What is the recommended approach for quantifying hemoglobinopathies, including laboratory tests and management strategies?

Quantification in Hemoglobinopathies

Primary Diagnostic Approach

For quantifying hemoglobinopathies, use high-performance liquid chromatography (HPLC) or capillary electrophoresis as the primary 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, 2, 3

Initial Screening Methods

First-Line Testing

• Hemoglobin electrophoresis at alkaline pH (pH 8.6) serves as the initial screening technique to detect abnormal hemoglobin bands compared with reference samples 2, 4
• Isoelectric focusing (IEF) offers superior resolution and may be preferred over standard electrophoresis for detecting variants 5, 4
• HPLC (cation-exchange) is now considered the gold standard for both screening and quantification, as it separates hemoglobin variants based on charge differences 6, 3, 5
• Capillary zone electrophoresis (CZE) provides an alternative high-resolution method for variant detection 3

Quantification Methods

• HPLC or microcolumn chromatography should be used to quantify HbA2 levels (critical for beta-thalassemia trait diagnosis when MCH <27 pg) 5
• HPLC quantifies HbF, HbA2, and variant hemoglobins with high precision 6, 2
• For HbA1c measurement specifically, laboratories must use only methods certified by the National Glycohemoglobin Standardization Program (NGSP) as traceable to the DCCT reference 6

Confirmatory Testing Algorithm

When Abnormal Pattern Detected

1. Perform acid electrophoresis (pH 7.1 or citrate agar) to confirm variant identity, as different hemoglobins may co-migrate at alkaline pH 1, 2, 4
2. Conduct targeted DNA sequencing of α- and β-globin genes for definitive identification, particularly for HbM variants or electrophoretically silent variants 6, 1
3. Measure methemoglobin levels using spectrophotometric method (Evelyn-Malloy) when HbM variants are suspected (normal <1%) 6

Specialized Functional Tests

• Oxygen affinity testing (p50 determination) when high or low oxygen affinity variants are suspected based on phenotype 2
• Stability testing (isopropanol or heat stability tests, Heinz body preparation) for suspected unstable hemoglobin variants 2
• Solubility testing for HbS confirmation, but never use as sole diagnostic method as it cannot differentiate heterozygous from homozygous states 7

Critical Laboratory Parameters

Complete Blood Count Interpretation

• Test for beta-thalassemia trait when MCH <27 pg in all ethnic groups 5
• Consider alpha-thalassemia trait testing when MCH <25 pg in selected ethnic groups (exclude Northern European Caucasians) 5
• Evaluate reticulocyte count to assess bone marrow response to hemolysis 7, 2
• Assess red blood cell morphology for microcytosis and hypocromia patterns 1, 2

Supporting Laboratory Tests

• Iron status assessment must be completed before interpreting hemoglobin patterns, as iron deficiency can mask thalassemia 4
• Bilirubin (total and direct) as markers of hemolysis 7
• Hemoglobin electrophoresis should be performed in absence of recent blood transfusion to avoid misinterpretation 4

Genetic Confirmation Methods

When Phenotypic Methods Are Insufficient

• PCR-based methods for detecting common deletions and mutations 2, 3
• DNA sequencing of genomic DNA for unknown variants 2, 3
• RT-PCR with cDNA sequencing for comprehensive globin gene analysis 2, 3
• DNA microarray technology for high-throughput screening 3
• Mass spectrometry for definitive structural characterization 3

Special Populations and Circumstances

Neonatal Screening

• Comprehensive hemoglobinopathy screen using HPLC, capillary electrophoresis, mass spectrometry, or gel electrophoresis should be confirmed by a second test 7
• Note that solubility tests can give false-negative results in neonates 7

Genetic Counseling Requirements

The screening must identify: 5

• Hemoglobins S, C, D-Punjab, O-Arab, E, Lepore, and H
• Carriers of α⁰-thalassemia and β-thalassemia
• All ethnic groups except Northern European Caucasians should be screened for variant hemoglobins

Methemoglobinemia-Specific Quantification

• Measure CYB5R enzyme activity using ferricyanide as receptor (residual activity <20% of normal confirms congenital deficiency) 6
• Express methemoglobin as percentage (MetHb concentration ÷ total Hb concentration), not absolute concentration 6
• Cyanosis appears when MetHb >1.5 g/dL 6

Common Pitfalls to Avoid

• Never rely on electrophoresis alone for variant identification, as some variants are electroforetically silent 6, 1
• Do not use sickle solubility test in isolation for diagnosis 7
• Avoid interpreting HbA1c in patients with hemoglobinopathies unless using assays without interference from abnormal hemoglobins (check www.ngsp.org/interf.asp for updated interference list) 6
• Do not skip family studies when hemoglobinopathy is identified, as this is crucial for genetic counseling 1
• Never interpret results without knowing baseline values in patients with known sickle cell disease, as many have chronic abnormalities 7
• Ensure iron deficiency is excluded or corrected before final interpretation, as it can confound thalassemia diagnosis 4

Quality Control Standards

• Intralaboratory CV should be <1.5% for HbA1c measurement 6
• Interlaboratory CV should be <2.5% for HbA1c measurement 6
• Use at least 2 control samples with different HbA1c levels for quality assurance 6
• Whole blood samples are stable for up to 1 week at 4°C for most methods 6