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Biological diagnosis of haemoglobinopathies

Pictogramme horloge Dominique BRUNENGO Pictogramme horloge March 2018

A few simple haematological and biochemical tests, combined with knowledge of the clinical context and family background, frequently enable diagnosis of haemoglobinopathy on the basis of a phenotypic analysis. Sometimes, in a second-line analysis, the characterisation of a rare variant requires the use of molecular biology for a genotypic diagnosis.

Haemoglobinopathies are divided into two types

  • Quantitative anomalies: mainly represented by thalassemias of the α, β (or γ) types, caused by defects in one of the globin chains. The thalassemia is referred to as an alpha type if the defect is on the alpha chain and as a beta type when the defect is on the beta chain.
  • Qualitative anomalies: synthesis of an abnormal haemoglobin (or haemoglobin variant): HbS, HbC, HbE, … resulting in general from a localised mutation on a gene (more than 1500 mutations have been inventorised to date).

Conditions for haemoglobinopathy screening

Screening for a haemoglobin abnormality is very often carried out after the diagnosis of haemolytic anaemia or isolated microcytosis without known iron deficiency, but also in the context of polyglobulia or if morphological abnormalities are identified in red blood cells. Such screening may also be ordered in the context of a family survey, in patients with a high risk ethnic or geographical origin, in connection with neonatal screening or, more rarely, in the event of incidental discovery of a variant in an HbA1c assay or in relation to clinical/biological signs of haemolysis (splenomegaly, high LDH, high bilirubin or a drop in haptoglobin levels, …).

The diagnosis of haemoglobinopathy is based on:

  • knowledge of the geographical origin of the patient, clinical and therapeutic information (treatment with an HbF inducer such as hydroxycarbamide or erythropoietin (EPO)?), family history, haematological data with haemogram performed a considerable time interval after any recent transfusion (4 months): GR, Hb and erythrocyte indices (MCV and MCH), as well as iron levels and, if possible, vitamin B12/folate assays.

    CBC results must be interpreted according to age (especially for MCV), but it should be remembered that the leading cause of microcytosis (worldwide) is iron deficiency (not haemoglobin abnormalities).  If there is a suspicion of iron deficiency, it is advisable to repeat the electrophoresis of Hb, 3 months after correction of any iron deficiency, in particular to exclude a minor beta thalassemia.


  • Compliance with pre-analysis conditions is essential: the preferred method is collection is a total blood sample collected in an EDTA tube, which is not stable for more than 7 days when refrigerated. Thereafter, degraded haemoglobin fractions may appear that prevent correct interpretation of the results. Freezing or keeping in ice must be prohibited to prevent haemolysis.


  • Haemoglobin analysis: based on first-line techniques, forming the phenotypic diagnosis. According to code 1120 of the NABM (French national list of reimbursable of biological tests), this analysis is defined as follows: “A standard analysis for the detection of a haemoglobin abnormality” must include 3 distinct phenotypic techniques, at least one of which must be electrophoretic technique. It is essential to use efficient and complementary techniques not only to detect and recognise thalassemias, but also the main variants of clinical interest. In other words, if a variant of haemoglobin is present, 3 techniques are required to confirm the identification of this variant, whether or not these are of separative type (electrophoresis techniques, high performance liquid chromatography (HPLC)).

    At the Eurofins-Biomnis laboratory, we use a capillary electrophoresis technique as the first-line technique. If a variant is detected, this is confirmed by HPLC and acid gel electrophoresis, the objective being to reliably identify HbS, HbC and HbE, which are the most frequently encountered variants in current clinical practice, and to also permit detection of “rare” variants.

    Specialised molecular biology techniques, referred to as second-line techniques, are available which make it possible to confirm or identify “rare” variants detected by phenotypic testing. They also support genetic counselling of couples at risk of passing on haemoglobinopathies to their children. The informed consent of the patient (or of the parents for a minor) is a prerequisite for the performance of the genotypic diagnosis.

For more information


  1. Baudin B. Les maladies de l’hémoglobine. Rev Fr Laboratoires 2016;481:25-41.
  2. Couque N. De Montalembert M. Diagnostic d’une hémoglobinopathie. Feuillets de Biologie 2013; LIV(311):5-17
  3. Aguilar-Martinez P, Badens C, Bonello-Palot N, Cadet E. Arbres décisionnels pour le diagnostic et la caractérisation moléculaire des hémoglobinopathies. Ann Biol Clin 2010;68 (4):455-64

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