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The clinical use of Ret-He in the differential diagnosis of anaemia

Anaemia is a global public health problem affecting populations in both developing and developed countries. According to the World Health Organization (WHO) anaemia affects 1.62 billion people, which corresponds to 25% of the world population. It is assumed that 50% of the cases of anaemia are due to insufficient iron content in the diet, especially in young children, vegetarians and women in child-bearing age with large menstrual blood loss or during pregnancy. Iron loss in women averages 1 to 3 mg per day, and dietary intake is often inadequate to maintain a positive iron balance. Moreover, pregnancy adds to demands for iron, with requirements of up to 6 mg per day by the end of pregnancy.

Although the primary cause of anaemia is iron deficiency, it frequently coexists with other causes. The risk of anaemia may be increased by deficiencies in other micro-nutrients, including vitamins A and B12, folic acid, riboflavin, or copper. Furthermore, the impact of haemoglobinopathies on anaemia prevalence needs to be considered within several populations. Consequently, once the diagnosis of anaemia has been established, further investigations are needed in order to identify the underlying cause.

Iron-deficient erythropoiesis and thalassaemia are both associated with mild to moderate microcytic anaemia, which frequently leads to an incorrect diagnosis. It is important to discriminate between iron-deficiency anaemia and thalassaemia, and to avoid unnecessary iron therapy to prevent the development of haemosiderosis, which may result in serious complications like cardiomyopathy, liver fibrosis or endocrine dysfunctions.

A wide range of laboratory parameters is available for anaemia screening and assessment of iron status. However, no single marker or combination of tests is optimal for discrimination between iron deficiency, functional iron deficiency and thalassaemia. The available indicators do not provide sufficient information and must be used in combination to obtain reliable information.

The first test for the identification of anaemia is haemoglobin. Subsequently, the mean cell volume (MCV) is commonly used for the classification of the anaemia.
For the cause of anaemia, microscopic evaluation of the red blood cells provides a rapid screening in the differentiation of anaemia:

        • iron-deficient erythropoiesis: almost empty red blood cells
        • thalassaemia: typical Target-cells
        • vitamin deficiency: macrocytic red blood cells

Red blood cell and reticulocyte haemoglobin content

In the past years, new parameters for eythropoiesis became available for investigation of thehaemoglobin content of (immature) red blood cells and for the production of red blood cells.

Haemoglobin content in reticulocytes (Ret-He) is a sensitive indicator for monitoring short term deteriorations in iron and vitamin availability for erythropoiesis. Reticulocyte maturation coincides with progressive decrease in RBC volume and Hb content. If compared with the interpretation of haemoglobin content of mature RBCs (RBC-He), the interpretation of haemoglobin content of reticulocytes yields additional information concerning various states of decreased availability of iron for the haemoglobinisation of erythroid precursors. If iron supplements are administered in subjects with depleted iron stores, Ret-He already increases within a few days.

The parameters involved are respectively denoted as RBC-He, Ret-He, Delta-He (Ret-He minus RBC-He) and IRF (immature reticulocyte fraction).

In normal circumstances, results of Ret-He are approximately 10% higher than RBC-He. Delta-He results below the reference range indicate poor erythropoiesis. In contrast, results above the reference range indicate normal or improved erythropoiesis.

Hypochromic red cells: %MicroR and %HypoHe

More recently, haemocytometric methods became available for establishing the percentage of hypochromic and microcytic RBC. The percentage hypochromic and microcytic red blood cells are parameters derived from the haemoglobin content of the mature red blood cells, measured in the reticulocyte channel, and expressed as %HypoHe and %MicroR respectively. %HypoHe and %MicroR are identified by the percentage of hypochromic red blood cells with an Hb content of <17 pg (= 1062 amol) and the percentage microcytic red blood cells with a volume <60 fL respectively.  Measurement of %HypoHe and %MicroR has demonstrated to be useful for the detection of small changes in the amount of red blood cells with inadequate haemoglobinisation. In subjects with b-thalassaemia results for %HypoHe are below the reference range as a result of decreased production of haemoglobin. Results for %HypoHe are not different from those observed in subjects with iron-deficiency anaemia. Results for %MicroR are increased in b-thalassaemia subjects if compared with results obtained in subjects with iron-deficiency anaemia.

Several clinical studies revealed the usefulness of the innovative haemocytometric parameters for haemoglobinisation of red blood cells (RBC) and reticulocytes, RBC-He, Ret-He and delta-He (Ret-He minus RBC-He) in the differential diagnosis of anaemia.

Subjects with microcytic anaemia

In subjects with microcytic anaemia and suspected iron deficiency anaemia (IDA) or thalassemia, results of Ret-He and RBC-He results are both decreased if compared with a group of apparently healthy subjects.

However, using combinationof Ret-He and Delta-He,a clear distinction can be made between subjects with iron deficiency and thalassemia.

In case of iron deficiency, a decreased Ret-He (cut-off value <1850 aMol) is demonstrated in combination with a decreased Delta-He. In the presence of thalassemia, usually a decreased Ret-He with a normal Delta-He is demonstrated. This is, with respect to healthcare costs, an important tool to prevent unnecessary and expensive laboratory diagnostics.

Other investigators evaluated the %MicroR and %HypoHe in subjects with microcytic anaemia. In subjects suspected for IDA or thalassemia, increased results for the %MicroR and %HypoHe were demonstrated. However, when combining both parameters to a M/H ratio, a clear difference was demonstrated between these groups.

In contrast to subjects with iron deficiency anaemia, in which the M/H ratio was normal to slightly increased, the M/H ratio in subjects with thalassaemia was strongly increased.

In summary, the laboratory screening for anaemia has been improved by using the new haemocytometric parameters and by the development of new discriminating algorithms for the diagnosis of iron-deficient erythropoiesis and thalassemia.


In pregnant women, the prevalence of iron-deficiency anaemia ranges from 6 to 30% in developed countries. Although a mild degree of anaemia is common in the third trimester of pregnancy, it remains a challenge to establish whether a decreased haemoglobin concentration (Hb) is physiological (due to haemodilution) or pathological (due to insufficient availability of essential nutrients, such as iron, folic acid of vitamin B12). The World Health Organization indicates a Hb below 6.8 mmol/L in the third trimester of pregnancy, as cut-off for iron supplementation. We recommend, in accordance with Ervasti and Wheeler, the application of Ret-He (cut off <1850 aMol) as a useful tool for screening on decreased iron availability during the pregnancy. A decreased Ret-He result is considered to be indicative for insufficient red blood cell haemoglobinisation, especially within a haemoglobin range suspicious for anaemia in pregnancy (Hb £6.8 mMol/L, MCV 80-100 fL).

In women with borderline haemoglobin concentration in the third trimester of pregnancy, the effect of iron supplementation on Ret-He was evaluated. After 4 weeks of iron supplementation, Ret-He results obviously increased towards the lower region of the reference interval (1900-2300 aMol). The effect of iron supplementation was also reflected by the red blood cell distribution width (RDW-SD) and the dimorphic red blood cell population.

To conclude, we strongly advise the discriminating parameters for anaemia discrimination in order to reduce diagnostic testing for confirmation and to proper diagnose the underlying cause(s) in several categories of patients, such as subjects with microcytic anaemia, women in the third trimester of pregnancy, adolescents and elderly people (age >75 year).

References available on request.

TAGS: Immunology
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