Haema 2019; 10(1):2-14
MD, Department of Laboratory Medicine, Boston Children’s Hospital Harvard Medical School, Boston, U.S.A.
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Iron deficiency anemia is the most common hematological disorder worldwide and a considerable source of morbidity: it impairs cognitive development in children and substantially affects quality of life in adults. Diagnostic approaches for biochemical iron deficiency in the absence of anemia and iron deficiency anemia share a reliance on both biochemical and haematological markers. While the diagnosis of iron deficiency anemia is straightforward in uncomplicated cases, it is more challenging in the presence of other diseases, or of chronic inflammation/infection, or when erythropoietic stimulating agents are used. In these cases, novel parameters like reticulocyte haemoglobin content and serum hepcidin are useful both for diagnosis and for prediction of response to iron replacement therapy. This review examines a variety of hematological and biochemical biomarkers currently in use or considered for use in the diagnosis and treatment of iron deficient states.
Key words: Iron deficiency, Anemia, Diagnosis, Hematological, Biochemical biomarkers
Corresponding author: Carlo Brugnara, Department of Laboratory Medicine, Boston Children’s Hospital, Harvard Medical School, 300 Longwood Avenue, Bader 760, Boston, MA 02115 U.S.A., Tel.: (617) 355-6347, Fax: (617) 730-0383, e-mail: firstname.lastname@example.org
Assessment of iron status is based on a combination of hematologic and biochemical markers, geared toward identifying iron deficient erythropoiesis and/or unusually low iron stores. Iron deficiency progresses through 3 states, which have their origin in a discrepancy between iron requirements for erythropoiesis and iron availability from body stores and diet/gastrointestinal absorption.*1 The 3 stages are:
Storage iron depletion: the iron stores are depleted below the normal range but there are no significant hematological changes. Biochemical markers are useful at this early stage.2,3
Iron deficiency with no anemia: biochemical and other hematological markers indicate that iron availability to the erythroid marrow is limited, but Hb still remains within the normal range or begins to decrease slightly form baseline steady state values.4
Iron deficiency anemia: anemia is present, in conjunction with classical biochemical and hematological signs of iron deficiency.5
Diagnosis of the third stage, iron deficiency anemia, is straightforward in most cases, based on the patient’s clinical history (poor iron intake and/or blood loss) and the presence of hematologic hallmarks of iron deficient erythropoiesis (reduced Hb, MCV and MCH, inappropriate reticulocyte response for the degree of anemia and low reticulocyte Hb content) in conjunction with biochemical signs of iron stores depletion (low serum ferritin, serum iron and transferrin saturation).
Prompt identification of the earlier stages of this condition can be challenging, especially when other conditions may alter the signals provided by the “traditional” diagnostic criteria, or in infants or toddlers, where progression to iron deficiency anemia may be missed if proper tests are not used. When iron may be present in the body stores, but is not made available for erythropoiesis, diagnosis is especially difficult: most frequently this is due to the presence of a chronic inflammatory state or other pathologies, often combined into the single category of anemia of chronic disease (ACD). In addition, iron supply that meets the baseline requirements of the erythron may not meet the demands of enhanced erythropoiesis. This condition has been defined as iron restricted erythropoiesis or functional iron deficiency.6 Functional iron deficiency has emerged as an important factor limiting the hematological response to recombinant human erythropoietin (r-HuEPO) or other ESAs.4,7
In rare, challenging cases, diagnosis depends on demonstrating responsiveness to IV iron.In the majority of uncomplicated cases, changes in laboratory parameters can be easily identified in individual patients over time.Markers with high biological/analytical variability are obviously less reliable in this setting, and diagnosis must be based on markers with lower variability.Next follows a discussion of individual biochemical and hematologic markers and how they can be properly integrated for optimal disease identification and management.
A.1. Plasma/Serum ferritin
Intracellular ferritin is a molecular complex formed by the interaction of twenty four H (heavy/heart, 21 kd) and L (light/liver, 19.7 kd) subunits assembled in a spherical form, with thousands of iron atoms stored in its central core. Ferritin in serum is either secreted from iron storage sites or is liberated as a consequence of cell death/lysis; it consists almost exclusively of the L-chain subunits, which are also glycosylated. Serum or plasma ferritin values are measured by immunoassays, with a normal range varying between 20 and 300 ng/mL (or µg/L). The assay is insensitive to mild hemolysis; however, complete hemolysis of the sample may result in a marked increase in serum ferritin due to the release of ferritin contained in the erythrocyte. Serum ferritin has no circadian variation and it is quite stable in the same subject over time (low intra-individual variability).Ferritin values, on the other hand, change with age: they are higher at birth, decrease during childhood and increase with age in adults. It is of interest that from screening in large populations it was shown that in the absence of known mutations associated with hemochromatosis, serum ferritin appear to be higher in Asians and Pacific Islanders both males and females.8 Several patients have been described with hyperferritinemia in the absence of iron overload, most likely due to mutations not involving the ferritin gene.9
Serum ferritin is a reliable and useful marker of iron stores, in the absence of acute or chronic inflammation (serum C-reactive protein < 0.5mg/dL). A value below 12 mg/L is typically diagnostic of complete depletion of iron stores. The sensitivity of such a threshold has been questioned, however, and there is evidence supporting the notion that a threshold of 30 ng/mL would yield better sensitivity with unchanged specificity (positive predictive value of 92% versus 75% for the lower threshold).10
In rare conditions, hypothyroidism and vitamin C deficiency may lower serum ferritin levels independently of iron depletion.On the other hand, hyperthyroidism, liver disease (especially due to hepatitis C virus), alcohol consumption and oral contraceptives increase serum ferritin values independently of iron status.11 In the case of damage of ferritin-rich tissues by acute or chronic processes, serum ferritin increases as a direct reflection of cellular damage. This can be seen in liver disease, leukemias, pancreatic and bronchial cancer, and neuroblastomas. While serum ferritin is normally glycosylated, ferritin released from damaged tissues are not. Serum ferritin is also an acute-phase reactant, which becomes elevated in the presence of acute or chronic inflammatory states. Furthermore in the hyperferritinemia-cataract syndrome, mutations in the iron responsive element of the L-ferritin gene result in elevated serum ferritin values in the absence of iron overload.12
In children, serum ferritin is not helpful in identifying iron deficient states and/or the development of iron deficient erythropoiesis.13,14 In the anemia of chronic kidney disease (CKD), serum ferritin is also not a reliable marker of iron stores, since it is elevated due to the concomitant chronic inflammatory state. Ferritin values > 200 µg/L are usually recommended for dialysis patients,5 and values of 100 µg/L are considered the lower limit of normal for CKD patients.16 The sensitivity for ruling out iron deficiency was determined to be 90% for a ferritin cut-off of 300 µg/L and 100% for a 500 µg/L cut-off in CKD patients.17
A.2. Serum iron, transferrin, and transferrin saturation
Iron circulates in plasma as transferrin-bound iron, which allows the transport of iron to cell types endowed with specific cell-surface receptors. Most of the assays used to measure serum iron are spectrophotometric methods based on the complexing of the iron with a chromogenic substrate. Iron is measured after its release from transferrin by acid pH treatment. These methods require the use of either serum or heparinized plasma; samples collected in EDTA are unsuitable for serum iron determination. Less commonly, iron can be directly measured with atomic absorption.It’s important to note that serum iron values reveal diurnal variations and respond to dietary intake, resulting in higher serum iron values early in the day or following the ingestion of iron-rich food or dietary supplements. Measurements are best performed in the morning, after > 8 hrs. fasting, to avoid these transient increases in serum iron due to food or multivitamin supplements containing iron.Other situations can influence the measurements as well: serum iron decreases with infection and inflammation;5 hemolysis may affect some of the biochemical methods used to measure iron and show falsely elevated values;18 some serum iron assays perform poorly in dialysis patients.19 A potential reason for the poor performance of some assays may be the release of iron from ferritin, which significantly affects serum iron measurements when ferritin is above 1200 ng/mL.
As its name denotes, transferrin is the carrier for almost all iron circulating in plasma. As an 80-kd glycoprotein, it’s capable of binding iron in two homologous domains in the N- and C- terminals of the molecule, and is produced and secreted mostly by the liver. Under normal conditions, transferrin is only 30% saturated by iron.
.Total iron binding capacity (TIBC) is no longer directly measured, but is estimated on a molar basis as being two-fold the concentration of transferrin, which is directly measured with immunoassays.
Transferrin synthesis and values in plasma are increased in cases of iron deficiency, and augmented iron needs, such as those of pregnancy Serum transferrin can be found elevated with the use of oral contraceptives and decreased with inflammation/infection. It is also increased in the presence of parenchymal liver damage.
Several genetic polymorphisms have been described in the transferrin gene, with Caucasians carrying almost uniformly the C allele, and West Africans carrying most frequently the D allele. A study in Zimbabwe, for example,showed that 90% of subjects had the TF CC genotype, and 10% had the TF CD genotype.20 In normal males with TF CD heterozygotes, significantly lower values for serum iron, TIBC, and TF saturation were observed, suggesting that these individuals may be somehow protected against iron overload.20
Due to an anti-transferrin immunoglobulin, elevated serum iron, transferrin, and monoclonal gammopathy of undetermined significance are seen in transferrin-immune complex disease, a rare and acquired disease.21
Bainton and Finch showed that a transferrin saturation below 16% is insufficient to meet the functional requirements of the erythroid marrow.24 The conditions that may alter either serum iron or transferrin values to this degree have been listed above: de-coupled values of iron and transferrin from iron metabolism substantially limit the value of these parameters in assessing the adequacy of iron supply to the marrow.
A low baseline serum iron has been shown to be an independent predictor of increased mortality and hospitalization in dialysis patients,25 whereas increased TSAT was associated with lower mortality.26 Fishbane et al have examined 34,782 NHANES III patients for whom TSAT and ferritin data were available:27 more than 50% of the patients had values below either the ferritin (100 ng/mL) or TSAT (20%) thresholds. Additionally, they found that,the prevalence of iron deficiency increased with deteriorating renal function in men but not in women.
A.3. Serum values of soluble Transferrin Receptor (sTfR)
The soluble transferring receptor (sTfR)is particularly useful in distinguishing the anemia of chronic disease, which has normal or near normal sTfR values from iron deficiency anemia (elevated sTfR).10,28-31
sTfR is a truncated form of TfR, consisting of the extracellular transferrin-binding domain of the molecule, which is cleaved from the rest of the molecule by a specific protease between aa 100 and 101. sTfR loss from the maturing erythroid cells is an indirect expression of the activity of the erythron and is not directly affected by inflammation. Independently of iron status, sTfR will increase in hyperproliferative anemias, with the use of ESAs, and decrease in hypoproliferative states, due to either EPO deficiency (chronic renal failure) or marrow aplasia.31 Independently of erythroid marrow activity, sTfR values are abnormally elevated in iron deficient states, reflecting a upregulatory response driven by the limited availability of iron to the erythron;32 conversely, they are depressed in the presence of iron overload.33
A variety of methods have been employed to measure sTfR, all based on immunoassays and with one method suitable for both whole blood and serum/plasma assays.34 Unfortunately, there is not yet a single reference standard, and different methods generate values with different units and normal ranges, as highlighted in a recent commutability study.35 A whole blood assay is available for sTfR, but its utilization has been limited.34
The effort to measure sTfR values seems promising for some cases. sTfR values <6 mg/L (NV 3.8-8.5) were shown to be predictive of response to EPO therapy in anemic dialysis patients.36 Because increased erythropoiesis increases sTfR, however, this parameter does not detect functional iron deficiency. Other studies have failed to show a predictive value of sTfR in CKD anemia management.37,38 A decline in sTfR may reflect increases in iron availability, as seen when intravenous ascorbic acid is used to mobilize iron stores in dialysis patients.39
Transferrin receptor type 2 (TfR2) is a homologue of the classic type-1 TfR (discussed in section A.3) which exhibits lower affinity for transferrin, and together with TfR1 plays a crucial role in iron metabolism.22 mutations in the TfR2 gene
Are found in recessive hemochromatosis type 3.23
A.4. sTfR-Ferritin (sTfR-F) index
Efforts to measure sTfR were advanced by Skikne et al., whose research demonstrated that the log ratio of sTfR to ferritin correlates quite well with iron stores.32 This approach has yielded precise estimates of body iron in large epidemiological studies.1 The log ratio of sTfR to ferritin identified more children and fewer females as iron deficient compared with ferritin alone.40 A recent study has shown that sTfR/ferritin and ferritin are equally satisfactory in quantifying iron stores in population studies, with ferritin having a lower cost and a better availability of reference material.41,42 There is limited evidence to support the use of this or similar indexes, however, in dialysis patients,43,44 a slightly different ratio (sTfR/log ferritin) has also been used to identify iron deficient states,30,45,46 For the sTfR to log ferritin ratio, cut-off values of 1.8 (90% C.I. 1.55 to 1.99) and 2.2 (90% C.I. 1.81 to 2.53) have been proposed to identify iron storage depletion and iron deficient erythropoiesis, respectively.45 ICut-off values of 1.5 and 0.8 have been proposed for the sTfR/log ferritin for the diagnosis of iron depletion in the absence and presence of inflammation, respectively.46 However, since these cut-offs are sTfR assay-specific they can be as high as 3.8 and 2.0, respectively, when a different assay is used.
A.5. Urinary and serum Hepcidin values
The identification of hepcidin as a key regulator of iron homeostasis has added a new biomarker for the diagnosis of both iron overload and iron deficient states and thus serum hepcidin determination may help shed light in patients’ iron status.47 Hepcidin is a 25 aa peptide produced and secreted by the liver, which modulates iron availability by causing internalization and degradation of ferroportin, a key iron exporter, which is essential for both iron absorption in the duodenum and recycling of iron/iron efflux by macrophages. Hepcidin is a negative regulator of iron absorption and mobilization: high hepcidin values turn off both duodenal iron absorption and release of iron from macrophages; low hepcidin values promote iron absorption and heme iron recycling/iron mobilization from macrophages. Thus, hepcidin values are expected to be high in iron overload states and diminished in iron deficient states (Table 1). In normal subjects, an oral iron loading produces a measurable increase in hepcidin values.3 while EPO therapy produces a reduction in serum hepcidin values, which may be indicative of EPO-responsiveness. Early studies focused on quantifying hepcidin in the urine or serum with mass spectrometry detection.
An immunoassay for serum human hepcidin (lower limit of detection: 5ng/ml) produced normal range values of 29-254 ng/ml in men and 16-288 ng/ml in women.3 A new sandwich ELISA assay has brought the limit of quantification for hepcidin down to 10 pg/mL.48 The assay sensitivity allows for the detection of changes in serum hepcidin due to diurnal variation and in response to oral iron. Measurements of prohepcidin, the precursors of the biologically active 25 aa hepcidin, are poorly correlated with hepcidin and are unresponsive to known hepcidin regulators.49 In iron refractory iron deficiency anemia (IRIDA), an increased serum Hepcidin is accompanied by low serum ferritin values, a unique combination of high diagnostic significance.50,51 Studies have found that elevated serum hepcidin values may predict poor therapeutic response to oral iron51,52 and to ESA therapy.53,54
A.6. Erythroferrone (ERFE), Growth differentiation factor 15 (GDF15) and Transmembrane serine protease 6 (TMPRSS6)
The erythroid regulator erythroferrone (ERFE) has been shown to be a key mediator of the EPO effects on hepcidin production.55 With increased erythropoietic activity, ERFE down-regulates hepcidin production, potentially explaining the iron-overload characteristically associated with ineffective erythropoiesis.56 ERFE mobilizes iron stores, with Erfe-deficient mice exhibiting a stunted anemia recovery after hemorrhage or in states of inflammation.57
The erythroid form of TRF2 has emerged as akey component of the iron-sensing mechanisms involved in the feedback loop between iron availability and erythropoiesis, possibly as a modulator of the EPO sensitivity of erythroblasts.58,59
The expression of GDF15 is upregulated by hypoxia and iron depletion. GDF15 in turn down-modulates hepcidin values, promoting iron absorption and mobilization. It may play an important role in the iron overload associated with expansion of the erythropoietic marrow, since its serum concentrations are extremely elevated in severe thalassemias and congenital dyserythropoietic anemia type I.60,61
TMPRSS6 is cell membrane sensor of decreased iron availability, which suppresses hepcidin production and allows increased intestinal absorption of iron.62 Mutations in TMPRSS6 have been associated with IRIDA,63 and a TMPRSS6 allele associated with lower serum iron and Hb values has been identified in population studies.64
B. HEMATOLOGICAL MARKERS
B.1. Hb, CBC, and red cell indices
Hb is a late marker of iron deficiency, which develops in the three stages model but culminates with the appearance of anemia.WHO guidelines have selected different Hb thresholds for anemia identification based on age and other conditions:65 for children, values of 11.0, 11.5 and 12.0 g/dL have been established based on age (6 months to under 5 years, 5 to 12 years, and 12 to under 15 years, respectively). For women and men over age 15, Hb thresholds are 12.0 and 13.0 g/Dl, respectively, while a value of 11.0 g/dL was selected for pregnant women.
The red cell indices may help identify the presence of iron deficiency erythropoiesis at a much earlier stage than Hb: patients may still have normal Hb, but present MCV and MCH which are slightly reduced or at the lower end of normal values, with increased RDW. RDW can be noticeably elevated in iron deficiency anemia, at times > 20%, reflecting the marked anisocytosis seen with iron deficient erythropoiesis.A definite reduction of MCV can be appreciated in many cases, however, only after the Hb drops below 10-11 g/dL: changes in MCV and other cell indices develop over a long time, due to the low turnover of the erythrocyte population.66A clear relationship connects iron supply and erythrocyte volume, not only with cell volume decreasing when iron supply decreases, but also with cell volume increasing when both iron supply and marrow erythropoiesis are increased.6 Iron deficiency has no effect on WBC counts. PLT count is usually elevated above baseline values in iron deficiency and returns to normal with successful iron supplementation therapy. PLT counts are highest, at times above 1 million PLT/µL, in patients with iron deficiency and active bleeding. If blood is spun in a microhematocrit centrifuge, the plasma of patients with iron deficiency anemia will appear paler than usual, while it is usually much darker in thalassemia trait.
B.2. Hypochromic red cells
Iron deficient erythropoiesis causes an increase in hypochromic erythrocytes, which can be measured as the % of erythrocytes with MCHC lower than 28 g/dL (% HYPO), by certain hematology analyzers (i.e. Siemens Medical Solutions, Tarrytown, NY).67,68 Similar parameters (Low Hemoglobin density, LHD% and %Hypo-He) are available in Beckman-Coulter and Sysmex instruments, respectively, although experience with them is more limited.69-72 This parameter is best measured in samples less than 4 hrs. old if kept at room temperature, since storage leads to a progressive increase in hypochromic red cells because of the concomitant increase in MCV and reduction in MCHC. Refrigeration minimizes storage-induced volume changes: hypochromic red cells can be measured in samples refrigerated for up to 24 hrs. Hypochromic red cells increase with reticulocytosis, a reflection of the fact that reticulocytes have lower MCHC than mature red cells.4
A classic study by Macdougall et al.in dialyzed patients revealed that functional iron deficiency induced by EPO treatment and the response to IV iron could be detected by changes in %HYPO.73 Indeed, %HYPO appears to be a sensitive and early indicator of iron deficiency in a variety of studies.74-79 Hypochromic cells were also shown to increase in normal subjects undergoing an intensive autologous donation regimen with r-HuEPO support, indicating the appearance of iron restricted erythropoiesis despite normal baseline iron status and concomitant oral iron supplementation.80 In this study and in another, also involving normal subjects being treated with r-HuEPO,81 iron restricted erythropoiesis was associated with a marked decrease (~75%) in serum ferritin values, indicating depletion of the readily available iron pool.
Much of the information about the %HYPO parameter has been accumulated in dialysis patients, as some guidelines have incorporated this parameter into the management of iron and erythropoietin treatment. Significantly, a European study in dialysis patients found the %HYPO to be the only independent predictor of mortality among various other iron status parameters: mortality risk increased two-fold for values > 10% compared with values <5%.82 Patients with %HYPO > 6 are most likely to respond to IV iron therapy.16 The1999 European Best Practice Guidelines for anemia management were tested in a clinical study that recommended a %HYPO target of < 10%: this study prospectively increased the delivered dose of IV iron to 228 dialysis patients to achieve a % HYPO < 2.5% and a serum ferritin of 200-500 ng/mL.79 The median % HYPO decreased from 8% to 4%, median serum ferritin increased from 188 to 480 ng/mL, and median EPO dose decreased from 136 to 72 IU/kg/wk. Yet findings were mixed: this study showed the cost-effectiveness of a strategy aimed at reducing % HYPO values below 10%, although it resulted in serum ferritin values much greater than those recommended in guidelines for some of the patients. North American studies have so far failed to show usefulness of %HYPO in assessing iron availability in dialysis patients.83,84 The reasons for the discrepancy are not yet clear. Perhaps, shipping of samples to centralized US reference laboratories may affect the stability of the %HYPO parameter. With the diminished availability of Siemens ADVIA analyzers, the commutability of %HYPO-based studies with similar parameters generated by other hematology analyzers remains to be demonstrated. A recent study suggested a cut-off value of 0.9% for %Hypo-He in predicting iron deficiency and iron deficiency anemia.85 A new density-based cell fractionation method to quantify the presence of hypochromic red cells has shown promise as a tool to identify iron deficiency anemia in settings without access to advanced testing systems.86
B.3. Micro/Hypo ratioand the availability of analyzers
In the presence of a microcytic hypochromic anemia, the differential diagnosis includes iron deficiency and thalassemias, especially beta-thalassemia trait and two-gene alpha thalassemia trait.Other hemoglobinopathies with microcytosis, and congenital or acquired disorders of heme synthesis (sideroblastic anemias) should also be considered in the differential diagnosis.To aid this diagnosis, various CBC parameters have been suggested, including the Mentzer index (MCV/RBC), the Bessman index (RDW), the England and Fraser index [MCV- (5 x Hb)-RBC], the MCV/MCH ratio, the RDW/RBC ratio, and the Green and King formula [(MCV2 x RDW)/(Hb x 100).87,88 The most useful parameter in distinguishing iron deficiency from thalassemias is the micro/hypo RBC ratio, which is usually >1.0 in thalassemia, where microcytosis prevails, and < 1.0 in iron deficiency, where hypochromia is more pronounced.87 This parameter, however, is generated only by instruments marketed by Siemens and is not available on Coulter, Abbott, or Sysmex hematology analyzers.
B.4. Reticulocyte Hemoglobin content (CHr or RET-He)
Reticulocytes spend 18-36 hrs. in the circulatory system after being released from the marrow before maturing to erythrocytes. Determination of reticulocyte cellular indices and of Hb content in particular provides a real-time assessment of the functional state of the bone marrow.89,90 Measurements of reticulocyte Hb content,91 (CHr or RET-He, pg/cell) have been shown to identify iron deficient erythropoiesis in a variety of settings, which include pediatric and adult patients and patients treated with rHuEPO. Two studies in children have shown that CHr performs better than other traditional parameters in identifying both the presence and the future development of iron deficiency.13,14 CHr showed greater specificity and sensitivity in assessing iron status than traditional iron parameters, after exclusion of patients with thalassemias or macrocytosis (MCV > 100 fL).92 A reduction in CHr is the earliest indicator of functional iron deficiency: healthy subjects with normal iron stores produced a substantial fraction of hypochromic, low-CHr reticulocytes when treated with rHuEPO. Only subjects with baseline serum ferritin above 100 µg/L were able to produce normal reticulocytes.81 Intavenous iron suppresses the production of hypochromic reticulocytes that accompanies r-HuEPO therapy.93 In pregnant women at term, iron deficiency can be reliably identified based on either CHr and/or % HYPO.94
Several studies have shown the value of CHr in identifying iron deficiency in dialysis patients.83,84,95,96 100% sensitivity and ~70%-80% specificity were reported in one study,84 while other studies reported lower values.95,96 These initial studies led to additional large clinical trials in dialysis patients, to test the role of CHr in managing the dosing of IV iron and rHuEPO. A study by Fishbane et al97 randomized 157 patients to two different IV iron management strategies: one strategy was based on CHr, in which IV iron was started if CHr fell below 29 pg and one was based on traditional parameters, in which IV iron was started if ferritin fell below 100 ng/ml or TSAT below 20%. A significant reduction in exposure to IV iron was obtained in the CHr-based management, with no differences in weekly EPO dosing between the two groups.97 Tessitore et al78 compared the diagnostic power of a variety of hematological and biochemical markers in 125 dialysis patients to identify subjects with an hemoglobin response to IV iron. A combination of % HYPO >6% or CHr <29 pg showed the best diagnostic efficiency for iron deficiency (80%) based on Hb response to IV iron. Other studies have provided additional confirmation of the diagnostic value of CHr,98,99 although one study has questioned its superiority to TSAT,100 and only one study has shown that use of IV iron in patients with low CHr resulted in decreased weekly usage of r-HuEPO.101
Several studies have also validated reticulocyte Hb measurements (REF-He and Ret-Hb) generated by analyzers produced by Sysmex, which are now widely used worldwide.70,102,103-105 Reticulocyte Hemoglobin content is decreased in the presence of alpha or beta thalassemia, while it is increased after treatment with hydroxyurea or with folate or B12deficiency.
B.5. Erythrocyte ferritin
Very small amounts of ferritin are contained inside erythrocytes, in the form of two different ferritin species (alkaline and acid). They can be measured after complete lysis of the erythrocytes, and after careful removal of both plasma and contaminating WBCs, which contain approximately 1000 times more ferritin than erythrocytes. This assay is available on automated analyzers using the regular serum ferritin methodology,106 but the process is inefficient: it requires complete removal of white cells to avoid measuring leukocyte ferritin,107 it is insensitive to dynamic changes in iron status, and it is a rarely available diagnostic tool used by clinicians.
B.6. Erythrocyte zinc protoporphyrin
Incorporation of iron into protoporphyrin IX is the final step of heme biosynthesis: if iron is not available, Zn is inserted into the molecule to form zinc protoporphyrin (ZPP).108 Red cell values of ZPP can be measured with a dedicated instrument called a hematofluorometer, which determines the ZPP concentration of a whole blood sample in either µm/L or as a molar ratio with hemoglobin (µmol ZPP/mol heme).109-112 When ZPP is measured in whole blood, falsely elevated values are observed in dialysis patients, and these false values are also observed in the presence of bilirubin and various drugs. Careful washing of the red cells is required to remove these interferences, rendering this assay unsuitable for routine clinical care.113,114 ZPP is also elevated in the presence of lead poisoning. Recent studies have shown that the diagnostic value of ZPP is inferior to that of red cell or reticulocyte parameters.78,115 A non-invasive method for ZPP measurement in the lip has shown promise as a screening tool for iron deficiency in clinical settings without access to other resources.116
B.7. Peripheral Blood morphology
Red cell morphology is most informative in clear cases of iron deficient anemia, but at this stage it is solely confirmative in nature, since diagnosis can be established in many cases without looking at the peripheral blood smear. The washed-out appearance of erythrocytes and their enlarged area of central pallor are clear indicators of hypochromia. In the peripheral smear, erythrocytes show various degrees of anisocytosis, poikilocytosis, microcytosis and hypochromia, with occasional target cells, while platelets appear to be overabundant.Pencil cells and prekeratocytes are two additional abnormal red cell morphologies,which have been described in iron deficiency:117 pencil cells are hypochromic elliptocytes, with a long axis at least three times the length of the short one, while prekeratocytes are poikilocytes exhibiting both a central pallor and one or more sub-membrane vacuoles. Pencil cells and prekeratocytes are not exclusive of iron deficiency and can also be seen in ß-thalassemia trait and the anemia of chronic disease.118 Morphological distinction between iron deficiency and thalassemia trait is challenging: the two features considered distinctive of thalassemia minor, namely target cells and basophilic stippling, were found to be of limited usefulness in a recent study.119
B.8. Bone Marrow Iron determination
Iron staining of a bone marrow biopsy was regarded as a gold-standard method to assess iron stores, but this invasive, potentially painful procedure has yielded widely divergent estimates of the prevalence of iron deficiency.120-124 The technique is based on the identification with the Prussian blue reaction (Pearl’s stain) of intracellular iron deposits, which appear as round, clumped granules in macrophages (they are typically absent in iron deficiency). A recent study in 100 non-dialysis CKD patients showed that evaluation of iron stores by iron staining of a bone marrow sternal aspirate was no better than either TSAT or ferritin in correctly identifying responders to IV iron therapy.125 This technique also does not help doctors to identify patients at risk of developing functional iron deficiency with EPO therapy.
C.DIAGNOSTIC APPROACH TO IRON DEFICIENT STATES
Normal Hb values do not rule-out iron deficiency, especially if the prior baseline value for an individual patient is not known. Some patients may experience chronic fatigue prior to the development of anemia, although there is actually no evidence that this may be due to non-hematological effects of iron deficiency as argued by some clinicians. Iron deficiency should be suspected when Hb values over time trend downward in combination with increasing RDW and decreasing MCV/MCH. In many instances, a patient’s history of chronic bleeding will prompt clinicians to add a biochemical iron study to a standard CBC. In the absence of chronic inflammatory conditions, a low or borderline-low ferritin (15-30 ng/mL) may be the only sign of the developing iron deficiency. In very young children, a careful dietary history may elicit an excessive consumption of cow’s milk with a following CBC revealing an otherwise unsuspected, pronounced hypochromic microcytic anemia.
In the absence of prior laboratory encounters with an individual patient, the unexpected finding of hypochromic microcytosis with or without anemia will require that alpha or beta thalassemia traits be ruled-out. As discussed above, the micro/hypo ratio is the best parameter for this differential diagnosis. If this parameter is not available, some of the other, classic formulas may be used, although their performance is inferior to that of the micro/hypo ratio. Although informative, sTfR is not widely used and does not add significant information for simple iron deficiency cases. It should be reserved, either alone or as a ratio of sTfR/ferritin, for responding to more complex cases with associated inflammatory conditions or altered iron metabolism due to systemic disease. Since reliable hepcidin assays are only now becoming available, only limited information exists on how this parameter could be used and how much additional information it would add to serum ferritin values. It has been suggested that in the setting of ACD, the presence of significant iron deficiency component should result in lower hepcidin values.
The availability of a reticulocyte count with reticulocyte Hb content may provide an additional important tool for assessing both evolving, simple iron deficiency cases and more complicated situations like the ACD. A reduction of the reticulocyte Hb content below the lower limit of normal (usually ~ 28 pg/cell) provides direct evidence that the availability of iron to the erythropoietic marrow is limited. It may also provide a simple and reliable tool for rapid determination of the helpfulness of iron replacement therapies.
D. TREATMENT MONITORING
D.1. Oral Iron replacement therapy
Unless the patient has experienced diseases producing substantial blood loss or impaired gastrointestinal iron absorption, oral iron replacement therapy is expected to result in a measurable change in Hb values after 2-3 weeks, with Hb values approaching baseline values in 2 months, barring extremely severe cases of anemia. A widely used criterion for assessing hematological response to oral iron is a 2 gr/dl increase in Hb values after 3 weeks of therapy. Increases in absolute reticulocyte count and reticulocyte Hb content can be monitored as early as one week after initiation of oral iron therapy.126 Therapy is usually continued for a few months after the anemia is corrected, to ensure that the storage iron is loading adequately. Monitoring both Hb values and reticulocyte parameters such as absolute reticulocyte count and reticulocyte Hb content127 not only allows doctors to estimate the patient’s response to iron replacement therapy but also to alert caregivers about either poor adherence to treatment or the presence of a concomitant pathology which could limit the effectiveness of the oral iron regimen.5,91,128
Biochemical parameters are less helpful to monitor therapeutic response, since plasma iron values are highly variable and may return to the normal range just because a single iron pill was taken before the blood draw. However, changes in serum iron following the administration of oral iron supplements have been used to determine iron absorption.127 Serum ferritin is not influenced by daily variations in iron intake: thus, a progressive increase in serum ferritin (in the absence of inflammation) suggests that the patient is adhering to the therapy and that iron is effectively replenishing the body’s stores. Ferritin is one of the last parameters to normalize, because it remains steadily in the normal range only after the correction of anemia, which makes it an ideal aspect to study.
D.2. Intravenous (IV) iron therapy
Reticulocyte parameters change dramatically within 24-48 hrs. following IV iron administration, with marked increases observed in the reticulocyte Hb content (CHr) as well as in reticulocyte volume and absolute reticulocyte count.126 In normal subjects, the concomitant use of r-HuEPO and IV iron resulted in measurable increases in the reticulocyte Hb content (CHr) and serum ferritin, with no changes in absolute reticulocyte count values compared to R-HuEPO alone.93 These findings are consistent with the work of Finch et al, which shows that increased iron availability and increased erythropoiesis result in the production of larger cells with greater Hb content.6
In conclusion, integrating hematological and biochemical parameters allows for an earlier and more accurate diagnosis of most uncomplicated iron deficient states. In more challenging cases, accompanied by chronic inflammation or other alterations of iron handling, measurements of serum hepcidin values may provide additional useful information, with potential practical therapeutic applications as well.
Conflict of Interest
Dr. Brugnara wishes to disclose a consulting agreement with Sysmex America Inc.
1. Cook JD, Flowers CH, Skikne BS. The quantitative assessment of body iron. Blood. 2003 May;101(9):3359-64.
2. Cook JD, Boy E, Flowers C, Daroca Mdel C. The influence of high-altitude living on body iron. Blood. 2005 Aug;106(4):1441-6.
3. Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human serum hepcidin. Blood. 2008 Nov;112(10):4292-7.
4. Brugnara C. A hematologic “gold standard” for iron-deficient states? Clinical Chemistry. 2002 Jul;48(7):981-2.
5. Brugnara C. Iron deficiency and erythropoiesis: new diagnostic approaches. Clinical Chemistry. 2003 Oct;49(10):1573-8.
6. Finch CA. Erythropoiesis, erythropoietin, and iron. Blood. 1982;60(6):1241-6.
7. Goodnough LT, Nemeth E, Ganz T. Detection, evaluation, and management of iron-restricted erythropoiesis. Blood. 2010 Dec;116(23):4754-61.
8. Harris EL, McLaren CE, Reboussin DM, Gordeuk VR, Barton JC, Acton RT, et al. Serum ferritin and transferrin saturation in Asians and Pacific Islanders. Arch Intern Med. 2007 Apr;167(7):722-6.
9. Ravasi G, Pelucchi S, Mariani R, Casati M, Greni F, Arosio C, et al. Unexplained isolated hyperferritinemia without iron overload. Am J Hematol. 2017 Apr;92:338-43.
10. Mast AE, Blinder MA, Gronowski AM, Chumley C, Scott MG. Clinical utility of the soluble transferrin receptor and comparison with serum ferritin in several populations. Clin Chem. 1998 Jan;44:45-51.
11. Leggett BA, Brown NN, Bryant SJ, Duplock L, Powell LW, Halliday JW. Factors affecting the concentrations of ferritin in serum in a healthy Australian population. Clin Chem. 1990 Jul;36:1350-5.
12. Girelli D, Corrocher R, Bisceglia L, Olivieri O, De Franceschi L, Zelante L, et al. Molecular basis for the recently described hereditary hyperferritinemia- cataract syndrome: a mutation in the iron-responsive element of ferritin L-subunit gene (the “Verona mutation”) [see comments]. Blood. 1995;86(11):4050-3.
13. Brugnara C, Zurakowski D, DiCanzio J, Boyd T, Platt O. Reticulocyte hemoglobin content to diagnose iron deficiency in children. JAMA. 1999 Jun;281(23):2225-30.
14. Ullrich C, Wu A, Armsby C, Rieber S, Wingerter S, Brugnara C, et al. Screening healthy infants for iron deficiency using reticulocyte hemoglobin content. JAMA. 2005 Aug;294(8):924-30.
15. KDOQI Clinical Practice Guideline and Clinical Practice Recommendations for anemia in chronic kidney disease: 2007 update of hemoglobin target. Am J Kidney Dis. 2007 Sep;50(3):471-530.
16. Locatelli F, Aljama PA, Barany P, Canaud B, Carrera F, Eckardt KU, et al. Revised european best practice guidelines for the management of anaemia in patients with chronic renal failure. Nephrol Dial Transplant. 2004 May;19 Suppl 2:ii1-47.
17. Fishbane S, Kowalski EA, Imbriano LJ, Maesaka JK. The evaluation of iron status in hemodialysis patients. J Am Soc Nephrol. 1996 Dec;7(12):2654-7.
18. Lippi G, Luca Salvagno G, Montagnana M, Brocco G, Cesare Guidi G. Influence of hemolysis on routine clinical chemistry testing. Clin Chem Lab Med. 2006;44(3):311-6.
19. Nadkarni S, Allen LC. Comparison of the ames, randox and roche methods with the synermed method for the determination of serum iron concentrations on nondialysis and dialysis specimens. Clin Biochem. 1998 Mar;31(2):89-94.
20. Kasvosve I, Delanghe JR, Gomo ZAR, Gangaidzo IT, Khumalo H, Wuyts B, et al. Transferrin polymorphism influences iron status in blacks. Clin Chem. 2000 Oct;46(10):1535-9.
21. Forni GL, Pinto V, Musso M, Mori M, Girelli D, Caldarelli I, et al. Transferrin-immune complex disease: A potentially overlooked gammopathy mediated by IgM and IgG. Am J Hematol. 2013 Aug;88(12):1045-9.
22. Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to Tango: Regulation of Mammalian Iron Metabolism. Cell. 2010 Jul;142(1):24-38.
23. Pichler I, Minelli C, Sanna S, Tanaka T, Schwienbacher C, Naitza S, et al. Identification of a common variant in the TFR2 gene implicated in the physiological regulation of serum iron levels. Human Molecular Genetics. 2011 Mar;20(6):1232-40.
24. Bainton DF, Finch CA. The diagnosis of iron deficiency anemia. Am J Med. 1964 Jul;37:62-70.
25. Kalantar-Zadeh K, McAllister CJ, Lehn RS, Kopple JD. A low serum iron level is a predictor of poor outcome in hemodialysis patients. Am J Kidney Dis. 2004 Apr;43(4):671-84.
26. Kovesdy CP, Estrada W, Ahmadzadeh S, Kalantar-Zadeh K. Association of markers of iron stores with outcomes in patients with nondialysis-dependent chronic kidney disease. Clin J Am Soc Nephrol 2009 Feb;4(2):435-41.
27. Fishbane S, Pollack S, Feldman HI, Joffe MM. Iron indices in chronic kidney disease in the National Health and Nutritional Examination Survey 1988-2004. Clin J Am Soc Nephrol. 2009 Jan;4(1):57-61.
28. Ferguson BJ, Skikne BS, Simpson KM, Baynes RD, Cook JD. Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency anemia. J Lab Clin Med. 1992 Apr;119(4):385-90.
29. Punnonen K, Irjala K, Rajamaki A. Iron-deficiency anemia is associated with high concentrations of transferrin receptor in serum. Clin Chem. 1994 May;40(5):774-6.
30. Punnonen K, Irjala K, Rajamaki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood. 1997 Feb;89(3):1052-7.
31. Skikne BS. Serum transferrin receptor. Am J Hematol. 2008 Nov;83(11):872-5.
32. Skikne BS, Flowers CH, Cook JD. Serum transferrin receptor: a quantitative measure of tissue iron deficiency. Blood. 1990 May;75:1870-6.
33. Khumalo H, Gomo ZAR, Moyo VM, Gordeuk VR, Saungweme T, Rouault TA, et al. Serum transferrin receptors are decreased in the presence of iron overload. Clin Chem. 1998 Jan;44(1):40-4.
34. Vikstedt R, von Lode P, Takala T, Irjala K, Peltola O, Pettersson K, et al. Rapid one-step immunofluorometric assay for measuring soluble transferrin receptor in whole blood. Clin Chem. 2004 Sep;50(10):1831-3.
35. Fertrin KY, Lanaro C, Franco-Penteado CF, de Albuquerque DM, de Mello MRB, Pallis FR, et al. Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15. American journal of hematology. 2014 Apr;89(4):385-90.
36. Ahluwalia N, Skikne BS, Savin V, Chonko A. Markers of masked iron deficiency and effectiveness of EPO therapy in chronic renal failure. Am J Kidney Dis. 1997 Oct;30:532-41.
37. Singh AK, Coyne DW, Shapiro W, Rizkala AR, DRIVE Study Group. Predictors of the response to treatment in anemic hemodialysis patients with high serum ferritin and low transferrin saturation. Kidney Int. 2007 Jun;71(11):1163-71.
38. Fusaro M, Munaretto G, Spinello M, Rebeschini M, Amici G, Gallieni M, et al. Soluble transferrin receptors and reticulocyte hemoglobin concentration in the assessment of iron deficiency in hemodialysis patients. J Nephrol. 2005 Jan-Feb;18(1):72-9.
39. Tarng DC, Hung SC, Huang TP. Effect of intravenous ascorbic acid medication on serum levels of soluble transferrin receptor in hemodialysis patients. J Am Soc Nephrol. 2004 Sep;15(9):2486-93.
40. Cogswell ME, Looker AC, Pfeiffer CM, Cook JD, Lacher DA, Beard JL, et al. Assessment of iron deficiency in US preschool children and nonpregnant females of childbearing age: National Health and Nutrition Examination Survey 2003-2006. Am J Clin Nutr. 2009 May;89(5):1334-42.
41. Yang Z, Dewey KG, Lönnerdal B, Hernell O, Chaparro C, Adu-Afarwuah S, et al. Comparison of plasma ferritin concentration with the ratio of plasma transferrin receptor to ferritin in estimating body iron stores: results of 4 intervention trials. Am J Clin Nutr. 2008 Jun;87(6):1892-8.
42. Lee EJ, Oh EJ, Park YJ, Lee HK, Kim BK. Soluble transferrin receptor (sTfR), ferritin, and sTfR/log ferritin index in anemic patients with nonhematologic malignancy and chronic inflammation. Clin Chem. 2002 Jul;48(7):1118-21
43. Chen YC, Hung SC, Tarng DC. Association between transferrin receptor-ferritin index and conventional measures of iron responsiveness in hemodialysis patients. Am J Kidney Dis. 2006 Jun;47(6):1036-44.
44. Margetic S, Topic E, Tesija-Kuna A, Vukasovic I. Soluble serum transferrin receptor and transferrin receptor-ferritin index in anemia of chronic kidney disease. Dial Transplant. 2006 Aug;35(8):520-43.
45. Suominen P, Punnonen K, Rajamaki A, Irjala K. Serum transferrin receptor and transferrin receptor-ferritin index identify healthy subjects with subclinical iron deficits. Blood. 1998 Oct;92(8):2934-9.
46. Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. Clin Chem. 2002 Jul;48(7):1066-76.
47. Ganz T. Hepcidin and iron regulation, 10 years later. Blood. 2011 Apr 28;117(17):4425-33.
48. Butterfield AM, Luan P, Witcher DR, Manetta J, Murphy AT, Wroblewski VJ, et al. A Dual-Monoclonal Sandwich ELISA Specific for Hepcidin-25. Clin Chem. 2010 Nov;56(11):1725-32.
49. Dallalio G, Fleury T, Means RT. Serum hepcidin in clinical specimens. Br J Haematol. 2003 Sep;122(6):996-1000.
50. Hershko C, Camaschella C. How I treat unexplained refractory iron deficiency anemia. Blood. 2014;123(3):326-33.
51. Bregman DB, Morris D, Koch TA, He A, Goodnough LT. Hepcidin levels predict nonresponsiveness to oral iron therapy in patients with iron deficiency anemia. Am J Hematol. 2013 Feb;88(2):97-101.
52. Prentice AM, Doherty CP, Abrams SA, Cox SE, Atkinson SH, Verhoef H, et al. Hepcidin is the major predictor of erythrocyte iron incorporation in anemic African children. Blood. 2012 Feb 23;119(8):1922-8.
53. Steensma DP, Sasu BJ, Sloan JA, Tomita D, Loprinzi. The relationship between serum hepcidin levels and clinical outcomes in patients with chemotherapy-associated anemia treated in a controlled trial. J Clin Oncol. 2011;29(15):9031.
54. Ukarma L, Johannes H, Beyer U, Zaug M, Osterwalder B, Scherhag A. Hepcidin as a predictor of response to epoetin therapy in anemic cancer patients. Clin Chem. 2009 Jul;55(7):1354-60
55. Kautz L, Jung G, Valore EV, Rivella S, Nemeth E, Ganz T. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014 Jul;46(7):678-84.
56. Jung G, Nemeth E, Ganz T. The erythroid factor erythroferrone and its role in iron homeostasis. Blood. 2013;122(21):4.
57. Kautz L, Jung G, Nemeth E, Ganz T. Erythroferrone contributes to recovery from anemia of inflammation. Blood. 2014 Oct;124(16):2569-74.
58. Nai A, Lidonnici MR, Rausa M, Mandelli G, Pagani A, Silvestri L, et al. The second transferrin receptor regulates red blood cell production in mice. 2015 Feb;125(7):1170-9.
59. Liang R, Ghaffari S. Advances in understanding the mechanisms of erythropoiesis in homeostasis and disease. Br J Haematol. 2016 Sep;174(5):661-73.
60. Tanno T, Bhanu NV, Oneal PA, Goh S-H, Staker P, Lee YT, et al. High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med. 2007 Sep;13(9):1096-101.
61. Tamary H, Shalev H, Perez-Avraham G, Zoldan M, Levi I, Swinkels DW, et al. Elevated growth differentiation factor 15 expression in patients with congenital dyserythropoietic anemia type I. Blood. 2008 Dec;112(13):5241-4.
62. Du X, She E, Gelbart T, Truksa J, Lee P, Xia Y, et al. The serine protease TMPRSS6 is required to sense iron deficiency. Science. Science. 2008 May;320(5879):1088-92.
63. Finberg KE, Heeney MM, Campagna DR, Aydinok Y, Pearson HA, Hartman KR, et al. Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA). Nat Genet. 2008 May;40(5):569-71.
64. Benyamin B, Ferreira MAR, Willemsen G, Gordon S, Middelberg RPS, McEvoy BP, et al. Common variants in TMPRSS6 are associated with iron status and erythrocyte volume. Nat Genet. 2009 Nov;41(11):1173-5.
65. World Health Organization. Worldwide prevalence of anaemia 1993–2005. WHO Global Database on Anaemia. Geneva: WHO; 2008 Available from: http://whqlibdoc.who.int/publications/2008/9789241596657_eng.pdf.
66. England JM, Ward SM, Down MC. Microcytosis, anisocytosis and the red cell indices in iron deficiency. Br J Haematol. 1976 Dec;34(4):589-97.
67. Mohandas N, Kim YR, Tycko DH, Orlik J, Wyatt J, Groner W. Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering. Blood. 1986 Aug;68(2):506-13.
68. Mohandas N, Johnson A, Wyatt J, Croisille L, Reeves J, Tycko D, et al. Automated quantitation of cell density distribution and hyperdense cell fraction in RBC disorders. Blood. 1989 Jul;74(1):442-7.
69. Urrechaga E. The new mature red cell parameter, low haemoglobin density of the Beckman-Coulter LH750: clinical utility in the diagnosis of iron deficiency. Int J Lab Hematol. 2010 Feb;32(1 Pt 1):e144-50.
70. Maconi M, Cavalca L, Danise P, Cardarelli F, Brini M. Erythrocyte and reticulocyte indices in iron deficiency in chronic kidney disease: comparison of two methods. Scand J Clin Lab Invest. 2009;69(3):365-70.
71. Urrechaga E, Borque L, Escanero JF. Clinical Value of Hypochromia Markers in the Detection of Latent Iron Deficiency in Nonanemic Premenopausal Women. J Clin Lab Anal. 2016 Sep;30(5):623-7.
72. Urrechaga E, Boveda O, Aguayo FJ, De la Hera P, Munoz RI, Gallardo I, et al. Percentage of hypochromic erythrocytes and reticulocyte hemoglobin equivalent predictors of response to intravenous iron in hemodialysis patients. Int J Lab Hematol. 2016 Aug;38(4):360-5.
73. Macdougall IC, Cavill I, Hulme B, Bain B, McGregor E, McKay P, et al. Detection of functional iron-deficiency during erythropoietin treatment – A new approach. British Medical Journal. 1992 Jan;304(6821):225-6.
74. Schaefer RM, Schaefer L. The hypochromic red cell: a new parameter for monitoring of iron supplementation during rhEPO therapy. Journal of Perinatal Medicine. 1995;23(1-2):83-8.
75. Braun J, Lindner K, Schreiber M, Heidler RA, Hörl WH. Percentage of hypochromic red blood cells as predictor of erythropoietic and iron response after i.v. iron supplementation in maintenance haemodialysis patients. Nephrology, Dialysis, Transplantation. 1997 Jun;12(6):1173-81.
76. Bovy C, Tsobo C, Crapanzano L, Rorive G, Beguin Y, Albert A, et al. Factors determining the percentage of hypochromic red blood cells in hemodialysis patients. Kidney Int. 1999 Sep;56(3):1113-9.
77. Schaefer RM, Schaefer L. Hypochromic red blood cells and reticulocytes. Kidney International.1999;Suppl 69:S44-8.
78. Tessitore N, Solero GP, Lippi G, Bassi A, Faccini GB, Bedogna V, et al. The role of iron status markers in predicting response to intravenous iron in haemodialysis patients on maintenance erythropoietin. Nephrology Dialysis Transplantation. 2001 Jul;16(7):1416-23.
79. Richardson D, Bartlett C, Will EJ. Optimizing erythropoietin therapy in hemodialysis patients. American Journal of Kidney Diseases. 2001;38(1):109-17.
80. Brugnara C, Chambers LA, Malynn E, Goldberg MA, Kruskall MS. Red-blood-cell regeneration induced by subcutaneous recombinant erythropoietin – iron-deficient erythropoiesis in iron-replete subjects. Blood. 1993 Feb;81(4):956-64.
81. Brugnara C, Colella GM, Cremins J, Langley RC, Schneider TJ, Rutherford CJ, et al. Effects of subcutaneous recombinant-human-erythropoietin in normal subjects – development of decreased reticulocyte hemoglobin content and iron-deficient erythropoiesis. J Lab Clin Med. 1994 May;123(5):660-7.
82. Winkelmayer WC, Lorenz M, Kramar R, Horl WH, Sunder-Plassmann G. Percentage of hypochromic red blood cells is an independent risk factor for mortality in kidney transplant recipients. Am J Transplant. 2004 Dec;4(12):2075-81.
83. Bhandari S, Norfolk D, Brownjohn A, Turney J. Evaluation of RBC ferritin and reticulocyte measurements in monitoring response to intravenous iron therapy. American Journal of Kidney Diseases. 1997 Dec;30(6):814-21.
84. Fishbane S, Galgano C, Langley RC Jr, Canfield W, Maesaka JK. Reticulocyte hemoglobin content in the evaluation of iron status of hemodialysis patients. Kidney Int. 1997 Jul;52(1):217-22.
85. Buttarello M, Pajola R, Novello E, Mezzapelle G, Plebani M. Evaluation of the hypochromic erythrocyte and reticulocyte hemoglobin content provided by the Sysmex XE-5000 analyzer in diagnosis of iron deficiency erythropoiesis. Clin Chem Lab Med. 2016 Dec;54(12):1939-45.
86. Hennek JW, Kumar AA, Wiltschko AB, Patton MR, Lee SYR, Brugnara C, et al. Diagnosis of iron deficiency anemia using density-based fractionation of red blood cells. Lab on a Chip. 2016 Aug;16(20):3929-39.
87. d’Onofrio G, Zini G, Ricerca BM, Mancini S, Mango G. Automated measurement of red blood cell microcytosis and hypochromia in iron deficiency and ß-thalassemia trait. Arch Pathol Lab Med. 1992 Jan;116(1):84-9.
88. Lafferty JD, Crowther MA, Ali MA, Levine M. The evaluation of various mathematical RBC indices and their efficacy in discriminating between thalassemic and non-thalassemic microcytosis. Am J Clin Pathol. 1996 Aug;106(2):201-5.
89. Brugnara C, Hipp MJ, Irving PJ, Lathrop H, Lee PA, Minchello EM, et al. Automated reticulocyte counting and measurement of reticulocyte cellular indexes – Evaluation of the Miles-H-Asterisk-3-Blood-Analyzer. Am J Clin Pathol. 1994 Nov;102(5):623-32.
90. Mast AE, Blinder MA, Dietzen DJ. Reticulocyte hemoglobin content. Am J Hematol. 2008 Apr;83(4):307-10
91. Brugnara C, Zelmanovic D, Sorette M, Ballas SK, Platt O. Reticulocyte hemoglobin – An integrated parameter for evaluation of erythropoietic activity. American Journal of Clinical Pathology. 1997 Aug;108(2):133-42.
92. Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency. Blood. 2002 Feb;99(4):1489-91.
93. Major A, Mathez-Loic F, Rohling R, Gautschi K, Brugnara C. The effect of intravenous iron on the reticulocyte response to recombinant human erythropoietin. Br J Haematol. 1997 Sep;98(2):292-4.
94. Ervasti M, Kotisaari S, Heinonen S, Punnonen K. Use of advanced red blood cell and reticulocyte indices improves the accuracy in diagnosing iron deficiency in pregnant women at term. Eur J Haematol. 2007 Dec;79(6):539-45.
95. Cullen P, Söffker J, Höpfl M, Bremer C, Schlaghecken R, Mehrens T, et al. Hypochromic red cells and reticulocyte haemglobin content as markers of iron-deficient erythropoiesis in patients undergoing chronic haemodialysis. Nephrology Dialysis Transplantation. 1999 Mar;14(3):659-65.
96. Mittman N, Sreedhara R, Mushnick R, Chattopadhyay J, Zelmanovic D, Vaseghi M, et al. Reticulocyte hemoglobin content predicts functional iron deficiency in hemodialysis patients receiving rHuEPO. Am J Kid Dis. 1997 Dec;30(6):912-22.
97. Fishbane S, Shapiro W, Dutka P, Valenzuela OF, Faubert J. A randomized trial of iron deficiency testing strategies in hemodialysis patients. Kidney Int. 2001 Dec;60(6):2406-11.
98. Chuang C-L, Liu R-S, Wei Y-H, Huang T-P, Tarng D-C. Early prediction of response to intravenous iron supplementation by reticulocyte haemoglobin content and high-fluorescence reticulocyte count in haemodialysis patients. Nephrol Dial Transplant. 2003 Feb;18(2):370-7.
99. Kim JM, Ihm CH, Kim HJ. Evaluation of reticulocyte haemoglobin content as marker of iron deficiency and predictor of response to intravenous iron in haemodialysis patients. Int J Lab Hematol. 2008 Feb;30(1):46-52.
100. Kaneko Y, Miyazaki S, Hirasawa Y, Gejyo F, Suzuki M. Transferrin saturation versus reticulocyte hemoglobin content for iron deficiency in Japanese hemodialysis patients. Kidney Int. 2003 Mar;63(3):1086-93.
101. Tsuchiya K, Okano H, Teramura M, Iwamoto Y, Yamashita N, Suda A, et al. Content of reticulocyte hemoglobin is a reliable tool for determining iron deficiency in dialysis patients. Clinical Nephrology. 2003 Feb;59(2):115-23.
102. Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) and assessment of iron-deficient states. Clin Lab Haematol. 2006 Oct;28(5):303-8.
103. Ng HY, Chen HC, Pan LL, Tsai YC, Hsu KT, Liao SC, et al. Clinical Interpretation of Reticulocyte Hemoglobin Content, RET-Y, in Chronic Hemodialysis Patients. Nephron Clin Pract. 2009;111(4):c247-52.
104. Miwa N, Akiba T, Kimata N, Hamaguchi Y, Arakawa Y, Tamura T, et al. Usefulness of measuring reticulocyte hemoglobin equivalent in the management of haemodialysis patients with iron deficiency. Int J Lab Hematol. 2010 Apr;32(2):248-55.
105. van Wyck D, Alcorn H, Gupta R. Analytical and Biological Variation in Measures of Anemia and Iron Status in Patients Treated With Maintenance Hemodialysis. Am J Kidney Dis. 2010 Sep;56(3):540-6.
106. Novembrino C, Porcella A, Conte D, de Vecchi AF, Buccianti G, Lonati S, et al. Erythrocyte ferritin concentration: analytical performance of the immunoenzymatic IMx-Ferritin (Abbott) assay. Clin Chem Lab Med. 2005;43(4):449-53.
107. Galan P, Sangare N, Preziosi P, Roudier M, Hercberg S. Is basic red cell ferritin a more specific indicator than serum ferritin in the assessment of iron stores in the elderly? Clin Chim Acta. 1990 Aug;189(2):159-62.
108. Labbé RF, Dewanji A, McLaughlin K. Observations on the Zinc Protoporphyrin/Heme Ratio in Whole Blood. Clin Chem. 1999 Jan;45(1):146-8.
109. Fishbane S, Lynn R. The utility of zinc protoporphyrin for predicting the need for intravenous iron therapy in hemodialysis-patients. American Journal Of Kidney Diseases. 1995 Mar;25(3):426-32.
110. Braun J, Hammerschmidt M, Schreiber M, Heidler R, Horl WH. Is zinc protoporphyria an indicator of iron-deficient erythropoiesis in maintenance haemodialysis patients? Nephrology Dialysis Transplantation. 1996 Mar;11(3):492-7.
111. Baldus M, Salopek S, Möller M, Schliesser J, Klooker P, Reddig J, et al. Experience with zinc protoporphyrin as a marker of endogenous iron availability in chronic haemodialysis patients. Nephrology Dialysis Transplantation. 1996 Mar;11(3):486-91.
112. Baldus M, Walter H, Thies K, Anders C, Stein M, Hellstern P, et al. Transferrin receptor assay and zinc protoporphyrin as markers of iron-deficient erythropoiesis in end-stage renal disease patients. Clinical Nephrology. 1998 Mar;49(3):186-92.
113. Hastka J, Lasserre JJ, Schwarzbeck A, Strauch M, Hehlmann R. Washing erythrocytes to remove interferents in measurements of zinc protoporphyrin by front-face hematofluorometry. Clin Chem. 1992 Nov;38(11):2184-9.
114. Garrett S, Worwood M. Zinc protoporphyrin and iron-deficient erythropoiesis. Acta Haematologica. 1994;91(1):21-5.
115. Besarab A, Amin N, Ahsan M, Vogel SE, Zazuwa G, Frinak S, et al. Optimization of epoetin therapy with intravenous iron therapy in hemodialysis patients. J Am Soc Nephrol. 2000 Mar;11(3):530-8.
116. Hennig G, Homann C, Teksan I, Hasbargen U, Hasmüller S, Holdt LM, et al. Non-invasive detection of iron deficiency by fluorescence measurement of erythrocyte zinc protoporphyrin in the lip. Nat Commun. 2016 Feb;7:10776.
117. Harrington AM, Kroft SH. Pencil cells and prekeratocytes in iron deficiency anemia. American Journal of Hematology. 2008 Dec;83(12):927.
118. Ford J. Red blood cell morphology. Int J Lab Hematol. 2013 Jun;35(3):351-7.
119. Harrington AM, Ward PCJ, Kroft SH. Iron Deficiency Anemia, beta-Thalassemia Minor, and Anemia of Chronic Disease. American Journal of Clinical Pathology. 2008 Mar;129(3):466-71.
120. Fernández-Rodríguez AM, Guindeo-Casasús MC, Molero-Labarta T, Domínguez-Cabrera C, Hortal-Cascón L, Pérez-Borges P, et al. Diagnosis of iron deficiency in chronic renal failure. American Journal of Kidney Diseases. 1999 Sep;34(3):508-13.
121. Kalantarzadeh K, Höffken B, Wünsch H, Fink H, Kleiner M, Luft FC. Diagnosis of iron-deficiency anemia in renal-failure patients during the post-erythropoietin era. American Journal of Kidney Diseases. 1995 Aug;26(2):292-9.
122. Domrongkitchaiporn S, Jirakranont B, Atamasrikul K, Ungkanont A, Bunyaratvej A. Indices of iron status in continuous ambulatory peritoneal dialysis patients. Am J Kidney Dis. 1999 Jul;34(1):29-35.
123. Gotloib L, Silverberg D, Fudin R, Shostak A. Iron deficiency is a common cause of anemia in chronic kidney disease and can often be corrected with intravenous iron. J Nephrol. 2006 Mar-Apr;19(2):161-7.
124. Martin-Cabrera P, Hung M, Ortmann E, Richards T, Ghosh M, Bottrill F, et al. Clinical use of low haemoglobin density, transferrin saturation, bone marrow morphology, Perl’s stain and other plasma markers in the identification of treatable anaemia presenting for cardiac surgery in a prospective cohort study. J Clin Pathol. 2015 Nov;68(11):923-30.
125. Stancu S, Bârsan L, Stanciu A, Mircescu G. Can the Response to Iron Therapy Be Predicted in Anemic Nondialysis Patients with Chronic Kidney Disease? Clinical Journal of the American Society of Nephrology. 2010 Mar;5(3):409-16.
126. Brugnara C, Laufer MR, Friedman AJ, Bridges K, Platt O. Reticulocyte hemoglobin content (CHr): early indicator of iron deficiency and response to therapy [letter]. Blood. 1994 May 15;83(10):3100-1.
127. Hoppe M, HulthÈn L, Hallberg L. The validation of using serum iron increase to measure iron absorption in human subjects. Br J Nutr. 2004 Sep;92(3):485-8.
128. Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) and assessment of iron deficient states. Clin Lab Haem. 2006 Oct;28(5):303-8.
*This simplified model, useful as it is, applies only to uncomplicated iron deficient states due to either dietary insufficiency or excessive iron loss; it is not applicable to conditions in which the movement of iron across cellular compartments is altered, as in the case of chronic inflammation or the anemia of chronic disease.
Received 9 Jan 2019
Accepted 28 Jan 2019