Background

Hemoglobin (Hb) is a tetrameric polypeptide composed of two α-globin and two β-like globin chains (ϵ, ^Gγ, ^Aγ, δ, or β). Hb is expressed in the erythrocytes and carries the oxygen from the lung to the tissues. Globin genes are differentially expressed throughout the development. For instance, during fetal life, the most abundant Hb is composed of two α- and two γ-globin chains (HbF, fetal Hb), while in human adults HbA, consisting of two α- and two β-globin chains, is predominantly expressed. Disorders caused by mutations in the β-like globin genes (β-hemoglobinopathies) represent the most frequent inherited diseases worldwide. In particular, β-hemoglobinopathies are caused by mutations that reduce adult β-globin production (β-thalassemia) or generate a mutant β-globin (sickle cell disease, SCD).

β-hemoglobinopathies are the most common monogenic disorders, therefore they represent ideal candidates for gene therapy approaches. A key parameter for determining the severity of the disease and the curative potential of treatment is the abundance of the different Hbs and globin chains. Indeed, techniques evaluating this parameter give insights into the degree of anemia (e.g., the α-/non-α-globin ratio, which is altered in β-thalassemia), highlight the presence of aberrant or uncoupled, toxic globins (e.g., α-globin aggregates in β-thalassemia) and evaluate the therapeutic expression of endogenous or exogenous β-like globins (e.g., fetal γ-globin, which is beneficial for both β-thalassemia and SCD).

Different methods can be used for the characterization and the quantification of Hbs and globin chains for the diagnosis and prognosis of Hb disorders, and for evaluating the therapeutic potential of gene therapy approaches.

Electrophoresis-based methods were firstly used to analyze Hb tetramers and globin chains. Erythroid cell lysates are commonly submitted to a cellulose-acetate electrophoresis (CEA) at alkaline pH. In this method, Hb tetramers migrate differently depending on their charge and are identified based on known Hb references (Schneider and Barwick, 1978; Wajcman, 2003).

More recently, capillary electrophoresis (CE) has been adapted to the analysis of Hb molecules. The separation of Hb molecules is based on their electrophoretic mobility at alkaline pH and is directed by pH and endosmosis.

Another conventional technique is the isoelectric focusing (IEF) that allows the separation of Hb molecules based on their different isoelectric point. Hb molecules migrate across a gradient until they reach a position where the net charge is zero. This method can distinguish some Hb variants that cannot be separated by CEA.

Finally, cation exchange-HPLC (CE-HPLC) can distinguish the different Hb molecules by chromatography using a salt gradient to elute them at a specific retention time.

All these methods give limited information on the amount of individual globin chains, in particular the γ-globin chains, ^Aγ and ^Gγ, which differ by a single amino acid. Moreover, in partially differentiated cell culture samples or chimeric models, these limitations are exacerbated by the presence of additional proteins or hybrid hemoglobin species. Finally, they often require a high amount of globin protein, which is difficult to obtain in in vitro generated RBCs or BFU-Es (Aprile et al., 2016).

Robust detection and relative quantification of individual globin chains can be achieved by reversed-phase HPLC (RP-HPLC), which separates proteins according to their hydrophobicity. RP-HPLC can be used for measuring the α-/non-α-globin ratio that is altered in β-thalassemia because of the reduced β-globin expression. Furthermore, this technique allows the measurement of both types of fetal γ-globin chains (^Aγ and ^Gγ). These globins are highly expressed in adult life in genetic conditions known as hereditary persistence of fetal hemoglobin (HPFH). Fetal γ-globin chain expression can also be induced by targeting known γ-globin silencers to alleviate the clinical severity of both β-thalassemia and SCD (Cavazzana et al., 2017). Importantly, this method can be applied to in vitro generated erythroid cells as it requires a relatively low amount of globin chains to evaluate the therapeutic potential of gene therapy approaches (Antoniani et al., 2018; Weber et al., 2020). As for the above-mentioned techniques, RP-HPLC does not allow an absolute quantification of globin chains.