Coupland Sarah E, Perez-Canto Alexandra, Hummel Michael, Stein Harald, Heimann Heinrich
Department of Pathology and Haematopathology Reference Centre, Charité-Medical Faculty Berlin, Campus Benjamin Franklin, Berlin, Germany.
Graefes Arch Clin Exp Ophthalmol. 2005 Sep;243(9):847-52. doi: 10.1007/s00417-005-1166-1. Epub 2005 May 21.
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.
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.
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.
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.
对于特发性类固醇抵抗性慢性葡萄膜炎患者，快速转运的未固定玻璃体标本的细胞学检查被认为是排除原发性眼内淋巴瘤（PIOL）的金标准。这些标本难以解读，“假阴性”或“假阳性”报告很常见。使用HOPE溶液（疱疹 - 谷氨酸缓冲介导的有机溶剂保护效应）固定已成功应用于细胞涂片制备的免疫细胞学、原位杂交和聚合酶链反应（PCR）研究。本研究的目的是比较HOPE固定后临床反应性玻璃体炎和PIOL患者玻璃体切除标本与传统细胞涂片的细胞学特征，并识别任何诊断陷阱。
对15例慢性葡萄膜炎患者进行了玻璃体切除术。将玻璃体样本一分为二：一半固定在HOPE溶液中；另一半保持未固定。所有样本均进行常规染色、免疫细胞学检查以及使用聚合酶链反应（IgH-PCR）和基因扫描进行克隆性分析。由两位对细胞学制备方法不知情的病理学家对标本进行评估。评估标本的细胞数量、细胞外观、细胞质和细胞核特征以及免疫染色质量。
15个玻璃体样本中有12个被诊断为反应性玻璃体炎；在3个标本中，诊断为B细胞型原发性眼内淋巴瘤。与未固定的玻璃体标本相比，HOPE固定标本的细胞形态学和免疫细胞学质量有所改善。IgH-PCR和基因扫描分析显示反应性病例中有多克隆扩增产物，而B-PIOL中有单克隆B细胞群体。
我们的结果表明，HOPE固定后玻璃体标本的细胞形态学和免疫反应性得到了很好的保存。可以从HOPE固定的玻璃体活检标本中提取质量足够的DNA，以进行克隆性分析。HOPE固定在简化这些标本的运输方面似乎很有前景，并且可能提高伪装综合征患者玻璃体标本的诊断可靠性。
