Conjunctival Impression Cytology Samples Research Articles

The Correlation of Routine Tear Function Tests and Conjunctival Impression Cytology in Dry Eye Syndrome

PurposeTo establish the strength of the association between routine tear function tests and conjunctival impression cytology (CIC) and to determine whether they simulate the morphological and cytological changes that occur on the ocular surface in dry eye. What are the sensitivity, specificity and positive predictive values of these tests when CIC is considered the gold standard?MethodsThe tear film profile included tear film break up time (TBUT), Schirmer's-1, Rose Bengal scores (RBS), and impression cytology. CIC samples were obtained from the inferior bulbar conjunctiva and stained with periodic acid-Schiff and counter stained with hematoxylin and eosin.ResultsThe mean Schirmer's value was 11.66 ± 5.90 in patients and 17.17 ± 2.97 in controls (p < 0.001). The mean TBUT in participants was 8.88 ± 3.54 and 13.53 ± 2.12 in controls (p < 0.001). Patients had a mean goblet cell density (GCD) of 490 ± 213, while the value for controls was 1,462 ± 661 (p < 0.001). Abnormal CIC was observed in 46.7% cases of dry eye and in 32.8% of controls. The correlation coefficient (L) for Schirmer's was 0.2 and 0.24 for participants and controls, respectively, while TBUT values were 0.26 and 0.38, RBS were 0.5 and 0.5, and GCD was 0.8 and 0.6 in cases and controls, respectively.ConclusionsGCD, RBS, and TBUT were better predictors of morphological and cytological changes in the conjunctiva than Schirmer's in dry eye syndrome. The sensitivity of tear function tests in diagnosing dry eye was TBUT > Schirmer's > RBS, and the specificity was Schirmer's > TBUT > RBS in decreasing order when CIC was considered the gold standard.

Novel Molecular Diagnostic System of Limbal Stem Cell Deficiency Based onMUC5ACTranscript Detection in Corneal Epithelium by PCR-Reverse Dot Blot

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. 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. 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. 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.

Ocular Mucin Gene Expression Levels as Biomarkers for the Diagnosis of Dry Eye Syndrome

To evaluate mRNA levels of the ocular mucins MUC1, MUC2, MUC4, MUC5AC, and MUC7 in conjunctival impression cytology samples from patients with moderate to severe dry eye syndrome (DES) compared with a population of healthy subjects; and to investigate the use of the levels of these mucin genes as biomarkers of DES and subsequently as a potential diagnostic test for DES. This prospective study commenced in the year 2000 and ended in the year 2009. Thirty-eight patients with DES and 43 age- and sex-matched healthy subjects completed the initial part of the study. Investigations were repeated at a later stage in 16 healthy subjects and 30 patients with DES, which were used as external validation data. Conjunctival impression cytology was performed in all subjects to test gene expression of ocular mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC7. Statistical analysis was performed to determine whether there was a difference in the levels of mucin gene expression between the two groups of subjects. Sensitivity and specificity of mucin gene expression for the diagnosis of DES was calculated. Expressions of MUC1, MUC2, MUC4, and MUC5AC (P < 0.0001) were significantly lower in conjunctival epithelium of patients with DES compared with that in normal subjects. These results were replicated in the external control subject and patient groups. MUC1 expression levels demonstrated the greatest sensitivity (83.3%) and specificity (87.5%) among all genes tested. The data strongly suggest that the expression levels of MUC1 may be used as a diagnostic test in DES for investigational and selective clinical trials.

A Novel Epithelial Proallergic Cytokine IL‐33 Serves As A Biomarker For Ocular Allergic Inflammation

PurposeTo explore a novel role of interleukin (IL)‐33 in Th2‐dominant allergic inflammation on ocular surface.MethodsIL‐33 and its signaling molecules were evaluated in patients with atopic conjunctivitis and in experimental allergic conjunctivitis (EAC) mice induced by short ragweed (SRW). Human corneal epithelial cells (HCECs) were cultured for IL‐33 regulation.ResultsIL‐33 and ST2 were highly detected in conjunctival impression cytology samples from atopic conjunctivitis patients. In the SRW‐induced EAC mice, IL‐33 and ST2 transcripts and immunoreactivity significantly increased in the corneal and conjunctival epithelia with largely infiltrated CD11c+/OX40L+ dendritic cells. Th2 cell infiltration was evidenced by increased transcripts and immunoreactivity of CD4, IL‐4, IL‐5 and IL‐13 in the conjunctiva, with increased expression of OX40, STAT6 and GATA3. In culture model, IL‐33 was dramatically stimulated in HCECs exposed to microbial ligands through toll‐like receptor (TLR) and NF‐kB pathways.ConclusionsOur findings demonstrate an important role of mucosal epithelial cells in initiating Th2‐dominant ocular allergic inflammation by IL‐33‐ST2 signaling pathways.

Assessment of agreement for assignment of a normal grade to human conjunctival impression cytology samples

To assess the level of agreement for assignment of a normal grade for human ocular surface bulbar conjunctival cells as collected by conjunctival impression cytology. Suitable images from published articles that included a scale marker were subjected to the same planimetry method to assess cell area, nucleus area, long (L) and short (S) dimensions of the cells and the nucleus. These measures, along with the nucleus-to-cytoplasmic (N/C) ratio, were compared. Area measures on normal grade images indicated a broad unimodal distribution (250-275 μm(2)), and distribution of nucleus area values was heterogeneous, with neither being normally distributed. The inter-sample variance for any particular area value ranged from 11 to 90% (group averages of 49%). The L:S dimension ratio averaged 1.288, consistently showing these cells are not round. N/C values showed a wide range (from 0.151 to 0.655), as did nucleus-to-cytoplasm dimensions (from 0.332 to 0.603). Current criteria for subjective assignment of a normal grade to bulbar conjunctival cells do not appear to be robust enough to allow for inter-sample comparison. Quantitative measures need to be developed further.

Glycogene Expression in Conjunctiva of Patients with Dry Eye: Downregulation of Notch Signaling

Glycoconjugates regulate a variety of biological events in mucosal surfaces, such as differentiation of postmitotic epithelial cells and maintenance of the wet-surfaced phenotype. This study aimed to identify the repertoire of genes (glycogenes) involved in biosynthesis of glycoconjugates in conjunctiva of normal subjects and patients with dry eye. RNA from conjunctival impression cytology samples was amplified and hybridized to a custom-designed glycogene microarray. Intensity data were converted to expression values and analyzed by ANOVA. Microarray data for selected Notch glycogenes were confirmed by quantitative real-time PCR. Notch receptors and ligands were immunolocalized on conjunctival biopsies by confocal microscopy. By microarray, 424 glycogenes were identified in normal conjunctival epithelium; galectins, glycosyltransferases, mucins, Notch signaling molecules, and proteoglycans were among the most highly expressed. In dry eye, 46 glycogenes were significantly downregulated, including five members of the Notch signaling pathway (Notch1, Notch 2, Notch 3, Jagged1, Delta1), four Wnt signaling molecules (Wnt4, -5A, Frizzled6, -7), and three heparan sulfate glycotransferases (HS2ST1, HS3ST6, EXTL2). Only interferon-induced transmembrane protein 1 was upregulated. By real-time PCR, expression ratios of Notch1, Notch 3, and Jagged1 in dry eye were 0.43, 0.56, and 0.50, respectively, compared to controls (P < 0.05). Notch1, Notch3, and Jagged1 were immunolocalized throughout the conjunctival epithelium, whereas Notch2 and Delta1 were distributed apically. This study revealed the differential glycogene expression profiles in normal subjects and patients with dry eye. Downregulation of Notch signaling in dry eye may result in abnormal differentiation of the conjunctival epithelium and have implications in the pathogenesis of the disease.

Pathogen or commensal: a PCR based study of ocular surface bacteria in normal and dry eyes

Abstract Purpose: To determine the ocular surface bacteria in normal and dry eye subjects and investigate whether there was any association between the bacterial population and goblet cell density (GCD). Methods: Fifty‐seven normal and 34 dry eye subjects were recruited. Conventional culture, 16S rDNA PCR and DNA sequencing were used to identify bacteria. The association between reduced GCD and bacterial numbers in a sub‐group of 27 subjects was assessed. Conjunctival impression cytology samples were stained with PAS and GCD graded as follows: Grade (1), &gt;30 goblet cells/4 high power fields; (2), 15‐30 GC/4HPF; (3), 5‐15 GC/4HPF; (4), &lt;5 GC/4HPF. Grades 3 and 4 indicated reduced GCD. Results: The majority of cultured bacteria were coagulase‐negative Staphylococci, while molecular methods demonstrated additional atypical bacterial species. Bacterial levels were higher overall in subjects displaying reduced GCD compared to normals. A statistically significant difference (P = 0.005) was noted between mean bacterial counts in IC Grade 4 samples of dry eye subjects (35 cfu/swab) and normals (5 cfu/swab). Conclusions: Molecular analysis revealed a diverse ocular surface bacterial population with identification of potentially pathogenic bacteria presenting a diagnostic dilemma. A trend of increasing bacterial numbers with reduced GCD was observed and studies are ongoing to investigate whether bacterial colonisation of the ocular surface may alter the number and function of goblet cells. The clinical relevance of such results and whether they should prompt intervention with therapy is not yet fully determined. A fuller definition of the normal ocular flora is needed to determine bacteria implicated in ocular surface disease.

Conjunctival Cytokine Expression in Symptomatic Moderate Dry Eye Subjects

To compare ocular surface cytokine expression in healthy controls and subjects with moderate dry eye and to study the ability of interleukin (IL)-1beta to modulate cytokine expression in cultured human conjunctival epithelial cells (CECs). Subjective (symptom questionnaire) and objective (tear osmolality, fluorescein tear break-up time [TBUT]) measures of dry eye were determined in five healthy controls and five subjects with moderate dry eye. Tear clearance rates were measured with a fluorophotometer. Enzyme immunoassay and a cytokine bead assay were used to quantify IL-1beta in tear fluid. RT-PCR was performed to detect expression of IL-1beta, IL-6, IL-8, growth-related oncogene (GRO)-beta, intercellular adhesion molecule (ICAM)-1, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and ephrin A5 in conjunctival impression cytology (CIC) samples and in CECs (IOBA-NHC cell line, n=3; primary cultured CEC, n=3) exposed to 10 ng/mL IL-1beta for 6 hours. Subjects with moderate dry eye had significantly higher symptom scores, higher tear osmolality, and shorter TBUT than healthy controls. Subjects with dry eye demonstrated slightly slower tear clearance (13.1% per minute) than healthy controls (15.4% per minute). Very low levels of IL-1beta protein were detected in the tear fluid of both groups. TRAIL was constitutively expressed in CIC samples, whereas IL-1beta, IL-6, and GRO-beta were absent. Weak expression of IL-8 (two healthy, four dry eye), ICAM-1 (four healthy, four dry eye), and ephrin A5 (one healthy, two dry eye) was observed. IL-1beta upregulated its own expression and that of IL-6, IL-8, GRO-beta, and ICAM-1 in cultured CECs but not that of ephrin A5 or TRAIL. The lack of major differences in ocular surface cytokine expression between the two groups of subjects implies other inflammatory pathways or etiologies are involved in moderate dry eye. Although IL-1beta modulated the expression of various cytokines in cultured CECs, its absence in tear fluid and CIC samples suggests that IL-1beta does not play a modulatory role in moderate dry eye.

Human epithelium from conjunctival impression cytology expresses MUC7 mucin gene.

To prove that noninvasive methods of obtaining conjunctival tissue, such as conjunctival impression cytology (CIC), could be valid alternatives that are simpler, faster, and more convenient for patients than biopsy to analyze mRNA levels of mucin genes. Using the semiquantitative reverse-transcriptase polymerase chain reaction, we studied the presence of the mucin genes described on the ocular surface thus far and attempted to detect the presence of MUC7 in CIC samples from 10 healthy donors. Conjunctival cells recovered by CIC expressed all the genes studied. There were no statistically significant differences between male and female subjects, and there was a significant correlation between the two eyes of the same donor only in the expression of MUC7. CIC is a valid, noninvasive technique to detect the mRNAs of ocular genes in healthy individuals. MUC1, MUC2, MUC4, MUC5AC, and MUC7 mucin genes could be all detected in each CIC sample. This technique may be a useful tool to study the expression of some genes in ocular diseases.

Levels of mucin gene expression in normal human conjunctival epithelium in vivo

Purpose. Conjunctival impression cytology (CIC) samples were used to determine the mean and normal range of mRNA levels of human MUC1, MUC2, MUC4, MUC5AC, and MUC7 mucin genes. Methods. Real time PCR was performed to determine normal mRNA levels in CIC samples of 24 male and 19 female healthy donors. Correlation coefficients between gene expression levels were obtained. Results. All five mucin genes were expressed in the CIC samples. MUC1 and MUC4 were present at the highest level and MUC2 was at the lowest. There were no gender differences. Significant positive correlations existed between MUC2 and MUC4 and between MUC2 and MUC7 levels. Conclusions. Normal levels and ranges of mRNAs for MUC1, MUC2, MUC4, MUC5AC and MUC7 conjunctival mucin genes have been established for the first time. These data may serve as the normal threshold values for future comparisons in different experimental and pathological conditions involving the ocular surface.

A Survey of Trachoma: The Histopathology and the Mechanism of Progressive Cicatrization of Eyelid Tissues

The aim of this study is to demonstrate the spectrum of conditions encompassed by the term ‘trachomatous cicatrization of eyelid tissue’, to discuss the mechanisms of scar tissue formation and to describe sequelae in this potentially blinding condition. Specimens of eyelid tissues were taken from 27 upper eyelids of 21 patients with entropion who underwent surgical procedures and 2 post-mortem upper eyelids with severe trachomatous entropion. Upper palpebral conjunctival swabs and biopsy specimens were taken from 5 patients with active trachoma and they were examined by fluorescence microscopy and routine histopathological methods. Conjunctival impression cytology samples were collected in all patients. In specimens taken from patients with active trachoma the inflammatory infiltrate was organized as lymphoid follicles in the underlying stroma and impression cytology showed cytoplasmic elementary bodies. In specimens taken from patients with scarring trachoma light microscopy studies showed subepithelial fibrous membrane formation, squamous metaplasia and loss of goblet cells, pseudogland formation in conjunctiva, degeneration of orbicularis oculi muscle fibres, subepithelial vascular dilatation, localized perivascular amyloidosis and subepithelial lymphocytic infiltration. Accessory lachrymal glands and the ducts of glands were compromised by subepithelial infiltration and scarring. The contraction of the subepithelial fibrous tissue formed by collagen fibres and anterior surface drying are the main factors contributing to the chronic cicatrization and entropion formation.

Comparison of grading schemes to quantitative assessments of nucleus-to-cytoplasmic ratios for human bulbar conjunctival cells collected by impression cytology

Purpose. To quantitatively assess the measured nucleus-to-cytoplasm (N/C) ratios for sets of bulbar conjunctival cells categorised by a subjective grading scheme (0-3). Methods. Impression cytology samples were taken from the nasal bulbar conjunctiva of 37 individuals (aged 22 to 74 years) without any significant eye disease. The sheets of cells were generally stained with Giemsa, photographed and 35 mm slides prepared. The images were graded 0 to 3 according to the scheme of Nelson [Cornea 7, 71–81 (1988)] and approximately 35 contiguous cells from each image measured by planimetry from an overlay. Results. Analysis of 1097 cells revealed that the N/C ratios ranged from 1.395 to 0.056 and correlated well with the grading. The average N/C ratio (range for ± 1.96 SD) for each grade was 0.607 (0.983 to 0.531) for grade 0, 0.533 (0.759 to 0.333) for grade 1, 0.291 (0.427 to 0.155) for grade 2, and 0.154 (0.228 to 0.080) for grade 3. Conclusions. N/C ratios can be assessed from conjunctival impression cytology samples and appear to be sufficiently consistent to allow for a designated range of cell N/C ratios to be specified for each grade.

Conjunctival impression cytology in the preterm infant and it's relation to outcome.

The preterm infant is deficient in vitamin A (retinol) and this has been implicated in the pathogenesis of chronic lung disease of prematurity. Conjunctival impression cytology (CIC) has been used in adults to assess retinol status. We aimed to assess the feasibility of performing CIC in the preterm infant and to determine the significance of abnormal CIC findings. CIC samples were collected during routine retinopathy screening, and classified as inadequate, normal, borderline normal or abnormal. Ninety preterm infants were studied. Seventy-four (82%) CIC specimens produced a positive yield, whereas 16 (18%) were inadequate. Of the 74 adequate samples, 61 (82%) were normal or borderline normal and 13 (18%) abnormal. Seventy-three CIC specimens were assessed by a second histopathologist with complete agreement on 64 (88%) samples and disagreement on 9 (12%) samples. Ten sets of conjunctival impressions, taken from both eyes, gave identical results in all adequate samples. Birth weight was significantly lower in this abnormal group. Four infants (32%) in the abnormal group required treatment for retinopathy compared to two (3%) in the normal/borderline normal group, (P < 0.01). Conjunctival impression cytology is simple and reproducible technique which maybe easily applied to the preterm infant. Abnormal CIC is associated with retinopathy of prematurity requiring treatment.