Electrophoresis

Overview of Hemoglobin Electrophoresis

Definition: Hemoglobin electrophoresis is an analytical technique used to separate and identify different types of hemoglobin (Hb) based on their electrical charge and migration patterns in a supporting medium when subjected to an electric field
Clinical Significance:
- Detection and Identification of Hemoglobin Variants: Helps identify normal (HbA, HbA2, HbF) and abnormal (HbS, HbC, HbD, HbE) hemoglobins
- Diagnosis of Hemoglobinopathies: Essential for diagnosing sickle cell anemia, thalassemias, and other inherited hemoglobin disorders
- Prenatal Screening: Can be used to screen for hemoglobinopathies in pregnant women or to diagnose affected fetuses
- Monitoring Transfusion Therapy: Useful for tracking changes in hemoglobin composition after blood transfusions
Key Principle: Exploits the differences in electrical charge among hemoglobin variants to separate them into distinct bands on a supporting medium

Components of a Hemoglobin Electrophoresis System

Sample: A hemolysate (lysed red blood cells) containing hemoglobin
Supporting Medium: A solid or semi-solid matrix that provides a surface for hemoglobin migration
- Common media: Cellulose acetate, agarose gel, capillary
Buffer: An electrolyte solution that provides a constant pH and ionic strength, influencing the charge and migration of hemoglobin
Electrophoresis Chamber: A device that holds the supporting medium and provides electrodes for applying the electric field
Power Supply: A device that generates and controls the electric current
Stain: A dye that binds to hemoglobin and makes the separated bands visible
Densitometer: A device that measures the optical density of the stained bands, allowing for quantification of each hemoglobin type

Methodologies for Hemoglobin Electrophoresis

Cellulose Acetate Electrophoresis
- Principle: Separates hemoglobin variants on a cellulose acetate membrane at alkaline pH
- Supporting Medium: A thin, flexible cellulose acetate membrane
- Buffer: Tris-EDTA-borate (TEB) buffer (pH 8.4 - 8.6)
- Procedure:
  1. Prepare Hemolysate: Lyse red blood cells to release hemoglobin
  2. Saturate Membrane: Soak the cellulose acetate membrane in the buffer
  3. Apply Sample: Apply a small amount of the hemolysate to the membrane
  4. Electrophoresis: Place the membrane in the electrophoresis chamber, apply the electric current, and allow the hemoglobin to migrate for a specific time (typically 20-30 minutes)
  5. Staining: Remove the membrane and stain it with Ponceau S or other suitable stain to visualize the hemoglobin bands
  6. Destaining: Remove excess stain with a destaining solution
  7. Interpretation: Examine the membrane visually or using densitometry to identify and quantify the hemoglobin bands
- Migration Pattern (from fastest to slowest migration):
  1. HbA1
  2. HbF
  3. HbS
  4. HbC
  5. HbA2
- Advantages:
  - Simple and relatively inexpensive
  - Good resolution for common hemoglobin variants (HbA, HbS, HbC, HbF)
- Limitations:
  - Some hemoglobins comigrate (e.g., HbS and HbD, HbC and HbE) at alkaline pH
  - Cannot be used to detect unstable hemoglobins
  - Lower sensitivity compared to other methods
Agarose Gel Electrophoresis
- Principle: Separates hemoglobin variants on an agarose gel at acidic pH
- Supporting Medium: Agarose gel
- Buffer: Citrate buffer (pH 6.0 - 6.2)
- Procedure: Similar to cellulose acetate electrophoresis, but using an agarose gel as the supporting medium and an acidic buffer
- Migration Pattern (from fastest to slowest migration):
  1. HbF
  2. HbA
  3. HbS
  4. HbC
- Advantages:
  - Can be used to confirm the identity of certain hemoglobin variants that comigrate on cellulose acetate (e.g., HbS and HbD)
- Limitations:
  - Not as commonly used as cellulose acetate electrophoresis for routine screening
  - Lower resolution for some hemoglobin variants
Capillary Electrophoresis (CE)
- Principle: Separates hemoglobin variants based on their charge-to-size ratio as they migrate through a capillary tube filled with an electrolyte solution under the influence of an electric field
- Procedure:
  1. Capillary Filling: The capillary tube is filled with an appropriate electrolyte solution
  2. Sample Injection: A small amount of the hemolysate is injected into the capillary
  3. Electrophoresis: An electric field is applied, causing the hemoglobin variants to migrate through the capillary
  4. Detection: A detector at the end of the capillary measures the absorbance or fluorescence of the separated hemoglobin variants
  5. Data Analysis: The data are analyzed by specialized software to identify and quantify the hemoglobin types
- Advantages:
  - High resolution and sensitivity
  - Automated and rapid
  - Can quantify small amounts of abnormal hemoglobins
  - Reduced manual handling
- Limitations:
  - More expensive than cellulose acetate electrophoresis
  - Requires specialized equipment and training

Common Hemoglobin Variants and their Electrophoretic Patterns

Here’s a table summarizing the electrophoretic mobility of common hemoglobin variants at alkaline pH (cellulose acetate electrophoresis):

Hemoglobin Variant	Mobility at Alkaline pH
HbA	Most common, migrates normally
HbF	Migrates near HbA
HbS	Slower than HbA but faster than HbC
HbC	Slowest migration
HbA2	Slowest migration (same as HbC)
HbD	Migrates with HbS
HbE	Migrates with HbC

Important Note: Remember that these migration patterns are at alkaline pH. The order may differ at acidic pH (agarose gel electrophoresis).

Quality Control (QC) Procedures

Run Controls with Known Hemoglobin Patterns:
- Include normal and abnormal controls with each electrophoresis run
- The control results should fall within established reference ranges
Check Reagents and Buffers:
- Use high-quality reagents and buffers
- Prepare reagents according to the manufacturer’s instructions
- Monitor the pH and expiration dates of buffers
Monitor Temperature and Humidity:
- Maintain the recommended temperature and humidity in the electrophoresis laboratory
Inspect the Electrophoresis Equipment:
- Regularly inspect the electrophoresis chamber, power supply, and other equipment for damage or malfunction
Document All Quality Control Procedures:
- Record the dates, lot numbers, and results of all quality control procedures

Troubleshooting

Poor Band Resolution:
- Check the buffer pH and ionic strength
- Ensure proper staining and destaining techniques
- Check for contamination of the sample or reagents
Distorted or Uneven Bands:
- Ensure that the supporting medium is properly hydrated and free of defects
- Check the electrode polarity
- Check for proper sample application
Weak or Faint Bands:
- Ensure that the sample concentration is adequate
- Check the staining procedure
Unexpected or Unidentified Bands:
- Consider the possibility of rare hemoglobin variants or hemoglobin derivatives
- Perform additional testing (e.g., HPLC, globin chain analysis) to confirm the identity of the unknown band
Instrument Errors:
- Consult the instrument’s troubleshooting guide or contact the manufacturer for assistance

Reporting Results

Report the percentage of each hemoglobin type (e.g., HbA, HbA2, HbF, HbS, HbC)
Include a visual representation of the electrophoresis pattern (if possible)
Provide an interpretation of the results, noting any abnormal findings
Correlate the laboratory findings with the patient’s clinical information

Key Terms

Hemoglobin Electrophoresis: Technique to separate and identify hemoglobin types
Hemoglobinopathy: A genetic disorder affecting the structure or synthesis of hemoglobin
Hemoglobin Variant: An abnormal hemoglobin molecule
Cellulose Acetate Electrophoresis: A common method for hemoglobin electrophoresis
Agarose Gel Electrophoresis: Another method for hemoglobin electrophoresis
Capillary Electrophoresis (CE): An automated method for hemoglobin separation
HPLC: High Performance Liquid Chromatography
Isoelectric Focusing (IEF): A high-resolution electrophoretic technique
HbA, HbA2, HbF, HbS, HbC: Common hemoglobin types
Sickle Cell Trait (HbAS): Heterozygous carrier of HbS
Sickle Cell Anemia (HbSS): Homozygous for HbS
Migration Pattern: The order in which different hemoglobin types move across the electrophoresis medium

Qualitative

HPLC