A novel zinc based fixative in tissue banking for molecular biological research
Cardiff Metropolitan University
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Fixation is a succession of complex chemical modifications affecting the macromolecules in cells preserving morphology, nucleic acids, proteins and other cell components. There are a wide range of fixatives available, however none are ideal for preserving both morphology and the integrity of DNA, RNA and protein. Combination of the preservation of intact nucleic acids combined with preservation of morphology in paraffin embedded tissue has proved to be extremely difficult. The solution to this problem could improve the development of molecular testing in the routine histopathology laboratory. The aim of this study was to compare two different fixatives, one commercial, FineFIX (Milestone SrL, Bergamo, Italy) and one non-commercial, Z7 (Lykidis D et al., 2007) with respect to their ability to preserve morphology when embedded into paraffin and to maintain the integrity of RNA and DNA. Snap frozen tissue samples were taken as the gold standard for maintenance of nucleic acid integrity and samples fixed in neutral buffered formalin (NBF) and processed to paraffin as the gold standard for morphology. The effects of these fixatives were investigated using 22 human tumour tissue samples (breast, kidney and pancreas) obtained from the Wales Cancer Bank. Results were obtained from H&E staining, immunohistochemistry (IHC), RNA Agilent bioanalyser, DNA gel electrophoresis and multiplex polymerase chain reaction (PCR). One-way analysis of variance (ANOVA) and t-tests showed that NBF was significantly better at preserving morphology and IHC, Z7 was better at preserving RNA integrity, however only marginally and although FineFIX performed better during the DNA gel electrophoresis the multiplex PCR results showed that quality of DNA extracted was not significantly different between the three fixatives. In conclusion, neither Z7, the novel zinc based fixative, nor FineFIX could be applied in the routine laboratory. Although FineFIX was the better fixative at preserving DNA integrity, it is still not as good as DNA retrieved from fresh frozen tissue and is therefore unlikely to replace fresh frozen samples for molecular biological techniques.
MSc Biomedical Science (Cellular Pathology)
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