Diagnosis of Hemoglobinopathies
Primary Diagnostic Method
Use high-performance liquid chromatography (HPLC) or capillary electrophoresis as your primary diagnostic method, with hemoglobin electrophoresis at alkaline pH (pH 8.6) as the initial screening tool, followed by confirmatory testing with acid electrophoresis (pH 7.1 or citrate agar) and genetic sequencing when variants are detected. 1
Initial Screening Algorithm
Step 1: Complete Blood Count and Red Cell Indices
- Obtain CBC with specific attention to mean cellular hemoglobin (MCH) 2
- Test for beta-thalassemia trait when MCH is <27 pg 2
- Consider alpha-thalassemia trait testing when MCH is <25 pg 2
- Evaluate reticulocyte count to assess bone marrow response to hemolysis 1
- Assess red blood cell morphology for microcytosis and hypochromia patterns, particularly in patients from appropriate ethnic groups to prevent unnecessary gastrointestinal investigations 1, 3
Step 2: Primary Screening Test
- Perform HPLC (cation-exchange), which is now the gold standard for both screening and quantification, separating hemoglobin variants based on charge differences 1
- Alternatively, use hemoglobin electrophoresis at alkaline pH (pH 8.6) as initial screening 1, 4
- HPLC quantifies HbF, HbA2, and variant hemoglobins with high precision and can separate more than 45 commonly encountered hemoglobin variants within 12 minutes 1, 5
Confirmatory Testing Protocol
Step 3: Confirm Variant Identity
- Perform acid electrophoresis (pH 7.1 or citrate agar) to confirm variant identity, as different hemoglobins may co-migrate at alkaline pH 1, 4
- Never rely on electrophoresis alone for variant identification, as some variants are electroforetically silent 1, 3
Step 4: Genetic Testing When Indicated
- Conduct targeted DNA sequencing of α- and β-globin genes for definitive identification, particularly for HbM variants or electrophoretically silent variants 6, 1
- For HbM variants specifically, DNA sequencing is technically simpler, more widely available, and more specific than electrophoresis alone 6, 3
Special Testing for Methemoglobinemia
When to Suspect HbM Variants
- Cyanosis present at birth suggests α-globin variants 6
- Cyanosis appearing at 6-9 months suggests β-globin variants 6
- Long-life history of cyanosis or family history of dusky skin or blue sclera suggests congenital forms 6
Specific Tests for Methemoglobinemia
- Measure methemoglobin levels using spectrophotometric method (Evelyn-Malloy) when HbM variants are suspected (normal <1%) 6, 1
- Measure CYB5R enzyme activity using ferricyanide as receptor (residual activity <20% of normal confirms congenital deficiency) 6, 1
- Express methemoglobin as percentage (MetHb concentration ÷ total Hb concentration), not absolute concentration 1
- Cyanosis appears when MetHb >1.5 g/dL 1
Additional Laboratory Parameters
Hemolysis Markers
- Measure bilirubin (total and direct) as markers of hemolysis 1
- Assess iron status before interpretation, as iron deficiency can confound results 7
Quality Control Requirements
- Comprehensive hemoglobinopathy screen using HPLC, capillary electrophoresis, mass spectrometry, or gel electrophoresis should be confirmed by a second test 1
- Intralaboratory CV should be <1.5% for HbA1c measurement 1
- Interlaboratory CV should be <2.5% for HbA1c measurement 1
- Use at least 2 control samples with different HbA1c levels for quality assurance 1
- Whole blood samples are stable for up to 1 week at 4°C for most methods 1
Critical Pitfalls to Avoid
Testing Errors
- Do not use sickle solubility test in isolation for diagnosis 1
- Never interpret HbA1c in patients with hemoglobinopathies unless using assays without interference from abnormal hemoglobins (check www.ngsp.org/interf.asp for updated interference list) 6, 1
- For patients with abnormal hemoglobin but normal red cell turnover (such as sickle cell trait), use an A1C assay without interference from abnormal hemoglobins 6
- In conditions with increased red cell turnover (pregnancy second and third trimesters, recent blood loss or transfusion, erythropoietin therapy, or hemolysis), only blood glucose criteria should be used to diagnose diabetes—never A1C 6
Clinical Management Errors
- Do not skip family studies when hemoglobinopathy is identified, as this is crucial for genetic counseling 1, 3
- Never interpret results without knowing baseline values in patients with known sickle cell disease, as many have chronic abnormalities 1
- Do not administer medicinal iron unless iron deficiency is biochemically proven, due to potential risk of lifetime iron overload from repeated red blood cell transfusions 6