title: Effective application of the methanol-based PreservCyt(™) fixative and the Cellient(™) automated cell block processor to diagnostic cytopathology, immunocytochemistry, and molecular biology.
abstract: We studied the feasibility of immunocytochemistry (ICC), in situ hybridization (ISH), and polymerase chain reaction (PCR) after Cellient(™) automated cell block processing, and tested whether methanol-based PreservCyt(™) fixation could replace formalin fixation, in an attempt to eliminate toxic formaldehyde vapors. Immunostaining with 30 different antibodies was performed on cell blocks from 73 FNA specimens and 42 body cavity fluid specimens prepared by Cellient(™) automated processing that uses the methanol-based fixative (PreservCyt(™) ). For each antibody we evaluated ICC in at least three different cell block specimens and compared it with immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) histological sections from the corresponding tumors. The quality of DNA and RNA in Cellient(™) blocks was analyzed by ISH, applying a SYT gene break-apart assay and EBER probes, respectively. Moreover, DNA quality was analyzed by PCR by using primer sets for DNA products of 100, 200, 300, 400, 500, and 600 base pairs, and evaluated by gel electrophoresis. When compared with IHC results in corresponding FFPE tumor tissue from the same patient, 24 out of 30 antibodies showed concordant ICC results. With FISH, distinctive hybridization signals were observed for SYT DNA sequences and EB virus RNA sequences. With PCR, DNA products, up to 600 base pairs in size, were readily observed after gel electrophoresis. The antibodies that showed concordant immunostaining in Cellient(™) blocks could be applied to diagnostic algorithms that proved to be helpful in the discrimination of major tumor types (carcinoma, lymphoma, melanoma, and germ cell tumors), discrimination of carcinoma subtypes, and determination of primary tumor site in cases of metastatic carcinoma. In a separate study, we found that the application of ICC to this cell block technique provided additional diagnostic and clinically important information in 24% of 100 consecutive cases. The high quality of DNA and RNA in Cellient(™) cell blocks allowed sensitive and specific molecular biologic analysis, in particular FISH and PCR.
authors: Department of Pathology and Medical Biology, Cytology Laboratory, University Medical Center Groningen, University of Groningen, The Netherlands.
authors: Bettien M
authors: BM
authors: van Hemel
authors: Albert J H
authors: AJ
authors: Suurmeijer
mesh_terms: D003581:Cytodiagnosis; D005240:Feasibility Studies; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D000432:Methanol / Q000737:chemistry*; D025202:Molecular Diagnostic Techniques; D009369:Neoplasms / Q000175:diagnosis / Q000473:pathology*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005404:Fixatives; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C093961:SS18 protein, human; D005557:Formaldehyde; D000432:Methanol
keywords: Cellient; MOC31; cell block; cytopathology; immunocytochemistry; in situ hybridization; polymerase chain reaction