Essentials of Blood Banking SR Mehdi
INDEX
×
Chapter Notes

Save Clear


ImmunohaematologyChapter 1

The immune system has evolved as a highly specialized function of human beings and 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 it's role in haematology. To understand the principles 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 exceed 10000, daltons. The ABH antigens are glycolipids while Rh antigen is a protein. The HbsAg is a lipoprotein.
A number of characteristics influence the antigenicity. These include the molecular size, charge and the solubility. The inheritance of Ir genes and occurrence of disease also influence the antibody response.
All the blood group substances are not equally immunogenic. Approximately 50 percent of Rh negative recipients of Rh positive blood are expected to be sensitized to the D antigen. Other Rh antigens like C and E and other blood group systems 2are much less immunogenic. That is the reason that only D and not other Rh antigens are routinely typed in the blood bank.
The number of antigen sites on the RBC vary according to specificity. There are approximately 1 million ABO antigen sites and 25000 Rh (D) antigen sites on RBC.
Some of the specificities are poorly developed at the time of birth.
 
ANTIBODY
The antibodies are immunoglobulin in nature. Approximately, 82-96 percent of antibodies are polypeptide and the rest 4-18 percent are carbohydrates.
 
Production
The antibodies are produced in the circulation of those individuals who lack that particular antigen. The production may be as a result of either blood transfusion or foeto-maternal leak of incompatible blood.
 
Immunoglobulin Structure
All immunoglobulin share a common chemical structural configuration. Each basic antibody unit is composed of four polypeptide chains: two identical light chains (M.W approximately 22500 daltons) and two identical heavy chains (M.W approximately 50000-75000 daltons). The four chains are held together by covalent disulfide bonds. 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 while the light chains are kappa and lambda common to all immunoglobulins.
 
Immunoglobulin Classes
The isotypes of the heavy chains determine the class of immunoglobulin. There are basically five class of immunoglobulins designated as IgA, IgD, IgE, IgG and IgM.
The blood group antibodies are usually, IgM, IgG or IgA.3
 
IgA
IgA class of antibodies exist both as monomer and polymers. The M.W is approximately 160000 daltons.
 
IgG
The IgG costitutes approximately 75 percent of total serum immunoglobulins. 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.
They react at 37°C.
The MW is 150000 daltons which is the lowest of all immunoglobulins, and enables IgG to cross the placental barrier.
 
IgM
The IgM constitute approximately 10 percent of the total serum immunoglobulins. They are pentamer in shape. The MW is 900000 daltons which makes it the heaviest of all classes of immunoglobulins. It does not cross the placental barrier.
They react at room temperature (20-25°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 they are called natural or complete antibodies.
The antibodies which require a bridge like the Coomb's sera 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.4
 
Monoclonal and Polyclonal Antibodies
The antibodies which are derived from multiple ancestral clones of antibody producing cells and which carry both kappa and lambda light chains are termed as polyclonal antibodies. In contrast some of the antibodies contain exclusively kappa or lambda light chains are known as monoclonal antibodies. Because monoclonal antisera have the ability to recognise single antigenic epitopes, this provides greater diagnostic precision than polyclonal antisera.
 
Identification and Estimation of Immunoglobulins
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.
 
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 is added to 4 drops of serum, it increases the sensitivity of the test. The other factors affecting the binding of antigen antibody are pH, temperature and incubation period.
 
COMPLEMENT
The complements are serum proteins which interact with bound antibodies resulting in cell lysis and enhanced phagocytosis.
The nine components of complements are designated C1 to C9. The C4 acts in between 1 and 2, the rest in numerical order.
The complements are destroyed when heated with anticoagulants to 56°C for 30 minutes.5
 
Sensitization
The sensitization is defined as binding of antigen and antibody, in vitro or in vivo with or without agglutination.
 
AGGLUTINATION
Whenever the sensitized cells come into contact of each other the end result is clumping of red cells known as agglutination.
 
Grades of Agglutination
The agglutination results are graded from 1+ to 4+. The AABB recommends the following grading system:
4+
=
One solid aggregate of red cells
3+
=
Several large aggregates
2+
=
Medium sized aggregates, clear background
1+
=
Small aggregates, turbid background
w
=
Tiny aggregates
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 and ultimately reducing the total charge called Zeta Potential.
 
Albumin or Enzymes
The type of the medium used affect the agglutination. The bovine albumin or enzyme papain reduces further the zeta potential. The IgG molecules form bridges between red cells resulting in agglutination.6
 
Effect of Coomb's Serum
The Coomb's or antihuman globulin (AHG) serum forms bridge between different molecules of IgG immunoglobulin and approximate the sensitized cells bringing about agglutination.
 
Haemolysis
The antigen and antibody reaction where complement is activated and leads to breakdown of red cells and release of Haemoglobin is called haemolysis.