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Beckman Coulter Launches Soluble Transferrin Receptor Assay - A Differentiator for Diagnosing Iron Deficiency Anaemia

Anaemia is a worldwide health issue which can have a profound effect on their quality of life. The most common cause is iron deficiency, estimated to be responsible for 50% of all anaemia cases

Iron Deficiency Anaemia (IDA) negatively impacts the cognitive and physical development of children as well as the work productivity of adults. In surgical patients, it may increase the risk of postoperative morbidity and death. IDA is the most common nutritional deficiency, affecting a quarter of the world's population. In most cases it can be treated simply with iron once the body's iron status is ascertained. The standard tests for establishing IDA include measuring serum ferritin, serum iron and iron binding capacity.

However, these analytes are unreliable when there is inflammation present, as commonly occurs in chronic diseases. In fact, inflammation is a frequent confounder because chronic disease is the second most common cause of anaemia. Anaemia of Chronic Disease (ACD), also known as anaemia of chronic inflammation, occurs with infections, cancer, autoimmune disease and chronic kidney disease. ACD can present with or without iron deficiency and its treatment will depend on its cause.

A highly accurate automated laboratory test detecting a biomarker that is not affected by inflammation could be a more reliable method of assessing the body's iron status in the presence of ACD. One such assay would measure serum soluble Transferrin receptor (sTfR) concentration. This marker increases in patients with IDA in response to the demand for iron by the erythroid progenitor cells, making it better suited than other available markers for investigation of IDA.

Regulation & transport of iron

Iron balance is maintained by regulation of intestinal iron absorption. After absorption by enterocytes, iron binds to the blood protein transferrin, which transports the iron in the blood and delivers it to target cells. The iron-laden transferrin binds to specific membrane receptors on these cells - the Transferring Receptor (TfR).

TfR is a homodimer of two 95 kDa subunits linked by disulphide bonds [Figure 1]. Each monomer has a 61 amino acid N-terminal cytoplasmic domain, a short transmembrane region, and a large extracellular domain of 671 residues. Each TfR can bind up to two iron-laden transferrin molecules.

Transferrin receptors are present on nearly every cell type, with the largest numbers found in the erythron, placenta and liver. When a cell needs iron, transferrin receptor production increases to augment receptor expression on the cell surface, thereby facilitating iron uptake. The major site of iron utilisation is the bone marrow, where erythrocyte precursors absorb the iron and incorporate it into haemoglobin. In normal adults, approximately 80 per cent of transferring receptors are associated with erythroid progenitor cells in the bone marrow.

The number of receptors decline as erythroid progenitors mature, lower levels are present during the reticulocyte stage and they are essentially absent in mature red cells.

When transferrin binds to the TfR, both the receptor and the iron-transferrin complex is internalised, and iron is released into the cytoplasm of the cell. In erythroid cells, most iron is incorporated into protoporphyrin (haeme). In nonerythroid cells, iron is stored as ferritin and haemosiderin, a complex of ferritin and denatured ferritin. Both transferrin and TfR are recycled to the cell surface for additional iron binding and uptake.

At the end of their life, red blood cells are destroyed by macrophages, which recycle iron from the haeme fraction of haemoglobin and transferrin once again transports the iron to target cells.

Current Diagnostic Challenges

The diagnosis of iron deficiency includes the evaluation of the body's iron status using laboratory tests to measure serum iron, transferrin, ferritin and transferrin saturation, as well as assessing haematological parameters.

Serum iron levels do not fall until iron stores are depleted. Extra iron is stored in hepatocytes and macrophages of the reticuloendothelial system. When these stores are depleted, serum iron level will fall below 50 µg/ dL, while transferrin increases linearly. As transferrin increases, transferrin saturation falls below 20per cent.

Serum ferritin is a marker of iron storage, and therefore levels will fall in IDA. A value lower than 22 ng/mL is generally considered an indicator of depleted iron stores in a healthy individual, but some researchers have suggested that a higher cut-off of 30 ng/mL may be better for identifying IDA. However, several clinical conditions are known to trigger an increase in ferritin levels irrespective of iron status— such as hyperthyroidism, hepatocellular disease, acute bacterial infections, as well as alcohol consumption or the use of oral contraceptives.

IDA can be masked if it presents with ACD because ferritin values are not decreased, as they would be with IDA alone, and may be as high as 51-100 ng/mL [11]. Ferritin levels cannot therefore be used to diagnose IDA in the presence of ACD where the anaemia is not only caused by a lack of iron, but also because the iron stored in the hepatocytes and macrophages of the reticuloendothelial system is prevented from being loaded onto transferrin.

Role of Soluble Transferrin Receptor

Another parameter that can be used to assess iron status is soluble transferrin receptor (sTfR). sTfR results from the proteolysis of TfR at a specific site in the extracellular domain, producing fragments that circulate in the blood complexed to ferritin.

The amount of total, cellular TfR is directly proportional to the concentration of sTfR in plasma or serum, and so sTfR in the plasma accurately reflects the total TfR. Because most TfRs are located on erythroid progenitors, the concentration of sTfR is believed to reflect erythroid turnover, and is determined by the erythroid proliferation rate and iron demand.

sTfR increases in haemolytic anaemia, reflecting the raised number of erythroid cells, and in IDA due to an increased iron demand for erythropoiesis. A reduced level of sTfR is typically seen in aplastic anaemia and renal failure as erythroid cell mass is reduced.

The inclusion of an sTfR assay when assessing a patient with suspected anaemia can provide a clearer picture of both the cause and the most appropriate treatment. The only automated immunoassay system to offer sTfR as part of a complete panel for speed and consistency is the Beckman Coulter Access Immunoassay System.

Differential Diagnosis of IDA & ACD

ACD is associated with mild to moderately decreased haemoglobin. Red cell production is suppressed by inflammatory cytokines and in some cases lower production of erythropoietin, resulting in a low reticulocyte count. However, a definitive diagnosis may be difficult because of coexisting conditions, such as iron deficiency, blood loss, effects of medications or the presence of variant haemoglobin forms such as in the thalassaemias.

The ACD may be microcytic and, particularly in conditions such as rheumatoid arthritis, gastric bleeding due to use of aspirin or other Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) may be a factor. With both anaemia of chronic disease and IDA as well as a combination of both conditions, the serum concentration of iron and transferring saturation may be reduced due to the cytokine effects.

To rule out coexisting IDA, whole-body iron status needs to be assessed, but the results of standard measures, such as the acute phase reactant ferritin, total iron-binding capacity and serum iron may be affected by the chronic disease state. In contrast, sTfR is not correlated with inflammatory parameters like erythrocyte sedimentation rate and C-reactive protein (CRP). sTfR will be elevated only if there is iron deficiency or increased red cell production as in haemolytic anaemia.

The sTfR/log ferritin index has an even higher specificity and sensitivity for IDA in ACD than sTfR alone. This calculation can be carried out in the ‘derived result function’ on the Access systems, or manually. Only the Access system offers automated on-board calculation of the sTfR index from the sTfR and ferritin results, giving the index value quickly and accurately, and removing the chance of clerical error.

Patients with ACD alone do not necessarily need iron therapy. The cause of the inflammation should be promptly identified and treated due to the critical nature of this issue. A physician runs the risk of missing a bleeding ulcer or other significant disorder— at great cost to the patient.

A useful algorithm indicating how the different causes of anaemia can be identified has been developed [Figure 2]. Table 1 shows the likely results of standard laboratory assays used to determine the iron status of patients with a chronic disease, and highlights the difficulties in determining the cause of anaemia.

Identifying Subclinical (latent) Iron Deficiency

As well as being a useful marker for determining whether or not iron deficiency exists in ACD, sTfR can also confirm suspected iron deficiency early in the condition. Again, the sTfR Index provides an even better indicator for subclinical iron deficiency than sTfR alone. Groups most at risk of this include women of child bearing age, young children and the elderly.

These populations may have normal haemoglobin but low or borderline ferritin and/or high or borderline transferrin. Elevated sTfR concentration (or index) will be a good indicator of early iron-deficient erythropoiesis until the storage deficit becomes sufficient to restrict synthesis of haemoglobin.

sTfR monitoring of erythropoiesis & other applications

As well as helping identify iron deficiency, sTfR is useful for monitoring erythropoiesis in malignancy and chronic renal disease. Development of erythropoiesis following bone marrow or stem cell transplantation is determined by the overall marrow proliferative capacity, which can be monitored with sTfR [16, 18]. During the aplastic period prior to transplantation, sTfR levels decline. Once erythropoiesis has recovered, sTfR levels return to normal values.

In anaemia of chronic renal failure, the early increase in sTfR values after starting recombinant human erythropoietin therapy is useful for predicting and assessing haematologic response to therapy. The change in sTfR levels occurs well before any change in haematocrit or haemoglobin values can be detected, allowing early adjustments in erythropoietin dosage and iron supplementation therapy.

In one clinical study of haemodialysis patients, a 20 per cent increase in sTfR was strongly associated with a response to recombinant erythropoietin therapy. A second study demonstrated that baseline and testing of sTfR in two week increments, combined with baseline testing of serum erythropoietin, increased the overall accuracy of predicting response to therapy.

sTfR and the sTfR/ferritin ratio also appear to have other useful application. A recent study indicated that the sTfR/ferritin ratio can discriminate between anaemic patients with and without coeliac disease. Subclinical iron deficiency in early pregnancy is strongly associated with bacterial vaginosis, and therefore sTfR and the sTfR/log ferritin index also might have a role in highlighting the risk of this condition. The sTfR/log ferritin index has also been shown to be superior to routine tests for predicting the response to iron therapy in long-term haemodialysis patients, and in discriminating between patients with iron deficiency and various haemoglobinopathies.

The Impact of sTfR on Patient Outcomes

Although most cases of anaemia can be treated easily, the condition still has a negative impact on the patient's quality of life. This is because, historically, pinpointing the cause to allow effective treatment could be difficult. Standard biological markers of iron status are influenced by inflammatory processes, so in patients with ACD any coexisting iron deficiency can remain masked. In addition, these assays are not sensitive enough to identify IDA in its earliest stages.

Serum sTfR also reflects the rate of erythroid proliferation and iron demand but as sTfR is not an acute phase reactant it is unaffected by inflammatory process. sTfR is therefore a useful addition to the existing anaemia assays. Systems able to calculate the sTfR/log ferritin ratio automatically provide a statistically superior diagnostic aid when compared to sTfR alone. It also has an important role in monitoring erythropoiesis as a determinant of response to therapy following bone marrow or stem cell transplantation and in chronic renal disease. The automated sTfR assay offering clinical cut-off information enables clinicians to obtain a more complete picture of the body's iron status, and so improve the speed of diagnosis and recovery.

Contact: sshah@beckman.com

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