Hemoglobin (Hb) Variant Analysis


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Subject: Hemoglobin (Hb) Variant Analysis

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  • Hb variant analysis is a separation process used to identify normal and abnormal forms of Hb. HbA is the main form of Hb in the normal adult. HbF (fetal) is the major Hb in the fetus, and the remainder is HbA2. Approximately 800 mutant forms of hemoglobin have been identified. Some are asymptomatic, especially in heterozygotes. Some may cause major morbid effects, especially in homozygotes. Globins found in different hemoglobins during fetal and adult life are indicated by Greek characters: α, β, γ, and δ. Variations in the amino acid composition of the globin chains cause the hemoglobinopathies.

  • Normal range: In healthy adults, 95–98% of the total Hb is HbA (α2 β2), 2–3% is HbA2 (α2 δ2), and 0.8–2.0% is fetal Hb (HbF) (α2 γ2). Note that the reference range is different in individuals younger than age 1. See Table 16.39.

TABLE 16–39
Normal Values for Hemoglobin Variants by Age


  • Evaluate a positive sickle cell screening test to differentiate sickle cell trait from sickle cell disease.

  • Once there is a high clinical suspicion and the preliminary hematologic and genetic information point toward a hemoglobinopathy, an investigation for definitive diagnosis of an abnormal Hb is warranted. Such a diagnosis will

    • Assist in the diagnosis of thalassemia, especially in patients with a family history positive for the disorder

    • Evaluate Coombs negative hemolytic anemia of unknown etiology

  • Measurement of HbA2 and HbF has great clinical value in the diagnosis as well as in characterization of some Hb structural variants and other hemoglobinopathies.

  • Increase in the HbA2 level is considered the most characteristic diagnostic feature of β-thalassemia trait and represents an essential test in the screening programs for β-thalassemia prevention.

  • Hemoglobin A2 prime is a delta chain variant Hb occurring in a small percentage of individuals of African ancestry. On HPLC, Hb A2 prime falls in the Hb S window, but its retention time differs from that of Hb S. When quantifying Hb A2 for the diagnosis of β-thalassemia heterozygosity, it is essential to add together the A2 and A2 prime to obtain a “total Hb A2.”

  • There are two methods in use to screen for Hb variants:

    • HPLC is used as the primary screening tool because it readily quantitates HbA, HbA2, and HbF; in addition, it presumptively identifies three of the most common Hb variants seen in North America: HbS, HbC, and HbD. All other abnormal variants will be flagged and need to be identified by hemoglobin electrophoresis (HE).

    • Alkaline and acid HE is used to investigate the whole array of Hb variants. A practical method for HE is cellulose acetate at alkaline pH. Hb molecules in an alkaline solution have a net negative charge and move toward the anode. The method separates HbA, HbA2, HbS, HbF, and HbC. Citrate agar gel electrophoresis at an acid pH separates hemoglobin variants that migrate together on cellulose acetate: HbS from HbD and HbG, HbC from HbE and HbO. Many of the hemoglobin forms that are difficult to differentiate by gel HE can be differentiated by HPLC. For instance, on gel electrophoresis, HbA2 is difficult to distinguish from HbC because they migrate together. Testing by HPLC allows the quantification of HbA2 in the presence of HbC. The two methods complement each other.

    • All Hb variants that are not diagnosed by these two methods require further testing with mass spectroscopy, capillary isoelectric focusing, or sequencing of DNA fragments generated by PCR.



  • HbA2: megaloblastic anemia, β-thalassemias

  • HbF: acquired aplastic anemia, hereditary persistence of fetal Hb, hyperthyroidism, leakage of fetal blood into maternal circulation, leukemia (acute or chronic), myeloproliferative neoplasms, sickle cell disease, thalassemias, β chain substitutions

  • HbC (second most common variant in the United States)

  • HbD (hemoglobinopathy that may also be found in combination with HbS or thalassemia)

  • HbE

  • HbS (sickle trait or disease)

Decreases In HbA2

  • α-Thalassemia

  • Erythroleukemia

  • Iron deficiency anemia (untreated)

  • Sideroblastic anemia


  • HbA2 and HbF levels should be considered in conjunction with family history plus laboratory data, including serum iron, TIBC, ferritin, red cell morphology, hemoglobin, Hct, and MCV.

  • Blood transfusions may temporarily obscure or dilute abnormal Hb.

  • When using HPLC, when HbA2 exceeds 10%, the presence of HbE or other Hb with similar resolution should be investigated.

  • Quantitation of hemoglobins is performed optimally after 1 year of age.

  • Hb Lepore arises from an unequal crossing-over and recombination event between adjacent δ and β globin genes. The resulting Hb has the mobility of HbS on alkaline electrophoresis and HbA on acid electrophoresis.

  • HbH is composed of a tetramer of normal β chains, resulting in a markedly reduced production of α chains. HbH has mobility much faster at alkaline pH than HbA. (HbH disease is a severe form of α-thalassemia with only one α chain.)