Soluble transferrin receptors and reticulocyte ...

O RIGINAL INVESTIGATION

J NEPHROL 2005; 18: 72-79

Soluble transferrin receptors and reticulocyte hemoglobin concentration in the assessment of iron deficiency in hemodialysis patients Maria Fusaro1, Giorgio Munaretto1, Michela Spinello1, Mirca Rebeschini1, Gianpaolo Amici2, Maurizio Gallieni3, Antonio Piccoli4 Nephrology and Dialysis Department, Hospital of Chioggia, Venice - Italy Nephrology and Dialysis Department, Hospital of Treviso, Treviso - Italy 3 Nephrology and Dialysis Department, San Paolo Hospital, Milan - Italy 4 Nephrology and Dialysis Department, University of Padua, Padua - Italy 1 2

ABSTRACT: Background: Diagnosing iron deficiency in hemodialysis (HD) patients is crucial for correct anemia management. Hypochromic erythrocytes appear to be the best available marker, but they are often unavailable. Transferrin saturation (TSAT) and ferritin are also indicated as reference markers by guidelines. We evaluated the usefulness of soluble transferrin receptor (s-TfR) and reticulocyte hemoglobin concentration (CHr), which have been recently proposed as more sensitive functional iron deficiency indicators. Methods: A single-center unselected cohort of 39 chronic HD patients underwent a cross-sectional determination of hemoglobin (Hb), hematocrit (Hct), CHr, transferrin, iron, TSAT, ferritin, folate, vitamin B12 and s-TfR. Twenty-nine patients (74.4%) were treated with subcutaneous erythropoietin (EPO) at a dose of 122 ± 98 U/kg/week and 24 patients (61.5%) were treated with intravenous (i.v.) iron gluconate, 62.5 mg/week. Results: Hb was 11.1 ± 1.2 g/dL, Hct 34.4 ± 3.7%, CHr 32.7 ± 3.8 pg, transferrin 170 ± 31 mg/dL, iron 60.2 ± 25.9 mg/dL, TSAT 30 ± 18%; ferritin 204 ± 219 ng/mL, folate 4.2 ± 1.0 mcg/L, vitamin B12 0.58 ± 0.15 mcg/L, and s-TfR 1.94 ± 0.83 mg/L. Both TSAT and s-TfR significantly correlated with CHr, but no relationship could be found between s-TfR and TSAT or between s-TfR and ferritin. Dividing the population into two groups based on iron repletion (ferritin >100 ng/mL and TSAT >20%) we found no differences for CHr levels and significantly lower levels of s-TfR in the replete group (s-TfR 1.71 ± 0.70 vs. 2.29 ± 0.90 mg/L; p=0.033). Analysis of 2x2 tables demonstrated that 44% of patients with TSAT >20% had elevated (>1.5 mg/L) s-TfR, indicating a possible functional iron deficiency, but covariance analysis showed that TSAT had a better correlation to CHr. Conclusions: No clear-cut advantages in the use of CHr content and s-TfR levels as single diagnostic tests could be demonstrated by this cross-sectional study. However, our results suggest that the combined use of TSAT 1.5 mg/L (therefore, including all patients with low TSAT, but also patients with high s-TfR despite normal TSAT) could improve functional iron deficiency detection in dialysis patients suspected of having inflammatory conditions. Key words: Soluble transferrin receptor, Reticulocyte hemoglobin concentration, Iron, Anemia, Dialysis

The diagnosis of iron deficiency in hemodialysis (HD) patients is crucial for correct anemia management. It has been traditionally based on a restricted panel of biochemical iron metabolism indicators, which includes the determination in serum or plasma of iron, transferrin, transferrin saturation (TSAT) and ferritin. Many other indicators have been proposed.

Among them, the percentage of hypochromic erythrocytes (defined as a hemoglobin (Hb) content 100 ng/mL). The remaining 16 patients had inadequate TSAT and/or ferritin levels, as illustrated in Figure 1. Figure 2 shows simple correlations between CHr and TSAT (Fig. 2A); CHr and s-TfR (Fig. 2B); s-TfR and 73

New markers of iron deficiency in dialysis patients

Fig. 1 - Patient population divided by iron status according to DOQI guidelines.

TABLE I - ERYTHROPOIETIC AND IRON METABOLISM PARAMETERS IN 39 HD PATIENTS Red blood cells indexes

Mean ± SD

Normal range

0..11.1 ± 1.2 g/dL 34.4 ± 3.7%. ..95.4 ± 10.7 fL 30.9 ± 3.6 pg ..1.45 ± 0.49 % 32.7 ± 3.8 pg 0029.0 ± 1.7 g/dL 113.4 ± 11.9 fL

11.7-15.5 35-47 80-95 27-34 0.5-2.5 25.9-30.6 23.5-28.5 103.2-126.3

Iron status indexes

Mean ± SD

Normal range

Serum transferrin Serum iron TIBC TSAT Serum ferritin Serum folate Serum vitamin B12 s-TfR

000.170 ± 31 mg/dL 0000.60.2 ± 25.9 mg/dL 000.212 ± 38 mg/dL .30 ± 18% 0000.204 ± 219 ng/mL 00004.2 ± 1.0 mcg/L 0000.0.58 ± 0.15 mcg/L 000.1.94 ± 0.83 mg/L

200-360 49-151 260-440 20-50% 3-151 2.5-15 0.2-1.1 1.0-2.9

Hemoglobin Hematocrit MCV MCH Reticulocytes CHr CHCMr MCVr

TSAT (Fig. 2C); and s-TfR and ferritin (Fig. 2D). There was no correlation found between s-TfR and CRP (not shown). Both TSAT and s-TfR significantly correlated with CHr (respectively, positive correlation: r=0.426; p=0.008 and inverse relationship: r=-0.380; p=0.018), but no relationship could be found between s-TfR and TSAT or between s-TfR and serum ferritin. The inverse relationship between s-TfR and CHr indicated that in iron deficient erythropoiesis a reduced CHr content is associated with increased s-TfR levels. Based on a cutoff value of 1.5 mg/L, we obtained a statistically significant separation (t-test 3.105; p=0.0037) of our patient population in two groups: 16 patients with s-TfR ≤1.5 mg/L, (low s-TfR patients, who had larger iron availability), and 23 patients with s-TfR >1.5 mg/L, (high s-TfR patients, who had smaller iron stores). In these two groups of patients, CHr levels were, respectively, 34.9 ± 2.8 and 31.3 ± 3.8 pg; TSAT levels were 39.1 ± 24.2% and 23.7 ± 7.6% (t-test 2.861; p=0.0069); ferritin was not statistically different (Tab. II). On the other hand, dividing the population into two groups based on the traditional iron depletion parameters (serum ferritin 1.5 mg/L) s-TfR, indicating a possible functional iron deficiency (Tab. IV). When both criteria of iron repletion were considered (ferritin and TSAT), 10 patients (43.8% of iron replete patients) had an elevated s-TfR. However, covariance analysis (Fig. 3) showed that TSAT had a better correlation to CHr (whole model F=4.6, p=0.016; CHr F=4.8, p=0.035; s-TfR F=1.3, p=0.269), reducing the importance of s-TfR and ferritin as iron status markers. From this perspective, s-TfR can be considered an additional rather than an alternative marker: it is no more potent than TSAT, but can be a useful tool to uncover functional iron deficiency. Coupling TSAT >20% and s-TfR ≤1.5 mg/L could be a better iron repletion marker. In patients responding to this iron repletion criteria, CHr was 34.9

TABLE II - IRON STORES, CHr AND s-TfR, WHEN PATIENTS WERE DIVIDED IN TWO GROUPS ACCORDING TO LOW sTFR (≤1.5 mg/L) AND HIGH s-TFR (>1.5 mg/L) LEVELS

CHr (pg) s-TfR (mg/L) Ferritin (ng/mL) TSAT (%)

Low s-TFR (n=16)

High s-TFR (n=23)

p

34.9 ± 2.8 1.22 ± 0.2 ..273 ± 316 ..39.1 ± 24.2

31.3 ± 3.8 2.41 ± 0.7 155 ± 93 23.7 ± 7.6

0.004 0.0001 0.097 ns 0.007

A large proportion of patients (23/39, or 59%) had possible iron deficiency based on this cut-off value.

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Fig. 2 - Simple correlations: A, CHr and TSAT; B, CHr and s-TfR; C, sTfR and TSAT; D, s-TfR and ferritin.

A

B

C

D

Fig. 3 - Three-dimensional views of the relationship between TSAT, CHr and s-TfR, analyzed by ANCOVA.

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± 2.8 pg, statistically different from iron depleted patients, whose CHr levels were 31.3 ± 3.8 (t-test 3.105; p=0.0037). Table V illustrates mean levels of iron stores, CHr and s-TfR when the patient population was divided based on the treatment received for anemia, specifically rHuEPO and iron. With the exception of a higher CHr level in patients treated by rHuEPO, we did not find any statistical difference. We also tested the usefulness of the ratio s-TfR/serum ferritin, as described by Punnonen et al (16), but no advantages over the single iron status markers could be found in our patient population.

DISCUSSION The main limitation of our study was its observational nature, with a cross-sectional design, in a limited number of patients. However, we included all patients treated in a single dialysis unit, therefore, allowing the study of an unselected population found in most dialysis centers, and better reflecting daily clinical practice. This study indicated that CHr, an iron status marker at the reticulocyte level, and s-TfR, a measure of erythropoiesis primarily derived from erythroid precursor cells, do not offer by themselves significant advantages over the more traditional iron status markers, TSAT

TABLE III - IRON STORES, CHr AND s-TfR.

CHr (pg) s-TfR (mg/L) Ferritin (ng/mL) TSAT (%)

Iron replete (n=23)

Iron depleted (n=16)

p

33.3 ± 4.6 1.71 ± 0.7 275 ± 261 35.6 ± 21.1

31.9 ± 5.0 2.29 ± 0.9 101 ± 47 22.0 ± 6.9

0.291 ns 0.033 0.012 0.018

Here iron depletion was traditionally defined by the presence of serum ferritin 20% was used as a guide to iron administration, while no significant changes in rHuEPO doses were observed when trying to maintain CHr levels >32.5 pg. This is in contrast with the results presented by Fishbane et al (19), indicating that CHr is a more stable indicator than ferritin and TSAT, and that iron management based on CHr results in similar Hct and rHuEPO dosing, while significantly reducing i.v. iron exposure. CHr determination provides a real time evaluation of bone marrow activity, reflecting the balance between iron and erythropoiesis of the preceding 48 hr (20). This is a clear advantage in detecting iron deficiency at an earlier stage, but CHr values can be difficult to interpret in patients who are often treated by i.v. iron, as in our patient population (>60% were receiving i.v. iron gluconate). Therefore, prompt but short-term increases in CHr levels could lead to a premature discontinuation of i.v. iron, reducing our ability to manage iron metabolism adequately in dialysis patients. In keeping with this suggestion, Cullen et al (12) demonstrated that CHr is superior to the percentage of hypochromic red cells in detecting iron deficiency in HD patients, but they also found that CHr levels did not change as predicted during treatment with rHuEPO and iron. Mean CHr levels in our patients (32.7 ± 3.8 pg) were similar to the Japanese data, 33.1 ± 2.1 pg (18), than to the cohorts described by Fishbane et al, 27.5 ± 2.8 pg (11), and by Cullen et al, 26.6 ± 2.8 pg (12). This makes a comparison of data more difficult: the cut-off proposed by Fishbane et al (11) of a CHr level 20%, together with ferritin, as ferritin often rises because it is an acute-phase reactant (2, 3). 77


Therefore, an evaluation of the clinical usefulness of s-TfR should consider whether it offers advantages over TSAT, rather than ferritin, in patients with possible functional iron deficiency. This is particularly important because an intensified iron substitution in patients with elevated s-TfR and normal TSAT levels could improve the cost-efficacy of rHuEPO treatment. We found no correlation between s-TfR and TSAT or between s-TfR and serum ferritin. The rather high intra-assay and inter-assay coefficients of variance for s-TFR, as described above, could have contributed to a lower reliability of this marker. However, when our patient population was divided into two groups based on the traditional iron depletion parameters (cut-off values, serum ferritin 100 ng/mL; TSAT 20%) we found significantly lower levels of s-TfR in the iron replete group (Tab. III). In addition, almost half the patients with TSAT >20% had elevated s-TfR, indicating a possible functional iron deficiency (Tab. IV). We also evaluated the possible role of inflammation on sTfR levels, and we confirmed that s-TfR is not correlated to CRP, as suggested by Tarng and Huang (9). Therefore, elevated s-TfR levels indicate iron deficiency, regardless of whether inflammation is present. If erythropoietic activity is in a steady-state, not influencing s-TfR expression, s-TfR levels could, therefore,

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Part of this work was presented at the American Society of Nephrology 35th Annual Meeting and Scientific Exposition - Renal Week, 2002 in Philadelphia, PA, USA.

Address for correspondence: Maria Fusaro, M.D. Department of Nephrology and Dialysis Ospedale di Chioggia Strada Madonna Marina, 500 Chioggia, Venezia 30015, Italy [email protected]

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Received: February 04, 2004 Revised: August 18, 2004 Accepted: October 18, 2004

© Società Italiana di Nefrologia

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