Guideline for the laboratory diagnosis of iron deficiency in adults (excluding pregnancy) and children

Abstract

This Good Practice Paper was compiled according to the British Society for Haematology (BSH) process at: https://b-s-h.org.uk/media/16732/bsh-guidance-development-process-dec-5-18.pdf. The BSH produces ‘Good Practice Papers’ to recommend good practice in areas where there is a limited evidence base but for which a degree of consensus or uniformity is likely to be beneficial to patient care. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org. Up to June 2020 MEDLINE and PUBMED were searched systematically for publications in English from 1966 to 2020, with additional ad hoc searches up to June 2021 for any new evidence using key words: iron deficiency, anaemia and each of the parameters discussed. Review of the manuscript was performed by the BSH Guidelines Committee General Haematology Task Force, the BSH Guidelines Committee and the General Haematology sounding board of the BSH. It was made available on the members’ section of the BSH website for comment. The laboratory diagnosis of iron deficiency is difficult because iron homeostasis is dynamic. No single test can provide an accurate assessment of iron absorption, transport, storage, and utilisation. The different assays available to assess iron and its stores will each be discussed, and recommendations made pertinent to practice within the UK. Iron metabolism in adults and children can be considered equivalent and these recommendations are applicable to both paediatric and adult practice. Iron deficiency is the most common cause of anaemia worldwide. The World Health Organization (WHO) defines anaemia as a condition in which the number of red cells (and consequently their oxygen-carrying capacity) is insufficient to meet the body’s physiological needs.1 In addition to this there is a recognised definition of haemoglobin concentration below age- and gender- specific thresholds: <130 g/l for adult men, <120 g/l for adult non-pregnant women and paediatric values that start at <110 g/l for ages 6–59 months and increase with age.1 The prevalence of iron deficiency anaemia in the UK population ranges between 0% and 6% according to age and sex, higher rates being recognised in certain demographic groups such as menstruating women and adults aged >85 years.2 Prevalence of iron deficiency in the absence of anaemia is not well documented. Laboratory tests should be performed in the context of a detailed history and clinical examination with an awareness of how results will direct onward investigation and management based on national gastrointestinal and gynaecology guidelines and local pathways within individual healthcare settings. This good practice paper should benefit all healthcare professionals investigating paediatric and non-pregnant adult patients with suspected iron deficiency. It supplements existing BSH guidelines for the specific situations of diagnosing functional iron deficiency and the diagnosis and management of iron deficiency in pregnancy.3, 4 A suggested algorithm for the investigation of iron deficiency anaemia is included below (Fig 1). Calculation of red cell values haematocrit (Hct), mean corpuscular volume (MCV), mean cell haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and red cell distribution width (RDW) can be found in Dacie and Lewis Practical Haematology.5 The full blood count (FBC) will confirm anaemia if haemoglobin (Hb) is below the laboratory reference range. Modern laboratory spectrophotometric (or colorimetric) measurement of Hb is accurate and reliable. Hb and its related parameter Hct do not provide information with regard to iron status. At population level there is significant overlap in the distribution of Hb between iron-replete and -deficient individuals.6 Automated FBC analysis includes the validated red cell parameters MCV, MCH and MCHC, as well as RDW, although in the UK this latter index is not yet quality assured. Each can be reduced by an iron deficient state, but not to a consistent enough degree for diagnostic clarity. Iron deficiency is classically associated with microcytic hypochromic anaemia, but low MCV, MCH or MCHC can be seen in other conditions, notably the thalassaemias. MCV can be normal in up to 40% of unselected iron-deficient patients and also in states of mixed haematinic deficiency.7 MCV is well known to be affected by pre-analytical variables such as sample temperature and storage times.8, 9 MCV, MCHC and RDW cannot be directly compared between analysers. Although the MCH retains accuracy and stability between analysers and is somewhat less affected by pre-analytical variables, it is not specific for iron deficiency. None of these parameters alone is able to predict response to oral iron.10-12 Erythrocytes contain approximately half the body iron at any one time.13 The red cell indices offered by more recent analysers can be predictive in diagnosing iron deficiency and response to iron (Table I). These advanced parameters include %HRC and the reticulocyte mean Hb content. Methods and nomenclature for these indices vary by analyser, but a high %HRC or low reticulocyte mean Hb content in persons without thalassaemia indicates that iron is not adequately available to the newly developed red cells. This can be due to iron deficiency or iron restriction.10 The %HRC is defined as the proportion of erythrocytes with cellular Hb of <280 g/l (HYPO%, Siemens Advia; %HPO, Abbott), or <17 pg (%Hypo-He; Sysmex), or as a calculated transformation of the MCHC [low haemoglobin density (LHD)%, Beckman-Coulter]. It relates to iron status over the preceding 3 months. A value for %HRC of >5% suggests iron deficiency. The %HRC is affected by sample transport time and should only be used in laboratories that can reliably ensure analysis within 3-4 h of phlebotomy. This limits its usefulness to some extent, but there is cost-benefit in not having to use a reticulocyte reagent.14 Measurement of mean reticulocyte Hb content is less dependent on pre-analytical factors, although reference intervals and diagnostic thresholds are method dependent.10There is evidence in the setting of chronic kidney disease (CKD) that these measures can predict iron response even in the face of raised serum ferritin.15 Suggested thresholds for predicting iron deficiency vary from 25 to 30 pg.16, 17 A cut-off of 28 pg has been shown to predict iron response in paediatric patients with cancer.18 Until further data is available a threshold of 29 pg is a pragmatic recommendation. Morphological changes develop with iron deficient states although they are not diagnostic. In early deficiency anisocytosis precedes hypochromia and microcytosis, with more classical red cell changes (elliptocytes, pencil cells and some target cells) occurring when the Hb drops below 100–110 g/l.19 The value of inspecting a peripheral blood film in anaemia is to identify specific changes not detected by automation.10 Microscopy can suggest alternate or concurrent diagnoses including microangiopathy, myelodysplasia, thalassaemias and other haemoglobinopathies. Morphological interpretation can be affected by sample storage time, staining quality and operator skill. Ferritin represents the main bulk of protein-bound tissue iron storage. A small proportion leaks into plasma and can be quantitated accurately by immunoassay. In health there is a direct relationship between reticuloendothelial iron stores and serum ferritin,20 making it a powerful laboratory test when free of confounders. Levels of <15 µg/l predict a high likelihood of iron deficiency.21 Levels up to 30 µg/l can still be consistent with deficiency, although is less specific.22 Reference ranges can vary by analytical method and by patient population, so local parameters should be established and regularly reassessed.22, 23 Normal values are not significantly different between those aged <5 years compared to children aged >5 years and adults.22 The behaviour of serum ferritin as an acute phase protein means that levels rise in response to inflammatory states as well as kidney disease, liver disease and malignancy. These variables confound clinical interpretation, a problem reflected by the difference in ranges suggested by international guidelines across a range of conditions.24 Formulae for interpreting ferritin in the context of raised inflammatory markers, either C-reactive protein (CRP) or erythrocyte sedimentation rate (ESR), have been proposed but we do not consider the evidence base robust enough to apply a ‘corrected’ assessment of iron status in current practice.25 Any clinical or laboratory evidence for an active inflammatory state should provide impetus for considering further investigation for iron deficiency regardless of a serum ferritin quantitation that lies within the normal range.26, 27 Normal ranges will vary by laboratory; for clarity we suggest a value of <150 µg/l be used as a trigger to consider further investigation for iron deficiency. Serum iron measures only the oxidised ferric form bound to transferrin and not the functional iron component of Hb. An individual with iron deficiency will usually exhibit a low concentration of serum iron, but it is a dynamic parameter with well-established day-to-day variability.28 Serum iron measurement is required to allow calculation of percentage TSAT. Measuring serum iron in isolation is not helpful and its inclusion in the iron profile report to clinicians should be assessed by each individual laboratory.3 The TIBC is calculated by adding excess iron to serum, removing unbound iron then measuring the iron concentration in the remaining sample. An alternative index, the UIBC, is more easily automated, and gives a TIBC when the value is added to the serum iron concentration. The concentration of transferrin, the predominant plasma iron transport protein, can be measured by direct immunological assay. TIBC and transferrin rise in iron deficiency. Transferrin is a negative acute phase protein so its concentration may be reduced in inflammation. TIBC and transferrin have less variability than serum iron quantitation, but specificity remains poor.29 The TSAT is the ratio of serum iron to transferrin (or TIBC) expressed as a percentage, representing the proportion of serum transferrin binding sites occupied with iron molecules.30 It relies on serum iron concentration so carries a similar caveat regarding variability and lack of specificity.29 Diurnal fluctuations can be up to 70%. Clinical disorders such as malnutrition and chronic disease cause a decrease in transferrin synthesis and therefore increase the TSAT, reducing its usefulness.31 A diagnostic threshold for TSAT has not been well established, with some guidelines suggesting <20% as a screening threshold. This is based on limited evidence. We prefer the increase in specificity provided by a lower target of <16%, which has some support in the literature.32, 33 It may be useful when the measurement of serum ferritin is equivocal. Developing erythroid cells adjust the number of TfRs on their surface, a proportion of which can be detected in the serum. Iron deficiency prompts an increase in receptor density and an associated rise in the sTfR concentration.34 The sTfR has been suggested as an alternative to conventional laboratory tests of iron deficiency.35 Braga et al.34 compared the diagnostic accuracy of sTfR versus ferritin, concluding that sTfR has a potential clinical value in detecting iron deficiency, but there is a lack of robust studies defining its overall accuracy in addition to lack of standardisation and reference material.36 Choi37 advises that it is not a useful test in early-stage iron deficiency and is more appropriate in advanced cases, suggesting it has no advantage over ferritin. Increased levels are also seen in states of erythroid hyperplasia, such as haemolysis, megaloblastic anaemia, thalassaemia, and hypoxia.38 sTfR is not useful in identifying iron deficiency in patients with beta thalassaemia trait in the early stages of deficiency but is abnormal in later stages.39 Iron is incorporated into porphyrin during the final steps of haem synthesis. A reduction in available iron allows uptake of zinc instead, with ZPP produced as a measurable by-product. Haematofluorometric measurement of ZPP has important limitations; misleading elevations are found in the presence of hyperbilirubinaemia40 and chronic renal failure.41 A progressive spurious increase in ZPP is seen as Hb falls to <100 g/l, so many subjects with moderate or severe anaemia have high ZPP irrespective of cause. These shortcomings can be overcome by washing red cells prior to testing or adjusting the Hb concentration to a standard value, but sample manipulation is time-consuming. Even without laboratory limitations the value of ZPP as an index of iron deficiency is questionable. Raised values also occur in functional iron deficiency, lead poisoning and conditions that stimulate excess porphyrin synthesis such as the thalassaemias.42 In simple iron-deficiency states, it adds no further information beyond the serum ferritin. In multifactorial anaemia it lacks specificity, as demonstrated in a population study where malaria and haemoglobinopathies were prevalent alongside iron deficiency.43 The advantage of ZPP needing only a small sample volume (0.2 ml) and being an easily performed test are not enough to recommend its use in a developed country.44-46 The predominant form of hepcidin in humans is hepcidin-25 produced in the liver in response to iron levels, inflammation and erythropoietic demand.47 Inflammation triggers a rise in hepcidin concentration, inhibiting iron release from macrophages, enterocytes, and hepatocytes. Conversely in iron deficiency, as iron levels fall, synthesis of hepcidin is inhibited.47-49 Such measurements make hepcidin a useful putative diagnostic tool to assess iron status and response to oral iron50 although, as with other iron indices, there are confounders such as diurnal variation.51 Hepcidin assays tend to be mass spectrometry (MS) or radioimmunoassay techniques. These methods have good specificity, but their ability to detect low levels of serum hepcidin limits their sensitivity.52 Their use is limited in routine clinical laboratories mainly due to the expense of the equipment, availability and unsuitability for high throughput testing,52 and may be more appropriate in establishing a reference standard.53 Work is on-going to improve standardisation material and normal ranges.54 In recent years, enzyme-linked immunosorbent assay (ELISA)-based methods have become available. These are simple but still relatively expensive although more suitable for use in routine clinical laboratories. Very few commercial assays are currently available. However, development of the hepcidin-25 (bioactive) ELISA immunoassay has future clinical promise in a routine clinical laboratory. The present good practice paper has summarised up-to-date investigations available in the developed world for assessment of iron deficiency in children and adults, including situations where results should be viewed with caution. It has been designed to work in conjunction with relevant specialty-specific clinical guidelines like those related to pregnancy or gastroenterological bleeding. We expect the evidence base for use of other tests such as hepcidin to evolve over the next few years allowing further development of this algorithm. The BSH General Haematology task force members at the time of writing this good practice paper were Dr Mamta Garg (Chair), Dr Charlotte Bradbury, Dr Barbara De La Salle, Dr Emmy Dickins, Dr Savio Fernandes, Dr John Grainger, Ciaran Mooney, Dr Naomi Roy, Dr Sara Stuart-Smith, and Nicola Svenson. The authors would like to thank them, the BSH sounding board and the BSH guidelines committee for their support in preparing this good practice paper. The BSH paid the expenses incurred during the writing of this good practice paper. All authors have made a declaration of interests to the BSH and Task Force Chairs which may be viewed on request. The members of the writing group have no conflicts of interest to declare. Members of the writing group will inform the writing group Chair if any new pertinent evidence becomes available that would alter the strength of the recommendations made in this document or render it obsolete. The document will be archived and removed from the BSH current guidelines website if it becomes obsolete. If new recommendations are made an addendum will be published on the BSH guidelines website (www.b-s-h.org/guidelines). While the advice and information in this guidance is believed to be true and accurate at the time of going to press, neither the authors, the BSH nor the publishers accept any legal responsibility for the content of this guidance.