A rapid colorimetric assay for measuring low concentrations of haemoglobin in large numbers of bovine plasma samples

The aim of this study was to develop a microplate colorimetric assay that could measure low levels (<2mg/mL) of haemoglobin in bovine plasma as a result of haemolytic trauma. The method presented uses the pseudo-peroxidase activity of haemoglobin to catalyse a redox reaction between tetramethylbenzidine (TMB) and hydrogen peroxide in an acidic environment. There is a good linear relationship (r2=0.999) between the optical density of the reaction product and haemoglobin concentration. The assay is highly repeatable (F=1.219, P=0.303) with an intra- and inter-assay variation ranging from 2.3% to 3.9% and 5.8% to 11.3% respectively for three control samples containing high, medium and low levels of haemoglobin. In conclusion, the method described in this paper provides an accurate, precise measurement of low concentrations of haemoglobin in bovine plasma that enables correction for haemolysis.