King Khalid University Journal of Health Sciences

: 2020  |  Volume : 5  |  Issue : 2  |  Page : 62--66

The clinical utility of red blood cells and reticulocyte-related indices for early detection of latent Iron deficiency

Husain Y Alkhaldy 
 Department of Internal Medicine, College of Medicine, King Khalid University, Abha, Saudi Arabia

Correspondence Address:
Dr. Husain Y Alkhaldy
Department of Internal Medicine, College of Medicine, King Khalid University, P.O. Box: 641, Postal Code 61421, Abha
Saudi Arabia


Background: Iron deficiency (ID) with and without anemia is common among young women of childbearing age. Screening and early diagnosis of ID before anemia develops are relevant to manage before the symptomatic stage. Several automated red blood cell (RBC) and reticulocyte indices have been investigated to identify early ID. Objectives: To explore the clinical utility of both old (MCV, MCH, and MCHC) and new (Ret-He and IFR) indices for detecting latent ID before the symptomatic anemia stage develops. Methods: A group of female college students at high altitude southern Saudi Arabia was assessed for iron status and associated anemia. Three groups were identified, ID anemia (IDA) group, ID without anemia (i.e., latent ID), and normal group. The lab generated RBC related indices (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], and mean corpuscular hemoglobin concentration), And reticulocyte count and their derived indices (reticulocyte hemoglobin content [RET-He] and immature reticulocyte fraction [IRF]) were collected. The RBC and Reticulocyte indices were compared between the normal, ID and IDA groups in order to identify possible distinguishing parameters of early ID state. Descriptive analysis and significance testing were performed. Results: Ninety-two of two hundred participants (46%) were found to have a latent ID and 30 (15%) had IDA. Hemoglobin, MCV, and MCH showed progressive decrement on their values from the iron replete through iron deplete state to full-blown anemia state. Reticulocytes count and MCV/MCH were significantly lower in the latent ID group compared to the normal group. RET-He and IRF showed no statistical difference between the latent ID and normal groups. Conclusion: Latent ID, before anemia develops, is associated with significant RBC indices, MCV/MCH, changes. These changes can be used to follow-up and detect early recurrence of ID. At our setting of high altitude, Ret-He shows no clinical utility in early diagnosis of ID state. Reticulocyte count drops early before qualitative reticulocyte indices like RET-He, a finding that could be attributed to baseline high-altitude-associated erythrocytosis.

How to cite this article:
Alkhaldy HY. The clinical utility of red blood cells and reticulocyte-related indices for early detection of latent Iron deficiency.King Khalid Univ J Health Sci 2020;5:62-66

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Alkhaldy HY. The clinical utility of red blood cells and reticulocyte-related indices for early detection of latent Iron deficiency. King Khalid Univ J Health Sci [serial online] 2020 [cited 2021 May 14 ];5:62-66
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Iron-deficiency anemia (IDA) is a global health problem that affects about one-fifth of the world population.[1] All countries – regardless of their economic status – suffer from it with significant additional health consequences. Young women of childbearing age are among those particularly affected.[2] In female students attending colleges in Saudi Arabia, a prevalence ranging from 12.5%–64% has been reported.[3],[4],[5]

In adults, iron deficiency (ID) has significant effects on cognitive, physical performance, and psychological well-being.[6] Depending on the underlying etiology, IDA usually develops gradually and progresses from an initially iron-replete state to iron deplete without anemia, also known as latent ID or iron restricted erythropoiesis, toward a full-blown picture of IDA. Clinical symptoms are usually subtle in the early stages. It is thus prudent to assume that early identification and management of ID before developing a full-blown state will be beneficial to the patients.

Clinical symptoms of anemia are largely vague and nonspecific. Thus, laboratory confirmation of anemia is essential. First described in 1929 by Wintrobe, the traditional red blood cell (RBC) indices, namely mean corpuscular volume (MCV) and mean corpuscular hemoglobin/hemoglobin concentration (MCH/MCHC), are essential parts of any complete blood count (CBC) report. These indices have important clinical utility in the classification of anemia. The reticulocyte count, now fully automated, has expanded the classification and pathway of diagnosing anemia.[7] A newer generation of automated hematology analyzers provides more derived indices of various cell counts with presumed clinical utility for various blood cell types. For example, immature granulocyte, reticulocyte, and platelet fractions can be estimated with some reported clinical utility like early detection of sepsis and early bone marrow engraftment after stem cell transplantation.[7] Other reticulocyte-related indices have also been extensively studied in various types of anemia. These have been explored as possible easy and less costly methods to screen, detect, and distinguish between different types of anemias and to follow-up the body response to the followed therapy protocol.[7]

The reticulocytes represent the final stage of RBC maturation with 2–3 day life span, mainly in the bone marrow and then briefly in the peripheral blood before maturing into RBCs. This character has drawn interest in using reticulocyte measurements to evaluate bone marrow erythroid activities. The automated analyzers can now estimate the reticulocyte count and derive other measurements including reticulocyte hemoglobin content/equivalent (RET-He), reticulocyte mean cellular volume, and reticulocyte Hb concentration. The maturity index of reticulocyte can also be estimated based on the strength of fluorescent induced light scatter, generating an immature reticulocyte fraction (IRF) that can be further divided based on the maturity stage into low fluorescence, middle fluorescence, and high fluorescence. Several studies suggest that RET content is useful for screening of IDA.[8],[9],[10],[11] A level of 28 pg or less has classically been used to suggest the presence of ID in children, adults, and pregnant ladies. Ret-He has also been suggested for detecting ID in complicated clinical scenarios where biochemical iron-derived results are difficult to interpret, like functional ID[12] and patients with chronic kidney disease or patients undergoing hemodialysis.[13],[14] Studies also suggest the utility of immature reticulocyte fraction (IRF) and its subsets for the classification of anemias.[7] The rise in the IRF has also been suggested to monitor bone marrow recovery after transplant and IDA response after treatment.[15]

High altitude represents a special challenge to the erythroid bone marrow. At a high altitude, the physiological increased demand induced by a decrease in the barometric pressure results in increased hemoglobin (Hb) concentration. This increase in Hb is mediated initially by the decrease in the plasma volume followed by a true increase in the red cell mass, associated with an increase in the reticulocyte percentage, mediated by the hypoxia-associated increase in the erythropoietin level with resulting erythrocytosis.[16] Since changes in Hb content of RBC are not possible, RBC and reticulocyte quantity, i.e., RBC count and reticulocyte count, but not quality, like Hb content or size might better reflect the erythroid activity, during conditions that affect erythroid activity.

This study aims at exploring the clinical utility of both old (MCV, MCH, and MCHC) and new (Ret-He and IFR) indices for detecting latent ID before the symptomatic anemia stage develops. High altitude might represent different settings for which reported literature of clinical utility of some of these indices needs to be validated.


The study was conducted at high altitude Abha City, Southern Saudi Arabia, residing at 2270 m above sea level. Participants' consents were collected, and the study protocol was approved by the ACH-IRB (REC#200181004) and REC of College of Medicine, King Khalid University (HA-06-B-001) in October 2018.

A sample of 200 female medical college students was evaluated for anemia and iron store status. Based on the results, three groups were defined, i.e., normal group, with normal Hb concentration and iron stores (defined as Hb more than 12.7 g/dL and serum ferritin more than 20 μg/l), latent ID group (defined by absent iron stores [serum ferritin less than 20 μg/l] when Hb level is normal [Hb > 12.7 g/dL]), and IDA group (low Hb levels, i.e., <12.7 g/dL along with serum ferritin <15 μg/l).

The use of Hb cutoff of 12.7 g/dL is based on the WHO recommendations for adjustment of high altitude.[17] Since our cohorts were healthy young females with no associated chronic illnesses, serum ferritin level is considered an adequate measure for the iron store status. Blood samples for CBC samples were analyzed using the Sysmex XN-1000 hematology analyzer. The serum ferritin was analyzed using the UniCel DxI 600 Access Immunoassay System.

Standard CBC report for Hb- and RBC-related indices were collected. These include Hb concentration, hematocrit (Hct), RBCs, RDW, MCV, MCH, and MCHC. Reticulocyte count expressed as a percentage of the total RBCS was also generated by the automated analyzer along with the IRFs. The RET-He measures the Hb content of the reticulocyte and reported in the picogram (pg.). The Reticulocyte-related indices are generated using fluorescent flow cytometry in the reticulocyte channel of the Sysmex XN analyzers. Absolute reticulocyte count was manually calculated by multiplying the reticulocyte count percentage with the total RBC count.

De-identified data were fed into an Excel sheet, revised, and verified. The analysis was done using software IBM SPSS version 22 (SPSS, Inc. Chicago, IL). Variables were compared among the three groups and reported using standard descriptive analysis of mean, standard deviation (SD), median, and range. Continuous variables were compared using two-tailed independent t-test. Differences between the groups were compared using the ANOVA test, and the significance level was set at P < 0.05 for all statistical comparisons.


Of all 200 participants, serum ferritin levels, CBC data, and reticulocyte count were available. The derived reticulocyte indices, RET-He and IRFs, were available for 148 participants. [Table 1] summarizes the statistical characteristics of the CBC and reticulocyte indices for the three groups.{Table 1}

Seventy-eight of two hundred participants (39%) of the sample have normal Hb concentration and iron stores, while 61% have an ID. This includes 92 (46%) with latent ID and 30 (15%) with IDA. The group of IDA shows mild anemia with a mean (SD) Hb concentration of 11.9 (0.8) g/dL.

Among the old indices, RBCs, MCV, MCHC, and RDW showed no statistical difference between the normal group and the group with latent ID. MCH showed a statistical difference between the two groups and showed the best correlation with Hb level (r2 = 0.60) that could be useful clinically (P = 0.018). While Hb concentration level is statistically different between the two groups (P ≤ 0.001), the amount of difference is small (0.65 g/dL) which could be clinically not useful.

Reticulocyte count expressed as a percentage or absolute count showed a statistically significant difference between the three groups and was statistically different (P ≤ 0.02) between normal and latent ID groups. RET-He, while significantly different between the normal group and the group with IDA, was not significant between the normal group and group with latent ID. Ferritin level once in the latent stage (<20 ng/ml) was a poor indicator to follow the extent or progression of latent ID toward the anemia stage.

Using receiver operator curve (ROC) analysis [Figure 1], the MCH level of 27.8 pg recorded a sensitivity of 71% and 57.5% for specificity to distinguish ID from the normal state.{Figure 1}


This study aims at exploring the utility of an automated hematology analyzer report of old and new indices to early identify an ID, known as latent ID or iron-restricted erythropoiesis. The present study clarified that the old RBC indices were poor to discover early ID. This comes as no surprise as RBCs indices are known to be normal or late to be affected in ID without anemia. Although still within the reference range, Hb concentration, MCH, and MCV do decrease early and significantly compared to those with normal Hb and iron stores. Using ROC analysis, MCH may be better than MCV for the detection of latent ID. In the appropriate setting, this trending down on the MCH could be used to monitor the recurrence of IDA in known patients who were treated before.

The reticulocyte count showed a statistically significant difference between the three groups, which was evident between normal and latent ID groups. In contrast, RET-He was significantly decreased in the IDA group but did not prove to be useful to distinguish between the group of latent ID and the normal groups. This comes in contrast to several studies that reported the clinical utility of RET-He for early detection of ID.[18],[19],[20],[21] Some studies also suggested that the decrease in RET-He correlates with the degree of ID.[22] A possible explanation could be attributed to physiological erythrocytosis at high altitude. Due to baseline erythrocytosis, reduction in the number of reticulocytes might precede the reduction in the Hb content/quantity before quality. However, one may wonder why the more mature RBC Hb content (MCH) showed the strongest correlation with Hb level at the latent ID group, while the younger recently produced reticulocyte did not. This could be explained by the fact that at normal conditions, younger reticulocytes are larger and contain more Hb (28–36 pg) compared to the mature RBCs (26.5–32.5 pg). Another explanation should be explored that discrepancy is related to the absence of locally defined reference range suitable for high altitude. At our high altitude, higher Hb, Hct, and RBCs values are physiological. This means that the reference range for these parameters and their derived indices should be determined. A corrected reference range, usually lower, will likely abolish this discrepancy.

It should be noted that RBC and reticulocyte indices are also affected by other types of anemia. Carriers of thalassemia represent a common clinical scenario for which differentiation is important. RET-He, among many other indices and discriminant formula, has been explored with variable results. Moreover, some formula using RET-He has been suggested to pick up a thalassemia patient who are also iron deficient.[23] [Table 2] demonstrates a cohort of our B-thalassemia carrier patients. The figure shows that thalassemia patients have markedly lower RET-He and higher reticulocyte count compared to ID and IDA groups. Our thalassemia patients also have discordant RET-He values as compared to what has been reported elsewhere.[24] This could be explained by the fact that our IDA group has milder anemia with a mean Hb of 11.9 g/dL. Hematological parameters in thalassemia patients are also known to be heterogeneous and reflect the underlying globin chain imbalance. A formula using the two variables of RET-He and absolute reticulocyte count can easily distinguish the thalassemia carrier from ID though that seems to be of academic interest only. By the time the RET-He drops as low as 25 pg or less, the IDA will be both clinically and laboratory apparent, and standard workups are usually requested to exclude one from another or identify coexistent IDA and thalassemia.{Table 2}

The current study still has some limitations. For instance, the sample is relatively small. High altitude represents a special setting for which control is needed, especially when altitude-specific derived reference range is not yet established. Other laboratory tests such as Hb electrophoresis were not available to exclude possible co-existent thalassemia or other nutrients deficiencies that might affect the CBC parameters. Further studies were initiated to both determine reference range values for CBC and reticulocyte parameters at a high altitude and to further study the utility of reticulocyte indices for early diagnosis of latent ID and IDA.


Screening and identification of latent ID are clinically relevant to initiate therapy before the advanced stage and anemia develop. CBC and reticulocyte indices can be used as a screening method for the detection of latent ID because of their availability, less variability, and cost than the biochemical assessment of iron status. While RBC and reticulocyte indices are usually reported normal in the latent ID stage, progressive decline in the Hb concentration and changes on these indices are clearly noticeable. At a high-altitude setting, bone marrow response for conditions with iron-restricted erythropoiesis may be different from elsewhere due to baseline erythrocytosis. Reticulocyte count may drop early and can be used for early detection of early latent ID. RET-He content, using the current reference range and the IRF, showed no clinical utility for discovering latent ID. If carefully assessed, changes on other RBC and Reticulocytes indices, though subtle, can suggest the presence of early ID. Finally, clinicians should be aware that other types of anemia can affect the RBC/reticulocyte indices, and the appropriate workup should be dedicated based on the clinical assessment.


I wish to thank all the medical students who participated in this study and Dr. Khalid Alghamdi for help with data entry. I thank Dr. Shehata F. Shehata for assistance with statistical analysis and Dr. Mohammed Saboor for Proofreading the final manuscript.


The author declares no conflicts of interest

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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