Immunohematology

Dr. Setalbanat Hassan Omer

 Immunohematology definition

• Immunohematology is the study of blood group

antigens and antibodies, HLA antigens and
antibodies, pre-transfusion testing, identification
of unexpected alloantibodies, immuno
hemolysis, autoantibodies ,drugs, blood, blood
transfusion practice, safety ,quality assessment,
cord blood.
IMMUNOLOGIC
PRINCIPLES
• primary immunological components: antigens &
antibodies  provides basis for blood bank
testing and reactions
• Cardinal rule in blood bank:
The antigens are found on the surface of red blood
cells and the antibodies are found in serum or
plasma
 Characteristics of Antigens

• Can also be called immunogens because they

elicit an immune response
• Combine with an antibody or T cell
• Proteins are the most immunogenic, followed by
glycoproteins and glycolipids.
• There are 23 antigen systems containing over 200
RBC antigens.
 Specific Classes of Antigens

1. Red cell antigens

2. Platelet antigens: membrane have protein antigens, plt.
Antibodies occur less frequently in the general
population because of less antigen variability.
3. Human leukocyte antigens(HLA) are found on the
surface of leukocytes and tissue cells. Gene that encode
the HLA antigens are parts of the major
histocompatibility complex (MHC)
 Characteristics of Antibodies

• Consist of two pairs of polypeptide chains

• Contain one pair of heavy chains is responsible for the
immunoglobulin group specificity.
• Five classifications: IgG, IgA, IgM, IgD, and IgE
• One pair of light chains
• Light chains are of two types: kappa or lambda
 IgM Antibodies

• The first class of antibody produced by B cells or plasma

cells when an antigen is encountered
• Is referred to as a pentamer because it is made up of five
basic units held together by a J chain
• Capable of agglutination of red cells in saline at room
temperature.
• Constitute 5% to 10% of total serum antibody
• Can activate complement.
• Usually clinical insignificant.
 IgG Antibodies
• Consist of a single four-chain unit
• Not effective in agglutinating red cells in saline
• Can cross the placenta and cause hemolytic disease of the
fetus and newborn (HDFN)
• Constitute 80% of antibody concentration in serum
• React optimally at 37c
• Clinically significant.
• Cannot activate complement unless two molecules are
present.
• A primary response is elicited on first exposure to a
foreign antigen
• Lag phase of 5 to 7 days before antibody can be detected
in the host
• Following the lag period, antibody levels rise, plateau,
and then decline
• IgM antibodies are produced first; then IgG are produced

Primary and Secondary

Immune Response
• Also known as an anamnestic response because there are
memory cells
• Within 1 to 2 days of antigen exposure, there is a
significant amount of antibody production
• The principal antibody is IgG, unlike a primary response,
in which IgM is the principal antibody
• 1000 times more antibody and a slow decline

Secondary Immune
Response
• Goodness of fit
• Strength and rate of reaction
• Size, shape, and charge
• Affinity: The strength of binding between a single
antibody and epitope of an antigen
• Avidity: The overall strength of reactions between several
epitopes and antibodies

Properties That Influence

Antigen-Antibody Binding
• Transfusion of blood and blood products can expose a
recipient to potentially foreign antigens on red cells,
white cells, and platelets
• Immunization can also occur during pregnancy when
fetal blood cells cross the placenta
• Antigens antibodies complexes are removed by the
reticuloendothelial system.

Antibody-Antigen
Reactions In Vivo
• Reactions in laboratory testing are detected by visible
agglutination or hemolysis
• The first stage of in vitro antibody-antigen binding is
usually not visible.The first stage is influenced by:
a. cell-to-serum ratio
b. Temperature 37 c
c. incubation time, and ionic strength
d. pH

Antigen-Antibody
Reactions In Vitro
• Random collisions of antibody coated RBCs link
antibodies together to form visual agglutination.
• Crosslinking of antibody-coated red cells is necessary for
visualization of red cell agglutination; for this to occur,
the following factors are important:
• Distance between red cells must overcome the zeta potential
(the force of repulsion between red cells in a saline
solution)
• Optimal amounts of antibody and antigen

Lattice Formation
• Maximum agglutination occurs when the concentrations
of antibodies and antigens fall within the zone of
equivalence
• A prozone occurs when the concentration of antibody
exceeds antigen concentration
• A postzone occurs when the concentration of antigen
exceeds antibody concentration

Zone of Equivalence
 Grading Agglutination Reactions
• Time and speed of centrifugation are important
• Reactions are graded on a 0 to 4+ scale
• Hemolysis is also an indicator of antigen-antibody
reactions caused by complement activation.
 Hemolysis
• When antibody (IgG or IgM) has activated Complement
to completion.
• The complement (C’) cascade is the mechanism that
actually destroys the red blood cell.
• Antibody (immunoglobulin) by itself cannot tear a hole in
the RBC membrane. It is the C’ that does that.
• Seen when supernatant is CLEAR and RED.
 Antibody Coating RBC Membrane without
Agglutination

• Attachment of antibody to corresponding antigen on RBC

membrane, ONLY.
• This reaction is NOT SEEN at the Immediate Spin (IS),
Room Temperature (RT), or 37oC (LISS) phases.
• This reaction requires the use of an Antihuman Globulin
reagent in the Direct (DAT) or Indirect Antiglobulin Tests
(IAT) to observe
 Separation of Red Blood Cells
• red blood cells are built in such a way as to keep
their distance from each other. It is a good thing.
Theories include:
• Waters of Hydration: Water bound by RBC
membrane glycoproteins helps maintain the
distance between rbc’s. (Water envelope)
• Electrostatic charges: Electron cloud surrounds
RBC with a net negative charge called the zeta
potential.
• To observe Ag/Ab reactions in vitro these forces
need to be overcome to enable Ab’s to attach to
their corresponding Ag.