CHr and Iron Deficiency in End Stage Renal Disease

The Use of Reticulocyte Hemoglobin Content in the Diagnosis of Iron Deficiency in Patients with End Stage Renal Disease

Beckie Michael, DO
Biographical Information

This is an excerpt from Bloodline Reviews, Volume 1, Issue 2-R, 2001, Diagnostic Advances in Hematology.

This coverage is supported by a grant from

Overview:

The anemia of end stage renal disease (ESRD) is a consequence of decreased production of erythropoietin. This is often accompanied by iron deficiency in patients on hemodialysis (HD) due to ongoing blood losses related to phlebotomy, blood retained in the dialyzer and tubing, and occult blood loss. Iron deficiency is the major cause of inadequate response to erythropoietin in HD patients. The traditional parameters used to assess iron status - serum iron, percent transferrin saturation and serum ferritin - are often unreliable in patients with ESRD. Problems with these markers include a decrease in transferrin in patients with hypoalbuminemia and chronic disease, and increased ferritin as an indication of inflammation. Absolute iron deficiency in ESRD had been defined as an iron saturation of <20% and/or a ferritin <100 ng/mL. The term "functional iron deficiency" has been used to describe the inability to mobilize stored iron from the reticuloendothelial system and meet the iron needs of erythropoietin driven hemoglobin synthesis. This diagnosis is made in patients with ESRD who have an iron saturation of 20-50% and a ferritin of 100-800 ng/mL and respond to intravenous iron with an increase in hemoglobin and/or a decrease in erythropoietin requirements.

Reticulocyte hemoglobin content (CHr) is a direct measure of iron status at the level of the reticulocyte. It has been studied as a potentially better indicator of iron deficiency in patients with ESRD. The benefits of CHr include not only a very high specificity and sensitivity for iron deficiency in ESRD, but also the ease of measuring CHr on a hematology autoanalyzer.

Core indicators from the Healthcare Financing Administration, which pays for the majority of hemodialysis in the U.S. and which follows outcomes of the treatment, show that in 1998, 21% of dialysis patients in the United States had a hematocrit level less than 30%, and that an even higher percentage had a hematocrit level of less than 33%, which is the target maintenance level for these patients. The data also show that 30% of dialysis patients had a transferrin saturation of less than 20% and that 6% of patients had what is agreed upon as absolute iron deficiency with a transferrin saturation of less than 20% and a ferritin of less than 100 ng/mL.

Iron deficiency is common in hemodialysis patients for several reasons. One is that even though all efforts are made to return blood to the patient at the end of dialysis, there is a small amount of blood loss with every procedure. Furthermore, there are occasions when the whole membrane clots, with the membrane and the tubing containing about 200 mL of blood. Therefore, these patients have significant obligate blood losses related to the dialysis procedure itself. These patients also get frequent phlebotomies, undergoing blood work at a minimum of once a month. The obligate blood losses in these patients are estimated to be equivalent to 1,500 to 2,000 milligrams of iron annually. Another issue is that there is probably poor dietary iron intake and dietary iron absorption in this population, as well as the fact that these patients take many medications and often do not take oral iron supplements as prescribed.

Iron deficiency is difficult to diagnose in hemodialysis patients. Bone marrow aspiration, the gold standard, can only diagnose absolute or storage iron deficiency and, therefore, cannot really help us to detect the functional iron deficiency present in many patients with ESRD. It is also impractical to perform bone marrow aspiration on hemodialysis patients just to assess their anemia. Further, the transferrin saturation and the ferritin tests, which are commonly used to assess iron status in hemodialysis patients, are indirect measures and often inaccurate, with false negative and positive rates of over 30%.

In the U.S., the National Kidney Foundation has published guidelines for the management of anemia in ESRD patients. These Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines¹ define both absolute and functional iron deficiency in this patient population and include recommendations on how to treat the conditions. Under these guidelines, absolute iron deficiency is defined as a transferrin saturation of less than 20% and a ferritin less than 100 ng/mL. Functional iron deficiency occurs when iron stores are present, but cannot be mobilized rapidly enough to maintain maximal erythropoietin driven erythrogenesis. Functional iron deficiency occurs when there is an increase in hemoglobin and/or decrease in erythropoeitin requirements in response to intravenous iron in patients with a transferrin saturation of 20-50% and a ferritin of 100-800 ng/ml.

The tests to assess iron status include the transferrin saturation and the ferritin. It is also possible to use percent hypochromic red cells - a technique commonly used in Europe - or a test for soluble transferrin receptor, which is increased in iron deficiency but not increased in the anemia of chronic disease. These tests, as stated previously, can be performed, but are not commonly used at this time in dialysis patients in the U.S. Finally, there is the test for reticulocyte hemoglobin content, which will be reviewed in further detail.

Reticulocyte hemoglobin content (CHr) is a direct measure of iron status at the level of the reticulocyte, and is an accurate and time-sensitive test. CHr is time-sensitive because reticulocytes circulate for approximately 24 hours before becoming mature red blood cells. Studies in both non-uremic and uremic patients have shown the value of CHr testing in diagnosing iron deficiency.

Two studies in non-uremic patients, both published by Carlo Brugnara, show the applicability of CHr testing in this population. The first study 2 involved the treatment of healthy volunteers with erythropoietin. In this study, the reticulocyte hemoglobin content sensitively detected changes in the iron status that occurred with erythropoietin administration. The second Brugnara study 3 involved the screening of 210 children for iron deficiency with traditional parameters, as well as soluble transferrin receptor and reticulocyte hemoglobin content. CHr was found to be the strongest predictor of iron deficiency, which was defined as transferrin saturation of less than 20%. Plasma ferritin had no predictive value in this study.

A study published by Mittman in the American Journal of Kidney Disease⁴ examined the use of CHr in patients with ESRD. In the first part of the study, in which CHr testing was performed in 364 hemodialysis patients, it was found that CHr had a normal distribution across the population, and that it correlated directly with hemoglobin, iron, transferrin saturation and ferritin. The second part of the study involved the administration of 500 mg of intravenous iron dextran in 79 patients, after which repeat CHr and hemoglobins were performed.

Of these 79 participants, only 47 had what could be defined as absolute iron deficiency, with a transferrin saturation < 20% or a ferritin < 100 ng/ml. 82% of patients shown to be iron-deficient by standard tests had an increase in CHr of greater than 2 picograms after intravenous iron therapy. Notably, 60% of those patients who were not defined as iron-deficient by standard tests also responded to intravenous iron with an increase in CHr of greater than 2 picograms.

In this study a CHr less than 28 picograms at baseline predicted functional iron deficiency, which was defined as a corrected reticulocyte increase of greater than 1% in response to intravenous iron. Also, the CHr predicted iron deficiency more accurately than iron saturation or ferritin.

A final point to consider in this study is that Mittman used the H*3 analyzer, which had slightly different methodology for calculating CHr, resulting in CHr results lower than those in the following studies, in which the ADVIA 120 autoanalyzer was used.

Another study addressing the use of reticulocyte hemoglobin content in dialysis patients was published by Stephen Fishbane in Kidney International in 1997⁵ . This study looked at CHr distribution in a population of dialysis patients, followed by an analysis of the responses of 32 patients to a single 1000 mg dose of intravenous iron. Of the seven patients classified as iron-deficient due to a greater than 1% increase in corrected reticulocyte production index within two weeks of iron administration, CHr predicted iron deficiency with a sensitivity of 100% and a specificity of 80%.

Based on these preliminary studies, Fishbane performed a long-term study using CHr to assess anemia in hemodialysis patients. This study followed 128 hemodialysis patients at three hemodialysis centers for six months. Participants were randomized either to conventional treatment with intravenous iron based on transferrin saturation of less than 20% or a ferritin of less than 100 ng/mL, or to intravenous iron if their CHr was less than 29 pg, as measured by the ADVIA 120 autoanalyzer. Overall, 80% of participants in the first group received intravenous iron over the six-month study period, compared to only 50% of patients in the second group.

In terms of outcomes, it was found that the positive predictive value for reticulocyte hemoglobin content was 71%, as compared to 57% for transferrin saturation and only 45% for ferritin. Further, because the CHr group received less intravenous iron over the six-month period, they ended up with lower ferritin levels at the end of the study than the control group. In addition, there was no difference in transferrin saturation, reticulocyte hemoglobin content, or hematocrit at the end of the study. Finally, as mentioned previously, the CHr group received less intravenous iron, and including the costs of lab analysis, the weekly cost of treating these patients was significantly lower than for the patients in the traditional iron management group.

These findings led Dr. Fishbane to conclude that CHr is a more accurate measure of iron status than existing tests in the hemodialysis population, and that iron management based on CHr leads to an equivalent hematocrit compared to standard tests, with a reduced utilization of intravenous iron, and a reduction in the overall cost of anemia care.

At Thomas Jefferson University, an ongoing prospective study of 93 hemodialysis patients is making use of the ADVIA 120 to assess the efficacy of reticulocyte hemoglobin content in optimizing the anemia management of this patient population. Only interim results from this study are available. Patients were excluded if they had active bleeding, a known malignancy, were HIV-positive, or had severe secondary hyperparathyroidism.

Researchers involved with this study want to look not only at CHr, hemoglobin and iron studies, but also to measure soluble transferrin receptor, making this perhaps the first direct comparison in dialysis patients of soluble transferrin receptor and reticulocyte hemoglobin content. Also being examined are levels of C-reactive protein (CRP), because several studies have suggested that CRP, a marker of inflammation, can actually predict mortality in dialysis patients, and that it is associated with erythropoietin resistance and hypoalbuminemia. Both baseline and post-intravenous iron administration soluble transferrin receptor levels were obtained and the samples were frozen.

In terms of the baseline patient characteristics, 36 patients had CHr levels less than 30 pg and ferritins less than 800 ng/mL. The control group consisted of the remaining 57 patients who had a CHr greater than 30 pg. The ages and sexes were similar across the two groups, but there was a higher percentage of African-American patients in the low CHr group. Baseline hemoglobin levels in the two groups were almost identical. The mean CHr in the study group was 27.75 pg with a standard deviation of 1.78, compared to a mean CHr of 32 pg with a standard deviation of 1.5 in the control group. The ferritin values and the erythropoietin doses were similar in both groups.

Table 1: Baseline characteristics of participants in the Thomas Jefferson University study of 93 hemodialysis patients.

Over the course of one month, study patients' hemoglobin levels increased from 11.15 g/dL to 11.82 g/dL, which was significant, and reticulocyte hemoglobin content increased from 27.75 to 29.3 picograms, which was also statistically significant.

59% of the patients who were treated had a greater than 1 picogram increase in CHr. Also, the CHr treatment cutoff of less than 30 did not pick up all the patients with absolute iron deficiency; only 4 of 7 with an iron saturation less than 20% and a ferritin less than 100 ng/mL were picked up with a CHr cutoff of less than 30. If the cutoff had been increased to 32, all patients with absolute iron deficiency would have been detected.

Table 2. Interim results of the Thomas Jefferson University study of 93 hemodialysis patients.

This ongoing trial demonstrates that the use of CHr is effective in assessing iron stores in hemodialysis patients and that intravenous iron therapy based on reticulocyte hemoglobin content results in a significant increase in hemoglobin. Further analysis will determine the effects on erythropoietin dose and iron utilization, and will compare the relationship of soluble transferrin receptor to reticulocyte hemoglobin content.

In conclusion, several studies have demonstrated the efficacy of reticulocyte hemoglobin content in assessing the iron status of hemodialysis patients. CHr has been found to be more sensitive and more specific than current methods of assessing iron status, and intravenous iron therapy based on CHr has been shown to improve hemoglobin concentrations in this patient population. Further, the use of CHr has been shown to be cost-effective and easy to perform, allowing more frequent assessment of a patient's iron status.

Overall, it appears that the ability to routinely perform CHr testing should allow for optimization of anemia management in patients with ESRD.

References:

1. K/DOQI Clinical Practice Guidelines For Anemia Of Chronic Kidney Disease: Update 2000. Am J Kidney Dis 2001 37: 182S-238S.
2. Brugnara C, Colella GM, Cremins J, Langley RC, Schneider TJ, Rutherford CJ, Goldberg MA: Effects of subcutaneous recombinant human erythropoietin in normal subjects: development of decreased reticulocyte hemoglobin content and iron-deficient erythropoiesis. J Lab Clin Med 123(5):660-7, 1994.
3. Brugnara C, Zurakowski D, DiCanzio J, Boyd T, Platt O: Reticulocyte hemoglobin content to diagnose iron deficiency in children. JAMA 281(23):2225-30, 1999.
4. Mittman N, Sreedhara R, Mushnick R, Chattopadhyay J, Zelmanovic D, Vaseghi M, Avram MM: Reticulocyte hemoglobin content predicts functional iron deficiency in hemodialysis patients receiving rHuEPO. Am J Kidney Dis 30(6):912-922, 1997.
5. Fishbane S, Galgano C, Langley RC, Canfield W, Maesaka JK: Reticulocyte hemoglobin content in the evaluation of iron status of hemodialysis patients. Kidney Int 52:217-222, 1997.

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