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Technical Briefs |
Departments of
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Clinical Chemistry,
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Medicine,
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Pediatrics,
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Pediatric Surgery,
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Hematology, and
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Paavo Nurmi Centre, Sports and Exercise Medicine Unit, Department of Physiology, Turku University Central Hospital, FIN-20521 Turku, Finland
aaddress correspondence to this author at: Department of Clinical Chemistry, Turku University Central Hospital, Central Laboratory, PL 52, FIN-20521 Turku, Finland; fax 358-2-2613920, e-mail pauli.suominen{at}tyks.fi
Serum soluble transferrin receptor (sTfR) has been established in recent years as a powerful tool for detecting iron deficiency (ID) in adults, especially in distinguishing between iron deficiency anemia (IDA) and anemia of chronic disease (1)(2)(3)(4)(5)(6)(7)(8). Investigations regarding sTfR as a measure of iron status in infants and children have provided promising results, including evidence that, in infants, sTfR concentrations may be superior to ferritin measurements in diagnosing ID (9). However, to date, concrete reference values and other decision-supporting limits for the commercially available methods have been virtually absent, and the age-relatedness of sTfR concentrations, although introduced as a concept, has not been unequivocally modeled statistically (10)(11)(12)(13)(14)(15).
In this study we measured the sTfR concentrations from a selection of 301 healthy children, 6 months to 18 years of age, using a commercially available automated immunoturbidimetric assay. We then used a regression-based method to construct age-dependent 2.5% and 97.5% reference limits for sTfR as well as 95% confidence intervals for these limits in our population (16). The purpose was to demonstrate consistent age-dependent changes in sTfR concentrations and to establish appropriate reference limits to enable the use of sTfR measurements in routine pediatric clinical practice.
A total of 301 children (130 boys and 171 girls; age range, 6 months to 18 years) were included in the healthy population of this study. The selection was made on the basis of detailed anamnesis and laboratory tests. Febrile episodes during the preceding 6 weeks, diet restrictions, chronic inflammatory or renal diseases, hematological diseases, recent iron supplementation, ongoing inflammation (C-reactive protein >10 mg/L; erythrocyte sedimentation rate >6 mm/h), anemia or abnormal red cell indices (17), and low ferritin concentrations (<10 µg/L) were considered criteria for exclusion from the study population. A population of 64 healthy boys and 38 girls (6 months to 8 years of age) was selected from patients undergoing elective short-term surgery or procedures (superficial hemangiomas, nevi, and other cosmetic surgeries; vesico-ureteral reflux, cystoscopies) in the Turku University Central Hospital. Additionally, 133 healthy girls and 66 boys (8–18 years of age) were recruited from local schools and athletic clubs.
We obtained bone marrow and serum samples from 24 children (13 boys and 11 girls; age range, 1.25–16.5 years), who were admitted to the hospital because of severe anemia, and analyzed them for iron and sTfR, respectively. The results were used to evaluate the validity of the reference limits for each age group by means of a "gold standard".
The blood samples were obtained before any intravenous infusions. The normality of the hemoglobin and ferritin was evaluated according to Dallman and Siimes (17) and Lockitch et al. (18). Written informed consent was obtained from the parents of all subjects, and the study design was approved by the Joint Committee of Ethics of the Turku University Central Hospital and University of Turku.
Blood counts were measured using an automated analyzer (Technicon H2; Bayer Diagnostics). sTfR assays were performed using an automated immunoturbidimetric assay (IDeA® sTfR-IT; Orion Diagnostica) on a Hitachi 917 analyzer. The method has been described in detail elsewhere (19).
P values for between-gender difference and differences between age groups were derived by the Student t-test using the Windows for WorkgroupsTM software package (Microsoft Corporation). The reference limits and 95% confidence intervals were calculated using SAS® 6.10 software, as was the P value for the significance of age. Logarithmic transformation was applied to sTfR concentrations. The Shapiro–Wilk statistic and graphic analysis of residuals were used to assess the normality and constancy of residuals. Diagnostic measures (Cook’s statistic, high leverage points, dffits, and dfbetas) were also calculated (16). Reference limits and confidence intervals were constructed as described by Virtanen et al. (16).
No significant between-gender difference in sTfR concentrations was
observed (P = 0.37) in infants and children up to 10
years of age. A significant between-gender difference (P
<0.001) was observed in adolescents (10–16 years), but this
difference was evened out by excluding subjects with ferritin
concentrations <10 µg/L (P = 0.28). A consistent
age-related decrease in sTfR concentrations could be demonstrated (Fig. 1
and Table 1
). Differences between children 0.5–4 and 4–10 years of age
(P <0.001) and between children 4–10 and 10–16 years of
age (P <0.01) were significant. The decrease was such that
the 95% interval in the children 16 years of age paralleled the
respective adult values obtained in a previous study (0.9–2.3 mg/L)
(19). The effect of age was statistically significant
(P <0.0001) with a coefficient of determination
(R2) of 19% (P <0.0001).
Of the 24 anemic patients whose iron status was verified by a bone
marrow iron stain, 10 presented with exhausted bone marrow iron stores.
The iron status was correctly determined in 23 of 24 patients by their
sTfR concentrations (area under ROC curve, 0.9196; SE, 0.0843).
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Measurement of sTfR has been brought up as a potentially useful tool for the pediatrician for several reasons. ID is known to be a common condition in infants, children, and adolescents, although the prevalence of frank IDA has decreased significantly. Because of their relatively rapid growth, the amount of storage iron in children often is low, thus causing ferritin concentrations to frequently lie close to the iron-deficient range. Measurement of sTfR offers an advantage over ferritin in that it accurately detects iron-deficient erythropoiesis and can therefore be used to signal the point where subclinical ID progresses from storage iron depletion to depletion of the functional compartment (8). The importance of this property is underlined by the recent associations made between subclinical ID and impairments in psychomotor development (20)(21)(22)(23). Furthermore, the results are not influenced by acute-phase reactions, and the sample volume required to perform the test in the commercially available assays is small (6)(19). It is therefore not surprising that sTfR has recently been shown to be superior to ferritin in distinguishing ID in infants in a study by Olivares et al. (9).
It has been reported that sTfR concentrations decrease with age (14)(15). In this study, our aim was to construct an accurate statistical model for the suggested age relatedness of sTfR concentrations and to derive decision-supporting cutoff values for ID on this particular assay. We investigated whether the method for regression-based 2.5% and 97.5% limits and confidence intervals recently introduced by Virtanen et al. (16) could be applied to calculating pediatric reference limits and confidence intervals of sTfR. A gold standard, namely bone marrow examination, was used to test the clinical validity of these reference limits.
The regression-based model showed that the age dependency of sTfR concentrations is quite substantial (R2 = 19%; P <0.0001). The decrease was such that the reference limits merged with adult values at the age of 16 years, which was therefore established as the age limit between adults and adolescents. A relatively small (n = 301) population seems to have been statistically sufficient to establish the 2.5% and 97.5% reference limits as implied by the relatively narrow 95% confidence intervals. Conventional partitioning methods would have required a considerably larger population size to achieve a sufficient number of well-represented subgroups (16).
Because borderline iron status (subclinical stages of ID) is known to be quite prevalent in the pediatric population, using hemoglobin concentrations as the only criterion for normality could have unduly compromised the sensitivity of sTfR reference values for ID (9). We used a cutoff value for ferritin concentrations (<10 µg/L) to exclude subjects with obvious subclinical storage iron deficits (18). Using the upper (97.5%) reference limits of sTfR as cutoff values for ID in the separate age groups, we achieved a good distinction between IDA and anemia of other causes in the 24 patients who were subjected to bone marrow examination. The results indicated that although the sensitivity of sTfR was probably improved, the specificity remained uncompromised by the exclusion of patients with decreased ferritin concentrations. Although the population was small, the clinical accuracy (area under the ROC curve, 0.9196; SE, 0.0843) was similar to that reported for adults (6)(19), which indicates that the reference limits presented here may be useful in clinical work.
We conclude that these age-dependent reference intervals could be implemented for sTfR in children 6 months to 16 years of age on the IDeA sTfR-IT assay to enhance the differential diagnosis of IDA.
Acknowledgments
This study was supported in part by grants from the Orion Research Foundation and the Finnish Foundation of Blood-related Diseases.
References
The following articles in journals at HighWire Press have cited this article:
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  H. Knox-Macaulay, D. Gravell, and F. Elender Serum Transferrin Receptor Status of Healthy Adult Arabs Ann. Clin. Lab. Sci., January 1, 2007; 37(1): 57 - 62. [Abstract] [Full Text] [PDF]
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 R. Crowell, A. M. Ferris, R. J. Wood, P. Joyce, and H. Slivka Comparative Effectiveness of Zinc Protoporphyrin and Hemoglobin Concentrations in Identifying Iron Deficiency in a Group of Low-Income, Preschool-Aged Children: Practical Implications of Recent Illness Pediatrics, July 1, 2006; 118(1): 224 - 232. [Abstract] [Full Text] [PDF]
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 J. M Schneider, M. L Fujii, C. L Lamp, B. Lonnerdal, K. G Dewey, and S. Zidenberg-Cherr Anemia, iron deficiency, and iron deficiency anemia in 12-36-mo-old children from low-income families Am. J. Clinical Nutrition, December 1, 2005; 82(6): 1269 - 1275. [Abstract] [Full Text] [PDF]
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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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P. Halonen, J. Mattila, P. Suominen, T. Ruuska, M. K. Salo, and A. Makipernaa Iron Overload in Children Who Are Treated for Acute Lymphoblastic Leukemia Estimated by Liver Siderosis and Serum Iron Parameters Pediatrics, January 1, 2003; 111(1): 91 - 96. [Abstract] [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. Wasen, P. Suominen, R. Isoaho, K. Mattila, A. Virtanen, S.-L. Kivela, and K. Irjala Serum Cystatin C as a Marker of Kidney Dysfunction in an Elderly Population Clin. Chem., July 1, 2002; 48(7): 1138 - 1140. [Full Text] [PDF]
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