Taiwo R. Kotila
Department of Haematology, University of Ibadan, Nigeria
The haemoglobinopathies are prevalent genetic disorders in Nigeria, it is therefore imperative that diagnosis is made accurately and promptly so that affected individuals can seek timely medical help. There is no structured modality for the investigation of the haemoglobinopathies in the country. Haemoglobin electrophoresis at alkaline pH is solely relied upon by both the secondary and tertiary health centres in the diagnosis of the haemoglobinopathies. It is also necessary to know and understand the various diagnostic tests available for this genetic disorder and to interpret the results correctly. This review discusses the different diagnostic tests appropriate for the different levels of health care in the country.
Dr. T.R. Kotila
Department of Haematology,
University College Hospital,
PMB 5116, Ibadan, Nigeria
The haemoglobinopathies are a group of inherited disorders of haemoglobin synthesis and are divided into five broad groups which include, structural (qualitative), quantitative (Thalassaemias), unstable haemoglobins, haemoglobins with low oxygen affinity and the M haemoglobins which are associated with familial cyanosis while haemoglobins with high oxygen affinity are associated with erythrocytosis (polycythaemia). Accurate diagnosis of the haemoglobinopathies is necessary in this environment not because it is a prevalent condition but also because even though there are over 400 haemoglobin variants not all are symptomatic. Most haemoglobinopathies are due to one or two amino acid substitutions thus affecting the mobility of the haemoglobin on electrophoresis. This aids easy diagnosis of these disorders. There are a number of clinically significant haemoglobins which do not alter the overall charge of the protein so are detected by other methods other than electrophoresis, e.g. isoelectric focusing, high- performance liquid chromatography (HPLC), and immunologic techniques.
Sickle cell disease (SCD) is a haemoglobinopathy with structural defect and it is very common in Nigeria. The prevalence of homozygous SCD (HbSS) and HbS+C disease is 3.1% and 1.1% respectively thus giving a combined prevalence of 4.1% while the trait (HbAS) has a prevalence of 23.7%1. This high prevalence requires that accurate diagnosis of the disorder is made, not only at the tertiary hospitals but also at the secondary level while primary health centres should be able to screen patients for the disorder.
The thalassaemias are quantitative disorders of haemoglobin synthesis which are classified into two; the alpha and the beta thalassaemias. The single deletion alpha thalassaemia which is common2 in this environment is the mild type but beta thalassaemia which is of more clinical significance was initially thought to be of low prevalence3 but it is now known to be of high prevalence4,5. There is therefore the need to be able to differentiate these various haemoglobinopathies and treat the patients’ accordingly. Accurate diagnosis will also prevent subjecting the patients to unnecessary investigations.
The objective of this write up is to provide a guideline for the screening and diagnosis of the haemoglobinopathies in the Nigerian setting and to delineate what tests are appropriate for the different level of health care.
Patients who present with unexplained anaemia should be screened for haemoglobinopathy and it should be mandatory to rule out a haemoglobinopathy especially in patients presenting with moderate or severe anaemia in the presence of jaundice5. Patients noticed to have microcytosis and hypochromia on blood film review, should be investigated for thalassaemia more so if a trial of iron therapy does not correct this blood film appearance. The nation is however ripe for newborn screening of these prevalent disorders; this will help in reducing the morbidity and mortality associated with them.
Microscopy: A review of the peripheral blood film is a good initial screening test, the presence of target cells and sickled erythrocyte or microcytic cell is a pointer to sickle cell disease or thalassaemia respectively.
Sickling test: This is a simple screening test that can be done especially in a primary health care facility. A drop of blood is mixed with two drops of 2% sodium metabisulphite, this is then covered with a coverslip and sealed with petroleum jelly. The slide is then examined under the microscope after 15 minutes, one hour and 24 hours6. The presence of sickled erythrocytes in a patient with symptoms of sickle cell disease is suggestive of the disorder. False positive results may occur if sodium metabisulphite of greater than 2% is used while false negative results may occur if the metabisulphite is stale and also in the neonatal period because of the low percentage of HbS.
Solubility test: This is based on the fact that HbS is less soluble than HbA in the deoxygenated state. One drop of the patient’s blood is mixed with a solution prepared using potassium dihydrogen phosphate, dipotassium hydrogen phosphate and sodium metabisulphite. The mixture is spun in a centrifuge; HbS if present, precipitates as a red opaque band on the surface of the test solution while other haemoglobins remain in solution6. Control blood samples with HbA, HbAS and HbS should be tested at the same time. There are commercial test kits now available for this procedure. Solubility test should be performed on all samples which showed the “S” band on alkaline electrophoresis especially in our environment in which haemoglobins G and D which comigrate with HbS on alkaline electrophoresis is present.
In an alkaline buffer, HbA is a negatively charged protein which moves toward the positive electrode (anode) in an electric field. Most haemoglobin variants are separated from HbA during electrophoresis because the structural abnormality usually involves changes in electrical charge while haemoglobin without a change in charge will not separate. The pH and medium used are varied in order to identify the different haemoglobin variants; electrophoresis for haemoglobin identification is commonly run with cellulose acetate paper at alkaline pH and/or citrate agar at acidic pH.
Cellulose acetate electrophoresis at alkaline pH: Cellulose acetate paper as support medium and at a pH of 8.4- 8.9 has the advantage of rapid separation of haemoglobins, ease of handling and minimal preparative work. Its disadvantages are that haemoglobin fractions in low concentration like HbA2, HbH and Hb Bart’s may be missed. Also when small haemoglobin fractions occur near a broad band, the minor fraction may be missed especially in neonates when high levels of HbF can cause the presence of either HbA or HbS to be missed. Certain haemoglobins which comigrate at this pH include HbS, HbD and HbG, at this pH HbA2, HbC, HbE and HbO also comigrate.
Citrate agar electrophoresis at acidic pH: Separation of haemoglobin variants by this method is determined by the relative affinity for agaropectin by certain haemoglobin surface groups. It is performed at a pH of 6.0- 6.2 and it delineates haemoglobins that comigrate at alkaline pH and other cases that cannot be resolved at alkaline pH. It is particularly useful in diagnosing sickle cell disease at birth when the high concentration of HbF may pose a problem. It is however technically difficult to handle. Haemoglobins A,D,E,G, H all comigrate at this pH.
Isoeletric focusing: This is a high resolution method for separating proteins according to their isoeletric points, i.e. points at which the net charge is zero. Separation of haemoglobins by this method is similar to that obtained by electrophoresis on cellulose acetate except that the bands are sharper and the resolution of some haemoglobins are better.