Advanced Concepts in Fixation: 1. Effects of Fixation on Immunohistochemistry, Reversibility of Fixation and Recovery of Proteins, Nucleic Acids, and other Molecules from Fixed and Processed Tissues. 2. Developmental Methods of Fixation

Eltoum, Isam A.; Fredenburgh, Jerry; Grizzle, William E.

doi:10.1179/014788801794812462

Cited by 17 publications

(25 citation statements)

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“…Formalin fixation produces methylene bridges between free amino groups and other functional groups of proteins, resulting in conformational changes in the tertiary and quaternary structures within proteins and cross-linking between tissue proteins, thus modifying the epitopes recognized by antibodies. 3,15,21,22,43,71 However, it needs to be emphasized that adequate fixation is essential in IHC. Underfixation of tissues for IHC testing is one of the major problems in human pathology.…”

Section: Fixationmentioning

confidence: 99%

Suggested Guidelines for Immunohistochemical Techniques in Veterinary Diagnostic Laboratories

et al. 2008

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Abstract. This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

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Section: Fixationmentioning

confidence: 99%

Suggested Guidelines for Immunohistochemical Techniques in Veterinary Diagnostic Laboratories

et al. 2008

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“…It also interacts with nucleic acids but has little effect on carbohydrates. 91,194 It is a good preservative of lipids if the fixative contains calcium. 176 The Chemistry of Formalin Fixation.…”

Section: Fixationmentioning

confidence: 99%

“…108 The deleterious effects of formaldehyde on immunoreactivity in cells and tissues can be partially reversed, 92 whereas glutaraldehyde fixation is considered irreversible. 91 Prolonged washing of fixed tissues reduces further fixation by removing unbound formaldehyde, 142 although established cross-links remain. 92 Long-term storage of formalin-fixed tissues in alcohol halts the formation of cross-links and, therefore, facilitates antigen detection should the tissues be needed subsequently for IHC.…”

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confidence: 99%

When Tissue Antigens and Antibodies Get Along

2013

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Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.

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“…Formalin fixation is a time-dependent process in which increased fixation time results in continued formaldehyde group binding to proteins to a point of equilibrium (Fox et al 1985). Studies have shown that formalin fixation, especially if prolonged, results in decreased antigenicity (Battifora and Kopinski 1986;Arnold et al 1996; Shi et al 1998), which limits the use of formalinfixed tissues for diagnostic IHC (Arnold et al 1996;Eltoum et al 2001a;Ramos-Vara 2005). Enzymatic digestion and heat-induced epitope retrieval (HIER) protocols have improved antigen detection following prolonged fixation (Battifora and Kopinski 1986;Shi et al 1998;Boenisch 2005); however, studies evaluating antigen detection after prolonged formalin fixation have been limited.…”

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Effects of Prolonged Formalin Fixation on Diagnostic Immunohistochemistry in Domestic Animals

Webster

Miller

DuSold

et al. 2009

J Histochem Cytochem.

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S U M M A R Y Immunohistochemistry (IHC) is routinely used in diagnostic pathology to detect infectious agents, to immunophenotype neoplastic cells, and to prognosticate neoplastic diseases. Formalin fixation is considered a limiting factor for IHC because formalin can cross-link antigens and mask epitopes. Prolonged formalin fixation is presumed to result in decreased antigen detection; however, this effect has only been evaluated with a few antibodies. The goal of this study was to evaluate the effect of prolonged formalin fixation on the immunohistochemical detection of 61 different antigens. Approximately 5-mm-thick tissue slices were fixed in 10% neutral-buffered formalin. Tissue slices were removed from formalin, processed, and paraffin-embedded at 1-day, 3-day, and then at ?1-week intervals. IHC was performed on all sections in tandem after all tissues were processed. Immunoreactions were evaluated by three pathologists according to a four-tier grading system. Immunoreactivity of cytokeratin 7, high-molecular-weight cytokeratin, and laminin was diminished by prolonged formalin fixation. However, immunohistochemical reactivity remained moderate to strong with up to 7 weeks of fixation for all other antibodies. These results suggest that prolonged formalin fixation has minimal effects on antigen detection for most commonly used antibodies. These results further validate the use of IHC in diagnostic pathology. IMMUNOHISTOCHEMISTRY IS AN ESSENTIAL TOOL for diagnostic pathology. Routine uses of diagnostic IHC include immunophenotyping and prognosticating neoplastic diseases, detecting or identifying pathogens associated with histologic lesions, and characterizing cellular infiltrates. The primary advantage of IHC over other protein detection techniques is that antigens can be detected in the context of tissue and cellular morphology. Therefore, antigen expression is not only detected in the diseased tissue, but associations between the presence of antigen and specific cell types or histologic lesions can be assessed with IHC.Many tissues used in diagnostic histopathology and IHC are routinely fixed in 10% neutral-buffered formalin, which is a 4% formaldehyde solution buffered to a neutral pH. Formalin is a cross-linking fixative that forms hydroxymethyl groups on reactive amino acid side chains and subsequently cross-links peptides. Formalin can also react with nucleotides and some unsaturated fatty acids (Eltoum et al. 2001b;Ramos-Vara 2005). Formalin inhibits cellular processes, prevents tissue degradation, preserves tissue architecture, and kills pathogens within lesions (Eltoum et al. 2001b;Ramos-Vara 2005). The use of formalin-fixed paraffinembedded tissues for IHC eliminates the need for fresh or fresh-frozen tissues, and allows the use of archival paraffin-embedded tissues in diagnostic cases and retrospective studies.Formalin provides excellent preservation of tissue architecture; however, formalin fixation can mask epitopes and result in decreased immunoreactivity (Arnold et al. 1996;Werner et al. 200...

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Advanced Concepts in Fixation: 1. Effects of Fixation on Immunohistochemistry, Reversibility of Fixation and Recovery of Proteins, Nucleic Acids, and other Molecules from Fixed and Processed Tissues. 2. Developmental Methods of Fixation

Cited by 17 publications

References 0 publications

Suggested Guidelines for Immunohistochemical Techniques in Veterinary Diagnostic Laboratories

Suggested Guidelines for Immunohistochemical Techniques in Veterinary Diagnostic Laboratories

When Tissue Antigens and Antibodies Get Along

Effects of Prolonged Formalin Fixation on Diagnostic Immunohistochemistry in Domestic Animals

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