Comparing DNA damage caused by formaldehyde, glutaraldehye [sic], Carnoy's and Methacarn in cancer tissue fixations

by Tsai, Chia-Jui.

Scott O. Rogers, Advisor Many molecular biotechniques are useful in detecting macromolecular changes. Disease stages may be analyzed accurately based on these molecular changes. Therefore, molecular methods have the potential to be applied as routine and highly informative clinical diagnostic tools. Fixation methods become an essential consideration while preserving tissues. This study investigates the frequency of DNA change caused by two aldehyde-based fixatives and two alcohol-chloroform-acetic acid fixatives. Aldehyde-based fixatives, especially 10% neutral buffered formalin (NBF) and 1% glutaraldehyde, are widely used in histological studies. Among these two fixatives, 1% glutaraldehyde preserves cellular structure better than formalin, and recent studies indicate that it preserves DNA better than formalin. In addition to the aldehyde-based fixatives, Carnoy’s, which is made with ethanol (60%), chloroform (30%) and acetic acid (10%), and Methacarn, which shares the same formula but uses methanol instead of ethanol were tested DNA changes for each fixation were compared this study. Human tissues (normal white blood cells, sarcomas, leukemias, and carcinomas) were fixed, separately using 10% NBF, 1% glutaraldehyde, Carnoy’s and Methacarn. DNA from the fixed tissues was extracted and segments (418 bp and 597 bp) of the nuclear small subunit ribosomal DNA (SSU rDNA) were amplified by polymerase chain reaction (PCR) and sequenced. The DNA sequences were analyzed by comparing the DNA changes between four iii fixative-treated and unfixed tissues. According to the results of the DNA statistical analyses of sequence alignments, we report that 10% NBF causes more DNA change than the other fixatives. Alcohol-chloroform-acetic acid-based fixatives, in general, caused less DNA changes and maintained the DNA integrity better than aldehyde-based fixatives. iv