Effects of Prolonged Formalin Fixation on Diagnostic Immunohistochemistry in Domestic Animals

Abstract

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...

Immunohistochemical Detection of CD34, E-cadherin, Claudin-1, Glucose Transporter 1, Laminin, and Protein Gene Product 9.5 in 28 Canine and 8 Feline Meningiomas

“…In our experience, immunoreactivity for all the markers examined is not significantly affected by formalin fixation up to 1 month. 62 For CD34, immunoreactivity is not reduced in tissues fixed for at least 2 weeks (personal observations). When multiple slides of the same tumor were available, the most representative sample was selected by 1 author (J.A.R.).…”

When Tissue Antigens and Antibodies Get Along

“…3). 288,297,403,404 Automated stainers are designed to duplicate manual procedures and ensure uniform application of each step of the process. Thus, the use of automated equipment creates a uniform and standardized testing environment, which will improve intralaboratory run-to-run consistency.…”

“…205 However, significant reduction of immunoreactivity is detected in only a few markers after several weeks' fixation ( Fig. 3), 238,403,404 so it seems that the negative effects of overfixation are antigen and antibody dependent and can be overcome in many instances with an appropriate antigen retrieval procedure. 6,22,164,242 Vimentin was once used as an internal control of antigenicity loss in overfixed tissues.…”

Infecção por Mycobacterium sp. em herbívoros selvagens de cativeiro no Rio Grande do Sul: estudo retrospectivo e detecção imuno-histoquímica (2003-2015)

“…A coloração de ZN mostrou distribuição semelhante à observada na IHQ, porém os anticorpos comerciais existentes não distinguem os diferentes tipos de micobactérias, com reações cruzadas entre elas (micobactérias tuberculosas, micobactérias não tuberculosas e bacilos da lepra) (Alves et al 1999). O tempo de fixação em formol é considerado um fator limitante na técnica de IHQ, pois a fixação prolongada em formalina pode resultar em falha de detecção do antígeno (Webster et al 2009). Esta situação não foi evidenciada no presente estudo, visto que houve imunomarcação na maioria dos casos.…”