Iron deficiency

Recommended indicators of iron status for determining iron deficiency include serum ferritin, serum soluble transferrin receptor, and total body iron 1,2.

Ferritin

Serum ferritin is the most specific, non-invasive biochemical test to quantify total body iron stores. In the absence of inflammation, the concentration of serum ferritin is positively correlated with the size of the total body irons stores, with a low serum ferritin concentration reflecting depleted iron stores and therefore iron deficiency 3. However, serum ferritin is an APP and is elevated in response to infectious or inflammatory processes. In population- based surveys, this may artificially lower the prevalence of iron deficiency, thus It is recommended that serum ferritin be assessed along with measures of inflammation (e.g. CRP and/or AGP).
There are several methods to account for the increase in ferritin values caused by inflammation. One method is to adjust serum ferritin concentrations based on CRP and AGP using regression or arithmetic correction factors, such as the BRINDA 4 or Thurnham methods 5, respectively. When using these correction approaches to adjust ferritin concentrations, you can apply the cutoff values recommended for apparently healthy populations. Another method is to apply a higher serum ferritin cutoff value that defines deficiency, 30 μg/L or 70 μg/L, depending on the age group, to individuals with infection or inflammation 3.

Specimen collection and management: Most commonly, ferritin is measured in serum or plasma samples that are obtained by centrifugation from whole blood collected by venipuncture or finger prick. The whole blood needs to be refrigerated as soon as possible and processed to serum or plasma within 48 hours of blood collection. Ferritin in serum is stable for at least one week at 4˚C and for at least one year at -20˚C 6.

Biomarker analysis: Ferritin is measured using immunoassays, including methods that can be conducted either with a fully-automated clinical analyser or a manually executed ELISA assay. Commercial assay kits are available for both types of assay. There is no significant difference in within-run imprecision, between-run imprecision, limit of detection, recovery rate or linearity between commercial and home-made assays, or between automated multiple-analytes detection equipment and single laboratory apparatus, showing, overall, that the most common methods used for ferritin determinations are comparable 7.

The required analysis volume is typically <25 µL serum, however a minimum specimen volume of >150 µL serum may be needed to fill the sample cup for the clinical analyser. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can use EDTA or heparin plasma. Sandwich ELISAs are also available to measure serum ferritin along with other indicators, including those assessing other iron indicators and vitamin A and inflammation status 8.

Quality control measures are required to ensure high quality results 9. Commercial assay kits include calibration materials and may also include quality control materials. It is nonetheless recommended to establish “in-house” quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision is typically 5–10% for clinical analyser assays and around 10% for ELISA assays. Ferritin is also part of the CDC VITAL-EQA program and of CDC’s Performance Verification Program for Serum Micronutrients 10.

A WHO-developed serum-based international standard (recombinant ferritin RM 94/572) 11, used to verify method accuracy, is available through the National Institute of Biological Standards and Control (NIBSC). However, not every assay may be able to use this material because the assay performance may differ between native patient samples and reference materials that have undergone some processing. Quality control materials for serum micronutrients including ferritin are available from CDC to support in-house quality assurance programs for laboratories engaged in public health work 12. Moderate assay differences (e.g. 2.0–5.0% variability depending on the assay) are common in proficiency testing programmes, such as the US College of American Pathologists (CAP) Chemistry Survey 13.

Approximate budget requirements for analysis: Instrumentation needed for this method includes either a clinical analyser (approximately US$ 100 000) or a plate-washer, plate-reader, and various pipettes (approximately US$ 30 000). The cost for materials and supplies is around US$ 2–5 per sample for a commercial kit assay. Material costs may be slightly lower for laboratory-developed ELISA assays that measure serum ferritin in addition to other micronutrients.

Interpretation of results: The classification of iron stores based on serum ferritin concentrations by the presence or absence of inflammation are presented in Table 3.5. The generally accepted serum ferritin cutoff value for defining depleted iron stores is <15 μg/L for children over 5 years of age, adolescents, adults and women in the first trimester of pregnancy, while a cutoff value of <12 μg/L is used for children under 5 years of age (60). These cutoffs are appropriate when a regression or arithmetic correction approach has been applied to the ferritin concentrations to account for inflammation. If no mathematical correction for inflammation is applied to the ferritin concentrations, then the higher concentrations may be used to define deficiency (e.g., <30 μg/L or <70 μg/L).

Thresholds for elevated serum ferritin to identify risk of iron overload should be used only in clinical care with additional indicators and evaluation to establish the underlying cause.

Serum ferritin (μg/L)b
Apparently healthy individualsc Individuals with infection or inflammation
Infants and young children 0-4 years of age <12 <30
Children and adolescents 5-19 years of age <15 <70
Adults ≥20 years <15 <70
Pregnant women first trimesterd <15 <70

a Source: reference 3.

b Markers of inflammation should be assessed along with the ferritin concentration, and ferritin adjusted as necessary.

c For the purposes of this guideline, an apparently healthy individual is defined as an individual with physical well-being for their age and physiological status, without detectable diseases or infirmities.

d There are several physiological changes occurring in pregnancy that may contribute to the variation in thresholds of iron deficiency in pregnancy as defined by serum ferritin, including a physiological rise in acute phase proteins secondary to pregnancy; second trimester plasma volume expansion; and changes in inflammatory measures in the final trimester of pregnancy.

Transferrin receptor

Transferrin receptor is found on the cell membrane and allows iron-bound transferrin to enter the cell. When the iron supply is inadequate, the number of transferrin receptors on a cell surface increases. This enables the cell to compete more effectively for iron. The number of membrane receptors is in proportion to the soluble transferrin receptor (sTfR), a truncated form of transferrin receptor found in plasma. An increase in sTfR levels is seen in patients with iron deficient erythropoiesis or iron deficiency anaemia with increased erythropoiesis 14.

Because sTfR is not an acute phase protein, it may be less influenced by inflammation than ferritin. Other advantages of sTfR are that cutoff values do not vary with age or gender, or by physiologic state. However, the BRINDA project found that the relationship between sTfR and AGP was consistently significant and therefore recommended adjusting sTfR for AGP and malaria 15. The BRINDA project reported that the relationship between sTfR and CRP was most often not statistically significant. The decision to include malaria in the BRINDA adjustment was based on the physiologic response of sTfR during infection. Circulating sTfR levels may be elevated when there is increased red blood cell production or turnover or both, such as in the case of haemolytic anaemia 14.

Specimen collection and management: Most commonly, sTfR is measured in serum samples that are obtained by centrifugation from whole blood collected by venipuncture or capillary sample. Whole blood should be refrigerated as soon as possible and processed within a few days of blood collection. The serum is stable for at least one week at 4˚C, and for at least one year at -20˚C 6.

Biomarker analysis: sTfR is measured by immunoassays (clinical analyser or ELISA assay), most often using commercial assay kits. The required analysis volume is typically <25 µL serum, however a minimum specimen volume of >150 µL serum may be needed to fill the sample cup for the clinical analyser. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can utilize EDTA or heparin plasma. Sandwich ELISAs are also available to measure sTfR along with other indicators, including those assessing other iron indicators, vitamin A and inflammation status 8. The method imprecision is usually around 10%.

A WHO-developed serum-based reference reagent (recombinant sTfR RR 07/202) is available through the NIBSC. This reagent has an assigned value based on protein content because assays have not yet been standardized and assay results are not comparable. The US College of American Pathologists proficiency testing offers a performance programme for laboratories performing sTfR measurements. CDC’s Performance Verification Program for Serum Micronutrients 10 covers sTfR and CDC also offers quality control materials for serum sTfR to support in-house quality assurance programmes for laboratories engaged in public health work 12.

Approximate budget requirements for analysis: sTfR is measured on the same instrumentation as serum ferritin, however the cost for materials and supplies is higher (approximately US$ 5–10 per sample for a commercial kit assay). Material costs may be slightly lower for in-house developed ELISA assays that measure serum sTfR in addition to other micronutrients.

Interpretation of results: Assay-specific normal ranges for sTfR are available (for example, 2.9–8.3 mg/L for one brand of ELISA), however, there is no universally agreed normal range for sTfR. Similarly, there are no WHO definitions of public health problems based on sTfR prevalence. Using data generated with the Roche sTfR assay for the United States population in the National Health and Examination Survey (NHANES), cutoff values indicating iron deficiency were proposed as ≥6.0 mg/L for children 1-5 years and ≥5.3 mg/L for non-pregnant women 15-49 years 16.

Body iron index

The body iron index provides a quantitative assessment of body iron stores (index value >0 mg/kg) and indicates the size of the functional iron deficit. The functional deficit can be described as the amount of iron needed before it can be accumulated in the body’s stores, in an individual who is iron deficient (index value ≤0 mg/kg). The index is not a measure of total iron in the body. Previous terms used to describe this measure include “body iron”, “total body iron”, and “total body iron stores”.

In a controlled phlebotomy study, the sTfR only increased once the iron stores (measured by serum ferritin) were completely exhausted 17. When serum ferritin fell below 12 μg/L, the sTfR began to rise, roughly in proportion to the deficit in functional iron. This indicates that sTfR measures the deficit in tissue iron once stores are depleted. The combination of serum ferritin and sTfR levels can portray the spectrum of iron status from normal to severe deficiency. The formula to calculate the body iron index using ferritin and sTfR values (adjusted for measurement using the Ramco ELISA assay) in µg/L is as follows 18:

Body iron index (mg/kg) = –[log(Ramco sTfR/ferritin) – 2.8229]/0.1207

If the sTfR has been measured by the Roche assay instead of the Ramco ELISA assay, the relationship of that assay to the Ramco ELISA assay needs to be taken into consideration. The adjustment equation is as follows 19:

Ramco sTfR = 1.5 × Roche sTfR + 0.35 mg/L.

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  19. Pfeiffer CM, Cook JD, Mei Z, Cogswell ME, Looker AC, Lacher DA. Evaluation of an automated soluble transferrin receptor (sTfR) assay on the Roche Hitachi analyzer and its comparison to two ELISA assays. Clin Chim Acta. 2007;382:112–6.