Usefulness of Reticulocyte Hemoglobin Equivalent for Diagnosis of Iron Deficiency

Background: Anemia is a significant worldwide health problem, and approximately 30% of world people suffer from anemia, the half of which is iron deficiency (ID). The diagnosis of anemia requires the confirmation of a decrease in hemoglobin (Hb) concentration. For the diagnosis of iron deficiency anemia (IDA), the determinations of serum ferritin and iron related parameters must be necessary even if microcytic hypochromic anemia is confirmed. With recent technological advances, the Hb content of reticulocytes can be quantified by flow cytometry. Reticulocytes exist for 1-2 days in the peripheral blood and its Hb levels might be a good index of ID.There are several markers for the assessment of Hb content in reticulocytes, including reticulocyte Hb equivalent (RET-He) and reticulocyte Hb content (CHr). RET-He, which can be measured in the same sample used for complete blood count tests by the latest automated hematology analyzers, is considered to reflect iron content in reticulocytes. If RET-He is capable of evaluating ID, it must be useful for immediate diagnosis of IDA. Therefore, we evaluated the usefulness of RET-He for determining of ID.

Methods: This prospective study was approved by the ethics committee of Asahikawa Medical University (authorization numbers 1356, 1679, and 1356-3). Blood samples were obtained from 211 patients (63 males and 148 females) from 14 to 91 years old. RET-He levels were determined using an automated hematology analyzer (XN-3000^® or XE-5000^®, Sysmex, Kobe, Japan). Serum iron, total iron binding capacity (TIBC), serum ferritin, and biochemical data were measured using an automated chemical analyzer. Soluble transferrin receptor (sTfR) was measured by an enzyme-linked immunosorbent assay. Anemia was defined as Hb level of <12 g/dL. ID state was defined as serum ferritin level of <12 ng/mL. Patients were classified into four groups which are IDA, ID, control, and anemia without ID groups according to their Hb and serum ferritin levels (Table 1). Laboratory parameters were compared among four groups. The changes of RET-He during oral iron administration were also determined for 21 IDA patients.

Results: There were 72 (14 males and 58 females), 28 (12 males and 16 females), 67 (23 males and 44 females), and 44 (14 males and 30 females) patients in the IDA, ID, control, and anemia without ID groups, respectively. As shown in Table 1, The median RET-He levels were 22.3 pg (15.1-35.6 pg), 29.7 pg (19.2-34.9 pg), 34.0 pg (25.9-38.0 pg), and 32.5 pg (19.1-46.3 pg) in the IDA, ID, control, and anemia without ID groups, respectively. Patients in not only IDA but ID groups had significantly lower RET-He levels than those in control group (p < 0.001) while there was no significant difference in RET-He levels between anemia without ID and control. RET-He correlated positively with serum iron (r = 0.654) and transferrin saturation (TSAT) (r = 0.666), and correlated negatively with TIBC (r = -0.617) and sTfR (r = -0.655). There was no correlation between RET-He and serum ferritin when all patients were included in the analysis (r = 0.287); however, analysis of groups according to their iron status revealed a positive correlation between RET-He and serum ferritin in the IDA and ID groups (r = 0.604). The area under the ROC curve (AUC) detecting ID for RET-He was 0.902, whereas AUC for serum iron, TIBC, TSAT, and sTfR were 0.889, 0.879, 0.922 and 0.821, respectively. The cutoff value of RET-He with maximal sensitivity and specificity was 30.9 pg, and the cutoff RET-He value of 28.5 pg had a specificity of >90% (sensitivity, 68%; specificity 91%). Among patients receiving iron treatments, the Hb levels increased in 14 patients, whereas Hb values decreased or did not change in 7 patients. Serum ferritin and RET-He values seemed to change in parallel with changes in Hb levels.

Conclusions: In the present study, our data showed the efficacy of RET-He for diagnosis of IDA and the usefulness for monitoring drug iron administration. Because other parameters related to ID such as iron and ferritin should be measured biochemically in serum, it takes a longer time to measure serum iron and ferritin levels when compared with complete blood count tests. We would therefore suggest that measurement of RET-He might be useful to diagnose IDA because its assessment is rapid, fully automated, and can be measured in same sample used for complete blood count test.

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