Abnormal haemoglobins

Abnormal haemoglobins are variations in the haemoglobin molecule that result from genetic mutations affecting the globin chains. These abnormalities can lead to various haematological disorders, including sickle cell disease, thalassemia, and other hemoglobinopathies. Identifying and estimating abnormal haemoglobins involves specific diagnostic techniques. Here’s a detailed look at these abnormal haemoglobins and how they are identified and estimated:

Abnormal haemoglobins

1. Hemoglobin S (HbS)

• Genetic Basis: Caused by a single nucleotide substitution (A to T) in the β-globin gene (HBB) on chromosome 11, resulting in the replacement of glutamic acid with valine at position 6 of the β-chain.
• Pathophysiology: The valine substitution causes haemoglobin molecules to polymerize under low oxygen conditions, forming sickle-shaped red blood cells. These rigid cells can obstruct blood flow, causing pain and tissue damage.
• Diagnosis:
  • Haemoglobin Electrophoresis: HbS migrates differently from normal HbA, allowing its identification.
  • HPLC: HbS has distinct chromatographic properties compared to HbA and other haemoglobins.

2. Hemoglobin C (HbC)

• Genetic Basis: Caused by a single nucleotide substitution (A to T) in the β-globin gene, leading to the replacement of glutamic acid with lysine at position 6 of the β-chain.
• Pathophysiology: HbC forms crystals within red blood cells, leading to mild to moderate hemolytic anaemia and splenomegaly.
• Diagnosis:
  • Hemoglobin Electrophoresis: Hb C appears as a separate band on electrophoresis, different from HbA.
  • HPLC: Hb C has unique chromatographic characteristics, allowing its differentiation.

3. Haemoglobin D (Hb D)

• Genetic Basis: Various mutations in the β-globin gene. HbD is less likely to affect red cell morphology than HbS or HbC.
• Pathophysiology: Generally, HbD does not cause significant clinical problems but can be associated with other hemoglobinopathies.
• Diagnosis:
  • Hemoglobin Electrophoresis: HbD migrates differently from HbA and HbS.
  • HPLC: Distinct from other haemoglobins, especially in the presence of other haemoglobin variants.

4. Hemoglobin E (HbE)

• Genetic Basis: Caused by a single nucleotide substitution (A to G) in the β-globin gene, replacing glutamic acid with lysine at position 26 of the β-chain.
• Pathophysiology: This can cause mild anaemia and is often found in Southeast Asian populations. It may co-occur with other hemoglobinopathies like thalassemia.
• Diagnosis:
  • Haemoglobin Electrophoresis: HbE migrates in a specific manner, different from HbA and HbC.
  • HPLC: Differentiates HbE from other haemoglobins based on its chromatographic profile.

5. Hemoglobin F (HbF)

• Genetic Basis: Composed of two alpha (α) and two gamma (γ) chains. Normally present during fetal development and decreases after birth.
• Pathophysiology: Elevated adult levels can indicate conditions like β-thalassemia or sickle cell disease. HbF has a higher affinity for oxygen compared to HbA.
• Diagnosis:
  • Hemoglobin Electrophoresis: HbF appears as a separate band.
  • HPLC: HbF elutes at a different time than HbA and other haemoglobins.

6. Hemoglobin M (HbM)

• Genetic Basis: Mutations in the globin gene that stabilise the iron in the ferric (Fe³⁺) state.
• Pathophysiology: Results in methemoglobinemia, where haemoglobin cannot bind oxygen effectively, leading to cyanosis.
• Diagnosis:
  • Co-oximeter: Measures methemoglobin levels and distinguishes HbM from normal haemoglobin.

Identification and Estimation Techniques

1. Hemoglobin Electrophoresis

• Technique:
  • Preparation: Blood is mixed with a buffer and applied to an electrophoresis medium (e.g., agarose or cellulose acetate).
  • Separation: An electric field is applied, causing haemoglobins to migrate based on their charge and size.
  • Visualization: Separated haemoglobin fractions are stained or visualized to identify different types.
  • Quantification: Band intensity is compared to known standards or reference curves to estimate the proportion of each haemoglobin type.
• Advantages: Reliable for identifying and quantifying abnormal haemoglobins; used for screening and diagnostic purposes.
• Disadvantages: Requires expertise and can be affected by multiple haemoglobin variants.

2. High-Performance Liquid Chromatography (HPLC)

• Technique:
  • Preparation: Blood is processed to separate haemoglobin from other blood components.
  • Separation: Hemoglobin is separated as it passes through a chromatographic column under high pressure.
  • Detection: Detected using UV or fluorescence spectroscopy as they exit the column.
  • Quantification: Peak areas or heights are used to estimate the concentration of each haemoglobin type.
• Advantages: Highly sensitive and specific; capable of distinguishing between various haemoglobin variants.
• Disadvantages: Requires specialized equipment and trained personnel.

3. Capillary Electrophoresis

• Technique:
  • Preparation: Blood is processed and loaded into a capillary tube.
  • Separation: An electric field is applied, causing haemoglobins to migrate through the capillary based on size and charge.
  • Detection: Hemoglobins are detected as they exit the capillary tube.
  • Quantification: Peak areas or heights are analyzed to estimate the amount of each haemoglobin type.
• Advantages: Provides high-resolution separation and rapid results.
• Disadvantages: Requires specialized equipment and interpretation.

4. DNA Analysis

• Technique:
  • Extraction: DNA is extracted from blood or tissue samples.
  • Amplification: Specific regions of the β-globin gene are amplified using PCR.
  • Sequencing or Mutation Detection: PCR products are sequenced or analyzed for known mutations using restriction fragment length polymorphism (RFLP) or allele-specific PCR techniques.
• Advantages: Provides a definitive diagnosis by identifying genetic mutations; useful for carrier screening and prenatal diagnosis.
• Disadvantages: Requires advanced laboratory facilities and technical expertise.

5. Solubility Test for Hemoglobin S

• Technique:
  • Preparation: Blood is mixed with a reagent that causes HbS to precipitate while HbA remains soluble.
  • Detection: The appearance of a turbid solution indicates the presence of HbS.
• Advantages: Simple and quick screening test for sickle cell disease.
• Disadvantages: Less specific; positive results should be confirmed with more precise methods like electrophoresis or HPLC.