A simple, rapid and improved colorimetric assay for non transferrin thalassemia patients bound iron estimation in thalassemia patients

Non transferrin bound iron comprises the forms of plasma iron which are not bound to its traditional transporter plasma protein transferrin. Such forms of iron have been suggested to be toxic due to its redox activity through Haiber weiss and Fenton chemistry. Non transferrin bound iron has been widely studied in patients suffering from iron overload conditions like hemochromatosis and thalassemia receiving blood transfusion treatment due to obvious higher transferrin saturation. NTBI has been suggested to be studied by various researchers to determine the efficacy of chelators used to chelate iron in iron overload patients. Several researchers suggested a variety of methods based on diverse approaches like chromatography, fluorimetry, atomic absorption spectroscopy, spectrophotometry etc. to determine the level of Non transferrin bound iron. However none of them is still widely accepted or considered gold standard due to high complexity of methodology, requirement of sophisticated specialized instruments and accessories as well as debatable reliability. We have experimented different variation in bathophenanthroline based colorimetric method for non transferrin bound iron estimation to make it simple, rapid, cost effective and suitable to be opted in laboratories with limited resources without compromising the reliability of the results. From all the trials, protocol giving most consistent results was used to determine the NTBI value in β thalassemia patients as well as control group. We found that the high speed centrifugation with in between sample pre incubation had significantly decreased the background noise and generated the most consistent results. The mean Non transferrin bound iron value we got in β thalassemia patient group was significantly higher than the respective mean of control group.