Application of alternative fixatives to formalin in diagnostic pathology

Fixation is a critical step in the preparation of tissues for histopathology. The aim of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine stainings), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved to be the best fixative for morphology. The morphology obtained with Bouin was comparable to the one with formalin. Hollande was the best fixative for histochemistry. Bouin proved to be equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved the possibility to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis

Periodontal Conditions of Sites Treated With Gingival Augmentation Surgery Compared With Untreated Contralateral Homologous Sites: An 18- to 35-Year Long-Term Study

BACKGROUND: The aim of this split-mouth study is to compare long-term (18 to 35 years) periodontal conditions of sites treated with gingival augmentation procedures (GAPs) and untreated homologous contralateral sites. METHODS: Forty-seven patients with 64 sites (test group), with lack of attached gingiva associated with recessions, were treated with marginal or submarginal free gingival grafts. Sixty-four contralateral homologous sites (control group), with or without gingival recession (GR) and with attached gingiva, were left untreated. Patients were recalled every 4 to 6 months during follow-up period. GR depth, keratinized tissue (KT) width, and probing depth were measured at baseline (T0), 1 year after surgery (T1), during follow-up (10 to 27 years, T2), and at the end of the follow-up period (18 to 35 years, T3). Multilevel and regression analyses were conducted. RESULTS: At the end of T3, 83% of the 64 treated sites showed recession reduction (RecRed), whereas 48% of the 64 untreated sites experienced increase in recession. Treated sites ended with gingival margin (GM) 1.7 mm (P = 0.01) more coronal and KT 3.3 mm (P <0.001) wider than untreated sites. In grafted sites, KT at T3 remained stable compared with T1 value (4.1 mm, P <0.001). CONCLUSIONS: Sites treated with GAPs resulted in coronal displacement of GM with RecRed up to complete root coverage, whereas contralateral untreated sites showed a tendency to increase in existing recession or develop new recession during the 18- to 35-year follow-up