|Year : 2020 | Volume
| Issue : 6 | Page : 1263-1272
|Diagnosis of iron deficiency in hemodialysis patients: Usefulness of measuring reticulocyte hemoglobin equivalent
Dawlat Sany1, Yasser El Shahawi2, Jenan Taha3
1 Department of Nephrology, Kalba Hospital, Fujairah, United Arab Emirates
2 Department of Nephrology, KhorFakan Hospital, Sharjah, United Arab Emirates
3 Department of Clinical Pathology, Kalba Hospital, Fujairah, United Arab Emirates
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|Date of Web Publication||29-Jan-2021|
| Abstract|| |
The evaluation of iron status in dialysis patients provides information essential to the planning of adequate recombinant human erythropoietin treatment. To diagnose iron deficiency in patients undergoing hemodialysis (HD), reticulocyte hemoglobin content and percentage of hypochromic red cells are incorporated into the European best practice guidelines on anemia management in chronic kidney disease (CKD), the mean reticulocyte hemoglobin content (Ret-HE) was proposed as alternatives to standard biochemical tests. Reticulocyte hemoglobin content and percentage of hypochromic red cells are incorporated into the European best practice guidelines on anemia management in CKD. Our aim was to assess the value of Ret-HE parameter, in terms of the sensitivity and specificity for detecting iron deficiency, in HD patients. We studied 50 patients undergoing HD three times weekly , to clarify the accuracy of Ret-HE in diagnosing iron deficiency in dialysis patients, we initially compared Ret-HE with such iron parameters as serum ferritin levels, transferrin saturation, and hypochromic red blood cell (Hypo%) which has been established as indicators of functional iron deficiency. Ret-HE mean value in anemic patients was (25.84 ± 4.23 pg) and had good correlation (P <0.001) between Ret-HE, serum iron, ferritin, transferrin, and transferin saturation in dialysis patients. Receiver operating characteristic curve analysis revealed, values of the area was 0.887, and at a cutoff value of 27.0 pg, a sensitivity of 90.4% and a specificity of 80.8% were achieved. The newly proposed Ret-HE can provide clinicians with information equivalent to iron deficiency anemia markers. Ret-HE is a new parameter that is easily measurable is suggested as reliable parameters for the study of erythropoiesis status in HD patients.
|How to cite this article:|
Sany D, El Shahawi Y, Taha J. Diagnosis of iron deficiency in hemodialysis patients: Usefulness of measuring reticulocyte hemoglobin equivalent. Saudi J Kidney Dis Transpl 2020;31:1263-72
|How to cite this URL:|
Sany D, El Shahawi Y, Taha J. Diagnosis of iron deficiency in hemodialysis patients: Usefulness of measuring reticulocyte hemoglobin equivalent. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2021 Mar 2];31:1263-72. Available from: https://www.sjkdt.org/text.asp?2020/31/6/1263/308335
| Introduction|| |
Anemia is a common problem among people affected by chronic kidney disease, especially among those patients undergoing hemodialysis (HD). Correction of the anemia yields numerous benefits: a higher tolerance for physical activity,, an improvement of cognitive and cardiovascular functions,, a better quality of life,, reduced hospitalization, and lower mortality. Anemia is corrected with the administration of erythropoiesis-stimulating agents (ESAs). The therapeutic goal is to reach a hemoglobin concentration between 11.0 and 12.0 g/dL (110–120 g/L).,,
In those undergoing HD and is treatment with ESAs, iron-deficient erythropoiesis frequently develops. The iron deficiency can be absolute from factors such as malnutrition, gastrointestinal bleeding, chronic blood retention in the dialysis circuit, and frequent blood collections. In addition, functional deficiency also can occur due to limitation of bone marrow erythropoietic activity by inability to mobilize sufficient iron from body storage sites. In this situation, the body’s total iron stores may be normal. The iron deficit limits the effectiveness of the therapy with ESAs, and, to optimize the treatment, patients must receive an intravenous (IV) iron supplement.,, It is essential to select patients who need iron supplementation because parenteral iron administration has potential immediate risks such as toxic effects and anaphylactic reactions and also long-term effects such as decreased polymorphonuclear leukocyte function, increased risk of infections, and organ damage. Best-practice guidelines have been developed for the use of potentially useful parameters for identifying and managing iron deficiency in patients undergoing HD; the European Best Practice Guideline, and the US Kidney Disease Outcomes Quality Initiative (KDOQI). The main differences concern the threshold value of the serum ferritin concentration (100 vs. 200 ng/mL) and the use of the percentage of erythrocytes with cellular hemoglobin concentration lower than 280 g/L (HYPO%), which represents the percentage of hypochromic red blood cells (RBCs). Even though HYPO% has been demonstrated as one of the best predictors of iron deficiency, it is not included in the KDOQI guidelines because the measured value depends on the time elapsed between collection and analysis. In fact, the erythrocytes in samples stored at room temperature tend to swell progressively, with consequent reduction of the cellular hemoglobin concentration and an increase of HYPO%. In contrast, the mean reticulocyte hemoglobin content (Ret-HE) parameter that indicates the average hemoglobin content of the reticulocytes remains stable over time. When HYPO% is used, the suggested threshold is 10%, but more recent studies indicate better diagnostic efficiency using values at a lower threshold, around 6% to 7%. Recently, the Sysmex (Sysmex, Kobe, Japan) was marketed. It provides a reticulocyte parameter, the equivalent of the mean Ret-HE.
| Aim of the Study|| |
The main goal of this work is the study of Ret-HE parameter, to assess their performance in the detection of functional iron deficiency in terms of sensitivity and specificity in HD patients and to verify the optimal cutoffs in comparison with anemia biochemical indices.
| Subjects and Methods|| |
The present study evaluates Ret-He to verify whether these parameters could help the correct classification of patients suffering functional iron deficiency, who will benefit from therapy, and other patients at risk of iron overload. The data were cross-sectional sample. As administration of IV iron is not recommended if serum ferritin level >500 ng/mL (KDOQI guidelines), we have excluded the patient with ferritin >800 ng/mL, patients with hemoglobin concentrations of more than 12.0 g/dL (120 g/L), recent bleeding, clinically evident inflammatory or infectious disease, malignancy, hemoglobinopathy, and a requirement for blood transfusion.
We investigated 50 HD patients who were undergoing long-term HD and managed according to the recommendations of the National Kidney Foundation, KDOQI guidelines objective of reaching and maintaining a hemoglobin concentration between 11.0 and 12.0 g/dL (110–120 g/L) and transferin saturation (TSAT) >20%. All of them were treated with intermittent in center HD (standard 4 h bicarbonate dialysis, 3 times per week), receiving epoetin therapy for at least three months, and were in the maintenance phase of their treatment, with stable doses for at least four weeks. There were 50 patients, 20 males and 30 females). The mean age was 67.4 ± 14.7 years. The causes of renal failure were diabetes mellitus in 35, and 15 were non-diabetics. These patients had received HD for 4.3 ± 10.1 (years).
Informed consent was obtained from participants before enrollment. Data obtained from these tests will be analyzed using the appropriate statistical method samples.
Blood specimens were obtained before the start of the first HD session of the week. Whole blood for the blood counts was collected by venipuncture into tubes containing trisodium ethylenediaminetetraacetic acid. Serum samples were prepared simultaneously and stored at -80°C until the measurements were made. Other serum chemical parameters, i.e., iron, ferritin, total iron binding capacity (TIBC), and transferrin were also measured. As indicators of iron, TSAT, and serum ferritin level were measured. Serum ferritin level was measured with a Roche Modular Analytics (Roche Diagnostics, Basel, Switzerland). TSAT was calculated after measurement of serum iron level and TIBC using a Hitachi automated analyzer 7700 (Nitoroso PSAP, Hitachi High-Technologies Corp., Tokyo, Japan). Ret-HE was obtained with the Sysmex XE-5000. These parameters were obtained using a “forward light scatter” measure that, because of the particular characteristics of the system, correlates to the cellular hemoglobin content. Because it is not a direct measurement of the cell hemoglobin content, the term equivalent was proposed by the manufacturer for this parameter. The instrument defines cells with a low hemoglobin (equivalent) content as those erythrocytes with a value lower than an arbitrary preset threshold equivalent to 17 pg. Ret-HE represents the average of the distribution of the values of the hemoglobin content of individual reticulocytes.,
| Statistical Analyses|| |
Using the software DR SPSS II (SPSS Inc., Chicago, IL, USA), Pearson’s correlation coefficient measurement and receiver operating characteristic curve (ROC) analysis were carried out. The significance of intergroup differences was tested by analysis of variance. P <0.05 was regarded statistically significant. To identify optimal tests and the cutoff point level for detecting iron deficiency and threshold values to predict iron deficiency, ROC was applied. Sensitivity was calculated as probability that a test result would be positive when the iron deficiency was present, and specificity was defined as probability that a test result would be negative when the iron deficiency was not present. Two-tailed P <0.05 was considered to indicate a statistically significant difference.
| Results|| |
Values of parameters and correlation in HD patients [Table 1] and [Table 2] summarize the basic statistical data on each parameter analyzed. In anemic HD patients, Ret-HE 25.84 ± 4.23 pg (mean ± SD). RBC and hemoglobin level in mature erythrocytes were 10.75 ± 1.43 g/dL. There was a significant correlation between Ret-HE and S. iron, ferritin, transferrin, and TSAT% in HD patients (P <0.001) as shown in [Table 3] and [Figure 1]. The coefficient of correlation (r) between these two parameters was 0.419 for patients with TSAT below 25% and 0.342 for patients with TSAT below 50%. Thus, the correlation between Ret-HE and TSAT tended to be stronger as TSAT became lower. A positive correlation was noted between Ret-HE and serum ferritin (r = 0.374, P <0.049). Like the correlation between Ret-HE and TSAT, the correlation between Ret-HE and serum ferritin became stronger as serum ferritin level became lower, with the coefficient of correlation (r) being 0.218 (P <0.045) in patients with serum ferritin below 500 ng/mL and 0.372 (P <0.01) in patients with serum ferritin below 100 ng/mL.
|Figure 1: Correlation between Ret-HE and each parameter in hemodialysis patients. (a) Ret-HE and serum iron, (b) Ret-HE and serum ferritin, (c) Ret-HE and serum transferrin and (d) Ret-HE and TSAT%|
Ret-HE: Reticulocyte hemoglobin equivalent, TSAT: Transferin saturation.
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|Table 2: Baseline characteristics of hemodialysis patients classified as non iron deficiency and iron deficiency.|
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|Table 3: Correlation between reticulocyte hemoglobin equivalent with all parameters in the hemodialysis group patient.|
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Cut-off level for diagnosis of iron deficiency
ROC analysis was carried out to evaluate the capability to detect iron deficiency using Ret-HE value, according to the abovementioned criteria for the diagnosis of iron deficiency (TSAT <20% and serum iron <9 umol/L), data from these cases were analyzed to determine the cutoff level of Ret-HE [Table 4] and [Figure 2]). When the cutoff level of Ret-HE was set at 26.0, 27.0, 28.0, 29.0, 30.0, and 31.0 (pg), the sensitivity was 76.8%, 90.4%, 92.2%, 94.1%, 95.9%, and 97.9%, and the specificity was 97.0%, 80.8%, 76.0%, 71.4%, 57.1%, and 43.1%, respectively. The product of sensitivity and specificity was maximal when the cutoff level was 27.0 (pg). The best cutoff level of HYPO% was 7.9%, with sensitivity 70% and specificity 58%.
|Figure 2: Receiver-operating characteristic curve of Ret-HE for prediction of iron deficiency.|
Ret-HE: equivalent of the mean reticulocyte hemoglobin content.
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|Table 4: Diagnostic characteristics of Ret-HE for predicting iron deficiency anemia.|
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| Discussion|| |
Absolute iron deficiency in HD patients is defined on the basis of TSAT and serum ferritin levels, whereas functional iron deficiency results when there is a need for a greater amount of iron to support erythro-poiesis than can be supplied. Thus, the conventional methods of estimating iron stores such as serum ferritin and TSAT are inadequate to evaluate functional iron deficiency. Functional or absolute iron deficiency is a limiting factor for the efficacy of ESAs in patients undergoing HD. The administration of IV iron can cause unwanted effects, immediate, and long term, so it becomes important to select patients who need iron supplementation and possibly have the parameters useful in predicting an effective response. Results from the multi-national Dialysis outcomes and practice patterns study add important new information in that they show an increase in mortality as well as hospitalization rates in patients whose monthly IV iron dose was higher than 300 mg. High doses are associated with an increased risk for all-cause mortality in patients with hemoglobin 100–120 g/L and ≥120 g/L. Several national and international guidelines suggest using some widely available biochemical markers for recognizing iron deficiency: TSAT% and serum ferritin. However TSAT% is strongly influenced by the daily fluctuation of serum iron levels, and the serum ferritin value is an acute phase protein and, thus, can be increased in chronic inflammatory diseases such as uremia. The guidelines for treating anemia in patients undergoing HD and receiving ESAs and IV iron agree on the lower values of TSAT (<20%) at which therapy has to be started but disagree on the upper value of ferritin, which should not be exceeded to avoid the risk of acute and chronic toxicity. Thus, the conventional methods of estimating iron stores such as serum ferritin and TSAT are inadequate to evaluate functional iron deficiency.,,,, Thus, a need to use additional new parameters with careful interpretation of results is required. Development of new parameters may allow the complete scope of disorders of iron metabolism to be identified quickly and managed. Hematological indices and the new parameters measured with automated counters are sensitive for detecting small changes in subpopulations of red cells. They hold promise for the evaluation of iron-restricted erythropoiesis and the status of iron metabolism. However, more studies are needed. Stimulation of erythropoiesis by EPO therapy increases the demand for instantly available iron, which often proves insufficient even in patients whose whole body iron store is not significantly depleted., Several parameters become available to evaluate iron deficiency, in addition to the conventional markers. The percentage of hypochromic cells (HYPO%) is a test that identified a subpopulation of mature RBCs exhibiting evidence of insufficient iron content. Although HYPO% represents direct measurement of iron status, diagnostic performance may be hindered by the long half-life of mature erythrocytes in the circulation,, also HYPO% is strongly influenced by the time between sampling and analysis, and it is not recommended by the KDOQI guidelines. The concentration of circulating soluble transferrin receptors (sTfR) has also been used to diagnose iron deficiency. The sTfR level is affected by both iron status and erythropoietic status, i.e., expression of these receptors increases in iron deficiency states, while newly formed erythrocytes shed many receptors into the circulation. Thus, it may be difficult to distinguish the status of iron deficiency and the expansion of receptors expressed by newly produced erythrocytes. The marketed XE-5000 analyzer offers some RBC and reticulocyte parameters (Hypo%-He and Ret-HE) with the same clinical applications, extending the adoption of the use of these parameters. Recent advances in automated flow cytometer methods have made it possible to perform accurate quantitative and qualitative analyses of reticulocytes. Ret-HE is a direct index of iron availability and reflects the cellular availability of iron. In this study, we have tried to show clinical utility of the Ret-HE parameter as an index for iron status in the anemic HD patients. In this study, strong correlation is observed between Ret-HE and Hb, serum iron, serum ferritin, serum transferrin, and TSAT%. The best cutoff level of Ret-HE was 27 pg for prediction of iron deficiency anemia in HD patients as the product of sensitivity and specificity was maximal. Ret-HE and CHr cutoff values reported for the identification of iron deficiency vary widely in the literature, from 26 to 33 pg, depending on the study patient population.,,,,,,,,, Urrechaga et al reported in their study that among HD patients, Ret-HE area under curve (AUC) was 0.84 (95% CI 0.64–0.93), at cutoff 30.8 pg, sensitivity 78.7%, and specificity 87.2%, thus concluded that Ret-HE are reliable parameters for the study of erythropoiesis status in HD patients. Nowadays, the development of new techniques and hematological parameters has revealed important information about functional integrity of bone marrow, diagnosis of anemia, and recombinant human erythro-poietin monitoring therapy used in HD patients. The newly proposed Ret-HE can provide clinicians with information equivalent to CHret and HYPO%. Ret-HE may be considered to be useful as an indicator of iron deficiency in HD patients. This parameter is advantageous in that it can be measured rapidly, full automatically, and simply in peripheral blood that it requires no additional reagent and that this parameter can be added to an existing automatic blood analyzer by instalment of a software. Ret-HE is considered as new parameter, equivalent value to CHr, and is easily measurable.
A limitation of the study is that we have focused on parameters of iron status, factors other than low iron stores have been recognized to hamper the efficacy of ESA therapy, that is high turnover bone disease and hyper-parathyroidism, so prospective studies are needed including more variables, to verify these results in dialyzed patients in different clinical situations.
| Conclusion|| |
Ret-HE may be considered as useful indicator of iron deficiency in HD patients. This is to be expected, as the Ret-HE parameter as an index of reticulocyte hemoglobin might predict future changes in hemoglobin levels. Potential may exist for the Ret-HE parameter to be used as a tool to diagnose iron deficiency in HD patients and may be used to monitor responses to IV iron in dialysis patients, in a way that is more meaningful than the current approaches.
| Acknowledgment|| |
We would like to thank Wafaa Moosa and Hessa Ibrahim laboratory technician in charge of the hematology department.
Conflict of interest: None declared.
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Department of Nephrology, Kalba Hospital, Fujairah
United Arab Emirates
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
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