Assessment of HOPE fixation in vitrectomy specimens in patients with chronic bilateral uveitis (masquerade syndrome)

Background: Cytological examination of rapidly transported, unfixed vitreous specimens is considered the gold standard in exclusion of primary intraocular lymphoma (PIOL) in patients with idiopathic steroid resistant chronic uveitis. These specimens are difficult to interpret, and reports of "false negatives" or "false positives" are common. Fixation using HOPE solution (Herpes-glutamic acid buffer mediated Organic solvent Protection Effect) has been successfully applied in the investigation of cytospin preparations using immunocytology, in situ hybridisation and polymerase chain reaction (PCR). The purpose of this study was to compare the cytologic features of vitrectomy specimens from patients with clinical reactive vitritis and PIOL prepared following HOPE fixation with those in conventional cytospins and to identify any diagnostic pitfalls. Methods: Pars plana vitrectomy was performed in 15 cases of patients with chronic uveitis. The vitreous samples were halved: one half was fixed in HOPE solution; the other half remained unfixed. All samples were subject to conventional staining, immunocytology and clonality analysis using polymerase chain reaction (IgH-PCR) and GeneScan. The specimens were assessed following by two pathologists who were masked to the cytological preparation method. The specimens were evaluated for cellularity, cellular appearance, cytoplasmic and nuclear features as well as quality of the immunostains. Results: Twelve of the fifteen vitreous samples were diagnosed as reactive vitritis; in three specimens, a primary intraocular lymphoma of B-cell type was diagnosed. Compared to the unfixed vitreal specimens, the quality of the cytomorphology and immunocytology improved in the HOPE-fixed specimens. IgH-PCR and GeneScan analysis demonstrated polyclonal amplification products in the reactive cases, and monoclonal B-cell populations in the B-PIOL. Conclusion: Our results demonstrate that cytomorphology and immunoreactivity of vitreous specimens are well preserved following HOPE fixation. DNA of sufficient quality could be extracted from HOPE-fixed vitreous biopsies, in order to perform clonality analyses. HOPE fixation appears to be promising in simplifying the transportation of these specimens, and may improve the diagnostic reliability of vitreous specimens in patients with masquerade syndrome.