135INTHE IMPORTANCE OF THE PREANALYTICAL PHASE FOR IMMUNOHISTOCHEMISTRY

ABSTRACT The importance of the preanalytical phase for immunohistochemistry Preanalytical treatment of tissues was studiedto clarify pitfalls in immunohistochemistry (IHC) and, in general, in molecular analysis in fixed and paraffin-embedded tissues, which are the only tissues available for any patient for diagnostics and for clinical research. Specific attention was given to this issue because of the important role recently played by the biomarkers predictive of treatment efficacy, and not only for diagnosis definition and prognosis evaluation. The preanalytical phase is quite complex and is not only related to pathologists but also to surgeons. The warm ischemia time, the use of specific techniques or simply the traction and distortion of tissues can influence gene expression modification, protein coagulation or their diffusion into extracellular spaces with consequences for IHC results. Warm ischemia and tissue transport conditions can differently influence genes with increased or decreased expression, with loss of reactivity in IHC. On the other hand, many genes can be totally indifferent to these conditions. IHC reliability can also be affected by common pathological processes such as hemorrhagic diffusion in tissues, necrosis, inflammation or apoptosis. Antigenetic epitopes of proteins are modified by fixation in formalin with the formation of methylenic bridges among different aminoacid residues, but also tertiary and quaternary structures are affected. Protein alterations are quantitatively related to time of fixation and enzymatic or heat related demodification procedures for antigen retrieval. This variability should be carefully taken into account. An insufficient time of fixation can also affect IHC results, because tissue protein alteration is related to over-fixation, but at the same time the inner part of the tissue is altered by hypoxia with initial autolytic phenomena due to released lysosomal enzymes. These hypoxic areas can also be affected by inclusion procedures and alcohol treatment coagulates the proteins. Inclusion process temperatures can also affect the same more sensitive antigens. All these possible alterations, together with methods and interpretation pitfalls, make internal and external quality controls and audit procedures mandatory for IHC laboratories. Disclosure: The author has declared no conflicts of interest.