Haemoglobin Electrophoresis

Overview

Haemoglobin Electrophoresis is one of the most reliable diagnostic methods used for the demonstration and separation of abnormal hemoglobin variants based on their electrophoretic mobility and electric charge differences. It is especially useful for identifying hemoglobinopathies such as sickle cell disease and thalassemia. The technique separates hemoglobins on supporting media like cellulose acetate electrophoresis at pH 8.6 and agar gel electrophoresis at pH 6.0. Normal adult hemoglobin (HbA) is composed of two alpha and two beta chains, while abnormal variants differ in chain composition, leading to disease. HbF (fetal hemoglobin) and other variants can also be detected. Clinically, haemoglobin electrophoresis is crucial in screening, diagnosis, prenatal testing, and genetic counseling, particularly in populations where hemoglobin disorders are prevalent.

Symptoms

(Clinical conditions prompting haemoglobin electrophoresis)

1. Unexplained hemolytic anemia, with persistent fatigue and pallor.
2. Microcytic anemia not due to iron deficiency, lead poisoning, or chronic disease.
3. Abnormal red cell morphology on peripheral smear, including target cells, sickle cells, or nucleated RBCs.
4. Family history of hemoglobinopathies, requiring confirmatory testing.
5. Neonatal screening, especially in regions with high prevalence of sickle cell or thalassemia.
6. Positive sickling tests or cases with elevated fetal hemoglobin (HbF).
7. Recurrent infections or delayed growth in children with suspected thalassemia.

Causes

(Underlying factors leading to abnormal haemoglobin electrophoresis results)

1. Genetic mutations causing structural variants such as HbS (sickle cell hemoglobin), HbC, HbD, HbE, and HbO.
2. Beta-thalassemia major and minor, due to reduced or absent beta-globin chain production.
3. Sickle cell disease or trait, where HbS polymerization causes abnormal RBC morphology.
4. Hereditary persistence of fetal hemoglobin (HPFH), with HbF production continuing into adulthood.
5. Other hemoglobinopathies, including those producing rare variants that migrate differently under electrophoresis.
6. Transfusions may temporarily alter results if mixed populations of normal and abnormal hemoglobin are present.

Risk factors

(Groups most at risk of abnormal findings on haemoglobin electrophoresis)

1. Individuals with family history of hemoglobinopathies, particularly sickle cell disease and thalassemia.
2. Patients from endemic regions, where abnormal hemoglobin variants are more prevalent.
3. Newborns in high-risk populations, for whom early screening prevents complications.
4. Children with unexplained anemia, growth delays, or recurrent infections.
5. Pregnant women with family history of hemoglobin disorders, requiring prenatal testing for fetal risk.
6. Individuals with abnormal blood smear results, showing anisopoikilocytosis or sickling.
7. Patients with recurrent hemolysis, fatigue, or chronic anemia unresponsive to standard treatment.

Prevention

(Strategies for accurate testing and management of hemoglobin disorders)

1. Accurate blood sample collection:
   1. Draw 2.0–2.5 ml blood via venipuncture.
   2. Collect in EDTA tubes (lavender top) and mix gently.
   3. Send samples to the laboratory immediately to avoid degradation.
2. Choice of electrophoresis method:
   1. Cellulose acetate electrophoresis (pH 8.6): reliable and rapid, separates HbA, HbS, HbC, and HbF.
   2. Citrate agar electrophoresis (pH 6.0): differentiates hemoglobins that migrate closely together in cellulose acetate (e.g., HbS vs HbD and HbC vs HbE).
   3. Agarose gel electrophoresis: alternative system suitable for laboratories processing fewer samples.
3. Interpretation guidelines:
   1. Presence of HbA and HbS with HbA dominant indicates sickle cell trait.
   2. HbS only indicates sickle cell disease.
   3. HbS and HbA with HbS dominant indicates sickle cell disease with recent transfusion.
4. Quality control: ensure proper sample application, prompt electrophoresis, and correct staining techniques to avoid misinterpretation.
5. Genetic counseling and prenatal testing: recommended for families with a history of hemoglobinopathies to reduce transmission risks.
6. Ongoing monitoring: patients with confirmed hemoglobin disorders should undergo regular testing to guide management and treatment decisions.
7. Prevent misdiagnosis: interpret results alongside iron studies, serum ferritin, and other relevant lab findings to differentiate from conditions like iron deficiency anemia or lead poisoning.