On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions

Abstract

Formalin has been recommended as an innocuous fixative for immunohistochemistry. However, several studies demonstrated impairment or blocking of antigenic activity of certain proteins. Formalin fixation was discovered accidentally by F. Blum in 1893 and its deleterious effects on various tissue structures were discussed extensively during the following decades. More recently, some authors assumed that formaldehyde bound to tissues can be largely or completely removed by washing and dehydration. According to chemical data, formaldehyde forms highly reactive methylols with uncharged amino groups. Such methylol groups yield methylene bridges with suitably spaced amides, arginine and aromatic amino acid sidechains. Only loosely bound formaldehyde is removed by washing for several hours. Residual bound formaldehyde cannot be dislodged by washing for weeks, but some formaldehyde is gradually removed when tissues are stored in water for an extended number of years. Methylene crosslinks resist treatment with high concentrations of urea, and can be broken only by drastic hydrolysis. It appears unlikely that such firmly bound formaldehyde is removed by conventional washing and dehydration procedures used in histochemistry. The superiority of methacarn, alcohol or acetone over formaldehyde fixation for immunohistochemical demonstration of prekeratin, myosin, type I and type IV collagen, laminin and fibronectin can be ascribed to the irreversible alterations of tissue proteins by formaldehyde.

Delay to formalin fixation effect on breast biomarkers

“…It is known that formaldehyde immediately produces reactive hydroxymethyl groups on amino acids that cross-link proteins and large molecules that are necessary for immunohistochemistry analysis. [32][33][34] Moreover, proteolysis often causes nonspecific binding to unrelated molecules. 35 These factors are most likely the reason behind decreased estrogen and progesterone receptor expression with tumor ischemia.…”

Immunohistochemical Detection of Prion Protein in Sheep with Scrapie

“…8 The marked improvement in amount of PrP-res detected with our fixative, as compared with either formalin or PLP with 0.5% paraformaldehyde, reflects the propensity of aldehyde-based fixatives to cross-link proteins and "mask" important antigenic epitopes in IHC reactions. 27 This problem is of special concern when tissues are stored in fixative for long periods of time. The under field conditions.…”

International Society of Urological Pathology (ISUP) Consensus Conference on Handling and Staging of Radical Prostatectomy Specimens. Working group 1: specimen handling

“…Fixation may be broadly categorized as the effects of dehydrants, heating, crosslinking agents, acids and any combination of these. 31 Formaldehyde as a 10% neutral buffered formalin solution is the most widely used fixative and the speed of fixation depends on the rate of diffusion and the rate of chemical reaction between the fixative and the various tissue components. It is recommended that specimens be fully immersed in at least 20 times their own volume of buffered formal saline with at least 1 h of fixation time per mm of tissue thickness being required.…”