Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen-retrieval Technique: From Experiments to Hypothesis

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Abstract
From a practical point of view, one of the most difficult issues in the standardization of IHC for FFPE tissue is the adverse influence of formalin upon antigenicity, as well as the great variation in fixation/processing procedures. Based on previous study, an additional study using four markers demonstrated the potential for obtaining equivalent IHC staining among FFPE tissue sections with periods of formalin fixation ranging from 6 hr to 30 days. On this basis, the following hypothesis is proposed. “The use of optimized AR protocols permits retrieval of specific proteins (antigens) from FFPE tissues to a defined and reproducible degree (expressed as R%), with reference to the amount of protein present in the original fresh/unfixed tissue”. This hypothesis may also be presented mathematically: the protein amount in a fresh cell/tissue, expressed as Pf, produces an IHC signal in fresh tissue of ∫ (Pf). When the identical IHC staining plus AR treatment is applied to a FFPE tissue section, the IHC signal may be represented as ∫ (Pffpe). The degree of retrieval after AR (R%) is calculated as follows: R% = ∫ (Pffpe)/ ∫ (Pf) × 100%. The amount of protein in the FFPE tissue may then be derived as follows: Pffpe = Pf × R%. In a situation where optimized AR is 100% effective, the IHC signal would then be of equal strength in fresh tissue and FFPE tissue, and Pffpe = Pf. Further studies are designed to test the limitations of the proposed hypothesis.

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