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The reticulocyte haemoglobin equivalent (RET_He) and laboratory screening for iron deficiency.


Iron deficiency (ID) and iron deficiency anaemia (IDA) affects approximately 2 billion people globally making it the most common of the nutritional deficiencies (1,2). Iron is essential for normal biological function and untreated IDA has been associated with developmental delays in the young (2). Iron deficiency can be difficult to diagnose using traditional biochemical markers of iron metabolism and the presence of the morphologically characteristic hypochromic microcytic red blood cells (RBC) in the blood film are typically only apparent once iron deficient erythropoiesis is advanced (3).

The complete blood count (CBC) is the most frequently ordered of all laboratory tests (1) and so expansion of the clinical utility of results produced as part of the CBC could be beneficial for patient diagnosis and management. The reticulocyte haemoglobin equivalent (RET_He) is a red cell parameter available on the XN-2000 and other Sysmex haematology analysers and provides a measure of the bioavailability of iron during erythropoiesis (4,5). Reticulocytes have a short life-span of 1-2 days in the peripheral blood before full maturation, and during iron deficient erythropoiesis, reticulocytes have reduced levels of haemoglobin production. The RET_He parameter has the potential for greater clinical use as an adjunct to current biochemical-based assays for ID. It has previously been proposed as a laboratory tool to distinguish between IDA and anaemia of chronic disease (ACD) with both aetiologies producing morphologically similar hypochromic microcytic RBC populations (1). ACD results from the inability of erythropoiesis to utilise body iron while IDA results from the lack of body iron.

The aim of this study was to establish a reference range for the RET_He at Taranaki Base Hospital and to assess its clinical value as a tool for the identification of ID.


The study utilised samples submitted to LabCare Pathology and Taranaki MedLab for iron studies over a period of eight weeks. Results were collated twice daily from the laboratory information systems and EDTA anticoagulated blood samples were run on the XN-2000 (Sysmex, Japan) using the RET channel analysis feature. Testing was performed twice daily to ensure samples were less than six hours old to reduce any effects of sample ageing. A total of 178 patient samples were included in the study and all laboratory testing results and patient clinical details were collated in Microsoft Excel.

In the RET channel, blood cells are exposed to a surfactant reagent that lightly perforates the membrane of the RBC, WBC and platelet populations. In the machine the blood sample, surfactant and a fluorescent dye (Fluorocell RET) are incubated together for a short period, allowing the dye to penetrate the cells (1). The stained cells are then cycled through a flow cell and past a beam of high intensity laser light. Reticulocytes containing RNA, fluoresce producing forward and side scattered light that is captured by light-detectors producing results that are presented graphically in the form of a 2D-scatterplot. Total cell numbers are counted with the forward light-scatter providing cell size and side-scattered light indicating the presence of cytoplasmic nucleic acid (DNA/RNA). In the RBC population the degree of fluorescence is proportional to the cytoplasmic RNA and provides the reticulocyte population. Results are presented as picograms (pg) of Hb per reticulocyte (1).

The biochemistry analyser used for iron studies at LabCare Pathology was the Cobas 6000 (Roche Diagnostics, Germany) with the iron panel providing results for serum ferritin, serum iron, serum transferrin and transferrin saturation. Ferritin was measured by electrochemiluminescent immunoassay, iron by colorimetric assay and transferrin using an immunoturbidimetric assay (6-8).

A RET_He reference range was constructed from the results of 66 EDTA anticoagulated peripheral blood samples. The samples were selected from patients presenting to Taranaki Base Hospital with a normal CBC and medical conditions unlikely to impact on their iron status or reticulocyte parameters.

The statistical software program MedCalc[R], was used to provide the reference range with a 95% confidence interval and an online Clinical Calculator software package (9) used to calculate sensitivity, specificity, positive and negative predictive values for selected RET_He cutoff values.


In the RET channel of the Sysmex XN-2000 analyser, normal and ID samples vary in the scatterplots they produce as a result of the differences in the RBC and reticulocyte populations. In Figures 1 and 2 the reticulocyte population (pink/red) is presented along with the mature RBC population in blue. Figure 2 shows the presence of microcytic RBC & reticulocyte populations lower on the Y axis of the scatterplot in contrast to the iron replete example in Figure 1. The microcytic RBCs in Figure 2 are the result of iron deficient erythropoiesis caused by reduced cellular Hb levels.

Participants had an average age of 44 with a male to female ratio of 1:2. The RET_He results were stratified against the iron studies, CBC results and relevant clinical information. This divided patients into normal, ID/ACD, IDA and haemoglobinopathy clinical groupings. The diagnostic cutoff values for ID were those used at LabCare Pathology with ferritin <20 ug/L and saturation <16%. Using the World Health Organisation (WHO) criteria, patients were classified as anaemic if the Hb was <120g/L for non-pregnant females and <130g/L for males (9). A C-reactive protein assay (ref range <5 mg/L) was used to establish cause when clinical details were not available for some apparent ID patients.

A summary of the results of the laboratory testing divided participants into four groups and is presented in Figure 3.

The data used to establish rates of True and False positives for the patient groups is presented in Tables 1 and 2. A RET_He cutoff of <26 pg favoured the detection of more true positives (21) but also more false positives (10). The cutoff of <25pg detected less true positives (19) but also had less false positives (7).

Sensitivity, specificity and positive and negative predictive values for the RET_He were calculated for the two cutoff options <25pg and <26pg rf. Table 3 (10). Results showed that a cutoff of <26pg had a small advantage for the detection of ID.

The results for the RET_He from 66 patient samples were used to calculate a reference range with a 95% confidence interval of 30.3-35.0 pg with a mean of 32.7 pg. The coefficient of skewness (-0.034) showed the data set was normally distributed (Figure 4b) with support from a low coefficient of Kurtosis or Z score of (-0.335) (Figure 4a).


The gold standard for the assessment of body iron is a bone marrow biopsy but it is an invasive procedure rarely used in the diagnosis of ID related disorders (11). Instead the quantitation of iron is traditionally performed using biochemistry-based assays. Ferritin is the long term storage form of iron and in the plasma it reflects total body iron stores. Its use as a marker for ID is complicated, as together with transferrin, both are also acute phase reactants that are elevated in infection, chronic disorders and other inflammatory states. As a result, the diagnosis of ID using iron studies is not always straight forward.

This study correlated patient clinical information against the results of iron studies, CBC data and the RET_He parameter. Results indicated that a RET_He cutoff of <26pg was able to identify ID patient groups and best supported the use of the RET_He as a screening test for iron deficiency. The RET_He uses the mean cell volume (MCV) and so results can be affected when there is microcytosis unrelated to IDA, such as, in double RBC populations, in cases of RBC aggregation and when there is hyper or hyponatraemia (12). To aid interpretation, the RET_He results should be considered together with the results of the red cell distribution width (RDW).

In this study the RET_He failed to provide a clear division for the ID and ACD groups affecting the overall specificity of the parameter. Given this, the follow-up of patients with a RET_He less than or close to the 26pg cutoff should include iron studies (13).

The RET_He reference range for the population in this study was 30.3-35.0 pg with a 95% confidence interval and is comparable to the range of 28.9-36.3 pg developed by LabPlus in New Zealand for a demographically similar population group (14). That study recommended a RET_He cutoff value for ID of <25pg slightly lower than the cutoff of <26pg in this work. A possible limitation of this study could have been the participant group selected for the reference range. Made up of selected inpatients at Taranaki Base Hospital instead of healthy members of the public, the reference range data could have been skewed. This does not appear to have been the case with other researchers producing reference ranges comparable to this study (2,5,11,12).

Laboratory cost containment has always been an important consideration in laboratory testing. A cost advantage for the RET_He as a screen for ID may be significant as compared to traditional iron studies. In this study iron study costs were estimated to be approx 1.5 times that of the CBC + RET. A closer consideration of the cost/benefit of the RET_He over the use of traditional iron studies as a screen for ID may or may not support the findings of this study.


This study highlighted the clinical potential of the RET_He. Its use as part of a screening algorithm together with Hb, MCV and RDW, could better guide laboratory recommendations for iron studies, reducing costs when iron studies are not warranted. The study showed that the RET_He with a cutoff of <26pg was highly sensitive but not specific for the detection of ID. Its future clinical utility could be as a screening test for ID but also as a negative predictor of ID when the RET_He results fall within the reference range in anaemic patients. Its utility as an early marker for ID has been previously reported and has been confirmed with the demographic investigated in this study. The future acceptance of the value of the RET_He by clinical staff may be hampered by a lack of awareness of its potential for patient diagnosis and treatment. The education of clinical staff could start by reporting the parameter in anaemic patients with results below the RET_He cutoff, triggering a comment about additional laboratory testing to rule out possible ID .


The authors wish to thank the laboratory staff at Taranaki Medlab, New Plymouth for their assistance in the study.


Charlotte Poffenroth, 4th year student BMLSc [1]

Craig Mabbett, BMLSc PGDipHSM MNZIMLS, Charge Scientist Haematology [2]

Chris Kendrick, LMNZIMLS, DipSci, MSc(Dis), Senior Lecturer [2]

[1] College of Health, Massey University, Palmerston North

[2] LabCare Pathology, New Plymouth.


(1.) Sysmex America, Inc. Reticulocyte hemoglobin content (RET-He): A parameter with well-established clinical value. 2013. Retrieved from

(2.) Hatoun J, Sobota A, Meyers A. Using reticulocyte haemoglobin equivalent to screen for iron deficiency may be problematic. Glob Pediatr Health 2014; 1: 2333794x14557030.

(3.) Brugnara C, Zurakowski D, DiCanzio J, Boyd T, Platt O. Reticulocyte hemoglobin content to diagnose iron deficiency in children. JAMA 1999; 281: 2225-2230.

(4.) Mehta S, Goyal LK, Kaushik D, Gulati S, Sharma N, Harshvardhan L, et al. Reticulocyte hemoglobin vis-a-vis serum ferritin as a marker of bone marrow iron store in iron deficient anemia. J Assoc Physicians India, 2016; 64: 38-42.

(5.) Mast AE, Blinder MA, Dietzen DJ. Reticulocyte haemoglobin content. Am J Hematol 2007; 83: 307-310.

(6.) Roche. (2016). Ferritin [Cobas Package Insert]. Germany.

(7.) Roche. (2015). Iron [Cobas Package Insert]. Germany.

(8.) Roche. (2013). Transferrin ver.2 [Cobas Package Insert]. Germany.

(9.) World Health Organisation. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. Vitamin and Mineral Nutrition Information System. Geneva, World Health Organization, 2011 (WHO/NMH/NHD/ MNM/11.1). Retrieved from indicators/haemoglobin.pdf, accessed 2017.

(10.) Vassarstats: Website for Statistical Computation http:// accessed 2017.

(11.) Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret-He) and assessment of iron-deficient states. Clin Lab Haematol 2006; 28: 303-308.

(12.) Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency. Blood 2002; 99: 1489-1491.

(13.) LabPLUS Test Guide. accessed 2017.

(14.) Ryan K, Bain BJ, Worthington D, James J, Plews D, Mason A, et al. Significant haemoglobinopathies: guideline for screening and diagnosis. Br J Haematol 2010; 149: 35-49.

Copyright: [C] 2017 The authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Charlotte Poffenroth [1], Craig Mabbett [2] and Christopher Kendrick [1]

[1] Massey University, Palmerston North and [2] LabCare Pathology, New Plymouth

Caption: Figure 1. RET scatter gram--normal iron.

Caption: Figure 2. RET scatter gram--iron level deficiency.

Caption: Figure 3. Clinical diagnoses vs RET_He results.

Caption: Figure 4a. Z score and population distribution for the RET_He reference range.

Caption: Figure 4b. Population distribution for the RET_He reference range.
Table 1. True and false positives using a <25pg cut-off for the

             True        False
RET_He     positives   positives   Totals

< 25pg        19           7         26
> 25pg         2          150        152
Totals        21          157        178

Table 2. True and false positives using a <26pg cut-off for the

RET_He       True        False     Totals
           positives   positives

< 26pg        21          10         31
> 26pg         0          147        147
Totals        21          157        178

Table 3. Summary of sensitivity, specificity,
positive (PPV) & negative predictive values
(NPV) for two cut-off values.

RET_He    Sensitivity   Specificity    PPV      NPV

< 25pg       0.905         0.955      0.730    0.987
< 26pg         1           0.936      0.677      1
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Author:Poffenroth, Charlotte; Mabbett, Craig; Kendrick, Christopher
Publication:New Zealand Journal of Medical Laboratory Science
Article Type:Report
Date:Nov 1, 2017
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