Microplate Assay for the Determination of Hemoglobin Concentration

摘要

The conversion of all hemoglobin species to cyanmethemoglobin (CNMetHb) by the addition of potassium ferricyanide and potassium cyanide (Drabkin s reagent) and the subsequent absorbance measurement at 540 nm is used widely in clinical and research laboratories as the standard method for hemoglobin quantitation. In 1984, however, Zander et al. described a spectrophotometric assay based upon the conversion of hemoglobin species to an alkaline hematin detergent complex designated D-575 (AHD), which absorbs maximally at 575 nm and is very stable. The AHD reagent contains 2.5% Triton X- 100 in 0.1 mol/l sodium hydroxide (NaOH), and converts all hemoglobin species, including COHb, to AHD within 5 min. Both protocols are carried out in cuvettes, and are, therefore, time intensive and difficult to manage when many samples are quantified. This impedes acquisition of triplicate values for each sample necessary to improve accuracy and determine statistical significance.