Clinical Significance of Reticulocyte Hemoglobin Content in the Diagnosis of Iron Deficiency Anemia

Objective: The aim of this study was to evaluate the clinical significance of reticulocyte hemoglobin content (CHr) in the diagnosis of iron deficiency anemia (IDA) and to compare it with other conventional iron parameters. Materials and Methods: A total of 32 female patients with IDA (serum hemoglobin <120 g/L and serum ferritin <20 ng/ mL) and 18 female patients with iron deficiency (serum hemoglobin > 120 g/L and serum ferritin <20 ng/mL) were enrolled. Results: CHr was 24.95±3.92 pg in female patients with IDA and 29.93±2.96 pg in female patients with iron deficiency. CHr showed a significant positive correlation with hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, serum iron, and transferrin saturation and a significant negative correlation with transferrin and total iron-binding capacity. The cut-off value of CHr for detecting IDA was 29 pg. Conclusion: Our data demonstrate that CHr is a useful parameter that can be confidently used in the diagnosis of IDA, and a CHr cut-off value of 29 pg predicts IDA. Conflict of interest:None declared.


Introduction
Iron defi ciency anemia (IDA) is the most common form of nutritional anemia worldwide [1]. Various biochemical parameters are used to diagnose IDA, including ferritin, transferrin saturation (TS), serum iron, and mean corpuscular volume (MCV). Despite the availability of these parameters, their validity for the diagnosis of IDA is still debatable. Serum ferritin, the most specifi c indicator of iron defi ciency, is an acute phase reactant and its level is affected by infl ammation. TS fl uctuates due to the diurnal variation of serum iron, and serum iron levels decrease with infection, infl ammation, and malignancy and increase with liver disease [2].
Reticulocytes are the youngest erythrocytes released from the bone marrow into the blood and they circulate for 1-2 days before becoming mature erythrocytes. The reticulocytes' hemoglobin content refl ects the amount of iron available for hemoglobin production in the bone marrow. Therefore, reticulocyte hemoglobin content (CHr) has been proposed as an iron status marker [3]. Several studies have indicated that CHr measurement in peripheral blood samples is useful for diagnosis of iron defi ciency [4,5,6,7,8]. It has been shown to be an accurate measure of iron status and a reliable iron marker for monitoring iron therapy's effectiveness [3,9].
In this study, we aimed to evaluate the signifi cance of CHr in the diagnosis of IDA and compare it with other conventional iron parameters.

Patients
The study was conducted at Eskişehir Osmangazi University, Faculty of Medicine, Department of Hematology. After obtaining the approval of the ethics committee and informed consent, 32 female patients with IDA (serum hemoglobin <120 g/L and serum ferritin <20 ng/mL) and 18 female patients with iron defi ciency (serum hemoglobin >120 g/L and serum ferritin <20 ng/mL) were enrolled.

Sample Collection and Laboratory Methods
Samples for complete blood count and CHr were collected in K3EDTA tubes and analyzed with an automated hematology analyzer, ADVIA 2120i (Siemens, New York, USA). Serum iron and total iron-binding capacity (TIBC) were measured with a LISA 500 Plus automated chemical analyzer (Hycell Diagnostics, Paris, France). Serum ferritin was measured with a Hitachi E170 automated analyzer (Hitachi, Tokyo, Japan). TS was calculated by dividing serum iron by TIBCx100. Transferrin was measured with a BN II automated chemical analyzer (Siemens, Marburg, Germany).

Statistical Analysis
Data were analyzed using IBM SPSS 20. The independent samples t-test was applied for normally distributed variables and results were given as mean ± standard deviation. The Mann-Whitney U test was applied for abnormally distributed variables and results were given as median (quartiles) values. Receiver operating characteristic (ROC) curve analysis was performed to identify the optimal CHr cut-off value for predicting IDA. P<0.05 was accepted as signifi cant.

Results
There was no statistically signifi cant difference between the 2 groups in terms of age and red blood cell (RBC) count. Hemoglobin, MCV, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum iron, and TS were signifi cantly lower in female patients with IDA in respect to iron-defi cient female patients. Transferrin and TIBC were signifi cantly higher in female patients with IDA compared to female patients with iron defi ciency. CHr was 24.95±3.92 pg in the IDA group and 29.93±2.96 pg in the iron defi ciency group, and a statistically signifi cant difference was observed between the 2 groups in respect to CHr (Table 1)

Discussion
Various biochemical parameters are being used for the diagnosis of IDA. However, there might be some diffi culties in the assessment of these conventional parameters. For example, ferritin behaves as an acute phase reactant, which limits its diagnostic accuracy greatly. The serum ferritin level is frequently increased independently of iron status by factors such as acute/chronic infl ammation, infection, malignancy, liver disease, and alcohol use. Serum iron levels also decrease with infection, infl ammation, and malignancy and increase with liver disease. TS is a calculated parameter, and therefore it refl ects confounding effects on individual components [2].
Measurement of CHr provides an indirect measure of the functional iron available for new RBC production. In a study performed by Mast et al., it was reported that CHr of <28 pg had an optimal sensitivity (74%) and specifi city (73%) for diagnosis of iron defi ciency, using Prussian blue staining of the bone marrow aspirate to defi ne iron defi ciency. In this study, the area under the curve of CHr exceeded that of ferritin, TS, and MCV, showing that CHr is a useful marker for diagnosis of iron defi ciency in adults [4].
Several studies have assessed the value of CHr as an indicator of iron defi ciency in dialysis patients. In a recent study by Thomas et al., functional iron defi ciency was defi ned as CHr < 28 pg [10]. Fishbane et al. also reported that CHr of <28 pg predicted iron defi ciency more accurately than did serum ferritin and TS in dialysis patients receiving erythropoietin [2]. Mitsuiki et al. reported that a CHr index for iron defi ciency with 100% high sensitivity was 32 pg [11]. In another study, a CHr cut-off value of 32 pg was found to be appropriate for the assessment of iron defi ciency in hemodialysis patients, in which an ethnic effect on CHr levels was considered to explain the higher cut-off [3]. In our study, a CHr cut-off value of 29 pg with 90.6% sensitivity and 66.7% specifi city was determined to predict IDA.
With the present investigation, we have identifi ed the value of CHr in the diagnosis of IDA. CHr showed the strongest correlation with hemoglobin, MCV, MCH, and TS with r>0.7, which was considered meaningful. Therefore, CHr in conjunction with these conventional parameters appears to be useful and reliable in identifying IDA.
In conclusion, besides the current conventional parameters that we use in routine practice to diagnose IDA, there is still a need for more sensitive and powerful parameters. CHr is a useful parameter that can be confi dently used in the diagnosis of IDA, and a CHr cut-off value of 29 pg predicts IDA.

Confl ict of Interest Statement
The authors of this paper have no confl icts of interest, including specifi c fi nancial interests, relationships, and/or affi liations relevant to the subject matter or materials included.