Early prediction of response to intravenous iron supplementation by reticulocyte haemoglobin content and high-fluorescence reticulocyte count in haemodialysis patients

Chiao-Lin Chuang3, Ren-Shyan Liu4, Yau-Huei Wei2, Tung-Po Huang1,3 and Der-Cherng Tarng1,3,

1 Faculty of Medicine and 2 Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University School of Medicine, 3 Division of Nephrology, Department of Medicine and 4 Department of Nuclear Medicine, Taipei Veterans General Hospital, Taipei, Taiwan



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Optimal response to recombinant human erythropoietin (rHuEpo) in haemodialysis (HD) patients requires provision of sufficient available iron. However, a balance between iron requirements and supplements remains a challenge in clinical practice. Reticulocyte parameters, i.e. reticulocyte haemoglobin content (CHr) and reticulocytes in a high-fluorescence intensity region (HFR), have been shown to be accurate predictors of iron-deficient erythropoiesis as compared with traditional markers. Therefore, the aim of this study was to appraise the diagnostic power of these two parameters in the early prediction of response to intravenous iron (IVFE) medications in HD patients receiving rHuEpo.

Methods. Sixty-five HD patients with a serum ferritin level of <500 µg/l and on rHuEpo therapy for >6 months were enrolled for IVFE supplementation (100 mg iron saccharate three times a week for 4 weeks, then 100 mg every 2 weeks for 5 months). Haemoglobin, haematocrit, serum ferritin, transferrin saturation, reticulocyte count, percentage of hypochromic red cells, CHr and HFR were measured before and following iron supplementation. Response was defined as a rise in haematocrit of >3% and/or a reduction in rHuEpo dose of >30% over the baseline values at the end of the study.

Results. Forty-two patients had a dramatic response to IVFE therapy with a 13.5% increase in mean haematocrit and a 38% reduction in rHuEpo dose at the end of the study (P<0.001). This paralleled a statistically significant rise in CHr and HFR (P<0.001). Univariate analyses showed that ferritin (P<0.010) and CHr (P<0.001) at baseline, changes in CHr ({Delta}CHr2W, P<0.001) and HFR ({Delta}HFR2W, P<0.010) at 2 weeks, as well as changes in CHr ({Delta}CHr4W, P<0.001) and HFR ({Delta}HFR4W, P<0.001) at 4 weeks, strongly correlated with response to IVFE supplementation. Stepwise discriminant analysis disclosed that {Delta}CHr4W in conjunction with {Delta}HFR4W exhibited an r2 value of 0.531 (P<0.001) to predict response to IVFE therapy. Analyses by receiver operating characteristic curves and logistic regression further revealed that {Delta}CHr4W at a cut-off value of >1.2 pg and {Delta}HFR4W of >500/µl were more specific to the status of iron-deficient erythropoiesis following IVFE medications. Combined use of the two cut-off values allowed for the highest accuracy in the early prediction of the response to IVFE therapy, with a sensitivity of 96% and a specificity of 100%.

Conclusions. Our study shows that changes in CHr and HFR at either 2 or 4 weeks are superior to the conventional erythrocyte and iron metabolism indices and may serve as reliable parameters to detect iron-deficient erythropoiesis in HD patients undergoing rHuEpo therapy. During aggressive IVFE treatment, early identification of non-responsiveness and subsequent discontinuation of treatment can avoid the inadvertent iron-related toxicity due to over-treatment.

Keywords: functional iron deficiency; haemodialysis; high-fluorescence reticulocyte; intravenous iron therapy; recombinant human erythropoietin; reticulocyte haemoglobin content



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Recombinant human erythropoietin (rHuEpo) can effectively alleviate or correct anaemia of end-stage renal disease (ESRD) and reduce the need for blood transfusion [1]. However, the rHuEpo effect is diminished due to provision of insufficient available iron for the accelerated erythropoiesis [2]. Absolute iron deficiency is defined as a decrease in total body iron stores and functional deficiency exhibits defective iron mobilization/utilization that cannot keep pace with the demands for the accelerated erythropoiesis, even though the iron reserve is normal or increased [2,3]. Accordingly, a large amount of research has been conducted to assess the mechanisms for iron-deficient erythropoiesis and the indications of iron supplementation. It has been well established that intravenous iron (IVFE) medication is essential for the great majority of haemodialysis (HD) patients undergoing rHuEpo therapy [4,5]. Nevertheless, IVFE therapy to maximize rHuEpo response must be weighed in the context of iron-associated risks, including anaphylaxis, haemosiderosis, hepatic dysfunction and cardiovascular and infectious morbidities [6,7]. To balance between optimizing the rHuEpo effect and avoiding the adverse effects of iron toxicity, it is of paramount importance to establish a monitoring system for the early prediction of who will or will not get an improvement in erythropoiesis following IVFE supplementation. Furthermore, early prediction of responsiveness to IVFE medications would avoid the emotional burden and the inadvertent hazards of iron overload.

Serum ferritin, transferrin saturation (TSAT) and percentage of hypochromic red cells (% HRC) are highly recommended for assessing the iron status of patients receiving rHuEpo therapy [4,5]. However, their validity for the diagnosis of iron-deficient erythropoiesis is still debatable [8,9], particularly in those with normal or even increased iron stores. Serum ferritin levels are affected by inflammation. Circulating inflammatory markers and cytokines are always increased in ESRD patients [10]. For these reasons, the threshold ferritin level below which iron depletion can be considered in HD patients is probably higher than that seen in individuals with normal renal function. Therefore, precisely where that threshold lies remains a subject of some debate [8,9]. Transferrin saturation fluctuates widely due to a diurnal variation in serum iron and transferrin affected by the nutritional status. This may lead to a lack of sensitivity and specificity for TSAT in assessing the iron availability/utilization during rHuEpo treatment [8,9,11]. A HRC is an individual erythrocyte haemoglobin content of <28 g/dl and a subpopulation of HRC above 10% indicates iron-deficient erythropoiesis [11]. Currently, % HRC has been introduced as a sensitive tool in diagnosing iron deficiency in dialysis patients [11]. However, % HRC can also be affected by inflammation [12] and its cut-off value for functional iron deficiency varies from 3.7 to 10% in different series [11,12]. In addition, HRC represents a time-average measurement of the degree of haemoglobinization in red cells. Because of the longer life-span of mature erythrocytes, % HRC failed to provide the relevant information of a rapid change in iron utilization [13].

Thus, an ideal assessment of iron status should be the direct evaluation of an erythropoietic responsiveness to iron therapy at the level of erythrocyte precursors. Since reticulocytes have a more rapid turnover in circulation than mature red cells (1–2 vs 120 days), it is hypothesized that reticulocytes may be more sensitive in detecting erythropoietic activity. With the advent of a novel automated flow-cytometry technique, measurements of reticulocyte cellular characteristics allow extremely early and objective information to be collected on erythropoietic activity in anaemia [14]. Reticulocyte haemoglobin content (CHr) has been proposed as a surrogate marker of iron status and an early predictor of response to iron therapy in HD patients [15,16]. Flow cytometry further assesses the maturation of reticulocytes by quantifying the fractions of reticulocytes in low-fluorescence, middle-fluorescence and high-fluorescence (HFR) intensity regions [17]. Reticulocyte fluorescence intensity is directly proportional to the quantity of intracellular RNA and thus expresses a function of the cellular maturity [18]. High-fluorescence reticulocytes with more RNA, which corresponds to immature reticulocytes, have been measured as an indicator of erythropoietic activity following various therapies for anaemia of different causes [18,19]. In this study, we therefore applied CHr and HFR, as well as conventional iron metabolism indices, to early predict the responsiveness to IVFE therapy in HD patients being treated with rHuEpo.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and study protocol
A prospective study was conducted at two dialysis centres of the affiliated hospital of National Yang-Ming University. Initially, 92 patients (49 men and 43 women, mean age of 58 years) agreed to participate in this study. The inclusion criteria were as follows: on HD treatment for >=6 months, on rHuEpo therapy for >=6 months, serum ferritin of <500 µg/l, no haematological disorder other than renal anaemia, no blood transfusions or iron supplementation in the preceding 3 months and no inflammatory diseases or infections that might affect the erythropoietic response to rHuEpo therapy. Patients were excluded if any of the following events occurred during the study: clinically significant bleeding or blood transfusions, hospitalizations, surgery, infections, treatment modality shifted to peritoneal dialysis or renal transplantation, as well as severe adverse reactions or poor compliance to IVFE therapy. Finally, 65 patients (33 men and 32 women, mean age of 60 years) completed the study. All patients were dialysed for 4.0–4.5 h three times a week, using a single-use dialyser with a 1.5 m2 effective surface area of cellulose diacetate membrane, blood flow of 250–350 ml/min and dialysate flow of 500 ml/min. This study was approved by the local medical ethics committee and informed consent was obtained from each of the patients.

All patients were supplied with 100 mg ferric saccharate (Ferrum Hausmann; Hausmann Lab. Inc., Switzerland) three times a week for 4 weeks (total dose of 1200 mg elemental iron) and then 100 mg ferric saccharate every 2 weeks for 5 months (total dose of 1000 mg elemental iron). Iron supply was administered intravenously at the end of each HD session. Before the first dose was administered, a test dose of 25 mg of ferric saccharate was administered for 30 min to observe any adverse reaction development. If no adverse reactions occurred, the IVFE supplementation protocol would then be begun. A response to IVFE therapy was defined as a rise in haematocrit of >=3% and/or a reduction in rHuEpo dose of >=30% over the baseline values at the end of the study. Those who did not fulfil the above-mentioned criteria were defined as non-responders. Iron metabolism indices (i.e. serum ferritin and iron, TSAT), erythrocyte indices (i.e. haematocrit, haemoglobin, RBC count and % HRC) and reticulocyte parameters (i.e. reticulocyte count, CHr and HFR) were examined at baselines and then followed at 2 weeks and monthly for the following 5 months. The rHuEpo (Eprex; Cilag, Schaffhausen, Switzerland) was administered subcutaneously two or three times a week. The dose was decreased by 3x15 U/kg if haematocrit increased to >32%. The rHuEpo dose was titrated biweekly to maintain a target haematocrit value of 30–32%.

Laboratory measurements
Blood was drawn pre-dialysis after an overnight fast. Haematocrit, haemoglobin, RBC count and CHr were determined by a Technicon H*3 automated cell counter (Bayer Laboratory, Germany). Reticulocyte count and HFR were measured by a flow cytometric analyser (Sysmex R-2000; TOA Medical Electronics, Kobe, Japan). Serum iron was measured by a calorimetric method (Hitachi 736-60 autoanalyzer; Hitachi, Naka, Japan), total iron-binding capacity (TIBC) by the TIBC Microtest (Daiichi, Tokyo, Japan) and ferritin by radioimmunoassay (Incstar, Stillwater, MN, USA). Transferrin saturation was calculated by dividing serum iron by TIBC x100.

Statistical analysis
Statistical analysis was performed using the computer software Statistical Package of Social Science (SPSS 7.5, 1996; SPSS Inc., IL, USA). Data were expressed as means±SD. Comparisons of data for responders and non-responders were performed by using the t-test or Mann–Whitney U-test. Pearson's chi-square test was used for frequency measures. A within-group comparison among post-treatment values and baselines was analysed by analysis of variance (ANOVA) for repeated measures, followed by pair-wise multiple comparison. Correlations between quantitative variables and the qualitative variable ‘response’ were calculated as point-biserial correlations. The stepwise discriminant analysis used stepwise selection of independent variables that contributed significantly to the discriminatory power of the model as measured by Wilks' lambda, the likelihood ratio criteria. In multiple regression analysis, we firstly used the receiver operating characteristic (ROC) analysis to calculate the cut-off values for the independent variables and then applied logistic regression analysis to calculate the odds ratios according to Mantel–Haenszel for the independent variables entered into the model. A P-value of <0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Sixty-five patients completed the study. There were 42 responders and 23 non-responders. No adverse reactions associated with IVFE medications were observed. Baseline characteristics of the patients in these two groups were similar with regard to age, gender distribution, time on HD and the causes of chronic renal failure. Baseline values of serum ferritin and CHr were significantly higher in the non-responders and basal rHuEpo doses were higher in the responders (P<0.05). The other parameters, such as serum ferritin, TSAT and % HRC as well as other factors affecting the rHuEpo responsiveness showed no significant differences between the two groups of patients (Table 1Go).


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Table 1.  Characteristics of the haemodialysis patients recruited in this study

 
Erythropoietic response following IVFE supplementation
In the responders (Table 2Go), the erythrocyte indices, including RBC count and haemoglobin values, showed no statistically significant increase until 2 months following iron therapy (repeated measured ANOVA, P<0.001). The reduction of rHuEpo doses started at 4 weeks and continued for all subsequent months in the responders as compared with the baseline values (repeated measured ANOVA, P<0.001). Impressively, there was a significant increase in reticulocyte indices, such as reticulocyte count, CHr and HFR, at 2 weeks and throughout the subsequent follow-up period (repeated measured ANOVA, P<0.001). Among the responders, 13 patients only had an increase in haematocrit, 11 only had a decrease in rHuEpo dosing and 18 had both. The changes in reticulocyte count, CHr and HFR in the three groups of patients were similar and exhibited the same trend as the changes in the responders as a whole. In contrast, the erythrocyte indices, reticulocyte parameters and rHuEpo doses in the non-responders showed no significant changes for 6 months following IVFE supplementation. Serum ferritin levels significantly increased in the two groups of patients for 6 months (repeated measured ANOVA, P<0.001), but % HRC did not show any changes in both groups. There was a significant increase in TSAT of the responders at 2 weeks and all subsequent follow-up periods (repeated measured ANOVA P<0.001), but only a transition rise in TSAT was observed at 2 weeks in the non-responders.


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Table 2.  Changes of erythropoiesis and rHuEpo doses in haemodialysis patients with response and non-responsiveness to intravenous iron therapy

 


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Table 3.  Univariate correlations between responsiveness to intravenous iron supplementation and different indices

 

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Table 4.  Stepwise discriminant analysisa in prediction of response to IVFE medications

 


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Table 5.  Results of logistic regression analysis and ROC curvesa

 

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Table 6.  Predictive values of chosen cut-off points for iron metabolism and erythropoietic indices in haemodialysis patients

 
Predictive value of single factors
Univariate analyses (Table 3Go) for different indices of erythropoietic activity and iron status showed that basal values of serum ferritin, rHuEpo dose, CHr, HFR and reticulocyte count significantly correlated with response to IVFE supplementation. There was an inverse relationship of response to IVFE treatment with CHr (r=-0.566, P<0.001) and serum ferritin (r=-0.356, P<0.01) at baseline. Thereafter, responsiveness to IVFE medication exhibited a strongly positive correlation with {Delta}CHr (r=0.530, P<0.001) and {Delta}HFR (r=0.359, P<0.01) at 2 weeks and then with {Delta}CHr (r=0.646, P<0.001) and {Delta}HFR (r=0.60, P<0.001) at 4 weeks.

Prediction of response to IVFE supplementation
Stepwise discriminant analyses showed that {Delta}CHr in conjunction with {Delta}HFR at 4 weeks possessed a superior accuracy in predicting the response to IVFE supplementation as compared with basal serum ferritin and CHr alone (r2=0.466, P<0.001) or {Delta}CHr with {Delta}HFR at 2 weeks (r2=0.356, P<0.001) (Table 4Go). The combined use of {Delta}CHr4W and {Delta}HFR4W allowed for the highest accuracy in predicting the responsiveness in HD patients (r2=0.531, P<0.001).

Each of these parameters was further analysed by ROC curves to obtain the cut-off value for the best sensitivity and specificity in prediction of response to IVFE supplementation. The best threshold values for detecting iron deficiency were: serum ferritin <300 µg/l, CHr <28 pg, {Delta}CHr2W >1.2 pg, {Delta}HFR2W >35/µl, {Delta}Chr4W >1.2 pg and {Delta}HFR4W >500/µl (Table 5Go). Logistic regression analysis displayed that factors such as serum ferritin and CHr at baseline, {Delta}CHr and {Delta}HFR at 2 weeks and {Delta}CHr and {Delta}HFR at 4 weeks entered the model and exhibited significant odds ratios in predicting the response to IVFE supplementation, respectively. The results were similar to those in Table 4Go.

Individual tests and their combinations demonstrated the different diagnostic performances in Table 6Go. Baseline serum ferritin as well as CHr had borderline sensitivity and specificity, and their combinations, either ferritin <300 µg/l plus CHr <28 pg or ferritin >300 µg/l plus CHr>28 pg, exhibited a high specificity but a low sensitivity. The use of {Delta}CHr2W in conjunction with {Delta}HFR2W produced an improvement in the positive and negative predictive values. The combined use of {Delta}CHr4W and {Delta}HFR4W further achieved the highest predictive powers (minimal false positive and negative results) and were superior to all tests at the cut-off values of >500/µl and >1.2 pg, respectively. The positive predictive value was 100% and the negative predictive value increased to 92% when the two markers at the corresponding cut-off values were combined.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Current available guidelines advocate an aggressive IVFE therapy in the hope of achieving and maintaining target haemoglobin of 11–12 g/dl and haematocrit of 33–36% in HD patients receiving rHuEpo therapy [4,5]. Although iron loss of 1–2 g/year from dialytic procedures is common in most HD patients, the repetitive parenteral iron administered as recommended may expose the patients to risk of iron overload when overt iron deficiency is, in fact, absent. The deleterious effects of iron include its oxidative properties, haemosiderosis, cardiovascular and infection morbidities [6,7]. Optimal iron balance between demand and supply, thus, remains a dilemma. With the risk/benefit consideration, ‘early’ recognition of poor response to IVFE supplementation is of great importance to avoid the inadvertent hazards from unnecessary treatment.

Fishbane et al. [20] and Macdougall et al. [21] proposed that it needs at least 4 or 8 weeks to observe a significant response to IVFE supplementation by monitoring the erythrocyte indices, including haemoglobin and haematocrit. Some of our results (Table 2Go) are in agreement with their findings. The main reason for the late diagnosis (>4 weeks) might be the long life-span of mature RBC. Since erythrocyte indices cannot provide information on the rapid change in erythropoietic activity, there is a need to seek an earlier and more sensitive and reliable marker for functional iron deficiency or iron-limited erythropoiesis in rHuEpo-treated patients. A growing body of evidence suggests the ideal method for visualizing the erythropoietic activity should focus on the reticulocyte, the newly produced erythrocyte that has just been released from the bone marrow. Recently, measurements of reticulocyte cellular characteristics by automated flow cytometry have been demonstrated to afford extremely sensitive and early information for both diagnosis and treatment of iron-deficient anaemia in normal subjects [22] and in patients with reticulocytosis [15,16,19].

In the responders (Table 3Go), baseline CHr of <28 pg with an inverse correlation with response to IVFE supplementation, indicating iron-deficient erythropoiesis stimulated by rHuEpo, is similar to the findings of Mittman et al. [16]. Intriguingly, the changes in CHr at 2 and 4 weeks exhibited a strong correlation with response to IVFE therapy in our study. Iron is utilized for synthesis of haemoglobin by the erythroid progenitors in bone marrow. Reticulocytes exist in the circulation for only 1–2 days and signal the marrow erythropoietic activity 3–4 days after iron was actively incorporated into haemoglobin. Thus, CHr reflects the amount of iron availability during RBC production. Investigators have indicated that CHr is a surrogate indicator for iron supply during rHuEpo therapy in both normal subjects [23] as well as in ESRD patients [15,16].

Fishbane et al. [15] and Mittman et al. [16] proposed the diagnosis of iron deficiency at the level of the reticulocyte within 48 h after a single IVFE dose infusion (500–1000 mg of elemental iron). The present study demonstrated a sustained CHr change for 6 months in the responders (Table 2Go). Our results corroborate the findings of Besarab et al. [2] but differ from the reports showing a time course of 2–11 days for CHr changes [15,16]. Such discrepancy might be partly attributed to the different IVFE dosing schedules leading to variances in the restoration of iron availability. As compared with a single large dose of iron administered at one time, the divided dosage of iron supply in our study necessitates a longer period to observe the changes in CHr, which depend on the adequacy of iron supply. In addition, the definition of responders in our study is quite different from that defined as an increase in the corrected reticulocyte index of >1 unit at any time point during the first 2 weeks after a single dose of iron administration [15,16]. Early changes in reticulocyte count may simply reflect the release of immature reticulocytes from the marrow rather than the true expansion of erythropoiesis [23]. For this reason, it might be more feasible to show a change in erythroid response to additional iron administration than to demonstrate an increase in the corrected reticulocyte index only.

Reticulocyte analysis with fluorescence-based methods provides the opportunity to quantify immature reticulocyte based on their increased mRNA content. The most immature reticulocyte, HFR, allows for prompt identification of erythropoietic activity following various therapies for anaemia of different diseases [18,19] and serves as a cost-effective assessment of iron status in clinical utility. Consistent with investigations in non-uraemic populations [19,22], we found that {Delta}HFR at 2 and 4 weeks strongly correlated with response to IVFE therapy and permitted a reliable assessment of erythropoietic activity in the HD patients following IVFE medications. Receiver operating characteristic curves were further used to calculate the cut-off value of each index that could maximize the sensitivity, specificity and predictive values. We found that {Delta}CHr4W and {Delta}HFR4W at cut-off values of >1.2 pg and >500/µl, respectively, were more predictive of the status of iron-deficient erythropoiesis in HD patients. When the two markers at the corresponding cut-off values were combined, they provided the highest accuracy in predicting responsiveness to IVFE supplementation, with a sensitivity of 96% and a specificity of 100%.

A perfect test should be accurate, simple and inexpensive for clinical utility. Nowadays, no single test of iron parameters achieves all these criteria to conclusively exclude the presence of functional iron deficiency in rHuEpo-treated HD patients [8,9]. Simultaneous measurements of a group of markers indicating iron availability and erythropoietic activity may afford a better diagnostic value. Based on our findings, a trial of IVFE supplementation accompanied with monitoring of CHr and HFR within 1 month is worthwhile for clinical practice. By means of early recognition of non-responsiveness to IVFE supplementation, the goal of improving therapeutic efficacy as well as avoiding the inadvertent hazards related to over-treatment with iron can be achieved. We herein propose an algorithm to guide IVFE treatment in HD patients (Figure 1Go), according to the analysed data in Tables 4Go–6GoGo.



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Fig. 1.  Algorithm for early prediction of response or unresponsiveness to intravenous iron therapy in haemodialysis patients receiving rHuEpo therapy.

 
Strategy 1
For patients with basal serum ferritin of <300 µg/l and CHr of <28 pg, IVFE replacement should be routinely administered due to the high predictive power of responsiveness (96%). In contrast, IVFE therapy is not highly recommended for those with serum ferritin of >300 µg/l and CHr of >28 pg because the probability of non-responsiveness is 92%.

Strategy 2
The possibility of functional iron deficiency in patients with either basal serum ferritin of >300 µg/l and CHr of <28 pg or basal ferritin of <300 µg/l and CHr of >28 pg remains controversial. For these two conditions, IVFE therapy may be tried by monitoring the changes in CHr and HFR at 2 or 4 weeks. At 2 weeks, IVFE medications should be continued in patients with status of {Delta}CHr2W >1.2 pg and {Delta}HFR2W >35/µl due to a high predictive power of 91% for response to IVFE treatment. Moreover, conclusive diagnosis of iron-deficient erythropoiesis can be established early by {Delta}CHr4W of >1.2 pg in conjunction with {Delta}HFR4W of >500/µl because of a predictive power of 100% for response to IVFE 5 months following supplementation.

We conclude that CHr and HFR are superior to the conventional erythrocyte and iron metabolism indices and have potential to accurately assess iron status as well as erythropoietic activity in HD patients following IVFE medications. Basal ferritin and CHr had borderline sensitivity and specificity in assessing iron deficiency in HD patients on rHuEpo treatment. Changes in CHr and HFR, at 2 and 4 weeks, respectively, may serve as an early predictor of response to IVFE medications during aggressive iron supplementation.



   Acknowledgments
 
The study was supported by grants nos VGH 91-067, VGH 91-284 and VGH 91-376-14 from the Research Programs of Taipei Veterans General Hospital, Taiwan.



   Notes
 
Correspondence and offprint requests to: Der-Cherng Tarng, MD, PhD, Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, No. 201, Section 2, Shih-Pai Road, Taipei 112, Taiwan. Email: dctarng{at}vghtpe.gov.tw Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 23. 6.02
Accepted in revised form: 12. 9.02