Methods of Hemoglobin Electrophoresis Used in Singapore
In Singapore, High-Performance Liquid Chromatography (HPLC) is the primary method used for hemoglobin electrophoresis, specifically cation-exchange HPLC, which offers superior resolution and accurate quantitation for hemoglobin variant analysis. 1
Primary Methods Used
Cation-Exchange HPLC
- Cation-exchange HPLC is the method of choice for initial screening and diagnosis of hemoglobinopathies in Singapore 1, 2
- This method quantifies hemoglobin based on charge differences between glycated and non-glycated components 1
- It provides high resolution separation of more than 45 commonly encountered hemoglobin variants within 12 minutes 3
- The method offers accurate quantitation of hemoglobin concentrations, particularly for HbF and HbA2, which are crucial for diagnosing conditions like beta-thalassemia 3, 2
Capillary Electrophoresis
- Capillary electrophoresis is also used as an alternative method in some laboratories 4
- It separates hemoglobin variants based on their charge differences at alkaline pH 1, 4
- This method shows good separation for most common hemoglobin variants but may have limitations in separating HbA2 from HbC 4
Technical Specifications
- Modern HPLC systems for hemoglobin analysis use porous silica coated with polyaspartic acid to improve elution time while maintaining high sensitivity 3, 5
- These systems are fully automated, making them ideal for routine clinical laboratory use 3, 2
- Sample preparation is simple, requiring minimal handling before analysis 3
- The HPLC method provides complete separation of common hemoglobin variants such as HbS, HbC, HbD, HbE, and others 5
Advantages of HPLC Over Traditional Methods
- Superior resolution compared to traditional gel electrophoresis methods 2
- More accurate quantitation of hemoglobin variants 3, 2
- Complete automation reduces human error and increases throughput 3
- Better distinction between similar hemoglobin variants that may be misidentified by conventional electrophoresis 2
- Capable of detecting both common and rare hemoglobin variants 3, 5
Quality Control Standards
- Laboratories follow international standards for HbA1c measurement with intralaboratory CV <1.5% and interlaboratory CV <2.5% 1
- Systems are certified by the National Glycohemoglobin Standardization Program (NGSP) as traceable to the Diabetes Control and Complications Trial (DCCT) reference 1
- Manufacturers also demonstrate traceability to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method 1
Clinical Applications
- Diagnosis of hemoglobinopathies including thalassemias and structural hemoglobin variants 2, 6
- Monitoring glycemic control in diabetes patients through HbA1c measurement 1
- Neonatal screening for sickle cell disease 6
- Prenatal diagnosis of thalassemia by fetal blood analysis 6
Limitations and Considerations
- Hemoglobin variants can interfere with HbA1c measurements, potentially causing spuriously high or low results 1
- Conditions that alter red blood cell lifespan can affect HbA1c results independent of the assay method 1
- For samples with suspected hemoglobin variants, boronate affinity chromatography may be more reliable for HbA1c measurement 1
- Proper sample collection and handling are crucial for accurate results 1