Summary

Iron is an essential element to our body, participating in many metabolic functions. Both iron deficiency and overloading can lead to severe clinical consequences. The body iron status can be measured using haematological and biochemical indices. In this paper, we discuss the clinical usefulness and limitations of the common biochemical markers relating to iron metabolism, namely serum iron, serum ferritin, total iron binding capacity, the derived transferrin saturation percentage and a more recently introduced parameter, soluble transferrin receptor. Common misconceptions regarding the interpretation of these markers would be highlighted.

摘要

鐵是許多人體代謝活動中的基本元素。無論鐵缺乏或過量都會引致嚴重病症。體內的鐵狀況可藉血液及生化指數去衡量。本文將討論與鐵代謝相關的常用生化標記在臨床上的應用及局限性。它包括血清鐵，血清鐵蛋白，總鐵結合力，從計算得來的轉鐵蛋白飽和度，以及近期開始引用的可溶性轉鐵蛋白受體。同時亦強調在詮釋標記量值時的常見謬誤。

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Introduction

Iron plays a vital role in many metabolic functions, especially in oxygen transport and energy metabolism. Both iron deficiency and overloading can lead to severe consequences. The iron status of the body can be measured using haematological and biochemical indices. There is no single ideal marker and awareness of associated limitations is critical in the correct interpretation and assessment of body iron status. Iron is distributed into a number of physiological compartments, namely in the red blood cells (RBCs) in the form of haemoglobin, as a component of many oxidative enzymes, in muscles in the form of myoglobin and stored in cells in the form of ferritin. Parameters for the investigation of iron status reflect changes in different body iron compartments and are affected in different manners to varying extents even for a given level of iron depletion or overload. Commonly available laboratory tests for the investigation of body iron status can be broadly categorised into haematological and biochemical markers. Haematological markers, such as haemoglobin (Hb), mean cell volume (MCV), mean cell haemoglobin (MCH), haematocrit, and red-cell distribution width (RDW), are useful in the assessment of the severity of iron deficiency. However, these markers are insensitive to early changes of iron status (Table 1). Therefore, a normal complete blood picture (CBP) does not rule out iron deficiency, but reflects the absence of functional haematological consequences at that stage. Biochemical markers, on the other hand, are the direct indices of the body iron status and include serum iron, serum ferritin, total iron binding capacity (TIBC) or serum transferrin, the derived transferrin saturation percentage (Sat%) and the more recently introduced soluble transferrin receptor (STFR). Because of the complicated array of laboratory tests, proper interpretation of an iron profile is not an easy cake. In this paper, we shall discuss the clinical utilities and the potential pitfalls of the aforementioned biochemical markers in relation to the investigation of iron deficiency and iron overload.

Serum ferritin

Case 1

A 65-year old female, with chronic peptic ulcer, complained of dizziness. Faecal occult blood testing was positive. CBP showed haemoglobin of 8.6 g/dL and a hypochromic microcytic red cell picture.

CollectioSerumn Time 9:30am
Serum						Unit		Reference range
Iron		4.2		Low		mmol/L		5 - 28
Ferritin		5		Low		pmol/L		22 - 640
TIBC		89		High		mmol/L		41 - 78
Sat%		5		Low		%		16 - 45
Question: Does the patient have iron deficiency?

Answer: This is a typical picture of longstanding iron deficiency (Stage III) and presents no diagnostic difficulty. Typically, as iron deficiency develops, ferritin stores would gradually deplete followed by an elevation in TIBC with consequential reduction in the Sat%. Eventually, there would be inadequate supply of iron for haemoglobin synthesis, resulting in anaemia. Absence of iron staining in bone marrow is the gold standard test for iron deficiency. However, this procedure at the bone marrow is too invasive for routine practice. The alternative is a therapeutic trial of oral iron. A positive response to iron therapy would further confirm the diagnosis. The patient was given parental iron with haemoglobin and red cell morphology normalised after three months.

Case 2

A 36-year old female complained of menorrhagia for three months with a normal complete blood picture.

Collection Time 10:15am
Serum						Unit		Reference range
Iron		8.1				mmol/L		5 - 28
Ferritin		13		Low		pmol/L		22 - 640
TIBC		46				mmol/L		41 - 78
Sat%		18				%		16 - 45
Question: Does the patient have iron deficiency?

Answer: Stage I iron deficiency. Ferritin is the major iron storage protein in tissues. A small amount is present in the circulation. Serum ferritin concentration varies directly with the body iron stores. Therefore, serum ferritin is the most sensitive marker of iron deficiency in metabolically stable patients. It is the first marker to fall in stage I iron deficiency when other markers remain normal. The only reason for a low serum ferritin is iron deficiency. Iron deficiency is the most common nutritional deficiency worldwide. About 10% of the pre-menopausal adult female population has early iron deficiency without anaemia.¹ In patients with obvious cause of excess iron loss or increased body iron requirement (e.g. pregnancy and lactation), iron status should be checked even in the presence of a normal blood picture in order to document early iron deficiency and treat accordingly.

Case 3

A 42-year old healthy female had pneumonia recently. Her CBP revealed leucocytosis, Hb 12.1 g/dL (reference range 11.7 - 14.8), MCV 66.7 fL (82.0 - 96.9), MCH 21.1 pg (27.5 - 33.4), RDW 14.1 % (11.7 - 14.0) and presence of microcytosis and hypochromasia.

Collection time 8:40am
Serum						Unit		Reference range
Iron		4.2		Low		mmol/L		5 - 28
Ferritin		1450		High		pmol/L		22 - 640
TIBC		30		Low		mmol/L		41 - 78
Sat%		12		Low		%		16 - 45
Question: Does the patient have iron deficiency?

Answer: Acute phase reaction without iron deficiency. Serum ferritin is a positive acute phase reactant and its concentration rises significantly in acute illness and acute liver pathology. On the other hand, serum iron and transferrin are negative acute phase reactants and therefore serum iron and TIBC, which is an indirect measure of transferrin concentration, may decrease in the event of acute illness. This patient was later found to have thalassemia trait.

Case 4

An 82-year old female with history of congestive heart disease and hypothyroidism on thyroxine replacement is found to be anaemic with Hb 8.4 g/dL (11.7 - 14.8), MCV 81.2 fL (82.0 - 96.9), MCH 26.2 pg (27.5 - 33.4), RDW 15.9 % (11.7 - 14.0) and presence of anisocytosis, microcytosis and hypochromasia.

Collection time 8:40am
Serum						Unit		Reference range
Iron		3.2		Low		mmol/L		5 - 28
Ferritin		856		High		pmol/L		22 - 640
TIBC		36		Low		mmol/L		41 - 78
Sat%		8		Low		%		16 - 45
Question: Does the patient have iron deficiency?

Answer: Anaemia of chronic disease (ACD) without iron deficiency. This condition remains a class of anaemia that is difficult to distinguish from anaemia due to iron deficiency. ACD can be seen in patients with chronic infective, inflammatory or neoplastic diseases. Typically, ACD is associated with reduced concentrations of serum iron, transferrin, TIBC, raised ferritin and erythrocyte sedimentation rate or C-reactive protein. Much of the reason for the observed pattern of the iron profile is due to acute phase reaction as explained in Case 3. Patients with ACD in fact have adequate body iron stores which, however, are compartmentalised in the reticuloendothelial system. It is thought that iron is retained within the reticuloendothelial system and not made available for erythropoiesis, hence anaemia ensues.² (Please also see Cases 11 and 12 below.)

Case 5

A 75-year old female has rheumatoid arthritis and anaemia. CBP showed Hb 7.6 g/dL (11.7 - 14.8), MCV 73.9 fL (82.0 - 96.9), MCH 24.1 pg (27.5 - 33.4), RDW 16.1 % (11.7 - 14.0) and presence of anisocytosis, microcytosis and hypochromasia.

Collection Time 8:30am
Serum						Unit		Reference range
Iron		3.8		Low		mmol/L		5 - 28
Ferritin		143				pmol/L		22 - 640
TIBC		30		Low		mmol/L		41 - 78
Sat%		11		Low		%		16 - 45
Question: Does the patient have iron deficiency?

Answer: ACD with concomitant iron deficiency. ACD and iron deficiency can coexist in some patients with chronic diseases. These may pose a diagnostic confusion because of the inflammatory effects exerting on the biochemical iron markers. The optimal cut-off values of serum ferritin to distinguish iron deficiency from ACD were examined in a landmark study in 259 anaemic patients over 65 years of age tested against bone marrow examination.³ The authors suggested that co-existing iron deficiency is likely when serum ferritin is less than 225 pmol/L (100 mg/L) in the presence of ACD. (Table 1) This cut-off is in fact widely adopted.

Case 6

A 45-year old female suffers from hypochromic microcytic anaemia.

Collection Time 7:45am
Serum						Unit		Reference range
Iron		2.0		Low		mmol/L		5 - 28
Ferritin		30				pmol/L		22 - 640
TIBC		80		High		mmol/L		41 - 78
Sat%		3		Low		%		16 - 45
Question: Does the patient have iron deficiency despite a "normal" ferritin?

Answer: Stage III iron deficiency. Many clinicians rely heavily on the reference ranges provided by the laboratories for their decision making. Unfortunately, reference ranges provided on the laboratory reports can sometimes be misleading. First of all, reference ranges are not equivalent to normal ranges. In reality, when establishing reference range for serum ferritin, it is difficult to exclude all asymptomatic subjects with early iron deficiency from a reference sample. There is also a considerable overlap between serum ferritin concentrations of normal and iron deficient subjects. Reference ranges for individual analytes should be used as a guide whenever appropriate. The most important thing is to consider the full test profile in the context of the clinical condition. In this case, we recommend the use of clinical decision cut-offs. In general, serum ferritin concentrations below 34 pmol/L (15 mg/L) and below 68 pmol/L (30 mg/L) are diagnostic of iron deficiency in patients without and with anaemia respectively.⁴

Case 7

A 6-month old baby girl was admitted for persistent fever for 1 week. Iron profile was checked for her decreased MCV 72.0 fL (74.0 - 105), HCT 0.28 (0.33 - 0.39), Hb 9.8 g/dL (9.5 - 16.5), MCH 25.3 pg (22 - 33), RDW 13.9 % (11.5 - 14.5). Later, she was diagnosed to have chronic benign neutropenia.

Collection Time 9:30am
Serum						Unit		Reference range
Iron		4.7		Low		mmol/L		5 - 28
Ferritin		664		High		pmol/L		22 - 640
TIBC		38		Low		mmol/L		41 - 78
Sat%		11		Low		%		16 - 45
Question: Does the patient have iron deficiency?

Answer: No. The reference ranges provided are not appropriate for this age group. The results are in fact normal when interpreted against age-specific as well as method-dependent reference ranges (For 1 - 6 months old infants: Iron 4.5 - 22.6 mmol/L; Ferritin 81 - 740 pmol/L; TIBC 24.7 - 65.3 mmol/L and Sat% 7 - 44 % respectively⁴). Therefore, one should observe if the laboratory report indicates that age-specific reference ranges are provided.

Case 8

A 26-year old pregnant lady at 28th week of gestation with Hb 11.5 g/dL.

Collection Time 8:46am
Serum						Unit		Reference range
Iron		7.9				mmol/L		5 - 28
Ferritin		55				pmol/L		22 - 640
TIBC		85		High		mmol/L		41 - 78
Sat%		8		Low		%		16 - 45
Question: Does the patient have iron deficiency?

Answer: No. The above reference ranges provided are for non-pregnant subjects only.

In our body, many biochemical markers are indeed age and sex dependent. Unfortunately, most of the time, reference ranges provided on laboratory reports are only suitable for adults aged 18 to 60 years old and are not tailored for any other special patient groups, e.g. paediatric and pregnant women. Correct interpretations of the iron profiles rely on the use of age-specific and method-dependent reference ranges for children, elderly and pregnant women.^4-6 Clinicians who are not aware of such limitations may misinterpret the laboratory results leading to unnecessary investigation and treatment. Paediatric values of serum ferritin vary a lot from adult range. In pregnant women, serum ferritin can normally fall below 45 pmol/L (20 mg/L) during the second and third trimesters.⁷ Moreover, pregnancy and oral contraceptives can increase the TIBC up to 90 mmol/L.⁸ An increased TIBC can result in apparently low transferrin saturation, which does not necessarily indicate iron deficiency. Iron deficiency is considered unlikely when Hb is greater than 11.0 g/dL and serum ferritin higher than 68 pmol/L (30 mg/L) in the first trimester; Hb greater than 10.5 g/dL and serum ferritin higher than 45 pmol/L (20 mg/L) in the second trimester.⁹ Different clinical guidelines are available.^10,11 Serum ferritin concentrations rise remarkably after the age of 65 years. Only 55% of iron deficient elderly had a serum ferritin less than 40 pmol/L (18 mg/L).¹² Higher diagnostic cut-offs of iron deficiency are recommended for hospitalized elderly <100 pmol/L (45 mg/L)⁴ and for community based elderly <49 pmol/L (22 mg/L).⁶ The importance of appropriate reference ranges and use of clinical decision cut-offs cannot be overemphasized during laboratory results interpretation. Remarks on the appropriateness of the reference ranges should be provided by the testing laboratories. Advice from Chemical Pathologists should be sought if in doubt.

Case 9

A Caucasian man is incidentally found to have deranged liver function tests.

Collection Time 8:50am
Serum						Unit		Reference range
Iron		39		High		mmol/L		5 - 28
Ferritin		300				pmol/L		22 - 640
TIBC		28		Low		mmol/L		41 - 78
Sat%		57		High		%		16 - 45
Question: Does the patient have iron deficiency?

Answer: Yes.

The determination of the iron saturation of serum transferrin is useful in screening iron overload. It has a higher diagnostic predictive value and is more sensitive than that of serum ferritin. Sat% > 45% in female and > 55% in male and postmenopausal women indicate iron overload despite normal serum ferritin. It is important that fasting morning samples should be used to minimize the effects of dietary iron and diurnal variation. Serum ferritin levels > 450 pmol/L (200 mg/L) in women and 675 pmol/L (300 mg/L) in men indicate increased iron stores. Hypothyroidism and Vitamin C deficiency decrease the serum ferritin concentrations and may mask the iron overload. Hereditary haemochromatosis should be considered first in Caucasians patients with prevalence of about 1 in 300 of the Australian population.¹²

Serum iron, TIBC and transferrin saturation

Case 10

A 47-year old female went to have a body check in a clinic after work.

Collection Time 8:45pm
Serum						Unit		Reference range
Iron		2.8		Low		mmol/L		5 - 28
Ferritin		112				pmol/L		22 - 640
TIBC		44				mmol/L		41 - 78
Sat%		6		Low		%		16 - 45
CRP		0.3				mg/dL		<0.76
Question: Does the patient have iron deficiency?

Answer: Although it is not a common practice to include iron status in doing general body check-up, iron status should be considered in patients having high risk factors of iron deficiency, e.g. menstruating, dieting, pregnancy, lactation, vegetarian, infants and toddlers,¹³ preschool children,¹⁴ adolescents in rapid growth phases,¹⁵ and people with a poor dietary intake of iron e.g. those who do not eat red-meat. The above iron profile does not suggest iron deficiency. The blood sample was taken at night time, when serum iron is at its lowest concentration level, leading to an apparently low Sat%.

Care is required in interpreting serum iron results because they are influenced by a variety of physiological and pathological factors. Men have serum iron 10-20% higher than women. Morning levels are higher than from afternoon samples. The diurnal variation can fluctuate as much as 50%. For analytes showing significant diurnal difference, reference ranges provided are usually those referring to morning samples disregarding to the time of blood collection. Since Sat% is derived from dividing serum iron by TIBC and multiplying by 100, an apparently low serum iron in any evening samples can result in a low Sat% despite a normal TIBC. In contrast, both serum ferritin and transferrin do not show significant circadian rhythm. In Case 10, the normal serum ferritin and CRP (excludes acute phase reaction) concentrations confirm a normal body iron status. Serum iron is also sensitive to day-to-day fluctuation of dietary iron intake. To avoid confusion, fasting morning samples should be taken for iron profile whenever possible. In addition, very low values may be seen immediately prior to, and during, menstruation. In summary, serum iron on its own provides no useful information and it should be interpreted in conjunction with serum TIBC or transferrin. One should not rely on a single serum iron or transferrin saturation result to diagnose iron deficiency. In uncomplicated iron deficiency, low Sat% must be accompanied with a high TIBC.

Soluble transferrin receptor (STFR)

Case 11

An 81-year old female has tuberculosis of the lung and microcytic hypochromic anaemia with Hb 9.8 g/dL.

Collection Time 9:40am
Serum						Unit		Reference range
Iron		2.0		Low		mmol/L		5 - 28
Ferritin		201				pmol/L		22 - 640
TIBC		31		Low		mmol/L		41 - 78
Sat%		7		Low		%		16 - 45
CRP		28		High		mg/dL		<0.76
STFR		2.8				mg/L		1.9 - 4.4
STFR/log ferritin		1.0
Question: Does the patient have iron deficiency?

Answer: ACD 

Case 12

A 79-year old male has chronic obstructive pulmonary disease and microcytic hypochromic anaemia with Hb 8.8 g/dL.

Collection Time 9:30am
Serum						Unit		Reference range
Iron		2.4		Low		mmol/L		5 - 28
Ferritin		220				pmol/L		22 - 640
TIBC		20		Low		mmol/L		41 - 78
Sat%		10		Low		%		16 - 45
CRP		17.6		High		mg/dL		<0.76
STFR		6.1		High		mg/L		2.2 - 5.0
STFR/log ferritin		3.1
Question: Does the patient have iron deficiency?

Answer: ACD with true iron deficiency.¹⁶

Soluble transferrin receptor (STFR) is a good marker to differentiate ACD from iron deficiency in concurrent inflammation. STFR is the circulatory extracellular part of transferrin receptor which is expressed on the surface of human cells that require iron. Its concentration reflects cellular iron status. When the iron store is depleted, transferrin receptors are up-regulated to enable the cell to compete more effectively for iron. High STFR concentrations are associated with iron deficiency. The merit of STFR is that it is not affected by acute or chronic inflammatory conditions. Compared with ferritin, STFR is normal in patients with ACD and during inflammatory conditions. The use of STFR/log ferritin index is found to have a better diagnostic efficiency than STFR or ferritin alone.¹⁷ One of the limitations of STFR is that it is also elevated in any causes of increased effective or ineffective erythropoiesis, e.g. in haemoglobinopathies.

Conclusion

In summary, despite the iron profile being a regularly requested laboratory investigation, it is not uncommon that one falls short of achieving the original intent simply due to inappropriate interpretation. As illustrated by the above cases, proper interpretation of iron profile is essential for effective patient management.

Key messages

1. Iron deficiency is the most common nutritional deficiency worldwide. About 10% reproductive females,¹ 5-18% infants and toddlers,¹³ and 20% preschool children¹⁴ show early iron deficiency with decreased serum ferritin concentrations without anaemia. This imposes a significant public health concern in that potential negative functional consequences may be associated with even mild iron deficiency.
2. There is no single ideal marker for the investigation of iron status and each laboratory marker reflects different stages of iron deficiency.
3. Haematological markers fall in the late stage of iron deficiency; therefore, a normal complete blood picture does not rule out iron deficiency, but merely its haematological consequences.
4. Gold standard test for iron deficiency is the absence of iron staining in bone marrow or a positive response after a therapeutic trial of oral iron.
5. Age-specific reference ranges should be used for children, elderly and pregnant women when interpreting the iron status.
6. Iron deficiency should not be diagnosed by a single serum iron and/or transferrin saturation test result. In uncomplicated iron deficiency, decreased serum iron and low transferrin saturation must be accompanied with a high TIBC.
7. Anaemia of chronic disease and iron deficiency can coexist in some patients with chronic diseases. A serum ferritin less than 225 pmol/L (100 mg/L) suggests co-existing iron deficiency in the presence of chronic illnesses.
8. Ferritin is the most sensitive marker for iron deficiency in metabolically stable patients. However, its clinical usefulness is compromised in most hospitalized patients who have concomittent acute or chronic inflammatory conditions.
9. Compared with ferritin, soluble transferrin receptor concentration is normal in patients with anaemia of chronic disease and during other inflammatory conditions. Its use in patients with serum ferritin concentrations less than 225 pmol/L can enhance the diagnostic efficiency of iron deficiency in anaemia of chronic disease.

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References