Hemoglobin electrophoresis

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Ligament model of hemoglobin A (HbA), which is physiologically predominant in adults. α- and β-chains in red and blue, heme in green.

Hemoglobin electrophoresis (Hb electrophoresis) is one of the most important examination methods for clarifying hemoglobinopathies . This term refers to the separation of hemoglobins using different electrophoretic methods such as e.g. B. of gel electrophoresis , capillary electrophoresis (CE) or isoelectric focusing . The HPLC is often attributed to hemoglobin electrophoresis of, although the separation of this chromatography is performed. A hemolysate is examined in which the hemoglobins are free in solution and are no longer enclosed in the erythrocytes .

Structure variants

Hemoglobin electrophoresis is particularly well suited for clarifying hemoglobin structure variants. With these structural anomalies, hemoglobins with new properties are present on the basis of a changed amino acid sequence . In the case of many of these anomalies, the sequence variant changes the acid constant of the protein, so that at a given pH value, the total charge of the molecule changes, which influences the migration in the electric field or the interaction with the phases of HPLC. Examples with a clear change in the acid-base properties are hemoglobin C (HbC) and E (HbE), in which the acidic glutamic acid is replaced by the basic lysine .

However, depending on the method, not all variants can be clearly delineated from one another, since some hemoglobin variants have very similar migration properties at a certain pH value. The standard procedure for the initial examination is Hb electrophoresis in an alkaline medium. If this investigation reveals an indication of the presence of a structural variant that cannot be clearly specified, an investigation in an acidic environment is then possible. Structural anomalies which have an identical migration pattern in alkaline electrophoresis can have different charge properties at high proton concentrations and are therefore differentiated from one another in acid electrophoresis. For example, HbC and HbE comigrate in an alkaline environment and can be more clearly distinguished from one another in an acidic environment. In the case of various abnormal hemoglobins, separation using electrophoretic methods is not possible.

A reliable quantification of the individual hemoglobin entities present , which is usually carried out by means of CE or HPLC, is important. The proportion of a structural variant found and the ratio of the individual fractions to one another can give an indication of a (possibly additionally present) hemoglobinopathy from the thalassemia spectrum.

Thalassemias

For the diagnosis of β-thalassemia, a valid quantification of the hemoglobin fractions is essential: in thalassemia, no hemoglobins with changed properties are formed, but the production of physiological hemoglobins is sometimes significantly impaired. The hemoglobin of adults consists mainly of hemoglobin A (HbA, ~ 95%), hemoglobin A 2 (HbA 2 , <3.5%) and traces of fetal hemoglobin F (HbF, <0.5%) ). HbA is composed of a tetramer of two α - and two β-globin chains ( α 2 β 2 ), while HbA 2 α- of two and two δ-globin chains ( α 2 δ 2 is formed). HbF arises from two α and two γ chains ( α 2 γ 2 ). In β-thalassemia, the production of β-globins is reduced. This leads to a relative increase in HbA 2 and sometimes HbF, which can be quantified. In β-thalassemia minor, the HbA 2 percentage is usually> 3.5%. HbF is predominantly found in β-thalassemia major.

The diagnosis of α-thalassemia, on the other hand, is only possible to a limited extent with Hb electrophoresis. Since the α-globins are involved in all fetal and adult hemoglobins, there is no relative increase in a fraction that could be used diagnostically. In α-thalassemia, depending on the clinical picture, the excess of β chains creates a very unstable β-globin tetramer, HbH ( β 4 ). I.a. Due to its instability, it often escapes laboratory detection in routine diagnostics. In HbH disease, however, there are sometimes amounts that can be detected. Alpha thalassemias are therefore usually investigated by means of hemoglobin genotyping of the HBA locus. Hb genotyping is also suitable for the molecular genetic verification of hemoglobin structure variants, for clarifying unclear cases or for further processing of complex (e.g. compound heterozygous ) combination forms of hemoglobinopathy.

Diagnosis

Sequence of embryonic , fetal and adult hemoglobin synthesis

When evaluating the results of Hb electrophoresis, it is of great importance to take into account the other findings as well as the clinical situation and anamnesis of the patient. Knowledge of the parameters of the red blood count is essential here. The electrophoretic findings must be assessed together with the blood count. Hemoglobin and erythrocyte parameters should match. Any discrepancies that arise here should suggest other, additional pathologies. If there is a suspicion, further diagnostics can then be initiated. The most common example of such a constellation is α-thalassemia: if blood count parameters are indicative (e.g. erythrocytosis , hypochromasia , microcytosis ), Hb electrophoresis is normal. After excluding common causes for similar changes (especially Fe deficiency ) and taking into account the anamnesis (e.g. ethnic origin), the blood count should give rise to further clarification, e.g. B. be by means of Hb genotyping.

The patient's age must also be taken into account in the evaluation. A switch from fetal to adult hemoglobin takes place shortly before birth. Therefore, large amounts of HbF can still be detected physiologically in infants . However, all pathologies that affect the β-globin chain are only fully developed in the first months of life due to the gradually increasing β-globin synthesis.

Individual evidence

  1. ↑ Ronald JA Trent: Diagnosis of the hemoglobinopathies . In: The Clinical Biochemist. Reviews . tape 27 , no. 1 , February 2006, ISSN  0159-8090 , p. 27-38 , PMID 16886045 , PMC 1390791 (free full text).
  2. ↑ Enno Kleihauer with the collaboration of Elisabeth Kohne and Andreas E. Kulozik: Anomalous hemoglobins and thalassemia syndromes: Basics and clinics . Ecomed, Landsberg 1996, ISBN 3-609-62760-3 .
  3. ↑ Hemoglobin Diagnostics | University hospital Freiburg. Retrieved April 10, 2020 .
  4. ↑ Beta thalassemia. Retrieved April 10, 2020 .
  5. ^ GM Clarke, TN Higgins: Laboratory investigation of hemoglobinopathies and thalassemias: review and update . In: Clinical Chemistry . tape 46 , 8 Pt 2, August 2000, ISSN  0009-9147 , p. 1284-1290 , PMID 10926923 .
  6. ^ S1 guideline thalassemia AWMF 025/017. (PDF) June 30, 2016, accessed April 10, 2020 .
  7. ↑ Stage diagnosis if hemoglobinopathy is suspected. (PDF) Labor 28 Berlin, January 2018, accessed on April 10, 2020 .