Impression Cytology Samples Research Articles

On the Use of NIH Image J for Objective Assessment of Conjunctival Cell and Nucleus Dimensions of Impression Cytology Samples

To assess the use of a public domain software Image J (NIH Image, Bethesda, MD) to make dimensional measures of human bulbar conjunctival cells. Impression cytology samples were obtained from the nasal bulbar conjunctiva, and color images were taken at 200× magnification and projected and an overlay prepared or the image uploaded into Image J. The final image magnification for the overlays was approximately 2× that on the computer screen. For either overlays or screen images, linear measures were made from 30 or 25 cells of the cell longest dimension (LONG) and of the longest dimension of the nucleus (NUCLONG). The predicted variability in measures, from the calculated average values for any particular image, was systematically assessed, and the overall average results were compared. Image J measures were within ±2% agreement with overlays, with LONG and NUCLONG measures increasing with the grade of squamous metaplasia. The net difference in LONG measures was within -2.65 to + 2.93 μm, and NUCLONG measures were within -0.87 and +1.39 μm. There was a slight tendency for NUCLONG measures to be systematically overestimated on smaller nuclei when using Image J on the color images. The manual use of Image J on on-screen images can provide reasonably accurate objective measures of bulbar conjunctival cells, as compared with a higher magnification manual overlay technique. This should be suitable for comparisons between samples and to assess the effects of any disease-related changes or any interventions.

Corneal Intraepithelial Neoplasia: In Vivo Confocal Microscopic Study With Histopathologic Correlation

To ascertain in vivo confocal microscopic features of corneal/conjunctival intraepithelial neoplasia (CIN) and correlate these with histology of the same lesions. Observational case series with evaluation of diagnostic technology. Four patients with unilateral CIN (3 men and 1 woman) were examined with the Heidelberg Retina Tomograph II (HRT II) Rostock Cornea Module (RCM) confocal microscope before, during, and after treatment. Corneal epithelial samples were taken by alcohol delamination technique in 2 patients, while impression cytology (IC) samples were obtained in the other 2 patients. Morphometric analysis of confocal and histologic images was carried out and the findings correlated. Four controls (all men, 2 with limbal stem cell deficiency, 1 with a limbal lesion, and 1 with diffuse keratoconjunctival proliferation) were similarly examined. Two of these had biopsy for histologic examination. Main outcome measures comprised the degree of correlation between histology and confocal microscopic features of CIN. Dysplastic cellular changes were noticed on histopathology and correlated well with confocal microscopy, corresponding to the different corneal epithelial layers. Bright nucleoli within huge nuclei and ill-defined cell borders were a feature of the basal epithelium on histopathology and confocal microscopy. Subbasal corneal nerves were not visualized on confocal microscopy in areas affected by CIN. These features disappeared in response to treatment cycles as the basal epithelium reverted to its normal pattern, as seen by confocal microscopy. Confocal microscopy findings highly correlate with histologic features in CIN. Confocal microscopic features of CIN as defined in this study will enable a reliable diagnosis in a noninvasive manner. Confocal microscopy will also allow real-time monitoring of the condition during treatment.

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.

Analysis of Inflammatory Cytokines in the Tears of Dry Eye Patients

To determine the levels of 8 important cytokines and 1 chemokine in tears of patients with dry eye disease. Tear samples were collected from 7 patients with dry eye disease and 7 healthy volunteers, and impression cytology samples were collected from 3 of the dry eye patients and 3 of the normal controls. Tears were analyzed for the presence of 8 cytokines [interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-1beta] and 1 chemokine (IL-8). The cytokines and chemokine in each tear sample were measured using Invitrogen's Multiplex Bead Immunoassays. The impression cytology samples were analyzed for IL-1beta, IL-6, IL-8, and TNF-alpha mRNA expression using real-time reverse transcriptase polymerase chain reaction anlaysis. All cytokines and the chemokine measured were significantly increased in the tears of dry eye patients as compared to normal controls. mRNA of all four markers was increased, and the fold increase correlated well with the fold increase of the cytokine concentration found in the tear samples. Tears from dry eye patients contain significantly increased concentrations of cytokines that show correlation to severity of the disease. The upregulation of their respective genes in the conjunctiva suggests that the concentration increase is not the result of evaporative effects, but of overproduction. These findings suggest that cytokines may play an important role in dry eye disease and topical cytokine modulators may be explored as a therapeutic approach to dry eye disease.

Myofibroblasts in the normal conjunctival surface

To investigate the occurrence of myofibroblasts (MFBs) in the normal conjunctival surface and to evaluate any anatomical and time-related variations. MFBs were screened among healthy individuals (35 eyes) by collecting impression cytology (IC) samples from the bulbar conjunctiva. A cohort of volunteers (12 eyes) was followed for 1 year by taking two to five imprints every month. MFBs were identified by immunohistochemical localization of the MFB marker alpha-smooth-muscle actin (alpha-SMA). Using a filter imprint technique, MFBs were found consistently in 94% of samples from the conjunctival surface of participating individuals. The overall MFB levels, expressed as percentage of all cells on the filter, were highest in March-May [mean 4.1%, standard deviation (SD) +/- 1.5] and lowest in December-February (mean 1.2%, SD +/- 0.5). The difference was statistically significant [p < 0.0005, Friedman test, one-way repeated measures analysis of variance (anova)]. Moreover, there was a clear divergence of MFB density between the nasal, temporal, superior and inferior bulbar conjunctiva (mean 1.7%, 1.9%, 22% and 9.7%, respectively). MFBs, known as a cellular constituent of granulation tissue in wound healing, occur in the normal conjunctival surface, which is a novel finding. Our results also show that MFB level follows a seasonal variation pattern in a temperate climate, increasing in April-September and decreasing in October-March. This variation might reflect a degree of a transient or ongoing state of tissue repair after conjunctival trauma or stress caused by exposure to environmental factors.

Lymphocyte and markers of inflammation detection in the conjunctival epithelium of patients with dry eye by enhanced flow cytometry

Abstract Purpose The aim of our study was to test the hypothesis that the use of cell culture medium can increase the number of cells obtained by impression cytology (IC) sampling of the conjunctiva in dry eye patients, and make the analysis of epithelial and lymphocyte cell populations with flow cytometry possible. Methods IC specimens were collected in 15 normal subject and 15 dry eye patients. Samples collected from the right eye were placed in cell culture medium containing 10% foetal calf serum (FCS), and samples from the left eye in Phosphate Bufferred Saline containing 0.05% paraformaldehyde (PFA). The number of cells was analyzed by flow cytometry in both groups. Samples collected from a group of 30 dry eye patients were placed in FCS and stained for the expression of CK19, CD45, CD3, HLA‐DR, and analyzed by flow cytometry. A control group of 10 subjects was used as control. Results The number of cells counted in the FCS samples was statistically increased in the normal (12791 ± 11350 events per minute) and dry eye group (23468 ± 15596) when compared to PFA samples (2031 ± 2336, and 2608 ± 1814 respectively). In dry eyes the low number of cells in PFA samples made possible only the analysis of HLA‐DR expression, while in FCS samples epithelial (CK19+), leukocyte (CD45+), and lymphocyte (CD3+) cell populations were characterized, other than analyzed for HLA‐DR expression. Conclusion This study indicates that using this new method of preservation can enhance flow cytometry analysis of epithelial and immune cells of the conjunctiva in dry eye patients, opening new scenarios in the comprehension of its pathogenesis and in testing new therapies.

Predictive index to differentiate invasive squamous cell carcinoma from preinvasive ocular surface lesions by impression cytology

Background/aims:In the literature, no cytological features have been identified that reliably differentiate invasive squamous cell carcinoma (SCC) from preinvasive lesions in impression cytology (IC) samples. The aim was to identify...

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.

Alterations of the ocular surface epithelial MUC16 and goblet cell MUC5AC in patients with atopic keratoconjunctivitis

An increased understanding of the ocular surface at cellular level in the conjunctiva and the cornea may help explain the pathogenesis and the subsequent clinical appearance of atopic ocular allergies, which may be potentially blinding. To investigate the MUC16 and MUC5AC alterations, tear function and the ocular surface disorder in patients with atopic keratoconjunctivitis (AKC). Thirty-six eyes of 18 AKC patients as well as 28 eyes of 14 age- and sex-matched normal subjects were studied. The subjects underwent corneal sensitivity measurements, Schirmer test, tear film break-up time (BUT), fluorescein and Rose-Bengal staining of the ocular surface, conjunctival impression cytology and brush cytology. Impression cytology samples underwent periodic acid schiff and immunohistochemical staining with MUC16 and MUC5AC antibodies. Brush cytology specimens underwent evaluation for inflammatory cell numbers and quantitative real-time polymerase chain reaction (PCR) for MUC16 and MUC5AC mRNA expression. The mean corneal sensitivity and BUT values were significantly lower in patients with AKC, compared with controls (P < 0.001). Brush cytology specimens from AKC patients revealed significantly higher numbers of inflammatory cells (P < 0.001). Specimens from patient eyes showed positive staining for MUC5AC and MUC16. MUC16 mRNA expression was significantly upregulated with significant downregulation of MUC5AC mRNA expression in eyes with AKC compared with the eyes of control subjects. Ocular surface inflammation, decline in corneal sensitivity, tear film instability, changes in conjunctival epithelial MUC5AC and MUC16 mRNA expressions were thought to be important in the pathogenesis of atopic ocular surface disease.

Expression pattern of antimicrobial peptides (AMPs) in acanthamoeba keratitis

Abstract Purpose Acanthamoeba keratitis (AK) is a sight‐threatening ocular infection, which is more likely in contact lens wearer. Antimicrobial peptides(AMPs)are cationic polypeptides with wide‐spectrum activity against pathogens. We studied(1)the profile of AMP mRNA expression in ex‐vivo impression cytology (IC) specimen from normals and 5 AK patients &amp;(2)compared AMP gene expression between ex‐vivo and in‐vitro cell culture samples. Methods a) IC samples were collected for Total RNA extraction and cDNA synthesis. Quantitative gene expression(qPCR)was employed to evaluate expression of Defensins,Cathelicidin,&amp; Liver‐expressed AMPs (LEAPs).b) Immortalised human corneal epithelial cell line (HuCL) was incubated with trophozoites of Acanthamoeba castellanii for 24 hours and the supernatant analysed by BD cytometric bead array for pro‐inflammatory cytokines.The cells were processed for qPCR analyses. Results Variable expression of AMPs was observed in AK patients.Eight AMPs,Human Beta defensins(HBD)1 to 3,Cathelicidin (LL37,LEAP 1 and 2 and DEF 109 were detected.Of these HBD 1 to 3 and DEF 109 were shown to be reduced and whereas LEAP 1 &amp; 2 were induced in AK specimen. Similarly, increased mRNA expression of LEAP 1 &amp; 2 was also demonstrated in Acanthamoebae treated HuCL cultures.Whereas, HBDs showed the baseline expression when compare to untreated HuCLs. Conclusion This is a first study demonstrating AMP gene expression in AK. Differential expression pattern of these AMPs during AK may have important implications in understanding the pathogenesis of this blinding infection. Modulation of AMP defense barrier at the ocular surface or use of the AMP as a contact lens preservative agent may help to reduce the AK infections

Ocular Mucin Visualization by Confocal Laser Scanning Microscopy

To describe a new method of visualizing human conjunctiva goblet cell mucin secretion by using a combination of impression cytology and laser scanning microscopy. By assembling a Z-stack of confocal microscopy images taken from human impression cytology samples, we obtained 3-dimensional information about the release and spread of goblet cell secretions above the conjunctival surface. After reconstruction and rendering of these images, analysis of the shape and spreading characteristics of the mucins permitted definition of the following parameters related to goblet cell secretion: mucin cloud height as the height of the top of the cloudlike mucin structure visible above the goblet cell opening and spread mucin thickness, which is the thickness of the mucin layer distributed over the surface of the conjunctiva. Several impression cytology samples of control and muco-deficient patients have been analyzed through the confocal laser scanning technique, and significant differences between these groups were found. Mucin cloud height and spread mucin thickness values for controls were 8.81 +/- 4.00 and 2.77 +/- 1.00 microm, respectively (n = 25). These values decreased by approximately 70% and 40%, respectively, for moderately mucodeficient subjects and by 84% and 48% for those with severe mucodeficiency. Classifying those individuals having mucin-related pathology may thus be possible on the basis of application of these techniques. In summary, we present a method of objectively identifying those individuals with problems associated with either a lack of mucins or a reduction in the distribution of these proteins over the ocular surface.

Nucleus and cell size changes in human bulbar conjunctival cells after soft contact lens wear, as assessed by impression cytology

Purpose To specifically assess the nucleus size and its relationship to cell size for human bulbar conjunctival cells. Methods Impression cytology samples were taken from the nasal side of the intra-palpebral zone of the bulbar conjunctival surface from 20 young adult white European males, half of whom were successful daily soft contact lens wearers. A Millcell ®-CM filter was used, after topical anaesthesia with oxybuprocaine 0.4%, which was stained with Giemsa and colour images taken at 400× magnification by light microscopy. The images were graded and also a 35 mm was prepared. From the projected image, an overlay method was used to outline the borders such that the cell and nucleus areas could be measured by planimetry. Results The group mean cell area values were 267 ± 59 μm 2 ( n = 10, ±S.D.) and 1028 ± 357 μm 2 for the non-contact lens wearers and contact lens wearers, respectively. The cell nucleus areas were 64 ± 11 μm 2 and 99 ± 19 μm 2, respectively. Both the cell areas and nucleus area values were statistically different between the two groups ( p < 0.001). Conclusions These studies confirm that soft contact lens wear can result in cell enlargement (squamous metaplasia) of the bulbar conjunctival cells. With this cell enlargement, the nucleus-to-cytoplasm ratio also changes, but the nucleus size generally increases (rather than decreases).

A Novel Antimicrobial Peptide on the Ocular Surface Shows Decreased Expression in Inflammation and Infection

Antimicrobial peptides (AMPs) are cationic host defense peptides with microbicidal and cell-signaling properties. They show promise as potential therapeutic agents. In the present study, a beta-defensin AMP gene was isolated from the ocular surface for the first time, and its expression was characterized in the presence of ocular inflammation and/or infection. Total RNA was obtained from impression cytology samples of the conjunctiva and cornea of normal patients and of those with bacterial, viral, acanthamoeba, or dry eye disease. The expression of the beta-defensin AMP DEFB-109 was determined by using reverse transcription-polymerase chain reaction (RT-PCR). Relative quantification of the gene in the various groups was performed by means of real-time PCR. DEFB-109 was constitutively expressed in all samples. The gene showed significantly decreased expression in the presence of all types of inflammation/infection. Reduced expression featured most prominently in acanthamoeba infection; the least change from normal was in dry eye. The discovery of DEFB-109 on the ocular surface enhances our knowledge of the profile of AMPs at this important mucosal surface. The fact that its expression is significantly reduced in both inflammatory and infective ocular surface disease reflects not only an intimate balance between this host defense gene and microbes but indicates a role other than purely microbicidal. This discovery will enable the mechanisms behind the intriguing phenomenon of reduced gene expression of an AMP in disease states to be uncovered.

Filtering Blebs and Aqueous Pathway: An Immunocytological and In Vivo Confocal Microscopy Study

To characterize and understand, at the cellular level, the aqueous humor pathways after filtering surgery, using in vivo confocal microscopy and impression cytology (IC). Observational case series. Thirty-two blebs of 29 patients after trabeculectomy were retrospectively evaluated. In vivo confocal microscopy and immunofluorescence staining of IC samples taken on and around the bleb area were performed. Impression cytology samples were examined under confocal microscopy after goblet cell and inflammatory cell immunostaining with anti-MUC5AC and antivimentin antibodies, respectively. Eyes were classified into 3 groups: (1) functioning blebs (11 eyes), (2) nonfunctioning blebs (10 eyes), and (3) functioning blebs after mitomycin C application (12 eyes). Impression cytology specimens and in vivo confocal microscopy images were analyzed and compared in a masked manner. Conjunctival epithelium changes of each type of bleb were analyzed using both impression cytology specimens and in vivo confocal microscopy and correlated to clinical outcomes. In all IC specimens, numerous MUC5AC-positive cells were observed outside the edges of the blebs. Few MUC5AC-positive cells were observed at the surface of nonfunctioning blebs. Numerous goblet cells with immunostaining that was weak or limited to the membrane were clearly visible morphologically at the surface of functioning blebs (with and without adjunctive mitomycin C). Using in vivo confocal microscopy, all functioning blebs showed numerous intraepithelial optically empty microcysts, whereas nonfunctioning blebs had none or only a few. All blebs contained dendritiform inflammatory cells, especially after mitomycin C application. Impression cytology and in vivo confocal microscopy provide a new approach to filtering blebs. Microcysts observed at the surface of functioning blebs seemed to correspond to goblet cells, mostly containing aqueous humor instead of highly hydrophilic gel-forming mucins. Although this hypothesis requires further confirmation, the transcellular pathway of the aqueous humor could be hypothesized to occur at the level of goblet cells toward the ocular surface.

Ambient Levels of Air Pollution Induce Goblet-Cell Hyperplasia in Human Conjunctival Epithelium

BackgroundOcular mucosa is exposed constantly to the external environment, and chronic exposure to air pollution may affect the ocular surface.ObjectiveWe assessed the effect of air pollution on the ocular surface by combining determinations of individual exposure and conjunctival impression cytology.MethodsA panel study was conducted with 29 volunteers recruited in two locations with different pollution levels: São Paulo (n = 13) and Divinolândia (n = 16). We assessed mean individual levels of nitrogen dioxide (NO2) exposure for 7 days, using a passive sampler. Impression cytology samples were obtained from inferior tarsal conjunctiva. Comparisons between the two groups in terms of NO2 exposure and goblet-cell counts were performed using the Student t-test. Correlations between goblet-cells counts and corresponding individual NO2 exposure levels were determined using Spearman’s correlation.ResultsIndividuals living in São Paulo received a significantly (p = 0.005) higher dose of NO2 (mean 32.47; SD 9.83) than those living in Divinolândia (mean 19.33; SD 5.24). There was a steady increase in goblet-cell counts, proportional to NO2 exposure (Spearman’s correlation = 0.566, p = 0.001), with a dose–response pattern.ConclusionsA positive and significant association between exposure to air pollution and goblet-cell hyperplasia in human conjunctiva was detected. The combination of simple measurements of exposure and impression cytology was an effective and noninvasive approach for characterizing human response to ambient levels of air pollution.

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.

Antimicrobial peptides on the ocular surface in health and disease

Abstract Purpose: Antimicrobial peptides (AMPs) are the eukaryotic analogues of antibiotics and of paramount importance in host defence. We report herein the expression of a spectrum of AMPs at the ocular surface in infective conditons compared to controls. Methods: RNA obtained from impression cytology samples of conjunctival and corneal epithelial cells of normal persons and of patients with bacterial, viral, acanthamoeba and dry eye disease underwent reverse‐transcription‐ Polymerase Chain Reaction (PCR) to assess expression of the AMPs HBD‐1 to ‐4( Human Beta Defensin),Dermicidin, LL‐37 (or Cathelicidin), LEAP‐1 and LEAP‐2 (Liver Expressed Antimicrobial Peptide) and a number of putative AMPs. Results: HBD‐1 and ‐2 were universally expressed in both health and disease. HBD‐3 was seen only occasionally in infection and not in controls and seemed to be commonly expressed in dry eye disease. LL‐37 occurred in both normals and infective groups but the level of expression was variable and was less in acanthamoeba infection. Likewise LEAP‐1 and ‐2 were commonly seen both in control and infection and appeared to be more strongly expressed i the former. The latter AMPs, i.e., the putative AMPs were not expressed. Conclusions: AMPs have a distinct profile on the ocular surface as opposed to other mucosal surfaces and different disease conditions appear to further modify gene expression. In contrast to other surfaces HBD‐1 and ‐2 both appear to be constitutively expressed on the ocular surface and other AMPs are more storngly expressed in certain disease states than others whereas others are more common in control groups than in infective conditions.

The differences of tear function and ocular surface findings in patients with atopic keratoconjunctivitis and vernal keratoconjunctivitis

The pathogenesis of the ocular surface disease in atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) has not been fully understood. We tried to clarify the differences in the ocular surface status in patients with AKC, VKC, and healthy control subjects. Twenty-four eyes of 12 AKC patients, 12 eyes of six VKC patients, and 20 eyes of 10 normal control subjects were studied. The subjects underwent corneal sensitivity measurements, Schirmer test, tear film break-up time (BUT), vital staining of the ocular surface, conjunctival impression and brush cytology. Impression cytology samples underwent periodic acid Schiff staining for goblet cell density, squamous metaplasia grading, and immunohistochemical staining for MUC1, 2, 4, and 5AC. Brush cytology specimens underwent staining for inflammatory cell counting and Real Time PCR for MUC1, 2, 4, and 5AC mRNA expression. The mean BUT, corneal sensitivity, and conjunctival goblet cell density values in AKC patients were significantly lower compared with VKC patients and control subjects. The squamous metaplasia grades in eyes with AKC were significantly higher compared to eyes with VKC and controls. The inflammatory cell response in brush cytology specimens was different between patients with AKC and VKC. Eyes with AKC showed significantly higher MUC1, 2 and 4 and lower MUC5AC mRNA expression compared to eyes with VKC. Differences of the infiltrates, higher level of tear instability, lower corneal sensitivity, up-regulation of MUC1, 2, and 4, and down regulation of MUC5AC were important differential features of the ocular surface disease in AKC compared with VKC.

Citologia de impressão no diagnóstico de infecção corneana por Acanthamoeba: relato de caso

To describe three cases of corneal infection due to Acanthamoeba sp in which was possible to detect Acanthamoeba sp cysts by the corneal impression cytology technique. Three patients referred to the External Eye Disease Laboratory in 2004 with superficial corneal alterations were submitted to corneal specimen collection by impression cytology filter paper to investigate the presence of Acanthamoeba sp cysts. Two impression cytology samples were obtained from each patient and were stained by PAS, hematoxylin and Papanicolaou. Routine microbiological investigation and culture were also performed using corneal scraping. Positive culture and impression cytology for Acanthamoeba sp was observed in all patients while smears with Giemsa stain were positive in two. Impression cytology Acanthamoeba sp cysts were observed among sheets of corneal epithelial cells and as isolated cells. Cysts were also found in the superficial epithelium in one of these patients after treatment while corneal scraping did not reveal any cyst. Histopathology revealed cysts in the epithelium and stroma in a transplanted cornea in one of these patients. The first description of impression cytology as a diagnostic method for Acanthamoeba keratitis occurred recently. In this study corneal impression cytology detected Acanthamoeba sp cysts successfully in these patients with only superficial involvement. Impression cytology as a non invasive technique can be used to facilitate early recognition of Acanthamoeba infection playing a useful role in the follow-up of the disease.

Alterations of the ocular surface epithelial mucins 1, 2, 4 and the tear functions in patients with atopic keratoconjunctivitis

An increased understanding of the ocular surface alterations at the cellular level in the conjunctiva and the cornea, may help explain the pathogenesis and the subsequent clinical appearance of atopic ocular allergies, which may be potentially blinding. To investigate MUC 1, 2 and 4 alterations, tear function and the ocular surface disorder in patients with atopic keratoconjunctivitis. Twenty-eight eyes of 14 atopic keratoconjunctivitis patients as well as 22 eyes of 11 age-and sex-matched normal subjects were studied. The subjects underwent corneal sensitivity measurements, Schirmer's test, tear film break-up time (BUT), fluorescein and Rose Bengal staining of the ocular surface, conjunctival impression cytology and brush cytology. Impression cytology samples underwent periodic acid-Schiff and immunohistochemical staining with MUC 1, 2 and 4 antibodies. Brush cytology specimens underwent evaluation for inflammatory cell numbers and quantitative real-time-PCR for MUC 1, 2 and 4 mRNA expression. Patient eyes with fluorescein and Rose Bengal scores greater than four points were regarded to have significant epithelial disease in this study. The mean corneal sensitivity and BUT values were significantly lower in atopic patients with significant epithelial disease, compared with patients with insignificant epithelial disease and controls (P < 0.01). Brush cytology specimens from patients with significant epithelial disease revealed significantly higher numbers of inflammatory cells (P < 0.01). Specimens from patient eyes showed positive staining for MUC 1, 2 and 4. MUC 1, 2 and 4 mRNA expressions were significantly higher in eyes with significant epithelial disease compared with eyes with insignificant epithelial disease and eyes of control subjects. Ocular surface inflammation, decline in corneal sensitivity, tear film instability, changes in conjunctival epithelial MUC 1, 2 and 4 mRNA expressions were thought to be important in the pathogenesis of atopic ocular surface disease.