Hemoglobin A₂ (HbA₂) has a measure of unreliability in diagnosing β-thalassemia trait (β-TT)

Abstract

Introduction: Detection of β-thalassemia trait or carriers (β-TT) depends significantly on an increase in Hemoglobin A₂ (HbA₂) levels, which is found at low levels (<3%) in normal healthy individuals and elevated levels (≥3.5%) in β-TT individuals. The study was designed to evaluate the reliability of the diagnostic parameter HbA₂ in the differentiation of β-TT and non-β-TT in Saudis.

Methods: The widely used high performance liquid chromatography (Variant II Bio-Rad) was used to measure HbA₂ levels in blood. Sanger sequencing was used to screen the variation in globin genes (HBB, HBD, HBA1, and HBA2). All the study subjects were divided into βTT and non-βTT (wild) categories based on the presence or absence of HBB variations and further sub-divided into false positive, true positive, false negative, and true negative, based on HbA₂ values.

Results: Out of 288 samples, 96 had HBB gene mutations. Of the 96 β-TT samples, sickle cell trait (SCT) samples (n = 58) were excluded, while the remaining (38 β-TT) were included in the detailed analysis: seven subjects with the HBB mutation had normal HbA₂ (<3%), and three were borderline (3.1–3.9%). The remainder (n = 28) had an elevated HbA₂ level (>4%). Based on HbA₂ analysis alone, both these groups would be incorrectly diagnosed as normal. Similarly, of the 189 non-β-TT samples, 179 had normal HbA₂, eight had borderline HbA₂, and two had a HbA₂ level above 4%. Based on HbA₂ analysis alone, borderline and >4% HbA₂ individuals, negative for β-TT, can be incorrectly diagnosed as carriers.

Conclusion: Given the percentage of samples falling in the HbA₂ “borderline” and “normal” categories, it can be concluded that HbA₂ has a measure of unreliability in the diagnosis of β-thalassemia carriers.

Keywords:

• β-Thalassemia carriers
• Pre-marital screening
• Molecular diagnosis
• Globin genes
• Hemoglobin A₂ (HbA₂)
• Borderline HbA₂
• β-Thalassemia trait
• Mutation

Transparency

Declaration of funding

This study was supported by The Deanship of Scientific Research, Imam Abdulrahman Bin Faisal University (Grant No: 2012186; 2014024; 2015292; 2016-077-IRMC), the King Abdulaziz City for Science and Technology (Grant No. LGP-35-204; LGP-32-3 and LGP-36-132), and the National Science Technology and Innovation Plan (Grant No: 12-MED-2798-46).

Declaration of financial/other relationships

The authors have no financial/other relationships to disclose. CMRO peer reviewers on this manuscript also have no relevant financial or other relationships to disclose.

Acknowledgments

We would like to thank Ranilo M. Tumbaga, Horace T. Pacifico, and Jee Entusiasamo Aquino for their assistance and technical expertise.