YBLRE-00473; No of Pages 9

Blood Reviews xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Blood Reviews

journal homepage: www.elsevier.com/locate/blre

REVIEW

New insights into iron deficiency and iron deficiency anemia

Clara Camaschella ⁎
Vita Salute University and IRCCS Ospedale San Raffaele, Milan, Italy

a r t i c l e i n f o a b s t r a c t

Available online xxxx Recent advances in iron metabolism have stimulated new interest in iron deficiency (ID) and its anemia (IDA),
common conditions worldwide. Absolute ID/IDA, i.e. the decrease of total body iron, is easily diagnosed based
Keywords: on decreased levels of serum ferritin and transferrin saturation. Relative lack of iron in specific organs/tissues,
Iron deficiency and IDA in the context of inflammatory disorders, are diagnosed based on arbitrary cut offs of ferritin and trans-
Anemia ferrin saturation and/or marker combination (as the soluble transferrin receptor/ferritin index) in an appropriate
Inflammation
clinical context. Most ID patients are candidate to traditional treatment with oral iron salts, while high hepcidin
Hepcidin
levels block their absorption in inflammatory disorders. New iron preparations and new treatment modalities are
available: high-dose intravenous iron compounds are becoming popular and indications to their use are increas-
ing, although long-term side effects remain to be evaluated.
© 2017 Published by Elsevier Ltd.

1. Introduction
Iron deficiency (ID) and iron deficiency anemia (IDA) are common
medical conditions worldwide; still uncertainties exist on their correct
diagnosis and treatment. This insecurity is related at least in part to a re-
cent broader interpretation of ID, but especially to the explosive knowl-
edge of iron metabolism in the last 20 years, that makes physicians
uncertain of which approach to iron disorders is correct. A further
level of complexity derives from the increasing number of conditions
(with high prevalence) recognized as associated with altered iron me-
tabolism. While it is easy to diagnose ID and IDA due to decreased iron
intake, increased iron demands, chronic blood loss or decreased intesti-
nal absorption, it is definitely more complex to recognize ID when it is
masked by chronic inflammatory disorders. Inflammatory cytokines in-
fluence iron metabolism: they stimulate hepcidin production, cause
iron sequestration in macrophage stores and decrease saturation of cir-
culating transferrin and thus tissue iron availability. Variable degrees of
inflammation characterize several common human disorders, such as
atherosclerosis, obesity, diabetes and metabolic syndrome. ID together
with inflammation is common in many types of cancer and in chronic
kidney disease (CKD), both in patients undergoing dialysis and in pre-
dialysis state. ID and iron restriction may occur in the elderly, often af-
fected by multiple comorbidities, as well as in middle age overweight
subjects and in obese individuals at any age.
A further problem is related to the diagnostic approach. Classic tests
for diagnosis are blood cell count, serum ferritin, serum iron and
⁎ Vita Salute University & San Raffaele Scientific Institute, Via Olgettina, 58-20132
Milano, Italy.
E-mail address: camaschella.clara@hsr.it.
transferrin (or total iron binding capacity) and transferrin saturation
(or saturated iron binding capacity). The rapid advances of the field
lead physicians uncertain of the tests to be prescribed and of their inter-
pretation: which ferritin cut off levels are indisputable indexes of ID? Is
there room for testing soluble transferrin receptor (sTFRC) or red cell
zinc protoporphyrins in ID? Are serum hepcidin level measurements
useful in ID and by which method should they be evaluated? Which is
the role of the novel reticulocyte or red cell indexes, as reticulocyte he-
moglobin content (RHC) or percent hypochromic red cells (HRC), now
provided by automated counters? When have I to suspect a genetic
form of IDA? Moreover, once the diagnosis of IDA is established, how
have I to treat patients? Is oral iron therapy still up to date? What
about patients who are intolerant or non-compliant to oral iron thera-
py? Concerning intravenous iron, which is the best protocol: is it the
use of multiple low doses or a single high-dose injection? Finally, in
the era of disease prevention, uncertainties exist as to whether only
IDA should be treated or whether treatment should be started earlier
in ID before anemia develops. ID goes across all medical specialties of in-
ternal medicine from hematology to gastroenterology, nephrology, on-
cology, infectious diseases, cardiology, pneumology not to mention
obstetrics and gynecology, surgery etc.… Guidelines may vary according
to specialties and often, because of the above raised questions guide-
lines do not provide clear-cut answers [1].
This review aims at clarifying the general problem of ID and IDA and
at providing at least some clues to tackle the above questions. Etiology,
signs and symptoms, traditional diagnosis and treatment of IDA are ex-
tensively discussed in recent reviews [2,3]. Here the ambition is to ex-
plore and discuss the novelties in the field. However, the issue is in
rapid evolution; this explains the heterogeneity of guidelines and even
their lack of agreement.

http://dx.doi.org/10.1016/j.blre.2017.02.004
0268-960X/© 2017 Published by Elsevier Ltd.

Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
2 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx
2. Iron metabolism
Iron is a trace element indispensable for all cell life. As a heme con-
stituent it participates to hemoglobin, myoglobin, cytochromes and en-
zymes synthesis and is essential for respiration, mitochondrial function
and energy production. As a component of iron-sulfur clusters iron is es-
sential for the prosthetic group of enzymes involved in cell proliferation,
DNA repair, heme synthesis and many other functions, including the ac-
tivity of Iron Regulatory Protein 1 (IRP1) which, when acquires the iron-
sulfur cluster, ceases to control iron acquisition and acquires the role of
aconitase, the enzyme that converts citrate to isocitrate in the tricarbox-
ylic acid [4]. This inter-conversion strengthens the iron role in both me-
tabolism and energy production.
Iron is conserved in mammals. Iron derived from erythrocyte break-
down is quickly recycled to the circulating iron carrier transferrin by
macrophages. Iron from damaged muscles is locally recycled to
regenerating fibers [5] and iron released from damaged red cells in
the circulation, e.g. in acute intravascular hemolysis, is recycled by a
“on demand” transient population of liver macrophages [6]. Virtually
all body iron is reutilized prevalently in erythropoiesis, as only 1 mg
iron is lost daily and is replaced by an equal amount absorbed from du-
odenal enterocytes. The capacity to absorb iron may increase, but re-
mains lower than the capacity of erythropoiesis expansion; this is one
reason why IDA may easily develop after blood loss (or even blood do-
nation), if iron stores are limited.
2.1. Iron trafficking
The iron carrier transferrin is produced and secreted in the circula-
tion by all cells, mainly by the hepatocytes. In normal condition one
third of transferrin is bound to iron representing the “saturated” trans-
ferrin. However, only diferric transferrin (one transferrin molecule
bound to two molecules of iron) is the natural high-affinity ligand of
transferrin receptor 1 (TFRC) that imports iron in most cells including
the hemoglobin synthesizing erythroblasts in bone marrow. Transferrin
is itself a regulator of iron homeostasis, as assessed in animal models,
through its ability to bind, although with lower affinity than TFR1, the
second transferrin receptor, TFR2, which is an iron sensor in the liver
[7] and in erythroid cells [8]. Tfr1 KO mice die during embryonic life be-
cause of severe anemia and brain abnormalities [9], while Tfr1 selective
inactivation in different organs showed that this receptor is indispens-
able for heart [10], skeletal muscle [11] and intestine [12] function.
Tfr1 haplo-insufficient mice have ID in the absence of anemia, with re-
duced total body iron and also low MCV and MCH [9]. Additional players
in TFR1 endosomal cycle are Solute Carrier Family 11 Member 2
(SLC11A2) commonly called Divalent Metal-Transporter-1 (DMT1)
and the metalloreductase Six-Transmembrane Epithelial Antigen of
Prostate 3 (STEAP3). DMT1 on the apical membrane of the duodenal
enterocytes uptakes iron and other metals from the lumen and works
in tight association with the membrane ferrireductase DCYT-B. The ex-
pression of DMT1, DCYT-B as well as of ferroportin, are highly increased
by HIF-2alpha in hypoxia; thus the iron flux from the lumen and its
transfer to the circulation is enhanced [13]. Mutations of DMT1 lead to
a recessive disorder that causes an atypical form of microcytic anemia,
with increased transferrin saturation and increased total body iron
[14], also defined “anemia, hypochromic microcytic, with iron overload
1 (AHMIO1)” (OMIM #206100). Other iron transporters as SLC39A14
(Zip14), a member of metal-ion transporters family, known to transport
zinc [15] and low voltage calcium channels play a role in iron uptake in
specific tissues. Zip14 has been shown to uptake non-transferrin-bound
iron (NTBI), which increases in plasma when transferrin saturation is
high, and to import it in acinar cells of the pancreas and in hepatocytes
[16]. Low voltage calcium channels have been proposed to import iron
in the heart [17] in condition of iron overload.
Several other proteins are involved in intracellular iron trafficking.
SLC25A37 encodes mitoferrin 1, a mitochondrial iron transporter that
Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
mediates iron flux from cytosol to mitochondria in erythroblasts, in re-
sponse to the high requirements of iron for heme and hemoglobin syn-
thesis [18]. SLC25A28 encodes mitoferrin 2, which transfers iron to
mitochondria for heme and iron sulfur cluster biogenesis in non-
erythroid cells. Poly (rC) binding protein 1 (PCBP1), a ubiquitous cyto-
solic chaperone, delivers iron to the storage protein ferritin [19]. The
Nuclear Receptor Coactivator 4 (NCOA4) is emerging as the cargo pro-
tein that addresses ferritin to autophagosomes for degradation [20] in
a process called “ferritinophagy” and has been reported to be important
for the recovery of stored iron in case of need [21].
2.2. Iron regulation
A tight regulation of the available iron is operated at both cellular
and systemic level: the two systems cooperate to ensure perfect homeo-
stasis of the metal, providing adequate organs supply and avoiding both
ID and iron overload.
2.2.1. Cellular iron regulation
In all cells iron homeostasis is maintained through the function of
Iron regulatory proteins (IRP1 and IRP2). IRP1 is ubiquitous and works
at the normal oxygen concentration, while IRP2 is active in hypoxia
through a hypoxia responsive element (HRE) located in its promoter
[4]. Well-known effects of IRP2 are the induction of erythropoietin syn-
thesis in the kidney [22,23] and the increased expression of intestinal
DMT1, DCYTB and ferroportin in hypoxia [13]. High iron levels abolish
the IRP function: on one side iron favors the interconversion of IRP1 to
aconitase through the acquisition of the iron/sulfur cluster, on the
other side iron induces the degradation of IRP2 [24]. The high number
of genes regulated by IRPs indicates that a wide network of mRNAs
and proteins are iron-dependent [25]. IRPs are also essential for mito-
chondrial function. Combined inactivation of both IRP1 and IRP2 genes
in hepatocytes, the cells central to body iron homeostasis, cause hepatic
steatosis, due to a mitochondriopathy because of deficiency of the elec-
tron chain transport and of the tricarboxylic acid, liver failure and death,
strengthening that IRPs play a critical role for mitochondrial and liver
function [26].
2.2.2. Systemic iron regulation
Systemic iron homeostasis is ensured by the hepatic hormone
hepcidin. Hepcidin is an antimicrobial peptide mainly produced by the
liver, which controls both iron entering to plasma from absorptive
sites and iron released from stores. Hepcidin performs its function by
binding and degrading the sole cell iron exporter ferroportin [27], an
ubiquitous protein, which is highly expressed on macrophage surface
and enterocyte basolateral membrane. Hepcidin is mainly controlled
at transcriptional level and its expression increases in response to in-
creased circulating and tissue iron, inflammatory cytokines (especially
IL-6) and metabolic needs. Hepcidin transcription is suppressed by
erythropoiesis expansion, ID and hypoxia [4], testosterone [28] and like-
ly other stimuli. Among the proposed hepcidin inhibitors TMPRSS6,
encoding matriptase 2 [29], is the liver inhibitor, which, cleaving the
BMP co-receptor hemojuvelin [30], attenuates the BMP signaling
pathway and hepcidin transcription. Erythroferrone is considered the
erythroid regulator produced by maturing erythroblasts, which sup-
presses hepcidin when erythropoiesis is expanded, through a still un-
known mechanism [31].
In absolute ID hepcidin suppression is a compensatory mechanism
that allows increased iron acquisition from the intestinal lumen. Low
hepcidin also accounts for the positive response to orally administered
pharmacologic iron, provided that the intestinal mucosa is intact [32].
3. Classification of iron deficiency
Traditionally and based on etiology and pathophysiology we distin-
guish absolute and relative ID, based on laboratory and clinical results ID
 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx 3

(without anemia) and IDA [2]. Deficiency of iron is usually acquired, al- Table 2
though a genetic predisposition toward ID exists and a genetic form of Main causes of iron deficiency/iron deficiency anemia.

iron-refractory iron deficiency anemia [33] is recognized as a rare dis- Type of cause Condition Mechanism
ease. A tentative classification of the different types of ID is shown in Increased Infancy, adolescence Rapid growth
Table 1. requirements ESA treatment Acute expansion of
erythroid mass
3.1. Absolute iron deficiency and iron deficiency anemia Pregnancy: 2nd and 3rd trimester Expansion of maternal
and fetal erythroid
mass
The term ID defines the condition in which iron stores are reduced or Low intake Malnutrition Insufficient iron in the
even depleted in the absence of overt anemia, indicating that iron sup- diet
ply to erythropoiesis is still maintained. Relative ID refers to conditions Vegetarians, vegans Reduction of
bioavailable iron
in which iron is lacking in specific tissues/organs, but total body iron is
Decreased intestinal Gastrectomy, duodenal by pass, Decreased absorptive
not decreased, reflecting an altered iron distribution. It should be absorption bariatric surgery, celiac sprue, surface
strengthened that anemia is the easiest recognizable sign of ID. In prin- inflammatory bowel diseases
ciple lack of iron may affect tissues/organs other than erythropoiesis, as Atrophic gastritis Increased pH
skeletal muscles, heart, brain, but ID of tissues other than erythroid mar- Helicobacter pylori infection Increased pH
Proton pump inhibitors, H2 Drug-induced
row remains unrecognizable by using traditional tests. It is often
blockers
claimed that symptoms as fatigue, muscle weakness, reduced physical IRIDA High hepcidin levels
performance, cognitive impairment may be due to muscle or brain ID. (TMPRSS6 mutations)
However, at present the correct interpretation of these symptoms as Chronic kidney disease Reduced hepcidin
signs of ID is only retrospective, when they abate after iron therapy. excretion
Chronic blood loss Site of bleeding
Restless leg syndrome may be associated with neurological inflammato- Benign, malignant lesions, Gastrointestinal tract
ry disorders, drugs and in a proportion (about one third) of the cases has hookworm
been ascribed to systemic ID [34]. Salicylates, corticosteroids, Drug-induced
Depending on the etiology, ID may progress to frank anemia in some, non-steroidal anti-inflammatory
drugs
but not in all cases. When IDA is the result of progressive ID, usually de-
Uterine bleeding, hematuria, Genitourinary system
velops rather slowly and for this reason may be well tolerated. The diag- intravascular hemolysis (e.g.
nosis of IDA requires laboratory testing, since symptoms may be paroxysmal nocturnal
present, but are unspecific and often are overlooked. For discussion of hemoglobinuria)
all possible symptoms reported in IDA see [35]. Bleeding defects (hereditary Any organ/system
hemorrhagic telangiectasia)
Conditions that predispose to ID and IDA (Table 2) are increased iron Regular blood donors Blood letting
requirements, as occurs in children during growth and in young women Multiple Chronic kidney disease, Increased
during the fertile age, because of menses and pregnancies. Other condi- mechanisms inflammatory bowel diseases, proinflammatory
tions as reduced iron intake, chronic blood loss and defective absorption associated with obesity cytokines, increased
inflammation hepcidin
are well known and their assessment is part of the workout of ID, espe-
Chronic heart failure Uncertain
cially when anemia is present. IDA is highly prevalent in children and pathogenesis
young females in developing countries with prevalence attaining 40% Acute blood loss Major surgery Postoperative anemia
in preschool children, around 30% in young females and up to 38% in (concomitant ID)
pregnant women respectively [36,37]. Also in high-income countries Adapted from reference [2], http://www.nejm.org/doi/full/10.1056/NEJMra1401038.
children and females, especially pregnant females, remain the top risk
categories [37]. IDA may coexist with other nutritional anemias, espe-
cially in developing countries or in elderly people. of reduced absorption underlying conditions might be Helicobacter pylo-
Since chronic blood loss from the gut and reduced iron absorption ri infection, celiac disease, autoimmune gastritis and even Inflammatory
are the most frequent causes, the investigation of ID/IDA may lead to Bowel Diseases (IBD). A meta-analysis of ID in patients infected with
discover gastrointestinal disorders, even severe or malignant. In case Helicobacter pylori concluded that there is an “increased likelihood of
depleted iron stores in relation to Helicobacter pylori infection” and
that its eradication improves ID [38]. ID and IDA are more common in
celiac patients than in the general population, and, in the absence of
Table 1 specific symptoms, the condition often goes undiagnosed [32]. Correct
Classification of iron deficiency (and iron deficiency anemia).
diagnosis is important since the introduction of the gluten-free diet
Mechanisms may restore iron absorption. Autoimmune gastritis, a common cause
Based on pathophysiology of megaloblastic anemia may be a rare cause of IDA [32].
Absolute iron deficiency Decreased iron stores Several drugs interfere with iron absorption (proton pump inhibi-
Relative iron deficiency Iron deficiency in selected tissues with tors, H2 blockers) or favor blood losses (aspirin, anticoagulants, non-
normal/high iron stores
steroidal anti-inflammatory drugs) and should always be considered
(e.g. iron restricted erythropoiesis,
ID in chronic heart failure) in patient evaluation.
ID/IDA, in addition of other nutritional deficiencies, may complicate
Based on clinical data
both Roux-en-Y gastric bypass and gastric banding, procedures used in
Iron deficiency Decreased iron stores, absence of anemia
Other tissue deficiency manifestations? bariatric surgery to control severe obesity and diabetes. It should be
Iron deficiency anemia ID + anemia of variable degree considered that patients often are iron deficient before surgery and
Microcytic and hypochromic red cells that nutritional deficiencies may persist or exacerbate after surgery
Based on inheritance [39]. ID is commonly reported in obese subjects due to their high
Genetic (rare) serum hepcidin and reduced iron absorption because of adiposity-
IRIDA Constitutive excess hepcidin production related inflammation. Measuring erythrocyte incorporation of iron iso-
Acquired (common) Increased iron demands, decreased intake, topes has demonstrated that obese subjects do not show the physiologic
blood loss, reduced absorption
up-regulation of iron absorption that is typical of ID and that this

Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
4 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx
negative effect improves after bariatric surgery [40]. Monitoring nutri-
tion and iron levels is mandatory in these patients.
ID is reported in patients with chronic heart failure, likely secondary
to reduced iron absorption or decreased intake. The proportion of pa-
tients with ID/IDA (up to 30–50%) differs in the different series and ac-
cording to the cut-off of iron parameters used to define ID [41]. Both
anemia and ID are negative prognostic factors in chronic heart failure,
associated with increase in all causes of death and with specific cardio-
vascular mortality [42].
Why cardiomyocytes develop ID in heart failure remains unknown.
Recently studies in murine models of ID have provided some clues to
this problem. Cardiomyocyte targeted deletion of TFR1 that induces ID
exclusively in the heart irrespective of systemic iron levels, results in de-
fective heart contraction, ventricle dilation and heart failure in mice
[10]. Conditional deletion of IRPs in cardiomyocytes leads to decrease
iron-sulfur cluster synthesis and mitochondria electron transport
chain, decreased left ventricle systolic function and mitochondrial respi-
ratory capacity in response to stress. Interestingly, all defects are
corrected by intravenous infusions of high iron dose [43].
ID is common in blood donors, especially females, and limits the do-
nations frequency. ID (and mild anemia) are often diagnosed in young
endurance athletes, especially females, due to hemolysis, blood loss
and mild inflammation and may impair sport performance.
3.2. Anemia of chronic disease or anemia of inflammation - relationship
with ID
3.2.1. Anemia of chronic disease or anemia of inflammation
Anemia of chronic disease or anemia of inflammation (ACD) is a
moderate anemia, usually observed in patients with clinically relevant
pathologic conditions such as cancer, chronic infections, autoimmune
diseases, CKD and in general in all conditions characterized by persis-
tent and high production of pro-inflammatory cytokines. Anemia re-
flects the severity of the basal condition and reverses when the basal
disease is successfully treated.
Cytokines as IL-1β, γ-Interferon and TNF-α negatively influence
erythropoiesis: they reduce Erythropoietin (Epo) production, Epo re-
ceptor expression on erythroid cells and in general Epo responsiveness.
Moreover they induce alterations of iron metabolism to the aim of se-
questering iron from the circulation [44]. Hepcidin expression, in-
creased under the stimulus of IL-6, IL-1beta and IL-22 [45], causes
ferroportin degradation, decreased intestinal iron absorption, macro-
phage iron withholding and iron restricted erythropoiesis. For this rea-
son serum ferritin is usually high, while transferrin saturation is
decreased. However, true ID may develop in the same setting, challeng-
ing the correct diagnosis.
3.2.2. Iron deficiency anemia associated with anemia of chronic disease
Coexistence of IDA and ACD in the same patient is not unusual and
makes the diagnosis complex. To recognize ID in the context of ACD is
not an academic issue, because iron treatment may reduce patient
symptoms and ameliorate the clinical performance. Classic examples
are chronic infections (e.g. malaria, but also bacterial or viral infections),
CKD and IBD. In endemic areas malaria contributes to increase hepcidin
levels, preventing iron absorption in subjects who may have IDA for nu-
tritional causes. Control of malaria may ameliorate iron deficiency and
improve response to iron treatment [36]. Another example in the
same low-income areas is hookworm infection, an important cause of
ID, because of chronic blood loss that may be associated with inflamma-
tion; also this condition is reversible, at least in part, after deworming
treatment.
IDA with inflammation is a hallmark of CKD, because chronic blood
loss may be concomitant with inflammation in the late stages of the dis-
ease. IDA may be further worsened by the use of erythropoiesis stimu-
lating agents. IBD are a common cause of anemia, often due to
Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
multiple causes including ID, vitamin B12 and folic acid deficiency and
inflammation.
In chronic congestive heart failure the relationship of IDA and ID
with inflammation/hepcidin is less clear [42].
3.3. Genetic iron deficiency
3.3.1. Iron refractory iron deficiency anemia
Iron refractory iron deficiency anemia (IRIDA, OMIM #206200) was
first recognized in 2008, following the discovery of the TMPRSS6 gene
that encodes the liver hepcidin inhibitor transmembrane protease ser-
ine 6, also called matriptase 2 [29]. IRIDA is an autosomal recessive dis-
ease, due to mutations of TMPRSS6 gene [33]. Normally TMPRSS6 blocks
the hepcidin activation by the BMP/SMAD pathway, cleaving the co-
receptor hemojuvelin from hepatocyte plasma membrane [30]. High
hepcidin levels consequent to inactivating mutations of TMPRSS6 in
IRIDA block iron absorption from the gut and iron release from macro-
phages, leading to very low levels of circulating iron, which are insuffi-
cient to the erythropoiesis needs. Epidemiological data are lacking: from
the available reports the disease is spread all over the world, but is rare,
occasionally found in different countries and in different ethnic groups.
As gathered from its name, the disease cannot be corrected by oral iron
supplementation, and is Epo-resistant. It usually requires parenteral, in-
travenous iron especially when the iron needs are high as occurs in chil-
dren [46].
Other genetic disorders affecting iron genes, as DMT1 and STEAP3
deficiencies are characterized by defective iron utilization in erythropoi-
esis, while atransferrinemia, known since long time has a defect in iron
release to erythropoiesis. All strengthen the concept that the transferrin
receptor pathway is essential for hemoglobin production in erythro-
blasts, but may be dispensable in other cells (as hepatocytes or pancre-
atic cells) that acquire iron through other transporters. Accordingly IDA
in these disorders has atypical features, being associated with high
transferrin saturation and increased iron stores that reflect the reduced
use of iron in erythropoiesis [for review see [14]].
3.3.2. Genetic susceptibility to ID
The identification of IRIDA has suggested the existence of genetic
susceptibility to develop ID. Genome Wide Association Studies, includ-
ing a recent meta-analysis, indicate that TMPRSS6 genetic variants asso-
ciate with erythrocyte and iron traits in Caucasians and Asian
populations [47,48]. However, there are inter-ethnic variations in the
association [49]. Interestingly the most common TMPRSS6 variant,
rs855791, which causes a non-synonymous change in the catalytic do-
main of the protease, due to the substitution of valine for alanine at po-
sition 736 (A736V), has been shown to influence serum hepcidin levels
in normal individuals [50].
ID may represent a problem in blood donors after repeated dona-
tions and in female donors even after a single donation [51]. The genetic
susceptibility to ID has been shown to have implications for blood dona-
tion. As an example, female carriers of the 736A allele of rs855791 in
TMPRSS6, which is associated with low hepcidin levels [50], have a de-
creased risk to develop ID after blood donation, at variance from carriers
of the high-hepcidin 736V variant [51]. The evaluation of SNPs of iron
genes such as HFE, Transferrin (TF), TMPRSS6 in N14.000 Swedish
blood donors, carried out in order to define the effect on serum ferritin
levels, showed that TMPRSS6 736V was negatively associated to iron
stores in males [52]. In addition the allele G of rs1800562 in HFE, corre-
sponding to the normal cysteine at position 282, where the mutation of
type 1 hemochromatosis (cysteine to tyrosine, C282Y) occurs, associ-
ates with lower iron stores in both gender. In the same gene the allele
C of the other variant rs179945, corresponding to the histidine at posi-
tion 63 where the H63D (histidine-Naspartic acid) substitution occurs,
associates with lower iron stores only in males, while the homozygous
state for the same allele associates with ID in female donors [52]. Further
studies in progress will help to improve and personalized blood
 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx
donation and at the same to prevent the development of ID in donors,
especially if females.
4. Diagnosis of IDA and ID
The diagnosis of IDA is usually easy based on few biochemical
markers. Low levels of serum iron, transferrin saturation and serum fer-
ritin are a straightforward combination for diagnosis of ID. However, the
same diagnosis can become challenging in the presence of other dis-
eases, especially inflammatory conditions, or when it is caused by rare
inherited defects of iron metabolism [53]. The identification of ID with-
out anemia is also simple when ID is absolute, but difficult in association
with other conditions.
4.1. Conventional tests
ID can be diagnosed by simple tests. Interpretation of serum ferritin
levels may be problematic when levels are increased, but when in the
low range ferritin deficiency equals ID. In absolute ID, especially in the
presence of anemia (IDA) ferritin is usually b 15 ng/ml in adults and
b12 ng/ml in children [54]. Levels lower than 30 ng/ml are accepted
as a sensitive and specific cut off which correlates with the absence of
iron stores in the bone marrow and thus identifies absolute ID [55].
Serum iron and transferrin saturation (or saturated iron binding ca-
pacity, IBC) provide a measure of iron available for erythropoiesis, inde-
pendently from iron stores. However, if chronic inflammation is present
both parameters are reduced and thus of limited utility. Transferrin
saturation b 16% is an accepted cut off for ID [56], but transferrin satura-
tion may also decrease in inflammation.
In longstanding anemia, red cells are microcytic and hypochromic, as
shown by low MCV and MCH and increased RDW: however, the diag-
nostic value of red cell indexes is limited: their changes occur late be-
cause of long life span of red cells. Automated blood cell counters may
provide several indexes to assess hypochromia and microcytosis, not
only of mature red blood cells but also of young reticulocytes. Reticulo-
cyte hemoglobin content (RHC) estimates the amount of iron available
for erythropoiesis in the previous 3–4 days and is an early index of ID;
since the changes occur rapidly it is especially useful to identify re-
sponders to iron supplementation or individuals who develop iron defi-
ciency after erythropoietin injection. The percentage of hypochromic
red cells (% HRC) reflects and estimates recent iron reduction. Unfortu-
nately these red cell indexes, proposed and useful i.e. in patients with
CKD treated with erythropoietic stimulating agents, are of limited
value in chronic ID. In addition they are not extensively used in clinical
practice and suitable counters may not be available in low-income
settings.
4.2. Old and new tests
Perl's staining has been for longtime the gold standard for diagnosis
of ID, as it visually demonstrates the absence of stainable iron in the
bone marrow smears, both in erythroblasts and in macrophages. At
present it is exceptionally used, since it requires the minimally invasive
procedure of bone marrow aspiration. In addition the absence of iron in
erythroblasts may occur in inflammation, although in this case iron is
present, even abundant, in macrophages.
The dosage of erythrocyte zinc protoporphyrins is used for screening
ID, especially in low-income countries, because it is a cheap and simple
test. However, it is unspecific, since protoporphyrins are increased also
in hemoglobinopathies, sideroblastic anemia, inflammation and lead
poisoning.
Serum erythropoietin (EPO) is increased in IDA and its increase is
exponentially related to anemia severity. However, EPO measurement
is useless, since it increases in any type of anemia able to induce
hypoxia.
Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
5
Circulating soluble transferrin receptor and serum hepcidin are po-
tentially useful biochemical markers that are discussed separately.
4.2.1. Soluble transferrin receptor (sTFRC)
TFRC is cleaved by a membrane protease in the erythroid cells when
it is not stabilized by diferric transferrin: thus serum soluble transferrin
receptor (sTFRC) levels are increased in ID [57] and also in acute eryth-
ropoiesis expansion, that induces a condition of relative ID. Usually de-
termining sTFRC is not needed for the diagnosis of ID. However, a recent
meta-analysis of results related to its use have shown that the assay has
a high sensitivity (86%) and specificity (75%) [58], but lower than that of
serum ferritin cut off placed at b30 ng/ml [55].
The levels of serum soluble transferrin receptor (sTFRC) have been
proposed to help the differential diagnosis of ID with ACD and especially
in identifying ID in the context of inflammation, as sTFRC levels are not
increased when erythropoiesis is attenuated. However, general agree-
ment is lacking [59]. The test has been used in epidemiologic studies
in Africa; as an example it is utilized in malaria endemic areas to identify
children with ID because sTFRC levels at variance with ferritin are more
stable in inflammation [55]. However, it is scarcely used for individual
diagnosis. The ratio between sTFRC - whose levels are high in ID and
normal/low in inflammation - and log ferritin - whose levels are low
in ID and normal/high in inflammation - may help in recognizing IDA
in the setting of ACD. Several authors suggest that within this context
the ratio of sTFRC and log ferritin is the most useful test [44]. Unfortu-
nately the levels of sTFRC (and, thus, the sTFRC–ferritin index) depend
on the assay used and lack of standardization among different immuno-
assays hampers both the comparison among different studies and the
use of the test in clinical practice.
4.2.2. Serum hepcidin
Serum hepcidin is a promising novel biomarker for the diagnosis of
iron disorders. It is decreased in ID [60,61] and levels may become unde-
tectable in severe cases of IDA [62]. Hepcidin levels show a circadian
rhythm and thus the test should be done in the morning at fast. The re-
sults should be interpreted in the light of the clinical context. To exclude
inflammation CRP should be assessed. Also renal and liver function tests
should be measured [62], considering that hepcidin levels tend to in-
crease with the decrease of glomerular filtration rate and to decrease ac-
cording to hepatocytes damage. In chronic kidney disease hepcidin is
increased because of both decreased excretion and concomitant inflam-
mation. In chronic liver disease hepcidin levels are decreased, as albu-
min, reflecting the deficiency of the hepatocyte synthetic capacity.
The indications to hepcidin dosage remain limited to selected condi-
tions. There is a general agreement that the dosage may be useful to
confirm IRIDA [2,62,63], because levels are constitutionally high or nor-
mal, in any case inappropriate to ID where levels should be low/unde-
tectable. Hepcidin dosage may thus represent an alternative to
TMPRSS6 gene sequencing in IRIDA. A pilot study has suggested that
serum hepcidin levels might be useful to guide the choice between
oral or intravenous iron for therapy [64], but large studies are not avail-
able. Whether serum levels of hepcidin might be an index of ID even
within ACD, in condition of chronic inflammation [62] is an interesting
possibility still under investigation. Hepcidin levels in African children
in malaria areas have been proposed to identify those who would ben-
efit of iron supplementation [65].
As in the case of sTFRC also with hepcidin dosage different tests are
available. Hepcidin is measured either by Mass Spectroscopy, that is not
at hand in routine laboratories, or by immunochemical methods by
means of ELISA commercial kits. Both methods may reveal the bioactive,
25 amino acid peptide but the results may considerably differ according
to the procedure used. Lack of harmonization among the different tests,
as in the case of sTFRC, makes it difficult to compare different studies
and hampers the clinical use of hepcidin dosage, since reference values
remain method-dependent. A recent study has shown a good correla-
tion among the different methods [66]: this observation suggests that
6 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx

it is possible to utilize an available test when validated [62] and opens Table 3
the possibility of a clinical use of hepcidin testing in the near future. Indications to intravenous iron therapy.

Condition Defect Mechanisms or examples

5. Treatment Intolerance to Gastrointestinal side effects Mucosa iron toxicity
oral iron
5.1. Oral iron therapy Refractoriness Gastric-duodenal surgery Reduced absorption (high pH,
to oral iron decreased absorptive surface)
Helicobacter pylori Reduced absorption, blood loss
Low/undetectable hepcidin levels ensure effective absorption of oral
infection
iron in most cases of IDA. Iron salts, especially iron sulfate, remain the Celiac disease Reduced absorption
gold standard of oral treatment. High and divided doses are traditionally Autoimmune atrophic Reduced absorption
recommended in IDA, but recent studies are challenging these tradition- gastritis
al habits. Inflammatory bowel disease Mucosal damage by oral iron,
(IBD) reduced absorption
To limit common gastrointestinal side effects preparations other IRIDA: iron refractory iron TMPRSS6 recessive mutations,
than iron salts as liposomal iron, iron-based phosphate binders, heme deficiency anemia high hepcidin
iron polypeptide, seem to be better absorbed and tolerated and appear Rapid Severe IDA Example: pregnancy (2nd, 3rd
promising [67]. However, at present the limited published data require correction of trimester)
anemia Chronic bleeding not Example: congenital coagulation
confirmation in larger prospective studies; moreover the molecular
needed manageable with oral iron disorders
mechanism of absorption remains to be investigated. ESA in CKD Intravenous iron more Functional ID due to
As shown in a recent meta-analysis, gastrointestinal side effects are efficacious than oral iron erythropoietic expansion and high
more common with oral than with intravenous iron [68]: they may be hepcidin levels because of low
caused by non-absorbed iron, which is potentially toxic for the gastroin- excretion and inflammation
Chronic heart High iron local needs? Clinical trials in progress
testinal mucosa, because of its oxidative properties. Attempts are ongo-
failure
ing to reduce the dose of supplemented iron, without losing efficacy. Substitute for Refusal for personal reasons Jehovah witnesses
Changes of gut microbiota profile have been reported in African chil- blood Perioperative anemia Elective surgery (orthopedic,
dren, who underwent an iron fortified diet: from the benign Lactobacil- transfusions (Transfusion sparing abdominal…)
strategies)
lus species that do not need iron, to the unfavorable pathogen
ACD (selected Anemia of cancer patients Erythropoiesis expansion
enterobacteria and changes were accompanied by calprotectin increase, cases) treated with ESA after
as a sign of gut inflammation [69]. Different microbiota changes have chemotherapy
been reported after oral compared with intravenous iron in IBD [70]. Adapted from reference [2], http://www.nejm.org/doi/full/10.1056/NEJMra1401038.
Traditionally oral iron has been administered in divided doses. How-
ever, the rapid increase of hepcidin in response to iron administration
[71] has strengthened its negative influence on the absorption of the adverse events or infections [77]. However, based on post-marketing re-
second daily doses. Moretti et al. [72]measuring the absorption of an ports the European Medicine Agency (EMA) recommendations remain
iron isotope, in young non-anemic women with ferritin restrictive, suggesting that when considering parenteral iron the rela-
levels ≤ 20 ng/ml showed that the oral administration of 60 mg iron sul- tionship of risks/benefits should always be evaluated and several rules
fate on alternate day maximized fractional iron absorption, increased ef- adopted. Among them iron should be administered only by trained
ficacy and reduced gastrointestinal side effects, thereby improving staff, in a suitable location with resuscitation facilities access. Iron infu-
tolerance. On the contrary when administered daily but in refracted sion should be always slow, especially in the first minutes of administra-
doses, the second dose was less absorbed because blocked by high tion and the patient carefully monitored. Patients with history of iron or
hepcidin levels secondary to iron increase. This pilot study might pave drug allergies should be carefully evaluated before treatment and likely
the way to novel strategies of oral iron schedules [73]. However, the excluded. Because of lack of safety data women in the first trimester of
sample size was small, the investigation was limited to 2 days and the pregnancy should be excluded from intravenous treatment.
study included only ID non-anemic women. Traditional indications to intravenous iron are well established
This condition will likely benefit of intermittent treatment (e.g. when absorption is expected to be low or random, as after gastric-
60 mg iron on alternate days); it is not yet clear whether the same treat- duodenal surgery or in case of malabsorption [2]. We have learnt that
ment would benefit IDA where iron absorption is expected to be great- oral iron is useless if inflammation coexists and hepcidin levels are
er, because hypoxia magnifies the intestinal iron import by increasing high. Oral iron is less effective than intravenous iron when associated
the expression of iron transporters, especially DMT1 and ferroportin with erythropoietin stimulating agents (ESA) in CKD [78]. The use of
[13] in duodenal enterocytes. oral iron is potentially dangerous and should be avoided in active IBD
Iron should be cautiously used in special circumstances. In malaria [79] that represents an indication to intravenous iron.
endemic areas iron supplementation in the attempt to correct ID may Novel commercial compounds allow the administration of high iron
increase parasitemia or even clinical malaria in children [74] and dose (up to 1 g) in a single injection in order to replace the total amount
might have negative effects also in other types of infection like diarrhea of iron needed in one-two doses. This approach may be convenient in
and acute respiratory infection [36,75]. This iron/infections link [76] selected cases, allowing not only to correct the hemoglobin deficit, but
suggests that further studies should be implemented to define the pos- also to rapidly reintegrate the stores [35].
itive and negative effects of iron supplementation in developing coun- For the description of the different preparations and the details on
tries, which have the highest prevalence of ID and IDA. iron administration readers are referred to a previous review [35]. The
. issues of iron adverse effects, their prevention and management are
well discussed in [80]. It has been suggested that intravenous iron is
5.2. Indication to intravenous iron treatment underutilized because of the wrong perception of immediate harmful
side effects [35]. It is possible that the attitude will change in the future,
Indications to intravenous iron are increasingly recognized (Table 3) since the increased use of the novel preparations seem to confirm the
in parallel with the awareness that modern compounds have less side lack of immediate dangerous side effects in the clinical settings.
effects when compared to old preparations. In a meta-analysis of Long-term side effects remain to be assessed especially related to the
N10,000 patients enrolled in clinical trials i.v. iron (compounds available potential toxicity due of iron accumulation or the development of infec-
on the market) were not associated with increased risk of severe tions. Most randomized clinical trials seem reassuring. However,

Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx
patients included in trials have short follow-up hence the trials have not
the power to assess long-term consequences, even in the clinical set-
tings with more experience of intravenous iron treatment, as in CKD
[81].
5.3. Special conditions
Intravenous iron and especially a high dose iron schedule seem ef-
fective in ameliorating the physical performance in chronic heart failure,
improving NYHA (New York Heart Association) class and quality of life,
even independently from the correction of anemia. Results have been
obtained in prospective trials of intravenous iron versus placebo [82,
83]. Since serum ferritin is usually high in these patients (ID is diag-
nosed even with levels up to 300 ng/ml, when transferrin saturation is
b20%); considering the positive results of iron treatment the condition
is taken as a possible example of ID limited to the heart tissue.
Why cardiomyocytes become susceptible to ID in chronic heart fail-
ure is unclear and why iron is required in high dose to correct ID and im-
prove cardiac energy is intriguing and requires further studies. The
models of selective heart ID in transgenic mice [10,43] described in
Section 3.2 may likely provide experimental support to explain the clin-
ical response observed in patients.
Patient blood management is enforced worldwide to better use of
blood components. Postoperative IDA is an important problem for
transfusional medicine, recently addressed to spare blood transfusions.
In addition, anemia in the perioperative period is associated with in-
creased morbidity and mortality. It is traditionally treated with alloge-
neic blood transfusions that, in turn, carry the risk of a negative
outcome [84]. Thus recently the problem of using intravenous iron has
been addressed to correct intraoperative blood loss and to spare blood
transfusions. Ideally patients should be evaluated and treated preoper-
atively. However, a recent randomized clinical trial on patients who
underwent major orthopedic and abdominal surgery evaluated the ef-
fects of administering postoperative high dose of intravenous iron on
anemia, iron stores, need of blood transfusions and outcomes at
4 weeks after surgery. The study concluded that hemoglobin and iron
parameters are improved and need of transfusion decreased in the
iron arm versus standard care [85]. Treating patients who undergo elec-
tive surgery with high iron in a single injection is a sort of primary pre-
vention of ID/IDA in patients undergoing major surgery. Even in this
case it is likely that intravenous iron is able to overcome the effect of
high hepcidin secondary to postoperative cytokine activation.
6. Summary and future directions
ID due to increased iron requirements, decreased intake, poor ab-
sorption or blood loss, is associated with reduction of total body iron,
is common worldwide and its main well-known manifestation is ane-
mia (IDA). Relative ID, i.e. lack of iron in specific tissues/organs in the
presence of normal/high iron stores is increasingly recognized. Exam-
ples are either chronic inflammatory disorders because of the increased
levels of the key iron regulator hepcidin or chronic heart failure in which
ID may develop even in the absence of anemia. While the diagnosis of ID
is simple, it becomes complex in the context of inflammatory disorders
and requires a combination of tests.
Treatment of IDA should be individualized according to the underly-
ing cause. Oral iron remains the first therapeutic choice in most patients,
but compliance to a prolonged treatment is limited by gastrointestinal
side effects. New schedules of administration or new preparations
may improve tolerability and efficacy. Correcting ID in the absence of
anemia is often optional, but common practice is changing. Oral alter-
nate day iron will probably become an option in stable cases, while ID
in conditions as chronic heart failure or the prevention of postoperative
ID requires high dose intravenous iron. In general indications to intrave-
nous iron are increasing; some are well defined, others have been pro-
posed and will be implemented based on ongoing and future studies.
Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
7
Practice points
• Patients should be carefully evaluated to define the cause of ID (in-
creased iron requirements, defective iron intake, reduced intestinal
absorption or blood loss). In case a pathological cause is identified
iron supplementation should be combined with treatment of the un-
derlying cause.
• Absolute ID is diagnosed by serum ferritin b 30 ng/ml and may be ac-
companied or not by anemia (IDA).
• Diagnosis of ID in the setting of inflammatory diseases relies on higher
ferritin cut offs (b100 ng/ml) or even higher values, in the presence of
low transferrin saturation (b20%) in CKD and congestive heart failure.
The sTFR/ferritin index is proposed but scarcely used in clinical prac-
tice. Hepcidin dosage has limited indications; at present it is preva-
lently a research test.
• Oral iron salts are the first choice of treatment in most cases. The need
of a prolonged treatment and intestinal side effects are the major
drawbacks of oral iron therapy. Indications to intravenous iron are in-
tolerance or refractoriness to oral therapy, defective intestinal absorp-
tion including high hepcidin levels and need of rapid hemoglobin
(Hb) increase. Others will be added based on ongoing and future stud-
ies.
• Clinical trials have shown that intravenous iron preparations (includ-
ing single high dose injection) have an acceptable low rate of immedi-
ate side effects; in the lack of specific data, long- term safety of high
dose iron remains to be established.
Research agenda
• Studies aimed at better understanding the dynamic of intestinal iron
absorption might lead to develop better tolerated and efficacious
oral compounds and to define novel protocols of iron administration.
• The identification of biomarkers of selective tissues ID (muscles, heart,
brain) would provide useful tools to guide therapy.
• Large studies should assess whether hepcidin dosage may help the
identification of ID in the context of inflammation.
• Indications to intravenous iron therapy should be established based
on the results of randomized clinical trials comparing intravenous
versus oral compounds in specific clinical conditions and aiming at
evaluating anemia correction, iron stores, immediate and possibly
long-term side effects.
Conflict of interest
CC is a member of the Iron Core Steering Committee of Vifor Pharma
and has received honoraria from Vifor Pharma.
References
[1] Peyrin-Biroulet L, Williet N, Cacoub P. Guidelines on the diagnosis and treatment of
iron deficiency across indications: a systematic review. Am J Clin Nutr 2015;102:
1585–94.
[2] Camaschella C. Iron-deficiency anemia. New Engl J Med 2015;372:1832–43.
[3] Lopez A, Cacoub P, Macdougall IC, Peyrin-Biroulet L. Iron deficiency anaemia. Lancet
2016;387:907–16.
[4] Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to tango: regulation of
mammalian iron metabolism. Cell 2010;142:24–38.
[5] Corna G, Caserta I, Monno A, Apostoli P, Manfredi AA, Camaschella C, et al. The repair
of skeletal muscle requires iron recycling through macrophage ferroportin. J
Immunol 2016;197:1914–25.
[6] Theurl I, Hilgendorf I, Nairz M, Tymoszuk P, Haschka D, Asshoff M, et al. On-demand
erythrocyte disposal and iron recycling requires transient macrophages in the liver.
Nat Med 2016;22:945–51.
[7] Goswami T, Andrews NC. Hereditary hemochromatosis protein, HFE, interaction
with transferrin receptor 2 suggests a molecular mechanism for mammalian iron
sensing. J Biol Chem 2006;281:28494–8.
[8] Nai A, Lidonnici MR, Rausa M, Mandelli G, Pagani A, Silvestri L, et al. The second
transferrin receptor regulates red blood cell production in mice. Blood 2015;125:
1170–9.
[9] Levy JE, Jin O, Fujiwara Y, Kuo F, Andrews NC. Transferrin receptor is necessary for
development of erythrocytes and the nervous system. Nat Genet 1999;21:396–9.
8 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx
[10] Xu W, Barrientos T, Mao L, Rockman HA, Sauve AA, Andrews NC. Lethal cardio-
myopathy in mice lacking transferrin receptor in the heart. Cell Rep 2015;13:
533–45.
[11] Barrientos T, Laothamatas I, Koves TR, Soderblom EJ, Bryan M, Moseley MA, et al.
Metabolic catastrophe in mice lacking transferrin receptor in muscle. EBioMedicine
2015;2:1705–17.
[12] Chen AC, Donovan A, Ned-Sykes R, Andrews NC. Noncanonical role of transferrin re-
ceptor 1 is essential for intestinal homeostasis. Proc Natl Acad Sci U S A 2015;112:
11714–9.
[13] Mastrogiannaki M, Matak P, Peyssonnaux C. The gut in iron homeostasis: role of HIF-
2 under normal and pathological conditions. Blood 2013;122:885–92.
[14] Camaschella C. How I manage patients with atypical microcytic anaemia. Br J
Haematol 2013;160:12–24.
[15] Taylor KM, Morgan HE, Johnson A, Nicholson RI. Structure-function analysis of a
novel member of the LIV-1 subfamily of zinc transporters, ZIP14. FEBS Lett 2005;
579:427–32.
[16] Jenkitkasemwong S, Wang CY, Coffey R, Zhang W, Chan A, Biel T, et al. SLC39A14 is
required for the development of hepatocellular iron overload in murine models of
hereditary hemochromatosis. Cell Metab 2015;22:138–50.
[17] Tsushima RG, Wickenden AD, Bouchard RA, Oudit GY, Liu PP, Backx PH. Modulation
of iron uptake in heart by L-type Ca2+ channel modifiers: possible implications in
iron overload. Circ Res 1999;84:1302–9.
[18] Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE, et al. Mitoferrin is essen-
tial for erythroid iron assimilation. Nature 2006;440:96–100.
[19] Leidgens S, Bullough KZ, Shi H, Li F, Shakoury-Elizeh M, Yabe T, et al. Each member of
the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity to-
ward ferritin. J Biol Chem 2013;288:17791–802.
[20] Mancias JD, Wang X, Gygi SP, Harper JW, Kimmelman AC. Quantitative proteomics
identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature 2014;
509:105–9.
[21] Bellelli R, Federico G, Matte A, Colecchia D, Iolascon A, Chiariello M, et al. NCOA4 de-
ficiency impairs systemic iron homeostasis. Cell Rep 2016;14:411–21.
[22] Anderson SA, Nizzi CP, Chang YI, Deck KM, Schmidt PJ, Galy B, et al. The IRP1-HIF-
2alpha axis coordinates iron and oxygen sensing with erythropoiesis and iron ab-
sorption. Cell Metab 2013;17:282–90.
[23] Ghosh MC, Zhang DL, Jeong SY, Kovtunovych G, Ollivierre-Wilson H, Noguchi A, et al.
Deletion of iron regulatory protein 1 causes polycythemia and pulmonary hyperten-
sion in mice through translational derepression of HIF2alpha. Cell Metab 2013;17:
271–81.
[24] Zhang DL, Ghosh MC, Rouault TA. The physiological functions of iron regulatory pro-
teins in iron homeostasis - an update. Front Pharmacol 2014;5:124.
[25] Sanchez M, Galy B, Schwanhaeusser B, Blake J, Bahr-Ivacevic T, Benes V, et al. Iron
regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and
shaping of the cellular proteome by iron regulatory proteins. Blood 2011;118:
e168–79.
[26] Galy B, Ferring-Appel D, Sauer SW, Kaden S, Lyoumi S, Puy H, et al. Iron regulatory
proteins secure mitochondrial iron sufficiency and function. Cell Metab 2010;12:
194–201.
[27] Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin
regulates cellular iron efflux by binding to ferroportin and inducing its internaliza-
tion. Science 2004;306:2090–3.
[28] Latour C, Kautz L, Besson-Fournier C, Island ML, Canonne-Hergaux F, Loreal O, et al.
Testosterone perturbs systemic iron balance through activation of epidermal growth
factor receptor signaling in the liver and repression of hepcidin. Hepatology 2014;
59:683–94.
[29] Du X, She E, Gelbart T, Truksa J, Lee P, Xia Y, et al. The serine protease TMPRSS6 is
required to sense iron deficiency. Science 2008;320:1088–92.
[30] Silvestri L, Pagani A, Nai A, De Domenico I, Kaplan J, Camaschella C. The serine pro-
tease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane
hemojuvelin. Cell Metab 2008;8:502–11.
[31] Kautz L, Jung G, Valore EV, Rivella S, Nemeth E, Ganz T. Identification of
erythroferrone as an erythroid regulator of iron metabolism. Nat Genet 2014;46:
678–84.
[32] Hershko C, Camaschella C. How I treat unexplained refractory iron deficiency ane-
mia. Blood 2014;123:326–33.
[33] Finberg KE, Heeney MM, Campagna DR, Aydinok Y, Pearson HA, Hartman KR, et al.
Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA). Nat
Genet 2008;40:569–71.
[34] Mehmood T, Auerbach M, Earley CJ, Allen RP. Response to intravenous iron in pa-
tients with iron deficiency anemia (IDA) and restless leg syndrome (Willis-Ekbom
disease). Sleep Med 2014;15:1473–6.
[35] Auerbach M, Adamson JW. How we diagnose and treat iron deficiency anemia. Am J
Hematol 2016;91:31–8.
[36] Pasricha SR, Drakesmith H, Black J, Hipgrave D, Biggs BA. Control of iron deficiency
anemia in low- and middle-income countries. Blood 2013;121:2607–17.
[37] Kassebaum NJ, Jasrasaria R, Naghavi M, Wulf SK, Johns N, Lozano R, et al. A sys-
tematic analysis of global anemia burden from 1990 to 2010. Blood 2014;123:
615–24.
[38] Hudak L, Jaraisy A, Haj S, Muhsen K. An updated systematic review and meta-
analysis on the association between Helicobacter pylori infection and iron deficiency
anemia. Helicobacter 2016. http://dx.doi.org/10.1111/hel.12330.
[39] Zarshenas N, Nacher M, Loi KW, Jorgensen JO. Investigating nutritional deficiencies
in a group of patients 3 years post laparoscopic sleeve gastrectomy. Obes Surg
2016;26:2936–43.
[40] Cepeda-Lopez AC, Allende-Labastida J, Melse-Boonstra A, Osendarp SJ, Herter-
Aeberli I, Moretti D, et al. The effects of fat loss after bariatric surgery on inflamma-
tion, serum hepcidin, and iron absorption: a prospective 6-mo iron stable isotope
study. Am J Clin Nutr 2016;104:1030–8.
[41] Cohen-Solal A, Damy T, Terbah M, Kerebel S, Baguet JP, Hanon O, et al. High preva-
lence of iron deficiency in patients with acute decompensated heart failure. Eur J
Heart Fail 2014;16:984–91.
Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004
[42] Cleland JG, Zhang J, Pellicori P, Dicken B, Dierckx R, Shoaib A, et al. Prevalence and
outcomes of anemia and hematinic deficiencies in patients with chronic heart fail-
ure. JAMA Cardiol 2016;1:539–47.
[43] Haddad S, Wang Y, Galy B, Korf-Klingebiel M, Hirsch V, Baru AM, et al. Iron-
regulatory proteins secure iron availability in cardiomyocytes to prevent heart fail-
ure. Eur Heart J 2016 (pii: ehw333).
[44] Weiss G, Goodnough LT. Anemia of chronic disease. New Engl J Med 2005;352:
1011–23.
[45] Smith CL, Arvedson TL, Cooke KS, Dickmann LJ, Forte C, Li H, et al. IL-22 regulates
iron availability in vivo through the induction of hepcidin. J Immunol 2013;191:
1845–55.
[46] De Falco L, Silvestri L, Kannengiesser C, Moran E, Oudin C, Rausa M, et al. Functional
and clinical impact of novel TMPRSS6 variants in iron-refractory iron-deficiency
anemia patients and genotype-phenotype studies. Hum Mutat 2014;35:1321–9.
[47] Benyamin B, Ferreira MA, Willemsen G, Gordon S, Middelberg RP, McEvoy BP, et al.
Common variants in TMPRSS6 are associated with iron status and erythrocyte vol-
ume. Nat Genet 2009;41:1173–5.
[48] Benyamin B, Esko T, Ried JS, Radhakrishnan A, Vermeulen SH, Traglia M, et al. Novel
loci affecting iron homeostasis and their effects in individuals at risk for hemochro-
matosis. Nat Commun 2014;5:4926.
[49] Gichohi-Wainaina WN, Towers GW, Swinkels DW, Zimmermann MB, Feskens EJ,
Melse-Boonstra A. Inter-ethnic differences in genetic variants within the transmem-
brane protease, serine 6 (TMPRSS6) gene associated with iron status indicators: a
systematic review with meta-analyses. Genes Nutr 2015;10:442.
[50] Nai A, Pagani A, Silvestri L, Campostrini N, Corbella M, Girelli D, et al. TMPRSS6
rs855791 modulates hepcidin transcription in vitro and serum hepcidin levels in
normal individuals. Blood 2011;118:4459–62.
[51] Kiss JE. Laboratory and genetic assessment of iron deficiency in blood donors. Clin
Lab Med 2015;35:73–91.
[52] Sorensen E, Rigas AS, Thorner LW, Burgdorf KS, Pedersen OB, Petersen MS, et al. Ge-
netic factors influencing ferritin levels in 14,126 blood donors: results from the Dan-
ish Blood Donor Study. Transfusion 2016;56:622–7.
[53] Archer NM, Brugnara C. Diagnosis of iron-deficient states. Crit Rev Clin Lab Sci 2015;
52:256–72.
[54] WHO. Iron Deficiency Anaemia, Assesment, Prevention and Control, a guide for pro-
gramme managers. Available from: http://www.who.int/nutrition/publications/
micronutrients/anaemia_iron_deficiency/WHO_NHD_01.3/en/; 2001.
[55] Mast AE, Blinder MA, Gronowski AM, Chumley C, Scott MG. Clinical utility of the sol-
uble transferrin receptor and comparison with serum ferritin in several populations.
Clin Chem 1998;44:45–51.
[56] Beutler E, Waalen J. The definition of anemia: what is the lower limit of normal of
the blood hemoglobin concentration? Blood 2006;107:1747–50.
[57] Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum
ferritin in the diagnosis of iron deficiency. Blood 1997;89:1052–7.
[58] Infusino I, Braga F, Dolci A, Panteghini M. Soluble transferrin receptor (sTfR) and
sTfR/log ferritin index for the diagnosis of iron-deficiency anemia. A meta-analysis.
Am J Clin Pathol 2012;138:642–9.
[59] Jonker FA, Boele van Hensbroek M, Leenstra T, Vet RJ, Brabin BJ, Maseko N, et al. Con-
ventional and novel peripheral blood iron markers compared against bone marrow
in Malawian children. J Clin Pathol 2014;67:717–23.
[60] Traglia M, Girelli D, Biino G, Campostrini N, Corbella M, Sala C, et al. Association of
HFE and TMPRSS6 genetic variants with iron and erythrocyte parameters is only
in part dependent on serum hepcidin concentrations. J Med Genet 2011;48:629–34.
[61] Galesloot TE, Vermeulen SH, Geurts-Moespot AJ, Klaver SM, Kroot JJ, van Tienoven D,
et al. Serum hepcidin: reference ranges and biochemical correlates in the general
population. Blood 2011;117:e218–25.
[62] Girelli D, Nemeth E, Swinkels DW. Hepcidin in the diagnosis of iron disorders. Blood
2016;127:2809–13.
[63] Ganz T. Hepcidin and iron regulation, 10 years later. Blood 2011;117:4425–33.
[64] Bregman DB, Morris D, Koch TA, He A, Goodnough LT. Hepcidin levels predict
nonresponsiveness to oral iron therapy in patients with iron deficiency anemia.
Am J Hematol 2013;88:97–101.
[65] Prentice AM, Doherty CP, Abrams SA, Cox SE, Atkinson SH, Verhoef H, et al. Hepcidin
is the major predictor of erythrocyte iron incorporation in anemic African children.
Blood 2012;119:1922–8.
[66] van der Vorm LN, Hendriks JC, Laarakkers CM, Klaver S, Armitage AE, Bamberg A,
et al. Toward worldwide hepcidin assay harmonization: identification of a commut-
able secondary reference material. Clin Chem 2016;62:993–1001.
[67] Locatelli F, Mazzaferro S, Yee J. Iron therapy challenges for the treatment of
nondialysis CKD patients. Clin J Am Soc Nephrol 2016;11:1269–80.
[68] Tolkien Z, Stecher L, Mander AP, Pereira DI, Powell JJ. Ferrous sulfate supplementa-
tion causes significant gastrointestinal side-effects in adults: a systematic review
and meta-analysis. PLoS One 2015;10:e0117383.
[69] Zimmermann MB, Chassard C, Rohner F, N'Goran EK, Nindjin C, Dostal A, et al. The
effects of iron fortification on the gut microbiota in African children: a randomized
controlled trial in Cote d'Ivoire. Am J Clin Nutr 2010;92:1406–15.
[70] Lee T, Clavel T, Smirnov K, Schmidt A, Lagkouvardos I, Walker A, et al. Oral versus in-
travenous iron replacement therapy distinctly alters the gut microbiota and metab-
olome in patients with IBD. Gut 2016 (pii: gutjnl-2015-309940).
[71] Piperno A, Girelli D, Nemeth E, Trombini P, Bozzini C, Poggiali E, et al. Blunted
hepcidin response to oral iron challenge in HFE-related hemochromatosis. Blood
2007;110:4096–100.
[72] Moretti D, Goede JS, Zeder C, Jiskra M, Chatzinakou V, Tjalsma H, et al. Oral iron sup-
plements increase hepcidin and decrease iron absorption from daily or twice-daily
doses in iron-depleted young women. Blood 2015;126:1981–9.
[73] Schrier SL. So you know how to treat iron deficiency anemia. Blood 2015;126:1971.
[74] Sazawal S, Black RE, Ramsan M, Chwaya HM, Stoltzfus RJ, Dutta A, et al. Effects
of routine prophylactic supplementation with iron and folic acid on admission
to hospital and mortality in preschool children in a high malaria transmission
setting: community-based, randomised, placebo-controlled trial. Lancet 2006;
367:133–43.
 C. CamaschellaBlood Reviews xxx (2017) xxx–xxx 9

[75] Pasricha SR, Drakesmith H. Iron deficiency anemia: problems in diagnosis and pre-
vention at the population level. Hematol Oncol Clin North Am 2016;30:309–25.
[76] Drakesmith H, Prentice AM. Hepcidin and the iron-infection axis. Science 2012;338:
768–72.
[77] Auerbach M, Adamson J, Bircher A, Breymann C, Fishbane S, Gafter-Gvili A, et al. On
the safety of intravenous iron, evidence trumps conjecture. Haematologica 2015;
100:e214–5.
[78] Macdougall IC, Bock AH, Carrera F, Eckardt KU, Gaillard C, Van Wyck D, et al. FIND-
CKD: a randomized trial of intravenous ferric carboxymaltose versus oral iron in pa-
tients with chronic kidney disease and iron deficiency anaemia. Nephrol Dial Trans-
plant 2014;29:2075–84.
[79] Gomollon F, Gisbert JP. Intravenous iron in inflammatory bowel diseases. Curr Opin
Gastroenterol 2013;29:201–7.
[80] Rampton D, Folkersen J, Fishbane S, Hedenus M, Howaldt S, Locatelli F, et al. Hyper-
sensitivity reactions to intravenous iron: guidance for risk minimization and man-
agement. Haematologica 2014;99:1671–6.
[81] Del Vecchio L, Longhi S, Locatelli F. Safety concerns about intravenous iron therapy
in patients with chronic kidney disease. Clin Kidney J 2016;9:260–7.
[82] Anker SD, Comin Colet J, Filippatos G, Willenheimer R, Dickstein K, Drexler H, et al.
Ferric carboxymaltose in patients with heart failure and iron deficiency. New Engl
J Med 2009;361:2436–48.
[83] Ponikowski P, van Veldhuisen DJ, Comin-Colet J, Ertl G, Komajda M, Mareev V, et al.
Beneficial effects of long-term intravenous iron therapy with ferric carboxymaltose
in patients with symptomatic heart failure and iron deficiency. Eur Heart J 2015;
36:657–68.
[84] Musallam KM, Tamim HM, Richards T, Spahn DR, Rosendaal FR, Habbal A, et al. Pre-
operative anaemia and postoperative outcomes in non-cardiac surgery: a retrospec-
tive cohort study. Lancet 2011;378:1396–407.
[85] Khalafallah AA, Yan C, Al-Badri R, Robinson E, Kirkby BE, Ingram E, et al. Intravenous
ferric carboxymaltose versus standard care in the management of postoperative
anaemia: a prospective, open-label, randomised controlled trial. Lancet Haematol
2016;3:e415–25.

Please cite this article as: Camaschella C, New insights into iron deficiency and iron deficiency anemia, Blood Rev (2017), http://dx.doi.org/
10.1016/j.blre.2017.02.004