HuBMAP | Formalin Fixation and Paraffin Embedding of Tissue Samples v1 (protocols.io.bqp6mvre)

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral ( Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents ( Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.

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Effect of formalin fixation on thermal conductivity of the biological tissue

Journal of Biomechanical Engineering ◽

10.1115/1.4026559 ◽

2014 ◽

Cited By ~ 1

Author(s):

Steven C. Bauserman ◽

Jonathan W. Valvano

Keyword(s):

Thermal Conductivity ◽

Biological Tissues ◽

Formalin Fixation ◽

Fatty Tissue ◽

Fresh Tissue ◽

Fixed Tissue ◽

Tissue Samples ◽

Formalin Fixed Tissue ◽

Formalin Fixed

Effect of formalin fixation on thermal conductivity of the biological tissues is presented. A self-heated thermistor probe was used to measure the tissue thermal conductivity. The thermal conductivity of muscle and fatty tissue samples was measured before the formalin fixation and then 27 hours after formalin fixation. The results indicate that the formalin fixation does not cause a significant change in the tissue thermal conductivity of muscle and fatty tissues. In the clinical setting, tissues removed surgically are often fixed in formalin for subsequent pathological analysis. These results suggest that, in terms of thermal properties, it is equally appropriate to perform in vitro studies in either fresh tissue or formalin-fixed tissue.

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Paraffin Embedding Tissue Samples for Sectioning

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot4989 ◽

2008 ◽

Vol 2008 (6) ◽

pp. pdb.prot4989-pdb.prot4989 ◽

Cited By ~ 9

Author(s):

A. H. Fischer ◽

K. A. Jacobson ◽

J. Rose ◽

R. Zeller

Keyword(s):

Tissue Samples ◽

Paraffin Embedding

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Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system

Journal of Clinical Pathology ◽

10.1136/jclinpath-2012-200983 ◽

2012 ◽

Vol 66 (2) ◽

pp. 124-135 ◽

Cited By ~ 29

Author(s):

Benedetta Belloni ◽

Chiara Lambertini ◽

Paolo Nuciforo ◽

Jay Phillips ◽

Eric Bruening ◽

...

Keyword(s):

Molecular Analysis ◽

Mutational Analysis ◽

Staining Intensity ◽

Formalin Fixation ◽

Pathology Laboratory ◽

Tissue Samples ◽

Dna And Rna ◽

Immunohistochemical Markers ◽

Fresh Frozen ◽

Ffpe Tissues

Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device.AimsIn this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples.Methods12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry, DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used.ResultsMorphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues.ConclusionsThe switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.

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Formalin Fixation of Human Healthy Autopsied Tissues: The Influence of Type of Tissue, Temperature and Incubation Time on the Quality of Isolated DNA

Serbian Journal of Experimental and Clinical Research ◽

10.2478/sjecr-2019-0020 ◽

2019 ◽

Vol 0 (0) ◽

Cited By ~ 1

Author(s):

Danijela Todorovic ◽

Katarina Vitosevic ◽

Milos Todorovic ◽

Zivana Slovic

Keyword(s):

Incubation Time ◽

Formalin Fixation ◽

Tissue Temperature ◽

Dna Molecules ◽

Healthy Human ◽

Tissue Samples ◽

The Impact ◽

The Brain ◽

Integrity Of Dna

Abstract Formalin fixation is a widely used method in histopathology that has certain limits. Formalin often leads to the degradation of DNA molecules in cancer tissues, which makes tissues unusable for molecular analysis. The other factors may also affect the quality of DNA isolated from fixed tissues. The aim of this study is to determine the impact of the incubation time and temperature on the quality of DNA molecules isolated from various healthy human tissues. The brain, lung and kidney tissues, excluded during the forensic autopsies of people who died of violent death, were fixed in phosphate-buffered formalin from 24h to two months. After the completion of the incubation period, the DNA was isolated using phenol-chloroform-isoamyl alcohol extraction method and the concentration and purity of the samples were determined spectrophotometrically. The degree of degradation of DNA was assessed by PCR reaction, by amplification of gene fragments which lengths were 150bp (GPD1) and 262bp (β-actin). The highest concentration, purity and preserved integrity of DNA were obtained from the brain samples. With prolonged tissue incubation times in formalin, the concentration and integrity of DNA decreased in all tissue samples, especially in the brain tissue, while the purity of DNA remained unchanged. Also, tissue fixation at +4°C contributed to a better quality of isolated DNA compared to DNA isolated from tissue fixed at room temperature. We can conclude that the type of human healthy tissue, temperature and the incubation time of formalin fixation have important influence on the concentration, purity and integrity of DNA during fixation of tissues excluded in the course of forensic autopsy.

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Long-term stability of RNA from FFPE brian tissue post-sectioning on slides.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e22142 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e22142-e22142

Author(s):

Donald James Witt ◽

Steven M. Anderson ◽

Briana King ◽

Christina Borrego ◽

Marcia Eisenberg ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Room Temperature ◽

Storage Period ◽

Ffpe Tissue ◽

Formalin Fixation ◽

Tissue Preparation ◽

Rna Integrity ◽

Paraffin Embedding ◽

Mutational Status

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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