Iron Binding Test

Iron Binding Test: Methods and Techniques

Introduction to Iron Binding Tests

Iron binding tests are essential for evaluating iron metabolism and diagnosing various conditions such as anemia and hemochromatosis. These tests measure serum iron and the total iron-binding capacity (TIBC) of the blood, providing insights into the body's iron storage and transport mechanisms.

Traditional Methods for Serum Iron and Iron-Binding Capacity

Macro and Micro Methods

Traditional methods for determining serum iron and iron-binding capacity involve several steps. Blood serum is treated with ascorbic acid and hydrochloric acid to release protein-bound iron. Proteins are then precipitated using trichloroacetic acid in the presence of chloroform. The clear supernatant is reacted with tripyridyltriazine and ammonium acetate to form a blue-colored complex, which is measured at 590 nm. TIBC is measured by saturating the serum with ferric iron and removing excess iron with magnesium carbonate.

Direct Measurement Techniques

Direct measurement techniques for serum iron involve incubation in an acetate buffer with a reducing agent and sulfonated bathophenanthroline. Suitable reductants include mercaptoacetic and ascorbic acids. This method shows excellent correlation with traditional precipitation techniques and compensates for bilirubin interference. It also allows for the estimation of unsaturated iron-binding capacity (UIBC) and TIBC.

Automated and Advanced Methods

Fully Automated TIBC Measurement

A fully automated method for measuring TIBC without separating unbound excess iron involves saturating serum transferrin with iron, eliminating unbound iron using ferrozine, and measuring the absorbance at 570 nm. This method is highly precise, with within-run CVs of 0.66-2.43% and day-to-day CVs of 1.06-1.57%.

AutoAnalyzer Procedures

AutoAnalyzer methods using ferrozine allow for high-throughput analysis, with sampling rates of up to 120 samples per hour. These methods are rapid and require smaller sample volumes compared to traditional techniques. They also include procedures for masking high concentrations of interfering substances like copper .

Atomic Absorption Spectrophotometry

A novel method using atomic absorption spectrophotometry involves treating serum with 20% trichloroacetic acid and heating to 90°C. This process liberates ferric iron and precipitates proteins, which are then removed by centrifugation. This method is specific, accurate, and faster than many colorimetric or chelation-based methods.

Modified and Simplified Techniques

Deoxyribose Test Modification

The deoxyribose test, traditionally used for hydroxyl radical reactivity, has been modified to detect strong iron binding. This involves examining the concentration dependence of deoxyribose degradation on Fe2+ concentration in the presence of a chelating agent, providing insights into iron binding strength.

Direct Serum TIBC Assay

A direct automated assay for TIBC involves adding a saturating amount of iron to serum, recording a blank reading, and then adding a neutral pH buffer. The decrease in absorbance is proportional to the TIBC. This method shows strong correlation with other established methods and is suitable for routine clinical use.

Conclusion

Iron binding tests are crucial for assessing iron metabolism and diagnosing related disorders. Traditional methods, while reliable, are being supplemented and often replaced by automated and advanced techniques that offer higher precision, faster processing times, and reduced sample volumes. These advancements are enhancing the efficiency and accuracy of iron metabolism studies in clinical settings.

Summary

Not a yes or no question