Unusual Hemoglobin Pattern in a Patient with Microcytosis

The alkaline hemoglobin electrophoresis shows a single major band in the HbA position and a minor band in the HbA2 position at 3.5%, which is within the normal range (2-3.5%). However, the HPLC shows an HbA2 of 5.5%, which is elevated and suggestive of beta-thalassemia trait. The discrepancy between the two methods may be due to the presence of a variant hemoglobin that co-elutes with HbA2 on HPLC or co-migrates with HbA on electrophoresis. The small HbF peak (1.5%) is mildly elevated, which can be seen in beta-thalassemia trait. The patient's microcytic anemia with normal iron studies (ferritin 50, transferrin saturation 20%) and normal RDW (14%) is consistent with thalassemia trait rather than iron deficiency (which typically has elevated RDW). The family history of mild anemia in the father supports a hereditary condition. The most likely diagnosis is beta-thalassemia trait. However, the normal HbA2 on electrophoresis is unusual; it could be due to a variant hemoglobin that comigrates with HbA, such as HbS or HbC in the heterozygous state, but the solubility test is negative for sickle cell. Another possibility is alpha-thalassemia trait, which usually has normal HbA2, but the HPLC HbA2 is elevated, which points away from alpha-thalassemia. The elevated HbA2 on HPLC may be a false elevation due to a variant hemoglobin that elutes in the HbA2 window, such as HbE, HbC, or HbS, but these would show additional bands on electrophoresis. The electrophoresis showed no other bands, so a variant that comigrates with HbA is possible. For example, Hb Knossos or Hb Lepore can comigrate with HbA and affect HbA2 measurement. Alternatively, the patient may have a beta-thalassemia trait with a variant that affects the HbA2 level. Further testing should include: (1) Repeat electrophoresis at acid pH to detect variants that comigrate with HbA at alkaline pH. (2) DNA analysis for beta-thalassemia mutations and alpha-thalassemia deletions. (3) Family studies. (4) HPLC with a different column or method. (5) Testing for Hb variants by mass spectrometry or sequencing. The most likely diagnosis is beta-thalassemia trait, but the possibility of a co-inherited hemoglobin variant should be investigated.

Further testing should include: (1) Acid hemoglobin electrophoresis or isoelectric focusing to detect variants that comigrate with HbA at alkaline pH, such as Hb Knossos, Hb Lepore, or Hb Hofu. (2) DNA testing for common beta-thalassemia mutations (e.g., IVS1-110, IVS1-6, codon 39) and for alpha-thalassemia deletions (e.g., -α3.7, -α4.2, --SEA, --MED). (3) Multiplex ligation-dependent probe amplification (MLPA) or gap-PCR for alpha-thalassemia deletions. (4) Sanger sequencing of the beta-globin gene if common mutations are not found. (5) Testing of family members to determine inheritance. (6) Genetic counseling for the patient and her partner, especially if she is planning pregnancy, because beta-thalassemia trait can lead to beta-thalassemia major if the partner is also a carrier. The patient should be informed of the risk and offered carrier testing for her partner. (7) If a variant hemoglobin is identified, further characterization may be needed. The results will guide management, which for beta-thalassemia trait is usually no treatment, but iron supplementation should be avoided unless iron deficiency is documented. The patient should be monitored for anemia and advised about the risk of iron overload if transfused.