santa Cruz, CA Chemicon international, Inc, Temcula, CA Santa Cruz Biotechnology, Santa Cruz, CA Visionbiosystems, Novocastra, Nanterre, France Visionbiosystems, Novocastra, Nanterre, France
Dilution 1/100 1/100 1/100 1/100 1/100 1/100
epithelia (Fig. 1S). Thus, although the central corneal epithelium did not stain for this marker (Fig. 1T), K19 was not considered as specific of the conjunctiva. MUC5AC expression was restricted to the conjunctival tissue (Fig. 1L) and conjunctival cells collected by IC (Fig. 1K) but was never observed in any of the samples of limbal and corneal ECs (Figs. 1M–O). Thus, MUC5AC was considered as a specific marker of the conjunctiva. All IgG isotype negative controls were negative.
Assessment of Patients With Clinically Suspected LSCD In the second stage, we looked for keratin expression on cells harvested from patients with a clinical diagnosis of LSCD. Carrying out IC proved painless but uncomfortable for patients with LSCD. No side effects were found to be related to sampling. It was easy to identify the areas of maximum conjunctivalization with the naked eye. In patients 1 and 2, IC samples did not harvest enough cells for analysis (Fig. 2). Therefore, for both of them, immunostaining alone was not able to provide unfailing diagnosis. Except for these 2 cases, a carpet of cells was harvested, respecting the architecture of the epithelial tissue and intercellular junctions. The number of cells decreased with the number of impressions, leaving sufficient cells on the second impressed slide for analysis. Finally, on slides impressed too strongly, the cell cytoplasm appeared disorganized and the cell nuclei were poorly individualized. Of the 10 analyzable samples (patients 3–12), the cells collected strongly expressed K7, K13, and K19 with no expression of K12, confirming the replacement of corneal
TABLE 3. Secondary Antibodies Used During the Study Antigen
Secondary Antibodies
K7 validation step K13 validation step K19 diagnostic step MUC5AC all steps K19 validation step
Alexa Fluor 568 IgG TRITC
Rabbit
Mouse
Invitrogen Corporation, Carlsbad, CA
1/100
Alexa Fluor 488 IgG FITC Alexa Fluor 488 IgG FITC
Goat
Mouse
Invitrogen Corporation, Carlsbad, CA
1/100
Donkey
Goat
Invitrogen Corporation, Carlsbad, CA
1/100
K12 diagnostic step K13 diagnostic step
Host
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Immunogen
Manufacturer
Dilution
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Cornea Volume 34, Number 7, July 2015
Poli et al
FIGURE 1. Cellular expression of keratin both after cytological impression of the bulbar conjunctiva and analysis of corneoscleral buttons comprising corneal, limbal, and conjunctival epithelia (corneal tissue is the positive control of the cornea and the negative control of the conjunctiva; conjunctival tissues are positive controls of the conjunctiva and negative controls of the cornea). K12 is specific to corneal epithelium because it is present in 100% of corneal cells and absent from conjunctival cells (F–J); K7, K13, and MUC5AC are specific to conjunctival epithelium because they are present in conjunctival cells and absent from corneal cells (A–O). K19 is not a specific marker of the conjunctiva because it is expressed in suprabasal layers of the peripheral cornea (S). Scale bar: 100 mm.
epithelium by conjunctival epithelium (Table 1, Fig. 2). In these cases, immunostaining alone provided unfailing diagnosis of LSCD without quantification of the degree of LSCD severity. Concerning MUC5AC, the immunostaining was positive in 4 of 10 patients clinically suspected of having LSCD related to chemical burn (patients 3 and 11), idiopathic etiology (patient 10), and infection (patient 12) (Table 1, Fig. 2). Despite pathognomonic clinical features of LSCD and positive staining for conjunctival markers, MUC5AC staining was negative for the other patients, demonstrating a major lack of sensitivity with that method.
Unfailing diagnosis of LSCD is a determinant in the management of the disease and has a great influence on the
treatment outcome. However, it is currently based on clinical examination (medical history and slit-lamp biomicroscopy) looking for nonspecific signs characterizing the disease. Our aim was to evaluate and compare new ocular surface markers for minimally invasive LSCD diagnosis to improve patient management, looking for evidence of limbal stem cell niche impairment and/or corneal conjunctivalization. The first step in this study confirmed that cytological impression of the ocular surface is a very suitable minimally invasive technique for collecting conjunctival ECs, with no side effects or contraindications compared with biopsy. Furthermore, when LSCD is present, many cells can be collected. In contrast, in healthy subjects, it retrieves few corneal ECs, probably because the corneal epithelium has little desquamative tissue. As demonstrated in a previous study, we concluded that a rich cytological impression is
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DISCUSSION
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Cornea Volume 34, Number 7, July 2015
Immunocytochemical Diagnosis
FIGURE 2. Biomicroscopic characteristics, corneal IC, and RT polymerase chain reaction detection of MUC5AC mRNA of 4 of the 12 eyes with a clinical diagnosis of LSCD. Patient 1: no cells were collected by corneal IC, suggesting a normal corneal differentiation. Patients 4, 7, and 11: Collected cells strongly expressed K7, K13, and K19 with no expression of K12, confirming the replacement of a corneal epithelium by a conjunctival epithelium even for patient 4 who had a moderate form of the disease. MUC5AC staining was rarely positive (patient 11). Scale bar: 100 mm.
therefore a characteristic that is specific to the conjunctival epithelium, and the high retrieval rate of cells by corneal IC is in itself a criterion of conjunctival differentiation and thus a diagnosis of LSCD.13 Healthy corneal and conjunctival epithelia were assessed for specific immunostaining techniques. Among the markers that we selected in accordance with the current data in the literature, strong corneal specificity was demonstrated for K12 and strong conjunctival specificity for K7, K13, and MUC5AC, as recently confirmed by many studies12,21,25 (Fig. 1). In contrast, K19-positive staining in the basal layers of the peripheral cornea confirmed the lack of conjunctival specificity of this marker.4,12,21,26,27 The second step in this study demonstrated that in LSCD conditions, IC produces strong returns with 84% of samples (10 of 12) suitable for analysis. In 2 cases, patients 1 and 2, IC did not collect enough cells. Both patients were clinically suspected of mild LSCD, and biomicroscopic examination found peripheral mild corneal neovascularization and central opacification, which are major but unspecific Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
clinical signs of LSCD. Patient 1 suffered from neurotrophic keratopathy related to facial palsy and patient 2 from bilateral chemical burn, which are common etiologies of LSCD. According to previous results obtained from healthy donors, lack of cellularity could have been the hallmark of corneal epithelium. Nevertheless, it could also have been related to a lack of sensitivity of the cell collection technique. Of the 10 analyzable samples, all were strongly positive for K7, K13, and K19 (100% of collected cells), suggesting their great sensitivity in the moderate to severe form of the disease (Table 1, Fig. 2, patients 3–5), but the immunocytochemical search for both K7 and K13 should be more efficient than K19 in diagnosing LSCD given the K19’s lack of conjunctival specificity. The negative predictive value of the test was confirmed on healthy histological tissue. In those cases, immunostaining on IC could alone be sufficient to provide certain diagnosis of LSCD, but without any quantification of the degree of the disease. Recently, levels of fluorescence expression of the different markers were www.corneajrnl.com |
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Cornea Volume 34, Number 7, July 2015
Poli et al
quantified through quantitative fluorescence immunohistochemistry to provide a grading of LSCD extent with promising results.4,28 MUC5AC-positive staining indicates the presence of goblet cells and thus indicates the conjunctival phenotype of the collected ECs.2–4 Of the 10 analyzable samples, MUC5AC immunostaining was negative in 6 cases of moderate to severe LSCD, whereas keratin immunostaining proved the conjunctival phenotype for all of them, suggesting its low accuracy for LSCD diagnosis by lack of sensitivity (Table 1, Fig. 2). Indeed, it has been clearly established that goblet cell density could decrease by up to 99% from the ocular surface depending on the etiology of LSCD.5–8 Recently, a new method of MUC5AC screening by means of RT polymerase chain reaction and IC has been reported with 98% of sensitivity and 89% of specificity, overcoming the variability of goblet cell density.25 Finally, K12 staining, a marker of corneal differentiation, could be used to refute an LSCD diagnosis or to quantify the severity of the disease when combined with conjunctival markers, its detection defining incomplete LSCD. In this study, K12 staining was negative for all LSCD samples. Two main hypotheses could explain this result: first, in patients 6 to 12 suspected of severe LSCD, the corneal phenotype could have been totally replaced by a conjunctival phenotype. Second, for patients 1 to 5 suspected of mild to moderate LSCD, K12 negativity could be related to how difficult it was to collect normal corneal ECs using IC related to the low quantity of desquamative tissue. Indeed, it should be remembered that a significant sampling error does exist in the frequency of K12+ cells sampled as shown in healthy subjects, where few corneal ECs were retrieved by IC.13 Therefore, K12 immunostaining is not recommended. Recently, in vivo confocal microscopy of the ocular surface has been evaluated in the diagnosis and monitoring of LSCD with promising results.29–31 This technique could quantify the severity of the disease, detecting residuals corneal ECs and/or palisades of Vogt.
interpretation, limiting its expansion in clinical practice and reserving it for planned limbal reconstruction. REFERENCES
We demonstrated that clinical examination could lead to misdiagnosis of LSCD while unfailing diagnosis of the disease and quantification of its severity are essential in cases of planned limbal reconstruction. Immunocytochemical detection of conjunctival keratin on corneal ECs collected by IC is a reproducible, noninvasive, and highly accurate method to investigate LSCD. We recommend both K7 and K13 in this indication, 2 equivalent and highly specific biomarkers of conjunctival differentiation. This labeling overcomes the variability of goblet cell density and leads to unfailing diagnosis of LSCD in 84% of cases, but without any quantification of the severity of the disease. The use of K19 should be avoided because of its lack of conjunctival specificity, whereas both MUC5AC and K12 detection are not sensitive enough to be recommended. This time-consuming technique requires skilled technicians and laboratory facilities for processing, staining, and
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Cornea Volume 34, Number 7, July 2015
24. Wei ZG, Wu RL, Lavker RM, et al. In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci. 1993;34:1814–1828. 25. Garcia I, Etxebarria J, Merayo-Lloves 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. 26. Yoshida S, Shimmura S, Kawakita T, et al. Cytokeratin 15 can be used to identify the limbal phenotype in normal and diseased ocular surfaces. Invest Ophthalmol Vis Sci. 2006;47: 4780–4786. 27. Kim HS, Jun Song X, de Paiva CS, et al. Phenotypic characterization of human corneal epithelial cells expanded ex vivo
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Immunocytochemical Diagnosis
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from limbal explant and single cell cultures. Exp Eye Res. 2004; 79:41–49. Pisella PJ, Brignole F, Debbasch C, et al. Flow cytometric analysis of conjunctival epithelium in ocular rosacea and keratoconjunctivitis sicca. Ophthalmology. 2000;107:1841–1849. Lagali N, Eden U, Utheim TP, et al. In vivo morphology of the limbal palisades of vogt correlates with progressive stem cell deficiency in aniridiarelated keratopathy. Invest Ophthalmol Vis Sci. 2013;54:5333–5342. Araujo AL, Ricardo JR, Sakai VN, et al. Impression cytology and in vivo confocal microscopy in corneas with total limbal stem cell deficiency. Arq Bras Oftalmol. 2013;76:305–308. Goldberg MF. In vivo confocal microscopy and diagnosis of limbal stem cell deficiency. Photographing the palisades of vogt and limbal stem cells. Am J Ophthalmol. 2013;156:205–206.
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