Bladder biopsies were obtained from 63 spinal cord injury patients after obtaining written informed consent. The North Sefton Local Research Ethics Committee approved this study. All were adults with spinal cord injury and neuropathic bladder. They were registered with the Regional Spinal Injuries Centre, Southport, England; were not suffering from acute urinary infection and were undergoing an elective therapeutic procedure in the urinary tract such as endoscopic lithotripsy of bladder stone, insertion of a ureteric stent, or diagnostic cystoscopy.
After routine prophylactic intravenous gentamicin, cold cup biopsies of the bladder mucosa were taken from the trigone of the urinary bladder. Thereafter, the biopsy site was fulgurated with diathermy to achieve haemostasis. Indwelling urinary catheter drainage was maintained after the procedure, and all patients remained in hospital for at least 24 hours. Biopsies were fixed in neutral buffered formaldehyde 4%, and then embedded in Paraplast. (Paraplast is a commercial paraffin wax incorporating plasticisers, used for embedding formalin-fixed tissues for histology.)
A Consultant Cellular Pathologist recorded the histopathological features of each bladder biopsy after examining sections stained with haematoxylin and eosin. None of the 63 biopsies from spinal cord injury patients showed evidence of dysplasia or neoplasia. The classical morphological features of dysplasia in the urothelium comprise: increased nucleo-cytoplasmic ratio, nuclear hyperchromatism, abnormal chromatin pattern, prominent nucleoli, and nuclear pleomorphism; and loss of normal polarity leading to a haphazard spatial arrangement of urothelial cells. The affected urothelium is, by definition, flat rather than papillary. In the most severe form of dysplasia (carcinoma-in-situ), the above nuclear characteristics are present to a marked degree, comparable in severity to those in a grade 3 Transitional Cell Carcinoma. Lesser degrees are termed 'dysplasia not amounting to carcinoma-in-situ'. At the mild end of the spectrum, distinguishing dysplasia from inflammatory or regenerative atypia can be difficult; the abnormal chromatin pattern in dysplasia is usually the most reliable criterion.
In two biopsies, epithelium was scarce. Eight biopsies showed squamous metaplasia with no transitional epithelium present. Umbrella cells were identifiable in the remaining 53 biopsies at least in one microscopic field. Umbrella cells were identified by their morphological characteristics, namely: large, elongated cells with abundant eosinophilic cytoplasm, their long axes parallel to the basement membrane, covering several underlying intermediate urothelial cells. The umbrella cells may occasionally be binucleate.
Immunostaining for cytokeratin 20 was carried out on tissue sections in one lot using a mouse monoclonal antibody, clone Ks20.8 (Novocastra Laboratories Ltd, Balliol Business Park West, Benton Lane, Newcastle upon Tyne NE12 8EW, UK). This is an IgG antibody (IgG2a, kappa), with specificity for human cytokeratin 20 intermediate filament protein, raised against cytoskeletal preparations isolated from microdissected villi of human duodenal mucosa. The normal positive staining pattern for cytokeratin 20 is cytoplasmic. The manufacturer's instructions were followed for immuno-staining. Sections were pre-treated with proteinase-K, to unmask cytokeratin epitopes masked by the cross-linking effect of formaldehyde during fixation and tissue processing. The antibody was used at a working dilution of 1:25. Immunoperoxidase technique was employed, with primary antibody incubation at 25°C for 60 minutes. Each sample included a negative control, by omitting the primary antibody. Avidin Biotin Complex (ABC) kit was purchased from Vector Laboratories. The version of the kit used was Elite ABC.
The presence of immunostaining in the vesical epithelium was recorded either as negative or positive, and the location of any positively staining cells within the transitional epithelium was noted.