The sickle cell disorder (SCD) is the most common inherited blood disorder responsible for about 70% of the world’s blood disorders. Haemoglobin (Hb), a protein present in red blood cells (RBCs) which is responsible for carrying oxygen from the lungs to the tissues and organs in the body as well as transporting carbon dioxide back to the lungs,is so vital for life. However, not all haemoglobins are the same in the human body. This is because the formation of haemoglobins consists of a sequence of steps which can be altered in different ways as a result of mutation causing numerous changes in the structure of haemoglobins. There are two categories of haemoglobins: the usual or normal haemoglobins and the variant or unusual haemoglobins.
Usual or Normal Haemoglobins
Usual or normal haemoglobins are Haemoglobin A, (Hb A), Haemoglobin A2, (Hb A2) andFetal Haemoglobin, (Hb F).
Some Variant oOr Unusual Haemoglobins
Variant or unusual haemoglobins are mutants caused by variation in genetics. Some well-known haemoglobin variants are sickle cell haemoglobin,(Hb S), Haemoglobin C,(Hb C), Haemoglobin E,(Hb E), Haemoglobin O,(Hb O), Haemoglobin D,(Hb D), Haemoglobin G,(Hb G), with hybrids in some cases. It is worth nothing that thousands of other variants also exist.
When children are born, they inherit haemoglobin pairs or formations from both parents. These haemoglobin formations are known as haemoglobin genotypes. Genotype refers to the genetic makeup (set of genes) of a living organism. The genotype of a person, therefore, determines a person’s physical appearance, development, and behavior. These physical characteristics which we observe such as a person’s body shape, height, colour of eyes, sound of voice or type of disease are known as phenotypes. Some of these observable characteristics (phenotypes) such as blood cells can also be measured in the laboratory.
Examples of common clinical genotypes found in our environment are:
- Homozygous (same) forms: Hb AA, Hb SS (SCD), Hb CC and Compound Heterozygous (different) forms: Hb
AS (sickle cell trait), Hb AC, Hb AG, Hb AD, Hb AO, H6 SC (SCD), Hb SD (SCD), Hb SO (SCD), Hb Sβthal trait (β+/o) (SCD), Hb SG, Hb Aβthal trait.
Homozygousgenotypes, that is genotypes which have identical or the same forms: Hb AA, Hb SS (SCD), Hb CC and
Compound Heterozygous genotypes, that is genotypes which have different forms : Hb AS (sickle cell trait), Hb AC, Hb AG, Hb AD, Hb AO, Hb SC (SCD), Hb SD (SCD), Hb SO (SCD), Hb Sβthal trait (β+/o) (SCD), Hb SG, Hb Aβthal trait.
The Hb SS genotype is what results in the sickle cell disease (phenotype) which we observe in a person who is affected by SCD.
Nigeria is the country with the largest burden of SCD owing to the high frequency of the sickle cell trait (Hb AS) at 23.7% which is 20 per 1,000 births resulting in about 150,000 babies being born annually with SCD; 4.1% of the country’s population is affected. The role of laboratory medicine is therefore imperative and often decisive in contributing to the diagnosis of these usual and unusual haemoglobins. Laboratory analysis which ensures consistent test results of haemoglobin types is critical to ameliorating the burden of sickle cell disease in Nigeria. As effective members of the health care team, medical laboratory scientists are tasked with the vital role of preventing misdiagnosis of genotypes by adhering to standardised guidelines which employ a systematic approach and methods to arrive at a definitive laboratory diagnosis of the various genotypes/phenotypes.
These guidelines are as follows:
Clinical information: Collecting relevant information about the patient is essential and will assist with the investigation. Some of the important information required include the date of birth/age of patient; recent blood transfusion history in which degenerated red cells are removed from the circulation by the reticuloendothelial system (a network of different cells of the body that get rid of dead or abnormal cells, tissues and foreign substances) at the end of their life span which is about 120 days; relevant drug therapy; pregnancy status; family history with details of propositus, that is a person affected with a disorder who is the primary or index subject in a study or investigation as of a genetic character in a family lineage and so on.
- Unfortunately, and to a large extent, the information provided on the patient’s medical history prior to an investigation is often inadequate and this increases the likelihood of an incorrect diagnosis.
1.2. Sample Required: Avenous pre-transfused blood sample has to be collected and put in an EDTA anticoagulant sample tube. Using a post transfused (less than 90-120 days) blood sample is inappropriate.
2.3. Blood Count: Apart from neonatal screening, a blood count: Hb value, RBC count and its indices (MCV, MCH) are needed. MCV stands for Mean Corpuscular Volume and this test measures the average size of the red blood cells while MCH stands for Mean Corpuscular Haemoglobin and this refers to the average quantity of haemoglobin present in a single red blood cell. This is essential particularly in the investigation of suspected thalassaemia. Many studies have revealed that MCH less than 27fl with MCV less than 78pg increases the risk of thalassemia. Red cell indices are reliable and appropriate in the laboratory screening for thalassaemia. In patients with the Beta-thalassaemia trait, MCV and MCH values are notably reduced compared to people with Hb SS (Microcytic, hypochromic anaemia is essential to the diagnosis of β-thalassaemia trait with MCV and Hb A2 being important diagnostic tools).
3.4. Sickling Test: It is a simple screening test. It requires only one reagent, a reducing agent (RA). It is not specific. The reagent must be freshly prepared 2% W/V sodium metabisulphite or sodium dithionite solution.
4.5. Sickle Solubility Test: This test can be used to distinguish sickle cell anaemia (HbSS) from HbSDsickle cell disease in geographic regions where both HbS and HbD occur.It gives information about the different forms of the sickle cell disorder. Blood is mixed in a phosphate buffer-saponin solution containing sodium dithionite as the reducing agent. In its reduced form, HbS is insoluble. HbSS is indicated by red precipitate (an insoluble solid form), other forms of haemoglobin e.g. HbD are soluble when in a reduced state.
5.6. Haemoglobin Electrophoresis: Haemoglobin electrophoresis is utilised to separate and recognise the different haemoglobins by their movement within an electric field. A number of techniques are available to separate haemoglobin types by electrophoresis. Electrophoresis in alkaline buffer at pH8. 4-8.6 using cellulose acetate membrane is adequate for routine analysis. It gives good separation of HbA, HbF, HbS, and HbC. Hb S, Hb D, and Hb G have the same mobility. Hb C, Hb A2, Hb E, Hb O and hybrid of Hb G also have the same migration.
In specialist laboratories, agarose gel and citrate acid electrophoresis can also be used.
Hbphenotypes to be confirmed by other specific methods
- High performance liquid chromatography (HPLC): HPLC separates, identifies and quantifies components of a mixture based on chemical and physical properties. With respect to haemoglobins, separation, identification and quantification of Hb types is achieved by charge differences (cation exchange).Each haemoglobin variant has its characteristic retention time. If a peak eluted at a retention time that is not pre-defined, it is labeled as an unknown.The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional haemoglobin electrophoresis.
- Isoelectric Focusing (IEF): This has a higher resolution than electrophoresis but requires trained personnel for its use and interpretation of results. It is also very expensive and not used widely
- Molecular technique: specific for definite diagnosis of genetic disorders. Used mostly for prenatal diagnosis (determination of the Hb genotype of the fetus) and for confirmation of some difficult Hb genotypes after using other methods.
Sample case report:
Previous test results(brought in by the patients from another laboratory):
- Father: Hb AS
- Mother: Hb AA
- Child: Hb SS
In conclusion, a shift in attitude is necessary for the laboratory analysis of haemoglobin types. It is high time laboratories went beyond routine alkaline electrophoresis and sickling test. Diagnosis should include a solubility test, an assessment of blood count, red cell indices, stained thin films and HPLC as a method suited for the identification and quantification of other Hbvariants in order to prevent a misdiagnosis. A DNA analysis is highly desirable when indicated or necessary.
- Oyetunji sent this piece through the Sickle Cell Foundation.
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