Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals

Area of Science:

• Anatomic Pathology
• Immunohistochemistry
• Biochemical Analysis

Background:

• Immunohistochemistry (IHC) is crucial in diagnostic pathology for identifying infectious agents, immunophenotyping, and prognostication.
• Formalin fixation, a standard tissue preservation method, can impede IHC by cross-linking antigens and masking epitopes.
• The impact of prolonged formalin fixation on antigen detection is not well-established for a broad range of antibodies.

Purpose of the Study:

• To systematically evaluate the effect of prolonged formalin fixation on the immunohistochemical detection of 61 diverse antigens.
• To determine if extended formalin fixation compromises antigen detectability for routine diagnostic applications.

Main Methods:

• Tissue slices (5mm thick) were fixed in 10% neutral-buffered formalin.
• Sections were processed and paraffin-embedded at intervals from 1 day up to approximately 7 weeks.
• Immunohistochemistry was performed on all sections, with immunoreactions graded by three pathologists using a four-tier system.

Main Results:

• Immunoreactivity for cytokeratin 7, high-molecular-weight cytokeratin, and laminin decreased with prolonged formalin fixation.
• However, moderate to strong immunohistochemical reactivity was maintained for the majority of the 61 tested antigens up to 7 weeks of fixation.
• These findings indicate that most antibodies are robust to extended formalin fixation periods.

Conclusions:

• Prolonged formalin fixation demonstrates minimal adverse effects on the immunohistochemical detection of most commonly used antigens.
• The study validates the continued reliable use of IHC in diagnostic pathology, even with extended formalin fixation times.
• Standard IHC protocols are generally suitable for diagnostic purposes despite potential variations in fixation duration.