Diagnosis of Iron Deficiency Anaemia: History
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Yvonne Chibanda
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Matthew Brookes
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David Churchill
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Hafid Omar Al-Hassi

Iron deficiency is a condition that results from long-term depletion of iron stores. It has several causes including haemorrhage, inadequate dietary intake and malabsorption syndromes.

  • serum ferritin
  • hepcidin
  • inflammation
  • iron replacement therapy

1. Iron Deficiency Anaemia

Iron deficiency is a condition that results from long-term depletion of iron stores. It has several causes including haemorrhage, inadequate dietary intake and malabsorption syndromes. Adequate levels of maternal iron are vital throughout pregnancy to also account for increased demand from the growing foetoplacental unit [1]. The effects of iron deficiency on its functions depend on its severity. Severe iron deficiency affects healthy erythropoietin-mediated erythropoiesis and causes anaemia: a reduction in haemoglobin concentration and red blood cell count [2]. Therefore, iron deficiency can present with or without anaemia. Although anaemia can be caused by other deficiencies such as B12 and folic acid, iron deficiency is by far the most common cause during pregnancy [3].
Despite numerous options of iron replacement therapy [4], iron deficiency anaemia (IDA) remains highly prevalent across the globe and, according to the World Health Organisation (WHO), 2019, accounts for 45% in the most vulnerable groups of pregnant women and infants (<5 years old). The importance of this is stipulated in the WHO’s 2025 target to halve global anaemia in women of reproductive age. Iron deficiency without anaemia affects 18% of pregnant women with 7% in the first trimester and 30% in the third trimester [5].
IDA during pregnancy has been linked to aberrant neonatal neurodevelopment, e.g., autism spectrum disorder and attention deficit hyperactivity disorder [6][7][8][9]. Excess iron replacement therapy is thought to be associated with preterm birth and infection [9]. Essentially, there are negative pregnancy outcomes associated with haemoglobin levels lower 110 g/L or greater than 130 g/L. Maintaining adequate levels of iron throughout pregnancy is challenging due to changes in iron demand at each trimester; more dietary iron is absorbed in the third trimester (7.5 mg) than in the first trimester (0.8 mg) [10].

2. Diagnosis

Accurate diagnosis, treatment and maintenance of iron and haemoglobin levels in pregnant women are a work in progress. This is because current diagnostic tests are not nationally standardised [11], and interpretation is problematic in the presence of inflammation and infection [12]. Treatment is dependent on diagnosis; therefore, reviewing the limitations to interpretation of standard tests is necessary to assess their effectiveness. Current iron testing includes serum ferritin, which is an acute phase reactant whose levels are affected by inflammation; Transferrin (Tf) saturation, which fluctuates due to diurnal variation of serum iron; and serum iron, which decreases with malignancy, infection and inflammation and increases with liver disease [13]. Haemoglobin measurement tends to be more robust however, and establishing the cause of anaemia is important, i.e., caused by inflammation or iron deficiency [9]. This would help prevent unnecessary prescription of iron replacement therapy.
Serum ferritin: Serum ferritin (diagnostic cut off in pregnancy ≤ 30 ng/mL) has been the most commonly used non-invasive test in diagnosing IDA when interpreted with haemoglobin levels. However, this is complicated in the presence of inflammation and lacks evidence in pregnancy. During an inflammatory response to infection, cytokines such as interleukin 1 beta prevent the release of cellular iron into the circulation. This occurs by upregulating the expression of heavy chain ferritin (in liver cells), resulting in increased iron storage. This deprives the invading pathogen of the hosts’ iron which it needs to thrive [14]. It has been suggested that the increase in maternal cytokines during the acute inflammatory response is more detrimental to foetal neurodevelopment than (some/specific) the invading pathogen itself [15]. In addition, pathogens such as Salmonella are capable of hepcidin-independent ferroportin (FPN) downregulation within macrophages [16]. This further complicates diagnosis because it results in iron delocalisation (i.e., accumulation in secretions and tissue) while deficient in blood, akin to hereditary haemochromatosis phenotype.
Serum hepcidin is a 21st century discovery: Hepcidin is a hepatic-produced 25-amino acid peptide protein that functions as the main systemic mediator of iron homeostasis. It was first discovered in human urine and serum and described in the year 2000. The functional hormone, hepcidin-25, is the cleaved product of prohormone (60 amino acids), which is a divided product of preprohormone (84 amino acids) encoded by the HAMP gene. Within circulation, hepcidin-25 is bound to α-2-macroglobulin and albumin. Functionally, hepcidin binds to, internalises and degrades FPN, the cell membrane bound gatekeeper of transcellular iron transport. FPN is found on enterocytes, hepatocytes, reticuloendothelial macrophages and adipocytes. Therefore, an increase and decrease in levels of hepcidin causes opposing iron availability. Hepcidin is upregulated and downregulated by several factors illustrated in Table 1; a complete lack of hepcidin causes juvenile haemochromatosis, and overexpression causes iron-restricted anaemia.
Table 1. An illustration of factors influencing levels of circulating hepcidin.
Upregulated Downregulated
  • Post partum
  • First, second and third trimester of pregnancy
  • Inflammation (Cytokine IL-6 induced) (Host protection mechanism from infection by depriving microbes of iron supply)
  • IDA (Remains downregulated during inflammation in severe cases)
  • Raised serum iron levels
  • Haemolytic anaemias
  • Oral or IV iron intake
  • Anaemias with ineffective erythropoiesis
  • Infection
  • Chronic liver disease
  • Low GFR and CKD
  • Chronic HCV infection
Studies on serum hepcidin expression to see if it is a superior test over the current standard iron tests have not been conclusive. Research results vary in showing it to be comparable [17], superior [18] or advantageous only when it is used in conjunction with another test, such as haemoglobin concentrations [19].
Initially linked to iron homeostasis in 2001, there is yet to be a standardised, robust diagnostic laboratory test for hepcidin. Current options for the measurement of hepcidin consist of testing levels in urine or serum, both of which show correlating levels (excluding renal failure). Techniques such as enzyme-linked immunosorbent assay (ELISA) and mass spectrometry have been employed in research to measure hepcidin, with the first commercial ELISA test having been trialled in 2011 by Geerts, Vermeersch and Joosten [20], although on a geriatric cohort of patients not in pregnancy. There are two main differences between the two techniques: cost and specificity. Mass spectrometry has high specificity in distinguishing the different hepcidin isoforms; however, it is not cost-effective; ELISA and competitive enzyme-linked immunosorbent assay (C-ELISA) are cost-effective and relatively efficient in measuring hepcidin levels but do not distinguish between isoforms, rather portraying total hepcidin. Affordability and turnaround times are of importance when integrating such a test within the healthcare setting.
Ratios: In terms of IDA diagnosis, correlation of serum ferritin and hepcidin [1] does not necessary make serum hepcidin superior to serum ferritin, and it might not be cost-effective to use both tests. However, its usefulness may be when used in conjunction with other tests or as ratios to predict risk of IDA. Likelihood ratios, indices or quotients (referred to hereafter as ratio) are calculated using standard test results that have deter-mined sensitivity and specificity in ratio format to predict the likelihood of a positive or negative diagnosis in pathology. The idea of using ratios is not a new one [21] but would be a novel approach in predicting IDA risk in pregnant women. Most iron-status-related ratios have been studied in cohorts which are different to pregnancy, such as cirrhosis [22], blood donors [21], haemoglobinopathies [23], neurodegeneration [24] and athletes [25]. However, applying this approach to pregnancy would potentially be beneficial to further guide treatment allowing for a more cost-effective, non-invasive and preventative strategy.
Considering that both serum ferritin and hepcidin levels are affected by inflammation [26][27], it could therefore be useful to include cytokines and/or soluble transferrin receptor (TfR) testing to aid differential diagnosis. One such example is an animal study by Sangkhae et al., 2020, which included a ratio where PIDI determines the risk of iron deficiency to the foetus by measuring FPN and TfR1 levels on the placenta following parturition [28]. However, this has not tested on human samples, and more research is needed in this area.
Another example on using ratios in the diagnosis of iron deficiency is the Mentzer index, used to distinguish the likelihood of iron deficiency versus a haemoglobinopathy. This is calculated from dividing the mean corpuscular volume (MCV) by the red cell (RBC) count from a full blood count result. If the value is above 13, the likelihood is ID, whereas lower than 13 is indicative of beta thalassemia [29].
Total iron-binding capacity (TIBC) is also a diagnostic tool for IDA and iron overload. TIBC values are also derived from the calculation of the ratio of serum iron and total iron-binding capacity, which is an indirect measure of Tf levels [30].
Under similar principles, measuring the ratios of iron metabolism proteins can be applied in the early diagnosis of incipient IDA in pregnancy. It may be useful to investigate ratio combinations using different iron metabolism markers, such as hepcidin-to-ferritin ratio [31], hepcidin-to-Tf ratio and ferritin-to-Tf ratio [32].

This entry is adapted from the peer-reviewed paper 10.3390/ijms241713323

References

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