When should hemoglobin electrophoresis be ordered for a patient with unexplained anemia, hemolysis, family history, or high‑risk ethnicity, and what does the test involve?

Hemoglobin Electrophoresis: Indications and Testing Approach

Hemoglobin electrophoresis should be ordered for any patient with unexplained microcytic anemia (especially when iron studies are normal), hemolysis, or when screening individuals at risk for hemoglobinopathies based on ethnicity or family history. 1

When to Order Hemoglobin Electrophoresis

High-Priority Clinical Scenarios

• Unexplained microcytic anemia with normal iron studies – When a patient presents with low MCV and MCH but ferritin is normal or elevated, hemoglobin electrophoresis is essential to exclude thalassemia and hemoglobin variants before pursuing unnecessary gastrointestinal investigations 2

• Evidence of hemolysis – Order testing in patients with chronic anemia, elevated reticulocyte count, elevated bilirubin, low haptoglobin, or splenomegaly, particularly when red cell morphology appears normal or near-normal 2

• Pre-operative screening – All patients at risk should be screened before surgery unless they are of solely northern or eastern European, Jewish, or South-East Asian heritage, or have been previously screened 2

Ethnicity-Based Screening

• Individuals of African, Mediterranean, Middle Eastern, Southeast Asian, or Central Indian descent should be considered for screening given the high prevalence of hemoglobin S, C, E, and thalassemia variants in these populations 1, 3

• Pregnant women – When sickle cell trait is identified in a pregnant woman, the father must also be tested, as there is a 25% chance of having a child with sickle cell disease if both parents carry the trait 1

Special Populations Requiring Testing

• Individuals born before 1987 or outside the U.S. who lack documented newborn screening results should undergo hemoglobin electrophoresis, as universal newborn screening was not established until 1987 1

• Patients with unexplained priapism – Hemoglobin electrophoresis may be appropriate based on underlying risk factors, though most patients with sickle cell disease will have been diagnosed previously 2

What the Test Involves

Technical Methods

• Hemoglobin electrophoresis is the gold standard confirmatory test that separates and identifies different hemoglobin types including HbA, HbS, HbC, and HbF 1, 4

• Hospital laboratories use various platforms including high-performance liquid chromatography (HPLC), capillary electrophoresis, mass spectrometry, or gel electrophoresis – all are highly sensitive, reliable, and reproducible 2, 5, 6

• The test differentiates between heterozygous states (trait), compound heterozygous states, and homozygous states (disease) 2

Normal vs. Abnormal Patterns

• Normal adult hemoglobin: 95-98% HbA, <1% HbF, <3-5% HbA2 2

• Sickle cell trait (HbAS): 55-65% HbA, 30-40% HbS, <1% HbF, <3-5% HbA2 2, 1

• The test provides comprehensive hemoglobin phenotype information in a single analysis 1

Critical Pitfalls to Avoid

Do Not Use Solubility Testing Alone

• Rapid sickle solubility tests cannot differentiate between trait (HbAS) and disease (HbSS, HbSC) and should never be used in isolation for definitive diagnosis 2, 1

• Solubility tests can give false negative results in neonates or heavily transfused patients 2

• If a solubility test is positive, always perform full hemoglobin electrophoresis for definitive characterization 2

Timing Considerations

• Recent blood transfusion can interfere with results due to the presence of normal donor red cells, potentially masking the patient's true hemoglobin pattern 2

• In heavily transfused patients, molecular testing may be required for accurate diagnosis 2

Impact on Other Laboratory Tests

• Hemoglobin S variant lowers A1C values by approximately 0.3% compared to individuals without the trait at the same glycemic level 1

• For diabetes screening or monitoring in patients with sickle cell trait, use an A1C assay without interference from hemoglobin variants (updated list available at www.ngsp.org/interf.asp) 2, 1

• In conditions with abnormal red cell turnover (pregnancy second/third trimester, recent blood loss, hemolysis), only blood glucose criteria should be used to diagnose diabetes, not A1C 2

Clinical Algorithm for Testing

For individuals without documented newborn screening:

1. Order hemoglobin electrophoresis directly as the initial test 1
2. Do not waste time with preliminary solubility testing 2, 1
3. Ensure complete blood count and reticulocyte count are obtained concurrently 2

For patients with positive results:

1. Document findings in the medical record 1
2. Provide genetic counseling, especially for reproductive-age individuals 2
3. Test partners of pregnant women or those planning pregnancy 1
4. Refer patients with sickle cell disease to specialist care 2