Fe-Flobead: A Sensitive High Throughput Test for Iron Overload
An iron-sensitive, fluorescent, bead-based probe has been developed and incorporated into an assay for the accurate measurement of non-transferrin-bound iron (NTBI). The measurement of NTBI provides a valuable means for the early detection and follow-up of diseases associated with iron overload such as thalassaemia, sickle cell anaemia, myelodysplastic syndrome and haemochromatosis.
Introduction
NTBI refers to all forms of iron that are found in the plasma and that bind to ligands other than transferrin. NTBI is thought to appear when the rate and amount of iron entering the circulation exceeds the binding capacity of plasma transferrin. This is considered to be a major marker of iron toxicity.
The early identification of iron overload can be crucial to enable appropriate diagnosis and to establish a patient’s response following treatment for pathological conditions associated with iron toxicity. Measurement of NTBI can complement the standard parameters of serum transferrin saturation and serum ferritin.
A variety of analytical approaches for measuring NTBI have been reported. However, existing methods have been unable to achieve clinically relevant levels of sensitivity.
Here we present a highly sensitive, high-throughput assay which provides a useful tool for the rapid detection of iron overload in the clinical setting.
Commercial Opportunity
KCL is seeking a partner for the further development and validation of this assay. Both the patent application and clinical data are available for licensing. Beads can also be made available for evaluation purposes.
Assay Procedure
The measurement of NTBI is achieved by the addition of the iron-chelatable fluorescent beads to the serum sample. A standard curve of the relative fluorescence of the beads in relation to the added iron concentration (0-20 µM) is presented in Figure 1. Based on the calibration curve, the concentrations of the chelatable iron in different sera can therefore be calculated.
Figure 1: Example standard curve of the iron-chelatable fluorescent beads. Relative fluorescence (%) is presented on the y axis and added iron concentration (µM) on the x axis.
Key Benefits
• Simple, high-throughput system
• No requirement for ultrafiltration, chromatography or a separate scavenger or transferrin blocker
• No removal of iron from transferrin (a common issue following the use of auxiliary scavenging molecules such as NTA)
• Quick (capable of running 400 analyses per week)
• High sensitivity (can quantify down to 0.2 µM)
• Lower rates of false negatives/positives at high and low values, respectively (due to a lower dependence on transferrin saturations)
Results
NTBI values in the sera of clinical samples from 46 β-thalassaemia/HbE patients were determined by both the aforementioned bead-based method and a widely used NTA method (Figure 2). The values obtained by the NTA method were slightly higher than those by the bead-based method. This is due to the NTA removing a small proportion of iron from transferrin as discussed.
Figure 3 shows transferrin saturation against the NTBI values as determined by the present bead-based method. Significantly, when transferrin saturation was lower than 90%, NTBI was not detected by this method. A similar trend was observed for the NTA method. However, negative NTBI values were observed in sera with less than 80% transferrin saturation (Figure 4). This is disadvantageous as clinicians are unhappy with assays that yield both positive and negative values.
Figure 2: Comparison of the two methods for measuring NTBI in iron-overloaded serum samples.
Figure 3: Correlation between NTBI values and transferrin saturation as measured by the present bead-based method.
Figure 4: Correlation between NTBI values and transferrin saturation as measured by the NTA method.