Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells.

The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or γ-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without γ-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ΔNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and γ-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p<0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a γ-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. STATEMENT OF SIGNIFICANCE: Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.

Unusual ocular presentations of ocular phaeohyphomycosis.

PurposePhaeohyphomycosis are melanin-containing fungi that rarely infect the eye. We describe three cases of ocular infection with some unusual clinical features.MethodsSeries of three case reports describing three different presentations of phaeohyphomycosis of the eye, their histopathology, and management.ResultsCase 1 mimicked an inflamed conjunctival naevus and was excised on this basis, revealing a conjunctival retention cyst containing pigmented fungal hyphae. Case 2 showed a wooden foreign body incidentally associated with pigmented fungal hyphae, which required treatment with topical antifungal therapy. Case 3 clinically was thought to be a perforated uveal melanoma and comprised an extensive plaque of pigmented fungal hyphae over ulcerated cornea.ConclusionThe pigmented melanin containing hyphae of phaeohyphomycosis mimicked melanocytic lesions in two cases and was an incidental finding in the context of a surface foreign body.

Ocular surface foreign bodies: novel findings mimicking ocular malignant melanoma.

PURPOSE: Malignant melanoma of the eye is an uncommon condition that is important to recognise. We describe three cases in which ocular foreign bodies have masqueraded as ocular malignant melanoma. METHODS: Interventional case reports. RESULTS: Case 1 describes diathermy-induced carbon particle implantation, during plaque therapy for the treatment of uveal melanoma, mimicking recurrence with extra-scleral invasion. Case 2 shows a foreign body called 'mullite' mimicking conjunctival melanoma. Case 3 demonstrates a conjunctival foreign body called 'illite' that mimicked a limbal melanocytic lesion, clinically thought to be either melanocytoma or melanoma. CONCLUSION: This report highlights the importance of careful history taking, examination, and appropriate biopsy in cases of suspected malignant melanoma, to prevent unnecessary and potentially radical treatment.

Three cases of intraocular mesectodermal leiomyoma expressing progesterone and androgen receptors.

BACKGROUND/AIMS: A prospective study identified three patients between 2004 and 2010 with mesectodermal leiomyoma. The study was conducted to analyse the presence or absence of sex steroid hormone receptors in mesectodermal leiomyomas. METHODS: The clinical features were collated. All three patients had operative procedures to either remove or sample the mesectodermal leiomyomas. The tissue was fixed in formalin and exposed to conventional histological processing. Immunohistochemistry using antibodies to androgen (AR), oestrogen (ER), and progesterone (PR) receptors was performed, followed by stain scoring to assess for expression status. RESULTS: All three cases were confirmed by histology to be examples of mesectodermal leiomyomas. All three expressed sex steroid hormone receptors. One case expressed both PR and AR, one case PR only and another case AR only. None of the cases expressed ER receptors. CONCLUSION: All three cases displayed some sex steroid hormone receptor expression. This is supportive evidence that sex steroid hormones may have a role in the pathogenesis of this tumour and suggest that it may be amenable to hormonal manipulation therapy, in a manner similar to conventional uterine leiomyomas.

Histopathological reporting of corneal pathology by a biomedical scientist: the Sheffield Experience.

AIMS: The advanced specialist diploma in ophthalmic pathology was jointly ratified by the Royal College of Pathologists and the Institute of Biomedical Science in 2008. It results in extended roles for suitably qualified Biomedical Scientists (BMSs) in ophthalmic pathology specimen dissection and histological reporting of selected specimens, specifically non-neoplastic corneas. This study aimed to examine the reporting of corneal histology by a BMS training towards this diploma in comparison with a Consultant Ophthalmic Pathologist. METHODS: This report covers a non-interventional, prospective examination of BMS's diagnostic skills. After 6 months training, 50 consecutive corneal cases were reported by the BMS and the same case reported by the consultant ophthalmic pathologist. The BMS's diagnosis was compared with the final consultant's diagnosis. This exercise was then repeated after 24 months of training. RESULTS: After 6 months, the BMS's diagnoses matched the consultant diagnoses in 44/50 (88%) cases. After 24 months, the BMS's diagnoses matched the consultant diagnoses in 48/50 (96%) of cases. CONCLUSIONS: The results indicate that BMS's reporting of corneal histology is a real and safe possibility. Furthermore, it indicated deficiencies in BMS's diagnostic skills to inform further training/knowledge acquisition.

Diagnostic cellular yield is superior with full pars plana vitrectomy compared with core vitreous biopsy.

PURPOSE: Vitreous biopsy for the cytological assessment of suspected intraocular lymphoma and vitritis of uncertain aetiology is a standard investigation. The types of specimens generated and the diagnostic rate are variable within and between centres. There are many reasons for this but one observation that has not been considered previously is the differential distribution of cells in the vitreous gel. To test this possibility, five consecutive patients with suspected vitreous involvement by lymphoma or vitritis of uncertain aetiology underwent a core vitreous biopsy immediately before a planned full pars plana vitrectomy (PPV) and the cellularity of the two sampling techniques compared. METHODS: A prospective study of five consecutive patients requiring vitreous sampling to secure a firm diagnosis. For each of five patients, the core vitreous biopsy specimen was received in a universal tube and the PPV specimen was received in a vitreous cassette. Fluid (0.25 ml) was removed from both specimens, centrifuged and haematoxylin and eosin (H&E) stained slides prepared per sampling method. The slides were examined with a light microscope, the most cellular field selected and the number of cells per mm(2) counted and compared between sampling techniques. RESULTS: PPV specimen's, revealed a cellularity range that was 7.4 to 78 × (average 31 ×) greater than a core vitreous biopsy. In the two cases of a final diagnosis of intraocular lymphoma, the vitreous core biopsy was non-diagnostic. Furthermore, the PPV specimen generated additional cellular material for numerous ancillary investigations to permit a secure diagnosis. CONCLUSIONS: The results of this differential vitreous sampling study has strengthened our anecdotal slit lamp clinical observations that inflammatory cells and lymphoma cells are concentrated more in the cortical vitreous. Therefore, vitreous cells have less chance to be sampled if a single core vitreous biopsy is performed. Indeed, the two cases of confirmed lymphoma generated a non-diagnostic core vitreous biopsy. In our centre, this study has lead to PPV being performed as a gold standard on all patients with suspected intraocular lymphoma or vitritis of uncertain aetiology.

Improving the interaction between the ophthalmology and histopathology departments.

PURPOSE: The purpose of this study was to improve communication between the ophthalmology and histopathology departments at Royal Hallamshire Hospital, Sheffield, by effectively changing the structure and completion of the histopathology request form through the process of a successful audit. This aimed to ensure that comprehensive information was made available to the histopathologist. METHODS: An audit was performed by review of 710 histopathology request forms, completed by the ophthalmology department, over a 1-year period, between July 2005 and June 2006 inclusive. Results were used to re-model the ophthalmic histopathology request form. New forms were circulated and all forms completed over a 3-month period, between January 2008 and March 2008, were reviewed, thus closing the audit loop. RESULTS: On the basis of audit results of 710 histopathology request forms, a new histopathology request form was created, which was easier to complete. Review of the 224 new histopathology request forms showed improved percentages of completion of important sections of the form. CONCLUSIONS: Through the audit process we have created a new ophthalmic histopathology request form that is more user-friendly for the ophthalmologist and more consistently provides the necessary information for the ophthalmic histopathologist. This has improved efficiency and effectiveness of communication between the specialities, which should contribute to minimise the chances of medical error and improved turnaround times for the planning and delivery of patient care.

Conjunctival intra-epithelial neoplasia occurring in young patients with asthma.

BACKGROUND: Conjunctival intra-epithelial neoplasia (CIN) typically occurs in elderly individuals. A number of aetiological factors are implicated in CIN, including life-long exposure to ultra-violet light and immunodeficiency states, particularly HIV infection. Asthma is a common condition affecting more than 3.5 million individuals in the UK, and is associated with atopy in approximately 70% of cases. In this paper we describe CIN occurring in young patients with asthma. METHODS: Retrospective case series: A review of our ocular oncology database helped to identify 11 patients<55 years of age and presenting with CIN. Of these, seven (64%) were noted to have co-existent asthma. RESULTS: Seven patients were included in the study (six male and one female). Mean age at presentation was 44 years (range 36-54 years). Five patients showed unilateral disease, whereas two showed bilateral. Five patients showed local recurrence; however, there were no cases of metastasis. CONCLUSION: The occurrence of CIN, particularly bilateral CIN, in younger immunocompetent individuals is very unusual, and the presence of asthma in 64% of our patient cohort suggests that atopic asthma may be a further aetiological factor involved in the development of this rare neoplasm.

The in vivo modulatory effects of an anterior-chamber microenvironment on uveal melanoma.

BACKGROUND: Primary melanoma of the iris, for reasons unknown has a lower metastatic rate compared with primary ciliary-body melanoma. Six histology cases of ciliary-body melanoma were identified that had spread onto the iris surface and into the stroma, representing a change in tumour microenvironment from aqueous humour non-exposure (ciliary-body component) to aqueous humour exposure (iris surface component). This provided an ideal paradigm for investigating the effects of different environments on melanoma. METHOD: Conventional light microscopy was performed on stained paraffin sections of the identified cases, followed by immunohistochemistry to cell cycle proteins p27 and Cyclin D1. Fluorescence in situ hybridisation (FISH) analysis was conducted on the paraffin sections for changes of chromosomes 3 and 8, associated with poor uveal melanoma prognosis. RESULTS: Iris surface melanoma cells were smaller compared with the adjacent deeper iris stromal melanoma cells and with those in the ciliary body. Fewer iris surface melanoma cells expressed Cyclin D1 protein, but more expressed p27 protein, compared with the larger iris stromal melanoma cells (paired Wilcoxon signed ranks test: Cyclin D1 p = 0.028; p27 p = 0.046) and with the ciliary-body melanoma cells (paired Wilcoxon signed ranks test: Cyclin D1 p = 0.028; p27 p = 0.028). With FISH, chromosome 3 and 8 alterations were less common among the iris surface melanoma cells than the deeper iris stromal melanoma cells and the ciliary-body melanoma cells, which were consistently characterised by a relative genetic imbalance for chromosomes 3 and 8. CONCLUSIONS: These data suggest that there are tumour-modulatory factors within the anterior chamber environment that probably select populations of ciliary-body melanoma cells, with a less aggressive, better-differentiated status. Furthermore, it may help explain why iris melanomas generally have a less aggressive course than ciliary-body and choroidal melanomas.

What is the significance of vortex vein invasion in uveal melanoma?

PURPOSE: To correlate vortex vein invasion with established prognostic factors for uveal melanoma. METHODS: Enucleated eyes with a confirmed histopathological diagnosis of uveal melanoma with vortex vein invasion were identified, over a 10-year period. Established uveal melanoma prognostic factors, with tumour genetics were correlated with vortex vein invasion and patient survival. RESULTS: Microscopic vortex vein involvement was present in 29 of 244 (11.9%) uveal melanomas. Of 29, 6 (20.7%) tumours had macroscopic evidence of vortex vein invasion. Of 29, 14 (48.3%) tumours also showed evidence of non-vortex vein, 'direct' scleral invasion. 23 (79.3%) of 29 melanomas involved only the choroid. The mean maximum diameter of tumours with vortex vein invasion was 15.8 mm and the mean thickness was 9.7 mm. The uveal melanoma was a discrete nodule in 27 of 29 (93.1%) cases. Histologically, 8 of 29 tumours (27.6%) were spindle cell, 19 of 29 (65.5%) were mixed cell, and 2 of 29 (6.9%) were epithelioid cell type. Of 29, 22 (75.9%) uveal melanomas with vortex vein invasion contained extracellular matrix networks and loops. Genetic abnormalities correlated with poor prognosis were seen in 25 of 29 (86.2%) tumours with vortex vein invasion. Liver metastasis was confirmed in 19 of 29 (65.5%) patients with vortex vein invasion. No patients with uveal melanomas showing vortex vein invasion suffered orbital recurrence of disease following enucleation. CONCLUSIONS: The trends show that vortex vein invasion is associated with a choroidal location, large tumour size, spindle cell bias, presence of extracellular matrix loops/networks and genetic markers. A higher proportion of patients with vortex vein invasion progress to develop liver metastasis compared with the general uveal melanoma population.

Abscessed porous polyethylene (Medpor) orbital implant: a case report.

We report the evolution of clinical features of orbital implant infection in a 42-year-old man. Despite appropriate treatment recurrent conjunctival dehiscence could not be prevented. Explanation of the implant resulted in complete resolution of symptoms. Histopathological examination confirmed focal necrotising acute inflammation with the presence of colonies of the organism in the deep substance of the implant.

Early pathological features of the cornea in toxic epidermal necrolysis.

AIM: To describe the early pathological changes in the cornea during toxic epidermal necrolysis (TEN). METHOD: Demonstration of histological features of sequential corneal samples taken during management of complications of TEN in a young adult. RESULTS: Early vacuolation of basal keratinocytes and late infiltration of the cornea with CD 8 lymphocytes were observed. These changes are similar to those found in cutaneous TEN and may represent weakening of the stromal-epithelium interface with resultant recurrent erosion and chronic inflammation. CONCLUSIONS: Similar changes were found in avascular corneal tissue to those previously described in skin. The initial insult may be traumatic. We propose that a cytokine-mediated response contributes to the initial insult, either in response to and/or by accelerating severe inflammation. This precedes a cytotoxic infiltration which may exacerbate episodes of recurrent erosion. This provides a new insight into the mechanism of disease in the cornea following TEN.