Haemoglobin is an iron-containing protein that transports oxygen. There are two parts to this protein: 1) the heme component which consists of the iron and the globin component which consists of the globin protein. The heme groups were identical between HbA and HbS suggesting that the differences should be in the globin domain of the protein. Vernon Ingram and J.A. Hunt sequenced the sickled haemoglobin in 1956 to show that a single mutation in the protein causes Sickle cell disease.
Diagnosis of Sickle Cell Disease
- Sickle Solubility Test: Used to screen for the presence of Haemoglobin S in the blood. A chemical is added to the blood that reduces the amount of oxygen present in it. Individuals older than 6 months who are carriers of the trait will have various amounts of haemoglobin S present, depending of the severity of the disease in the affected person. The lack of oxygen in the blood will cause the red blood cells affected by haemoglobin S to sickle and form S-related polymers.
- Haemoglobinopathy: This method of testing involves a variety of tests used for screening, diagnosis, and confirmation of the disease. It subjects blood samples taken from the patient to a number of tests that isolate proteins in the blood based on their molecular properties. Blood proteins affected by sickle cell disease will have different properties than healthy blood proteins, and can therefore be isolated.
- Haemoglobin Electrophoresis: Used to isolate haemoglobin S and SC. It uses citrus agar gel and cellulose acetate to expose the patient’s blood to an electric field. This field will separate various blood proteins from each other on a gel based on their electric charges. Since the electric properties of these proteins are known, one can look for the presence of proteins at specific areas of the gel to confirm the presence of haemoglobin S and SC.
- Isoelectric Focussing: This method is more sensitive than electrophoresis. At birth, infants have large amounts of haemoglobin F. Those with the sickle cell trait find that their haemoglobin S levels rise as their haemoglobin F levels fall. The levels of these two variations of haemoglobin stabilize around the age of 2. Adult sickle cell carriers continue to produce regular haemoglobin A, while those with sickle cell anemia produce little to none of this haemoglobin variant. Each of these variants of haemoglobin have specific properties that allow them to be separated by isoelectric focussing. This method again requires the patient’s blood sample to be subjected to an electric field of a gel where the haemoglobin variants are separated by their electric charge. It is similar to electrophoresis but differs in the fact that the variants only migrate through a gel to a certain point (the isoelectric point, where the charge through the gel at that point equals the charge of the protein) rather than allowing the variants to arbitrarily migrate across the gel.
- High Performance Liquid Chromatography: This method is also more sensitive than electrophoresis. It is a chromatographic technique that separates haemoglobin variants according to their unique characteristics and properties. One method, called fractionation, involves changing the conditions of the blood plasma so that proteins that are usually soluble in the liquid become insoluble and are precipitated out in clumps. This technique can be used to precipitate haemoglobin variants out of the blood plasma.