What is the best test for diagnosing hereditary (inherited) spherocytosis?

Best Diagnostic Test for Hereditary Spherocytosis

The flow cytometric osmotic fragility test (FOFT) is the best diagnostic test for hereditary spherocytosis, with superior sensitivity (96.6%) and specificity (98.63%) compared to other available tests. 1

Diagnostic Options for Hereditary Spherocytosis

Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia. Several diagnostic tests are available:

Flow Cytometric Tests

1. Flow Cytometric Osmotic Fragility Test (FOFT)

   • Highest sensitivity (96.6%) and specificity (98.63%) 1
   • Measures percentage of residual red blood cells after exposure to hypotonic solution
   • Optimal cutoff for diagnosis: <25.6% residual red cells 1
   • Extended analysis time (300 seconds) further increases sensitivity to 90.0% 2

2. Eosin-5'-Maleimide (EMA) Binding Test

   • Directly targets the molecular defect in HS
   • Good sensitivity (86.2-93%) and specificity (93.9-98%) 1, 3
   • Optimal cutoff: >12.5% decrease in EMA fluorescence 1
   • Performance independent of clinical phenotype or molecular defect 3

Conventional Tests

1. Incubated Osmotic Fragility Test (IOFT)

   • Moderate sensitivity (79.31%) and specificity (87.67%) 1
   • Decreased sensitivity in compensated cases (64%) 3

2. Room Temperature Osmotic Fragility Test (RT-OFT)

   • Lower sensitivity (62.06%) and specificity (86.3%) 1
   • Even lower sensitivity in compensated cases (53%) 3

3. Acidified Glycerol Lysis Test

   • Good sensitivity (95%) 3
   • Less specific than EMA test

4. Cryohemolysis Test

   • Limited utility due to false positivity in iron deficiency anemia 4
   • Significantly worse performance (AUC 0.723) compared to EMA and FOFT 4

Optimal Diagnostic Strategy

The combination of FOFT and EMA binding test achieves 100% sensitivity with high specificity for diagnosing HS, including mild and compensated cases 1, 3. This combination is particularly valuable because:

1. They target different aspects of the disease:

   • FOFT measures membrane stability under osmotic stress
   • EMA binding directly assesses the molecular defect

2. They complement each other's limitations:

   • FOFT may have false positives in some hemolytic conditions
   • EMA may miss some rare variants of HS

Disease Severity Assessment

The Hemoglobin/Mean Corpuscular Hemoglobin Concentration (Hb/MCHC) ratio is the most useful parameter for assessing clinical severity of HS 4. Interestingly, neither the %EMA decrease nor the %residual red cells in FOFT correlate with disease severity 1.

Clinical Pitfalls and Considerations

1. False Positives:

   • Cryohemolysis test shows significant false positivity in iron deficiency anemia 4
   • Conventional osmotic fragility tests may be abnormal in other hemolytic conditions

2. Mild/Compensated Cases:

   • Conventional osmotic fragility tests have significantly reduced sensitivity in compensated HS 3
   • Flow cytometric tests maintain good sensitivity even in mild cases

3. Peripheral Blood Smear:

   • Combining FOFT with peripheral blood smear examination further improves diagnostic accuracy 5
   • Look for characteristic spherocytes, which appear as small, dense, round erythrocytes without central pallor

In summary, while multiple tests are available for diagnosing hereditary spherocytosis, the flow cytometric osmotic fragility test offers the best single-test performance, with EMA binding test as a close second. For optimal diagnostic accuracy, especially in mild or atypical cases, combining these two tests is recommended.