What test(s) can be done to satisfy regulatory requirements for abnormal hemoglobin results?

Confirmatory Testing for Abnormal Hemoglobin Results

When an abnormal hemoglobin result is detected, laboratories should verify the finding by repeat testing using a different analytical method, such as high-performance liquid chromatography (HPLC), capillary electrophoresis, or molecular genetic testing. 1

Primary Confirmatory Tests

Repeat Testing with Different Methodology

The primary approach to confirming abnormal hemoglobin results involves:

1. Repeat testing of the same specimen - This is mandatory for:

   • Results below the lower limit of the reference interval
   • Results greater than 15% (140 mmol/mol) HbA1c
   • Any result that doesn't correlate with clinical impression 1

2. Use of a different analytical principle - The repeat measurement should use a method based on a different analytical principle than the initial assay 1. Options include:

   • If initial test was charge-based (cation-exchange chromatography, electrophoresis), use a structural-based method
   • If initial test was structural-based (affinity chromatography, immunoassay), use a charge-based method 1

Specific Confirmatory Methods

• Boronate affinity chromatography - Particularly useful when hemoglobin variants are suspected, as this method is less affected by hemoglobin variants than ion-exchange methods 1

• Capillary electrophoresis - Provides excellent resolution for detecting various hemoglobin variants 2

• HPLC with different column chemistry - Using a different column chemistry can help confirm the presence of variants 3

Secondary Confirmatory Tests

If the primary confirmatory test still shows abnormal results, additional testing may include:

• Comparison with other glycated protein assays - Testing fructosamine or glycated albumin can provide supporting evidence when HbA1c results are questionable 1

• Molecular genetic testing - For definitive identification of hemoglobin variants:

  • PCR-based methods like Amplification Refractory Mutation System (ARMS)
  • DNA sequencing for specific mutations 4

When to Suspect Hemoglobin Variants

Suspect a hemoglobin variant when:

• HbA1c reading is surprisingly high or low
• HbA1c result changes significantly when laboratory method changes
• Patient has known hemoglobinopathy or family history of one
• Patient belongs to an ethnic group with higher prevalence of hemoglobinopathies 1

Algorithm for Confirmatory Testing

1. Initial abnormal result detected
2. Repeat test using same method to rule out technical error
3. If confirmed abnormal, test using different analytical principle
4. If still abnormal:
   • Compare with clinical picture and glucose measurements
   • Consider additional testing (fructosamine, glycated albumin)
   • Consider referral for molecular genetic testing if hemoglobinopathy suspected

Common Pitfalls to Avoid

• Relying on a single method - Different methods have different interferences with hemoglobin variants 1

• Ignoring clinical correlation - Always compare HbA1c results with clinical impression and other glucose measurements 1

• Overlooking concurrent conditions - Conditions affecting red cell turnover (anemia, blood loss, hemolysis) can affect HbA1c independent of the testing method 1

• Missing nutritional deficiencies - Iron deficiency can decrease HbA1c while megaloblastic anemia can increase it, potentially confounding results 5

By following this approach, laboratories can satisfy regulatory requirements for confirming abnormal hemoglobin results while ensuring accurate patient diagnosis and appropriate clinical management.