The role of conjunctival epithelial cell xanthine oxidoreductase/xanthine oxidase in oxidative reactions on the ocular surface of dry eye patients with Sjögren's syndrome

Previous papers examined lipid peroxidase levels and myeloperoxidase activity as products of oxidative and inflammatory reactions in the tear fluid of patients suffering from dry eye. The aim of the present paper was to investigate whether the enzymes xanthine oxidoreductase/xanthine oxidase known to generate reactive oxygen species contribute to oxidative reactions on the ocular surface. Xanthine oxidoreductase/xanthine oxidase were examined immunohistochemically as well as histochemically in conjunctival epithelial cells of patients suffering from dry eye. Patients with verified autoimmune dry eye (Sj?gren's syndrome) participated in our study; normal eyes served as controls. Conjunctival epithelial cells were obtained by the method of impression cytology using Millicell membranes. The results revealed a pronounced expression, as well as activity of xanthine oxidoreductase/xanthine oxidase in the conjunctival epithelium of dry eye. It is suggested that reactive oxygen species which are generated by this enzymatic system, contribute to oxidative reactions on the eye surface of patients with ocular manifestations of autoimmune disease (Sj?gren's syndrome).     

Decreased expression of antioxidant enzymes in the conjunctival epithelium of dry eye (Sjögren's syndrome) and its possible contribution to the development of ocular surface oxidative injuries

Previous studies have described elevated lipid peroxidase, myeloperoxidase and xanthine oxidoreductase/xanthine oxidase levels on the ocular surface of patients suffering from autoimmune dry eye (Sj?gren's syndrome, SS). Reactive oxygen species generated by various enzymatic systems may be dangerous to the eye if they are not sufficiently cleaved by antioxidants. Because antioxidants have not been investigated in dry eye, the aim of this study was to examine the expression of antioxidant enzymes that cleave reactive oxygen species and play a key role in antioxidant protection. Conjunctival epithelial cells of dry eye (SS) patients were obtained by the method of impression cytology using Millicell membranes. Normal eyes served as controls. In the conjunctival epithelium superoxide dismutase, catalase and glutathione peroxidase were examined immunohistochemically. The enzyme expression levels were determined by image analysis and statistical evaluation. In contrast to normal eyes, where antioxidant enzymes were highly expressed in the conjunctival epithelium, in dry eye their expression was much less pronounced in correlation with the increasing severity of dry eye symptoms. Our study suggests that the decreased expression of antioxidant enzymes in dry eye disease (SS) contributes to the development of anterior eye surface oxidative injuries.     

Identification of 9-O-acetyl-N-acetylneuraminic acid in normal canine pre-ocular tear film secreted mucins and its depletion in Keratoconjunctivitis sicca

O-Acetylated sialic acids have been reported in many sialoglycoproteins where they mediate a variety of immune and other biological events. We have previously demonstrated that the protective mucus barrier on the surface of the canine eye contains sialoglycoproteins. We have also investigated the occurrence of O-Acetylated sialic acids in these ocular mucins. Mucus aspirated from the surface of normal dog eyes and those with keratoconjunctivitis sicca (KCS) was fractionated into three pools by density gradient centrifugation. Sialic acids comprised 0.6–0.9% of the dry weight of the mucins isolated. The sialic acid profile in these pools was examined using HPLC. O-Acetylated sialic acids, mainly Neu5,9Ac₂, were detected in normal animals and made up 10–30% of the total sialic acids detected. A doubling of the sialic acid content was found in KCS mucins, but the level of 9-O-Acetylated sialic acid was reduced below 4% of total. Histological analysis of conjunctival tissue from normal and KCS dogs showed the presence of sialic acids, detected with the α(2–6) sialic acid-specific lectin Sambucus nigra, in the goblet cells and corresponding to the staining pattern for MUC5AC, the major ocular-secreted mucin gene product. In KCS animals a disruption of the normal pattern of conjunctival goblet cells was seen with preservation of the pattern of lectin binding observed in normal animals. Thus the data demonstrate the presence of mono-O-Acetylated sialic acids in normal canine ocular mucins and a loss of this population of sialic acids in dry eye disease in spite of a significant increase in total sialic acids in KCS mucin.     

Prostaglandin E receptor subtype EP3 expression in human conjunctival epithelium and its changes in various ocular surface disorders

Background

In our earlier genome-wide association study on Stevens-Johnson Syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), we found that in Japanese patients with these severe ocular surface complications there was an association with prostaglandin E receptor 3 (EP3) gene (PTGER3) polymorphisms. We also reported that EP3 is dominantly expressed in the ocular surface-, especially the conjunctival epithelium, and suggested that EP3 in the conjunctival epithelium may down-regulate ocular surface inflammation. In the current study we investigated the expression of EP3 protein in the conjunctiva of patients with various ocular surface diseases such as SJS/TEN, chemical eye burns, Mooren’s ulcers, and ocular cicatricial pemphigoid (OCP).

Methodology/Principal Findings

Conjunctival tissues were obtained from patients undergoing surgical reconstruction of the ocular surface due to SJS/TEN, chemical eye burns, and OCP, and from patients with Mooren''s ulcers treated by resection of the inflammatory conjunctiva. The controls were nearly normal human conjunctival tissues acquired at surgery for conjunctivochalasis. We performed immunohistological analysis of the EP3 protein and evaluated the immunohistological staining of EP3 protein in the conjunctival epithelium of patients with ocular surface diseases. EP3 was expressed in the conjunctival epithelium of patients with chemical eye burns and Mooren’s ulcer and in normal human conjunctival epithelium. However, it was markedly down-regulated in the conjunctival epithelium of SJS/TEN and OCP patients.

Conclusions

We posit an association between the down-regulation of EP3 in conjunctival epithelium and the pathogenesis and pathology of SJS/TEN and OCP, and suggest a common mechanism(s) in the pathology of these diseases. The examination of EP3 protein expression in conjunctival epithelium may aid in the differential diagnosis of various ocular surface diseases.     

Diagnostic impression cytology: a simple technique for the diagnosis of external eye disease

Diagnostic impression cytology: a simple technique for the diagnosis of external eye disease
The technique of impression cytology was used for cytologic examination of conjunctival samples from 128 patients with a variety of external eye diseases. The method of sample collection and the staining procedure are described. Microscopic examination showed 62 cases with allergic conjunctivitis, 23 with bacterial or viral infection, 26 cases with changes typical of dry eye and 17 cases with non-specific chronic inflammation. The cytologic features and numerous applications of impression cytology are discussed.     

Presence of snake-like chromatin in epithelial cells of keratoconjunctivitis sicca followed by a large number of micronuclei

OBJECTIVE: To evaluate the number of micronuclei in snake-like chromatin (SLC) cells in the conjunctival epithelium of keratoconjunctivitis sicca (KCS) patients. To elucidate possible correlations between SLC cell numbers and KCS intensity. STUDY DESIGN: Impression cytology specimens from the bulbar conjunctiva of healthy controls and KCS patients were harvested and divided into 3 groups: group 1, controls; group 2, KCS SLC-negative; and group 3, KCS SLC-positive. The number of micronuclei (MNi) in SLC-negative and SLC-positive epithelial cells of each group was counted. RESULTS: The number of MNi in SLC-negative cells of groups 1 and 2 did not exceed 1 MNi/1,000 cells. A significant increase in the frequency of micronuclei in the upper bulbar conjunctiva was noted in SLC-positive (14.75 +/- 8.09 MNi/1,000 cells) as well as SLC-negative cells (4.0 +/- 3.83 MNi/1,000 cells) of group 3. CONCLUSION: We demonstrate here that the presence of MNi in the conjunctival epithelium of KCS patients could be a characteristic feature accompanying SLC cells. The fact that increased numbers of SLC cells correlates with impaired values in clinical test as well as decreased goblet and epithelial cell densities confirms that the presence of SLC cells correlates with KCS intensity.     

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

M. J. Doughty Assessment of agreement for assignment of a normal grade to human conjunctival impression cytology samples Objective: 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. Methods: 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. Results: Area measures on normal grade images indicated a broad unimodal distribution (250–275 μm²), 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). Conclusions: 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.     

Conjunctival brush cytology

In order to collect conjunctival cells efficiently, we developed a special brush that is a modification of the Cytobrush used in cervical cytology. The conjunctival brush is small and made of nylon bristles. Cells collected by these brushes were rinsed into a buffered solution, from which filter preparations were made. This technique produced adequately cellular samples from temporal bulbar conjunctiva; these preparations stained well with the Papanicolaou stain. Under normal conditions, three cell types were observed in the brushing samples; one was the polygonal epithelial cell, the second type was a small rounded cell, and the third type was a mucus-secreting goblet cell. Samples from dry-eyed patients contained keratinized cells with or without a decrease in goblet cells. Elongated cells were seen in samples from postirradiation and postoperative patients. Irritation caused by the brushing was of the same intensity as irritation caused by collecting cytologic specimens by impression or by the use of cotton swabs. These findings suggest that brushing cytology of the conjunctiva is a relatively noninvasive technique and can provide valuable information for evaluation of conjunctival conditions.     

Evaluation of thin-layer methods in urine cytology

Conventional cytospin smears prepared from urinary tract specimens were compared with two new thin layer techniques, i.e. ThinPrep and AutoCyte PREP. Cellularity, cell preservation, background features, detection rate, screening time and ease of preparation were evaluated. Thin-layer techniques when applied to urine cytology were found to improve cell yield and cell preservation, and reduce background artefact. The reporting rate for abnormal urothelial cells was comparable to conventional cytospin smears, as was screening time. Laboratory staff found the methodologies to be practicable and easily incorporated into a large routine diagnostic service. We conclude that a one-slide thin-layer urine preparation is comparable to four cytospin slides in the detection of urothelial abnormalities, and that both ThinPrep and AutoCyte PREP have comparable features.     

Conjunctival impression cytology in contact lens wearers

The upper tarsal conjunctiva is in constant friction with the surface of the contact lens. The conjunctival surfaces of 80 soft and gas-permeable contact lens wearers (40 each) and 20 controls were studied using biomicroscopy and impression cytology. A filter dissolution technique was used to process the conjunctival imprints. Biomicroscopic and cytologic grading of the conjunctivae was performed using four-tier grading systems. Impression cytology is a non-invasive, painless procedure. The altered technique of processing yielded better cellularity and excellent cellular detail. On biomicroscopy and cytology, all controls showed Grade 1 appearances. Soft lens wearers who were symptomatic were found to have a significant increase in both biomicroscopic and cytologic grades, when compared with their asymptomatic counterparts. No correlation was found between duration of lens use and biomicroscopic or cytologic grades. All changes were found to be more severe in soft lens wearers.     

Conjunctival dysplasia in soldiers exposed to mustard gas during the Iraq-Iran war: scrape cytology.

OBJECTIVE: To describe the conjunctival scrape cytology findings in Iraq-Iran war veterans exposed to mustard gas and to detect malignant transformation at an early stage. STUDY DESIGN: Twenty-two male war veterans (29-54 years old) who were exposed to chemical bombing about 14 years earlier, formed the base of this study. All of them had chronic eye problems, such as dryness, conjunctival scarring and decreased visual acuity. Conjunctival scrape cytology was performed for cytologic study. RESULTS: Among 22 cases of conjunctival scarring, 9 were diagnosed as dysplasia. Mild inflammation and squamous metaplasia were also noted. Squamous cell carcinoma was not identified. CONCLUSION: Conjunctival scraping cytology is useful for the diagnosis of dysplasia in soldiers exposed to mustard gas during of the Iraq-Iran war. This method is also effective for follow-up study.     

Morphological and immunocytochemical characterization of snake-like chromatin cells

Snake-like chromatin (SLC) is a nuclear alteration occurring under various pathological conditions and in different tissues. The aim of this study was the morphological and immunocytochemical characterization of SLC-positive conjunctival epithelial cells from keratoconjunctivitis sicca (KCS) patients. Impression cytology specimens from the upper bulbar conjunctiva of 10 controls and 10 KCS patients with a high incidence of SLC cells were assessed, the morphology of SLC nuclei evaluated by light microscopy, and proliferation markers, nucleolar proteins, lamins and cytokeratin filaments detected immunocytochemically. In KCS patients, SLC cells with a normal nuclear shape, with nuclear membrane notching (2.3% of cells) and with binuclear dumb-bell structures (4.4% of cells) were observed. The most striking features of SLC cells were the absence of an A/C lamin signal, the redistribution of fibrillarin into two spots adjacent to SLC structures and cytokeratin 14 positivity in the strangulation belt of the dumb-bell structures. The deficiency of lamin A/C is the probable reason for the disintegration of chromatin from the nuclear lamina in SLC cells. The occurrence of SLC-positive cells, SLC-positive dumb-bell shaped nuclei and SLC-positive binucleated cells, together with the absence of mitotic markers, leads to the conclusion that the SLC phenomenon might be a form of nuclear segregation.     

A comparative study: conventional preparation and ThinPrep® 2000 in respiratory cytology

In order to compare and contrast conventional preparation (CP) with ThinPrep 2000 (TP) in respiratory cytology, 207 samples were divided equally and processed by the two different preparation methods, generating three CP and one TP slide per sample. No lesion identified by CP was missed by TP and there were no significant differences between TP and CP in the diagnostic categories. However, two cases of squamous cell carcinoma were detected on TP which had been classified as unsatisfactory and moderate squamous dyskaryosis, respectively, on CP. ThinPrep was found to be superior to CP in many respects as it provided standardized preparations in a greater proportion of cases and problems such as cell overlapping and background debris were markedly reduced. In several instances the diagnostic accuracy in CP was compromised by smears that were either too thick, too thin, or too scanty. Cell preservation was also better on TP when compared with CP, facilitating more accurate diagnosis and significantly reducing the primary screening and reporting time, especially of sputum samples. A major advantage of TP methodology is the fact that it facilitates optimal use of skilled cytotechnologists and streamlines the workflow in the laboratory.     

Impression Cytology In Patients With Keratoconjunctivitis Sicca

Impression cytology is a simple and painless procedure that allows the collection of the superficial layers of the conjunctival epithelium. Each sample is assigned a grade of epithelial metaplasia, and goblet cell density is calculated in each one. We have studied the superior and temporal bulbar conjuctiva of dry eyes and have compared it with that of normal controls. In normal and dry eyes we find a statistically significant difference both in goblet cell density and grade of metaplasia, between superior and temporal bulbar conjunctiva. the differences in grade of metaplasia and goblet cell density between normal and dry eyes are significant in the superior conjunctiva, but in the temporal conjunctiva we only find significant differences in grade of metaplasia.     

Preoperative brush and impression cytology in ocular surface squamous neoplasms

OBJECTIVE: Preoperative cytologic diagnoses of ocular surface squamous neoplasms were evaluated and compared with histologic diagnoses. STUDY DESIGN: Impression cytology (Millipore filter paper) and brush cytology were applied to 32 patients who had conjunctival neoplasms. Papanicolaou-stained cytologic preparations and hematoxylin and eosin-stained histologic sections were examined by light microscopy. RESULTS: The brush technique was used on 27 patients; impression cytology was applied in 5 cases. Cytologic and histologic diagnoses were concordant in 26 cases. Squamous cell carcinoma or carcinoma in situ was diagnosed in 18 and dysplasia in 4 cases. Squamous metaplasia and normal-appearing conjunctival epithelial cells were diagnosed cytologically in four cases; of those histologic diagnoses, one was pterygium and three, conjunctival nevus. Four cases revealed discrepancies between the cytologic and histologic preparations. There was one false positive result, and one case was subconjunctival invasion of basal cell carcinoma of the eyelid. CONCLUSION: Impression and brush cytology are fast, cost-effective, reliable and noninvasive diagnostic tools for ocular surface squamous neoplasms. However, the brush technique has several advantages over impression cytology.     

Cytologic and DNA cytometric diagnosis and therapy monitoring of squamous cell carcinoma in situ and malignant melanoma of the cornea and conjunctiva

OBJECTIVE: To investigate the diagnostic accuracy of exfoliative cytology of the cornea and conjunctiva and DNA image cytometry for quality control and monitoring of therapy for malignant neoplasms. STUDY DESIGN: Conjunctival or corneal smears from six cases clinically suspicious for malignant melanomas and eight suspicious for carcinomas in situ were investigated. Smears from 18 cases clinically nonsuspicious for neoplastic diseases served as negative controls. Repeated smears were obtained during and after local mitomycin C (MMC) therapy. RESULTS: In none of 18 nonsuspicious cases, cytology revealed abnormal cells. DNA cytometry showed nonaneuploidy in all of these. All smears from patients with histologically proven malignant melanomas (MM) and squamous cell carcinomas in situ revealed abnormal cells. Image cytometry demonstrated DNA aneuploidy in 66.6% of patients with MM and 80% with carcinoma. Sensitivity of cytology thus was 100% for both MM and carcinoma; specificity also was 100%. DNA measurements after MMC therapy revealed euploid polyploidization of nonneoplastic squamous cells. DNA cytometry provided an objective identification of tumor cell regression. CONCLUSION: Cytologic examination of corneal and conjunctival smears is a noninvasive tool with high diagnostic accuracy for detection of epithelial neoplasms. DNA image cytometry can serve for quality control and for objective monitoring of the effect of local chemotherapy.     

Analysis of urine cytology tests in 120 paired cases

OBJECTIVE: To evaluate the smear quality and diagnostic accuracy of ThinPrep processing in comparison to conventional Cytospin technique for urinary cytology. STUDY DESIGN: ThinPrep and Cytospin techniques were retrospectively evaluated by 2 observers in a double-blinded, randomized fashion. Each quality parameter was scored using a semi-quantitative score of 1-3. Diagnostic accuracy indices were calculated with biopsy histology as the gold standard. RESULTS: Quality of cellular distribution and cell preservation were better with Cytospin preparations, whereas ThinPrep smears were superior in terms of stain distribution and cleaner slide background. However, the only significant differences observed were in cellular distribution and a clean background (p < 0.05). Sensitivity and positive and negative predictive values were higher with Cytospin than the ThinPrep technique (90.0%, 94.7% and 71.4% vs. 80.0%, 94.1% and 55.6%, respectively). Conversely, the specificity of both techniques was comparable. CONCLUSION: The Cytospin smears were of better quality than those prepared by the ThinPrep technique. Although both techniques resulted in similar diagnostic accuracies in negative cases, the ThinPrep preparations were not found to be superior to Cytospin smears in diagnosing positive urinary cytology.     

New aspects of vulvar cytology

Smears were taken from vulvar skin in 203 cases (83 normal-appearing, 23 benign, 12 precancerous and 85 malignant conditions). Three cases were analyzed by electron microscopy. The appearance of dyskeratotic cells was regarded as a sign of malignancy; this was supported by ultrastructural study. Smears from normal vulvar skin revealed a high correlation between age and presence of nucleated keratinized squames, which occurred most often in the third and rarely in the eighth decade. Benign conditions showed cellular features like those of the normal vulva. In precancerous lesions, 58% of the smears showed dyskeratotic cells. Malignant conditions showed dyskeratotic cells in 66% of the smears and true cancer cells exclusively in an additional 14%. Our observations indicate that vulvar cytology can be a major diagnostic aid in all suspicious cases, especially in the elderly. Vulvar cytology cannot replace an exact histologic examination, but it can provide a reason for taking a biopsy earlier than might otherwise be the case.     

Bile duct brushings cytology – improving sensitivity of diagnosis using the ThinPrep® technique: a review of 113 cases

OBJECTIVE: Common bile duct (CBD) brushings have been recognized as a technique of moderate sensitivity and high specificity in identifying carcinoma of the ampulla and pancreatico-biliary regions. This study evaluated the increase in sensitivity of this technique using the ThinPrep technique of specimen preparation when compared with conventional cytology smears. METHODS: A total of 113 bile duct brushings were included in the study (38 conventional smears and 75 slides prepared using the ThinPrep technique). All slides were reviewed by one cytologist. Five categories of reporting were used: inadequate, negative, atypia, suspicious and malignant. RESULTS: The inadequate category of reporting disappeared in the ThinPrep group with improved specimen fixation and preparation and hence reduced artefact. Sensitivity of diagnosis of malignancy increased from 39% in conventional smears to 53% in the ThinPrep group. Specificity, positive and negative predictive values and accuracy were 100%, 100%, 60% and 68% for conventional smears and were 100%, 100%, 60% and 72%, respectively, for ThinPrep specimens. CONCLUSIONS: ThinPrep technique was associated with increased sensitivity of diagnosis, in part due to improved specimen fixation and reduced artefact. Cytology of bile duct brushings is an important diagnostic tool for sites from which it can be difficult to obtain a histology biopsy. It may therefore provide the only opportunity for tissue diagnosis of carcinoma from these sites, hence the importance of optimizing sensitivity.     

Diagnostic value of fine needle aspirates processed by ThinPrep® for the assessment of axillary lymph node status in patients with invasive carcinoma of the breast

X. Jing, E. Wey and C. W. Michael Diagnostic value of fine needle aspirates processed by ThinPrep® for the assessment of axillary lymph node status in patients with invasive carcinoma of the breast Objective: To evaluate the utility of ThinPrep® as an optional specimen processing method for the detection of axillary lymph node metastasis of invasive breast carcinoma. Methods: A computer SNOMED search from the file at our institution between January 2003 and August 2011 retrieved a total of 209 fine needle aspiration (FNA) specimens of axillary lymph nodes prepared by ThinPrep and followed by axillary lymph node biopsy and/or dissection. Original cytological diagnoses and corresponding histological diagnoses were documented. Using the histological diagnoses as the gold standard, the diagnostic parameters including sensitivity, specificity, positive (PPV) and negative predictive values (NPV) and diagnostic accuracy were calculated. Both cytology and histology slides from cyto‐histologically discrepant cases were reviewed. Results: Out of a total of 209 specimens, 193 (92%) had adequate diagnostic material while the remaining 16 specimens (8%) were inadequate for cytological assessment. The diagnostic specimens included 168 invasive ductal carcinomas (IDC), 15 invasive lobular carcinomas (ILC) and 10 mixed carcinomas (IDC and ILC). Excluding 19 cases with malignant cells on FNA in which no residual tumour was found in fibrotic lymph nodes after neoadjuvant therapy (cytology and histology confirmed on review) ThinPrep detected nodal metastasis with an overall sensitivity of 77.5%, specificity of 100%, PPV of 100% and NPV of 53.7%. Diagnostic accuracy was 82.2%. There was no difference in Bloom–Richardson grade or the number or size of metastases between tumours with true‐positive and false‐negative cytology. Sampling error was the sole factor contributing to cyto‐histological discrepancy. Conclusions: ThinPrep is a good alternative to the conventional smear for cytological assessment of axillary lymph node status in patients with invasive breast carcinoma, particularly when specimens are collected at remote sites or when cytologists are not available for assistance during FNA.