Serum Transferrin Receptor Concentrations Correlate More Strongly with Red Cell Indices than with Iron Parameters in Iron-Deficient Adolescents

Iron deficiency develops in sequential stages over a period of negative iron balance. These stages include the iron depletion phase (stage I), iron-deficient erythropoiesis (stage II) and iron deficiency anemia (stage III) [1]. During the iron depletion phase, iron stores are exhausted; however, neither anemia nor decrease of serum iron is present. In the stage of iron-deficient erythropoiesis, serum iron and serum ferritin levels are decreased; however, anemia and hypochromia are still not detectable. Increased serum transferrin receptor (sTfR) concentrations are usually observed in iron deficiency or in cases of iron depletion in tissues [2]. Because erythropoietic cells of bone marrow and circulating reticulocytes shed their transferrin receptors into the blood during their maturation sequence, measurement of sTfR has provided a new laboratory parameter of erythropoiesis [3, 4]. Effective erythropoiesis can be monitored by flow cytometric analysis of reticulocytes and reticulocyte subpopulations, such as low-, middle- and high-fluorescence reticulocytes (LFR, MFR and HFR, respectively) [5]. The relationship between sTfR concentrations and iron markers has been extensively studied. However, few studies have closely examined associations between sTfR concentrations and red cell indices, especially as compared to erythropoietic activities. In the present study, we investigated the correlation coefficients of red cell indices, reticulocyte subpopulations and iron markers versus sTfR concentrations, to ascertain which index most accurately reflects sTfR concentrations during three stages of iron deficiency in adolescents.A total of 475 adolescents (age range 13–16 years; male to female ratio 0.8:1) were investigated for the measurement of hemogram, iron profiles, reticulocyte subpopulations and sTfR concentrations. The subjects were divided into four groups: iron depletion phase (stage I, n = 127), iron-deficient erythropoiesis (stage II, n = 74), iron deficiency anemia (stage III, n = 105), and healthy controls (n = 169). This survey was explained to, and was approved by, both parents and directors at each educational center, and only volunteers were included in the study population. The study was approved by the Committee of Ethics of the Inha University Hospital, and informed consent was obtained from all subjects. Nonanemic subjects with a normal serum iron level (>50 µg/dl), but with a decreased ferritin concentration (<12 µg/l), were classified as being in the iron depletion phase (stage I). Iron-deficient erythropoiesis (stage II) was defined as serum ferritin concentration <12 µg/l and serum iron level <50 µg/dl without overt anemia.When the subjects who showed a decreased serum ferritin concentration and a decreased serum iron level also had a decreased hemoglobin level (<12 g/dl), we considered them to have iron deficiency anemia (stage III). Venous blood was drawn into iron-free evacuated tubes. Complete blood cell count and reticulocyte subpopulations were measured with EDTA-anticoagulated blood within 3 h after collection. Routine complete blood cell count and red cell indices were determined with an electronic counter (SE 9000, Sysmex, Kobe, Japan). Reticulocytes and their subpopulations were analyzed by flow cytometry (R-3000, Sysmex). The reticulocyte maturity index (RMI), which is known to be the earliest and most sensitive predictor of erythropoiesis [6], was calculated from the equation RMI = [(MFR + HFR) × 100]/LFR, and was expressed as a percentage [7]. Serum iron and total iron-binding capacity (TIBC) were assayed with a chemical analyzer (Hitachi 747, Hitachi, Tokyo, Japan), and ferritin was measured by the chemiluminescence method (ACS 180, Chiron, Mass., USA). Soluble transferrin receptor was measured by an immunoenzymometric method (IDeATM sTfR, Orion Diagnostica, Espoo, Finland). The sTfR test was performed as described previously [8]. The Mann-Whitney U test was used to determine the differences in mean values between two groups. Correlation coefficients were analyzed by Spearman’s method. A p value ≤0.01 was considered statistically significant.There were no significant differences in reticulocyte parameters and hematologic markers between the iron depletion group (stage I) and healthy controls, nor between the iron depletion group (stage I) and iron-deficient erythropoiesis group (stage II). However, the iron deficiency anemic group (stage III) did show significantly lower red cell indices and significantly higher MFR and RMI values than did the iron depletion or iron-deficient erythropoiesis groups (table 1). These results suggest that a decrease in serum ferritin or serum iron concentrations does not affect reticulocyte subpopulations; however, once a subject has attained a state of frank anemia, immature reticulocyte production seems to be elevated. These findings are in accordance with our previous study showing that reticulocyte subpopulations and RMI rise as iron deficiency proceeds [9].Skikne et al. [10]reported that there was no change in sTfR concentrations during the phase of storage iron depletion. Similarly, in our study, there were no significant differences in sTfR concentrations between the iron depletion group (stage I) and healthy controls. The sTfR concentrations began to increase significantly from the iron-deficient erythropoiesis group (stage II); the sTfR concentrations averaged 3.1 ± 0.7 mg/l in the iron-deficient erythropoiesis group, which were significantly higher than those of the iron depletion group (2.2 ± 0.8 mg/l; p < 0.01). However, no significant changes in immature reticulocyte fractions and red cell indices were observed in the iron-deficient erythropoiesis group compared to the iron depletion group. These results suggest that the sTfR concentration may be a more sensitive guide for body iron status than reticulocyte parameters or red cell indices.As shown in table 2, the correlation coefficients between sTfR concentrations and red cell indices were higher than those of iron markers and reticulocyte subpopulations versus sTfR concentrations through three stages of iron deficiency. These associations were also observed even in healthy controls with no evidence of iron deficiency or iron deficiency anemia, although the correlations were weak. Among the red cell indices, the sTfR correlated closely with mean corpuscular volume (MCV) (r = –0.52, p < 0.01) during the iron depletion phase and correlated strongly with mean corpuscular hemoglobin (MCH) during iron-deficient erythropoiesis (r = –0.63, p < 0.01) and iron deficiency anemia (r = –0.69, p < 0.01). The correlation coefficients between sTfR concentrations and red cell indices increased gradually over a period of negative iron balance, as iron deficiency progressed. Therefore, it is conceivable that changes in sTfR concentrations are more closely associated with red cell indices than with iron markers or reticulocyte subpopulations during adolescence. Our data are in general agreement with the results of a previous report that sTfR concentrations showed a significant correlation with serum ferritin, TIBC, MCV and MCH [11]. In contrast, Kivivuori et al. [12]demonstrated that there were no associations between sTfR concentrations and the values of hemoglobin, packed cell volume, MCV, MCH, serum iron or serum ferritin. These discrepancies may have derived from the differences in age, gender and nutritional status in the different study populations. Kivivuori et al. [12]studied healthy prepubertal and early pubertal boys without iron deficiency and did not take into account the different stages of iron deficiency, while in the present study, we investigated boys and girls aged 13–16 years who were mostly in the stages of iron deficiency or iron deficiency anemia.In conclusion, sTfR concentrations were more strongly correlated with red cell indices than with iron markers or reticulocyte subpopulations in iron-deficient adolescents, suggesting that red cell indices seem to more accurately reflect sTfR concentrations in these subjects, at least when compared to iron markers or reticulocyte parameters.