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Biomarkers for the differentiation of anemia

and their clinical usefulness

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Journal of Blood Medicine
19 March 2013
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Christine A Northrop- Abstract: The World Health Organization defines anemia as the point at which the amount of
Clewes 1 hemoglobin in the circulation falls below World Health Organization cutoffs for specific age and
David I Thurnham 2 sex groups. Anemia is a worldwide problem of complex etiology and is associated with many
factors. The purpose of this review was to describe the biomarkers used to identify the nature of
1
Nutrition Consultant,
Cambridge, UK; 2Northern Ireland anemia in patients and in the community. The important biomarkers are the automated red cell
Centre for Food and Health, School counts, tests for nutritional deficiencies, hemoglobinopathies, and inflammation. Diseases are
of Biomedical Sciences, University of
Ulster, Coleraine, UK
important potential initiators of anemia, but biomarkers of specific diseases are not included in
this review, only the underlying feature common to all disease – namely, inflammation.
Keywords: iron deficiency, biological markers, blood cell count, inflammation, avitaminosis,
hemoglobinopathies

Introduction
The World Health Organization (WHO) estimates that 1.62 billion people (95%
confidence interval [CI]: 1.50, 1.74 billion), corresponding to 24.8% (95% CI: 22.9,
26.7) of the global population, are affected by anemia.1 However, although anemia
affects all population groups in all countries of the world, certain groups are more
vulnerable than others. The highest prevalence tends to be in preschool-age children
(ie, those aged 6–59 months), women of reproductive age (ie, those aged 15–49 years),
and pregnant women (Table 1). Data for lactating women do not seem to be available
at the national level.

Anemia
Anemia is defined as a hemoglobin (Hb) concentration below certain thresholds estab-
lished for specified population groups living at sea level (Table 2).2 Hb is the main
component of erythrocytes or red blood cells (RBCs). Red cell synthesis takes place
in the bone marrow and is under the control of erythropoietin produced by interstitial
fibroblasts in the kidney. There are several intermediate stages in the development of
the mature erythrocyte during which Hb is synthesized and the nucleus is catabolized.
Limitations in the supply of several essential micronutrients can affect the production
of new cells. Vitamins B6 (pyridoxine)3 and B2 (riboflavin)4,5 are needed for the syn-
thesis of the protein globin and iron has to be incorporated into the final Hb molecule.6
Correspondence: Christine A Northrop-
Restrictions in the supply of these nutrients will produce small red cells (microcytosis)
Clewes with low Hb concentration (hypochromia). In addition, zinc may be substituted in the
46 High Street, Little Wilbraham,
Cambridge CB21 5JY, UK
absence of iron, giving rise to zinc protoporphyrin in the red cell.7 Limitations in the
Email christinaclewes@btinternet.com supply of folate and vitamin B12 (cyanocobalamin) reduce the number of red cells

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Table 1 Anemia prevalence in preschool-age children (pre-SAC)a these influences.2 The prevalence of subjects with Hb con-
and women of reproductive age (pregnant and nonpregnant centrations below the defined thresholds (Table 2) is used
women) by World Health Organization (WHO) regionb
to estimate the level of the public health problem within a
WHO region Prevalence [% (95% CI)]
country or region (Table 3). Infants younger than 6 months
Pre-SAC Pregnant Nonpregnant
were excluded from the WHO estimates.
women women
Although anemia is often the result of a complex interac-
Africa 67.6 (64.3, 71.0) 57.1 (52.8, 61.3) 47.5 (43.3, 51.6)
Americas 29.3 (26.8, 31.9) 24.1 (17.3, 30.8) 17.8 (12.9, 22.7) tion of a number of factors, the most significant contributor
Southeast Asia 65.5 (61.0, 70.0) 48.2 (43.9, 52.5) 45.7 (41.9, 49.4) has always been thought to be iron deficiency. In the litera-
Europe 21.7 (15.4, 28.0) 25.1 (18.6, 31.6) 19.0 (14.7, 23.3) ture, statements such as “at least half the anemia is due to
Eastern 46.7 (42.2, 51.2) 44.2 (38.2, 50.3) 32.4 (29.2, 35.6)
nutritional iron deficiency”9 and “up to a prevalence of 40%
Mediterranean
Western Pacific 23.1 (21.9, 24.4) 30.7 (28.8, 32.7) 21.5 (20.8, 22.2) iron-deficiency anemia, iron deficiency will be about 2.5 times
Global 47.4 (45.7, 49.1) 41.8 (39.9, 43.8) 30.2 (28.7, 31.6) that of anemia”2 have been widely reported. ­However, it is
Notes: aPre-SAC are those aged 6–59 months; bdata obtained from the WHO report evident that there are considerable variations in both of these
Worldwide Prevalence of Anaemia 1993–2005: WHO Global Database on Anaemia.1
Abbreviation: CI, confidence interval. ratios depending on the age and sex of the people being
studied, the region of the world in which they live, and the
prevalence rates of other causes of anemia. Table 4 illustrates
produced, so cells can be larger than normal (macrocytosis)
the major factors that contribute to anemia.7
and this may or may not be accompanied by hypochromia.8
Although diseases feature prominently in Table 4 – notably,
Where the rate of red cell production is higher than normal,
malaria, human immunodeficiency virus, and helminth infec-
a further feature may appear in some red cells – namely,
tions such as hookworm – it is not the purpose of this review
remnants of ribosomes and rough endoplasmic reticulum –
to describe biomarkers for different diseases. Diseases will
and the cells are then called reticulocytes.7 The presence of
be dealt with by physicians as determined by the clinical
reticulocytes is a feature of the hemolytic anemias. However,
need of their patients or, in the community, by distribution
as micronutrient deficiencies rarely occur singly, the effects
of prophylactic measures such as bed nets, albendazole, and
on the morphology of red cells during production can be
so forth. However, many diseases exert their effects on the
mixed, producing a wide distribution of cell types.
hemopoietic system through the inflammatory response.10
Anemia, therefore, is an indicator of both poor nutrition
This produces the type of anemia known as “anemia of
and poor health resulting in increased risks of maternal and
chronic inflammation” (ACI) and because of the widespread
child mortality. Hb is transported in the circulation within
distribution of endemic disease in much of the developing
the erythrocytes or RBCs, where its principal function is to
world, ACI may be an important etiological component of
carry oxygen to body tissues. The requirements for oxygen
anemia globally. Therefore, inflammation biomarkers will be
are influenced by environmental factors such as altitude and
included in this review, as they indicate the disease exposure
smoking and physiological factors such as gestation, and the
in the individual and the community.
body can change Hb concentrations and/or blood volumes to
Measuring the blood Hb concentration is the most reliable
accommodate the need for oxygen. For statistical purposes,
indicator of anemia at the population level and it is used in
correction factors can adjust Hb concentrations to remove
national demographic health surveys and by national govern-
ments surveying populations before making new policy for
Table 2 World Health Organization hemoglobin concentration nutritional interventions such as the distribution of supple-
thresholds used to define anemia at sea level2 ments or the fortification of staple foods. ­Measuring Hb
Population group Hemoglobin cutoff (g/L)
Table 3 World Health Organization classifications of anemia that
Children
define the extent of the public health problem within a country
Aged 0.50–4.99 years 110
or region2
Aged 5.00–11.99 years 115
Aged 12.00–14.99 years 120 Prevalence of anemia (%)a Category of public health significance
Nonpregnant women 120 #4.9 No public health problem
Pregnant women 110 $5.0–19.9 Mild public health problem
Men 130
$20.0–39.9 Moderate public health problem
Note: Hemoglobin cut-offs are adjusted upwards to compensate for altitude and
$40.0 Severe public health problem
smoking. In pregnant women, hemoglobin is adjusted variably according to the stage
of pregnancy.2 Note: aSee Table 2 for definition of anemia.

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Dovepress Biomarkers of anemia

Table 4 Major factors contributing to anemia and principal and centrifuges may be the only equipment available. In the
biomarkers for interpretation absence of automated cell counters, different hematological
Cause Biomarkers approaches may be needed; however, these approaches will
Iron deficiency Ferritin, soluble transferrin not be discussed here.6
receptors, serum iron, red cell
morphology, bone marrow
Other nutritional deficiencies Specific nutritional indicators, Red cell count
such as folate, vitamin B12, vitamin B6, dietary intake data, red cell The normal number of red cells per cubic microliter of blood
riboflavin, and vitamin A morphology is 5 million, with slightly more in men (4.5–6.5 million) than
Malaria Parasite antigen tests, thick and
thin blood films for parasite counts
in women (3.9–5.6 million).6 The introduction of equipment
Helminth infections, especially Stool and/or urine egg counts, with automated counting meant that all the indices described
hookworm and schistosomiasis immunological tests in Table 5 are now far more accurate and useful than when
Chronic infections (eg, HIV/AIDS, Specific clinical tests to identify
only the manual techniques were available.
tuberculosis, cancer, rheumatoid pathogens or pathological state
arthritis)
Hemoglobinopathies (eg, sickle-cell Genetic screening Mean corpuscular volume
disease and thalassemia) electrophoresis The mean corpuscular volume (MCV) and the mean cor-
Inflammation Acute-phase proteins, white
puscular Hb concentration (MCHC) provide identical infor-
blood cell count
Physiological blood losses in women Medical diagnoses mation and are interchangeable in identifying a microcytic
associated with pregnancy anemia. MCV has been used most widely and is a measure
and menstruation of the average size of the RBCs. A MCV below the reference
Gastric and intestinal diseases Fecal blood
value is usually an indication of iron deficiency (Table 5).
Abbreviations: HIV, human immunodeficiency virus; AIDS, acquired immune
deficiency syndrome. However, low values are not specific to iron deficiency and
can be found in those with thalassemia and in about 50%
of people with anemia due to inflammation. Conversely,
is easy and relatively cheap. In many surveys the portable
if MCV values are above the reference value, they indi-
HemoCue® (Hemocue AB, Ängelholm, Sweden) device is
cate macrocytic anemia caused by, for example, folate or
used to get an instant reading of the concentration; the result
B12 deficiency and also alcohol abuse, liver disease, and
can be given to the household straight away, and, if necessary,
hypothyroidism.
action can be taken if concentrations are low (eg, referral to
clinic for a course of iron tablets, test for malaria). In a clini-
Reticulocytes
cal setting or in continuous national cross-sectional surveys
Reticulocytes are young, anucleate erythrocytes, which
(eg, the UK National Diet and Nutrition Survey [NDNS]),
are released from bone marrow into the blood in increased
Hb is usually measured using automated red cell–counting
numbers as a response to anemia caused by hemolysis
­equipment. However, it must always be remembered that a
(destruction) or loss (hemorrhage) of erythrocytes. ­Detection
low Hb concentration is not necessarily a measure of iron
and identification of immature anucleate RBCs verifies
deficiency and that more detailed investigations are needed
whether the bone marrow is responding to the anemia by
to determine the cause of the anemia.
increasing RBC production in a regenerative response. The
reticulocytes are evaluated by the presence of ribonucleic
Biomarkers to differentiate anemias acid (RNA) in the cytoplasm of immature erythrocytes. The
Red cell–counting techniques more immature the cell, the more RNA it will contain. If
to differentiate anemias the anemia is regenerative, there will be an above-normal
Measurement of erythrocytes and reticulocyte numbers using number of reticulocytes and polychromasia (variation in the
automated flow cytometers is useful for evaluating anemia Hb content of erythrocytes) in peripheral blood. In contrast,
in both routine and clinically complex settings, particularly mature RBCs, which are no longer synthesizing Hb, contain
for the high degree of precision attainable. Automated flow very small amounts of or no RNA.
cytometers provide information on a number of red cell Because the life span of reticulocytes is longer in anemic
parameters (Table 5). However, access to complex equip- subjects, the reticulocyte proliferation index is normally
ment is more problematic in developing countries, where red calculated (Table 5). Low values indicate a low production
cell–counting chambers, stained blood films, photometers, of red cells possibly due to nutrient deficiency, whereas high

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Table 5 Biomarkers of anemia from red cell morphology

Biomarker Reference value Calculation or measurement Interpretation
Total RBC count × 10 /L 12 Men: 4.2–5.9 NR Low: anemia
Women: 3.6–5.3 High: dehydration, high altitude, smoking
Hematocrit (PCV)a Men: 0.45 L/L (-2SD 0.39) Proportion of RBCs as percentage Values , 0.39 L/L in men and ,0.34 L/L
Women: 0.4 L/L (-2SD 0.34) of total blood volume in women indicate anemia
MCV 83–101 fL 10 × PCV (%)/RBC count (106/μL) Microcytic: ,83 fL indicates iron deficiency
Macrocytic: .101 fL suggests folate or
vitamin B12 deficiency
RC ∼0.5%–1% of RBCs in peripheral Fluorescent dye marks cells with Higher values are due to increased production
blood RNA to distinguish from RBCs of RBCs in response to hemolysis or loss
of RBCs – regenerative anemia
RPI62 1%–2% RC × (Hbobs/Hbnormal) × 0.5 ,1% indicates low production of new RBCs
.2% indicates increased production of new RBCs
RDW 11%–15% SD of MCV as percentage .15% indicates iron-deficiency anemia or
of mean MCV anemias of mixed origin
MCHC 4.9–5.5 mmol/L or as mass fraction Mass fraction (mHb/mRBC) Low: ,4.9 mmol/L indicates iron deficiency
32–36 g/dL or percentage High: macrocytic anemia
Note: Hematocrit cut-offs are adjusted upwards to compensate for increases in hemoglobin caused by altitude and smoking and adjusted variably according to the specific
stage of pregnancy.2
Abbreviations: RBC, red blood cell; NR, not relevant; PCV, packed cell volume; 2SD, two standard deviations; MCV, mean corpuscular volume; RC, reticulocyte count; obs,
observed; RNA, ribonucleic acid; RPI, reticulocyte proliferation index; Hb, hemoglobin; RDW, red cell distribution width; SD, standard deviation; MCHC, mean corpuscular
hemoglobin concentration.

values indicate a high production of reticulocytes to replace initially presents with a varied size distribution of RBCs,
lost blood and healthy hematopoiesis. which show an increased RDW. In the case of a mixed iron
and B12 and/or folate deficiency, there will be a mix of both
Hematocrit or packed cell volume large and small cells, causing the RDW to be elevated.
The hematocrit is the proportion of the blood volume occu-
pied by RBCs and is determined by cell number and size. MCHC
Concentrations below the reference range may indicate MCHC is a measure of the concentration of Hb in a given
abnormal cell development. The normal hematocrit range volume of packed RBCs. MCHC is a useful index because it
used in a typical clinical setting is 0.4–0.5 L/L for men and incorporates red cell number, size, and Hb content. It is cal-
0.36–0.46 L/L for women.11 The WHO definition of a hema- culated by dividing the Hb concentration by the hematocrit,
tocrit for men aged 18–44.99 years is 0.445 L/L (minus two assuming a RBC concentration of 1 g/mL and negligible Hb
standard deviations: 0.39 L/L); the definition for women in plasma (Table 5). Low values indicate hypochromia and
of a similar age is 0.4 L/L (minus two standard deviations: impaired synthesis of Hb, while high values suggest folate
0.34 L/L).2 For international comparisons and statistical or vitamin B12 deficiency.
analyses, hematocrit is adjusted upward for gestation and
downward for altitude and in smokers.2 Hb
Hb is measured as a part of the automated red cell count
Red cell distribution width using the cyanomethemoglobin method. Cells are lysed using
Red cell distribution width (RDW) is a measure of the varia- Drabkin’s solution to release the Hb into the fluid and convert
tion of RBC width; the normal reference range is 11%–15% it to cyanomethemoglobin, which is measured at 525 nm.
(Table 5). Usually RBCs are a standard size of about 6–8 µm,
but certain disorders cause a significant variation in cell size. White cell count (leucocytes)
Higher RDW values indicate greater variation in size. If The normal total white cell count varies from about 4000 to
anemia is observed, RDW test results are often used together 10,000 per microliter with an average of 6000 per microliter.
with MCV results to determine the possible causes of the An increase in the total number of leucocytes to 15,000–25,000
anemia.12 The measure of RDW is mainly used to differenti- per microliter is termed a leucocytosis. The commonest cause
ate an anemia of mixed causes from an anemia of a single of a polymorphonuclear leucocytosis is bacterial or general-
cause. Vitamin B12 deficiency alone produces a macrocytic ized infection or tissue destruction by burns or surgery.6 Thus,
anemia with a normal RDW, while iron-deficiency anemia a leucocytosis may indicate subclinical inflammation and may

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Dovepress Biomarkers of anemia

influence the interpretation of serum ferritin concentrations (see 80% in ineffective erythropoiesis (red cell maturation defect
“Inflammation and anemia” section later in this review). such as thalassemia) or 100% in idiopathic hemochromatosis
(iron overload). In hemolytic anemia with effective erythro-
Measurements of iron status poiesis, transferrin saturation is slightly raised over normal
Iron deficiency to 45%. Effective erythropoiesis can also be assessed using
Iron deficiency is a state in which there is insufficient iron to the reticulocyte proliferation index (Table 5). A saturation of
maintain the normal physiological function of tissues such ,16% is inadequate to meet basal erythropoietic needs.17
as the blood, brain, and muscles. The total transferrin concentration in blood is usually
Serum ferritin is the most useful biomarker to measure expressed as the maximum iron-binding capacity of the
iron status, and concentrations , 12 µg/L for children plasma – namely, TIBC – and the normal concentration is
younger than 5 years of age and ,15 µg/L for those 5 years 60 µmol/L (95% CI: 45, 70). At birth, TIBC is ∼42 µmol/L
and older are an indication of low iron stores in the body and and progressively rises through infancy to reach adult ­values
a high risk of iron deficiency. Low iron stores in combination at the age of 2 years. There is no difference between males and
with anemia , as defined in Table 2, is termed iron-deficiency females at any age, but concentrations decline in older age
anemia. Iron-deficiency anemia is the final clinical stage of groups. TIBC is higher in iron deficiency but lower in infec-
iron deficiency. Iron deficiency in the absence of anemia tion, hemolytic anemias, hemochromatosis, and undernutri-
suggests that dietary iron intake is sufficient for metabolic tion. During pregnancy, TIBC rises to 72 and 90 µmol/L at
requirements but insufficient to accumulate any stores.2 the fifth and eighth months, respectively.17
Unfortunately, in the presence of inflammation the con- The generally high amount of unbound binding capacity
centration of ferritin is usually increased, even if iron stores on transferrin in normal subjects is believed to provide protec-
are low; hence, it can be difficult to interpret the true con- tion against unbound or “free” iron in the blood.17,18 In healthy
centration of ferritin in situations where exposure to inflam- subjects the unbound iron–binding capacity is around 65%, and
mation is common.13 If the major causes of the inflammation this can increase to 85% during infection. However, transfer-
are seasonal, and a national survey is needed, measurements rin is a negative acute-phase protein (APP) and TIBC can fall
should be performed in the season of lowest transmission or to 40 µmol/L during infection, although the lower transferrin
the concentration of ferritin can be adjusted for the influence saturation (15%) ensures the proportion of unbound iron–
of inflammation if the concentrations of serum C-reactive binding capacity is only slightly less than in healthy subjects
protein (CRP) and alpha-1-acid glycoprotein (AGP) are (∼55%) even though binding capacity is reduced.
measured (see “Inflammation and anemia” section).13,14
Storage iron
Serum iron, total iron-binding capacity, Storage iron is the pool of iron in the body that is not being
and transferrin saturation used by tissues. Healthy children and adults (apart from
Serum iron, total iron-binding capacity (TIBC), and calcu- infants aged 6–11 months and pregnant women) usually
lated transferrin saturation tests are used to screen for and have some iron stores to act as a buffer against iron defi-
monitor conditions of iron deficiency and iron overload. ciency during periods when dietary iron may be temporarily
Mean concentrations of serum iron, which is transported by ­insufficient.7 Methods to express the amount of stored iron are
transferrin, are approximately 16–18 µmol/L (95% CI: 9, 26). based on the log ratio of serum soluble transferrin receptor
Most people exhibit a morning peak and an evening nadir, (sTfR) to serum ferritin concentrations.19 The amount of body
and a consistent time of blood collection is recommended.15 iron based on body weight can be calculated using the fol-
High serum iron values can indicate hemolysis or iron over- lowing equation, where sTfR is measured in milligrams per
load but iron concentrations can be markedly (.50%) and liter and ferritin is measured in micrograms per liter:19,20
rapidly (24 hours) depressed by inflammation.16
The major source of iron for transferrin is the reticuloen- Body iron (mg/kg) =−[log10 (sTfR × 1000/
dothelial system, with some from hepatocytes and the ­intestine. ferritin) − 2.8229]/0.1207
Transferrin saturation also exhibits a daily fluctuation similar to
that of serum iron, with a mean morning saturation of 35% and Iron depletion
a normal range of 20%–50%. ­Transferrin saturation can fall to Iron depletion is the state in which storage iron is absent
5% in iron deficiency and 15% in infection, and it can rise to or nearly absent but the tissues that need iron are able to

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Northrop-Clewes and Thurnham Dovepress

maintain normal physiological functions – that is, iron intake anemia, thalassemia, and Hb E (see Table 6 and later section
may be just sufficient to cover requirements. Evidence from “Hemoglobinopathies associated with anemia”).
animals fed on iron-deficient diets indicates that iron defi- Although an international reference standard for sTfR
ciency becomes detectable at about the same time in the has been produced,25 there is still no reference range of
blood, brain, and tissue enzyme systems,21 and low serum ­concentrations. Skikne et al20 reported the mean sTfR con-
ferritin concentrations indicate when there is a high risk of centration in normal subjects was 5.6 mg/L with a range of
impairing these functions. 2.8–8.5 mg/L (plus or minus two standard deviations) and a
It is possible for a functional iron deficiency to develop commercial kit produced in the United States (human trans-
even when iron stores are present, if the normal physiological ferrin receptor catalogue number TFC-94-CE; Ramco Labo-
systems for transporting iron to target tissues or mobilizing ratories, Inc, Stafford, TX, USA) suggests a normal range of
iron from storage are impaired. This occurs most commonly 2.9–8.3 mg/L, which has been used in many national surveys.26
because inflammatory cytokines released during infection In healthy adults, sTfR concentrations did not differ by sex or
under the influence of hepcidin prevent the normal transport age between the ages of 18 and 80 years, but black subjects had
of iron.22 Neither iron supplementation nor iron fortification significantly higher concentrations than nonblack (Caucasian,
have any benefit in such circumstances, as iron absorption is Hispanic, and Asian) subjects, and black and nonblack subjects
inhibited.23 Deficiencies of other nutrients such as vitamin A living at high altitude had higher concentrations than those
may also cause a functional iron deficiency even when iron living closer to sea level.27 There is also evidence that sTfR
stores are adequate, as these deficiencies can increase the concentrations are higher in infants (mean 7.8 mg/L; 95% CI:
risk and prevalence of disease.24 4.5, 11.1) than in prepubertal boys (7.0 mg/L; 95% CI: 4.7,
9.2), and higher in prepubertal boys than in men (5.8 mg/L;
Zinc protoporphyrin 95% CI: 3.1, 8.5).28 The same authors also reported that lower
The presence of zinc protoporphyrin (ZPP) in blood reflects serum ferritin and iron concentrations, even within the normal
a shortage of iron in the last stages of making Hb so that zinc physiologic range, were associated with high sTfR concentra-
is inserted into the protoporphyrin molecule in the place of tions and that the lower the serum ferritin concentrations, the
iron. ZPP can be detected in RBCs by fluorometry and is higher the sTfR concentrations.28
a measure of the severity of iron deficiency.7 For national
surveys, a portable hematofluorometer is available (Aviv Disease and ACI
Biomedical, Inc, Lakewood, NJ, USA). The cutoff is a ZPP The distinction between iron-deficiency anemia and the
concentration of .61 µmol/mol heme.2 anemia that accompanies inflammation is difficult to iden-
tify because indicators such as ferritin are influenced by
Serum sTfRs the acute-phase response. Serum sTfR is not an APP, and
Serum sTfR concentrations are derived mostly from ­developing concentrations remain in the normal range in ACI, but sTfR
RBCs and so they reflect the intensity of ­erythropoiesis and concentrations may be slightly elevated (9 mg/L) in nonane-
the demand for iron; the concentration rises in iron-deficiency mic iron deficiency (ie, low ferritin concentrations) and more
anemia and it is a marker of the severity of iron insufficiency, elevated in iron-deficiency anemia (25 mg/L; ie, low ferritin
but only when iron stores have been exhausted, provided and low Hb). Because sTfR concentrations barely change in
that there are no other causes of abnormal ­erythropoiesis.7 ACI but are very much increased in iron-deficiency anemia,
The concentration of sTfR is also increased in hemolytic sTfR distinguishes between these anemias (Table 7).22,29

Table 6 Mean hematological variables for normal hemoglobin (Hb) and four major Hb variants in Cambodian children aged 24–59 months
Variable HbAA HbAE α-thalassemia Hb E trait and HbEE
trait α-thalassemia trait
Children (n) 531 325 204 122 77
Hb (g/L) 114 110a 111a 108a 98a
sTfR (mg/L) 7.1 7.4a 7.5a 7.5a 9.6a
Ferritin (μg/L)b 31.6 32.5 30.4 30.1 33.7
Notes: Adapted with permission from George J, Yiannakis M, Main B, et al. Genetic hemoglobin disorders, infection, and deficiencies of iron and vitamin A determine anemia
in young Cambodian children. J Nutr. 2012;142(4):781–787.42 © 2012 American Society for Nutrition. aDifferent from concentration in HbAA group (P , 0.05); bvalues for
ferritin were corrected for subclinical inflammation by methods in Thurnham et al.13
Abbreviations: HbAA, normal hemoglobin; HbAE, hemoglobin E trait; α, alpha; sTfR, soluble transferrin receptor; HbEE, homozygous hemoglobin E.

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Dovepress Biomarkers of anemia

Table 7 Changes in serum concentrations of markers of to quantify both red cell and serum folate concentrations; other
iron status in iron-deficiency anemia (IDA), anemia of chronic possible methods are mass spectrometry and protein-binding
inflammation (ACI), and the combination of botha
assays. Normal concentrations are $10 nmol/L for serum
Biomarkers IDA ACI IDA + ACI folate and $340 nmol/L for RBC folate.8
Hemoglobin ↓ ↓ ↓
Serum iron ↓ ↓ ↓
Vitamin B12 (cyanocobalamin)
Ferritin ↓ NC-↑ ↑
Soluble transferrin ↑ NC NC-↑ Vitamin B12 is only present in animal-source foods, so defi-
receptors (sTfR) ciency is prevalent when the intake of these foods is low
Ratio sTfR/log ferritin ↑↑ (ratio . 2) NC (ratio , 1) ↑ (ratio . 2) because of high cost, lack of availability, cultural or religious
Transferrinb ↑ ↓-NC ↓-NC
beliefs,8 or malabsorption due to lack of intrinsic factor in gas-
Inflammatory NC ↑ ↑
cytokinesc tric parietal cells of the stomach (this final example is called
Notes: aArrows indicate the direction of a change; btransferrin is a negative acute- pernicious anemia, a megaloblastic anemia). The metabolism
phase protein, so concentrations fall slightly during inflammation; cinflammatory of folate and B12 are linked, in that vitamin B12 is needed for
cytokines include hepcidin, interleukin 6, and others.
Adapted with permission from Northrop-Clewes CA. Interpreting indicators the metabolism of folate, and in the absence of vitamin B12,
of iron status during an acute phase response: lessons from malaria and human
folate is trapped as N5-methyltetrahydrofolate and cannot be
immunodefi­ciency virus. Ann Clin Biochem. 2008;45(Pt 1):18–32,22 and Beguin Y.
Soluble transferrin receptor for the evaluation of erythropoi­esis and iron status. Clin recycled back to the folate pool. A low RBC folate can there-
Chim Acta. 2003;329(1–2):9–22.29
Abbreviations: NC, no change; NC-s↑, no change or slight increase; NC-↓, no
fore indicate primary vitamin B12 deficiency. Inadequate intake
change or slight decrease; ↑↑, very strong increase. of vitamin B12 leads to low plasma concentrations, an elevated
plasma homocysteine concentration, and elevated urinary or
The production of inflammatory cytokines, such as serum methylmalonic acid concentrations. A deficiency of
tumor necrosis factor-alpha and hepcidin in infection can vitamin B12 is also associated with megaloblastic anemia as
suppress both hematopoiesis, by inhibition of ­erythropoietin well as neurological symptoms caused by defects in myelin
­production, and erythropoiesis, by inhibition of iron mobili- synthesis. Normal plasma concentrations for vitamin B12 are
zation.23,30 For example, in a chronic inflammatory condition $150 pmol/L, measured by a microbiological assay.8
such as rheumatoid arthritis, serum sTfR concentrations can
remain within the normal range even when iron stores are Vitamin A
depleted because of the aforementioned effects.31 Therefore, Vitamin A status is widely reported to influence iron metabo-
the relationship between iron deficiency and sTfR concentra- lism but the precise mechanism is not yet elucidated.33
tions in ACI is usually inversely dependent on the degree of Vitamin A deficiency (VAD) and anemia often coexist in
inflammation since inflammation will depress erythropoietin populations, and supplementation studies suggest that there
production and bone marrow activity.29,32 is an additive reduction in iron-deficiency anemia following
a combined supplement of vitamin A and iron in compari-
Other nutritional deficiencies associated son with the effects of the nutrients given alone.34 However,
with anemia inflammation depresses both plasma retinol concentrations
Folates and the mobilization of iron,14 so the role of infection in
Folates are a group of water-soluble vitamins that are the vitamin A–iron interaction should not be overlooked.
important in the transfer of one-carbon units in amino acid Biochemically, a risk of VAD is defined as a serum retinol
interconversions and which are therefore essential for protein concentration , 0.7 µmol/L.35 WHO estimates suggest that
synthesis and in hematopoiesis. Serum folate concentrations VAD is a global problem in preschool-age children (190 mil-
start to fall after about 3 weeks of folate deprivation, but red lion; 95% CI: 178, 202) and pregnant women (19.1 million;
cell folate concentrations remain stable for longer, as they 95% CI: 9.3, 29), equivalent to 33.3% and 15.3% of the global
more accurately reflect tissue stores of folate. ­Prolonged folate population, respectively.35 Vitamin A is found in two forms in
deficiency leads to megaloblastic anemia, which can result the diet: (1) as provitamin carotenoids, such as beta-carotene,
in increased levels of serum homocysteine concentrations in in vegetables and fruits and (2) as retinol in animal products.
adults and neural tube defects in the fetus. Measurements of Retinol or retinol esters from animal-source foods are more
both serum and red cell concentrations of folate provide the bioavailable than provitamin carotenoids but are relatively
best assessment of folate status (also see the following section, expensive; vegetables, on the other hand, are widely available
“Vitamin B12”). In general, the microbiological method is used and inexpensive but are undervalued and underutilized.

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Northrop-Clewes and Thurnham Dovepress

Vitamin B2 (riboflavin [7,8-dimethyl-10-ribityl- one gene in the nucleus is affected; in the homozygous form,
isoalloxazine; vitamin B2]) denoted by HbEE, two genes are affected.
Riboflavin-responsive anemia was first described by Foy and
Kondi4,5 about 60 years ago. This anemia is characterized by Alpha-thalassemia
erythroid hypoplasia (small number of red cells produced) In alpha-thalassemia (α-thalassemia), there is impaired
and reticulocytopenia (abnormal decrease in reticulocytes). synthesis of one or more of the α-globin chains. When the
Riboflavin deficiency alters iron metabolism and, although deletion affects one α-chain it is known as the α-thalassemia
the mechanism is not clear, research in animals suggests that trait, which can occur alone or be accompanied by HbAE.
riboflavin deficiency may impair iron absorption, increase Both the latter forms tend to be asymptomatic and are known
intestinal loss of iron, and/or impair iron utilization for the as thalassemia minor. Hb concentrations are lower in all
synthesis of Hb.36 Riboflavin is a water-soluble vitamin variant Hb groups and sTfR and ferritin concentrations are
present especially in dairy products but it is also found in all higher (Table 6). In Table 6 the ferritin concentrations
lesser amounts in other foods. Riboflavin functions in the indicated that most children had good iron status since mean
body as an integral part of two coenzymes, flavin adenine ferritin concentrations were well above 12 ug/L. This was
dinucleotide and flavin mononucleotide, which take part in in spite of downward adjustments to the ferritin values to
a range of oxidation-reduction reactions.36 compensate for the high prevalence of subclinical inflam-
In human studies, correction of riboflavin deficiency in mation.13 Anemias due to Hb E or α-globin do not respond
pregnant and lactating women, adult males, and school-age to iron therapy.42
children, all of whom were both riboflavin and iron deficient,
improved the response of iron-deficiency anemia to iron Sickle-cell disease
therapy.37–39 In addition, studies using 58Fe in adult Gambian Sickle-cell disease occurs mainly in African populations
men have suggested that improved riboflavin status increased and is associated with the presence of sickle-cell Hb, where
mobilization of stored iron to raise Hb concentrations but that it one amino acid residue is different from that in normal adult
had no measurable effect on iron absorption from the diet.40 Hb. Sickle-cell disease results from the severe, genetically
determined homozygous form, denoted by HbSS, where an
Vitamin B6 (pyridoxine) abnormal gene is inherited from each parent. In contrast, the
Vitamin B6 occurs naturally in a variety of forms among sickle-cell trait is the heterozygous form, denoted by HbAS,
foods of both plant and animal origin, but all are deriva- where only one abnormal gene is inherited. In sickle-cell dis-
tives of 2-methyl-3-hydroxy-5-hydroxymethyl-pyridine.41 ease, the degree of anemia caused is variable but often severe
Vitamin B6 is required as a coenzyme in the synthesis of and it is characteristically normochromic and ­normocytic.
delta-aminolevulinic acid,3 which is the first rate-limiting However, when in combination with iron, folate, or B12
step in the synthesis of heme. Deficiency of vitamin B6 deficiency, hypochromic or macrocytic megaloblastic ane-
is associated with microcytic hypochromic anemia but is mias may be found. The reticulocyte count is usually raised
rarely found. 10%–20% and polychromasia is marked. In the sickle-cell
trait, there is no anemia or evidence of increased red cell
Hemoglobinopathies associated destruction and the reticulocyte count is normal. Only on
with anemia exposure to anoxia do some of the erythrocytes take on the
Hb E sickle-cell shape.
In Southeast Asia, four main Hb variants have been identified
linked with Hb E and thalassemia, and all are major risk factors Haptoglobin
for anemia. The anemia observed is predominantly microcytic During intravascular hemolysis (eg, malaria and hemoglo-
hypochromic anemia, which is the form commonly attributed binopathies), potentially tissue-damaging Hb is released
to iron deficiency. In the normal adult Hb, the globin molecule into the plasma, where it is bound by the protective APP,
is made up of four polypeptide chains, two alpha and two beta, haptoglobin (Hp). One molecule of Hp binds virtually irre-
and is denoted by HbAA. Hb E disease arises from a genetic versibly with two molecules of Hb43 to form a compound of
alteration in the physical structure of Hb – specifically, a single higher molecular weight that cannot pass the renal glomeruli
amino acid substitution in one of the beta-globin chains. The and which reduces the risk of iron loss and renal tubular
heterozygous form, denoted by HbAE, is present when only injury.6,17 Serum Hp concentrations are a good indicator of

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Dovepress Biomarkers of anemia

hemolytic anemia, as normal Hp concentrations (0.5–2.6 g/L)6 drugs, including antimalarial treatments, cause Hb denatur-
rapidly become undetectable if Hb is present, since the ation, ultimately resulting in hemolytic anemia.
complex is rapidly cleared from the plasma by binding the
Hp-Hb complex to the specific receptor CD163 on circulating Inflammation and anemia
monocytes and macrophages and it is internalized.44,45 The Serum ferritin and the acute-phase response
iron-loaded macrophages migrate to the reticuloendothelial In 2004, the WHO working group recognized ferritin as prob-
system, where they can lodge for long periods until the ably the most useful biomarker of iron status,7 but, because it
inflammatory response subsides and the iron component can is also a positive APP, the group recommended that ferritin
be recycled for erythropoiesis.45 measurements should be accompanied by the analysis of one
Three phenotypes of Hp are found in humans: Hp1-1, or more APPs to detect the presence of subclinical infection
Hp1-2, and Hp2-2. These phenotypes have been shown to or inflammation.
bind Hb with different affinities: Hp1-1 . Hp1-2 . Hp2-2. At the onset of an infection, serum ferritin concentrations
Hp2-2 is the weakest Hb binder, and recent studies in rise rapidly and in parallel with CRP concentrations.49 During
the Gambia found that the fall in mean Hb concentration the clinical infection, ferritin concentrations remain high,50 but
through the malaria season in children aged 2–6 years with they do not fall when CRP concentrations fall as clinical signs
the Hp2-2 phenotype was significantly greater by 4g/L than disappear.49 Instead, ferritin concentration can remain high
in those with the other phenotypes.46 The Hp2-2 phenotype into the convalescent period, similar to the behavior of the
is associated with higher levels of oxidant stress, which may “chronic” APP biomarker, AGP.13,22 For the physician handling
confer antimalarial benefits at the expense of Hb loss. Thus, diseased patients, a high ferritin concentration associated with
while the Hp2-2 phenotype is a risk factor for anemia, its anemia is of little surprise and his or her main objective is to
antimalarial properties made it comparable with glucose-6- cure the patient to reestablish normal hematopoiesis. However,
phosphate dehydrogenase (G6PD) deficiency and HbAS in although patients in convalescence may be apparently healthy
malaria-endemic areas. The Hp-Hb phenotype may protect and no longer in clinical care, to interpret their ferritin concen-
against life-threatening malaria at the expense of impaired trations correctly it is important to establish whether there is
hematological recovery from mild asymptomatic malaria.46 any evidence of residual subclinical inflammation.

G6PD Correcting serum ferritin concentrations

Because G6PD deficiency is inherited as a sex-linked char- for inflammation
acter and is carried on the X chromosome, deficient variants Correcting ferritin for subclinical inflammation can be done
are expressed more commonly in males than in females. using the methods described by Thurnham et al.13 At the onset
­Worldwide, an estimated 400 million people are G6PD defi- of an infection, CRP concentrations rise rapidly over the first
cient, with the distribution corresponding to areas in which 24–48 hours before clinical evidence of disease appears (the
malaria is or has been prevalent; it is most common in West incubation period). In contrast, AGP rises much more slowly
Africa, Central Africa, the Mediterranean region, the Middle and it only reaches a maximum after 4–5 days. When the clini-
East, and Southeast Asia.47 There is some evidence that hemi­ cal phase of the disease subsides, CRP falls but AGP remains
zygous males may be protected against severe malaria. elevated. In a meta-analysis, using these inflammation proteins
G6PD deficiency is characterized by abnormally low levels the present authors allocated subjects to three inflammatory
of G6PD, an enzyme in the pentose phosphate pathway of groups: (1) incubation (only CRP is raised), (2) early convales-
RBCs, which is the only biochemical pathway for generat- cence (both CRP and AGP are raised), and (3) late convales-
ing energy in the form of NADPH and reduced glutathione.47 cence (only AGP is raised). The authors defined inflammation
The G6PD gene is highly polymorphic, with almost 400 using cutoff values . 5 mg/L for CRP and .1 g/L for AGP.
reported variants, but in sub-Saharan Africa there are three Subjects with no raised APP formed the reference group.
main variants: G6PD-B (wild type, full enzyme activity) and Correction factors were calculated from the ratios of the
two mutations, G6PD-A+ and G6PD-A−. G6PD-A+ retains geometric means of the inflammatory/reference groups and,
90% of full activity but G6PD-A− retains only 8%–20% of from a meta-analysis of 22 studies, correction factors for the
the wild-type enzyme activity. Individuals with G6PD-A− are incubation, early convalescent, and late convalescent groups
normally asymptomatic; however, erythrocyte exposure to were 0.77, 0.53, and 0.75, respectively.13 ­Ferritin concentra-
oxidative stress such as infections and certain foods48 and tions are corrected by multiplying the ferritin concentration of

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Northrop-Clewes and Thurnham Dovepress

subjects in the three inflammatory groups by their respective Gambia support this suggestion, and the present authors
correction factors. Using this technique, the authors13 and observed an increasing prevalence of both acute and chronic
others51 showed that removing the effects of inflammation inflammation in Gambian infants through the first 12 months
increased the prevalence of iron deficiency. of life, with anemia (Hb , 90 g/L) increasing in parallel from
20% at 3 months of age to 48%, 62%, and 67% at 6, 9, and
Clinical usefulness of biomarkers 12 months of age, respectively.57,58 The increase in prevalence
In the preceding sections, the authors have described the of moderate anemia was not linked to seasonal factors, since
main biomarkers that are associated with anemia. Use of infants were recruited over 3 years and the effects spanned
these biomarkers will help identify the factors that potentially both wet and dry seasons and seasonal diseases. Instead, the
may have caused anemia in patients and groups of subjects authors believe the increase in moderate anemia is at least
in the community. It has to be remembered, however, that partially the result of increasing exposure to pathogens with
anemia is the final stage of a set of circumstances involving age and the inability to completely overcome the effects of
dietary deficiencies, parasites such as malaria or hookworm, one infection before exposure to the next.
or metabolic factors such as hemoglobinopathies and so forth. Iron supplementation has been considered standard
Dietary deficiencies of iron, folate, vitamin B12, pyridoxine, treatment for anemia over many years in both the developed
or riboflavin can limit the synthesis of Hb or RBCs,41 or eryth- and the developing worlds but it has often not been very
rocytes can be destroyed so rapidly that the body is unable ­effective. The reasons given have included lack of bioavail-
to carry out red cell replenishment quickly enough – as, for ability, insufficient dose, side effects, and compliance.59,60
example, in malaria52 or in an oxidative crisis in people with However, it is now increasingly recognized that other factors
congenital G6PD deficiency.48 Furthermore, when there is no may be equally as or more important than iron deficiency in
overt cause of a severe anemia, examining potential biomark- the etiology of anemia. Infectious diseases, contaminated
ers in patients may not be particularly helpful in establishing water, and poor hygiene are widespread in the developing
the etiology of the anemia. In a case-control study in rural world and prevalence rates for anemia are also high. In
Malawi of 381 children under the age of 2 years with severe contrast, in the developed world, prevalence rates for anemia
anemia (Hb , 50 g/L) and 757 similar children without are much lower and infectious diseases have been largely
severe anemia, there were seven factors that contributed controlled by vaccinations, clean water supplies, access
significantly to explaining the severe ­anemia: (1) bacteremia, to medical care, and improved hygiene. Improvements
(2) malaria, (3) hookworm, (4) human immunodeficiency in public health in developing countries are taking place
virus, (5) G6PD deficiency, (6) vitamin A deficiency, and slowly,61 but poverty and limited opportunities to break the
(7) vitamin B12 deficiency.53 No one factor explained more disease-malnutrition cycle will prevent any great reductions
than 5.3% of the severe anemia. The authors concluded in anemia prevalence in many developing countries for the
that there were multiple causes of severe anemia but folate foreseeable future.
and iron deficiencies were not prominent among them.
­Additionally, the authors cautioned that even in the presence Conclusion
of malaria parasites, additional or alternative causes of severe Anemia is of multifactorial etiology, but it is to be hoped that
malaria should be considered. by using the biomarkers described in this review, investiga-
It is noteworthy that four of the seven factors contributing tors will obtain a fuller understanding of the potential causes
to the severe anemia in the Malawian infants were infections. of anemia. Automated cell counters provide much useful
There is a common feature of infections – namely, that they information to point to potential causes of anemia, but no
activate the acute-phase response.10 An acute-phase response one method will ever be sufficient to describe the full etiol-
will restrict the supply of iron to the bone marrow, as previ- ogy of anemia. Specific tests may confirm the underlying
ously described,23,54 and reduce concentrations of several causes but anemia is the result of a complex interaction of
other nutrients involved directly and indirectly in red cell events, which may be difficult to unravel. This review lists
synthesis, including plasma retinol55 and vitamin B6.56 Low the biomarkers that can be used to determine the most likely
concentrations of plasma retinol have been associated with causes of anemia, but the authors emphasize that anemia is
increased morbidity, so one infection may increase suscepti- the final stage in a deterioration in red cell synthesis to a point
bility to another, especially when disease is highly endemic, when anemia is defined. At that stage the factors associated
as it is in much of the developing world.10,24 Studies in the with anemia may not fully reflect the circumstances that were

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Dovepress Biomarkers of anemia

responsible for the initial or the progress of deterioration in 19. Cook JD, Flowers CH, Skikne BS. The quantitative assessment of body
iron. Blood. 2003;101(9):3359–3364.
red cell synthesis. 20. Skikne BS, Flowers CH, Cook JD. Serum transferrin receptor:
a quantitative measure of tissue iron deficiency. Blood. 1990;75(9):
1870–1876.
Disclosure 21. Beard JL. Iron biology in immune function, muscle metabolism and
The authors report no conflicts of interest in this work. neuronal functioning. J Nutr. 2001;131(2 Suppl 2):S568–S579.
22. Northrop-Clewes CA. Interpreting indicators of iron status during an
acute phase response: lessons from malaria and human immunodefi-
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access, online journal publishing laboratory, experimental and clinical aspects blood related medicine; Legal aspects of blood medicine; Historical perspec-
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Transfusion Medicine; Blood collection, Donor issues, Transmittable diseases, a very quick and fair peer-review system. Visit http://www.dovepress.com/
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