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Annals of Clinical & Laboratory Science 33:279-284 (2003)
© 2003 Association of Clinical Scientists

Associations Between Serum Transferrin Receptor Concentrations and Erythropoietic Activities According to Body Iron Status

Jong Weon Choi and Soo Hwan Pai
Department of Laboratory Medicine, College of Medicine, Inha University, Inchon, South Korea

Address correspondence to Jong Weon Choi, MD, PhD, Dept. of Laboratory Medicine, Inha University Hospital, 7-206, 3-ga, Shinheung-dong, Jung-gu, Inchon, 400-711, South Korea; tel 82 32 890 2503; fax 82 32 890 2529; e-mail jwchoi{at}inha.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study investigated the associations between serum transferrin receptor (sTfR) concentrations and erythropoietic activities during 3 stages of iron deficiency in humans. Serum iron markers, fluorescent intensity of reticulocytes, and sTfR concentrations were measured in 227 prepubescent children, age 9 to 12 yr. Reticulocyte subpopulations were analyzed by flow cytometry and sTfR concentrations were measured by enzyme immunoassay. Mean values of middle-fluorescence reticulocytes (MFR), reticulocyte maturity index (RMI), and sTfR concentrations were significantly higher in iron-deficiency anemia subjects than in healthy controls. Reticulocyte subpopulations increased gradually, as body iron status diminished; the mean values of MFR and RMI in subjects with serum ferritin concentrations <4.0 µg/L were 3-fold higher than those in healthy controls (p <0.01). Correlation coefficients of MFR and RMI vs log ferritin values (r = 0.43 and r = 0.42) were higher than those of MFR and RMI vs sTfR concentrations (r = 0.24 and r = 0.27) in iron-deficiency anemia subjects. In summary, iron deficiency leads to increased production of immature reticulocytes. Erythropoietic activity is more closely associated with log ferritin values than with sTfR concentrations in iron-deficiency anemia.

(received 30 April 2003; accepted 15 May 2003)

Keywords: serum transferrin receptor, erythropoietic activity, ferritin, reticulocyte subpopulations


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transferrin receptor (TfR) is a transmembrane protein that mediates iron delivery from the extracellular pool into erythroblasts by receptor-mediated endocytosis [1]. Nearly all mammalian cells have TfR on their surfaces, but TfR is mostly located in the erythroid precursors in bone marrow [2]. Serum TfR (sTfR) concentration is proportional to cellular expression of the membrane-associated TfR and increases with elevated cellular iron needs and cellular proliferation [3]. Thus, measurement of sTfR concentration can be used as a diagnostic tool for evaluating body iron status and provides a new laboratory parameter of erythropoiesis [4–6].

Erythropoiesis can be monitored precisely by quantitative measurement of reticulocytes using flow cytometry. The degree of reticulocyte maturation is determined by the relative concentration of residual RNA, to which the fluorescent intensity of reticulocytes is directly proportional. Reticulocytes can be divided into 3 subpopulations based on fluorescent intensity: low-, middle-, and high-fluorescence reticulocytes (LFR, MFR, and HFR, respectively) [7]. A reticulocyte maturity index (RMI), calculated from the proportion of reticulocyte subpopulations, appears to be the earliest and most sensitive predictor of erythropoiesis [8].

Iron deficiency develops in sequential stages during a period of negative iron balance. These stages include the iron-depletion phase (stage I), iron-deficient erythropoiesis (stage II), and iron-deficiency anemia (IDA, stage III) [9]. During the iron-depletion phase, iron stores are exhausted; but anemia or decrease of serum iron is not present. In the stage of iron-deficient erythropoiesis, serum iron and serum ferritin levels are decreased; but anemia and hypochromia are still undemonstrable. The relationship between sTfR concentrations and iron parameters in IDA or anemia of chronic diseases has been extensively studied, but few studies have closely examined associations between sTfR concentrations and reticulocyte subpopulations during the 3 stages of iron deficiency, especially as compared to log ferritin values. In the present study, we determined which index most accurately reflects erythropoietic activities during iron deficiency, based on the correlation coefficients of sTfR, log ferritin, and sTfR/log ferritin ratio (sTfR-F index), in respect to each reticulocyte subpopulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Complete blood cell count (CBC), iron profiles, reticulocyte subpopulations, and sTfR concentrations were measured in 227 prepubescent children, aged 9 to 12 yr (male 51.7%, mean 10.8 yr). The subjects included were all volunteers and were South Korean children from middle-class families. This survey was explained to and approved by the parents and by the directors at each participating school.

The subjects were divided into 4 groups based on their body iron status: iron-depletion phase (stage I, n = 41), iron-deficient erythropoiesis (stage II, n = 25), IDA (stage III, n = 72), and healthy controls (n = 89). Non-anemic subjects with a normal serum iron level (>50 µg/dl), but with decreased serum ferritin concentration (<12 µg/l), were classified 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. Subjects showing a decreased serum ferritin concentration, decreased serum iron level, and decreased blood hemoglobin level (<12 g/dl) were considered to have IDA (stage III). The IDA subjects and healthy controls were divided into 4 or 3 subgroups, respectively according to serum ferritin levels: IDA subjects (serum ferritin <4.0 µg/L, n = 16; 4.1–6.0 µg/L, n = 19; 6.1–9.0 µg/ L, n = 17; 9.1–12.0 µg/L, n = 20) and healthy controls (serum ferritin 30.0 µg/L, n = 34; 20.0–29.9 µg/L, n = 31; 12.0–19.9 µg/L, n = 24).

Venous blood was drawn in iron-free evacuated tubes. CBC and reticulocyte subpopulations were measured with EDTA-anticoagulated blood within 3 hr after collection. CBC 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 corrected reticulocyte count was calculated, based on a normal hematocrit of 45%, from the following formula: corrected reticulocyte count (%) = (subject’s hematocrit/45) x reticulocyte count (%). RMI was calculated from the equation, RMI = [(MFR + HFR) x 100]/LFR and was expressed as the percentage [10]. 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, MA, USA). The sTfR concentrations were measured by an immuno-enzymometric method (IDeATM sTfR, Orion Diagnostica, Espoo, Finland). The intraassay coefficients of variation (CVs, n = 15) for 3 samples (mean sTfR, 1.3–6.5 mg/L) were 3.2–5.4%; the interassay CVs calculated from duplicate results in 10 consecutive runs were 3.1–5.9%.

Data analysis was performed with the SAS 6.12 software package (SAS Institute, Cary, NC). Non-parametric tests were used because the distributions of most variables were non-Gaussian by the Kolmogorov-Smirnov test. The Mann-Whitney U test was used to evaluate the differences in mean values between 2 groups. Correlation coefficients were analyzed by Spearman’s method. All p values 0.01 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mean values of reticulocyte subpopulations and iron parameters based on three stages of iron deficiency are summarized in Table 1Go. There were no significant differences in reticulocyte parameters between the iron-depletion phase (stage I) and the control group, nor between the iron-depletion phase (stage I) and iron-deficient erythropoiesis (stage II). However, mean proportions of MFR and RMI in IDA (stage III) were 2.3 ± 1.1% and 2.5 ± 1.3%, which were significantly higher than those of the iron-depletion phase (stage I, 0.9 ± 0.6% and 1.0 ± 0.6%), or iron-deficient erythropoiesis (stage II, 1.1 ± 0.8% and 1.2 ± 0.7%, p <0.01, respectively). The mean sTfR concentrations began to increase significantly at iron-deficient erythropoiesis (stage II) and reached the maximal values at IDA (stage III).


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  		Table 1. Mean values of reticulocyte and iron parameters based on 3 stages of iron deficiency.
 
Among the iron-deficiency anemia subjects, the children with serum ferritin concentrations of 9.1–12.0 µg/L showed no elevation in the values of reticulocyte subpopulations, compared to the control group. However, when the serum ferritin fell to the level of 6.1–9.0 µg/L, MFR and RMI reached 1.9 ± 0.8% and 2.2 ± 1.0%, which were significantly higher than those (1.1 ± 0.6% and 1.3 ± 0.6%) of the subjects with serum ferritin levels of 9.1–12.0 µg/L (p <0.01, respectively).

The mean values of MFR and RMI in the subjects with serum ferritin concentrations <4.0 µg/ L were 3-fold higher than those in healthy controls (p <0.01) (Table 2Go). Reticulocyte parameters of healthy controls according to serum ferritin concentrations are summarized in Table 3Go. There were no significant differences in reticulocyte subpopulations between the subjects with serum ferritin 30.0 µg/L and with serum ferritin concentrations of 12.0–19.9 µg/L.


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  		Table 2. Reticulocyte and iron parameters in 72 iron-deficiency anemia subjects, based on serum ferritin concentrations.
 

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  		Table 3. Mean values of reticulocyte and iron parameters in 89 healthy controls based on serum ferritin concentrations.
 
As shown in Table 4Go, no significant correlations were observed between reticulocyte parameters and the values of ferritin, log ferritin, sTfR, and sTfR-F index in healthy controls. In iron-deficiency anemia subjects, however, the correlation coefficients of LFR (r = - 0.41, p <0.01), MFR (r = 0.43, p <0.01), and RMI (r = 0.42, p <0.01) vs log ferritin values were higher than those of corresponding reticulocyte subpopulations vs sTfR or sTfR-F index.


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  		Table 4. Correlation coefficients of serum ferritin, log ferritin, sTfR, and sTfR-F index vs reticulocyte parameters in iron-deficiency anemia subjects and healthy controls.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the changes in reticulocyte subpopulations and RMI as the body iron store was depleted and we also evaluated the associations between reticulocyte production and sTfR concentrations during negative iron balance. We found that erythropoietic activity more strongly correlates with log ferritin values than with sTfR concentrations or sTfR-F index in iron-deficiency anemic children. Our data for relationships between reticulocyte subpopulations and iron deficiency are in accordance with the results of some investigators, who demonstrated that reticulocyte mean channel fluorescence was elevated in patients with IDA [11].

In a previous study, we reported that immature reticulocyte fractions began to increase from the time that serum iron and ferritin levels both decreased, reaching their peak when the subjects acquired overt IDA [12]. In the present study, no significant elevation of reticulocyte subpopulations was noted in the iron-deficient erythropoietic group. Furthermore, among the subgroups of IDA populations, there was no increase in the mean values of RMI even in subjects with serum ferritin of 9.1–12.0 µg/L; however, RMI began to increase significantly when the serum ferritin fell to the range of 6.1–9.0 µg/L. These results suggest that a serum ferritin concentration <9.0 µg/L may be a critical level, which induces elevated production of immature reticulocytes in children with iron-deficiency anemia. The discrepancies between the results of the present and the previous studies may be due to differences in body iron requirement according to subject’s age during a period of growth spurt: in the previous work we investigated only female adolescents, whereas in the present study we evaluated prepubescent children.

In normal erythropoiesis, reticulocytes become gradually mature red blood cells in the peripheral blood, losing both RNA and TfR [13,14]. Reticulocytes continue to synthesize hemoglobin, provided there is a sufficient supply of both iron and mRNA. Iron deficiency restricts hemoglobin synthesis and increases TfR production rate [14]. In the present study, sTfR concentrations in iron-deficient erythropoiesis (stage II) were significantly higher than those in healthy controls. However, no significant changes in immature reticulocyte fractions were observed in iron-deficient erythropoiesis (stage II), compared to healthy controls. Similar findings were observed in healthy controls who had normal hemoglobin levels: mean values of sTfR concentrations were significantly higher in the subjects with serum ferritin levels of 12.0–19.9 µg/ L than in the subjects with serum ferritin 30.0 µg/ L; however, there were no significant differences in reticulocyte parameters between the 2 groups. These results suggest sTfR reflects more sensitively the body iron status than do the reticulocyte subpopulations. Our data imply that decreased ferritin concentration in non-anemic subjects does not affect reticulocyte production; however, once a subject has attained a state of frank anemia, decreased ferritin levels may influence erythropoietic activity. Because serum ferritin reflects the storage iron compartment and sTfR reflects the functional iron compartment, the sTfR-F index, based on these two values, has been suggested as a good estimate of body iron [15]. Some investigators have used the sTfR-F index as an additional biochemical marker for identification of iron-deficient erythropoiesis [16].

In this study, we investigated to what extent sTfR concentrations correlate with reticulocyte subpopulations, compared to the values of ferritin, log ferritin, and sTfR-F index. In healthy controls, who had no evidence of anemia or iron depletion, there were no significant correlations between reticulocyte parameters and the values of sTfR, ferritin, log ferritin, and sTfR-F index. Interestingly, in IDA subjects, log ferritin values were more strongly correlated with reticulocyte subpopulations and RMI than serum ferritin, sTfR, or the sTfR-F index. These results suggest that the sTfR or sTfR-F index are not superior to log ferritin values for the evaluation of erythropoietic activity during iron deficiency.

The main sources of the sTfR are known to be the erythroblasts and reticulocytes that eventually shed their receptors into the circulating blood during maturation sequence [17]. In our study, however, significant elevation of sTfR concentrations was observed in a considerable numbers of subjects with no increase of reticulocytes or their subpopulations. Therefore, it is conceivable that elevated sTfR concentrations in subjects without an increase of reticulocytes may be derived from erythroblasts in the bone marrow. In contrast, one group of investigators has reported that the turnover of erythroblasts was markedly reduced in iron deficiency and the reduction was caused by a progressively decreasing rate of erythroblast proliferation and maturation [18]. Because the present study measured only sTfR concentrations and reticulocyte subpopulations in the circulating blood, it does not provide direct evidence of a relationship between intramedullary erythroblasts and sTfR concentrations.

In conclusion, reticulocytes and their subpopulations appear to be more closely associated with log ferritin values than with sTfR concentrations or the sTfR-F index. Serum ferritin concentration <9.0 µg/L seems to be a critical level to induce to elevate immature reticulocyte production in iron-deficiency anemic children. To verify the relationships of sTfR concentrations and reticulocyte production to intramedullary erythroid precursors, further studies are needed, especially in regard to examination of bone marrow samples.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Feelders RA, Kuiper-Kramer EP, van Eijk HG. Structure, function and clinical significance of transferrin receptors. Clin Chem Lab Med 1999;37:1–10.[Medline]
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  4. Cook JD, Dassenko S, Skikne BS. Serum transferrin receptor as an index of iron absorption. Br J Haematol 1990;75:603–609.[Medline]
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  6. Kohgo Y, Niitsu Y, Kondo H, Kato J, Tsushima N, Sakaki K, Kirayama N, Numata T, Nishisato T, Urushizaki I. Serum transferrin receptor as a new index of erythropoiesis. Blood 1987;70:1955–1958.[Abstract/Free Full Text]
  7. Chang CC, Kass L. Clinical significance of immature reticulocyte fraction determined by automated reticulocyte counting. Am J Clin Pathol 1997;108:69–73.[Medline]
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  12. Choi JW, Pai SH. Reticulocyte subpopulations and reticulocyte maturity index (RMI) rise as body iron status falls. Am J Hematol 2001;67:130–135.[Medline]
  13. Rouault T, Rao K, Harford J, Mattia E, Klausner R. Hemin, chelatable iron, and the regulation of transferrin receptors. J Biol Chem 1985;260:14862–14866.[Abstract/Free Full Text]
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