Haemoglobin Defects: Qualitative and Quantitative

• Haemoglobin is a tetrameric protein with 4 globin polypeptides and 4 haem groups.
• This protein is the main component of red blood cells, and transports oxygen.
• Problems in its synthesis can cause disease.
• Abnormal haemoglobin may or may not have altered function.
• Structural abnormality causing disease is called haemoglobinopathy, resulting in poor oxygen delivery.
• Significant abnormalities can be qualitative (abnormal molecules) or quantitative (abnormal amount)

Qualitative Haemoglobin Defects

• These result in abnormal haemoglobin molecules, potentially leading to premature red blood cell lysis such as Haemoglobin S.

Quantitative Haemoglobin Defects

• Also known as thalassemias, arise due to decreased synthesis of globin chains.
• This results in decreased haemoglobin formation and content in red blood cells.
• Other forms are caused by failure to switch from fetal to adult haemoglobin after birth, known as Hereditary Persistence of Fetal Haemoglobin (HPFH).

Pathophysiology

• 98% of red blood cell protein is haemoglobin, made of 4 globin chains and 4 haem molecules.
• Normal adult red blood cells have mostly Haemoglobin A (α2β2).
• Minor adult haemoglobins are Haemoglobin A2 (α2δ2) and Haemoglobin F (α2γ2).
• At birth, Haemoglobin F is predominant, shifting to adult proportions (97% A, 2% A2, 1% F) in the first year.
• Haemoglobin A1c forms from glucose addition to Haemoglobin A β-chains.
• Increased Haemoglobin A1c indicates diabetes mellitus.
• Each globin chain (α, β, δ, γ) has its own genetic locus.
• Genetic mutations in these genes cause qualitative alterations, typically point mutations substituting a single amino acid.
• This can lead to haemoglobinopathy if structural abnormalities cause clinical issues.
• Common haemoglobinopathies involve mutations in the beta chain, examples being Haemoglobin S, C, and E.

Amino Acid Substitutions in Beta Chains of Common Haemoglobin Variants

• Haemoglobin S has a substitution at position 6 (Glutamic Acid to Valine), found in Nigeria, Ghana, Zaire, Gabon, and Saudi Arabia.
• Haemoglobin C has a substitution at position 6 (Glutamic Acid to Lysine), found in West Africa, Ghana, and Upper Volta.
• Haemoglobin E has a substitution at position 26 (Glutamic Acid to Lysine), found mostly in Southeast Asia.
• Haemoglobin D has a substitution at position 121 (Glutamine to Glutamic Acid), found in India and Pakistan.
• Homozygous state is referred to as the trait.
• The variant with the higher concentration is listed first.

Quantitative Defects (Thalassemias)

• Mutations reducing globin chain synthesis cause quantitative decreases in normal haemoglobin production, leading to thalassemic syndromes.
• Thalassemias are classified by the affected chain (α or β), with α-thalassemias most commonly caused by deletions of alpha globin genes .
• β-thalassemic disorders result from genetic mutations affecting RNA synthesis, processing, or stability, leading to reduced beta globin proteins.
• β thalassemia is classified as minor (heterozygous) or major (homozygous).
• The most common combination is sickle β-thalassemia disease.
• Beta thalassemia is related to defects in beta-globin gene
• Sickle cell disease is related to defects in hemoglobin with abnormal hemoglobin S

Investigation of Common Haemoglobinopathies

Tests for Sickling

• Blood film: Involves thin blood smear and staining with blood smear.
• Dithionite Reduction Test: Erythrocytes with Haemoglobin S assume sickle shape under low oxygen tension with dithionite reducing oxygen tension.
• Performing the test: Freshly prepared dithionite reagent is added to blood on a microscope slide, covered with a thin film of Vaseline, and examined immediately and at intervals under high power.
• A known Haemoglobin S sample and a normal sample can be used as control
• Positive result: Erythrocytes with Haemoglobin S have a sickle shape.
• Solubility Tests for Haemoglobin: Sickle cell Haemoglobin is insoluble when deoxygenated in high phosphate buffer
• The solution becomes turbid as a result
• Performing the test: Blood is added to high phosphate buffer reagent, mixed, and turbidity is assessed against a white card with black lines.
• Positive and negative tests are used as controls
• Positive for sickling Haemoglobin test: Turbid solution
• Negative Test: black lines on the white card cannot be seen through the solution.
• Most haemoglobins including A, C, D, F, G, and A2 give negative results.

Monoclonal Antibodies Method for HaemoglobinS

• This method uses monoclonal antibodies specific to the amino acid at position 6 of the β chain.
• Antibodies form a red complex with specific haemoglobin molecules.
• The bound complex is indicative of a positive test

Haemoglobin Electrophoresis

• It Identifies abnormal haemoglobins by separating and detecting them based on charge.
• In basic solution (pH>8), haemoglobins have a negative charge and move towards the anode.
• The mobility is proportional to the net negative charges.
• HaemoglobinS contains valine instead of glutamic acid resulting in a smaller negative charge and slower movement.

CELLULOSE ACETATE ELECTROPHORESIS

• Procedure for initial electrophoretic testing is to use cellulose acetate at pH 8.4-8.8
• Patient and control red blood cells are haemolysed and subjected to electrophoresis for 15 to 30 minutes.
• On completion, the membrane is stained and the haemoglobins are identified by their position on the cellulose acetate paper and quantitated with Tris EDTA Boric Acid buffer.
• Electrophoresis in citrate agar at pH 6.2 complements cellulose acetate electrophoresis.
• In Alkaline electrophoresis Haemoglobin D, S, and G have the same electrophoretic mobility but acid (citrate Agar) electrophoresis separates HaemoglobinS from HaemoglobinD and HaemoglobinG.
• Also citrate agar differentiates Haemoglobin C from Haemoglobin A2.
• In alkaline pH electrophoresis Haemoglobin S and D move together.
• Haemoglobin C & E migrate together but separated in acid (Agarose gel) electrophoresis.

RED BLOOD CELLS Appearance In Disorders of Haemoglobin Synthesis

• Haemoglobin S shows sickle cells
• Haemoglobin C shows target cells and Haemoglobin C crystals after splenectomy
• Haemoglobin E shows Microcytosis, hypochromia, and target cells
• Unstable Haemoglobin shows Red blood cells inclusions with supravital dyes
• Thalassemia shows Microcytosis, target cells, and basophilic stippling

Explore haemoglobin defects, including qualitative abnormalities like Haemoglobin S that cause premature red blood cell lysis. Also learn about quantitative defects such as thalassemias, caused by decreased synthesis of globin chains. Understand the impact of these defects on oxygen delivery and haemoglobin formation.