The diagnostic plot

The Diagnostic PlotA Concept for Identifying Different States of Iron Deficiency

and Monitoring the Response to Epoetin Therapy

Christian Thomas,1 Andreas Kirschbaum,2

Dieter Boehm,3 and Lothar Thomas4

1Urologische Klinik und Poliklinik der Universität Mainz Langenbeckstrasse 1 55131 Mainz,2Klinik und Poliklinik für Herz- und Thoraxchirurgie Universitätsklinik Würzburg

Josef-Schneider Strasse 6 97080 Würzburg, 3Klinik für Operative Intensivmedizin Krankenhaus Nordwest Steinbacher Hohl 2-26 60488 Frankfurt, Germany, and 4Laboratoriumsmedizin,

Krankenhaus Nordwest, Steinbacher Hohl 2-26 60488 Frankfurt

Abstract

Iron balance is regulated by the rate of erythropoiesis and the size of the iron stores. Anemia that accom-panies infection, inflammation, and cancer (anemia of chronic disease) features normal or increased ironstores, although patients may have functional iron deficiency, namely, an imbalance between iron require-ments of the erythroid marrow and the actual supply. The proportion of hypochromic red cells and the hemo-globin content of reticulocytes are direct indicators of functional iron deficiency. Biochemical markers,especially the soluble transferrin receptor/log ferritin ratio (ferritin index), are useful indicators of the iron sup-ply to erythropoiesis. The relationship between functional iron deficiency (reticulocyte hemoglobin content)and iron supply to erythropoiesis (ferritin index) can be described in a diagnostic plot. In normoproliferativeand hypoproliferative erythropoiesis, the plot allows the differentiation of classic iron deficiency from anemiaof chronic disease and the combined state of functional iron deficiency with anemia of chronic disease. Thetherapeutic implications of the plot are to differentiate patients into those who should be administered ironsupplements, epoetin, or a combination of epoetin and iron. In patients receiving epoetin therapy, the plot isan important tool for monitoring erythropoietic activity, functional iron deficiency, and adequate iron storesfor new red cell production. Enhanced erythropoiesis is reflected quantitatively by the ferritin index vector. Atransgression of the 1.5 (3.2) cut-off value for the ferritin index indicates that extra doses of iron need to beadministered to increase the body’s iron stores. A lack of increase or a reticulocyte hemoglobin content below28 picograms indicates functional iron deficiency. The diagnostic plot is a model for differentiating iron-defi-cient states and predicting those patients who will respond to epoetin therapy.

Key Words: Iron deficiency; anemia of chronic disease; soluble transferrin receptor; ferritin index; diag-nostic plot; epoetin therapy.

23

Review

Received 05/25/05; Accepted 06/17/05Corresponding author: Prof. Dr. med. Lothar Thomas, Krankenhaus Nordwest, Laboratoriumsmedizin, Steinbacher Hohl 2-26,

60488 Frankfurt, Germany E-mail: [email protected].

Medical Oncology, vol. 23, no. 1, 23–36, 2006© Copyright 2006 by Humana Press Inc. All rights of any nature whatsoever reserved. 1357-0560/(Online)1559-131X/06/23:23–36/$30.00

IntroductionThe treatment of anemia that accompanies

chronic infection and inflammation, cancer, and end-stage renal failure, commonly termed anemia ofchronic disease (ACD), remains a significant prob-lem (1) with low hemoglobin levels potentiallyimpacting on the outcome of cancers treated bychemotherapy (2). Currently, patients with a malig-nant tumor and hemoglobin (Hb) <100 g/L aretreated mainly with banked blood (3). However, notonly is there a shortage of banked blood in industri-alized countries, chronic blood transfusions alsoimpose numerous disadvantages on the patient.Treatment with recombinant human erythropoietin isa way of solving this problem (4–12), with ≤100 g/Loften being the Hb threshold for initiating epoetintherapy (13,14). However, optimum hematologicbenefits will only be produced if a significant bonemarrow response can be predicted and iron avail-ability is able to keep pace with the iron demandsmade by erythropoiesis enhanced by epoetin. It istherefore important to recommend diagnostic teststhat consistently reflect the erythropoietic state andare able to indicate the advances in iron deficiency(ID) from an early stage, especially in patients withcombined ID and ACD (ID/ACD).

This review will discuss some of these key issues,in particular, our studies with the aim of answeringthe following three questions:

1. How good is the clinical efficacy of standard bio-chemical markers for ID in anemic patients, andwhich marker is the best for detecting ID pro-gression?

2. Can the erythropoietic state of anemic patients becategorized into various pathologic conditionsusing biochemical markers and hematologicindices of iron metabolism?

3. Is it possible to develop a predictive model able toadequately identify patients who respond to epo-etin or iron therapy according to their erythropoi-etic state?

Anemia of Chronic Disease (ACD)The following pathologic processes in ACD can

be explained by the effect of inflammatory cytokines(15–21):

• Alteration of epoetin production and decreasedsensitivity of erythroid precursors to the stimulat-ing effect of epoetin: In patients with inflammationin response to anemia, epoetin secretion is low forthe degree of anemia present and is inadequate tomaintain normal Hb levels. Their blunted epoetinresponse is a result of proinflammatory cytokines,such as transforming growth factor-β (TGF-β),interferon-γ (IFN-γ), and tumor necrosis factor-α(TNF-α) that inhibit epoetin production.

• Suppression of erythropoiesis by cytokines associ-ated with inflammation, especially IFN-γ: Thesehave the opposite effect to epoetin on erythroidprecursors at the CFU-E stage of development.IFN-γ in bone marrow causes early death of ery-throid precursor cells, thus antagonizing the anti-apoptotic effect of epoetin (22).

• Reduced responsiveness to epoetin caused by rela-tive resistance: The mechanism by which proin-flammatory cytokines elicit resistance to epoetin inred cell precursors is unknown.

• Interference with the iron metabolism: Inflammatorycytokines decrease the availability of iron for ery-thropoiesis despite adequate body stores of iron, andcause the output of hypochromic red cells (23). Thiscondition is known as functional iron deficiency(FID) (24) and is defined as a relative imbalancebetween iron delivery to erythroblasts and epoetinstimulation caused by the hepcidin/inflammatorycytokine mechanism (25). Restriction of iron deliv-ery must be more severe than suppression of marrowprecursors and epoetin output.

Iron balance is fundamentally regulated by the rateof erythropoiesis and the size of iron stores. In irondeficiency anemia (IDA), the iron supply depends onthe quantity of the body’s stored iron, whereas in FIDthe supply depends on the rate of iron mobilization(26). Biochemical markers and hematologic indicesare used to determine the iron status.

Biochemical Markers of IDConventional biochemical markers for differential

diagnosis of anemia include the following indicatorsof iron turnover (24,27–29): ferritin (the iron-storageprotein), total iron-binding capacity (TIBC), andtransferrin saturation. These parameters, althoughwidely used, are affected by acute-phase response

24 Thomas et al.

Medical Oncology Volume 23, 2006

(APR), which complicates the clinical interpretationof results (30). Ferritin is an acute-phase reactant,while transferrin is a negative one. Serum transferrinreceptor (sTfR) is also a useful marker for differenti-ating anemias. sTfR levels increase in tissue ID,reflecting the degree of ID in the erythroid precursors.As sTfR concentrations do not increase in anemia sec-ondary to inflammatory disorders, they have becomea popular way of differentiating ACD from ID andID/ACD (31–33). The measurement of sTfR for cal-culating the sTfR to ferritin ratio (sTfR/log ferritin;ferritin index) displays an inverse relationship to ironstatus and covers the entire range from repleted ironstores to FID (34–40). However, sTfR levels also risein response to proliferation of red cell precursors. Thisreflects a greater degree of receptor shedding fromincreased number of precursors. Therefore, elevatedvalues cannot be interpreted unless proliferation oferythroid marrow precursors can be ruled out and theferritin fit the picture of ID (31–33).

Many patients with ACD demonstrate a character-istic pattern of low TIBC, where the percentage trans-ferrin saturation is between 10% and 20% coupledwith a rise in ferritin concentration (23). It is impor-tant to note that this pattern is distinctly differentfrom classical ID, where a rise in TIBC is accompa-nied by a very low ferritin concentration. In ACD, therelationship to the severity of illness is also mirroredby biochemical iron markers. The more pronouncedthe inflammatory component of the patient’s illness,the lower the transferrin concentration and TIBC, andthe higher the serum ferritin concentration. It is wellknown that patients with ACD have repleted ironstores with normal to elevated ferritin levels (41).Nevertheless, around 20% of these patients sufferfrom FID (42). In ACD, as seen in end-stage renaldisease, serum ferritin concentrations of <200 µg/Lmay be related to depleted iron stores. Ferritin con-centrations of 200–2000 µg/L are a function of bothiron and inflammation, while levels >2000 µg/L aremuch more likely to be an indicator of iron overload(43). In such patients, a ferritin range of 200–2000µg/L shows a significant correlation to CRP. Thissuggests that serum ferritin is a marker of iron storesregardless of the range, but correlates with inflamma-tion between 200 and 2000 µg/L (43).

sTfR increases once serum ferritin falls below30–40 µg/L, making it a sensitive measure of ID

(44,45). However, none of the studies demonstratingexclusion of ID in the presence of inflammation areable to indicate a clear cut-off separating inflamma-tory states from non-inflammatory states. This is animportant point because depending on the severity ofAPR, erythropoiesis is strongly hypoproliferative inthe combined state of ID/ACD resulting in a lowsTfR concentration (46). Often patients with com-bined ID/ACD only show an sTfR increase withinthe reference range; this small change in sTfR neverexceeds the cut-off for ID.

The ferritin index is a valuable parameter for dis-tinguishing between iron-repleted and iron-depletedanemic patients when diagnostic classification of thepatient is based on either changes induced by ironsupplementation (36,37) or examination of the bonemarrow using iron staining as the gold standard foriron depletion (35,38–40). The ferritin index is help-ful at predicting erythroid response to epoetin ther-apy in patients with ACD. A low ferritin index [lowsTfR combined with a relatively normal (upper endof range) to an above normal ferritin concentration],indicates decreased erythroid activity with adequateiron stores. This suggests that a response to epoetinis likely, whereas a high ferritin index predicts a lackof response to epoetin because of bone marrow unre-sponsiveness or underlying iron deficiency (47). Thevalue of this parameter is only limited to a smalldegree by inflammation as the logarithm of ferritin isused to calculate the ferritin index.

Hematologic Markers of IDDiagnosis of ID is challenging when the complete

blood count (CBC) is used. This is because the longlifespan of erythrocytes requires a prolonged ID statefor diagnosing hypochromic red cells. In addition toCBC, other new hematologic indices that have gainedmerit for assessing iron status include reticulocytehemoglobin content (Ret-Hb) (48) and the proportionof hypochromic red cells (%HYPO) (49).

Because erythrocytes have a lifespan of approx120 d, %HYPO is able to provide information over aseveral month period, and is a time-averaged indica-tor of ID. Modern hematology analyzers are capableof identifying small subpopulations of erythrocyteswithin the total RBC population; they offer appro-priate tools for measuring %HYPO. A HYPO value

Diagnostic Plot in Iron Deficiency and Epoetin Therapy 25


of <10% combined with low serum ferritin isassumed to indicate the marrow’s iron supply isbeing maintained at a rate sufficient for normal redcell hemoglobinization, even though the body’s ironstores may have been depleted considerably (50).%HYPO is recommended as a meaningful and inex-pensive marker for the integrated effects of ironstores, inflammation, erythropoietic stimulation, andiron availability in hemodialyzed patients (51,52).

Ret-Hb is a real-time marker of FID, as reticulo-cytes only exist in the circulation for 1–2 d. Ret-Hbcan be estimated by measuring CHr with the BayerAdvia 120 analyzer (48) or by measuring Ret-Y withthe Sysmex NE analyzer series (53,54). The Adviaanalyzer measures the mean concentration of reticu-locytes (CHCMr) and the mean cell volume(MCVr). CHr is a calculated index [CHr (pg) =MCVr (fL) × CHCMr (g/L)]. Ret-Y is the meanvalue of the forward scattered light histogram of thereticulocyte population expressed in arbitrary chan-nel numbers (AC). There is good comparabilitybetween CHr and Ret-Y. A Ret-Y of 1630–1860 ACcorresponds to a CHr range of 28–35 pg. Using theregression formula (function y = 5.5569 e0.001x), ACnumbers can be used to transform Ret-Y into itshemoglobin equivalent expressed as a picogram anddenoted as Ret-He (55). Ret-Hb is recommended fordiagnosing ID in children (56) and for iron manage-ment in hemodialysis patients (57–59).

Evaluation of Biochemical Markers of Iron Status in the Diagnosis of ID

Red cell hemoglobinization, measured as %HYPOor Ret-Hb, provides direct evaluation of bone marrowactivity, and reflects the balance between iron anderythropoiesis. The central 95% ranges for personswith no clinical laboratory signs of anemia for%HYPO and Ret-Hb (CHr, Ret-He) are 1–5% and28–35 pg, respectively (46,55). Because of the stronginfluence of APR on the iron supply for erythro-poiesis, we compared the iron metabolism of patientsboth with and without APR. The CRP assay was usedas a marker of APR; all patients with a CRP level of>5 mg/L are considered to have an APR (60). To eval-uate the impact of biochemical markers on indicatingFID, ferritin, sTfR, and the ferritin index were mea-sured in anemic patients both with and without APR.

A Ret-Hb of <28 pg and a %HYPO of >5% wereused as gold standards for FID. As shown in Fig. 1,ferritin, sTfR, and ferritin index displayed greatersensitivity, specificity, positive likelihood ratio, andarea under ROC curve in patients without APR. Fromthese studies, we found that the ferritin index was themost accurate marker for biochemical identificationof FID. This agrees with investigations that show theferritin index to be a good marker of iron supply forerythropoiesis because it reflects iron turnover fromthe stores to the marrow (35,36,39,40). Optimum cut-offs (highest sensitivity and specificity from ROCcurves) for ID in anemic patients with reduced redcell hemoglobinization are shown in Table 1. The fer-ritin cut-off indicating ID increased 2- to 3-fold inpatients with APR compared to those without APR(46). Patients with CRP levels of 34 ± 21 mg/L (x ±SD) had a lower ferritin index because ferritin is anacute-phase protein that is elevated in inflammatorydiseases independently of the body’s iron stores (47).As a result, the decision point was moved to a lower

26 Thomas et al.


Fig. 1. ROC plot showing the ability of ferritin, sTfR,and sTfR/log ferritin (ferritin index) to indicate func-tional iron deficiency in patients both with and withoutAPR. As references, a reticulocyte hemoglobin concen-tration (CHr, Ret-He) of <28 pg was used. The solid linesrepresent patients who did not exhibit acute-phaseresponse (CRP ≤ 5 mg/L), while the dotted lines repre-sent patients with acute-phase response (CRP > 5 mg/L).The ROC plots using a HYPO of >5% as reference areavailable from the online version of the original article athttp://www.clinchem.org/content/vol48/issue7/.

value (46). The decision point for the ferritin indexwas dependent on the sTfR assay. Ratios of 1.5 and0.8 (Dade Behring assay) corresponded to values of3.2 and 2.0 (Roche assay). According to this classifi-cation, 57 out of 269 (26.8%) ACD patients withAPR and FID had repleted iron stores, but only 14out of 145 (9.2%) patients without APR had repletedstores. The use of different cut-offs for the ferritinindex is very important. Using %HYPO or Ret-Hb asreferences, only 37% of patients with ACD and aCRP value of >5 mg/L were evaluated as functionallyiron deficient if ratios of 1.5 (3.2) instead of 0.8 (2.0)were used as indicators of ID (46). ROC plots usinga %HYPO of >5% as reference are available in theonline version of the original article at http://www.Clinchem.org/content/vol48/issue7/.

Development of a Diagnostic Plot for Identifying Erythropoietic Stateand Different Phases of Advancing ID

The rationale behind the diagnostic plot was tocombine the best iron supply marker for erythro-poiesis with Ret-Hb as the indicator for FID. Patientswith a Ret-Hb of ≥28 pg (normal hemoglobin contentof reticulocytes) do not have FID, whereas those witha Ret-Hb of <28 pg (reduced hemoglobin content ofreticulocytes) have FID because of iron demand onthe bone marrow. It is also important to know whetheror not an inflammatory disorder co-exists. The ferritinindex separating normal iron supply from reducediron supply to the marrow was 1.5 (3.2) in patientswithout APR (simple ID) and 0.8 (2.0) in patients with

inflammation, as previously stated. The relationshipbetween Ret-Hb and the ferritin index can bedescribed in a plot (46) divided into quadrants thatcorrespond to the following four states (Fig. 2):

1. Normal iron supply and normal red cell hemoglo-binization. This quadrant contains patients withcancer-related anemia, acute and chronic inflam-mation, and end-stage renal failure without ID

2. Patients with reduced iron supply where erythro-poiesis is not yet iron deficient and red cell hemo-globinization remains normal. This quadrantincludes anemic or non-anemic patients withlatent ID, patients with ID shortly after com-mencing oral iron therapy, and patients withhyperproliferative erythropoiesis due to acutehemorrhage, hemolysis, and in the third trimesterof pregnancy with increased sTfR, but no FID

3. Reduced iron supply to erythropoiesis withdecreased red cell hemoglobinization attributableto FID in depleted iron stores that typicallyoccurs in classical ID.

4. FID in iron-repleted state with decreased red cellhemoglobinization as the cause in ID/ACDpatients who have anemia accompanying infec-tion, chronic inflammation, and cancer. Patientswith data points in quadrant 4 have at least two offour criteria indicating FID (Ret-Hb <28 pg,HYPO >5%, Ret-Hb inversion, and elevatedsTfR). An inverted Ret-Hb/erythrocyte-Hb ratioshows evidence of recently developed FID.However, patients possessing a β-thal trait likethose with ID/ACD also have data points in quad-rant 4, and must be separated by determining the



Table 1Cut-off Values for Biochemical Markers in Patients

with (CRP >5 mg/L) and without (CRP ≤5 mg/L) Acute-Phase Response Using a Ret-Hb of <28 pg as the Reference for Functional Iron Deficiency (27)

Marker CRP ≤5mg/L CRP >5mg/L

sTfR (mg/L)(Dade Behring) M 2.0 F 1.7 M 1.5 F 1.8(Roche Diagnostics) M 4.3 F 3.7 M 3.2 F 3.9

sTfR/log ferritin (ferritin index)Dade Behring Diagnostics M + F 1.5 M + F 0.8Roche Diagnostics M + F 3.2 M + F 2.0

M, male; f, female.

ratio of the percentage of microcytic to hypo-chromic red cells. Patients with a ratio of >0.9and >18% microcytic red cells are suspected ashaving β-thal trait (61).

The diagnostic plot shows good selectivity forassessing iron status in disease-specific anemias suchas classical ID, end-stage renal failure, cancer-relatedanemia, and anemia of infection and inflammation(46). The diagnostic plot recorded the following dis-tributions of 211 patients with cancer-related anemia.The majority (47.9%) were placed in quadrant 1, indi-cating normal hemoglobinization in the iron-repletedstate. In 12.8% of patients, data points were localizedin quadrant 2, indicating latent ID. Approximately40% of patients showed data point distributions inquadrants 3 and 4, representing decreased red cellhemoglobinization in the iron-depleted state (24.7%)or iron-repleted state (14.7%), respectively (46).Instead of Ret-Hb, %HYPO can be used in the diag-nostic plot, utilizing a cut-off of >5% as the referencefor FID with no loss of diagnostic accuracy.

Predictive Model for AdequateIdentification of Responders to Epoetin or Iron Therapy According to the Erythropoietic State

The therapeutic implication of the diagnostic plotis to differentiate patients into those who should beadministered iron, epoetin, or a combination of epo-etin and iron (Fig. 3). We recommend the clinicianprovides iron supplementation to anemic patientswith data points in quadrants 2 and 3. Patients withadequate iron supplementation usually respond witha shift in their data points from quadrant 3 to quad-rant 2 within 10 d, and to quadrant 1 after 4 to 20 wk.

We recommend anemic patients with data points inquadrants 1 and 4 are administered epoetin therapy.However, use of epoetin should be restricted to spe-cific clinical situations that are not expected toimprove by simply treating the cause of ACD.According to the diagnostic plot, patients with datapoints in quadrant 4 have FID requiring combined

28 Thomas et al.


Fig. 2. Diagnostic plot for identifying the different erythropoietic states of advancing ID combining the biochemicalindicator for iron supply (sTfR/log ferritin ratio, ferritin index) with Ret-Hb, the hematologic indicator for iron demandby erythropoiesis (CHr, Ret-He). The plot is also used for evaluating the erythropoietic state prior to commencing epo-etin therapy. A Ret-Hb of <28 pg indicates functional iron deficiency. In patients with CRP values of ≤5 mg/L, a ferritinindex of >1.5 (3.2) indicates depleted iron stores, and a ferritin index of <1.5 (3.2) indicates repleted iron stores. Inpatients with a CRP concentration of >5 mg/L, the cut-off value for the ferritin index is 0.8 (2.0). Ferritin index cut-offvalues for the Dade Behring sTfR assay are shown without parentheses, while those for the Roche assay are shown withparentheses. ACD, anemia of chronic disease; ERF, end-stage renal failure; IDA, iron deficiency anemia.

intravenous iron supplementation along with their firstepoetin dosage. Patients with data points in quadrant1 should be treated primarily with epoetin only.

Prediction of Response to Epoetin in Patients with ACD

Anemia, especially in patients with end-stagerenal failure (62) and patients with multiplemyeloma (4,5), has been shown to be effectivelyreversed by epoetin, as the drug is able to speed uperythropoiesis by several multiples. The response ofanemia to epoetin is dose-related; it varies frompatient to patient, and cannot always be predicted.Patients often undergo sequential increases in epo-etin dosage over the course of therapy until their cor-rect dose is identified, leading to a hematologicresponse and potential increase in Hb to desirablelevels (63). Identification of a significant hemato-logic response may take months, because in mostclinical settings effectiveness of each sequentialincrease of epoetin dosage is evaluated by monitor-ing the increase in Hb 4–5 wk after escalating theepoetin dosage. Thus, it would be useful to identifylaboratory tests that change early on in the hemato-logic response to an increase in epoetin dosage, and

are able to predict a pending erythroid response at agiven epoetin dose (47).

Enhanced erythropoiesis induced by epoetinadministration is closely tied to iron metabolism, asthere is a concurrent requirement of an adequate sup-ply of iron for new red cell production. Increasing cir-culating Hb by 10 g/L uses 150 mg of stored iron (64).In such a state, iron turnover is a function of ironstores and erythropoietic activity of the bone marrow.Iron availability may not keep pace with the irondemand of erythropoiesis enhanced by epoetin, a sit-uation known as FID. Therefore, assessment of ironrequirements and an adequate iron supply are prereq-uisites for an optimum response to epoetin therapy.However, a number of questions remain regarding theappropriate use and efficacy of iron treatment (65).Intravenous iron supplementation has been shown toimprove Hb response to epoetin and overall quality oflife in patients with cancer-related anemia (66,67).

Laboratory tests designed to predict the hemato-logic response of cancer patients receiving epoetintherapy are recommended (68–70). However, notests have so far been evaluated for differentialassessment of erythropoietic activity and progres-sion of ID in real time. Therefore, valid methods arestill debatable. To optimize epoetin therapy with



Fig. 3. Therapeutic implications for the treatment of different phases of iron deficiency. ACD, anemia of chronic dis-ease; ID, iron deficiency.

proper laboratory management, we recommend thefollowing procedure:

1. Classification of patients with anemia of cancerinto four different categories of erythropoieticstate in accordance with Fig. 2 as the preliminaryinvestigation. We recommend that the clinicianprovide iron supplements to patients with datapoints in quadrants 2 and 3. Patients with datapoints in quadrant 1 should be treated primarilywith epoetin only, and patients with data points inquadrant 4 should be given iron supplementsalong with their first dose of epoetin.

2. Monitoring epoetin therapy in accordance withthe modified diagnostic plot two and six weeksafter starting therapy, as shown in Fig. 4.

According to our studies, a Ret-Hb of <28 pg is asensitive indicator of FID. This is reported in com-parison with biochemical markers of iron status(46,56) and in end-stage renal failure patients treatedwith epoetin (57–59). However, the dosage of ironneeded to sustain normal Ret-Hb is the minimumdose needed to satisfy iron demand at a reticulocytelevel, and total body iron stores remain unsatisfactory(71). sTfR levels are a good marker of erythropoietic

activity (63,72,73), although they are unable to dif-ferentiate iron availability status of patients on main-tenance epoetin doses (74). We therefore prefer theferritin index as this reflects the effect of erythropoi-etic activity (sTfR) and iron reserves (ferritin).

Evaluation of the Diagnostic PlotThe following study was performed to demon-

strate that the diagnostic plot is able to detectpatients whom will benefit from epoetin therapy. Theintention was to establish under epoetin therapywhether an increase in erythropoietic activity andadvancing FID can be predicted from the erythro-poietic state using the diagnostic plot. Twenty-sixcritically ill anemic patients were enrolled in thestudy. Twenty patients had newly diagnosed solidmalignant tumors (five bronchogenic carcinoma,three pancreatic carcinoma, three gallbladder carci-noma, three prostate carcinoma, two colonic carci-noma, two esophageal carcinoma, two gastriccarcinoma) and six patients with different nonmalig-nant abdominal surgeries. After surgical treatment,all patients were cared for by a surgical intensivecare unit as they were suffering from postoperative

30 Thomas et al.


Fig. 4. Diagnostic plot for monitoring erythropoietic activity, iron entering red cell precursors, and adequate ironstores. An increase in ferritin index vector of ≥0.25 (0.60) indicate the effectiveness of the epoetin dosage, a Ret-Hb of≥28 pg or an increase ≥2 pg excludes functional iron deficiency, and a ferritin index of <1.5 (3.2) demonstrates adequateiron supply for new red cell production.

complications such as inflammatory disorders. Theyhad thus fulfilled the criteria of ACD for the past 4–8wk. Baseline patient characteristics included Hb lev-els of <105 g/L for more than 4 wk, elevated CRPconcentrations, and an erythropoietic state in quad-rant 1 or 4, apart from two patients who had datapoints in quadrants 2 and 3. None of the patients hadreceived blood transfusions in the previous 2 wk.Epoetin (Roche NeoRecormon) doses of 150 U/kgsc were administered twice a week. Only one patientwith data points in quadrant 3 received iron iv sup-plementation. The erythropoietic states and Hb con-

centrations were evaluated at baseline, and moni-tored after the initial epoetin dosage twice weekly.The following conditions were defined for optimumresponsiveness to epoetin:

1. Increase in Hb of ≥10 g/L.2. Increase in the ferritin index as an indicator of

stimulated erythropoietic activity.3. Increase in Ret-Hb of ≥2 pg or Ret-Hb of ≥28

picograms as an indicator of iron turnover beingable to satisfy iron demand at a reticulocyte level;a Ret-Hb of <28 pg characterizes FID.



Table 2|Changes to Ferritin Index, Ret-Hb, Hemoglobin (Hb), and CRP Concentrations

According to Erythropoietic State before Epoetin and 14 d after Epoetin Administration

Ferritin ∆Ret-Hb Pat. No ES ∆Hb (g/L) index vector (pg) 0 d CRP 14 d CRP Result

1 1 –6 – 0.10 + 2 90 38 NR2 4 + 14 + 0.40 + 4 117 22 R3 4 + 15 + 0.34 – 1 78 65 R, FID4 4 + 16 + 0.33 + 4 214 204 R5 1 + 14 + 0.10 – 1 28 77 NR6 1 + 7 + 0.09 – 1 69 41 NR7 1 + 1 + 0.03 – 2 29 69 NR8 1 + 31 + 0.38 + 3 89 20 R9 4 + 11 + 0.31 + 4 103 193 R10 4 + 9 + 0.06 – 3 58 36 NR11 2 – 4 – 0.55 – 1 178 287 NR12 1 + 7 + 0.16 – 2 75 77 NR13 4 + 10 + 0.34 – 2 157 353 R, FID14 4 + 25 + 0.51 0 65 35 R, FID15 4 + 11 + 0.58 + 2 78 45 R, FID16 1 + 16 + 0.27 + 3 91 41 R17 4 + 31 + 0.35 + 2 74 16 R, FID18 1 – 6 + 0.18 + 4 386 152 NR19 4 + 24 + 0.76 + 2 167 20 R20 4 + 11 + 0.66 + 2 222 105 R21 4 + 10 + 0.73 – 1 225 62 R, FID22 1 + 11 + 0.34 + 2 60 18 R23 1 + 14 + 0.30 + 1 20 193 R24 3 + 24 – 0.47 + 8 15 12 R25 1 + 2 + 0.11 – 2 56 97 NR26 1 + 6 + 0.24 + 1 236 221 NR

Abbreviations: Pat. No, patient number; ES, erythropoietic state (quadrant); ∆Hb, (+) increase or (–) decrease of Hb value 2 wksafter starting epoetin therapy; Ferritin index vector increase (+) or decrease (–) of ferritin index 2 wk after starting epoetin therapy;∆Ret-Hb, increase (+) or decrease (–) of reticulocyte hemoglobin content 2 wk after starting epoetin therapy; CRP, C-reactive proteinconcentration (mg/L) before and 2 wk after starting epoetin therapy; R, response to epoetin therapy; NR, no response to epoetin ther-apy; FID, functional iron deficiency; pg, picograms

4. No increase in the ferritin index to >1.5 (3.2), thethreshold ferritin concentration at which irondepletion is predictable.

Sixteen out of the 26 patients responded to epo-etin and demonstrated a significant increase in Hbconcentration and ferritin index (∆FI) within 2 wk(Tab. 2). The lowest ferritin index vector ∆FI indi-cating erythropoietic activity stimulated by epoetinwas 0.25 using the Dade Behring sTfR assay and0.6 by the Roche assay. Six of the 12 responderswith an erythropoietic state of 4 had FID, and mostof the quadrant 4 responders progressed to a ferritinindex range of 1–1.5 (2.0–3.2) indicating that aniron-depleted state would soon develop (Fig. 5).This was also demonstrated in multiple myelomapatients treated with epoetin, using %HYPOinstead of Ret-Hb as the indicator for FID (75). Tenpatients did not respond to rHuEPO. Their ferritinindex vector was <0.25 (0.6). Seven of thesepatients had a Ret-Hb of <28 pg or showed anadvancing decrease (Fig. 6).

Based on our preliminary data, the following pre-dictions can be made for epoetin therapy:

1. The ≥0.25 (0.6) increase in the ferritin index vectorfor erythopoietic states (quadrants 1 and 4) indi-cates erythropoietic activity in response to epoetin.

2. A decrease in Ret-Hb in the range of 35–28 pgindicated advancing FID, while a Ret-Hb of <28pg demonstrates persistent FID

3. Iron depletion is predictable if the ferritin indexexceeds a threshold of 1.5 (3.2) as correspondingferritin concentrations are between 20 and 200µg/L in hyperproliferative erythropoiesis wherethe sTfR concentration is 1.5- to 3-fold the upperreference limit.

The question arises as to the advantages of thediagnostic plot over Hb measurement and knownpredictive models for monitoring epoetin response.The combination of ferritin index and Ret-Hb deter-mines the erythropoietic state as basic investigationand in the early stage of epoetin treatment differenti-

32 Thomas et al.


Fig. 5. Patients with anemia of chronic disease responding to epoetin with an increase in hemoglobin concentrationof >10 g/L within 2 wk of rHuEPO therapy. The arrow-head marks the value of the Ret-Hb and the ferritin index after 2wk. All responders showed a ferritin index vector of ≥0.25 (0.60). The broken arrows characterize patients with functionaliron deficiency. It can be suggested that patients in whom the ferritin index has advanced to values of between 1 and 1.5will soon have an inadequate iron supply. The patient in erythropoietic state 3 received iron iv supplements in combina-tion with epoetin. This patient responded with a decrease in the ferritin index and a large increase in Ret-Hb.

ation of FID from intrinsic nonresponsiveness of themarrow because of inadequate epoetin dosage ispossible. An increase in Ret-Hb indicates responseof erythropoiesis to epoetin and the absence of FID.A decrease in Ret-Hb occurs within 2–4 d in FID(76) and responders to epoetin begin to exhibit anelevation in the ferritin index >0.25 (0.60) 4–6 d ear-lier as an increase in Hb level.

Limitations of the Diagnostic PlotThe following limitations should be considered in

order to effectively use the diagnostic plot:

1. An increase of erythroid precursor mass (hemolyticsyndrome, myelodysplastic syndrome, pregnancy)may displace data points from quadrant 1 to quad-rant 2. This is caused by sTfR which correlates witherythroid precursor mass.

2. Patients with β thalassemia trait may have datapoints in quadrant 4 even though they do nothave FID.

3. A ferritin index cut-off of 2.0 (Roche sTfR assay)or 0.8 (Dade Behring sTfR assay) in patients withAPR should only be used where there is persis-tently elevated CRP for at least 2 wk, as changesin the iron stores and erythroid precursor masscaused by APR need this amount of time tochange the ferritin index.

4. Patients with ACD on iron supplements may havedata points in quadrants 2 or 3, but near the fer-ritin index cut-off. The reason is that in somecases of ACD iron supplements may increase ery-thropoiesis maturation and elevate sTfR, mostlywithin the reference range.

5. In cancer patients with chemotherapy-inducedanemia and HYPO in the 5–25% range, Ret-Hbmay have inadequately high values, regardless ofwhether Ret-Hb is measured as CHr or Ret-He.

6. A low reticulocyte count may cause large varia-tions in the calculation of Ret-Hb as hematologyanalyzers only measure fixed red cell counts (sumof erythrocytes and reticulocytes) in the sample.



Fig. 6. Patients with anemia of chronic disease not responding to epoetin with an increase in hemoglobin concentra-tion of ≥10 g/L within 2 wk of epoetin therapy. All non-responders had a ferritin index vector of <0.25 (0.60), with 2 outof 10 patients demonstrating an increase in Ret-Hb of ≥2 pg. Until now no data have been available for demonstrating ifpatients with an increase in Ret-Hb and no significant increase in ferritin index vector and hemoglobin concentration willrespond to a higher dosage of epoetin or after a longer time of treatment.

ConclusionsBiochemical markers of ID demonstrate weak-

nesses in diagnosing ID, especially in patients withAPR. The combination of Ret-Hb and ferritin indexin a diagnostic plot offers diagnostic value in differ-entiating ID from ACD and the combined state ofID/ACD. Identification of an early significant ACDresponse to epoetin can be assessed by evaluating thecourse of ferritin index and Ret-Hb in the plot. A fer-ritin index vector of ≥0.25 (0.60) indicates that theepoetin dosage is effective, a Ret-Hb of ≥28 pgexcludes persistent FID, and a ferritin index of <1.5(3.2) demonstrates an adequate iron supply for newred cell production. Until now only limited data havebeen available for demonstrating the value of thediagnostic plot as a predictive model to adequatelyidentify patients who respond to epoetin accordingto erythropoietic state (75); however, several studiesare currently undergoing investigation.

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