Optimal fixation for total preanalytic phase evaluation in pathology laboratories. A comprehensive study including immunohistochemistry, DNA, and mRNA assays

Abstract

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. Key words: fixation, pathology, preanalytic phaseThe numerous components of laboratory test processes can be categorized into preanalytic, analytic, and postanalytic phases. Many laboratory tests using formalin-fixed and paraffin-embedded tissue samples in the preanalytic phase have been developed. Fixation is one of the most important steps in the preanalytic phase in pathological testing. Multiple factors including kinds of fixative, fixation duration, and acidity levels have been reported to have an effect on immunohistochemical results and gene amplification. For these reasons, a standardized and optimized fixation protocol for pathology laboratories would allow for more consistent and accurate data to be produced and enable better informed decisions based on the results. [1][2][3][4][5][6][7][8] However, the current recommendations for preanalytic phase testing vary depending on what organ, therapy, or procedure (such as protein, DNA, or RNA assay) is being used.9-18 Furthermore, most studies investigating the effects of fixation in laboratory testing have dealt with only one kind of laboratory test target. A standardized fixation protocol for pathology laboratories should be made available. We analyzed Ki-67 immunohistochemistry (IHC) testing and polymerase chain reaction (PCR)-based gene amplification using DNA and mRNA from one samples that was fixed by different protocols to clarify how variations in fixation influence postanalytic results in different assays. In regards to IHC testing, we also evaluated the effect of staining procedures, autostainer or manual staining, and different antibody clones, to search for the best way to reduce the effect of nonstandardized fixation protocols....

Tissue heterogeneity contributes to suboptimal precision of WHO 2010 scoring criteria for Ki67 labeling index in a subset of neuroendocrine neoplasms of the pancreascriteria for Ki67 labeling index in a subset of neuroendocrine neoplasms of the pancreas

“…In breast cancer, Ki67 scores are influenced by many pre-analytical and analytical factors, for example type of fixative, delay of fixation, prolonged fixation, cutting of the fresh tumour tissue aiming to improve fixation, storage of cut sections, methods of antigen retrieval, antibody clone, and counterstaining [39,57,60]. Significant inter-laboratory variability of Ki67 LI reporting was seen in breast cancer [53], but also in NEN [22].…”

Preanalytics and Precision Pathology: Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine

“…87,88 Tissue penetration is also profoundly affected by the pH of the formalin. 87,89 Fixatives other than formalin may be used electively for specific analyses, at the discretion of the pathologist, if appropriately validated, but should be recorded as a deviation from routine procedure in the pathology report. The use of acid decalcification, before or during the fixation process, results in hydrolysis of DNA and RNA and is contraindicated for molecular analyses of nucleic acids.…”

Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine. Part 3: Technical Validation of Immunohistochemistry (IHC) Assays in Clinical IHC Laboratories

“…(a) Preanalytical phase: While the preanalytical phase is difficult to control, it is reasonably well-defined and its importance is generally agreed upon. [44][45][46][47][48] There are numerous components in the preanalytical phase. 48 Of these, a subset can be identified for which changes would automatically trigger the need for primary revalidation of the IHC protocol.…”