Keratin 13 Immunostaining in Corneal Impression Cytology for the Diagnosis of Limbal Stem Cell Deﬁciency

Cornea Keratin 13 Immunostaining in Corneal Impression Cytology for the Diagnosis of Limbal Stem Cell Deﬁciency

Muriel Poli,1 He´le`ne Janin,1 Virginie Justin,2 Celine Auxenfans,2 Carole Burillon,1,3 and Odile Damour2,3

PURPOSE. The aim of this study was to develop a validated, lial healing, replacement of the corneal epithelium with con- reliable, and minimally invasive technique for diagnosing lim- junctiva (conjunctivalization), and corneal scarring that con- bal stem cell deficiency (LSCD) by immunocytochemical de- tribute to severe visual loss. In recent years, bioengineered tection of conjunctival and corneal keratins on epithelial cells corneal epithelial equivalents, developed from the ex vivo collected by impression cytology (IC). expansion of limbal stem cells, have been used to treat severe 2,3 METHODS. After validation of labeling techniques on a cohort of limbal stem cell deficiency, with promising results. To justify 10 healthy control patients, keratins K12, K13, and K19 were these expensive treatments, health service regulators require a labeled on corneal IC of 10 eyes suspected of LSCD. Positive reliable paraclinical diagnostic technique for this disease. scores for the conjunctival markers K13/K19, coupled with the The epithelial cells (ECs) of the ocular surface are charac- rarity of the corneal marker K12, were diagnostic proof of terized by markers with high tissue specificity, the keratin LSCD. antigens of intermediate filaments. The current dogma is that the expression of K3/12 (K3 basic keratin of 64,000 Da; K12 RESULTS. IC is a reliable and noninvasive technique for collect- acidic keratin of 55,000 Da) is thought to be a hallmark of ing epithelial cells. The labeling validation phase has permitted corneal ECs.4–9 However, the corneal specificity of K3 has K3 labeling to be eliminated due to lack of corneal specificity. recently been questioned, strongly suggesting that the actual Among patients with LSCD, nine samples were diagnosed with expression patterns of K3/12 in the ocular surface epithelium LSCD (K13ϩ/K19ϩ), which was severe (K12Ϫ) in eight cases 10–14 ϩ are not as clear cut as the current dogma suggests. and mild (K12 ) in one case. One sample could not be ana- The superficial layers of the conjunctival epithelium are lyzed due to lack of cells. characterized by specific keratins of nonkeratinized stratified CONCLUSIONS. K13 has shown to be a new marker of conjunctival epithelia (K4/K13), simple epithelia (K8/K19), and glandular differentiation. The immunocytochemical search for the K13/K19 epithelia (K7).15–17 The diagnosis of LSCD has long relied on couple by corneal IC provides a simple and reliable method for the detection of goblet cells after impression cytology (IC) of diagnosing LSCD, whereas the level of K12 could provide a score the corneal epithelium, either by periodic acid–Schiff staining, of disease severity. On the other hand, the authors question the or by more specific techniques, such as Mucin 5AC stain- corneal specificity of K3 as conventionally established. (Invest ing.14,18,19 However, the density of conjunctival goblet cells Ophthalmol Vis Sci. 2011;52:9411–9415) DOI:10.1167/iovs.10- can decrease by up to 99% under pathologic situations,20–23 7049 and at the early stages of LSCD, goblet cells may not even be detectable in the cornea, whereas there are already recurrent he limbus, specific source of stem cells for the corneal erosions with delayed corneal healing and neovasculariza- Tepithelium, is an anatomic and functional boundary be- tion.24 Thus, the absence of goblet cells within the corneal tween the conjunctiva and cornea. Limbal stem cell deficiency epithelium does not exclude the diagnosis of LSCD.25 syndrome (LSCD), which is often attributed to trauma and The aim of this study was to develop a validated, reliable, damage to the stem cells and their niche, is characterized by and minimally invasive technique for diagnosing LSCD by im- dystrophy of the corneal epithelium followed by invasion of munocytochemical detection of conjunctival and corneal ker- the corneal surface by a vascularized epithelium with conjunc- atins on epithelial cells collected by IC. tival differentiation, a vector of immune cells.1 Thus, it is a In this study, we validated the K12, K13, and K19 labeling major risk factor for graft rejection. techniques and refuted the K3 marker (most commonly used Diagnosis of the disease has long been based on clinical for the diagnosis of LSCD) on tissue and cells collected by IC examination, associating pathologic conditions thought to in- and used these markers for the diagnosis of LSCD. Our hypoth- volve stem cell deficiencies to classic biomicroscopic findings esis was that the expression of differentiation markers of the such as chronic inflammatory condition, poor corneal epithe- conjunctival epithelium by IC of the corneal epithelium was diagnostic proof of LSCD. The presence of differentiation markers of the corneal epithelium would make it possible to quan- From the 1Clinique Ophtalmologique, Hoˆpital Edouard Herriot, tify the severity of the disease. Lyon, France; and the 2Laboratoire des Greffes et Substituts Cutane´s, Hoˆpital Edouard Herriot, Lyon, France. 3These authors contributed equally to the work presented here MATERIALS AND METHODS and should therefore be regarded as equivalent authors. Submitted for publication December 13, 2010; revised July 20 and Human Samples September 14, 2011; accepted October 22, 2011. This prospective and monocentric study followed the tenets of the Disclosure: M. Poli, None; H. Janin, None; V. Justin, None; C. Auxenfans, None; C. Burillon, None; O. Damour, None Declaration of Helsinki and was approved by the institutional review Corresponding author: Muriel Poli, Clinique Ophtalmologique, board. Pavillon C, Hoˆpital Edouard Herriot, 5 place d’Arsonval, 69003 Lyon, The inclusion criteria for healthy patients were normal visual acuity France; [email protected]. and biomicroscopic examination. Pathologic patients enrolled should

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TABLE 1. Primary Antibodies Used during the Study

Antigen Clone Type Host Immunogen Manufacturer Dilution

K3/K76 AE5 Monoclonal Mouse Human K3 Progen, Biotechnik GmbH, Heidelberg, Germany 1/100 K12 12 (N-16) Polyclonal Goat Human K12 Santa Cruz Biotechnology, Santa Cruz, CA 1/100 K13 ks13.1 Monoclonal Mouse Human K13 Chemicon International, Inc., Temecula, CA 1/100 K19 NCL-CK19/b170 Monoclonal Mouse Human K19 Vision Biosystems, Novocastra, Newcastle upon 1/100 Tyne, UK

present biomicroscopic examination compatible with LSCD, consist- Thermo Scientific,) using light finger pressure. The same filter was thus ing in persistent epithelial defect, chronic inflammation, corneal neo- impressed on a maximum of three different slides. vascularization, conjunctivalization, and stromal scarring with corneal The cells were fixed in acetone at Ϫ20°C and kept frozen. opacity and loss of vision. Each patient should be older than 18 years of age. For both healthy and pathologic patients, exclusion criteria Immunostaining were sensitivity to drugs that provide local anesthesia or active infec- Primary antibodies were against K3/K76 and K12, both specifics of tion of the external eye. corneal EC, and K13 and K19, specifics of conjunctival EC (Table 1). We scored LSCD as mild when the visual axis was covered with The secondary antibody was a commercial IgG (HϩL) (AlexaFluor 488; normal corneal epithelium and peripheral neovascularization and con- Invitrogen, diluted 100-fold) for K3/K76, K13, and K12 immunostain- junctivalization and when the patient was relatively asymptomatic. ing. For K19 immunostaining, the secondary antibody was either of LSCD was severe when there was a central corneal involvement with two commercial IgGs (HϩL) in validation steps (AlexaFluor 488; Invit- complete conjunctivalization and 360° neovascularization with signif- rogen, diluted 100-fold) or IgG (HϩL) in diagnostic steps (AlexaFluor icant irritation. 568; Invitrogen, diluted 100-fold) (Table 2). For the corneal markers Tissue samples were collected from cadaveric tissues of deceased K3/K12, the positive control was the cornea with its collar and the donors: corneosceral buttons comprising corneal, limbal, and conjunc- negative control was the bulbar conjunctival tissue of a deceased tival epithelia, not suitable for therapeutic use, were obtained from the patient. For the conjunctival markers K13/K19, the positive control tissue and cell bank (Lyon, France). Permission to use the donated was the bulbar conjunctival tissue and cornea was the negative control. tissues for research was obtained from all donor families. In all immunostainings, anisotype negative control (IgG) was carried Cells were obtained by cytologic impression of cornea and bulbar out. conjunctiva of 10 healthy volunteers (3 males and 7 females; mean age, 61.0 Ϯ 25.0 years), and of patients with a clinical diagnosis of LSCD Design of the Study [n ϭ 10 eyes of 9 patients; 4 males and 5 females; mean age, 50.3 Ϯ 13.2 years with etiologies of aniridia (n ϭ 1), chemical burn (n ϭ 2), The single labeling techniques for K3, K12, K13, and K19 were vali- Lyell syndrome (n ϭ 2), infections (n ϭ 2), postradiotherapy (n ϭ 1), dated using both healthy cells from ICs and tissues from deceased rosacea (n ϭ 1), and exposure keratitis with facial palsy (n ϭ 1)] (see donors; the double-labeling techniques for K12/K13 and K12/K19 Table 3 in the following text). All patients were recruited consecu- were carried out on corneoscleral buttons unsuitable for transplanta- tively at the eye clinic at Edouard Herriot Hospital (Lyon, France), tion from healthy deceased donors (Step 1). between December 2009 and March 2010. Prior informed consent was The simple labeling of K13 and double labeling of K12/K19 were obtained from the subjects after the nature and possible consequences carried out on patients with a clinical diagnosis of LSCD (Step 2). of the study were explained. RESULTS Impression Cytology Validation of the Labeling Method The cells were collected by IC on nitrocellulose filters (Millipore, Cork, Ireland) after topical anesthesia with drops of 0.04% oxybuprocaine. The demographic and clinical data of patients clinically diag- The sampling sites chosen were the central cornea and the remote nosed with LSCD are summarized in Table 3. temporal bulbar conjunctiva (10 mm from the limbus) for healthy The IC was simple, fast, and easy to carry out for the patients, and areas of maximum corneal conjunctivalization for those operator, painless and atraumatic but uncomfortable for the 10 clinically suspected of LSCD. In healthy patients, two ICs were per- healthy patients: 3 males and 7 females, aged 61 Ϯ 25 years. formed at each corneal and bulbar conjunctiva site, ensuring that the The results obtained in terms of cell density were very repro- same site was not impressed twice, at the risk of collecting basal cells ducible for the same corneal or conjunctival tissue studied. whose immunocytochemical characteristics differ from those of su- Indeed, 100% of conjunctival ICs provided a high rate of prabasal cells. In diseased patients, only one sample per site was taken. return, giving carpets of many joint cells containing at least 200 Cellular contamination of the filter by the sample taker was avoided by cells per field, at a magnification ϫ200. Conversely, 100% of using nonsterile, powder-free gloves. The cells thus collected were corneal ICs yielded only a handful of isolated cells, a maximum immediately transferred to glass slides (Menzel-Gla¨ser, Superfrost Plus; 17 cells/field at a magnification ϫ200 (Fig. 1).

TABLE 2. Secondary Antibodies Used during the Qualiﬁcation of Markers (I) and Qualiﬁcation of the Diagnostic Test (II)

Step Antigen Secondary Antibodies Host Immunogen Manufacturer Dilution

1 K3/K76 Alexa Fluor 488 IgG FITC Goat Mouse Invitrogen Corporation, Carlsbad, CA 1/100 K13 K19 2 K13 Alexa Fluor 488 IgG FITC Goat Mouse Invitrogen Corporation, Carlsbad, CA 1/100 K19 Alexa Fluor 568 IgG TRITC Rabbit Mouse Invitrogen Corporation, Carlsbad, CA 1/100 1,2 K12 Alexa Fluor 488 IgG FITC Donkey Goat Invitrogen Corporation, Carlsbad, CA 1/100

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TABLE 3. Clinical and Immunocytochemical Characteristics of Patients with Clinically Suspected LSCD

Eye Sex Age (y) Laterality Score Visual Acuity Pathology K13 K19 K12

1 Female 71 Left Severe LPϩ Ocular rosacea ϩϩϪ 2 Male 41 Right Severe LPϩ Chemical burn (acid) ϩϩϪ 3 Female 53 Left Severe LPϩ Corneal herpetic infection ϩϩϩ 4 Male 71 Right Mild CF at 50 cm Trachoma ϩϩϪ 5 Female 51 Right Severe CF at 50 cm Facial palsy ϩ NA NA 6 Female 33 Left Severe LPϩ Aniridia ϩϩϪ 7 Female 43 Right Severe LPϩ Lyell syndrome ϩϩϪ 8 Female 43 Left Severe CF at 10 cm Lyell syndrome ϩϩϪ 9 Male 41 Right Severe LPϩ Postradiation burn ϩϩϪ 10 Male 49 Right Severe CF at 50 cm Chemical burn (acid) ϩϩϪ

LP, light perception; CF, counts ﬁngers; NA, not analyzable.

The cell density decreased significantly as the filters were cornea, we selected K19 too as marker of conjunctival differ- impressed, this operation not being renewable more than three entiation, considering the higher sensitivity of a double-stain- times. ing K13/K19 in detecting conjunctival EC. As shown in Figure 1, strong K3 expression was observed in On the contrary, the lack of corneal specificity of K3 led to the suprabasal layers of the conjunctival EC (Fig. 1A), and all the its abandonment. layers of the whole corneal EC (Fig. 1C,D), but absent of the The K12/K19 double labeling was successfully qualified on limbus (Fig. 1B), suggesting a lack of sensitivity of K3. On bulbar conjunctival and corneal tissue. the contrary, K12 expression was restricted to the whole corneal epithelium (all layers) (Figs. 1E, 1H, 1I) and the suprabasal layers Labeling of Cells of Patients with a Clinical of the limbal EC (Figs. 1E, 1G) only. As opposed to K3, K12 was Diagnosis of LSCD never observed in the conjunctival EC (Figs. 1E, 1F), confirming that K12 is a specific marker of the cornea. During the second stage, carrying out IC proved painless but Strong K13 expression was restricted to the conjunctival uncomfortable for patients with LSCD. No side effects were epithelium (all layers) (Figs. 1E, 1F) and the suprabasal layers of found linked to sampling. It was easy to identify the areas of the limbal EC (Figs. 1E, 1G) only, but was never observed in the maximum conjunctivalization by the naked eye. whole corneal EC (Figs. 1E, 1H, 1I), confirming that K13 is a In patient 5, the sampling had been made difficult by the specific marker of the conjunctiva. As the same, K19 expres- presence of a symblepharon, limiting the palpebral fissure (Fig. 2). sion was restricted to the conjunctival epithelium (all layers) This sample was not analyzable because of a lack of cellularity on (Fig. 1L) and the suprabasal layers of the limbal EC (Fig. 1M) the second printed blade (Fig. 2). only and was never observed in the central corneal EC (Fig. With the exception of patient 5, all samples included a large 1O). Nevertheless, as opposed to K13, some weak staining of number of cells, respecting the architecture of the epithelial K19 was observed in the basal cells of the peripheral corneal tissue and intercellular junctions. The second slide impressed EC (Fig. 1N). Thus, K19 was considered as a specific marker of often contained fewer cells than the first but still in sufficient the conjunctiva compared with the central cornea. All the IgG numbers to allow its interpretation. isotype negative controls were negative. Finally, on slides impressed too strongly, the cell cytoplasm The K12 and K13, thus qualified, were retained for the appeared disorganized and the cell nuclei were poorly individ- second stage. Because ICs were performed on the central ualized.

Conjunctiva Limbus Peripheral cornea Central cornea B C FIGURE 1. Expression of epithelial A D cell markers in corneoscleral buttons K3 of healthy deceased donors compris- (FITC) ing conjunctiva, limbus, and cornea. __ K3 __ K3 __ K3__ K3 The expression pattern of keratin 3 (K3) (green, A–D), keratin 12 (K12) ------Conjunctiva------Limbus------Cornea------ (green, E–I), keratin 13 (K13) (red, Double E–I), and keratin 19 (K19) (green, staining F E L–O) were analyzed. K13 is specific K12 K12/K13 GH I of conjunctival differentiation be- (FITC) cause it is specifically located in the limbus (E, G) and conjunctival epi- K13 F GHI thelia (E, F). The same pattern is (TRITC) found for K19 (L, M, O), although some weak staining is observed in __ the basal layers of the peripheral cor- __ K12/K13 _ K12/K13 K12/K13 __ K12/K13 nea (N). K12 is specific of corneal differentiation because it is specifically localized in the limbus (E, G) K19 L M N O and cornea (E, H, I). K3, present in (FITC) both corneal (C, D) and conjunctival (A) epithelia, is not a specific marker. _ __ __ Scale bar: 100 ␮m(A–D, F–O) and 50 __ K19 K19 K19 K19 ␮m(E).

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FIGURE 2. Biomicroscopic characteristics and corneal impression cytology of four of the stage 2 patients. Patient 1: there is a strong expression of conjunctival differentiation markers (K13, K19), whereas K12, a marker of corneal differentiation, is absent. Patient 4: These same characteristics are found at a clinically mild LSCD, demonstrating the sensitivity of the diagnostic test. Patient 3: the coexis- tence of corneal and conjunctival markers indicates a weak form of the disease. Patient 5: the existence of a symblepharon limiting the opening of the palpebral ﬁssure made cell collection impossible.

For 8 of 10 patients (patients 1, 2, 4, 6, 7, 8, 9, and 10), One hypothesis for this K3 nonspecificity is that, in this collected cells strongly expressed K13 and K19 but were study, we sampled from the bulbar conjunctiva, far from the negative for K12, confirming the presence of a conjunctival cornea to avoid contamination of our conjunctival impressions epithelium (Fig. 2). It should be noted that this conjunctival by corneal cells, whereas the bibliographic data indicating the phenotype was found in patient 4, who had a mild form of the specificity of K3 tend to focus on the study of the near-limbus disease (Fig. 2). Patient 3 presented a mixed phenotype, with conjunctiva.29 It has, in fact, been reported that K3 was ex- K13, K19, and K12 expression on almost 100%, 20%, and 80% pressed by noncorneal tissues, including bulbar conjunc- of cells, respectively (Fig. 2). tiva,11,14,30 nose, and oral mucosa.31 As shown in Figure 1, strong K3 expression was observed in the suprabasal layers of DISCUSSION the conjunctival EC (Fig. 1A) but absent of the limbus (Fig. 1B). A second hypothesis on the lack of K3 specificity is that the The first step in this study showed that the cytological impres- expression of our antibody AE5 in the conjunctiva is due to the sion technique is very suitable for collecting conjunctival epi- detection of K76. Indeed, today, the specificity of the AE5 thelial cells. It can collect many cells while preserving inter- clone that we used is in question, because the latter would cellular junctions. In contrast, in healthy subjects, it retrieves recognize the K3 and K76 isoforms. K76 is a keratin that is few corneal epithelium cells, the pauci-desquamative character specific to suprabasal cells of the masticatory epithelium (gum of which partly explains the difficulty of collecting them. A and palate),31 and its presence in conjunctival cells still re- rich cytologic impression is therefore a characteristic specific mains to be proven. to conjunctival epithelium and the high retrieval rate of cells by The second step in this study showed that IC produces corneal IC represents in itself a criterion of conjunctival differ- strong returns for LSCD with 90% of samples suitable for entiation and thus a diagnosis of LSCD. analysis. Unfavorable anatomic conditions, however, are a con- Among the markers that we selected in accordance with traindication to sampling. current data in the literature, K12 has been shown to be an This diagnostic test has proved very sensitive, with 8 posi- excellent marker for corneal differentiation, and K13 an excel- tive samples of 9 tested here, and capable of diagnosing LSCD lent new marker for conjunctival differentiation, as recently at a mild form of the disease (Fig. 2, patient 4). The negative confirmed by the work of Ramirez-Miranda et al.26 Moreover, predictive value of the test was confirmed on healthy histo- K13 is a more reliable marker for distinguishing between the logic tissue. corneal and conjunctival epithelia compared with K19. Indeed, In the case of patient 3, we identified a mixed corneal and we should carefully consider the conjunctival specificity of conjunctival epithelium (K12, K13, and K19ϩ). This patient K19 due to its staining in the basal cells of the peripheral had a herpetic keratitis with major corneal opacification and corneal EC, as reported too in several recent studies.14,17,26–28 advanced corneal neovascularization, which are not specific of Moreover, K3, the most widely used marker, has not been LSCD but can result from the viral infection. After reading the shown to be specific to the corneal epithelium. diagnostic test, we questioned our clinical diagnosis of severe

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