Fixation In Neutral Buffered Formalin Research Articles

A comparison of fixation and immunofluorescence protocols for successful reproducibility and improved signal in human left ventricle cardiac tissue.

Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh-frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised.

Glyoxal: a proposed substitute for formalin in H&E and special stains

ABSTRACT Neutral-buffered formalin (NBF) has been used as the primary fixative in anatomic pathology laboratories for decades. Although it yields excellent morphologic and staining results, NBF poses significant health hazards requiring tissue to be grossed under a grossing/chemical fume hood. Glyoxal fixatives offer far less toxic alternatives and do not necessitate use of a grossing hood. Using freshly extracted canine and feline testes, ovaries, and uteri, the effects of glyoxal and NBF fixation were compared. While NBF is still considered the gold standard, some glyoxal fixatives perform as well as NBF in regards to morphology, H&E staining properties, and histochemical staining properties.

Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples

Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts. We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies. Using matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data. We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation.

Determination of Optimum Formalin Fixation Duration for Prostate Needle Biopsies for Immunohistochemistry and Quantum Dot FISH Analysis.

Prostate biopsy is the key clinical specimen for disease diagnosis. However, various conditions used during biopsy processing for histologic analysis may affect the performance of diagnostic tests, such as hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), or in situ hybridization (ISH). One such condition that may affect diagnostic test performance is fixation duration in 10% neutral buffered formalin (NBF). For example, prostate needle biopsies are often <1 mm in diameter and thus overfixed. It is important to understand the impact of tissue fixation duration on diagnostic test performance to enable optimized assay procedures. This study was designed to study the effect of 10% NBF fixation duration of prostate needle biopsy on multiplexed quantum dot (QD) ISH assay of ERG and PTEN, 2 genes commonly altered in prostate cancer. The samples were also evaluated for H&E staining and ERG and PTEN IHC. H&E staining and ERG and PTEN IHC were acceptable for all the durations of fixation tested. For QD ISH, we observed good signals with biopsy samples fixed from 4 to 120 hours. Biopsy specimens fixed between 8 and 72 hours gave the best signal as scored by the study pathologist. In a separate cohort of 18 routinely processed prostate biopsy cores, all cores were stained successfully with the QD ISH assay, and results were 100% concordant to ERG and PTEN IHC. We conclude that 8 to 72 hours duration of fixation for prostate needle biopsies in 10% NBF results in optimal QD ISH assay performance.

Histochemical and immunohistochemical evaluation of mouse skin histology: comparison of fixation with neutral buffered formalin and alcoholic formalin

Histological analysis is a cornerstone of skin disease diagnosis, and the stratified nature of the skin presents many technical problems in the preparation of sections of acceptable quality. Furthermore, histological artifacts may be exacerbated in pathological states that compromise cutaneous integrity. Therefore, great care must be taken when selecting the conditions, particularly fixative type, to ensure that meaningful conclusions may be drawn. The goal of this work was to compare the morphology of mouse skin as revealed using a range of histological stains, and the antigenicity of key cutaneous proteins using immunohistochemistry (IHC) following fixation in either 10% neutral buffered formalin (NBF) or alcoholic formalin (AF). Although 10% NBF fixation is routinely used for histology, providing good retention of morphology and structure for special staining, AF fixation more effectively maintained antigenicity for the cutaneous markers investigated in this study. A comprehensive assessment of the two fixatives and histological techniques to optimize morphology, structure, and antigenicity in the analysis of mouse skin is presented.

Abstract 1265: Tissue is alive: Preserving phosphoproteins and tissue morphology in clinical trial samples

Abstract An urgent clinical goal is to identify subpopulations of cancer patients that may respond to targeted kinase inhibitors and/or their phosphorylated substrates. Consequently, phosphorylated signal pathway proteins have emerged as a critical class of analytes for therapeutic stratification. Technologies now exist that can measure protein phosphorylations and map cell signaling pathways in a single core-needle biopsy, thus relying on the inhibition of any kinase/phosphatase activity within the sample immediately following excision. Unfortunately applying these technologies to clinical samples has been hindered because phosphoprotein epitopes are not adequately preserved by formalin fixation and paraffin embedding, while freezing tissue samples may not be feasible in multi-center clinical trial sites and cannot adequately preserve morphology. To facilitate clinical trial molecular profiling, where immediate snap-freezing of tumor biopsies is not feasible, we have created a novel, one-step, room temperature preservative that stabilizes proteins/phosphoproteins equivalent to snap-freezing and tissue/cell morphology equivalent to neutral buffered formalin fixation. In addition, our preservative solution simultaneously fixes and decalcifies bony tissue, thus permitting molecular profiling of bony tissues that was never before possible. We have utilized our Biomarker and Histology Preservative (BHP-Cell and/or BHP-Tissue) in a breast cancer multi-site clinical trial (US Oncology 05-074/GSK LPT109096) and a clinical research multiple myeloma trial. BHP has been validated to a) function as a transport medium while preserving histomorphology, b) maintain full antigenicity for clinical immunohistochemistry (such as Ki-67, ER, PR, Her2, p63, and phosphorylated epitopes), c) preserve phosphoprotein epitopes for cell signaling pathway profiling by reverse phase protein microarray (RPMA), d) be compatible with frozen sections or paraffin embedding, and e) obviate the need for additional decalcification. Preservation of tissue morphology has been demonstrated in 25 mouse, feline, and human tissues with enhanced immunohistochemical properties for &amp;gt;20 antigens using standard IHC protocols. RPMA data from LPT10906 shows differentially deranged signaling networks in the pre-treatment biopsies for patients that did not have a pathologic complete response compared to responders. Our multiple myeloma trial quantifies cell signaling pathway expression pre/post kinase inhibitor treatment, in an ex vivo treatment model, on both myeloma and bone marrow cells, preserved in BHP. We are currently evaluating the integrity of nucleic acid preservation in parallel with proteins to provide a universal one step biospecimen tissue fixative for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1265. doi:1538-7445.AM2012-1265

Influence of Preservation in Two Kinds of Formaldehyde Solutions on the Fracture Characteristics of Bovine Femoral Compact Bones

For the evaluation of the physical properties of bones, specimens are often either deep-frozen or chemically fixed with reagents such as formaldehyde (formalin) or ethanol for antisepsis and sterilization. However, formalin contains formic acid, which dissolves bone minerals such as Ca and P. To suppress this bone mineral elution, we tested neutral buffered formalin as a fixative. In this study, we investigated the influence of formalin and the neutral buffered formalin fixation on the fracture characteristics of bovine femoral compact bones over a relatively long-term preservation period. In both the formalin and the neutral buffered formalin fixation, bone minerals migrated rapidly into the fixatives. Thus, some aqueous solutions such as a normal saline solution may also dissolve bone minerals. In the neutral buffered formalin fixation, fine calcium phosphate grains precipitated on the surface of the soft tissues of blood vessels in the Haversian canal, as observed by scanning electron microscopy. An element analysis with energy-dispersive X-ray spectroscopy also demonstrated the presence of Ca and P. Thus, the precipitated grains are assumed to be hydroxyapatite. In this study we evaluated the influence of formalin preservation, which reduces the fracture toughness of bovine femoral compact bones, and concluded that due to the formation of chemical bridges from the reaction of formaldehyde with the collagen fibers in the bones, the collagen fibers are cured and hardened, resulting in a reduction in the fracture toughness of the bovine femoral compact bones.

ウシ大腿皮質骨の破壊特性に及ぼすホルマリン保存液の影響

To evaluate the physical properties of a bone, a specimen is often deep-frozen or chemically fixed with reagents such as formaldehyde solution (formalin) or ethanol for antisepsis and sterilization. However, formalin contains formic acid and dissolves bone minerals such as Ca and P into the fixative. To suppress bone mineral solution, we used neutral buffered formalin as the fixative solution. In this study, we investigated the effect of formalin and of neutral buffered formalin fixation on the fracture characteristics of bovine femoral compact bone during relatively long-term preservation. With both formalin and neutral buffered formalin fixation, bone mineral migrates rapidly into the fixative solution. Thus, formic acid is not solely responsible for the dissolution of bone minerals, but some aqueous solutions such as saline can also dissolve bone mineral. With neutral buffered formalin fixation, small calcium phosphate grains precipitate at the surface of blood vessels in the Haversian canal, as observed by scanning electron microscopy. An energy-dispersive spectroscopy also demonstrates the presence of Ca and P. Thus, the precipitated grains are assumed to be hydroxyapatite. In this study we evaluate the effect of formalin preservation, which is to reduce the fracture toughness of bovine femoral compact bone, and to form chemical bridges by the reaction of formaldehyde with the collagen fiber of bone. The collagen fiber was cured and hardened, resulting in a reduction in the fracture toughness of bovine femoral compact bone.

Zinc-Based Fixative Improves Preservation of Genomic DNA and Proteins in Histoprocessing of Human Tissues

Advantageous preservation of histology and detailed cellular morphology has rendered neutral buffered formalin (NBF) the most widely used fixative in clinical pathology. Despite excellent morphology for routine diagnostics, a major drawback of NBF fixation is its detrimental effect on DNA and RNA quality. In addition to complicating analysis of genes and transcripts in complex tissues, NBF denatures proteins and thereby hampers immunohistochemical visualization of certain antigens. In the present study, we evaluated a zinc-based fixative (ZBF) regarding its effects on tissue morphology, quality of genomic DNA, and preservation of protein immunoreactivity in a broad spectrum of tissues. Four different modes of fixation were analyzed: ZBF-paraffin embedding, NBF-paraffin embedding, ZBF-fixation prior to snap-freezing, and immediate snap-freezing. Laser-assisted microdissection, allowing retrieval of a defined number of cells for PCR, was used to study DNA quality. Genomic DNA was analyzed using primers for β2-microglobulin and the transferrin receptor. Immunohistochemistry was performed using nine antibodies. Tissue microarray blocks were used for analysis of morphology and immunoreactivity. Only slight impairment of morphologic qualities was found after ZBF-paraffin embedding, whereas ZBF prior to freezing resulted in a more crisp morphology compared with routine cryosections. A significantly higher DNA yield was observed in samples isolated from ZBF-paraffin–embedded tissues compared with NBF-paraffin–embedded tissues. Both yield and quality of DNA was comparable in frozen tissues irrespective to prior ZBF fixation. Immunoreactivity in paraffin-embedded tissue was superior in ZBF-fixated tissue compared with NBF-fixated for a majority of tested antibodies. Furthermore, for seven out of nine antibodies, antigen retrieval pretreatment proved unnecessary in ZBF-fixated tissue. Thus, despite a slight impairment of morphology, ZBF preserves protein structures well. We conclude that ZBF is superior to NBF for analysis of DNA and protein expression. Fixation of tissues in ZBF may also be an alternative strategy to freeze storage of tissue specimens, eg, in future bio-banks.

Duration and method of fixation affects the sensitivity of a digoxygenin-labelled DNA probe in detecting Kudoa thyrsites in Atlantic salmon skeletal muscle

Abstract The effect of fixative and duration of fixation on the sensitivity of a non-radioactive in situ hybridisation (ISH) protocol to detect Kudoa thyrsites small subunit ribosomal deoxyribonucleic acid (DNA) was investigated. Strong ISH reactions were detected in 5-μm sections of paraffin-embedded Atlantic salmon muscle after fixation for 1 day in Davidson's solution (DS). Reactions were weak following 3 or 5 days fixation and absent after 17 or 28 days fixation. Strong ISH reactions were observed after 1, 3 or 5 days fixation in neutral buffered formalin (NBF). The reactions were weak after 17 days and weak to nonexistent after 28 days of fixation. Reactions were consistently strong after fixation in 95% ethanol for up to 28 days. Some mature spores reacted weakly or not at all by ISH. Parasite DNA was weakly amplified by polymerase chain reaction (PCR) from paraffin-embedded muscle after 1 day of fixation in DS but not after fixation for 3, 5, 17 or 28 days. Amplified DNA was detected after fixation in NBF for 1, 3 and 5 days, but not after 17 or 28 days. In contrast, PCR consistently amplified DNA from paraffin-embedded, ethanol-fixed muscle. Caution should be used in the choice of fixative and duration of fixation when preserving Atlantic salmon tissues for molecular diagnosis of K. thyrsites .

KP1 Immunohistochemical Demonstration of Macrophages in Colorectal Tissues: Effects of Different Fixatives and Different Fixation Times

AbstractThe effects of 10 fixatives and formalin fixation time were assessed on the immunoreactivity of KP1 (CD68 antigens) in colorectal tissues in paraffin embedded sections. A panel of fixatives including B5, periodate-lysine-paraformaldehydedichromate, periodate-lysine-paraformaldehyde, 4% paraformaldehyde (PF), zinc formalin, formol dichromate, 10% neutral buffered formalin (NBF), Bouin fluid, Carnoy fluid, and acetone were used to determine an optimal fixative. Another set of colorectal tissues were fixed in 10% NBF progressively at intervals of 1,2,4, and 20 hr; 2,7, and 14 days; and 4 wk to investigate the formalin-induced detrimental effect on CD68 antigens of macrophages.The best result was found after fixation in B5. Short fixation in NBF for 4 hr with trypsinization also gave satisfactory results. There was a distinct decrease in staining intensity after 20 hr exposure to NBF, and the reactivities were totally abolished after 2 days. Trypsinization did enhance the staining intensity significan...

A histological comparison of phase-partition fixation with fixation in aqueous solutions.

In phase-partition fixation, tissue is immersed in a non-aqueous solvent at equilibrium with an aqueous solution of a fixing agent to minimize osmotic effects. Preservation of morphology afforded by phase-partition fixation using formalin and glutaraldehyde and several organic solvents was compared to aqueous 10% neutral buffered formalin fixation for five tissues. It was shown that phase-partition fixation can provide excellent fixation for light microscopy if the proper combinations of fixatives and solvents are used.

A Simple Method for Histochemical Demonstration of Viral Inclusion Bodies in Cell Monolayers in Microtitration Plates

The present paper describes a Bouin's-formalin fixation and Giemsa staining procedure for demonstrating viral cytoplasmic inclusion bodies in cellular monolayers in microtitration plates. Monolayers are fixed in Bouin's fixative for 15 min followed by 10% neutral buffered formalin fixation overnight. After fixation, the monolayers are stained with 4% (v/v) Giemsa stain. The method is superior to the separate use of formalin, methanol or Bouin's fixation-staining methods and compares favorably to immunocytochemical techniques for sensitivity.

THE DEMONSTRATION AND DISTRIBUTION OF WATER SOLUBLE BLOOD GROUP O (H) ANTIGEN IN TISSUE SECTIONS USING A FLUORESCEIN LABELLED EXTRACT OF ULEX EUROPEUS SEED.

A fluorescein labelled extract of Ulex europeus seed was found to be satisfactory for demonstrating water soluble H antigen in formalin fixed paraffin embedded tissue sections. Commercially available anti-A typing serum was labelled with fluorescein and used to demonstrate water soluble A antigen in formalin fixed paraffin embedded tissues. The reactivity of A and H antigen after neutral buffered formalin fixation was superior to that observed after Zenker's, Helly's or Bouin's fixation; reactivity in paraffin sections of formalin fixed tissues were equal to that of fresh frozen sections. Autolysis did not prevent the demonstration of blood group antigen A or H in fixed paraffin embedded tissues but was associated with some diffusion of the antigens. The distribution of H antigen in formalin fixed paraffin embedded tissue section for patients with type A, B and O blood also was studied. In secretors blood group substances were widely distributed in the epithelial mucins of the body whereas in nonsecretors the antigens are largely confined to the deep portion of the pyloric mucosa of the stomach and Brunner's glands. The distribution of A and H antigen within a gland, such as Brunner's glands, was not homogeneous. That is, some acini contained A antigen but no H antigen; others contained H antigen but no A antigen: a few contained both A and H antigen. The significance of this mosaic distribution which previously has not been described was discussed.