Faculty of Allied Medical Science Blood Banking (MLBB 201)

FUNDAMENTALS OF IMMUNOLOGY FOR BLOOD BANKERS Prof.Dr Nadia A. Sadek Prof. in Hematology, Head of Blood Bank, MRI

Outcomes By the end of the lecture, the students will know the antigen-antibody reaction. By the end of the lecture, the students will know the antigen-antibody reaction. The stages of the reaction and factors affecting antigen-antibody reaction. The stages of the reaction and factors affecting antigen-antibody reaction.

Agglutination or lysis due to complement reaction is a visible end-point of an antigen-antibody reaction. Agglutination or lysis due to complement reaction is a visible end-point of an antigen-antibody reaction. The reaction occurs in 2 stages: The reaction occurs in 2 stages: 1- The first stage is sensitization, the antibody binds to the red cell antigen

2-The second involves agglutination of the sensitized red cells. The first stage is association of antibody with antigen = sensitization and it is “ reversible ”

Antibodies may be: 1- Warm antibodies = usually IgG, they react best at 37 degrees. 2- Cold antibodies = usually IgM, they react best at low temperatures.

The strength of binding of antibody to antigen (=equilibrium constant) depends on the “ exactness of fit “ between them. The strength of binding of antibody to antigen (=equilibrium constant) depends on the “ exactness of fit “ between them. It depends on:- It depends on:- 1- Temperature: Cold antibodies (usually IgM) generally bind best to the red cells at a low temperature (e.g. 4 degrees). Cold antibodies (usually IgM) generally bind best to the red cells at a low temperature (e.g. 4 degrees).

Warm antibodies (usually IgG) bind most efficiently at body temperature i.e. 37 degrees. 2- pH: Little change occurs in antibody binding over the pH range Little change occurs in antibody binding over the pH range It is preferable to buffer the saline in which serum or cells are diluted to a fixed pH, usually 7.0

Some antibody elution techniques depend on altering the pH to less than 4 or more than Ionic strength of the medium: Low ionic strength increases the rate of antibody binding. This is why we use low ionic strength saline (LISS). Low ionic strength increases the rate of antibody binding. This is why we use low ionic strength saline (LISS).

The red cell surface is negatively charged owing to sialic acid residues which keeps the individual cells apart. The minimum distance between red cells suspension in saline is 18nm.Agglutination occurs by antibody cross-linking between cells.

The span between antigen-binding sites on IgM molecules is 30nm, sufficient to allow IgM antibodies to bridge between saline suspended red cells (after settling) and so, they cause agglutination.

IgG molecules have a shorter span (15nm) and are usually unable to agglutinate sensitized red cells suspended in saline. Heavy IgG sensitization, owing to high density lowers intercellular repulsive forces and is able to promote agglutination in saline, e.g. IgG anti-A and Anti-B.

How to promote agglutination - Red cell agglutination is enhanced by centrifugation. - Reduce intercellular distance and surface negative charge by adding protease enzymes e.g. papain or bromelin. - Add polymers e.g. albumin.

- Bridging between sensitized cells with an antiglobulin reaction in the antiglobulin test. - Some complement-binding antibodies (especially IgM) may cause lysis in vitro without agglutination. This can be enhanced by adding fresh serum as a source of complement.

General points in serology Serum versus Plasma Serum is preferred to plasma for detection of red cell alloantibodies. Serum is preferred to plasma for detection of red cell alloantibodies. Plasma is used in microplate technology and automated system. Plasma is used in microplate technology and automated system. When plasma is used, complement is inactivated by EDTA anticoagulant. When plasma is used, complement is inactivated by EDTA anticoagulant.

Collection of blood samples - Positive identification for the patient and careful labelling of blood samples are essential to avoid misidentification errors. - Venous blood is desirable for blood grouping. - If serum is needed, withdraw 5-10ml blood in sterile glass tubes, better in a 37 degrees water bath and centrifuge as soon as the clot can be seen.

Storage of serum or plasma - Correctly label the serum or plasma separated from the patient ’ s original sample. - Whole blood samples will deteriorate over time:- red cell lysis and loss of complement red cell lysis and loss of complement decreased potency of antibodies especially IgM decreased potency of antibodies especially IgM bacterial contamination. bacterial contamination.

Study question Complete:-  The strength of binding of antibody to antigen depends on ……..  Red cell agglutination is enhanced by ……..

Assignments Apheresis Apheresis سرى افتخار عبد الفتاح