Thin-Layer/Isoelectric Focusing (IEF)
Thin-Layer/Isoelectric Focusing (IEF)
Isoelectric focusing is a high-resolution electrophoretic technique used to separate proteins based on their isoelectric points (pI). In pediatrics, it's particularly valuable for hemoglobin analysis and newborn screening.
Key Points
- Gold standard for hemoglobinopathy diagnosis
- Superior resolution compared to conventional electrophoresis
- Can separate hemoglobin variants differing by single amino acid
- Essential tool in newborn screening programs
Basic Principles
Physical Basis
- Utilizes pH gradient in electrophoretic medium
- Proteins migrate until reaching their isoelectric point
- Separation based on molecular charge differences
Technical Aspects
- pH gradient created using ampholytes
- Electric field applied perpendicular to pH gradient
- Proteins focus into sharp bands
- Resolution capability of 0.01 pH units
Advantages
- Higher resolution than conventional methods
- Can detect subtle hemoglobin variants
- Requires minimal sample volume
- Rapid analysis time (2-3 hours)
Methodology
Sample Preparation
- Blood collection in EDTA tube
- RBC lysis to release hemoglobin
- Sample dilution and standardization
- Quality control preparation
Procedure Steps
- Gel preparation with ampholytes
- Sample application
- Electric field application
- Focusing period (usually 2 hours)
- Fixation and staining
- Documentation and analysis
Quality Control
- Internal controls with known hemoglobin variants
- External quality assessment program participation
- Regular calibration checks
- Documentation of lot numbers and expiry dates
Clinical Applications
Primary Applications
- Newborn screening for hemoglobinopathies
- Diagnosis of thalassemia syndromes
- Identification of hemoglobin variants
- Monitoring therapeutic interventions
Specific Clinical Scenarios
- Neonatal Screening:
- Detection of Hb S, C, E, and other variants
- Early identification of sickle cell disease
- Beta thalassemia screening
- Diagnostic Workup:
- Unexplained anemia
- Family history of hemoglobinopathy
- Abnormal CBC findings
Result Interpretation
Normal Pattern
- HbA: 96-98%
- HbA2: 2-3%
- HbF: <1% (after 6 months of age)
Abnormal Patterns
- Sickle Cell Disease:
- Presence of HbS band
- Absence or reduced HbA
- Variable HbF levels
- Beta Thalassemia:
- Elevated HbA2 (>3.5%)
- Variable HbF elevation
- HbE Disease:
- Presence of HbE band
- Reduced or absent HbA
Important Considerations
- Age-specific reference ranges
- Impact of recent transfusion
- Need for family studies
- Correlation with clinical findings
