Technical methods. Use of 3,3',5,5'-tetramethylbenzidine (TMB) in the identification of erythroid colonies

Abstract

The peroxydatic activity of haemoglobin in the presence of hydrogen peroxide catalyses the oxidation of TMB to a coloured product. Haemoglobin peroxidase has been termed a 'pseudoperoxidase' because, unlike true peroxidases which exhibit specificity for phenols, erythrocytes contain a peroxidase (erythrocyte glutathione peroxidase) specific for oxidation of reduced glutathione. This causes the in vitro detoxification of hydrogen peroxide. Glutathione peroxidase activity has been found to be associated with a relatively stable, nondialysible, heat-labile, intracellular component which can be separated from haemoglobin by gel filtration and ammonium sulphate precipitation. The pH optimum of glutathione peroxidase has been shown to be pH 8.0 with negligibel activities below pH 6.0. Pretreatment of the fixed slides with resorcinol before staining with TMB and hydrogen peroxide inhibits myeloperoxidase staining. This increases specificity of the stain and reduces error in enumeration of erythroid colonies. By omitting counter staining only erythroid colonies and precursors are stained.