Defining functional reference limits for serum ferritin based on the association between iron deficiency and anaemia

Iron is an essential element required for physiological function and development. While necessary for humans, free-form iron is toxic and is therefore tightly regulated: it is either stored in a ferritin complex or circulates via transferrin. Increased loss or inadequate intake of iron can result in a state of malnutrition known as iron deficiency, during which circulatory ferritin levels decrease. Iron deficiency is the leading cause of anaemia and is a common nutritional deficiency. The World Health Organisation estimates almost a quarter of the global population have anaemia, most a result of iron deficiency. In Australia, anaemia is thought to affect 3% of the population, with 71% attributed to iron deficiency. 

Iron deficiency is diagnosed by laboratory testing of circulating serum ferritin. In Australia, serum ferritin is often requested as part of an iron panel, containing serum iron, transferrin, and transferrin saturation. Serum iron may incorrectly be assumed to represent iron homeostasis due to its vocabular similarity to ‘iron’. The first study of this thesis investigated the influence of serum iron test results on the diagnosis of iron deficiency by general practitioners among children. This study’s results showed that low serum iron results increased the likelihood of a diagnosis in patients with sufficient stored-iron levels, and normal serum iron results decreased the likelihood of a diagnosis in patients with low stored-iron levels. The inclusion of serum iron in the diagnostic process of iron deficiency therefore has the potential to lead to misdiagnosis. 

When diagnosing iron deficiency, serum ferritin test results are compared to a value representing ‘non-disease’ presented as either reference intervals or clinical decision limits. Reference intervals represent the distribution of results within the population, and clinical decision limits represent a threshold at which the risk of disease becomes high. Clinical decision limits for iron deficiency vary, and are generally based on expert opinion. The second study of this thesis presented a novel approach to identify an evidence-based serum ferritin level at which iron deficiency anaemia begins to occur, termed ‘functional reference limit’. This study’s results showed that the lower limit of the reference interval falls below the point at which the patient becomes anaemic, and provided a functional reference limit relevant to iron deficiency anaemia for children at different age and sex intervals. The third study of this thesis expands upon the concept of ‘functional reference limits’, drawing from other similar studies and presenting multiple approaches. Functional reference limits can be used as reference limits themselves, or as evidence for clinical decision limits. 

Collectively, the studies of this thesis provide important novel information for the diagnosis of iron deficiency and have considerable significance for its accurate diagnosis. Specifically, serum iron interpreted as a measure of stored-iron instead of serum ferritin confounds the diagnosis of iron deficiency, which may be improved if serum iron is removed from the diagnostic process. Furthermore, the laboratory interpretation of serum ferritin can be improved by using scientifically and clinically valid reference limits, with this thesis providing evidence for such a limit.