Clinical utility of reticulocyte hemoglobin equivalent in patients with heart failure

Anemia and iron deficiency (ID) are common in patients with heart failure (HF) and intravenous (IV) administration of iron to patients hospitalized for decompensated HF with ID improves outcome. The diagnosis of ID in routine practice is based on serum ferritin and transferrin saturation (TSAT) but both have limitations; alternatives should be considered. Reticulocyte hemoglobin equivalent (Ret-He) reflects iron content in reticulocytes but its clinical utility in patients with HF remains uncertain. We prospectively enrolled 142 patients hospitalized for decompensated HF. Sixty five percent had ID as defined in current international guidelines. Ret-He was directly correlated with serum iron and ferritin concentrations and with TSAT. There was a poor relationship between quartile of Ret-He and HF hospitalization or death but increases or decreases in Ret-He between admission and discharge were associated with a worse outcome. The clinical utility of Ret-He for identifying ID and predicting response to IV iron and prognosis for patients with HF requires further investigation.

. Patient characteristics on admission according to quartile of Ret-He levels. Continuous variables are presented as median and interquartile range, and categorical variables are presented as number (percentage). BMI body mass index, SBP systolic blood pressure, HR heart rate, Past HF past hospitalization for heart failure, HT hypertension, DM diabetes mellitus, AF atrial fibrillation, OMI old myocardial infarction, CVD cerebrovascular disease, eGFR estimated glomerular filtration rate, LVEF left ventricular ejection fraction, BNP brain natriuretic peptide, Alb albumin, Hb hemoglobin, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, TIBC total iron binding capacity, TSAT transferrin saturation, Ret-He reticulocyte hemoglobin equivalent, ACE-I angiotensinconverting enzyme inhibitor, ARB angiotensin II receptor blocker.     (Fig. 3C). And, the AUC for Ret-He to screen for IDA was 0.722. Similar to ID, the cut-off value for Ret-He to screen for IDA was 32.4 pg (true positive fraction: 0.716, 1-false positive fraction: 0.617) in HF patients (Fig. 3D).

Prognostic associations of Ret-He levels in HF patients. ID is associated with an adverse prognosis
in HF patients 3,5 , we thus assessed prognostic associations of Ret-He levels in HF patients. During a median follow-up of 21.7 months, composite outcomes (all-cause death or HF readmission) occurred in 61 patients. We first investigated prognosis in HF patients according to quartile of Ret-He levels; however, there was not significant different prognosis among the groups ( Fig. 4A-C). Then, we investigated prognostic associations of Ret-He levels in HF patients, whose Ret-He levels were measured both at admission and discharge, by change in Ret-He levels between admission and discharge (ΔRet-He). The distribution of ΔRet-He is shown in Supplemental Fig. 1. As there were patients with little change in Ret-He levels between admission and discharge, we performed a prognostic analysis based on a three-group classification of change in Ret-He levels (group 1: ΔRet-He ≦ − 2 pg, group 2: − 2 pg < ΔRet-He < 2 pg, and group 3: 2 pg ≦ ΔRet-He). Of note, a statistically significant worse prognosis was observed in group 1 than in the other groups ( Fig. 5A-C). The patient characteristics by a three-group classification of change in Ret-He levels are shown   Table 2. The median body mass index, TSAT, Ret-He, the prevalence of atrial fibrillation, and the use of Ca blockers were different among the groups.
To investigate the prognostic impact of ΔRet-He, we finally performed univariate and multivariate Cox regression analyses for prognosis in these patients. In the univariate model, ΔRet-He ≦ − 2 pg was associated with a worse outcome, while this tended to be associated with a worse outcome in a multivariate analysis (Table 3).

Discussion
This study showed for the first time that Ret-He has the clinical utility as a simple marker for ID in HF patients. Ret-He levels were directly correlated with serum iron and ferritin levels and with TSAT. Moreover, Kaplan-Meier analysis demonstrated that increases or decreases in Ret-He between admission and discharge were associated with a worse outcome in HF patients.
The gold-standard for measurement of ID is bone marrow histology. Although there are many definitions of ID based on circulating biomarkers, it is unknown which marker is best. Serum ferritin levels are commonly used to evaluate iron status; however, as ferritin is an acute phase protein, serum ferritin levels increase in the presence of chronic inflammation. As chronic inflammation is associated with the pathogenesis of HF, it should be taken into consideration. In fact, previous studies have shown that higher serum ferritin levels are associated with an adverse prognosis in HF patients 13,17 . Thus, cut-off values of these markers for ID should be reconsidered for HF patients. In this regard, Ret-He can be easily obtained, it thus may be advantageous for the screening for ID in patients with HF.
Ret-He can reveal the current iron status that are clinically significant. Of note, Ret-He levels were directly correlated with standard parameters of iron metabolism in HF patients. In this study, there were a few patients whose TSAT was about 90%. These patients had liver congestion or poor nutrition due to severe HF, which resulted in low transferrin production and high TSAT. In addition, the cut-off value for Ret-He to screen for both ID and IDA as defined in current international guidelines was 32.4 pg. These results indicate that Ret-He could reflect iron status and lead more easily applicable definition to screen for ID and IDA in HF patients. Ret-He can be easily measured in the same tube of cellular blood analysis, and the costs of Ret-He measurement are also economical. Taken together, these results suggest that Ret-He has the clinical utility as a simple marker for ID in HF patients. As the reports of improved clinical symptom with intravenous (IV) iron administration focus on anemia and ID in HF patients [14][15][16] , the measurements of Ret-He levels is considered to have an important perspective in HF patients.
A recent study has shown that there was no association between current guideline definition for ID and mortality in HF patients 17 . Similarly, in this study, ID defined by current guideline was not associated with prognosis in HF patients (P = 0.472 for all-cause death or HF readmission, P = 0.926 for all-cause death, P = 0.282 for HF readmission). Then, we evaluated the prognostic impact of Ret-He levels in HF patients. However, there was not a significant difference on prognosis according to quartile of Ret-He levels. We then investigated the prognostic impact of Ret-He in HF patients, by change in Ret-He levels between admission and discharge. Of note, by a prognostic analysis based on a three-group classification of change in Ret-He levels, a worse outcome was observed in patients with ΔRet-He ≦ − 2 pg than in the other groups. Also, multivariate analysis showed that ΔRet-He ≦ − 2 pg tended to be associated with a worse outcome. These results indicate that ΔRet-He ≦ − 2 pg even after optimal HF treatment is associated with a worse outcome in HF patients. To the best of our knowledge, this study showed for the first time that the change in Ret-He levels between admission and discharge was associated with prognosis in HF patients. Treatment of ID by monitoring Ret-He levels may lead to improve prognosis in HF patients. Recent clinical studies have shown that IV iron administration is beneficial for clinical symptom in HF patients with reduced ejection fraction with ID [14][15][16] . However, the effects of IV iron administration on prognosis in HF patients has been entirely unknown. Iron administration may provide a benefit for prognosis particularly in HF patients with ΔRet-He ≦ − 2 pg. In conclusion, these findings support the clinical utility of Ret-He to assess

Study limitations.
This study is a small single-centered and relatively small number study. Further validation in the future is needed.  Patients with renal failure with estimated glomerular filtration rate < 10 mL/min/1.73 m 2 or receiving hemodialysis (n = 18), leukemia (n = 1), autoimmune hemolytic anemia (n = 2), or active malignancy (n = 12) were excluded. In addition, we excluded 25 participants whose missing all the required iron indices and 7 participants who were lost to follow-up, which left a final analytical cohort of 142 participants (Supplementary Fig. 2). The patients' demographic data including co-morbid conditions, clinical signs, laboratory and echocardiographic data, in-hospital treatment including oral and intravenous medication, and length of hospital stay were obtained. Echocardiography was performed by sonographer and LVEF was defined with the modified Simpson method. Hypertension was defined as systolic blood pressure ≧ 140 mmHg or diastolic blood pressure ≧ 90 mmHg or receiving anti-hypertensive drugs. Diabetes mellitus was defined as glycated hemoglobin ≧ 6.5%, casual blood glucose ≧ 200 mg/dL, or fasting blood glucose ≧ 126 mg/dL or receiving oral hypoglycemic agents and/or insulin. The presence of old myocardial infarction and cerebrovascular disease was defined based on history, clinical presentation, examinations, and medications. Anemia was defined according to WHO criteria (Hb levels < 13 g/dL in men, Hb levels < 12 g/dL in women). ID was defined as serum ferritin levels < 100 μg/L, or serum ferritin levels 100-299 μg/L with TSAT (serum iron levels / TIBC × 100) < 20% 7,14,19 . IDA was defined by serum ferritin < 100 μg/L, or between 100 and 299 μg/L with TSAT < 20% and Hb levels below WHO criteria of anemia. Ret-He levels were determined by an automated hematology analyzer (XN-9100 ® or 1000 ® , Sysmex, Kobe, Japan). Blood samples were collected within 24 h from the admission. Laboratory parameters were measured at admission and discharge.
The primary outcome was defined as a composite endpoint of all-cause death or HF readmission and prospectively recorded. Follow-up was performed at discharge, and after discharge by direct contact with patients, telephone interview of patients or, if deceased, of family members, and mail, by investigators. Informed consent were written from all patients. These procedures for informed consent and enrolment were in accordance with the detailed regulations regarding informed consent described in the guidelines, and this study has been approved by the ethics committee of Hyogo Medical University (authorization numbers 3549).
Statistical analysis. Continuous variables were presented as median and interquartile range. Continuous variables were compared using 1-way analysis of variance or Kruskal-Wallis test. Continuous variables were tested for normality using the Shapiro-Wilk test. Categorical variables were made by chi-squared test or Fisher's exact test for dichotomous variables, when appropriate. The correlations of Ret-He levels with serum iron, TSAT, ferritin, and Hb levels were assessed by Spearman correlation analysis. The cumulative incidence of a composite outcome (all-cause death or HF readmission) was estimated using Kaplan-Meier analysis. ROC analysis was performed to assess the potential capabilities of Ret-He as a marker for ID and IDA, computing the ROC curve and its AUC. The results of this analysis can be shown as the ROC curve, in which the sensitivity is plotted against the value of 1-specificity. To investigate the prognostic impact of change in Ret-He levels, covariates which were closely related to change in Ret-He levels (age, sex, body mass index, BNP, the prevalence of atrial fibrillation, and the use of Ca blockers) were chosen in this study. All tests were two tailed, and a value of P < 0.05 was considered statistically significant. All analyses were performed with R version 3.3.2. All methods were performed in accordance with relevant guidelines and regulations.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.