Monitoring the content of reticulocyte hemoglobin (CHr) as the progression of anemia in nondialysis chronic renal failure (CRF) patients

Background: We previously showed that the content of reticulocyte hemoglobin (CHr) is a reliable measure of iron status in chronic dialysis patients with erythrocytopoiesis. The CHr was significantly correlated with conventional parameters of iron deficiency in dialysis patients. We attempted to utilize the measurement of CHr levels to monitor iron status and clarify the changes in iron levels that occur as renal anemia progresses in patients with chronic renal failure (CRF). Methods: We measured CHr, iron parameters, and the intrinsic erythropoietin (EPO) concentration in nondialysis CRF patients who visited our outpatient clinic (n = 211). Iron deficiency was defined according to the transferrin saturation (TSAT) and ferritin levels. Conventional red blood cell parameters and CHr levels were measured using an ADVIA120 autoanalyzer (Bayer Medical, USA). Results: The mean CHr value of the nondialysis CRF patients (creatinine clearance less than 70 mL/min) was 32.3 pg, which was not significantly different from that of the dialysis patients. Significant correlations were found between CHr and ferritin levels (r = 0.042, p < 0.0403) and CHr and TSAT levels (r = 0.040, p < 0.0157). A positive correlation was observed between the CHr and serum creatinine levels. Nondialysis CRF patients treated with recombinant human EPO (rHuEPO) at a dose of 24,000 U/month exhibited lower CHr levels, compared with those of other patients who received less than 24,000 U/month. Conclusion: CHr is an easily measurable and trustworthy marker of iron status in nondialysis CRF patients. Moreover, the CHr level was also sensitive to iron alterations in nondialysis CRF patients receiving rHuEPO treatment, and thus, the CHr value could likely provide useful information regarding the need for iron supplementation.

Keywords: Reticulocyte hemoglobin; Anemia; Chronic renal failure (CRF); Erythropoietin (EPO); Recombinant human erythropoietin (rHuEPO)

Anemia associated with deteriorating renal function, defined as renal anemia, is mainly attributed to a disturbance in the renal production of erythropoietin. Renal anemia is a major complication in end-stage renal failure that can lead to hepatitis virus infection or hemochromatosis, as a result of frequent transfusions, etc. Over the last 10 years, however, recombinant human erythropoietin (rHuEPO) therapy has allowed anemia to be treated not only in dialysis patients,[1], [2], [3] but also in nondialysis chronic renal failure (CRF) patients.[4], [5]

Since the improvement in anemia is frequently blunted by the presence of iron deficiencies, an adequate supply of iron is needed during the intense use of rHuEPO. Thus, determining the iron deficiency status in hemodialysis (HD) patients is essential for deciding on appropriate dosages of rHuEPO and iron.[6], [7], [8] The conventional method for determining the existence of an iron deficiency involves measuring the serum ferritin and transferrin saturation (TSAT) levels, but these markers are indirect. The ideal method for evaluating iron status would be to directly measure the iron content of erythrocytes, particularly in newly produced cells. Recently, a test that evaluates the hemoglobin level of reticulocytes using the flow cytometric technique has become available.[9] The content of reticulocyte hemoglobin (CHr) is a measure of hemoglobin in newly formed reticulocytes.

As mentioned above, rHuEPO treatment is now available for nondialysis CRF patients.[4], [5] Since an improvement in patient's QOL and, moreover, an attenuation in the deterioration of renal function have been reported,[4], [10] rHuEPO treatment has become widely used even among nondialysis CRF patients. However, information on iron status, dosage, and changes in iron dynamics were limited in clinical practice. The purpose of this study was to evaluate the significance of CHr in nondialysis CRF patients by comparing it with conventional examinations of iron status in these patients.

We measured CHr, iron parameters, and the intrinsic EPO concentration to evaluate the significance of CHr as an indicator of iron status in nondialysis patients. Iron deficiency was defined as a TSAT value of less than 20% and a serum ferritin level of less than 100 ng/mL, based on the Clinical Practice Guidelines—Anemia of Chronic Renal Failure in National Kidney Foundation: DOQI (Dialysis Outcomes Quality Initiative).[11] Forty-eight patients were selected for the treatment of renal anemia (as defined by an Hct of less than 30%) to compare the effects of rHuEPO treatment by subcutaneous injection with a maximum dosage of 24,000 U per month on the iron parameters.

The study was performed in the Tokyo Women's Medical University Hospital's outpatient clinic. The protocol of the study and the informed consent form were approved by the hospital's institutional review board in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects participating in the study.

We investigated 211 nondialysis CRF patients and 53 age-matched controls. Nondialysis CRF was defined as a serum creatinine level of more than 1.3 mg/dL or a creatinine clearance of less than 70 mL/min. The mean age of the CRF patients was 56.2 ± 7.2 years, while the mean age of the normal controls was 52.9 ± 9.9 years. One hundred six males and 105 females were enrolled in the study. The causes of renal failure were as follows: chronic glomerulonephritis (n = 93), gestosis (n = 6), polycystic kidney disease (n = 20), chronic pyelonephritis (n = 4), nephrosclerosis (n = 4), angitis (n = 8), diabetic nephropathy (n = 24), and other or unknown etiology (n = 49). Patients were excluded from the study if there were any changes in the rHuEPO or iron supplementation protocol, or if any episode of bleeding or blood transfusion had occurred in the 4 weeks prior to the commencement of the study.

Blood specimens were obtained in the outpatient clinic. Whole blood for the blood counts was collected by venipuncture into tubes containing trisodium EDTA. Serum samples were prepared simultaneously and stored at − 20°C until the intrinsic EPO concentration was measured. Other serum chemical factors, such as iron (Fe), ferritin, the total iron binding capacity (TIBC), albumin (alb), c-reactive protein (CRP), creatinine (Cr), and transferrin were also measured.

The conventional red blood cell parameters, percentage of hypochromic red blood cells (%HYPO), and the CHr level were measured using an ADVIA120 autoanalyzer (Bayer Diagnostic, Tarrytown, NY). This autoanalyzer quantifies blood cell hemoglobin and Hct using theautomated flow cytometric.[9] CHr, or the mean value of the hemoglobin mass in each cell, was determined by measuring the volume and hemoglobin concentration of each reticulocyte.

Serum chemistry values were determined using conventional analytical methods. The intrinsic EPO concentration was measured using an enzyme immunoassay.

Results are presented as the mean ± SEM. Data were evaluated using the ANOVA and post-hoc Bonferroni tests. A value of p < 0.05 was considered to be statistically significant.

A comparison of the patient characteristics and hematological indices between the control and CRF patients is shown in Tables 1 and 2. The CRF patients were divided into three groups according to their serum Cr levels, because the clearance data could not be obtained from all patients: group 1, under 2 mg/dL; group 2, 2 to 5 mg/dL; and group 3, more than 5 mg/dL. As shown in Table 1, no significant differences in gender, age, serum albumin, or CRP level were observed between the control group and the CRF subgroups.

Table 1. Patient Profiles and Laboratory Findings (Non-rHuEPO Recipients).

Table 2. Serum Iron Indices During the Progression of Renal Dysfunction.

406 ap < 0.05 vs. control. bp < 0.05 vs. Cr < 5 mg/dL.

Anemia had already appeared in group 1 (ANOVA, p = 0.002), and the mean hemoglobin (Hb) and Hct levels were lower overall among the CRF patients. Serum Fe and TIBC were significantly lower in the CRF patients than in the control subjects; however, no significant differences in serum ferritin levels were observed except for an elevated value in group 3. The TSAT in the CRF patients was significantly lower than that of the control subjects but was reciprocally elevated in group 3. The mean CHr in the CRF patients was 32.3 ± 2.2 pg, which did not differ significantly from that in the control subjects (32.5 ± 1.4 pg). On the other hand, when the patients were divided into two groups according to the iron deficiency criteria based on the DOQI guidelines, the mean CHr was slightly but significantly lower in the iron-deficient patients (32.1 ± 2.3 pg versus 32.9 ± 1.6 pg, p < 0.0159).

The intrinsic erythropoietin (iEPO) levels were measured in each patient with renal dysfunction to estimate how EPO affected the progression of anemia. The iEPO concentration and the serum creatinine level were not directly related in nondialysis patients. However, a negative relationship was observed between thelogarithm of the iEPO concentration and the serum creatinine level (r = − 0.093, p = 0.0146). No relationship was observed between iEPO or log iEPO and Hct. As to the changes in each iron parameter versus renal function, namely, the serum Cr level, a positive correlation was observed between the Cr and ferritin levels (r = 0.058, p = 0.0077) and the Cr and CHr levels (r = 0.029, p = 0.0468); the correlation between Cr and TSAT was not statistically significant. To evaluate the reliability of CHr as a marker of iron status, the correlations between CHr and other serum iron markers were examined in nondialysis patients who were not undergoing rHuEPO treatment. Conventional markers used to estimate iron storage, such as ferritin and TSAT, were estimated. Significant correlation was observed between CHr and serum ferritin levels (r = 0.0403, p = 0.0420) and between CHr and TSAT levels (r = 0.040, p = 0.0157).

The effects of external EPO administration on the iron parameters, including CHr, were studied in 48 patients; 20 patients without and 28 patients with subcutaneous rHuEPO treatment whose serum creatinine levels were more than 5 mg/dL. The patients were categorized into three groups according to the dosage of rHuEPO that they received. Group 1 was not treated with rHuEPO; group 2 was treated with a partial dosage of rHuEPO; and group 3 was treated with the maximum dosage of 24,000 U/month of rHuEPO. The mean values of CHr in each group were 33.2 pg, 33.7 pg, and 30.6 pg, respectively, and the change in group 3 was statistically significant (p = 0.0023 versus group 1, p = 0.001 versusgroup 2). Neither the TSAT nor the ferritin levels differed among the three groups (Figure 1).

Graph: Figure 1. Effects of rHuEPO treatment on iron parameters in patients with a creatinine level of more than 5 mg/dL.

To observe the influence of rHuEPO administration on CHr, the time course of the changes in CHr, %HYPO, and Hct after the start of rHuEPO treatment was studied in 10 patients. The patients received a subcutaneous administration of 12,000 U of rHuEPO twice a month. CHr, %HYPO, and Hct levels were investigated before and during the rHuEPO therapy. A time course of the data obtained for these patients is summarized in Figure 2. An improvement in Hct and a decrease in CHr were observed. One month after the start of the rHuEPO treatment, the CHr level decreased from 32.9 ± 0.8 to 30.2 ± 1.2 pg (p < 0.05), while the Hct level improved from 27.2 ± 1.3 to 28.6 ± 2.1% (p < 0.02) after 2 months. On the other hand, %HYPO tended to increase after the initiation of rHuEPO and became significant 3 months after the treatment.

Graph: Figure 2. Time course of iron parameters after starting the treatment with rHuEPO in nondialysis CRF patients (n = 10).

Renal anemia is a major complication of end-stage renal failure but first appears early during the clinical course of renal dysfunction, at a creatinine clearance level of around 60–70 mL/min.[12], [13], [14] The cause of anemia is thought to be mainly due to a disturbance in the production of erythropoietin, which disturbs the terminal differentiation of erythroblasts, resulting in a lower iron demand and an increase in iron stores. On the other hand, nondialysis CRF patients may develop an iron-deficient status for a variety of reasons, such as simple anorexia, low protein diet, etc.[15]

Nevertheless, the details of iron absorption and metabolism in nondialysis CRF patients have not been fully clarified, and the changes in iron-related parameters during the progression of renal dysfunction have not been adequately explained. In this study, the TSAT level tended to decrease in patients with a serum creatinine level of less than 5 mg/dL, suggesting that these patients may have an iron deficiency. Fe and TIBC decreased in CRF patients, as reported by Madore et al.,[16] but the cause of the decrease was not fully analyzed. Furthermore, TSAT and ferritin levels increased in patients with a serum creatinine level of more than 5 mg/dL. Overall, positive relations between serum creatinine and ferritin and between creatinine and CHr were observed, so it seems likely that iron was repleted in the erythrocytes as the deterioration of renal function advanced.

The CHr marker can be used to evaluate the hemoglobin level of reticulocytes using a flow cytometric technique. Reticulocytes are newly produced, immature red blood cells that can be detected in the circulation for 24 h, becoming mature cells when they lose their RNA.[9], [17] This test can be used to directly measure iron availability and may be an ideal test for evaluating iron status in dialysis patients. On the other hand, several new parameters have become available for evaluating iron deficiency. The percentage of hypochromic cells (%HYPO) is a test that seeks to identify a subpopulation of mature red blood cells exhibiting evidence of insufficient iron content. Although %HYPO reflects the direct measurement of iron status, the diagnostic performance of this test may be hindered by the long half-life of circulating mature red blood cells.[18], [19], [20] The concentration of circulating soluble transferrin receptors (sTfR) has also been used to diagnose iron deficiency.[21] However, the sTfR level is affected by both iron status and erythropoietic status, reflecting the increase in the number of newly synthesized red blood cells. Thus, distinguishing between iron deficiency and cell proliferation may be difficult using this marker.[22], [23]

We compared CHr with other conventional parameters for monitoring iron levels in nondialysis patients, as in HD patients.[24], [25], [26] CHr was well correlated with ferritin and TSAT levels, both of which have been utilized to determine iron deficiency in HD patients. These data indicate that CHr might be clinically useful as an iron parameter in nondialysis patients.

If nondialysis patients are treated with rHuEPO, these iron parameters are likely to be affected as in HD patients. Because improvements in anemia are frequently blunted by the presence of iron deficiencies, an adequate iron supply must be insured during the intense use ofrHuEPO.[27], [28], [29] In this study, the changes in the iron parameters of patients with a serum creatinine level of more than 5 mg/dL and who were undergoing rHuEPO treatment were analyzed to evaluate the usefulness of monitoring iron status. Our results demonstrated that only CHr showed a significant decrease in patients treated with the maximum rHuEPO dosage. In addition to absolute iron depletion, the stimulation of erythropoiesis by rHuEPO therapy increases the demand for instantly available iron, which often proves to be insufficient despite the patient having a body store of iron that may not be significantly depleted.[6], [7] Moreover, as these changes were not observed when ferritin or TSAT levels were analyzed, CHr measurements may be more sensitive at detecting functional iron deficiency. To confirm these results, the effects of rHuEPO treatment on CHr, TSAT, %HYPO, and Hct were observed over time in the same patients. Subcutaneous rHuEPO administration improved the Hct level, and the CHr level significantly decreased within 1 month of the start of therapy; in contrast, the TSAT and %HYPO levels did not change significantly (Figure 2).

Long-acting EPO is now available for these patients, but these patients may be more susceptible to iron deficiencies because of maintaining high serum EPO levels.[30] Whether iron levels affect the immune system,[31] such as in cases with chronic infections or chronic hepatitis or cardiovascular events,[32] is also controversial. Thus, iron supplementation should be strictly controlled. CHr may be a beneficial tool for real-time monitoring of iron levels, enabling a customized dosage of iron supplements to be administered.

This study was supported, in part, by the Foundation for renal anemia therapy.