Screening methods based on the measurement of erythrocyte osmotic resistance in progressively hypoosmotic saline solutions have been abandoned as they are nonspecific and have poor sensitivity. In some less equipped realities, techniques based on the hemolysis time in isosmotic glycerol solutions, the so-called GLT50, and its commercial variants (Green test, Osmored A, and others), are still used.
The diagnostic process for thalassemia suspicion is based on the use of hematological methods, such as the CBC and the reticulocyte count, electrophoretic methods, and HPLC (with the use of dedicated instruments, which are today more common than electrophoretic systems), with eventual mass spectrometry in particular cases.
In some situations, the use of molecular biology techniques based on nucleic acids is appropriate. In the α-thalassemic trait, the erythrocyte indices show alterations characteristic of microcytic anemia, that is, decreased total hemoglobin, MCH <80 fL, erythrocyte count mostly increased, normal or little increased reticulocytes, and normal or decreased HbF. Indeed, there is no HbA2 marker as in the case of the β-thalassemia trait because it is also affected by α gene deletion. Moreover, the observable effects depend on the number of nonfunctioning genes and on the techniques used. For example, the presence of HbH (or rarely of Hb Bart) can already be evident in sub jects with two deletions when sensitive methods like HPLC, and eventually mass spectrometry, are used, which is more difficult with electrophoresis. HbH is instead well evident on electrophoresis and HPLC in subjects with three nonfunctioning genes, together with HbH intraerythrocytic inclusion bodies.
In the β-thalassemic trait, the erythrocyte indices showing variations are as follows:
• Decreased hemoglobin (9–10 g/L)
• Decreased MCH (5.0 T/L)
• Normal or slightly increased RDW, in contrast to sideropenic anemia
• Normal or increased HbF
• Increased HbA2 due to increased relative to HbA
• Slightly increased reticulocytes
Stained smears may highlight morphological features, such as microcytes, target cells, F cells (rich in HbF), and included bodies. These elements are not determinative per se but contribute to the diagnosis.
Electrophoresis at alkaline pH shows the relative increase in HbA2 and, where increased, HbF.
HPLC shows the relative increase in HbA2 and, where increased, HbF.
HPFH and δβ-thalassemia both present high HbF values. The distinction between the two forms lies in the distribution of intraerythrocytic HbF, homogeneously diffused in HPFH and concentrated in erythrocytes particularly rich in HbF (F cells) in δβ-thalassemia. The microscopic observation after staining with Kleihauer’s method or a more recent method with specific antibodies in flow cytometry can provide differential diagnostic elements.
