Qualitative

Overview of Qualitative Hemoglobin Analysis

Definition: Laboratory tests used to identify and characterize different types of hemoglobin in a blood sample. These tests are crucial for diagnosing hemoglobinopathies, such as sickle cell anemia and thalassemia
Purpose
- Detect and quantify normal and abnormal hemoglobin variants (e.g., HbA, HbA2, HbF, HbS, HbC, HbD, HbE)
- Identify hemoglobinopathies in patients with anemia, jaundice, or a family history of hemoglobin disorders
- Screen newborns for sickle cell disease and other hemoglobinopathies
- Monitor treatment response in patients with hemoglobinopathies
Methods
- Hemoglobin Electrophoresis
- High-Performance Liquid Chromatography (HPLC)
- Sickle Solubility Testing

Principle
- Separates hemoglobin variants based on their electrical charge and migration patterns in an electric field
- Hemoglobin molecules are placed on a supporting medium (e.g., cellulose acetate or agarose gel) and an electric current is applied
- Different hemoglobin types migrate at different rates depending on their charge
- After electrophoresis, the separated hemoglobin bands are visualized using a stain

Principle * Separates hemoglobin variants based on their interaction with a stationary phase in a column, followed by detection using spectrophotometry
- Steps:
  - The hemolysate is injected into a column packed with a solid material (stationary phase)
  - A liquid solvent (mobile phase) is pumped through the column
  - Hemoglobin variants interact differently with the stationary phase based on their size, charge, and hydrophobicity
  - As each hemoglobin variant elutes from the column, it passes through a detector (spectrophotometer) that measures its absorbance at a specific wavelength
  - The detector generates a chromatogram, which shows a series of peaks corresponding to the different hemoglobin types

Principle
- Hemoglobin S (HbS) is insoluble in a concentrated phosphate buffer when deoxygenated, causing the solution to become turbid (cloudy)
- Other hemoglobins remain soluble in the buffer

Run Controls with Known Hemoglobin Patterns
- Include normal and abnormal controls with each electrophoresis or HPLC run
- The control results should fall within established reference ranges
Document All Quality Control Procedures
- Record the dates, lot numbers, and results of all quality control procedures
Follow Established Laboratory Procedures
- Adhere to standardized operating procedures for performing and interpreting hemoglobin analysis
Participate in Proficiency Testing Programs
- To assess the accuracy and reliability of the laboratory’s testing