American Association for Clinical Chemistry
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November 2009 Clinical Laboratory News: Differential Anemia Diagnosis

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November 2009: Volume 35, Number 11


Differential Anemia Diagnosis
What’s the Best Marker of Iron Status?
By Genna Rollins

Anemia compromises quality of life through fatigue and impaired cognitive function and has been linked to cardiovascular disease morbidity, increased hospitalization, and mortality. Despite the significant health consequences associated with the condition, anemia remains one of the most prevalent conditions worldwide and is the most common nutritional deficiency, affecting an estimated one-quarter of the world’s population. Even with such a profound disease burden, iron deficiency anemia (IDA) and anemia of chronic disease (ACD) often receive sub-optimal clinical management. Strategies to definitively diagnose and treat the conditions have not been widely implemented, and there is disagreement about the best combination of tests and cutoff points to distinguish between the two. At the same time, research advances have enhanced the field’s understanding of iron homeostasis, and ushered in a new generation of analytical tools with their own set of challenges.

“There needs to be some kind of better systematic approach to differentiating between IDA and ACD. We need evidence-based guidelines to help direct diagnosis and treatment, but they just don’t exist right now,” said Susan Clark, RD, PhD, associate professor of human nutrition, foods and exercise at Virginia Tech University in Blacksburg. “Until we do that we’re not going to help patients as much as possible and their outcomes will be more negative than positive.”

Lacking Attention to Anemia

The prevalence of anemia and iron deficiency varies according to demographic groups, but is most common in young children, women of childbearing age, racial and ethnic minorities, and in individuals with various chronic conditions. Prevalence increases with age and is quite common in chronic diseases like cancer and autoimmune disorders. For example, anemia occurs in an estimated 30% to 60% of rheumatoid arthritis patients and anywhere from 30% to 80% of patients with inflammatory bowel disease (IBD). IDA is the most common form of anemia, accounting for about half of all cases, while ACD is the second most common type with about 30% of all cases. Although they originate from different pathways, IDA and ACD both are readily treatable. Yet both tend to receive short shrift by clinicians. “The management of IDA is often sub-optimal with most patients being incompletely investigated if not at all,” according to the British Society of Gastroenterology guidelines for the management of IDA. Essentially the same has been said of ACD.

Some experts have argued that clinicians paradoxically may be less likely to intervene in patients with chronic diseases who are the most likely to have both IDA and ACD. “Gastroenterologists tend to tolerate reduced hemoglobin levels better than their patients. It is important to consider that anemia impairs quality of life even in the absence of specific symptoms and that its treatment leads to improvement in the quality of life. These simple facts are often unrecognized or neglected by gastroenterologists caring for patients with IBD,” according to recently published guidelines on the diagnosis and management of iron deficiency and anemia in IBD (Inflamm Bowel Dis 2007;13:1545–1553). Christophe Gasche, MD, lead the guideline effort because of the lack of attention to anemia in IBD patients. In the many extant IBD-related guidelines, “I’m not aware of a single word on anemia in any of them,” he explained. “So this is a huge problem that’s not being considered as a problem. Our goal was to make an awakening among our peers.” Gasche is professor of medicine and director of the Christian Doppler Laboratory on Molecular Cancer Chemoprevention at the Medical University of Vienna in Austria.

Gasche is not alone in advocating for better recognition and treatment of IDA and ACD. “There’s a sentiment that we think we can treat anemia by using the old standards, but if that’s the case, then why do we still have the problem?” said Clark. “It can take quite a while for someone with iron deficiency to become blatantly anemic. If the anemia is compounded by a chronic disease, then by the time you’re trying to discern what’s going on, you’ve got a really sick person on your hands, and sometimes that can be a comorbidity in their outcome.” Conversely, others have argued that ACD is a “beneficial and adaptive response to an underlying disease state” and that treatment of it poses distinct risks (CMAJ 2008;179:333–337).

A Tightly Regulated System

Iron homeostasis is a complicated, tightly regulated system centered around the rate of erythropoesis and the level of iron stores. Iron absorbed through the intestine binds to transferrin, a blood protein that transports iron to target cells, attaching to those cells via transferrin receptors (see figure, below). In healthy adults, most transferrin receptors are connected to erythroid progenitor cells in bone marrow, and this is where most iron is used as a component of hemoglobin. When cells need iron, transferrin receptor production rises, making more iron uptake possible. At the end of their typical 120 day life, red blood cells are destroyed by macrophages that recycle iron from hemoglobin. In non-erythroid cells, iron is stored as ferritin in hepatocytes and macrophages as part of the reticuloendothelial system.

Pathways of Iron Exchange

The largest flux of iron takes place in its recycling from senescent erythrocytes out of macrophages to incorporation in erythroid precursors. The liver and reticuloendothelial macrophages are major iron stores. Normally only 1–2 mg of iron is absorbed and lost every day. Total iron in the body is regulated by absorption, but iron loss occurs through sloughing of cells and blood loss. Hepcidin controls the plasma iron concentration by inhibiting duodenal absorption of iron. Hepcidin expression is regulated by iron concentration in hepatocytes, by inflammatory stimuli, by erythroid iron demand, and by hypoxia involving expression of certain genes.

Image courtesy of Dorine W. Swinkels, MD, PhD; adapted from Clin Chem 2006; 52:950–968. Used with permission.

With discovery of the peptide hormone hepcidin in 2000, a more nuanced understanding of iron homeostasis has emerged. Secreted primarily by hepatocytes, hepcidin negatively regulates two essential aspects of iron homeostasis, intestinal absorption and macrophage recycling. The level of iron stores influences the release of hepcidin: when stores are low, hepcidin expression decreases to facilitate iron absorption, and when they are replete, it increases to forestall iron overload. Hepcidin expression also is influenced by inflammatory cytokines, and its increase has been implicated in the development of ACD.

IDA occurs when there is not enough iron to maintain normal physiological functions, and is manifested on a continuum from negative iron balance to iron depletion, iron-deficiency erythropoiesis, and finally, full-blown anemia. Serum iron levels fall only when iron stores become depleted. As this happens, transferrin levels rise and transferrin saturation declines. Iron depletion exists when iron stores are low or empty but the tissues that need iron are maintaining normal function. The causes of IDA are increased iron demand, such as in pregnancy or lactation, inadequate dietary intake—most commonly through malabsorption—or as a result of intestinal bleeding.

In contrast, ACD involves immune and inflammatory mechanisms that cause disruptions in iron metabolism, erythropoesis, and erythrocyte survival. Iron may be retained in the reticular-endothelial cells, there may be inadequate erythropoietin production, or inhibited proliferation of erythroid progenitor cells in bone marrow. In ACD, the iron supply depends on its rate of mobilization, so if the mechanisms for transporting iron to tissue are disrupted there can be a functional iron deficiency even though iron stores are adequate.

The Diagnostic Work-up

Regardless of the type of anemia involved, labs play an essential role in pinpointing the exact problem. “It is unusual for patients to present with anemia so advanced that the clinical manifestations predominate,” according to a monograph by the National Anemia Action Council. “Anemia is almost always discovered through abnormal laboratory screening test results.” The first tip-off often is a low hemoglobin level. The World Health Organization (WHO) defines anemia as hemoglobin <13 g/dL for men, <12 g/dL for women, and <11 g/dL in pregnant women.

The conventional work-up for IDA is fairly clear-cut and typically involves assessing erythrocyte morphology along with serum ferritin, serum iron, and total iron binding capacity (see table, below). IDA morphology is microcytic, hypochromic, and all three serum markers are low. However, the picture is murkier when it comes to diagnosing ACD or the combined state of IDA and ACD, which occurs in an estimated 20% to 30% of patients who also have ACD. One of the key issues is that ferritin is an acute phase reactant with levels determined not only by iron stores but also by the degree of cytokine activation. So values can rise in the presence of inflammation, even when iron stores are depleted from IDA. Most clinicians would agree that high and low ferritin values argue for inflammation and iron deficiency, respectively, but the middle ground leaves room for confusion and misinterpretation.

Selected Biochemical Indicators of Iron Status

Measurement

Commonly Used Methods

Indicator of

Reticulocyte hemoglobin concentration

Automated flow cytometry

Concentration of hemoglobin in new RBCs

Serum or plasma iron

Colorimetry

Iron bound to transferrin in blood

Ferritin

Immunoassay, e.g. ELISA or
immunoturbidometry

Size of iron stores

Total iron binding capacity (TIBC)

Colorimetric assay of amount of iron that can be bound to unsaturated transferrin in vitro; determination from transferrin concentration measured immunologically

Total capacity of circulating transferrin bound to iron

Transferrin saturation

Calculated from: Serum iron/TIBC

Saturation of <15% with high TIBC indicates iron deficiency

Transferrin receptor

Immunoassay, e.g. ELISA or
immunoturbidometry

Reflects balance between cellular iron requirements and iron supply

Body iron stores

Ratio of transferrin receptor to ferritin–[log(TfR/ferritin ratio)–2.8229]/0.1207

Measure of body iron status including iron deficits, status of storage iron and iron overload

Hepcidin

Immunoassay; mass spectrometry

Regulator of iron absorption from gut

Adapted from “Assessing the Iron Status of Populations,” WHO/CDC Technical Consultation, 2007, Second Edition.

“In a person who is healthy with no active inflammation, diagnosing iron deficiency is pretty straightforward,” explained Robert Means, Jr., MD, a hematologist, professor and senior associate chair of internal medicine at the University of Kentucky College of Medicine in Lexington. “The difficulty is in a person who is sick from other reasons. Their iron and transferrin levels may be depressed as a result of the inflammation, and the ferritin may be falsely elevated.” While ferritin concentrations of ≤15 µg/L are indicative of iron deficiency, when inflammation is present, levels can rise to as much as 100 µg/L even when iron stores are depleted. In the IBD guidelines, Gasche and his colleagues recommended serum ferritin <30 µg/L as an indicator of depleted iron stores in patients with inactive IBD, and <100 µg/L in patients with active disease.

Since ferritin can confound a clear diagnosis in the presence of inflammation, many clinicians simply interpret ferritin levels in the context of inflammatory markers like C-reactive protein (CRP) or erythrocyte sedimentation rate. WHO has suggested that α-1-antichymotrypsin may better reflect change in ferritin concentration during infection (Assessing the Iron Status of Populations, WHO, 2007, Second Edition).

Another way around equivocal ferritin results in the presence of inflammation is measurement of soluble transferrin receptor (sTfR), a truncated fragment of transferrin receptor that reflects erythropoiesis and is not affected by inflammation. sTfR is increased in patients with either IDA or both IDA and ACD but not ACD alone. Recent unpublished research found that the combination of ferritin, sTfR and the sTfR/log ferritin index (sTfR index) improved the detection of IDA and aided in the differential diagnosis of IDA and ACD in comparison to ferritin or sTfR alone. However, one of the study’s authors, Kari Punnonen, MD, PhD, cautioned about the instrumentation used with these biomarkers. “In order to use both sTfR and the sTfR index, one needs both assays run on the same platform. Otherwise, because of the different reference units one might not be able to determine proper cut-off values,” he explained, adding that the study in question used the Beckman Coulter automated Access sTfR assay. Punnonen is general manager and medical director of Eastern Finland Laboratory Center in Kuopio.

sFtR assays have not been standardized, and that may be one reason why the test has not been implemented widely, even though it has been available for a decade or more. However, a standardization effort is underway. Scientists at the British Health Protection Agency (HPA) have evaluated a lyophilized preparation of recombinant sTfR (rsTfR) in a sTfR-depleted serum matrix, coded 07/202, and in October were expected to present to the WHO Expert Committee on Biological Standardisation a study that had been carried out with manufacturers of sTfR kits. The study suggested that use of rsTfR 07/202 as a reference reagent would significantly reduce inter-method variability if manufacturers would adopt it, according to Susan Thorpe, PhD, principal scientist in the parenterals section of the biotherapeutics group at HPA.

Whether standardization will boost the use of sTfR remains to be seen. Although Punnonen has been a leading researcher in the field and his lab now performs about 6,000 sTfR assays per year versus 8,000 of ferritin, getting to that point has been a slow process. “I’m happy to see that sTfR is ordered in almost the same numbers as ferritin, but it’s taken 10 years. It takes a long time to change any clinical process,” he observed. An algorithm incorporating the use of transferrin, ferritin, sTfR and sTfR index has been proposed (NEJM 2005;352:1011–23).

Newer Measures of Iron Status

Still other researchers argue that better measures of IDA and ACD are the reticulocyte hemoglobin content (CHr) and proportion of hypochromic red cells (HYPO) in combination with the sTfR index. “Typical biomarkers are only indicators of iron supply but not of iron demand. These markers give no information about whether the cell really uses the iron for the synthesis of hemoglobin or enzymes of the respiratory chain,” explained Lothar Thomas, MD, professor of medicine at University Hospital Nordwest in Frankfurt, Germany. “The only proof is an increase in the hemoglobin value in the CBC. But this will last several weeks. The CHr is a real time parameter that indicates changes in iron demand of the cell within a week. Therefore I feel that CHr and HYPO are better parameters than the typical biomarkers of iron metabolism.” Thomas reported in 2002 that use of a diagnostic plot of CHr and sTfR index demonstrates the progressive stages of iron deficiency, regardless of whether inflammation is present (Clin Chem 2002;48:1066–1076). His lab uses the plot and an accompanying explanation to guide physicians in diagnosing and treating anemia.

While Punnonen agreed that use of CHr and HYPO is a viable strategy for assessing iron demand, he cautioned that the two parameters can be produced only by one type of analyzer, the Siemens Advia 120. “There are other systems which provide what they call comparable measures, but they’re based on more or less different concepts. So there’s no way one could compare the results between manufacturers or analyzers,” he said.

Even as researchers and laboratorians debate the merits of various iron status biomarkers, they are in agreement that development of robust and reliable commercial hepcidin assays could transform the diagnostic landscape. “The field is moving to hepcidin. It may be as popular in three to five years as ferritin is now,” predicted Thomas. However, he cautioned that values reported by immunoassay and mass spectrometry methods vary considerably. A recent review article bore this out: the seven methods examined used a wide range of normal values and had variable intra-assay precision and lower limits of detection (J Prot 2009 doi:10.1916/j.jprot.2009.08.003). Meanwhile, Dutch researchers have proposed an algorithm using transferrin saturation, sTfR and CRP to predict measured hepcidin levels (Blood Cells Mol Dis 2008;40:339–346).

Whether or not hepcidin assays hit the mainstream, experts suggested that laboratorians can do much to improve the work-up of anemia. Clark would like to see overall evidence-based guidelines for the diagnosis and treatment of IDA, ACD, and concomitant IDA and ACD. Means suggested that lab directors put on their educator hats. “Labs could be helpful by explaining that low serum iron is not necessarily diagnostic of iron deficiency and by indicating that in the setting of inflammation ferritin can be raised even in the absence of iron,” he said. “They could also help when clinicians come to them with cases they believe may be iron deficient but with indeterminate results. In such cases, the clinician needs help deciding what additional tests to do.”

Both Means and Gasche urged labs to re-evaluate the normal values reported for ferritin. “There’s no reason why women should have lower ferritin levels than men, but in most labs there is a difference in the normal ranges for men and women,” Gasche explained. For his part, Punnonen worked to reduce the use of iron and transferrin measurements in the differential diagnosis between IDA and ACD, and saw the number of tests drop by about one-third. “It used to be routine here to measure serum transferrin and iron in patients who had anemia, but iron is low in both IDA and ACD, so it’s of no use to measure iron,” he explained. “When someone has to decide if a patient has iron deficiency, one should use sTfR and ferritin and forget iron and transferrin.”

The many indicators of iron status, combined with changing analytical challenges, underscore the need for laboratorians to keep abreast of new developments and maintain an active dialogue with clinicians.

Dr. Means is a consultant to Beckman Coulter, and Dr. Punnonen has conducted researched funded by Beckman Coulter.