(188bz) Microsphere Immunoassay and Cell Tracking Velocimetry to Diagnose Iron-Related Disorders in Point-of-Care Applications

Iron-Related Disorders in Point-of-Care Applications

Introduction

Around one third of the world’s population suffers from anemia, and half of these cases are due to iron (Fe) deficiency. This is the most common form of micronutrient malnutrition and causes physical and mental stunting as well as other serious organ diseases. Children under age 5, young or pregnant women and regular blood donors are at particularly high risk of iron deficiency anemia (IDA). It is estimated that 25-35% of the repeat blood donors in the United States are iron deficient as well as 7% of toddlers and 9-15% of females age 12-49 in the US. This epidemic is not exclusive to low-income countries and effective, low-cost treatment can be as simple as oral iron supplementation.

This proposal details the methodology for a new method to diagnose iron deficiency (ID) in point-of-care (POC) settings. Diagnosis of iron-related diseases in a POC application just before a blood donation takes place can return results quickly enough to be practical in a blood drive. This ensures that the donor’s iron stores are high enough to meet the recipient’s needs as well as continue to produce hemoglobin for the donor after donation.

Objectives

The novel method proposed in this study utilizes the intrinsic magnetic properties of iron, specifically in ferritin, to diagnose iron deficiency without anemia. Using a Cell Tracking Velocimetry (CTV) instrument, which has previously determined the iron content of red blood cells with femtogram level sensitivity, we propose the following specific aims:

• Determine the magnetic susceptibility of a label-less magnetic microsphere immunoassay (m-MIA) using a CTV instrument. Serum ferritin bound to a detection antibody with high specificity that is bound to a capture antibody on a 6.7μm polystyrene bead will have measurable magnetic moment. Then, determine the magnetic susceptibility and iron content of the ferritin protein population.
• Compute the ferritin concentration and iron content of the serum sample to determine if the patient is iron deficient.

The World Health Organization defines depleted iron stores as < 12 ng/mL and <15 ng/mL of serum ferritin for patients less than 5 years old and 5 years old or over, respectively. The proposed method goes a step further by determining the abundance of iron in ferritin rather than simply the presence of ferritin.

Background

Ferritin is a 450 kDa iron storage protein that is used to create new hemoglobin units and reticulocytes. Different types of ferritin are present throughout the body; each contains 24 of the heavy (H) or light (L) type subunits that have a different function and number of iron atoms in each protein. Ferritin can contain up to 4,500 iron atoms per molecule with varying reduction states (Fe²⁺ or Fe³⁺) within the mineral core. Serum ferritin is a particularly useful indicator of total body iron stores.

Enzyme linked immunosorbent assays (ELISA) utilize the interaction between an antibody and its corresponding antigen/protein for detection and quantitation. ELISA have been developed for thousands of biomolecules and diseases and can have sensitivities in the pg/mL range. However, performing an ELISA requires expensive materials, trained personnel, time-consuming optimizations and multiple incubation steps that can take several hours to days. Invasive bone marrow tests and liver biopsies are definitive but not practical in POC applications or developing countries that lack necessary testing equipment. Transport limitations in ELISA have led to the development of microsphere immunoassays (MIA) in which antibodies or antigens bind to monodisperse microspheres rather than a 96-well plate. The increased surface area and mixing of the microspheres can cut down the incubation time to 20 minutes. Direct, indirect, sandwich and competitive assays are possible with MIA as well as ELISA.

Currently, diagnosis requires both ferritin and soluble transferrin (sTfR) ELISA to provide a complete view of the body’s iron stores Often a colorimetric method is necessary to determine the total iron binding capacity (TIBC). Inflammation can complicate results and cause significant variability between samples. It is noteworthy that none of these commonly used methods directly measure the amount of iron in a patient sample, only the biomolecules used for iron storage and regulation.

The routine screening test performed at blood drives yields rapid results but does not provide a complete picture of a patient’s iron status. In order to screen donors, a small sample of blood is removed from the finger and tested to quickly measure the hemoglobin concentration. In the US, male and female donors must have a concentration greater than 125 g/L to donate blood. Although anemia is defined in terms of hemoglobin or red blood cell concentration, iron deficiency without anemia cannot be measured in this way. ID is still an issue without immediately altering a donor’s hemoglobin concentration. ID without anemia impairs hemoglobin synthesis and can cause cognitive and physical growth impairments in young children that may be irreversible.

We have previously published a mathematical relationship relating the antibody binding capacity (ABC) of immunomagnetically labelled cells to the antibody amplification, magnetic susceptibility, magnetic field strength and cell size. The proposed methodology simply replaces the aforementioned cells with antibody-coated PS microspheres conjugated to magnetic, iron-containing ferritin proteins.

Research Plan

The proposed research project aims to diagnose iron deficiency by measuring the magnetic susceptibility of ferritin bound to a m-MIA using a CTV instrument and confirming results with a sandwich ELISA bound to a microsphere (sandwich MIA). PS microspheres 6.7μm in diameter conjugated with capture antibodies will bind specifically to a ferritin detection antibody that is then bound to ferritin found in serum. This m-MIA is large enough in size to be measured by the CTV instrument (red blood cells are around 5μm in diameter) and the intrinsic magnetic properties of ferritin will produce a measurable magnetic velocity.

Additionally, in order to validate results with a standard method, some of the beads used for CTV will undergo another incubation step with the ferritin detection antibody (conjugated with a fluorescent enzyme) and used with a spectrophotometer in a 96-well plate.

Similar to the results obtained with red blood cells, a distribution of magnetic susceptibilities is expected from the proposed ferritin-bound m-MIA. There are several reasons for this anticipation.

First, there is a distribution in the size of the microspheres. Due to limitations in microbead synthesis, the size distribution of PS microspheres cannot be perfect. Nonuniformity in both the bead weight and the number of ferritin molecules bound to it will complicate results. Additionally, a distribution in the iron content of serum ferritin and oxidation states (Fe²⁺ or Fe³⁺) is expected due to age and functional differences. While all serum ferritin molecules have the same exact size and composition of H and L subunits, the age and recent hemoglobin production may affect the amount of iron stored in its mineral core. For these reasons, it may be sufficient to average the iron stores in the measured ferritin and report total body iron stores. However, we wish only to determine a patient’s total iron stores and the nonuniformity may be insignificant.

We expect nonspecific binding to the antibodies to be negligible as long as proper blocking and washing steps are followed. Lastly, polystyrene will introduce a small amount of background fluorescence (due to its aromaticity) while characterizing the sandwich MIA in the spectrophotometer. New polymer microshpheres are being developed for greater monodispersity without background fluorescence.

Impact and Significance of Proposed Work

This novel method to diagnose iron deficiency has the potential to improve the health of blood donors, recipients and people at high risk of IDA. ID repeat blood donors who pass the finger stick test remain at risk for IDA while passing hematological tests. Replacing the finger stick test with the proposed diagnostic method will alert patients of low or depleted iron stores who otherwise would not know without a more thorough, expensive test. Currently, ELISA tests must be conducted for ferritin as well as transferrin along with another test to determine TIBC to completely understand a patient’s iron status. A ferritin ELISA is insufficient by itself because the ferritin concentration can vary depending on multiple physiological factors. However, using an m-MIA with CTV may avoid this issue by determining the number of iron molecules per ferritin molecule. There are other, less prevalent iron-related diseases such as thalassemia, tissue iron overload, lack of hypoferremic response to infections and hemochromatosis that require more information (concentrations of hepcidin and transferrin, red blood cell size distribution). A MIA with hepcidin/transferrin, an iron regulation protein, or a single label on a red blood cell to determine size can be utilized with a CTV instrument to diagnose a much larger array of iron-related diseases.