Optimal fixation for total preanalytic phase evaluation in pathology laboratories: a comprehensive study including...

The purpose of this study was to set the optimal preanalytical fixation protocol to enhance analytical and postanalytical phase accuracy and consistency. Twenty-five normal colorectal tissues were fixed using various formalin concentrations, pHs, and fixation periods. All specimens were embedded in paraffin and 4 μm sections were used for immunohistochemistry of Ki-67, and extraction and amplification of DNA and RNA. The Ki-67 labeling index and the successful gene amplification rate for DNA and mRNA were evaluated and compared among variously fixed tissue samples. Ki-67 positivity was enhanced by low pH and short fixation time, and was influenced by the type of antibody, but not by the staining (with or without using an autostainer) method. DNA amplification by PCR was strongly influenced by pH of formalin. cDNA amplification could be accomplished only with the shortest PCR fragment of 142 bp, and longer fixation times impaired the amplification. These data suggest that multiple different factors influence immunohistochemical results and gene amplification using DNA and mRNA. We recommend, based on data from this comprehensive analysis, a 10% neutral buffered formalin and fixation times of no longer than 1 week to produce consistent immunohistochemical slides and DNA amplification within 500 bp in pathology laboratories.