The term sickle cell anemia refers to a collection of autosomal recessive conditions with Mendelian transmission characterized by the presence of the HbS mutation, also in combination with other Hb variants. Mixed heterozygotes are defined as individuals who have one copy of HbS and one copy of another variant, for example, HbDPunjab, or β-thalassemia. They usually present modest clinical mani festations, especially those with β-thalassemia, but there are mixed forms leading to sickle cell syndrome-like conditions, thus with severe clinical manifestations. Carriers have a single copy of HbS and a normal β gene (HbAS) or a variant that does not cause sickling (e.g., HbS/Hb Korle Bu); even in carriers, the clinical manifestations are usually mild or absent. The condition is referred to as sickle cell trait. In sickle cell anemia (HbSS genotype), the affected individual has two copies of the variant Hb, and this determines the greater severity of the condition. In circulation, the main form of hemoglobin is HbS, with variable levels of HbF and normal levels of HbA2. This is a qualitative hemoglobinopathy caused by a single missense substitution (GAG → GTG) that results in the insertion of a valine instead of a glutamic acid at position 6 of the β globin chain. The structural consequence of the substitution is the propensity of the molecule to form polymers under conditions of reduced oxygen pressure with a characteristic elon gated and curved shape (sickle). This causes deformation of the erythrocytes that contain the polymerizing molecules, a phenomenon that occurs above all in the microcirculation, slowing down to the point of stopping the circulation (Fig. 1). The main clinical features are as follows:
• Reduced erythrocyte survival
• Vaso-occlusion phenomena, which occur at the level of the cerebral vessels can cause strokes
• Painful seizures, respiratory complications, and organ damage
• Anemia
• Episodic hemolytic seizures
• Blood sequestration in organs like the spleen, lungs, and liver
• Tendency to infections of varying severity
Fig1. Blood smear showing the presence of sickle cell erythrocytes
From a clinical point of view, it is also important to deter mine the haplotype of the patient affected by sickle cell anemia. Indeed, some haplotypes favor the contextual expression of HbF, whose presence within the erythrocyte has an inhibiting effect on the polymerization of HbS and, therefore, on the sickling, with a consequently better prognosis. There are five haplotypes of HbS, named according to the regions/ populations in which they have been observed (Senegal, Benin, Cameroon, Bantu, and Arab-Indians). The Senegal and Arab-Indian haplotypes favor a higher expression of HbF, while the Cameroon haplotype favors a low expression of HbF and is therefore associated with a less favorable prognosis than the previous one.
Double heterozygosity for other hemoglobin variants has different effects on sickling. For example, HbS/HbOArab and HbS/HbDPunjab combinations are sickling, while HbS/Hb Korle Bu delays sickling. The HbS/HbC combination tends to reduce sickling as HbC has a dehydrating effect on eryth rocytes and thus causes an increased relative concentration of Hb with MCHC, which can reach 40%, and at that point, sickling is observed.
In areas where there is a higher prevalence of hemoglobinopathy S, infant mortality is high, with a peak between 1 and 3 years, mainly due to infections. In the most developed areas, there is better survival, guaranteed by neonatal screening programs, early vaccinations, prophylaxis with oral penicillin, and, in general, better treatment of infections. In the United States, for example, the median life expectancy for individuals with HbS is 42 years for males and 48 years for females.
The laboratory diagnosis of HbS is based first on the identification of the carrier (HbA/HbS), usually silent, by the following:
• Separation techniques:
– Cellulose acetate electrophoresis at alkaline pH and at acid pH
– Capillary electrophoresis
– Isoelectrophoresis, if other hemoglobin fractions are present
– HPLC
• Confirmatory function tests:
– Sick cell test
– Solubility test for HbS
• Identification of the mutation by molecular biology tests
During electrophoresis at pH 8.5, HbS migrates more slowly than HbA (Fig. 2). Noteworthy, other Hbs also migrate to the same position, such as HbDPunjab, which can coexist in heterozygosis with HbS (see above).
Fig2. Electrophoresis at alkaline pH of Hb lysates belonging to samples from subjects with HbAA (left), HbAS (center), and HbSS (left). (Copyright EDISES 2021. Reproduced with permission)
Electrophoresis at an acidic pH (5.2) in agarose gels can provide additional diagnostic criteria to identify HbS and differentiate it from other hemoglobins (Fig. 3).
Fig3. Electrophoresis at acid pH of lysates from samples with different hemoglobin variants
Isoelectrophoresis can better define the different fractions of hemoglobin (Fig. 4).
Fig4. Isoelectrophoresis pattern at pH range of 6.0–9.0 for lysates from samples with different hemoglobin variants
The use of HPLC is favored by the fact that it is an auto mated method for measuring HbA1c, making it widespread in laboratories and useful for separating and quantifying the remaining hemoglobin fractions as well. Presumptive identification of variants is made based on retention time (Fig. 5).
Fig5. HPLC pattern of lysate from sample with HbA/HbS genotype
The in vitro sickle cell test is performed by placing a drop of the patient’s blood on a slide and covering it with a cover slip. In a short time, the lowering of the oxygen level in the preparation causes the appearance of sickles visible under the microscope (Fig. 1). The test is made more rapid and sensitive by adding a small amount of sodium dithionite or tetrathionate powder.
The solubility test for HbS is done by adding a drop of blood to phosphate buffer pH 6.8–2.8 mol/L, deoxygenated with Na dithionite. A precipitate is observed in the presence of HbS alone (Fig. 6). The test is specific but does not distinguish heterozygous from homozygous status.
Fig6. Solubility test for HbS. Evident turbidity in the sample on the right. (1) Normal and (2) sample containing HbS
Molecular biology tests involve amplification by polymerase chain reaction (PCR) of the DNA tract with the first exon of the β-chain, the use of restriction enzymes, electrophoresis, and restriction fragment length polymorphism (RFLP) analysis. The A → T mutation eliminates a restriction site for various enzymes (e.g., MstII). Note that the method is not specific to HbS, as there may be other site mutations. PCR-ARMS (amplification refractory mutation system methods with allele-specific probes can also be used.
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