Factors Influencing the Degradation of Archival Formalin-Fixed Paraffin-Embedded Tissue Sections

The loss of antigenicity in archival formalin-fixed paraffin-embedded (FFPE) tissue sections negatively affects both diagnostic histopathology and advanced molecular studies. The mechanisms underlying antigenicity loss in FFPE tissues remain unclear. The authors hypothesize that water is a crucial contributor to protein degradation and decrement of immunoreactivity in FFPE tissues. To test their hypothesis, they examined fixation time, processing time, and humidity of storage environment on protein integrity and antigenicity by immunohistochemistry, Western blotting, and protein extraction. This study revealed that inadequate tissue processing, resulting in retention of endogenous water in tissue sections, results in antigen degradation. Exposure to high humidity during storage results in significant protein degradation and reduced immunoreactivity, and the effects of storage humidity are temperature dependent. Slides stored under vacuum with desiccant do not protect against the effects of residual water from inadequate tissue processing. These results support that the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Optimal tissue processing is essential. The parameters of optimal storage of unstained slides remain to be defined, as they are directly affected by preanalytic variables. Nevertheless, minimization of exposure to water is required for antigen preservation in FFPE tissue sections. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Novel zinc-based fixative for high quality DNA, RNA and protein analysis

We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT–PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4kb in length and RNA fragments up to 361bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT–PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6–14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.