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Technical Briefs |
1
Laboratory of Hematology; Bosch Medicentrum, Nieuwstraat 34, 5211 NL ‘s-Hertogenbosch, The Netherlands
2
Department of Clinical Chemistry; St. Anna Ziekenhuis, 5340 BE Oss, The Netherlands
3
Department of Clinical Chemistry; Akademisch Ziekenhuis,
3015 GD Rotterdam, The Netherlands
4
Department of Hematology; Vrije Universiteit, 1081 HV Amsterdam, The
Netherlands
a author for correspondence: fax 31-73-6162958
The conventional laboratory test for diagnosing iron deficiency anemia (IDA) is the measurement of serum ferritin. In healthy persons, serum ferritin is directly proportional to body iron stores. In the presence of anemia, a value <20 µg/L is regarded as evidence of IDA (1). In patients with anemia of chronic disease (ACD), an increase in serum ferritin, unrelated to iron storage status, occurs. Serum ferritin concentrations usually exceed 100 µg/L when iron stores are adequate, but they may still be within the reference interval in the presence of coexisting iron deficiency (1). The assay lacks sensitivity in diagnosing IDA in patients with ACD, as well as in patients with acute infections and with liver diseases.
The best established method for evaluating iron stores is examination of bone marrow iron, but this test is cumbersome and expensive.
Since the early 1970s, measurement of the zinc protoporphyrin concentration (ZPP) in erythrocytes has been used as test for iron deficiency. When there is insufficient iron for incorporation into protoporphyrin IX to form heme, ZPP increases, indicating iron-deficient erythropoiesis (2). Most authors have reported a high sensitivity in diagnosing IDA, irrespective of the presence of complicating diseases (3)(4)(5). However, disturbed incorporation of iron into protoporphyrin IX can also occur in other diseases, such as ACD, hemolytic anemias, bone marrow diseases, and lead intoxication. The specificity of ZPP in diagnosing IDA is limited (6)(7).
In recent years, the measurement of soluble transferrin receptor (sTfR) fragments has been introduced for the identification of iron deficiency (8). The concentration of sTfR is directly proportional to the number of red cell precursors. An exception to this rule is in patients with IDA. A reduction in iron supply leads to up-regulation of transferrin receptor synthesis, producing an increase in sTfR concentration. Several authors have reported a good sensitivity in diagnosing IDA in patients without complicating diseases (9)(10)(11)(12). However, the specificity of the test is negatively influenced by the fact that in cases of increased erythropoietic activity and in cases of disturbed iron transport to and in the cells, the sTfR concentration can also be increased (9)(10)(13).
ZPP measurements have been included in the anemia investigation protocol of our laboratory for several years. In this study, we also performed sTfR measurements and compared the results with ZPP.
In 232 adult patients (171 women, 61 men) referred for investigation of
anemia, hemoglobin (Hb), mean corpuscular volume (MCV), reticulocyte
count, ZPP, serum ferritin, and sTfR were measured. In patients with
reticulocyte counts >25
, haptoglobin was determined, and in
patients with MCV >100 fL, vitamin B12 and folic
acid concentrations were measured. Anemia was defined as Hb <137 g/L
in men and <121 g/L in women. Hb, MCV, and ZPP measurements were
performed in peripheral blood in EDTA. Hb and MCV were measured with an
automated cell counter (NE 8000; Sysmex). ZPP concentrations were
determined with a front-face hematofluorometer (AVIV Biomedical). To
avoid the influence of plasma components, the erythrocytes were washed
before ZPP was measured. ZPP measured as the ZPP/Hb ratio is expressed
in µmol/mol heme. The reference interval for ZPP in our hospital is
15–55 µmol/mol heme (mean ± 2 SD). sTfR assays were performed
in plasma, using a kit based on a polyclonal antibody in a sandwich
immunoassay (Eurogenetics). To find the reference values, plasma
samples of 70 healthy adult volunteers were analyzed. We found
reference values of 310–745 kilounits/L (mean ± 2 SD).
Serum ferritin measurements were performed using an enzyme-linked assay
(IMx; Abbott). Linear regression analysis was performed using
commercial software (SPPS).
We found decreased haptoglobin concentrations in three patients, indicating hemolysis. They showed increased sTfR concentrations, and two of these patients had increased ZPP. In seven patients, MCV was >100 fL. None of these patients showed decreased vitamin B12 or folic acid concentrations.
We divided the patients into four groups, according to serum ferritin
concentrations. Table 1
shows the number of patients with normal and increased ZPP and
sTfR concentrations in the four groups.
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Using a definition of IDA as anemia in combination with a serum ferritin concentration <20 µg/L, we found sensitivities for ZPP and sTfR in diagnosing IDA of 81% and 78%, respectively. When we assumed that patients with serum ferritin concentrations >100 µg/L did not suffer from IDA, the specificities of ZPP and sTfR in diagnosing IDA were 59% and 77%, respectively. Similar specificities were found when we took serum ferritin concentrations >200 µg/L as evidence of anemia not caused by iron deficiency (57% for ZPP and 76% for sTfR).
In Fig. 1
A, sTfR is plotted against ZPP in patients with serum ferritin
concentrations <20 µg/L. A linear correlation (r =
0.89; P <0.001) was found. In Fig. 1B
, ZPP and sTfR are
plotted against MCV in patients with serum ferritin concentrations <20
µg/L. A linear correlation between MCV and ZPP (r =
-0.75; P <0.001) as well as MCV and sTfR
(r = -0.72; P <0.001) was found. Likewise,
a linear correlation between Hb and ZPP (r = -0.71;
P <0.001) as well as between Hb and sTfR (r
= -0.70; P <0.001) was found in patients with serum
ferritin values <20 µg/L (data not shown).
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The linear correlation between ZPP and sTfR concentrations in the patients with serum ferritin values <20 µg/L indicates that both markers reflect the severity of iron-deficient erythropoiesis in a similar way in IDA.
Ten of the 86 patients with serum ferritin concentrations <20 µg/L showed both sTfR and ZPP concentrations within the reference values. None of these patients showed microcytosis. It is likely that in these iron-depleted patients, anemia was not caused by iron deficiency but by other factors, such as acute bleeding or hemodilution in pregnancy. When we excluded these patients, the sensitivities of ZPP and sTfR in diagnosing IDA increased to 92% and 88%, respectively. Nine patients with serum ferritin concentrations <20 µg/L and mild anemia showed increased ZPP concentrations (mean, 70 µmol/mol heme; range, 58–105 µmol/mol heme) with sTfR concentrations within the reference values. In IDA, the decrease in Hb concentration is caused by diminished erythropoietic activity and impaired hemoglobinization of the cells. The sTfR concentration in IDA is the net effect of the decrease in erythropoietic activity and the increase in density of surface transferrin receptors on the erythropoietic cells. Especially in patients with low erythropoietic activity and mild iron-deficient erythropoiesis, sTfR concentrations may not rise to abnormal concentrations. Six mildly anemic patients with serum ferritin concentrations <20 µg/L showed increased sTfR concentrations (mean, 875 kilounits/L; range, 752-1039 kilounits/L) without increased ZPP (mean, 45 µmol/mol heme). Because ZPP is fixed in circulating erythrocytes, its concentration in whole blood will change only when new erythrocytes enter the circulation. The ZPP concentration in the erythrocytes reflects the severity of iron deficiency of the erythropoietic cells during the prior 3 months; therefore, ZPP concentrations may be within the reference interval in recently developed IDA. The ZPP and sTfR results in the individual patient might give insight into the development of IDA for that particular patient.
In earlier studies, we found a sensitivity of 95% for ZPP in diagnosing IDA. Other authors have reported similar results (4)(5). For sTfR, a sensitivity of 90% has been reported (11)(12). In this study, we found a lower sensitivity for both markers. This may reflect the fact that some patients with severe IDA were not included in this study because some physicians did not request an anemia investigation according to the laboratory protocol for such patients.
Taking a serum ferritin concentration >100 µg/L as cutoff value for excluding IDA, the specificity in diagnosing IDA was 77% for sTfR and 59% for ZPP, and similar values were found for serum ferritin concentrations >200 µg/L. The low specificities of both tests render them insufficient to be used as a single test for identification of iron deficiency in unselected patients, as was reported by others (14)(15)(16).
As shown in Table 1
, 18 of the 31 patients with serum ferritin
concentrations >100 µg/L and 11 of the 30 patients with serum
ferritin concentrations of 20–100 µg/L showed increased ZPP with
normal sTfR concentrations, indicating that iron-deficient
erythropoiesis in these patients did not cause increased transferrin
receptor synthesis. In patients with ACD, an impaired ability to
respond to erythropoietin, blunted erythropoiesis, and decreased
transferrin receptor expression may cause low sTfR concentrations
(17). The sensitivity of sTfR in diagnosing concomitant IDA
in such patients may be negatively influenced by these phenomena.
Indeed, in patients with ACD and concomitant iron deficiency, mean sTfR
concentrations tend to be lower than in patients with uncomplicated IDA
(12)(18). More studies should be performed to
assess the sensitivity of sTfR in diagnosing IDA in patients with
concomitant diseases. If the sensitivity is as good as in patients with
uncomplicated IDA, it might be useful to perform this test in patients
in whom ZPP and serum ferritin concentrations are inconclusive. In
patients with sTfR concentrations within the reference interval,
examination of bone marrow iron then can be omitted.
References
The following articles in journals at HighWire Press have cited this article:
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  M. B Zimmermann, L. Molinari, F. Staubli-Asobayire, S. Y Hess, N. Chaouki, P. Adou, and R. F Hurrell Serum transferrin receptor and zinc protoporphyrin as indicators of iron status in African children Am. J. Clinical Nutrition, March 1, 2005; 81(3): 615 - 623. [Abstract] [Full Text] [PDF]
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