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Technical Briefs |
1
Clinical Chemistry and
2
Hematology, Turku University Central Hospital, FIN-20521 Turku, Finland;
3
Department of Clinical Chemistry, Kuopio University Hospital, 70210 Kuopio, Finland,
4
Orion Diagnostica, 02101 Espoo, Finland;
a author for correspondence: Turku University Hospital, Central Laboratory, P.O. Box 52, FIN-20521 Turku, Finland
Cellular iron uptake is mediated by transferrin receptors (TfRs), which are present on virtually all mammalian cells. A soluble form of TfR (sTfR) can be detected in serum, the concentration of which is closely related to erythroid TfR turnover. The prime determinants of sTfR concentration are cellular iron demands and the erythroid proliferation rate (1). The measurement of sTfR has been introduced as a powerful tool for the diagnosis of iron deficiency (ID) in a variety of clinical situations (1)(2)(3)(4)(5)(6)(7)(8). Up-regulation of the expression of cellular TfR occurs as a result of an inadequate tissue supply of iron or increased cellular demand for iron, for example, in the context of chronic loss of blood and compensatorily activated erythropoiesis. In a clinical setting, sTfR measurements offer an attractive amendment to the repertoire of indices of iron status because it has been shown to sensitively detect iron-deficient erythropoiesis early in developing ID and to retain its specificity to changes in iron status irrespective of the concurrent inflammatory status (2)(3)(6)(8)(9). It has been suggested, however, that in cases of noncomplicated ID, the use of sTfR measurements does not provide any relevant additional information compared with ferritin measurements (10). The sTfR concentration has also been shown to be a more sensitive and less variable index of iron status than the more conventional serum iron, transferrin, and total iron-binding capacity (8)(11)(12).
Several commercially available methods have been introduced to measure sTfR (7)(9). To date, the available methods have been manually performed enzyme immunoassays, which are fairly laborious and time-consuming and require special equipment. Another obstacle for wider implementation of sTfR measurements is the lack of uniform calibrators (7)(9). This study was undertaken to evaluate the analytical and clinical performance characteristics of a new automated immunoturbidimetric assay for sTfR (IDeA® sTfR-IT; Orion Diagnostica). This method is a successor of the manual enzyme immunoassay by the same manufacturer, namely the IDeA® sTfR IEMA assay (9). We believe that the automated method described here promotes a more widespread adoption of sTfR as a routine laboratory measurement and renders the method available even to small laboratories.
In this study, we tested the IDeA sTfR-IT application on the Hitachi 911 analyzer (Roche-Boehringer Mannheim). Applications are also available for the Hitachi 917 and Cobas Mira analyzers.
The method requires 20 µL of sample, 250 µL of IDeA sTfR-IT buffer, and 20 µL of IDeA sTfR-IT reagent. The reagent consists of polyclonal anti-human TfR F(ab)2 antibodies bound to SVBC-latex particles. In the presence of sTfR, the latex particles are agglutinated in a dose-dependent manner, causing increased turbidity. The increase in turbidity is detected at 660 nm. The amount of sTfR in the sample is determined by means of a calibration curve based on five ready-to-use calibrators, which may be also prepared by automatic dilution of the highest calibrator, which contains 8–9 mg/L sTfR. Two additional controls (IDeA sTfR-IT control low and control high) are also provided. Calibrators and controls are derived from human serum, and sTfR is therefore present bound to transferrin, forming complexes comprising two transferrin and two sTfR molecules. The insert states that suitable assay specimens are fresh serum, EDTA plasma, heparin-treated plasma, and citrate plasma. Volume correction for the sTfR result is needed if liquid anticoagulant-containing sample tubes (EDTA and citrate) are used. The results from the IDeA sTfR-IT method were compared with results from the Quantikine® IVD® sTfR assay (R&D), which has been approved by the Food and Drug Administration (13).
In addition, the clinical performance of the method was evaluated by using several previously described populations of healthy individuals as well as anemic patients. Sera from 78 patients with iron deficiency anemia (IDA), anemia of chronic disease, and ID in the presence of a complicating inflammatory condition (COMBI) were assayed to evaluate the ability to detect ID in the presence of COMBI. The patient material has been described in detail elsewhere (9). An adult reference interval (95%) was obtained from a population of healthy, nonanemic adults, consisting mainly of laboratory staff and other related personnel. The pediatric reference population consisted of 102 healthy males (n = 64) and females (n = 38), who were undergoing selective short-term surgery in the Turku University Central Hospital. The selection of patients was made on the basis of detailed anamnesis and laboratory tests to exclude the effect of manifest ID, acute or chronic inflammatory conditions, and dietary restrictions. The reference values were calculated by a regression-based method described elsewhere (14). The cutoff value for iron-deficient erythropoiesis was calculated from the population and as described in a previous paper from our group (8).
Statistical analyses were carried out using the SPSS 7.5 for Windows software (SPSS) and the GraphROC for Windows software (15). The Bland-Altman analysis was performed as described elsewhere (16).
The detection limit of the method was 0.05 mg/L, as determined by the concentration corresponding to mean 4 SD above absorbance value of the zero calibrator (9 g/L NaCl). The intraassay CV was 0.3–1.8% for four serum samples (0.6–7.7 mg/L) with 10 replicates. The interassay CVs calculated for four serum samples in 10 subsequent assays over 2 weeks were 2.4–3.5%. The linearity of the method was tested by serially diluting serum samples. The linear range spanned the entire reportable range of the assay (0.3–8.5 mg/L). The measured results were 90.5–107% of expected values within the linear range. Possible interference by potential interfering factors was investigated by adding up to 5 g/L hemoglobin (as hemolysate), up to 400 µmol/L nonconjugated bilirubin, up to 30 mg/L L-ascorbic acid, up to 23 mmol/L triglycerides (Intralipid), and up to 5.0 x 105 IU/L rheumatoid factor to serum samples. No interference was observed with these concentrations. Samples with up to 1 x 106 IU/L rheumatoid factor diluted linearly in IDeA sTfR-IT assay.
The 95% reference interval for healthy nonanemic adults was 0.85–2.30
mg/L, whereas the corresponding limits for the manual IDeA method by
the same manufacturer were 1.3–3.3 mg/L (9). The pediatric
age-related reference limits for the automated method are presented in
Table 1
. The cutoff concentration for iron-deficient erythropoiesis in
a population of 65 healthy adults, from which iron deficiency was
excluded by peroral iron supplementation (8), was 1.90 mg/L.
For the method's ability to distinguish between iron deficiency (IDA
and COMBI) and anemia related to other causes (anemia of chronic
disease), the area under the ROC curve (AUCROC)
was 0.987 (SE, 0.009), similar to the value observed with the manual
IDeA method (0.973; SE, 0.014) (9). The
AUCROC values for separating anemia of chronic
disease and COMBI patients were 0.950 (SE, 0.010) and 0.918 (SE,
0.047), respectively. The optimal cutoff value for ID as calculated by
the GraphROC for Windows software in this material was 2.40 mg/L. The
results from 50 patient samples by the sTfR IDeA-IT assay correlated
well with the results obtained with the Quantikine sTfR assay, which
thus far is the only sTfR assay that has been approved by the Food and
Drug Administration (Fig. 1
). However, the Bland-Altman analysis revealed differences
between the two methods that are unacceptable for direct comparison of
clinical data (Fig. 1
). The area between the lines of agreement
is wide, and separate reference values as well as cutoff values for the
two methods are therefore warranted.
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The IDeA sTfR-IT assay is an analytically adequate method, superior to its manual ELISA predecessors in several aspects. Assay time is reduced from ~3 h to 10 min, and the intra- and interassay CVs are two- to threefold lower than the CVs for commercial sTfR-ELISAs. The antibody (polyclonal) used in this new method is different from the antibody in the manual method (monoclonal) produced by the same manufacturer, which accounts for the different values of the results. No sign of matrix effects was seen in the IDeA sTfR-IT assay when patient samples presenting compromised linearity on the manual IDeA IEMA assay were tested with the IDeA sTfR-IT assay (data not shown).
The measurement of sTfR has become a widely used tool in assessing iron
status, but its use has mainly been restricted to academic and
scientific research. The need for special equipment has been a
major setback to the usefulness of the manually operated sTfR assays
based on enzyme immunoassay format. The method evaluated here can be
operated on several commonplace clinical chemistry automated analyzers
and is, therefore, an attractive upgrade to the repertoire of markers
of ID in virtually any laboratory. Although calibration in this new
automated IDeA sTfR-IT method is based on human serum sTfR, which is an
important step toward making sTfR even more widely accepted and
documented, the pressure toward establishing international guidelines
for the calibration of sTfR assays is obvious. This issue is
underscored by the results presented here. The good correlation between
the IDeA sTfR-IT and Quantikine assays suggests that they probably
measure the same complex (sTfR-transferrin heterodimer in calibrator);
however, the difference in results is clinically unacceptable, as
indicated by the wide area between the lines of agreement (Fig. 1
). We
believe that the difference observed between these two assays may be
attributable not only to the different antibodies used but to
differences in the preparation of the calibrator specimen and could,
perhaps, be overcome by establishing a common calibrator.
References
The following articles in journals at HighWire Press have cited this article:
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  R. Vikstedt, P. von Lode, T. Takala, K. Irjala, O. Peltola, K. Pettersson, and P. Suominen Rapid One-Step Immunofluorometric Assay for Measuring Soluble Transferrin Receptor in Whole Blood Clin. Chem., October 1, 2004; 50(10): 1831 - 1833. [Full Text] [PDF]
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T. I. Takala, P. Suominen, R. Isoaho, S.-L. Kivela, M. Lopponen, O. Peltola, A. Rajamaki, and K. Irjala Iron-Replete Reference Intervals to Increase Sensitivity of Hematologic and Iron Status Laboratory Tests in the Elderly Clin. Chem., September 1, 2002; 48(9): 1586 - 1589. [Full Text] [PDF]
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E. J. Lee, E.-J. Oh, Y.-J. Park, H. K. Lee, and B. K. Kim Soluble Transferrin Receptor (sTfR), Ferritin, and sTfR/Log Ferritin Index in Anemic Patients with Nonhematologic Malignancy and Chronic Inflammation Clin. Chem., July 1, 2002; 48(7): 1118 - 1121. [Full Text] [PDF]
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 S. Muslimatun, M. K. Schmidt, C. E. West, W. Schultink, J. G. A. J. Hautvast, and D. Karyadi Weekly Vitamin A and Iron Supplementation during Pregnancy Increases Vitamin A Concentration of Breast Milk but Not Iron Status in Indonesian Lactating Women J. Nutr., October 1, 2001; 131(10): 2664 - 2669. [Abstract] [Full Text] [PDF]
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P. Suominen, A. Virtanen, M. Lehtonen-Veromaa, O. J. Heinonen, T. T. Salmi, M. Alanen, T. Mottonen, A. Rajamaki, and K. Irjala Regression-based Reference Limits for Serum Transferrin Receptor in Children 6 Months to 16 Years of Age Clin. Chem., May 1, 2001; 47(5): 935 - 937. [Full Text] [PDF]
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S. Muslimatun Weekly Supplementation with Iron and Vitamin A during Pregnancy Increases Hemoglobin Concentration but Decreases Serum Ferritin Concentration in Indonesian Pregnant Women J. Nutr., January 1, 2001; 131(1): 85 - 90. [Abstract] [Full Text]
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 M. Olivares, T. Walter, J. D Cook, E. Hertrampf, and F. Pizarro Usefulness of serum transferrin receptor and serum ferritin in diagnosis of iron deficiency in infancy Am. J. Clinical Nutrition, November 1, 2000; 72(5): 1191 - 1195. [Abstract] [Full Text] [PDF]
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