J Lab Med 2004;28(5):391–399 2004 by Walter de Gruyter • Berlin • New York. DOI 10.1515/LabMed.2004.054

Ha¨ matologie Redaktion: T. Nebe

Diagnostic work-up of deficiency Diagnostisches Vorgehen bei Eisenmangel

Georgia Metzgeroth and Jan Hastka unterschiedliche Stadien erfassen. Durch Bestimmung von mehreren Parametern ist es mo¨ glich, den Eisensta- III. Medizinische Universita¨ tsklinik, Klinikum Mannheim, tus einer Person pra¨ zis zu charakterisieren und den Universita¨ t Heidelberg, Germany Schweregrad des Eisenmangels genau einzustufen. Von besonderem klinischem Interesse sind neuere Parameter Abstract der eisendefizita¨ ren Erythropoese: das Zinkprotoporphy- rin (ZPP), die lo¨ slichen Transferrinrezeptoren (sTfR), die Iron deficiency (ID) is defined as a diminished total body hypochromen Erythrozyten (Hypo) und das Retikulozy- iron content. Three degrees of severity have been tenha¨ moglobin (CHr). ZPP kann als ein zuverla¨ ssiger und defined: storage iron depletion (stage I), iron-deficient preiswerter Screeningparameter des gesamten Eisen- erythropoiesis (stage II), and iron deficiency anemia stoffwechsels verwendet werden, indem es das Endsta- (stage III). When assessing the patient’s iron status, it is dium der Ha¨ msynthese u¨ berwacht. Die Bestimmung der important to bear in mind that each of the various iron sTfR-Konzentration erlaubt die Differenzierung zwischen tests indicate something different in terms of ID. As they einem echten Eisenmangel und Eisenverwertungssto¨ run- detect different stages of ID, the parameters efficiently gen bei Ana¨ mien der chronischen Erkrankung (ACD). complement each other to characterize the iron status in Hypo und CHr scheinen die besten Parameter zu sein, the individual patient. Of particular clinical interest are um Eisenmangel bei Ha¨ modialysepatienten unter Eryth- newer iron tests of the iron-deficient erythropoiesis: zinc ropoietintherapie zu diagnostizieren. protoporphyrin (ZPP), soluble receptor (sTfR), hypochrome erythrocytes (Hypo) and reticulocyte hemo- Schlu¨ sselwo¨ rter: Eisenmangel; ; hypochrome globin (CHr). Supervising the final step of the heme pro- Erythrozyten; lo¨ slicher Transferrin-Rezeptor; Retikulozy- duction, ZPP can be used as a reliable and inexpensive ten-Ha¨ moglobin; Zinkprotoporphyrin. screening test of the whole iron metabolism. sTfR meas- urement is a useful tool to distinguish between real iron deficiency and functional iron deficiency in anemia of Iron deficiency (ID) probably represents the most com- chronic disorders. Hypo and CHr seem to be the best mon deficiency disease in humans. It is estimated that parameters to diagnose ID in hemodialysis patients treat- about 500 million people worldwide suffer from ID, and ed with recombinant human erythropoietin. it is by no means a problem confined to the so-called third world. In industrial countries, the estimated preva- Keywords: ferritin; hypochrome erythrocytes; iron defi- lence of ID in women of child-bearing age is 20% w1x. ciency; reticulocyte ; soluble transferrin This widespread occurrence results from different socio- receptor; zinc protoporphyrin. economic and physiological factors. But it is mainly pro- moted by the fact that ID is mostly regarded as a minor Zusammenfassung clinical problem, as the disease is not immediately life- Eisenmangel ist definiert als Verminderung des Ganzko¨r- threatening. Consequently, the assessment of the iron pereisens. Abha¨ ngig von dessen Schweregrad werden status is frequently performed with minimal effort, and drei Stadien unterschieden: Speichereisenmangel (Sta- unsuitable screening tests like the iron concentra- dium I), eisendefizita¨ re Erythropoese (Stadium II) und tion or the red cell indices are used. Eisenmangelana¨ mie (Stadium III). Wenn man den Eisen- It is of course true that ID per se is not immediately status einer Person beurteilt, muß man immer beru¨ ck- endangering life. Nevertheless, it is clinically important to sichtigen, daß die verschiedenen ‘‘Eisen-Parameter’’ diagnose ID as soon as possible. This is in particular true for pregnant women and small children, as neonates and Correspondence: Prof. Dr. Jan Hastka, III. Medizinische infants are at greatest risk of long-term or permanent Universita¨ tsklinik, Wiesbadenerstraße 7-11, 68305 Mannheim, damage. Even at the preanemic stage, insufficient iron Germany support causes developmental delays with cognitive, Tel.: q49-621-383-4110 Fax: q49-621-383-4201 neurodevelopmental and behavioral deficits in infants E-mail: [email protected] w2–6x. The early diagnosis of ID is also of vital interest in 392 G. Metzgeroth and J. Hastka: Diagnostic work-up of iron deficiency

individuals whose negative iron balance is caused by a Table 1 Reference ranges of the different iron parameters w9x. malignancy of the gastrointestinal tract. Finally, there is no reason whatsoever for a considerable number of Parameter Reference values women, many children and elderly to suffer from a dis- BM – hemosiderin* 2 ease which can be diagnosed and treated without any BM – sideroblasts 15–50% problems. Hemoglobin Women: 123–153 g/l Men: 140–175 g/l MCV 80–96 fl MCH 28–33 pg Stages of iron deficiency Hypochrome erythrocytes -2.5% Reticulocyte hemoglobin G26 pg Before speaking about the assessment of the iron status, Women: 6.6–26 mmol/l it must be clarified what the term ‘‘ID’’ means. ID is Men: 11–28 mmol/l defined as a state in which the total body iron content is Serum ferritin Women: 15–150 mg/l less than normal. But ‘‘iron deficiency’’ in itself does not Men: 30–400 mg/l mean very much, unless we take into account its severity, Transferrin 200–400 mg/dl 16–45% which is very important from the clinical point of view. sTfR** Women: 1.9–4.4 mg/l Depending on the severity, three stages of ID are distin- Men: 2.2–5.0 mg/l guished: storage iron depletion, iron-deficient erythro- TfR-F index** Women: 0.9–3.7 poiesis, and iron deficiency anemia w7x. The formerly Men: 0.9–3.4 used terms ‘‘prelatent-latent-manifest’’ are misleading Zinc protoporphyrin F40 mmol/mol heme and should be avoided. sTfR: soluble transferrin receptor; BM: bone marrow; MCH: The negative iron balance first leads to storage iron mean corpuscular hemoglobin; MCV: mean red cell volume. depletion (stage I), where the total body iron is *Scale 0–4. **The is test-dependent, here Tina- quant/Roche Diagnostics. decreased, without the synthesis of hemoglobin being affected. In this stage, there is no immediate clinical dys- individual parameters do not measure a single entity function. In stage II, the iron-deficient erythropoiesis,ID called ‘‘iron deficiency’’, but they are related to different becomes a disease, as the amount of iron is no longer stages of ID. Consequently, the ‘‘best parameter’’ of ID sufficient to meet the requirement of the erythropoietic does not exist. By detecting different stages, the various precursors in the bone marrow. The hemoglobin concen- tests, however, efficiently complement one another in tration, however, is still within the reference range. Finally, order to characterize the severity of ID in the individual when the iron supply is insufficient to maintain a normal patient w10x. hemoglobin concentration, stage III of ID, the iron defi- Prussian blue staining of the bone marrow and the ciency anemia, is reached. determination of ferritin reflect the iron stores. Parame-

Iron tests

There are numerous parameters of iron metabolism which can be used for the assessment of the patient’s iron status (Table 1) w8x. The gold standard is still Prussian blue staining of the bone marrow. As bone marrow exam- ination is not routinely practicable, other tests are required for the evaluation of the iron status in clinical practice. Numerous laboratory tests are used, such as the serum concentration of iron, ferritin and transferrin, the determination of hemoglobin and the red cell indices mean red cell volume (MCV) and mean corpuscular hemoglobin (MCH), and the measurement of the trans- ferrin saturation. In addition to these standard markers, Figure 1 Diagnostic sensitivity of various iron tests of iron defi- new iron tests have been established in the last years, ciency (ID). Bone marrow (BM) hemosiderin and serum ferritin e.g. the measurement of soluble transferrin receptor in assess the iron stores and allow the diagnosis of iron depletion the serum (sTfR), the percentage of hypochromic eryth- (ID, stage I). Pathological values of sideroblast count, transferrin rocytes (Hypo), the reticulocyte hemoglobin (CHr), and saturation, soluble transferrin receptor, hypochrome erythro- the concentration of erythrocytic zinc protoporphyrin cytes, reticulocyte hemoglobin, and zinc protoporphyrin indicate that an iron-deficient erythropoiesis (ID, stage II) has already (ZPP). developed. Finally, hemoglobin below reference range defines When assessing the iron status, it is important to iron deficiency anemia (ID, stage III), which in severe cases is always bear in mind that each of the various tests indi- accompanied by low red cell indices. cate something different in terms of ID (Figure 1). The I, II, III: iron deficiency, stage I, II and III, respectively. G. Metzgeroth and J. Hastka: Diagnostic work-up of iron deficiency 393

ters of the iron-deficient erythropoiesis indicate whether the ZPP concentrations are mostly )100 mmol/mol the ID in a given patient is clinically relevant or not. The heme w10, 11x. presence of iron-deficient erythropoiesis used to be eval- In assessing above-normal ZPP values, one must be uated either directly by counting the sideroblasts in a aware that increased ZPP concentration is not caused bone marrow smear or indirectly by determining the exclusively by real ID, but also by derangements of iron transferrin saturation. In recent years, new ‘‘stage-II- metabolism in chronic inflammatory and neoplastic dis- tests’’ have been established: soluble transferrin recep- eases, in lead poisoning and also in myelodysplastic dis- tor, hypochromic erythrocytes, reticulocyte hemoglobin, orders w14, 15, 19–21x. In contrast to cases with real ID, and zinc protoporphyrin. Hemoglobin is the defining ZPP values in myelodysplastic syndromes amount to parameter for stage III, the iron deficiency anemia. The -80 mmol/mol heme even in the most severe cases, but red cell indices MCH and MCV do not usually become are usually -60 mmol/mol heme w20x. Disturbances of pathological until the iron deficiency anemia is more iron metabolism in chronic disorders are usually associ- advanced w10x. ated with ZPP -80 mmol/mol heme; ZPP exceeds Iron test studies are mostly understood as a cham- 100 mmol/mol heme only in severe cases w19x. Clinically pionship to find the best iron test. Figure 1 shows that a relevant lead intoxication, on the other hand, is associ- simple correlation between the various markers is not ated with very high ZPP, mostly )1000 mmol/mol heme. appropriate for assessing their diagnostic value and that Acute inflammatory diseases do not influence the ZPP the sensitivity of an iron parameter cannot be defined measurement, as they do not induce an iron-deficient without taking into account the different stages of ID. erythropoiesis. Depending on the composition of the investigated group, In the mid 1980s, a new ELISA-based ‘‘iron test’’ was the correlation shows considerable variation if the com- introduced – the soluble transferrin receptor (sTfR) pared tests do not detect the same stage of ID. State- w22–30x. It was shown that the sTfR-concentration not ments such as ‘‘MCV detects ID in 50% of the cases’’ only depends on the erythropoietic activity, but also on are misleading, because this marker only works in stage- the iron metabolism. Elevated sTfR-values were meas- III-patients and shows no conclusive changes in the first ured in patients suffering from hematological disorders two stages of ID. Thus, the ‘‘sensitivity’’ of MCV obtained with increased erythropoiesis like , thal- without ID staging primarily depends on the composition assemia, spherocytosis, or polycythemia vera w22, 24, of the group of patients examined w10x. 26, 32x, but also in patients with ID, when the stage of the iron-deficient erythropoiesis was reached w24, 26, 29–31x. In individuals with simple storage iron depletion, Newer iron tests sTfR levels are normal w25x. At present, the widespread clinical use and understanding of this parameter is In recent years, there have been some new iron tests impaired due to a lack of reference material and stan- established which are of practical interest in the assess- dardized methods. Consequently, the individual sTfR ment of ID. All of these tests are sensitive in the second assays have their own reference ranges. The upper limit stage of iron deficiency, as they directly mirror the iron of the sTfR reference range is 1.75 mg/l with the Dade support to the erythropoiesis. Behring’s Latex-test, 3.05 mg/l with the Quantikine- The oldest of these parameters is zinc protoporphyrin sTfR-Immunoassay produced by R&D Systems, and (ZPP) w10–19x. ZPP is produced instead of heme when 5.0 mg/l with the Roche Diagnostics Tinaquant-assay. the iron support to the erythropoiesis becomes insuffi- The main advantage of sTfR is the differentiation cient and zinc instead of iron is incorporated into proto- between real ID and disturbances of iron metabolism. porphyrin IX. According to its fluorescence properties, While iron deficiency anemia is associated with increased ZPP can be measured fluorometrically. The development sTfR, the values are mostly normal in anemia of chronic of a front-face hematofluorometer, which allows rapid disorders (ACD) w28x. and inexpensive determination of the erythrocyte ZPP/ To increase the sensitivity and specificity of the sTfR- heme ratio, made the routine use of this parameter pos- measurement, the formation of the so-called TfR-F Index sible w16x. In storage iron depletion, the ZPP values are was proposed w33–36x. The TfR-F Index is the ratio of still within the reference range, which is F40 mmol/mol the serum concentration of soluble transferrin receptor heme for men, women, and children of all ages w18x. With wmg/lx and the logarithm of the serum ferritin value wmg/ the onset of iron-deficient erythropoiesis, there is a con- lx: sTfR/log ferritin. This parameter combines the advan- tinuous increase in the erythrocytic ZPP concentration. tages, but also the problems of the two individual iron High ZPP values are not only diagnostic for iron-deficient tests ferritin and sTfR. In individuals with iron deficiency, erythropoiesis, but also show its severity and clinical rel- the TfR-F Index is raised. The parameter appears to func- evance w10, 11, 17x. Values of 40–60 mmol/mol heme are tion not only in healthy individuals, but also permits the associated with mild iron-deficient erythropoiesis with no diagnosis of ID in patients with chronic diseases. When clinical symptoms or anemia. Clinical symptoms and evaluating the TfR-F Index, it is important to remember anemia usually first appear when ZPP is )80 mmol/mol that this parameter also depends on the erythropoietic heme. In patients with severe ID anemia (Hb-100 g/l), activity. Thus, raised values are also found in diseases 394 G. Metzgeroth and J. Hastka: Diagnostic work-up of iron deficiency

is )10%. The European Best Practice Guidelines for the management of anemia in patients with chronic renal failure recommend Hypo )10% as the most accurate marker for ID in hemodialysis patients treated with recombinant erythropoietin (rHuEPO) w43x. Other investigators, however, criticized the relative insensitivity of Hypo in rHuEPO-treated patients to detect rapid changes in iron status because of the long life-span of mature erythrocytes. Consequently, research activity focused on the reticulocytes, the newly produced eryth- rocytes which circulate in the bloodstream for only 1–2 days and signal the marrow erythropoiesis and hemoglobin production 3–4 days after the iron was incor- porated into protoporphyrin IX and hemoglobin was pro- Figure 2 Evaluation of the iron metabolism with the TfR-F duced. Thus, more advanced flow cytometers were Index and reticulocyte hemoglobin (CHr) as proposed by Tho- developed which are able to perform a selective evalu- masetal.w37x. The TfR-F Index is assay-dependent. With the Dade Behring’s sTfR-assay, TfR-F Index )1.5 points to iron ation of the reticulocytes by measuring the volume and deficiency. Decreased CHr indicates iron-deficient erythropoie- the hemoglobin content of each individual reticulocyte sis. In contrast to the above used threshold of -28 pg, the cur- (CHr). By quantifying the reticulocytes fractions in the rently accepted CHr cut-off value is -26 pg. low-, middle- and high-fluorescence intensity regions, it is even possible to assess the maturation of the reticu- with increased erythropoiesis. The main drawback for the locytes and to identify the most immature reticulocytes, diagnostic use of the TfR-F Index, however, is the lack which contain more RNA and are therefore localized in of uniform standards. The reference ranges depend on the high-fluorescence intensity region (HFR) w44, 45x. the sTfR assay used. With the Roche Diagnostics Tina- Reticulocyte measurements allow a snapshot of the quant-sTfR-assay, the reference range is 0.9–3.4 for men iron available for erythropoiesis by measuring only the and 0.9–3.7 for women. erythrocyte population just produced. In several studies Combining the TfR-F Index with other parameters, it is performed on hemodialysis patients, CHr proved to be a possible to distinguish between different forms of anemia reliable marker of iron-deficient erythropoiesis. It seems in ID, , or derangements of iron metabolism. to be even more sensitive than the determination of the Useful complements of the TfR-F Index proved to be the hypochromic erythrocytes in this patient group w40, inflammation marker CRP (C-reactive protein) and the 46–48x. The proposed CHr-threshold varies from 23 to reticulocyte hemoglobin content CHr (Figure 2) w37x. 29 pg, the currently accepted CHr cut-off, which indi- Using flow cytometry, cell volume and hemoglobin cates an iron-deficient erythropoiesis, is -26 pg w39, 48, concentration can be measured in individual red cells and 49x. Currently, one cannot judge the suitability of Hypo in reticulocytes. Based on these techniques, two para- and CHr as routine screening tests of ID, because these meters have proved to be particularly useful in identifying parameters have not been validated yet in the daily clin- iron-deficient erythropoiesis: the percentage of hypo- ical routine. Thus, the possible sources of error are not chromic erythrocytes (Hypo) and the content of reticu- known. Recently published studies show that Hypo and locyte hemoglobin (CHr). These new parameters seem CHr are affected by inflammation, hemoglobinopathies, very promising, but their diagnostic value for clinical rou- increased erythropoiesis, and diseases causing an tine is not yet clear, since studies addressing their diag- increase in the erythrocyte size, e.g. B12-deficiency or nostic accuracy in detecting ID are few. Furthermore, hemolytic anemias w40, 41, 50–53x. At present, CHr is these studies were mainly performed on hemodialysis accepted as the best predictor of response to iron sup- patients, and data derived from a large, well ‘‘iron-char- plementation therapy, being normalized within 48 hours acterized’’ population do not exist. after a single infusion of 500–1000 mg Fe w48x. In healthy individuals without iron deficiency, the per- centage of hypochromic erythrocytes (hemoglobin con- tent -28 pg) is less than 2.5%. If there is insufficient iron Relative iron deficiency in anemia of chronic available for erythropoiesis, the proportion of hypochro- disorders mic erythrocytes increases. Which cut-off value should be used in clinical practice as diagnostic for iron-deficient Patients with chronic inflammatory disorders often devel- erythropoiesis remains somewhat debatable, with values op anemia, associated with characteristic disturbances from 3.7 to 10% having been proposed w38–41x.In of iron metabolism and decreased iron supply for the hemodialysis patients, the best combination of sensitivity erythropoiesis w54–56x. In contrast to real ID, the anemia and specificity is possibly achieved at a Hypo-threshold of chronic disorders (ACD) is caused by impaired iron )6% w42x. However, the currently accepted cut-off, bioavailability, whereas the iron stores are repleted. The which is considered proof of iron-deficient erythropoiesis, typical findings of this relative iron deficiency are sub- G. Metzgeroth and J. Hastka: Diagnostic work-up of iron deficiency 395

Figure 3 Assessment of iron stores by ferritin and the inflammatory markers ESR (erythrocyte sedimentation rate) or CRP (C-reactive protein) w8x. normal values of serum iron concentration, low transfer- detects, but also quantifies the ACD-associated distur- rin saturation, normal or increased serum ferritin, an bances. By recording the impaired bioavailability quan- increased amount of bone marrow hemosiderin, and a titatively, chronic diseases and their response to therapy decreased number of sideroblasts. The hypoferremia has can be monitored reliably and inexpensively, without been suggested to represent a nonspecific defense bone marrow examination w19x. mechanism. According to this mechanism, the iron, which is necessary for all cells, is not available to the invading microorganisms, tumor cells, or other patholog- Screening for iron deficiency ical cells which cause the chronic inflammatory process. As a consequence of the impaired iron supply to the What screening strategy and sequence of tests to use bone marrow, the erythropoiesis becomes iron-deficient when the iron status is unknown depends on the individ- and the synthesis of hemoglobin is decreased. It takes ual patient, the clinical conditions, and especially the lab- about one to two months of sustained inflammation for oratory equipment available. Most clinicians prefer to use anemia to develop. Therefore, acute inflammations do ferritin as the first-line test of iron status: it is not invasive, not cause anemia, despite being associated with low it is widely available, and, from a theoretical point of view serum iron and decreased transferrin saturation. (being a stage-I-marker), it is more sensitive than the oth- Using the conventional laboratory tests, distinguishing er laboratory tests. However, that ferritin has a higher between real ID and ACD is difficult. Serum iron concen- sensitivity is true only for healthy individuals without tration, ferritin, and transferrin saturation are of limited inflammatory diseases. For patients with acute-phase value, being considerably influenced by acute phase response or with neoplastic and hepatic disorders, ferritin response. The ideal parameter for this purpose proved is frequently not useful, even in cases of severe ID ane- to be the sTfR measurement. In contrast to ID which is mia. Especially in patients with ID resulting from occult associated with elevated sTfR values, the sTfR concen- bleeding caused by neoplasia – the only patients in tration is normal in ACD w28, 29x. However, in diagnosing whom early detection of decreased body iron stores is ID and ACD using sTfR, one should always bear in mind of vital interest – the ferritin values are false normal. If that this test is also influenced by the erythropoietic ferritin is used as a screening parameter, it is important activity. to make sure (clinically, erythrocyte sedimentation rate, The diagnosis of ACD requires an extensive diagnostic CRP) that there is no significant inflammation present work-up, including bone marrow examination and count- (Figure 3). Values -12 mg/l always confirm ID w8x. The ing the sideroblasts. By determination of Hypo, CHr or practical significance of ferritin as a screening test for ID ZPP, which all supervise the hemoglobinization of the is further restricted by its costs which are too high – erythrocytes, a rapid detection of the impaired iron bio- especially for the use in developing countries. availability in ACD is possible w19, 38, 40x. Out of these Given the observed diagnostic uncertainty of ferritin in tests, we prefer the determination of ZPP which not only hospitalized patients, the use of one of the new stage-II- 396 G. Metzgeroth and J. Hastka: Diagnostic work-up of iron deficiency

Should ZPP be used supplementary to ferritin, or as a first-line test instead of ferritin, if appropriate laboratory equipment is available? We prefer the simultaneous measurement of ferritin and ZPP in patients at risk, such as young and pregnant women, blood donors, children, or persons with malabsorption – e.g. patients who fre- quently develop ID and for whom ferritin determinations usually deliver correct results, as they are not disturbed by inflammatory diseases. We also prefer this strategy in hematologic patients, particularly in those with anemia. The simultaneous determination of ferritin and ZPP is of practical interest in hemochromatosis patients treated by venesection as well, in order to guarantee iron depletion Figure 4 Supervising the final stage of the heme-production, and prevent the development of iron-deficient erythro- zinc protoporphyrin (ZPP) controls the whole iron metabolism. poiesis in such patients. Elevated levels are observed in real iron deficiency and also in derangements of iron metabolism in ACD (anemia of chronic dis- In our daily clinical routine, however, we only use ZPP orders), lead poisoning or myelodysplastic syndromes w19x. as the first-line test and determine ferritin and sTfR in a second step, if necessary, to distinguish between real ID and sideroachrestic disorders (Figure 5). Although this tests seems to be a suitable alternative. Even though strategy does not allow to detect cases with iron deple- they detect iron deficiency later than the storage indi- tion, we find it justifiable because, except for hemochro- cator ferritin, the tests of the iron-deficient erythropoiesis matosis, whether the iron stores are full or half full is of are often more sensitive ID-indicators, especially in little clinical relevance, and, except for neoplasia (where patients with inflammatory or liver diseases. ferritin values frequently do not work well), early detection As a screening test should not only be reliable but also of decreased body iron stores is not of vital interest. What cheap, the antibody-based sTfR-tests are not suitable for is relevant is whether there is an insufficient iron supply this purpose. In our experience, of the remaining stage- to the cells – especially to the erythropoietic marrow, II-tests, ZPP is the best alternative. It is true that ZPP is because only at that moment ID becomes a disease. And increased not only in real ID, but in all derangements of just this moment is reliably detected by ZPP. iron metabolism, supervising the final step of the heme Despite the described advantages of ZPP in diagnos- production (Figure 4). This reduces its diagnostic impor- ing and managing ID, we nonetheless emphasize that our tance as a screening parameter of ID. But at the same recommendation of ZPP as the only first-line test is main- time, it enables ZPP to serve as a screening parameter ly for financial reasons. If diagnostic costs do not matter, for the whole iron metabolism, which is clinically much we recommend measuring both ferritin and ZPP,together more important. In clinical practice, physicians want to with hemoglobin (Figure 6). This test combination has know whether or not a given patient has clinically rele- proved to be particularly useful for evaluating a person’s vant ID and whether the symptoms or pathological find- iron metabolism and, if iron deficiency is found, for deter- ings (e.g. anemia) are caused by ID or another mining its stage w11x. The determination of hypochromic derangement of iron metabolism. This very complex erythrocytes or CHr can be used as stage-II-tests instead question, which the physician encounters in each patient, of ZPP. can reliably and inexpensively be answered by determin- In rHuEPO-treated hemodialysis patients, the diagno- ing just one laboratory marker: ZPP always detects an sis of ID remains a challenge even with the new iron iron-deficient erythropoiesis, the only possible source of tests. The best currently available parameter to identify error is the rare congenital erythropoietic porphyria. Nor- patients who will improve their response to erythropoietin mal or only slightly increased ZPP values always exclude after intravenous iron seems to be Hypo )10% and CHr clinically relevant ID and show the physician that, what- -26 pg w43, 48x. Useful for clinical practice is also the ever the pathological findings for the given patient may pragmatic solution of this problem by CHr-based early be, they are not caused by ID or a disturbed iron metab- assessment of the response to intravenous iron therapy, olism. High ZPP values indicate an iron-deficient eryth- as proposed by Fishbane and others w45, 48x. ropoiesis, which can be caused by real ID or by disturbed Another diagnostic problem remains the identification iron metabolism in ACD, lead poisoning, or myelodys- of ID in ACD-patients. The only laboratory test which can plastic syndromes. Thus, when ZPP is above normal be used for this purpose is the determination of sTfR. range, the physician must ascertain the differential diag- The sTfR-measurement effectively identifies ID, even in nosis by clinical findings and further laboratory tests. This the presence of ACD. An increase in the sTfR concentra- clinically important statement makes – in our opinion – tion in ACD-patients indicates real ID, if increased eryth- the ZPP determination superior to the determination of ropoiesis is excluded w33x. The detection of ID in the hypochrome erythrocytes and the reticulocyte ACD-patients, however, is not really important from the hemoglobin. clinical point of view. It is important, though, to identify G. Metzgeroth and J. Hastka: Diagnostic work-up of iron deficiency 397

Figure 5 Proper sequence of tests if iron status is unknown. Elevated zinc protoporphyrin (ZPP) levels diagnose and quantify iron- deficient erythropoiesis, which can be addressed by ferritin and/or soluble transferrin receptor (sTfR) in real ID and derangements of iron metabolism (Hb: hemoglobin, n: normal). those patients who will clinically improve by iron supple- occurrence in ID and show no changes in the first two mentation. As they recognize the iron supplementation stages of iron deficiency. The iron tolerance test is not a effect early, CHr or HFR are the most interesting para- suitable screening test for clinical practice either. It meters in this clinical situation. By using these tests, both requires two venipunctures, oral administration of iron goals, the improvement of the general performance stat- and is further affected by gastrointestinal disorders. us or anemia in a given ACD-patient and the avoidance In conclusion, the ‘‘best’’ parameter of ID does not of the impairment of the underlying chronic inflammatory exist. ID is a dynamic process and cannot be defined by disease by over-treatment with iron, can be achieved w45, one test only. To understand the iron status in a given 48x. patient, one must know which stage of ID the individual Serum iron, iron saturation of transferrin and the mean tests detect and by which factors they are disturbed. By red cell volume MCV are still widely used as a screening detecting different stages, the various markers efficiently parameter for ID. However, it must be pointed out that complement one another to characterize the iron status these tests are not suitable for this purpose. Serum iron in an individual case. and transferrin saturation are influenced by the acute- phase response and further affected by the well-known circadian problem. The red cell indices are a rather late Acknowledgments

The authors thank Dr. B. Schultheis and P. Duisberg, Ph.D. for critically reading the manuscript.

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