Effects of Formalin Fixation, Paraffin Embedding, and Time of Storage on DNA Preservation in Brain Tissue: A BrainNet Europe Study

e22142 Background: Analysis of nucleic acids (NA) from formalin fixed paraffin embedded (FFPE) tissue can provide detailed information about gene sequence mutational status, which may be important for oncology treatment decisions. FFPE specimens also have utility for retrospective analyses. Potential degradation of NA during formalin fixation, paraffin embedding processes and possible continued deterioration during subsequent storage may diminish utility of FFPE tissue for these purposes. Using real-time PCR, this study investigated the functional stability of RNA from brain FFPE tissue sections on slides over an extended time period after sectioning. Methods: Brain biopsy specimens obtained from glioblastoma patients with informed consent were used to prepare blocks with standard formalin fixation and paraffin embedding techniques. Slides were made from the FFPE blocks and stored at room temperature until testing. RNA was extracted from sequential slides within one week of sectioning for a zero time and then at 4, 8 and 12 months. Reverse transcription PCR was performed, and real-time PCR was analyzed on the ABI7900 to detect EGFRvIII mutation and cABL gene. RNA Integrity Analysis was performed with an Agilent Bioanalyzer. Results: Consistent qualitative results were obtained with EGFRvIII mutant positive specimens (n =10) and wild type (wt) specimens (n =10) from slides stored up to twelve months at room temperature compared to the initial testing (95% agreement). One wt specimen showed negative results for the first three time points but a low positive result at 12 months, possibly due to tumor content change in the different sections of the FFPE block. Ct values for EGFR (wt and mutant) and cABL genes did not increase during the storage period. RNA integrity number (RIN) indicated the degradation of RNA during FFPE processing, although no further significant degradation occurred during the course of the experiment. Conclusions: The results of this study indicated that although the RNA was impacted by the tissue preparation, fixation, and processing steps, for the brain FFPE slide specimens, target genes with amplicon size up to 124bp could be detected with minimum degradation for up to 12 months when slides were stored at room temperature.

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Breast cancer and melanoma cell line identification by FTIR imaging after formalin-fixation and paraffin-embedding

The Analyst ◽

10.1039/c3an00246b ◽

2013 ◽

Vol 138 (14) ◽

pp. 4083 ◽

Cited By ~ 33

Author(s):

M. Verdonck ◽

N. Wald ◽

J. Janssis ◽

P. Yan ◽

C. Meyer ◽

...

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Melanoma Cell ◽

Melanoma Cell Line ◽

Formalin Fixation ◽

Ftir Imaging ◽

Paraffin Embedding ◽

Line Identification ◽

Cell Line Identification

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Chemical alterations to murine brain tissue induced by formalin fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis

The Analyst ◽

10.1039/c0an00269k ◽

2011 ◽

Vol 136 (14) ◽

pp. 2941 ◽

Cited By ~ 118

Author(s):

Mark J. Hackett ◽

James A. McQuillan ◽

Fatima El-Assaad ◽

Jade B. Aitken ◽

Aviva Levina ◽

...

Keyword(s):

Brain Tissue ◽

Formalin Fixation ◽

Disease Pathogenesis ◽

Murine Brain

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Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 1: Data acquisition, anatomical feature segmentation, tracking global volume and density changes

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2021.109354 ◽

2021 ◽

pp. 109354 ◽

Cited By ~ 1

Author(s):

Griffin Rodgers ◽

Willy Kuo ◽

Georg Schulz ◽

Mario Scheel ◽

Alexandra Migga ◽

...

Keyword(s):

Data Acquisition ◽

Mouse Brain ◽

Formalin Fixation ◽

Anatomical Feature ◽

Virtual Histology ◽

Paraffin Embedding ◽

Feature Segmentation

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Comparison of histomorphology and DNA preservation produced by fixatives in the veterinary diagnostic laboratory setting

10.7287/peerj.preprints.296v1 ◽

2014 ◽

Author(s):

Michael J. Dark ◽

William F. Craft ◽

Julia A. Conway

Keyword(s):

Dna Amplification ◽

The Other ◽

Formalin Fixation ◽

Laboratory Setting ◽

Diagnostic Laboratory ◽

Dna Preservation ◽

Diagnostic Practices ◽

Morphologic Characteristics

Histopathology is the most useful tool for diagnosis of a number of diseases, especially cancer. To be effective, histopathology requires that tissues be fixed prior to processing. Formalin is currently the most common histologic fixative, offering many advantages: it is cheap, readily available, and pathologists are routinely trained to examine tissues fixed in formalin. However, formalin fixation substantially degrades tissue DNA, hindering subsequent use in diagnostics and research. We therefore evaluated three alternative fixatives, TissueTek® Xpress® Molecular Fixative, modified methacarn, and PAXgene®, all of which have been proposed as formalin alternatives, to determine their suitability for routine use in a veterinary diagnostic laboratory. This was accomplished by examining the histomorphology of sections produced from fixed tissues as well as the ability to amplify fragments from extracted DNA. Tissues were sampled from two dogs and four cats, fixed for 24-48 hours, and processed routinely. While all fixatives produced acceptable histomorphology, formalin had significantly better morphologic characteristics than the other three fixatives. Alternative fixatives generally had better DNA amplification than formalin, although results varied somewhat depending on the tissue examined. While no fixative is yet ready to replace formalin, the alternative fixatives examined may be useful as adjuncts to formalin in diagnostic practices.

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Comparison of histomorphology and DNA preservation produced by fixatives in the veterinary diagnostic laboratory setting

10.7287/peerj.preprints.296 ◽

2014 ◽

Author(s):

Michael J. Dark ◽

William F. Craft ◽

Julia A. Conway

Keyword(s):

Dna Amplification ◽

The Other ◽

Formalin Fixation ◽

Laboratory Setting ◽

Diagnostic Laboratory ◽

Dna Preservation ◽

Diagnostic Practices ◽

Morphologic Characteristics

Histopathology is the most useful tool for diagnosis of a number of diseases, especially cancer. To be effective, histopathology requires that tissues be fixed prior to processing. Formalin is currently the most common histologic fixative, offering many advantages: it is cheap, readily available, and pathologists are routinely trained to examine tissues fixed in formalin. However, formalin fixation substantially degrades tissue DNA, hindering subsequent use in diagnostics and research. We therefore evaluated three alternative fixatives, TissueTek® Xpress® Molecular Fixative, modified methacarn, and PAXgene®, all of which have been proposed as formalin alternatives, to determine their suitability for routine use in a veterinary diagnostic laboratory. This was accomplished by examining the histomorphology of sections produced from fixed tissues as well as the ability to amplify fragments from extracted DNA. Tissues were sampled from two dogs and four cats, fixed for 24-48 hours, and processed routinely. While all fixatives produced acceptable histomorphology, formalin had significantly better morphologic characteristics than the other three fixatives. Alternative fixatives generally had better DNA amplification than formalin, although results varied somewhat depending on the tissue examined. While no fixative is yet ready to replace formalin, the alternative fixatives examined may be useful as adjuncts to formalin in diagnostic practices.

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Effects of Alzheimer’s disease and formalin fixation on the different mineralised-iron forms in the human brain

10.1101/2020.06.02.129593 ◽

2020 ◽

Cited By ~ 1

Author(s):

Louise van der Weerd ◽

Anton Lefering ◽

Andrew Webb ◽

Ramon Egli ◽

Lucia Bossoni

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Brain Tissue ◽

Formalin Fixation ◽

Magnetic Carriers ◽

Post Mortem Brain ◽

Brain Material ◽

Alzheimer’S Pathology ◽

The Brain ◽

Iron Forms

ABSTRACTIron accumulation in the brain is a phenomenon common to many neurodegenerative diseases, perhaps most notably Alzheimer’s disease (AD).We present here magnetic analyses of post-mortem brain tissue of patients who had severe Alzheimer’s disease, and compare the results with those from healthy controls. Isothermal remanent magnetization experiments were performed to assess the extent to which different magnetic carriers are affected by AD pathology and formalin fixation.While Alzheimer’s brain material did not show higher levels of magnetite/maghemite nanoparticles than corresponding controls, the ferrihydrite mineral, known to be found within the core of ferritin proteins and hemosiderin aggregates, almost doubled in concentration in patients with Alzheimer’s pathology, strengthening the conclusions of our previous studies. As part of this study, we also investigated the effects of sample preparation, by performing experiments on frozen tissue as well as tissue which had been fixed in formalin for a period of five months. Our results showed that the two different preparations did not critically affect the concentration of magnetic carriers in brain tissue, as observable by SQUID magnetometry.

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