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Wien Med Wochenschr (2016) 166:411–423DOI 10.1007/s10354-016-0505-7

Iron deficiency or anemia of inflammation?

Differential diagnosis andmechanisms of anemia of inflammation

Manfred Nairz · Igor Theurl · Dominik Wolf · Günter Weiss

Received: 19 January 2016 / Accepted: 30 May 2016 / Published online: 24 August 2016© The Author(s) 2016. This article is available at SpringerLink with Open Access.

Summary Iron deficiency and immune activationare the two most frequent causes of anemia, both ofwhich are based on disturbances of iron homeostasis.Iron deficiency anemia results from a reduction ofthe body’s iron content due to blood loss, inadequatedietary iron intake, its malabsorption, or increasediron demand. Immune activation drives a diversionof iron fluxes from the erythropoietic bone marrow,where hemoglobinization takes place, to storage sites,particularly the mononuclear phagocytes system inliver and spleen. This results in iron-limited erythro-poiesis and anemia. This review summarizes currentdiagnostic and pathophysiological concepts of irondeficiency anemia and anemia of inflammation, aswell as combined conditions, and provides a briefoutlook on novel therapeutic options.

Keywords Anemia of inflammation · Anemia ofchronic disease · Iron · Hepcidin · Macrophage

Eisenmangel oder Entzündungsanämie?Differenzialdiagnose und Mechanismen derEntzündungsanämie

Zusammenfassung Eisenmangel und Immunaktivie-rung sind die zwei häufigsten Ursachen der Anämie.In beiden Situationen besteht ursächlich eine Störung

M. Nairz (�) · I. Theurl · G. Weiss (�)Department of Internal Medicine VI, Infectious Diseases,Immunology, Rheumatology, Pneumology, MedicalUniversity of Innsbruck, Anichstraße 35, 6020 Innsbruck,Austriamanfred.nairz@i-med.ac.at; guenter.weiss@i-med.ac.at

D. WolfMedical Clinic III, Department of Oncology, Hematologyand Rheumatology, University Clinic Bonn (UKB), Bonn,Germany

der Eisenhomöostase. Die Eisenmangelanämie beruhtauf einer Verminderung des Gesamtkörpereisens in-folge von Blutverlust, unzureichender alimentärer Zu-fuhr oder intestinaler Absorption bzw. erhöhtem Be-darf an Eisen. Immunaktivierung führt bei norma-lem Gesamtkörpereisen zu dessen Umverteilung vomerythropoetischen Knochenmark, der primären Stel-le der Hämoglobinproduktion, in das mononukleärePhagozytensystem der Leber und Milz, die Hauptor-gane der Eisenspeicherung. Dies führt letztlich zurAnämie. In dem vorliegenden Übersichtsartikel wer-den aktuelle diagnostische und pathophysiologischeKonzepte von Eisenmangelanämie, Entzündungsan-ämie sowie kombinierter Anämie zusammengefasstund ein kurzer Ausblick auf neue Therapieoptionengeboten.

Schlüsselwörter Entzündungsanämie · Anämie beichronischer Erkrankung · Eisen · Hepcidin · Makro-phage

Introduction

Iron deficiency (ID) can occur in two major forms: ab-solute and functional ID. Both forms of ID can man-ifest either isolated or combined, and will result iniron-deficient erythropoiesis and, if unrecognized orleft untreated, in anemia [1, 2].

Absolute ID, as defined by a decrease in the body’siron content, usually develops when the absorptionof dietary iron in the duodenum and proximal je-junum (Fig. 1a) cannot compensate for an increasediron demand or blood loss. Despite adaptive induc-tion of expression of the transmembrane iron trans-porters divalent metal transporter (DMT)-1 and fer-roportin (FPN)-1 in enterocytes upon ID, iron absorp-tion can only be increased by 2- to 3-fold to approxi-mately 5 mg per day [3, 4]. Due to this relatively inef-

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ImpairedFe-Recycling

ReducedFe-Utilization

MPS Fe-sequestration

Erythroid progenitor damage/apoptosis

Premature RBC

aging

Reduced RBClifespan

Increased spleen

size

ReducedTf-Fe

RPM

ReducedEPO

production/effectReduced

Fe-Absorption

Increased Blood-/ Fe-Loss

IL-6

TNF

B cellMacrophage Auto-antibody

production

T cellCytokinesecretion

Impaired RBC maturation

Reduced RBCFe-content

MPSFe-

sequest-ration

Tf-Loss

KC

FT

IncreasedHAMP

production

IL-6

a b

Liver

Kidney

Duodenum

Erythroid progenitors

Aged RBC

Mature RBC 1500-2500 mg

Fe in Hb

Spleen

Tf-Fe3-5 mg

HAMP

EPOERFE

GDF15

HAMP

Fe-Absorption1-2 mg

Fe-Loss1-2 mg

Fe-Recycling20-25 mg

Fe-Utilization20-25 mg

KC

FT-associated storage-Fe200-1200 mg RPM

FPN1

Fe-AbsorptionFe-Recycling

Fe-Mobilization

Fe-LossFe-Utilization

Fe-Storage

BMP6

FPN1

FT

Fig. 1 aUnder homeostatic conditions, the absorptionof 1–2mgof ironper daycompensates for its loss viadesquamationof ep-ithelial cells fromskinandmucosalmembranesandduringmenstrual bleeding. Themajorityof the20–25mgof iron required fordailyerythropoiesis isprovidedby thedegradationof effeteRBCand the ironcontainedwithin their Hb (Hemoglobin).Bothduodenal ironabsorptionand iron recycling in spleenand liver are negatively regulatedbyHAMP (Hepcidin antimicrobial peptide).HAMP ismainlygeneratedbyhepatocytes in response toan increase inserum ironor storage ironwhileerythropoieticactivity inhibitsHAMPexpres-sionviasolublemediatorsincludingGDF15andERFE.bFollowingimmuneactivationbypathogen-ordamage-associatedmolecularpatterns, the interactionofmyeloid cellswithT andB lymphocytes results in thegenerationof pro- andanti-inflammatorycytokines.Thesedivert ironfluxesfromthecirculationtostoragesitesbycontrolling theexpressionofHAMP,of irontransporters,andof the iron-storageproteinFT.Therefore,duodenal ironabsorptionandmacrophageironrecyclingarereduced, theserumbecomesiron-starvedandtheerythron lackssufficient iron forproliferationandhemoglobinsynthesis. Therefore, zincmayreplace ironasthecentralheme-cation. Zincprotoporphyrin-IX (notdepicted) canbemeasured to confirm thepresenceof thismechanismof ironsequestration. Inaddition to cytokines, othermediators suchasauto-antibodiesand reactive intermediatescan tagmatureRBC for degradationordamagethemortheirprecursors,contributingtothehyporegenerativenatureofAI.Inparallel, renalEPOproductionisreducedandtheresponsivenessoftheerythrontoEPOisdampened. Intheend,amildtomoderatenormocyticanemiawithevidenceof iron-restrictederythropoiesis (lowTSAT, highFT, low reticulocytes, highZnPP-IX in reticulocytes, low tonormalEPO)occurs.Keypathways for thepathogenesis are inboldface. Putative additional pathwaysare in lightface.BMP6bonemorphogenetic protein-6,ERFEerythro-ferrone,EPOerythropoietin,FPN1 ferroportin-1,FT ferritin,GDF15growthdifferentiation factor-15,HAMPhepcidin anti-microbialpeptide, IL interleukin,KCKupffer cell,MPSmononuclear phagocyte system,PDGF-BBplatelet-derivedgrowth factor isoformBB,RBCredbloodcell,RPM redpulpmacrophage,Tf-Fe transferrin-boundiron,TNF tumornecrosisfactor,ZnPP-IXzincprotoporphyrin-IX

ficient process, iron stores, particularly ferritin-asso-ciated iron in liver and spleen, can become depletedduring chronic bleeding episodes, repetitive blood do-nations, helminth infestations, through materno-fetaltransfer, or during growth [5].

In very rare cases, genetic mutations of iron home-ostasis proteins such as DMT1 or TMPRSS6 (Trans-membrane Protease, Serine 6), the latter encoding formatriptase-2, can result in inadequate iron absorp-tion and development of anemia [6, 7]. Similarly, lackof the iron-carrying serum protein transferrin (TF),due to genetic deficiency, auto-antibody production,or proteinuria, can cause absolute ID [8]. Inadequateiron absorption has also been found in associationwith Helicobacter pylori infection, hypergastrinemia,celiac disease, or vitamin D deficiency [9, 10]. Pro-longed ID results in the inability to regenerate skinand mucosal membranes and in iron deficiency ane-mia (IDA) with its classical symptoms such as fatigue.Details on the clinical implications of ID are reviewedelsewhere in this special issue.

Functional ID has a more complex pathophysiol-ogy and is commonly defined as a redistribution of

iron from the key sites of its utilization (erythron,epidermis, mucosal surfaces) to storage sites, partic-ularly the hepatic and splenic mononuclear phago-cyte system (MPS). Moreover, in states of increasederythropoiesis such as during therapy with erythro-poiesis-stimulating agent (ESA) or after major bloodloss, erythropoiesis may become iron-restricted solong as the mobilization of storage iron cannot catchup with its demand for hemoglobin (Hb) synthesis(see the interpretation of CHr (Content of reticulocytehemoglobin), HYPO (Hypochromic erythrocytes), andZnPP (Zinc protoporphyrin) in diagnostic section).The ultimate consequence of these functional distur-bances of iron homeostasis is anemia, which is oftenreferred to as anemia of inflammation (AI) or anemiaof chronic disease (ACD).

Absolute and functional iron deficiency may alsocoexist. Such combined conditions render the in-terpretation of erythrocyte indices and parameters ofiron status challenging. While new diagnostic param-eters are not yet readily used in clinical routine, thisdifferential is important as the therapeutic approachvaries. In addition, the random detection of AI during

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FT < 30 ng/ml TSAT < 16%

LDH, haptoglobin, Coombs tests, blood smear,

FACS, Hb electrophoresis, osmotic resistance, Hx, physical exam,

endoscopy

Hb < 12 g/dl (female) Hb < 13 g/dl (male) w otherwise normal CBC

Thrombopenia Leucopenia/leucocytosis in the

absence of infection

MCH < 28 pg

Hyporegenerativemicrocytic hypochromic anemia

IDA

MCH 28-35 pg

Hyporegenerativenormocytic normochromic anemia

DD: AI

AI+IDA Familial sideroblastic anemia

Aluminium intoxication Lead intoxication

FT > 200 ng/ml sTFR normal

FTI < 1

AI

FT 30- 200 ng/ml FTI > 2

AI + IDADD:

Acute bleeding (prior to reticulocyte response)

Aplastic anemia Pure red cell aplasia

Anemia assocated w MDS (BM biopsy)

Renal anemia (GFR, EPO) Cardio-renal anemia syndrome

Anemia of the elderly Anemia due to combined

MCH > 35 pg

Hyporegenerative macrocytic hyperchromic anemia

Folate Vitamin B12

CBC Blood smear

Bone marrow biopsy

DD:

MDS (BM biopsy) Bone-marrow toxicity

(BM biopsy)

CBC

RPI

Erythrocyte indices

Iron indices, CH r, ZnPP

Additional tests (if applicable)

RPI < 2

MCH 28-35 pg

Regenerative normocytic normochromic anemia

Hyporegenerativeanemias

Regenerative anemias

DD: Bleeding (w yet

conserved iron stores) Hemolytic anemias Membrane defects Enzyme disorders

Hemoglobinopathies PNH (FACS)

Mechanical valve replacementMicroangiopathy

DD: Hematologic malignancy

Aplastic anemia

RPI > 3

RPI = Reticulocyte count 0.5Hb (normal)

Hb (observed)* *

FTI = sTFR / logFT

Note: Reference ranges may vary between countries, laboratories and assays

Fig. 2 For thedifferential diagnosisof IDAvs. AI vs. a combinationof both formsor other causesof anemia, a stepwiseapproach isproposed. ACBCenables thedifferentiationof isolatedanemias frombi- andpancytopenias [161]. The lattermay require amoreex-tensivework-up. Also, theRPIcanbeestimatedfromtheCBC.AnRPIof<2characterizeshyporegenerativeanemiaswhileamRPI>3isobserved in regenerative formssuchas thehemolytic anemias. Twooutof threeerythrocyte indicesare relevant, i.e., theMCVandtheMCH, as theyallow for theclassificationofmicrocytic hypochromic, normocytic normochromic, andmakrocytic hyperchromicanemias. In IDA,bothserumFTandTSATare reduced. Incontrast, an increasedFT is typicalofAI. Incombinedconditions, theFTI,ascalculated fromtheserumTFRdividedby the logarithmicserumFT,continues tobehelpful for thedifferential diagnosis. In the future,novel parameters suchasHAMPmaybe incorporated intodiagnostic algorithms. Note: Reference rangesmayvarybetweencoun-tries, laboratories, andassays. Hbcutoffs correspond toWHOdefinitions.AIanemiaof inflammation,BMbonemarrow,CBCcom-pletebloodcount,DDdifferentialdiagnosis,EPOerythropoietin,GFRglomerularfiltrationrate,FACSfluorescenceactivatedcellsort-ing,FT ferritin,FTI ferritin index,Hbhemoglobin,Hxhistory, IDA iron-deficiencyanemia,LDH lactatedehydrogenase,MCHmeancor-puscular hemoglobin,MCVmeancorpuscular volume,MDSmyelodysplastic syndrome,PNHparoxysmal nocturnal hemoglobin-uria,RPI reticulocyteproduction index, sTFR soluble transferrin receptor,TSAT transferrin saturation

routine blood sampling should prompt a search forthe underlying disease.

Iron deficiency anemia

While IDA poses a major public-health problem in de-veloping countries [11], it is also frequently observedin industrialized countries: in 5–10% of individuals,as detailed elsewhere in this special issue. IsolatedIDA can be detected by a complete blood count, andiron status based on the reticulocyte count or retic-ulocyte production index (RPI), erythrocyte indices,ferritin (FT), and transferrin saturation (TSAT). Typi-

cally, IDA is an isolated hyporegenerative microcytichypochromic anemia, with reduced FT concentrationand TSAT as indicators of a depletion of iron storesand serum iron, respectively [12–15]. The RPI can eas-ily be estimated by one of two established formulas(Fig. 2).

ID results in difficulties regenerating epidermis andmucosal epithelia, while also affecting the clinicalcourse of associated chronic diseases. For instance,ID has negative effects on mitochondrial respirationand tissue oxygen consumption and, thus, on cardiacfunction and the clinical course of congestive heartfailure (CHF) [16–18]. The importance of anemia for

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CHF is underscored by a linear increase of mortalitywith declining Hb levels [19–21]. Likewise, parenteraliron substitution has been found to improve the clin-ical course of CHF in patients with coexisting ID[22–24].

Anemia of inflammation

AI can be viewed as a spectrum of acute and chronicforms of anemia whose common pathophysiologicaldenominator is their occurrence as a result of immuneactivation [25, 26].

Acute and chronic infections, inflammatory disor-ders, and malignancies are the principal disease typesunderlying AI. However, AI shares features with the re-nal anemia observed in patients with chronic kidneydisease (CKD), the anemia in patients with chronicobstructive pulmonary disease (COPD), the anemia inpatients with CHF without or with cardio-renal syn-drome, and the anemia of the elderly [23, 27, 28].

The anemia of critical illness occurring after acuteevents such as major surgery, severe trauma, myocar-dial infarction or sepsis may be classified as a spe-cific acute form of AI. Moreover, some features of AIalso characterize the anemias occurring in hemato-logic disorders such as multiple myeloma or malig-nant lymphoma [29–32].

In addition, combined forms of IDA and AI maybe present. This scenario is typically observed in in-flammatory bowel disease (IBD) or gastrointestinal orurogenital malignancy. Mucosal erosions and ulcera-tions are associated with recurrent bleeding episodesand lead to a substantial loss of iron, since 0.5 mg ofiron are contained within the Hb of 1 ml of blood. Atthe same time, the underlying disease provides an in-flammatory stimulus for the sequestration of iron inthe MPS. Moreover, menstruation, hemodialysis, therequirement for repetitive blood sampling, and anti-coagulant or antiplatelet drugs may contribute to ironloss in CKD and other chronic diseases.

Multiple players in the pathophysiology of AI

Immune cells

The activation of immune cells by infectious agents,auto-antigens, or neoplastic cells initiates and main-tains the development of AI by several mechanismswhich coexist and are cross-regulatory (Fig. 1b). Theexcessive production of inflammatory mediators di-verts iron to the MPS, rendering it relatively unavail-able for erythroid progenitors [33]. A paradigm forsuch a mediator is hepcidin anti-microbial peptide(HAMP). HAMP is the hormonal negative-feedbackregulator of serum iron, as it limits iron-fluxes tothe circulation. Upon iron excess or inflammation,HAMP is produced by hepatocytes and, in muchsmaller quantities, by immune cells and other celltypes. HAMP’s specific receptor is FPN1, whose only

known function is to act as an export protein forionic iron. Binding of HAMP to FPN1 tags the latterfor internalization from the cell membrane and forlysosomal degradation [34].

Activation of pattern recognition receptors such asToll-like receptor (TLR)-4, as well as pro- and anti-inflammatory cytokines regulate HAMP expression,while similar pathways control transcriptional expres-sion of iron transporters transferrin receptor (TFR)-1,DMT1, and FPN1, as well as the iron storage proteinFT [26].

For instance, lipopolysaccharide as a component ofthe Gram-negative cell wall enhances HAMP produc-tion while stimulating DMT1 expression in myeloidcells, thereby favoring iron sequestration [35]. In par-allel, interleukin (IL)-10 increases TFR1 and FT tran-scription, whichmay aggravate AI in patients with IBD[36].

Increased HAMP levels are also well documentedin infections, rheumatoid disorders, and IBD. Further-more, in almost all patient cohorts, HAMP concentra-tion positively correlates with disease activity linkingthe extent of inflammation to the severity of iron se-questration in the MPS [37–43].

Liver

The liver is a key organ initiating and maintaining AI[12]. Hepatocytes are the key source of HAMP, whileKupffer cells (KC) are a major site of inflammation-driven iron storage. Interestingly, KC dampen HAMPproduction in homeostatic conditions but may berequired for inflammation-driven HAMP secretion[44, 45]. IL-6 is essential for the up-regulation ofHAMP upon inflammation and IL-6 blockade for thetreatment of rheumatoid arthritis lowers both dis-ease activity and circulating HAMP levels [46, 47].TF is a major product of hepatocytes and one ofa limited number of negative acute phase reactants.IL-6 and other pro-inflammatory cytokines result ina downregulation of TF expression in the liver, thusreducing the serum’s capacity to transport iron [48].This mechanism may additionally contribute to ironsequestration in the MPS. Since TF-bound iron andTFR1 form the key mechanism of iron uptake for ery-throid progenitors, a central role for the developmentof AI is implicit. TFR1 is also expressed by neoplasticcells in solid tumors and hematologic malignancies,including chronic lymphocytic leukemia, suggestingthat inflammation associated with malignant diseasesmay also limit iron availability for cancer cells [49,50]. However, potential functional consequences fortumor-associated monocytes/macrophages (TAM)are not sufficiently addressed. In addition, severalpathogens are able to acquire TF-bound iron [51–54].Therefore, the reduction of serum TF appears to beone of the mechanisms of microbial iron withdrawal[54–57].

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Serum iron (TF-bound iron), the amount of storediron (FT-stored iron), and the iron demand for ery-thropoiesis are key variables that are integrated byhepatocytes to adapt HAMP production to currentmetabolic needs. Serum iron levels are sensedby a machinery involving TFR1, TFR2, and thehemochromatosis-associated HFE protein [58]. How-ever, in being the primary iron source for erythro-poiesis, TF also indirectly regulates HAMP expressionvia erythroid progenitor-derived mediators, suggest-ing that the pathways of HAMP regulation are inter-connected [59–61].

An increase in the erythropoietic activity as ob-served after blood loss or erythropoietin (EPO) admin-istration suppresses HAMP production [62]. Part ofthis effect may be mediated via erythroferrone (ERFE),a lack of which delays the recovery from AI in a mousemodel [63]. Growth-differentiation factor (GDF)-15,whose levels are increased in thalassemia and AI withor without ID, also inhibits HAMP expression [60, 64].Hypoxia has a similar effect on HAMP that is mediatedvia platelet-derived growth factor isoform BB (PDGF-BB), which may enable the required increase of Hblevels at high altitude [65].

Iron accumulation in the liver induces bone mor-phogenetic protein (BMP)-6, which is essential tomaintain body iron homeostasis. BMP6 binds toa heterodimeric receptor complexed with hemoju-velin (HJV) and matriptase-2 (the gene product ofTMPRSS6), and stimulates HAMP expression [66,67]. Notably, BMP6 is primarily produced by non-parenchymal liver cells and may act in a paracrinemanner on adjacent hepatocytes [68].

In the context of inflammation, IL-6 and IL-22 stim-ulate HAMP expression via specific receptors signalingthrough signal transducer and activator of transcrip-tion (STAT)-3, while alpha-1 antitrypsin may do sovia HJV and matriptase-2 [69–71]. However, inflam-mation also feeds into the BMP6 signaling pathway,adding further complexity; not only to the regulationof iron homeostasis, but also to the pathophysiologyof AI and the clinical interpretation of iron indices[72].

In their reproductive years, women have an in-creased iron demand. Estradiol, whose levels increaseafter menstrual bleeding during the first half of themenstrual cycle (follicular phase) until ovulation, in-hibits HAMP transcription in hepatocytes, which mayallow for higher intestinal iron absorption to compen-sate for the average 20–80 ml of monthly menstrualblood loss [73, 74]. In contrast, progesterone, whichrises after ovulation and dominates the second halfof the cycle (luteal phase) until menstrual bleeding,rather stimulates HAMP expression [75]. Given the re-sulting fluctuations of HAMP and iron indices, the lastfive days of the menstrual cycle have been proposedfor blood sampling to allow for a more representativeevaluation of iron status in women [76].

Recently, the concept has emerged that drugs mayhave undesired side effects on iron homeostasis,since the mTOR inhibitor rapamycin may increaseHAMP levels after heart transplantation, thus induc-ing a functional ID and anemia [77].

Spleen

The spleen contributes to the pathogenesis of AI assite of iron retention in macrophages. Furthermore,splenomegaly may result in hypersplenism and a re-duced half-life of red blood cells (RBC) as a conse-quence of the increased RBC elimination by red pulpmacrophages (RPM). Similarly, evidence from mousemodels suggests that increased erythrophagocytosiscontributes to the rapid Hb drop in acute and sub-acute forms of AI [78, 79]. Under conditions of ex-cessive inflammation as seen in sepsis patients, reac-tive oxygen intermediates may further accelerate RBCdamage and their removal by complement-dependentmechanisms [80, 81].

Kidney

While hepatic HAMP formation is increased duringinflammation, EPO production in the kidney is sub-ject to inhibition by inflammatory mediators such astumor necrosis factor (TNF) and IL-1 [82–84].

CKD with a glomerular filtration rate (GFR) < 40ml/min/m2 results in insufficient or deregulated produc-tion of EPO and of 1, 25-dihydroxy-cholecalciferole,both of which are negative regulators of HAMP [85,86]. Theoretically, for the assessment of whether therenal EPO response is adequate in AI, the EPO con-centration as measured should be corrected for theactual Hb level (comparable to RPI for the correctionof reticulocyte counts). However, no consensus ex-ists on a correction formula for EPO for subjects withnormal renal function or for CKD patients [84, 87].

Independently, glomerulopathy may result in pro-teinuria and the loss of the 80-kD serum protein TF,which is the major shuttle between compartments ofiron absorption (intestine)/iron recycling (MPS) andthe erythron. While isolated antibodies to TF maylead to IDA, such auto-antibodies have not yet beenreported in systemic autoimmune diseases. However,it is known that a functionally distinct type of anti-TF antibodies in monoclonal gammopathies may re-sult in hyperferritinemia and increase of hepatic ironstorage [88, 89].

Erythron

A resistance of the erythron to EPO is another mecha-nism underlying AI, since it reduces the erythropoieticdrive even in the setting of normal or adequately in-creased serum EPO concentrations. Part of this maybe attributed to downregulation of the EPO receptoron erythroid cells by interferon (IFN)-γ [90]. Further-

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more, a range of inflammatory mediators includingTNF, IL-1, IFN-γ, and reactive intermediates inhibitsthe proliferation and differentiation of erythroid pro-genitors or induces their apoptosis [91–94]. Thesepathways ultimately culminate in an insufficient re-nal EPO response and hematopoietic EPO resistancefurther aggravating anemia in AI [95].

Numerous infectious agents (e. g., parvovirus B19and human herpes virus-6) and neoplastic cells mayinfiltrate the bone marrow, which eventually disturbserythropoiesis by several mechanisms, including di-rect damage to erythroid cells and putative negativeeffects on the microenvironment and the stem cellniche. In addition, there may be direct toxic ef-fects of drugs including chemotherapeutics and ofradiation therapy on hematopoietic stem/progenitorcells. Cytopenia, including anemia, is a concern ofmethotrexate treatment for rheumatoid arthritis [96].However, immunological deregulation induced by bi-ologics such as anti-TNF therapy may also induceaplastic anemia [97].

While in its classical form AI constitutes a hy-poregenerative anemia, hemolysis may contributeto the development of AI or aggravate its degree inseveral settings. For instance, several bacteria includ-ing Staphylococcus aureus produce hemolysins [98].These destroy RBC, liberating heme for its uptake intobacteria by specific receptors. Different mechanismsof heme iron acquisition are exploited by intraery-throcytic infectious agents such as Plasmodium [99].In addition, malaria induces HAMP, suggesting thatiron sequestration is a major contributing factor tomalarial anemia [100, 101]. Elevated HAMP levelshave also been reported in patients with HIV (Hu-man immunodeficiency virus) infection, in whichthey are associated with anemia and independentlypredict mortality [102]. While auto-antibodies againstRBC can be induced by acute Epstein–Barr virusand Mycoplasma pneumoniae infections resulting incold agglutinin disease, auto-immune hemolysis mayalso occur in the setting of chronic infections or asa side effect of medication [103]. In addition, the lifespan of circulating RBC may be negatively affectedby inflammatory mediators such as TNF and by me-chanical stress [104]. Therefore, hemolysis may alsocontribute to AI in conditions such as CHF associatedwith mechanical valve replacement or endocarditis,or when microangiopathy is present. However, dueto fluid retention, the degree of anemia tends to beoverestimated in CHF patients.

Others

Similar to the concurrent presence of absolute ID inthe setting of AI, deficiencies in other nutrients es-sential to erythropoiesis, such as folate and vitaminB12, may be contributory. For instance, celiac diseasemay cause profound malassimilation of various nu-trients or poor food intake may aggravate the anemia

of the elderly. Particularly in elderly patients, ane-mia due to clonal hematopoietic diseases, includingmyelodysplastic syndromes (MDS), has to be consid-ered as well.

Current and promising diagnostic tools

Complete blood count, reticulocyte productionindex, and red blood cell indices

Both IDA and AI typically manifest as isolated anemia.As detailed elsewhere in this special issue, both abso-lute ID and inflammation can also result in thrombo-cytosis due to the effects of altered thrombopoietin,EPO, and IL-6 levels on megakaryopoiesis [105–107].In addition, the disorders underlying AI or the im-mune-modulatory therapy required for their controlcan affect circulating leukocyte numbers [108]. A dif-ferential blood count can be recommended for un-clear cases of anemia where monoclonality may besuspected as an underlying disease (Fig. 2). Serumprotein electrophoresis and bone marrow aspirationor trephine biopsy may reveal additional diagnosticclues. The reticulocyte count allows for the differen-tiation of hyporegenerative anemias (disorders of ery-throid proliferation and maturation) vs. regenerativeanemias (hemolysis or hemorrhage). However, to ac-count for the increased proportion of reticulocytes inanemia and the increased presence of prematurely re-leased reticulocytes in the circulation, the RPI shouldbe calculated (Fig. 2).

Erythrocyte staining indices do not define the causeof anemia, but they may be helpful during the diag-nostic workup. IDA is a microcytic hypochromic ane-mia, while AI may be microcytic hypochromic or nor-mocytic normochromic in appearance. High normalto elevated MCV and MCH may be due to a complexmetabolic disorder (e. g., in alcoholism), severe nutri-ent deficiency (e. g., in celiac disease), or an alterna-tive diagnosis such as MDS.

In addition, clinical signs along with the measure-ment of TSH (Thyroid stimulating hormone) andPTH (Parathyroid hormone) will help to rule outendocrine disorders (specifically hyperthyroidism,hypothyroidism, panhypopituitarism, and hyper-parathyroidism) as the cause of a hyporegenerative,normocytic normochromic anemia.

Ferritin and transferrin saturation

In IDA, serum FT and TFS may enable an accurate in-terpretation of body iron status. A reduction in serumFT below 30 ng/ml shows ID with high diagnosticaccuracy because a strong correlation exists betweenserum FT and the body’s total iron storage. It is gen-erally assumed that for each 1 ng/ml of serum FT,10 mg of iron are stored in tissues and organs. SerumFT appears to be iron-poor and mainly derived frommacrophages [109].

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Serum FT levels in the setting of inflammation aremore difficult to interpret as a range of stimuli re-sult in altered production of FT. Therefore, the clin-ical presentation, along with markers of inflamma-tion such as C-reactive protein or IL-6, needs to betaken into account. The appearance of hyperferritine-mia >200 ng/ml in the context of a decreased TSAT issuggestive of immune-driven iron sequestration. Thismay be indicative of inflammation, cancer, infection,or liver disease. Extraordinarily high FT levels havebeen documented in patients with adult-onset Still’sdisease or hemophagocytic syndrome [110, 111].

In an attempt to transport the available iron as ef-ficiently as possible, serum TF is increased in ID, re-sulting in a TSAT < 16%. Similar levels are observed inAI because TF is a negative acute-phase reactant (seeabove).

Hyperferritinemia in the context of an increasedTSAT of >45% should prompt evaluation for primaryor secondary iron overload. In the context of mi-crocytic anemia and Mediterranean or Asian descent,thalassemia is a valid differential diagnosis. In the ab-sence of anemia, HFE-associated hemochromatosis ordysmetabolic iron overload are possible explanationsfor pathologically increased FT and TSAT.

Soluble transferrin receptor and ferritin index

TFR1 is the key receptor for iron acquisition by ery-throid cells. Its soluble form (sTFR) can be measuredin the serum and it reflects ID and erythropoietic ac-tivity. sTFR is increased in ID, hemolytic anemias, tha-lassemia, and some hematologic malignancies, whileits levels tend to be normal in AI [112]. Therefore, anincreased sTFR in the setting of AI suggests the pres-ence of additional absolute ID. However, the use ofsTFR is limited by the lack of its standardization andthe fact that age, ethnicity, and inflammation influ-ence its normal range [113].

The ferritin index (FTI) may also be helpful in thedifferential diagnosis of AI and combined IDA/AI.However, the lack of standardized tests for sTFRprevents its broad recommendation. The FTI is calcu-lated from the sTFR divided by the logarithm of serumFT (Fig. 2). In patients with chronic diseases and AI,an increased FTI suggests the concurrent presence ofabsolute ID requiring correction. However, the cutoffvalue is largely dependent on the specific diagnostictest used [114–116]. Therefore, at the current stageof research we are unfortunately not able to providea universally applicable algorithm for the differentia-tion between isolated AI and anemia with combinedfunctional and absolute ID.

Content of reticulocyte hemoglobin, percent ofhypochromic erythrocytes, and zinc protpoporphyrin

Content of reticulocyte hemoglobin (CHr), percent ofhypochromic erythrocytes (%HYPO), and zinc proto-

porphyrin (ZnPP) allow for the prediction of iron avail-ability for erythropoiesis, but have little to no role inthe differentiation between IDA and AI.

The content of Hb in reticulocytes correlates withthe availability of iron for erythropoiesis. A CHr< 26 pg suggests iron-limited erythropoiesis, as ob-served in both IDA and AI. In response to iron sub-stitution, it is one of the first parameters to respondwith an increase. A lack of this predicted responseraises the concern of an alternative diagnosis, unlessCKD is present and EPO deficiency awaits correction.

HYPO is defined as the relative number of hypo-chromic RBC with a Hb content <28 pg. A HYPO >10%indicates iron-deficient erythropoiesis due to IDA orAI.

As erythropoiesis becomes iron-deficient, the ery-throid enzyme ferrochelatase incorporates zinc in-stead of iron into protoporphyrin-IX. Since ZnPP andheme are analogues, an increase in the ratio of ZnPP/heme indicates ID for erythropoiesis and is observedin IDA, AI, MDS, and sideroblastic anemias, includ-ing the form secondary to lead intoxication [117].This highlights the lack of specificity of this set ofparameters for the differential diagnosis of anemias.

Hepcidin and its regulators

Hepcidin (HAMP) may be helpful in the differentialdiagnosis of anemias, as well as in the assessmentof therapeutic options. For instance, HAMP is sup-pressed in IDA, in the normal range in IDA/AI, andelevated in AI [112, 118]. High HAMP at the time ofinitiation of therapy with ESA may predict poor treat-ment response in AI [119]. In addition, high HAMPpredicts poor response to oral iron in IDA patients[120]. In CKD patients, the predictive power of HAMPfor the indication for iron therapy is limited [43, 121,122]. Attempts have been undertaken to harmonizethe different diagnostic methods for hepcidin deter-mination to allow the broad clinical use of this method[123]. GDF15 is normal in IDA and elevated in AI andIDA/AI [64].

In the future, information technology may provideus with software based on complex algorithms fora more accurate assessment of iron status and, just asimportantly, guide therapy for the most appropriatetreatment. For instance, we may witness that mobileapplications based on a combined panel of HAMP,EPO, ERFE, GDF15, BMP6, and other parameters,such as high sensitive CRP and IL-6, enter clinicalroutine.

Treatment options

Since the AI is a direct consequence of an active im-mune-driven disease, its first-line therapy is treatmentof the underlying condition. However, the subsequenttherapeutic approach to AI remains a matter of de-bate and ongoing clinical trials. Iron supplementa-

K Iron deficiency or anemia of inflammation? 417

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tion, ESA, and transfusion of packed RBC are the cur-rent specific treatment options for AI.

It is generally assumed, similar to the hypoferremiaof the acute phase response, that AI is the patho-physiological consequence of the body’s attempt toreduce the availability of iron for infectious agents.Therefore, there is the concern that iron supplemen-tation may stimulate pathogen proliferation or resultin a flare of an underlying inflammatory disorder ormalignancy [54, 124, 125]. Similarly, ESA and RBCtransfusions may have adverse immune-modulatoryeffects [126–129]. The target therapeutic Hb levelshave not yet been defined in prospective trials; how-ever, data from studies in patients with anemia andCKD or cancer suggest a slightly anemic target rangebetween 11–12 g/dl to be safe [130, 131].

Iron preparations

Isolated IDA can often be prevented by iron fortifica-tion/supplementation and, once it has manifested, ispreferentially treated by oral iron salts. For instance,approximately 100 mg of elemental iron contained in300–350-mg ferrous sulfate preparations can be pre-scribed as a daily dose. These iron salts are absorbedby the sequential action of DMT1 and FPN1 and as-sociated oxidoreductases, but have a low bioavailabil-ity. However, products using heme rather than ioniciron have entered the market. These are absorbedby alternative pathways that are incompletely char-acterized, since the proposed solute carrier SLC46A1absorbs folate more efficiently than heme [132, 133].A recent study in non-anemic young women with IDhas shown that a single morning dose of 40–80mg fer-rous sulfate resulted in adequate iron absorption yetelicited a transient rise in serum HAMP levels, whichargues against twice-daily dosage. Whether or not al-ternate day supplementation provides a benefit awaitsinvestigation in prospective trials [134].

Parenteral iron supplementation is an alternative toconsider, especially when a rapid correction is needed,or gastrointestinal (GI) malassimilation or active in-flammatory disease dampens dietary iron absorptionin AI [135, 136]. Also, in patients with intolerance tooral iron supplements, parenteral iron is the therapyof choice. Currently, six different forms of parenteraliron are available for clinical use, i. e., ferric carboxy-maltose, ferumoxytol, iron dextran, iron gluconate,iron isomaltoside, and iron sucrose. These representmacromolecules in which iron is complexed to sac-charides. The complexes are endocytotically taken upby the MPS and ionic iron is distributed into the cir-culation via FPN1 by macrophages [137]. Concernshave been raised regarding the risk of severe anaphy-lactic reactions when using intravenous iron prepa-rations. However, these are infrequent and specificprecautions are recommended in at-risk patients, tominimize the occurrence of such adverse events [138].In addition, parenteral iron supplements harbor an in-

trinsic risk of inducing hypophosphatemia. Therefore,serum phosphate may be measured when erythropoi-etic and iron indices are determined to evaluate theresponse to treatment [139].

Despite the fact that the MPS has a lower thresholdto respond to increased HAMP than have duodenalenterocytes, parenteral iron remains effective whenintestinal iron absorption is hampered by immune ac-tivation [140]. Since parenteral iron stimulates HAMPsecretion, as documented in hemodialysis patients,frequent administration of low doses may be bene-ficial [141]. Prospective trials are required to optimizetreatment regiments to ensure adequate efficiency ofparenteral supplementation in different clinical set-tings.

In the context of AI, parameters which predictthe subsequent response to EPO therapy are beingevaluated in prospective studies. In CKD patients,iron therapy is specifically recommended to replenishstores prior to initiation of ESA therapy. TSAT < 20%and FT < 100 ng/ml have been proposed as cutoffsfor absolute ID in non-dialysis CKD patients [142].Further details are reviewed elsewhere [130].

ESA

Currently, the arm of therapy with ESA remains lim-ited to EPO analogues, as the synthetic EPO receptoragonist peginesatide has been taken off the marketbecause of rare anaphylactic reactions [143].

Many subjects with AI who are under causativetreatment for their underlying condition do not havean adequate Hb response to iron therapy. EPO re-sistance of the erythron or renal EPO deficiency maybe present, such that ESA should be considered asadd-on therapy for anemia. Specific studies havebeen conducted in patients with AI in the settingof rheumatoid arthritis or HIV infection, in whichEPO levels <500 U/L predicted a response to ESAadministration [144, 145]. Standard starting doses ofEPO are 100–150 U/kg, administered subcutaneouslythree times a week, although higher doses may berequired for individual patients. As Hb levels in-crease during efficient EPO therapy, iron parametersshould be monitored and iron supplemented in orderto maintain a TSAT ≥20% and a FT ≥100 ng/ml fora sufficient Hb response. In dialysis patients, higherFT target levels have been suggested [146]. In MDS,ESA are in wide clinical use despite of the fact thatofficial approval of this approach is still pending [147].The specific regimes and pitfalls in ESA therapy arereviewed elsewhere [130, 131, 148, 149].

The HAMP–FPN1 axis as target

Given its important role for iron-sequestration inAI, HAMP and its receptor FPN1 are attractive tar-gets for therapeutic interventions. Different phar-maceutical preparations including antibodies, an-

418 Iron deficiency or anemia of inflammation? K

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tichalins, Spiegelmers, thiamine derivatives, and hep-arin derivatives can bind and neutralize HAMP [150,151]. Moreover, direct blockage of HAMP expressionvia sHJV or BMP signaling inhibitors have shown effi-ciency in blocking HAMP function and amelioratinganemia [152]. Some of these treatments are alreadybeing evaluated in clinical trials [153–158]. Moreover,an FPN1 stabilizing antibody is currently also underinvestigation [159].

Since the HIF–EPO axis forms an alternative tar-getable pathway, prolyl hydroxylase inhibitors haveentered clinical trials. This class of drugs can beorally administered and protects HIF from degrada-tion, which increases EPO levels and erythropoieticiron availability [160].

Summary

The precise differential diagnosis between IDA, AI,and a combination of both forms is of clinical impor-tance because of differing treatment strategies. Cur-rently, the lack of data from prospective clinical tri-als prevents definitive recommendations on diagnos-tic algorithms and prognostic indices. These prob-lems are aggravated by the lack of standardization inotherwise promising tests, such as measurement ofsTFR. However, within the next few years, standard-ized tests for novel parameters such as HAMP or ERFEwill become available for more accurate differentialdiagnosis, stratification of treatment indications, andprediction of therapeutic response. Furthermore, theHAMP–FPN1 axis continues to receive a lot of atten-tion as a therapeutic target. Blocking HAMP expres-sion or processing, neutralization of circulating HAMP,and blockage of its interaction with FPN1 are underactive investigation for the treatment of AI.

Moreover, different forms of AI may have to betaken into account. Dependent on the underlyingconditions and dominant pathophysiological path-ways, a more personalized approach to optimal man-agement of distinct forms of anemias will be required.

Funding information This work was supported by grantsfrom the Austrian Research Fund (FWF; project TRP-188to G.W.; projects P28302-B30 and P24749-B13 to I.T.), the“Theodor Körner Fonds” (to M.N.), the intramural fundingprogram of the Medical University Innsbruck for young sci-entists MUI-START (project 2012032003 to M.N.), and by the“Verein zur Förderung von Forschung und Weiterbildung inInfektiologie und Immunologie an der Medizinischen Uni-versität Innsbruck”.

Open access funding provided by University of Innsbruckand Medical University of Innsbruck.

Conflict of interest M. Nairz, I. Theurl, D. Wolf, and G. Weissdeclare that they have no competing interests.

Open Access This article is distributed under the terms ofthe Creative Commons Attribution 4.0 International License(http://creativecommons.org/licenses/by/4.0/), which per-mits unrestricted use, distribution, and reproduction in any

medium, provided you give appropriate credit to the origi-nal author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made.

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