Flow Cytometry-based Non-Transferrin-Bound Iron Assay

Fe-Flobead: A Sensitive High Throughput Test For Iron Overload

 

An iron-sensitive fluorescent probe attached to beads has been developed and incorporated into an assay for the sensitive and accurate measurement of non-transferrin-bound iron (NTBI). Measurement of NTBI should provide a valuable means of 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 in the plasma that bind to ligands other than transferrin, and is considered to be a marker of iron toxicity. This form of iron is thought to appear when the rate and amount of iron entering the circulation exceed the capacity of plasma transferrin to bind it efficiently.

Early identification of iron overload can be crucial for appropriate diagnosis and establishing response to treatment in 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, a clinically relevant level of sensitivity has not been acheived with exisiting methods.

Here we present a highly sensitive high throughput assay which can be a useful tool for rapid detection of iron overload in the clinical setting.


Commercial opportunity

 

KCL is seeking a partner for further development and validation of this assay. Patent application and clinical data are available for licensing.


Assay Procedure

 

The measurement of NTBI was performed by applying iron-chelatable fluorescent beads on the serum samples. A standard curve of the fluorescence of the beads related to the added iron concentration (0-20 mM) was performed (Fig 1). Based on the calibration curve, the concentrations of the chelatable iron in different sera were calculated.

 

Figure1: Standard curve of the iron-chelatable fluorescent beads

 

Key Benefits

  • Simple high throughput system.
  • Does not involve ultrafiltration, chromatography and the use of a separate scavenger or a transferrin blocker.
  • Time saving: capable of running 400 analyses per week.
  • High sensitivity :able to quantify NTBI down to levels of 0.2 µM.
  • Does not remove any iron from transferrin, a common problem associated with the use of auxiliary scavenging molecules such as NTA.

 

Results

 

Clinical samples from 46 B-thalassaemia/HbE patients were collected. NTBI values in the sera were determined by Fe-Flobead method and the NTA method (Figure 2). The values obtained by the NTA method are slightly higher than those by the Fe-Flobead. This difference is due to NTA removing a small proportion of iron from transferrin.

Figure 3 shows transferrin saturation against the NTBI values determined by the Fe-Flobead method. Significantly, when transferrin saturation is less than 90%, NTBI was not detected by this method. Although a similar trend was observed for the NTA method when transferrin saturation is above 80%, negative NTBI values of sera with less than 80% transferrin saturation were also detected, with a few reaching -3 µM (Fig 4). 

 

Figure 2:  Comparison of two methods for NTBI values of iron-overloaded serum samples

 

 

Figure 3: Correlation between NTBI values by “Beads method” and transferrin saturation

 

Figure 4: Correlation between NTBI values by NTA method and transferrin saturation

 

Patent Information:
Category(s):
Screening assay
For Information, Contact:
Jenny Worthington
King's College London
jenny.worthington@kcl.ac.uk
Inventors:
Robert Hider
Yongmin Ma
Ulrich Schaible
Maria Podinovskaia
Keywords: