The immune system has evolved as a highly specialised function of human beings, which is concerned with the substances considered “foreign” to the body. It consists of a cellular component and a humoral component. Although the field of blood group serology is associated mainly with the humoral component of the immune system, the mechanics of antibody production in vivo involves the cellular arm of the immune system or the cell-mediated immunity.
The science of immunohaematology deals with the basic principles of antigen and antibody structure, the genetics, the biochemistry, its mode of action and its role in haematology. To understand the principle of compatibility testing and transfusion reactions the basic knowledge of immunohaematology is essential.
Antigen
Antigen is a substance, which elicits immune response. It is a complex molecule whose molecular weight exceeds 10000 daltons. The ABH antigens are glycolipids while Rh antigens are protein. The
hepatitis B surface antigen (HbsAg) is a lipoprotein.
A number of characteristics influence the antigenicity. These include the molecular size, charge on the surface of cells and the solubility. The inheritance of Ir genes and occurrence of disease also influence the antibody response.
Not all the blood group substances are equally immunogenic. Approximately 50% of Rh-negative recipients of Rh-positive blood have the tendency to get sensitised to the D antigen. Other Rh antigens like C and E and antigens of other blood group systems are relatively less immunogenic. The number of antigen sites on the RBC varies according to specificity. There are approximately 1 million ABO antigen sites and 25000 Rh (D) antigen sites on a RBC.
Antibody
The antibodies are immunoglobulin in nature. Approximately 82–96%
of antibodies are polypeptide, and the rest 4-18% are carbohydrates in nature.2
Production
The antibodies are produced in the plasma of those individuals who lack the corresponding antigen. The production may be because of either blood transfusion or foetomaternal leak of incompatible blood.
Immunoglobulin structure
All the immunoglobulins share a common chemical structural configuration. Each basic antibody unit is composed of four polypeptide chains: two identical light chains having a molecular weight (M.W) of approximately 22500 daltons and two identical heavy chains with a M.W of 50000-75000 daltons. Covalent disulfide bonds hold the four chains together. Each heavy chain has 440 amino acids and each light chain 220 amino acids.
The chemical structure of heavy chains is responsible for the diversity of immunoglobulin classes. The light chains kappa and lambda are common to all immunoglobulins.
Immunoglobulin classes
The isotypes of the heavy chains determine the class of immuno-globulin. There are five classes of immunoglobulins designated as IgA, IgD, IgE, IgG and IgM.
The blood group antibodies are commonly, IgM, IgG or IgA.
IgA
IgA class of antibodies exists both as a monomer and as polymers. The M.W is approximately 160000 daltons.
IgG
The IgG constitutes approximately 75% of total serum immuno-globulins. It is a Y-shaped monomer. There are four subclasses of IgG; IgG1, IgG2, IgG3 and IgG4 based on the sequence of amino acids in the heavy chain.
The IgG antibodies react at 37°C.
The MW of IgG is 150000 daltons which is the lowest of all immunoglobulins. It enables IgG to cross the placental barrier.
IgM
The IgM antibodies constitute approximately 10% of the total serum immunoglobulins. They are pentamer in shape. The M.W is 900000 daltons which makes it the heaviest of all classes of immunoglobulins. It does not 3cross the placental barrier.
They react at room temperature (20-24°C).
The IgM are highly effective agglutinins and are capable of activating the complement. Plasma contains significant amounts of IgM.
Complete and incomplete antibodies
The antibodies, which are produced without any antigenic stimulus, are known as complete antibodies. Most IgM class antibodies fall in this category. They are capable of agglutinating red cells suspended in normal saline at 20-25°C. Most of the ABH antibodies are IgM in nature, and called natural or complete antibodies.
The antibodies that require a bridge like the Coomb's molecule for binding to the antigenic site are called incomplete antibodies. Most IgG antibodies are incomplete antibodies. They react at 37°C. The Rh (CDE) are incomplete or acquired antibodies.
Monoclonal and polyclonal antibodies
The antibodies, which are derived from multiple ancestral clones of antibody producing cells and carry both kappa and lambda light chains are termed as polyclonal antibodies. In contrast, the antibodies, which contain exclusively kappa or lambda light chains, are known as monoclonal antibodies. Monoclonal antibodies have the ability to recognise single antigenic epitope, and provide greater diagnostic precision than polyclonal antibodies.
Identification and estimation of immunoglobulin
The specificity of the blood group antibodies is determined by two methods. Either by 2-mercaptoethanol treatment or by separating the antibody on column chromatography. The haemagglutination inhibition technique is applied for estimation of IgG, IgM and IgA class of antibodies.
Antigen antibody ratio
The speed by which antigen and antibody bind, is dependent on number of antibody molecules in the medium and the antigen sites available on the cell. By raising the serum to cell ratio the number of molecules are increased. If 2 drops of cell suspension are added to 4 drops of serum that increases the sensitivity of the test. The other factors affecting the binding of antigen antibody are pH of the medium, temperature and incubation period.4
Complement
The complements are plasma proteins that interact with bound antibodies resulting in cell lysis and enhanced phagocytosis.
The nine components of complements are designated from C1 to C9. The C4 acts in between 1 and 2, the rest being in numerical order.
The complements are destroyed when heated with anticoagulants to 56°C for 30 minutes.
Sensitisation
The sensitisation is defined as binding of antigen and antibody, in vitro or in vivo, with or without agglutination.
Agglutination
Whenever the sensitised cells come into contact of each other, the result is clumping of red cells known as agglutination.
Grades of agglutination
The agglutination results are graded from 1+ to 4+. The American Association of Blood Banks (AABB) recommends the following grading system:
4+= One solid aggregate of red cells
3+ = Several large aggregates
2+ = Medium sized aggregates with a clear background
1+ = Small aggregates with a turbid background giving granular appearance.
Weak (w) = Tiny aggregates are seen only under microscope
Negative = All cells are free.
Factors influencing agglutination
The following factors affect the process of agglutination.
Charge on cells
The red cells carry negative charge on their surface and repel each other, but when the Na+ present in the normal saline medium is added the negative charge is reduced, ultimately reducing the total charge, called zeta potential.5
Albumin or enzymes
The type of the medium used affects the agglutination. The bovine albumin or enzyme papain reduces further the zeta potential. The IgG molecules form bridges between red cells, resulting in agglutination.
Effect of Coomb's serum
The Coomb's or antihuman globulin molecule (AHG) forms bridge between different molecules of IgG immunoglobulin and approximates the sensitised cells leading to agglutination.
Haemolysis
The antigen and antibody reaction where complement is activated leading to breakdown of red cells and release of haemoglobin is called haemolysis.