Haemoglobin Pigments

Haemoglobin pigments are crucial in the diagnosis and management of various haematological conditions. These pigments, primarily the different forms of haemoglobin and their derivatives, can be measured to assess overall blood health, oxygen-carrying capacity, and other aspects of red blood cell function. Here’s an overview of key haemoglobin pigments and their measurements:

1. Table of contents

   Toggle

   Haemoglobin Pigments

Haemoglobin Forms

1. Hemoglobin A (HbA)
   • Composition: Two alpha (α) and two beta (β) chains.
   • Function: The primary form of adult haemoglobin responsible for most oxygen transport.
2. Hemoglobin A2 (HbA2)
   • Composition: Two alpha (α) and two delta (δ) chains.
   • Function: Present in small amounts in adults (about 2.5% of total haemoglobin). Elevated levels can indicate beta-thalassemia.
3. Hemoglobin F (HbF)
   • Composition: Two alpha (α) and two gamma (γ) chains.
   • Function: Predominant form in fetuses and newborns, with a higher affinity for oxygen than HbA, facilitating oxygen transfer from the mother to the fetus.
   • Clinical Relevance: Elevated adult levels can indicate certain anaemia or disorders like sickle cell disease or thalassemia.
4. Hemoglobin S (HbS)
   • Composition: Two alpha (α) and two beta (β) chains substituting valine for glutamic acid in the beta chains.
   • Function: Causes sickle cell disease, characterized by abnormally shaped red blood cells that can lead to blockages in blood vessels and various complications.
5. Hemoglobin C (HbC)
   • Composition: Two alpha (α) and two beta (β) chains substituting lysine for glutamic acid in the beta chains.
   • Function: Associated with haemoglobin C disease, which can cause mild to moderate anaemia and splenomegaly.

Hemoglobin Derivatives

1. Methaemoglobin (MetHb)
   • Formation occurs when the heme group’s iron is oxidized from the ferrous (Fe²⁺) to the ferric (Fe³⁺) state.
   • Function: Cannot bind oxygen; affects the oxygen-carrying capacity of the blood.
   • Clinical Relevance: Elevated levels can result from exposure to certain drugs (e.g., nitrates), chemicals, or congenital conditions. Methemoglobinemia can cause cyanosis and hypoxia.
2. Carboxyhaemoglobin (COHb)
   • Formation: Formed when haemoglobin binds with carbon monoxide (CO), which has a much higher affinity for haemoglobin than oxygen.
   • Function: Reduces the amount of haemoglobin available for oxygen transport.
   • Clinical Relevance: Elevated levels are indicative of carbon monoxide poisoning, which can cause severe hypoxia and is a medical emergency.
3. Sulhemoglobin (SulfHb)
   • Formation: Results from sulfur binding to haemoglobin, often due to exposure to sulfur-containing drugs or chemicals.
   • Function: Does not participate in oxygen transport and can lead to cyanosis.
   • Clinical Relevance: Sulhemoglobinemia can occur from certain medications or exposure to industrial chemicals.

---

2. Measurement Techniques

Total Hemoglobin Measurement

1. Cyanmethemoglobin Method
   • Procedure:
     • Blood is mixed with a reagent containing potassium ferricyanide and potassium cyanide.
     • Hemoglobin is oxidized to methemoglobin, which then reacts with cyanide to form cyanmethemoglobin.
     • The concentration of cyanmethemoglobin is measured spectrophotometrically at 540 nm.
   • Advantage: Reliable and widely used method for routine haemoglobin measurement.
   • Disadvantage: Cyanide used in the reagent is toxic, though present in very small quantities.
2. Hemoglobinometer
   • Procedure:
     • A portable device that measures haemoglobin concentration directly from a small blood sample.
     • Often uses photometric methods to quantify haemoglobin concentration.
   • Advantage: Quick and convenient for point-of-care testing.
   • Disadvantage: It may not be as accurate as laboratory-based methods.
3. Hematology Analyzer
   • Procedure:
     • Automated devices that measure haemoglobin concentration as part of a complete blood count (CBC).
     • Utilize various techniques, including electrical impedance or laser-based methods.
   • Advantage: Provides a comprehensive blood profile, including red cell indices, white blood cell counts, and platelet counts.
   • Disadvantage: Requires calibration and maintenance.

Hemoglobin Electrophoresis

• Procedure:
  • Blood samples are treated to extract haemoglobin.
  • The haemoglobin is then loaded onto an electrophoresis medium (e.g., agarose or cellulose acetate).
  • An electric field is applied, causing different haemoglobin types to migrate at different rates based on their charge.
  • Separated haemoglobin fractions are visualized using staining techniques or specific dyes.
  • Quantification is done by comparing the intensity of the bands to known standards.
• Advantage: Effective for diagnosing hemoglobinopathies (e.g., sickle cell disease, thalassemia) and assessing abnormal haemoglobin variants.
• Disadvantage: Requires specialized equipment and expertise.

Measurement of Hemoglobin Derivatives

1. Methemoglobin Measurement
   • Method:
     • Often measured using a co-oximeter, which differentiates between oxyhemoglobin, deoxyhemoglobin, methemoglobin, and carboxyhemoglobin based on their absorption spectra.
   • Advantage: Provides an accurate assessment of methemoglobin levels and is useful for diagnosing methemoglobinemia.
   • Disadvantage: Requires specialized equipment and can be complex to interpret.
2. Carboxyhemoglobin Measurement
   • Method:
     • Measured using a co-oximeter or specific test kits that detect carbon monoxide bound to haemoglobin.
   • Advantage: Critical for diagnosing carbon monoxide poisoning and assessing exposure levels.
   • Disadvantage: Requires precise measurement and can be affected by other factors such as smoking history.
3. Sulhemoglobin Measurement
   • Method:
     • Specific assays are used to detect and quantify sulhemoglobin levels, often in specialized laboratories.
   • Advantage: Useful for diagnosing methemoglobinemia related to drug or chemical exposure.
   • Disadvantage: Less commonly available and may require specialized testing.

Measurement of Hemoglobin A1c (HbA1c)

• Method:
  • High-Performance Liquid Chromatography (HPLC): Separates haemoglobin A1c from other haemoglobin variants based on their different affinities to chromatographic media.
  • Immunoassay: Uses specific antibodies to detect and quantify HbA1c levels.
  • Capillary Electrophoresis: Separates HbA1c from other haemoglobin variants based on their charge and size.
• Advantage: Provides a measure of long-term glucose control, essential for managing diabetes.
• Disadvantage: Requires specific equipment and may be affected by conditions like anaemia or haemoglobin variants.