What laboratory tests are recommended to diagnose sickle cell disease or trait?

Laboratory Testing for Sickle Cell Disease and Trait

The diagnosis of sickle cell disease requires hemoglobin electrophoresis or high-performance liquid chromatography (HPLC) as the primary diagnostic test, with confirmation by a second method such as isoelectricfocusing, solubility testing, or citrate agar electrophoresis. 1

Newborn and Infant Screening

• All infants diagnosed by newborn screening (NBS) should be seen as soon as possible after birth to confirm diagnosis, with follow-up communication of confirmatory testing results when available 2
• Confirmatory testing must be performed after initial newborn screening to establish definitive diagnosis before initiating treatment 2
• A standard laboratory panel should be performed at 1 year of age (or prior to starting hydroxyurea) that includes:
  • Complete blood count with differential 2
  • Absolute reticulocyte count 2
  • Extended red cell phenotype (all patients) and genotype (preferred) 2

Primary Diagnostic Methods

First-Line Testing

• Hemoglobin electrophoresis remains a standard first-line test, though isoelectricfocusing provides superior resolution compared to conventional cellulose acetate electrophoresis at alkaline pH 1
• Cation exchange HPLC is increasingly used as the initial test in many laboratories and demonstrated 98.7% accuracy in identifying HbSS and HbSC in a large cohort 3
• Point-of-care lateral flow tests (such as Sickle SCAN™ and SICKLECHECK™) show 99% sensitivity and specificity for detecting hemoglobin A, S, and C, offering rapid bedside diagnosis without requiring electricity or advanced equipment 4, 5

Confirmatory Testing

• A second, more specific test must always be performed to confirm the presence of HbS 1
• Confirmatory options include:
  • Solubility testing (detects reduced sickle hemoglobin based on low solubility) 6
  • Electrophoresis on agar in citrate buffer 1
  • Isoelectricfocusing 1
  • Automated pyrosequencing for HBB gene mutations (particularly useful for differentiating Sβ⁰ thalassemia from HbSS) 3

Distinguishing Trait from Disease

Complete Blood Count Parameters

• Anemia is significantly more common in sickle cell disease compared to trait (P < 0.001) in children ≥3 months old, but lacks high sensitivity and specificity in children <4 years 7
• The combination of hemoglobin level (adjusted for age), RBC morphology, and reticulocyte count >2% approaches 100% sensitivity for differentiating disease from trait 7
• Absence of anemia alone does not exclude sickle cell disease in young children 7

Solubility Testing Limitations

• The sickle cell solubility test (SCST) is highly sensitive and specific for detecting HbS presence but cannot quantify HbS levels or distinguish between trait and disease 8
• SCST requires confirmatory testing for definitive diagnosis 8
• Intermediate results between trait and disease may occur in unusual cases with high fetal hemoglobin 6

Ongoing Monitoring in Established Disease

• Complete blood count with differential and absolute reticulocyte count should be performed 2-4 times per year in the first 2 years of life to establish baseline values 2
• These tests should be completed prior to first transfusion 2
• Additional laboratory evaluation should assess factors interacting with HbS, including HbF level, sickle cell restriction haplotype, and association with alpha-thalassemias 1

Critical Pitfalls to Avoid

• Never accept HbS diagnosis based on a single electrophoretic test alone—always confirm with a second method 1
• In children <4 years presenting with acute conditions and positive sickle screen, determine hemoglobin, RBC morphology, AND reticulocyte count—not just hemoglobin alone 7
• Hemoglobin electrophoresis may not easily differentiate Sβ⁰ thalassemia from HbSS; in these cases, HBB gene sequencing may be necessary 3
• The most frequent beta thalassemia mutations in Sβ⁰ and Sβ⁺ are HBB: c.118C>T (Gln40Stop) and HBB c.92+6T>C, respectively 3