Diagnostics

Hemoglobinopathies (overview)

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Blood Count
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Hemoglobin Differentiation
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Iron Status
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Molecular genetics
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Overview
Symptoms
Diagnostics
Background

Based on current guidelines and the most recent research, different diagnostic recommendations arise for patients with a hemoglobinopathy, such as thalassemia or sickle cell disease. We have summarized the most important information on the classification and diagnostic methods at MLL. In addition, we have compiled further links on the various diseases so that you can obtain more in-depth information.

Hemoglobinopathies: Overview

Hemoglobinopathies are hereditary hemoglobin diseases caused by different gene variants resulting in impaired synthesis of globin chains.

There are quantitative (thalassemias) and qualitative (hemoglobin structure variants) hemoglobinopathies.

MLL offers further information on the following entities including diagnostics and therapy:

Hemoglobinopathies: Symptoms

The leading symptom of thalassemias and thalassemic structural hemoglobin variants is microcytic hypochromic anemia.

Sickle cell disease (sickle cell anemia) is characterized by symptoms due to vaso-occlusive events. Clinically, there is often also a chronic hemolytic anemia that accompanies these patients throughout their lives.

Otherwise, symptoms of structural hemoglobin variants are not uniform and depend on the underlying structural hemoglobin variant.

The following are indications for the workup of a hemoglobinopathy:

Hypochromia and/ or anemia with prior exclusion of iron deficiency
Chronic hemolytic anemia
Vascular occlusions of unexplained etiology
Recurrent pain crises
Unexplained severe infections
Hydrops fetalis syndrome
Positive family history

Hemoglobinopathies: Diagnostic methods

The diagnosis of hemoglobinopathies is based on an interplay of various biochemical and molecular genetic methods and is usually performed as a step-by-step work-up. The first step is the full blood count and hemoglobin separation. Especially in hypochromia, the iron status should always be determined to exclude an underlying iron deficiency. In a second step, molecular genetic tests are used to characterize abnormal findings and to verify the molecular genetic cause of the clinical picture and the changes in the blood count and hemoglobin separation.

Furthermore, the molecular genetic findings can be used to estimate the risk for offspring to develop a severe homozygous form of hemoglobinopathy.

Hemoglobin separation is performed using high-resolution capillary electrophoresis. The different hemoglobin fractions present in the patient's blood are separated and quantified. The composition of the different fractions of hemoglobin is age-dependent and changes mainly before and during the first six months after birth until a distribution as seen in adults is established. At birth, HbA and HbF are present in approximately equal proportions. During the first months of life, the proportion of HbA increases continuously and the proportion of HbF decreases. A slight physiological increase in HbA2 is also seen during this period, which is why β-thalassemia minor (HbA2 > 3.5% and/or HbF increased) cannot always be detected with certainty before six months of age with hemoglobin electrophoresis alone. Depending on the relative distribution of physiologically occurring globin chains, evidence for β-thalassemia and severe α-thalassemia (HbA2 decreased, HbH and/or Hb Bart's detectable), among others, can thus be found in hemoglobin electrophoresis.

Iron deficiency is the most important differential diagnosis of clinically asymptomatic thalassemia, which in most cases is characterized only by blood count changes. In addition to ferritin, CRP and soluble transferrin receptor are measured to determine iron status.

Ferritin correlates with iron stores in the body and is a marker for detecting iron deficiency. Since ferritin can be normal or elevated in inflammatory diseases (e.g. infections or malignancies) despite iron deficiency, CRP is measured simultaneously as an inflammatory marker. Furthermore, soluble transferrin receptor is measured to assess iron status, which is not affected by chronic diseases such as tumors, inflammatory conditions or liver dysfunction, thus allowing differentiation of iron deficiency from anemia of chronic diseases.

Sequencing of the α- and β -globin complex.

In the presence of a structural hemoglobin variant in hemoglobin electrophoresis or suspected β-thalassemia, the β-globin gene complex is sequenced to verify the type of structural hemoglobin variant or identify the underlying variant of β-thalassemia.

Complementary to deletion assays of the α-globin complex (MLPA and gap-PCR, see below), the α-globin complex is sequenced to detect α-thalassemia based on molecular genetic alterations such as single base variants or minor deletions.

MLPA for the α- and β -globin complex.

Large deletions in the region of the α-globin gene complex are the most common causes of α-thalassemia and can be detected by multiplex ligation-dependent probe amplification (MLPA). Triplications or other multiplications of the α-globin gene are also detected by this method.

β-thalassemia can also be caused by deletions of the β-globin gene in part, in whole, or in its regulatory regions. These deletions can be detected with a MLPA assay of the β-globin gene complex. Larger deletions causing, for example, (γ)δβ-thalassemia are also detected by MLPA of the β-globin gene complex. In cases where almost exclusively a structural hemoglobin variant is present in the hemoglobin separation and HbA cannot be detected, MLPA is also used to distinguish whether the structural hemoglobin variant is present in the homozygous state or as a compound heterozygous state with a β-thalassemia.

Hemoglobinopathies: Background

Physiologically, hemoglobin is composed of two identical globin chains, to each of which a heme molecule is bound.

The predominant types of hemoglobin in adults are:

Hemoglobin type	Globin chains	Proportion of total hemoglobin
HbA	α2β2	96,7 - 97,8 %
HbA2	α2δ2	2,2 - 3,2 %
HbF	α2γ2	< 0,5 %

The α-globin gene complex on chromosome 16 contains, among others, the two α-globin genes HBA1 and HBA2, which encode the α-globin chains. The β-globin gene complex is located on chromosome 11 and includes, among others, the β- (HBB), two γ- (HBG1 and HBG2), and the δ-globin gene (HBD). These genes encode the corresponding β-, γ-, or δ-globin chains that, with two α-globin chains, form the different types of hemoglobin. Physiologically, each of these genes exists twice - once on the maternal and once on the paternal chromosome.

Status: April 2024

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Hemoglobinopathies (overview)

Hemoglobinopathies: Overview

Hemoglobinopathies: Symptoms

Hemoglobinopathies: Diagnostic methods

Hemoglobin differentiation

Iron status

Molecular genetics

Hemoglobinopathies: Background

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