Unit 4 Principles of Serological
Testing in Immunohematology
Terry Kotrla, MS, MT(ASCP)BB
Blood Group Antigens and Antibodies
 Antigens are embedded structures in or protruding from
RBCs, WBCs or platelets.
 Exposure to blood group antigens through transfusion or
pregnancy.
 Individual may form antibodies to antigens they lack.
 Most concerned with IgG and IgM class antibodies.
 IgM naturally occurring, react at RT, easily detected.
 IgG are “immune” requires exposure to the actual antigen and
requires special serological test (Coomb’s) to detect.
 ALWAYS testing for antibodies in serum/plasma and antigens
on cells.
Detection Tests
 Detection of antigens routinely done to determine
individuals’ ABO and D type.
 Antibody detection tests used to
 Confirm ABO antigen typing.
 Detect unexpected antibodies in serum/plasma which may
cause destruction of transfused cells containing antigen.
Dynamics of Antigen-Antibody
Reactions
 Union of antigen with antibody depends on structure and
charge of molecule.
 Lock and key
 Opposite charges on antigen and antibody create attraction
forces.
 Reactions is REVERSIBLE .
Dynamics of Antigen-Antibody
Reactions
 Physical forces hold antigen and antibody together.
 Weak
 Attractive forces vary in strength with changes in pH, ionic
strength, temperature and nature of solven.
 Blood group antigens and antibodies bind until dynamic
equilibrium is reached.
 Represented by bell shaped curve based on concentration.
 Prozone
 Postzone
 Zone of equivalence.
Antigen Antibody Reactions
Detection of Antigen-Antibody
Reactions
 Agglutination
 RBCs have antigens which combine with antibodies present in serum (either
patient or reagent).
 Antibody bridges form with antigens on adjacent cells resulting in
agglutination.
 Hemolysis caused by antibody binding, activation of complement which
results in destruction of RBC membrane.
 Solid Phase to detect antigens or antibodies
 Primarily done in donor centers.
 Microplates coated with antigen or antibody.
 Positive, cells adhere to sides of well.
 Negative, cells fall to bottom and form a button
Agglutination – Principle and Variables
 TWO stages involved
 First stage is sensitization, attachment of an antibody to
corresponding antigen on RBC membrane.
 Second stage is lattice formation, bridges form between
sensitized cells.
 IgG have 2 antigen binding sites, one binds to antigen on one cell, the
other binds to antigen on adjacent cell.
 IgM has 5 antigen binding sites and can bind to several antigen sites on
different cells.
 Lattice formation will result in hemagglutination.
Agglutination
 IgG agglutinating RBCs
 IgM agglutination RBCs.
Variables Affecting First Stage
 Antigen-antibody ratio – CRITICAL
 Optimal amount of serum/plasma must be determined.
 Increase serum/plasma increase amount of antibody – more
sensitive.
 pH of 6.5 to 7.5, for routine procedures 7.0 used.
 Ionic strength of suspending medium
 Saline partially neutralizes oppositely charged sites on antigen
and antibody molecules which hinders association.
 Low ionic strength saline (LISS) with lower salt concentration
enhances uptake of antibody onto cells.
 Albumin, unless used under low ionic conditions, does little to
affect antibody uptake onto cell, usually affects second stage.
Variables Affecting First Stage
 Temperature requirements depend on antibody class
detected.
 IgG reacts optimally with antigens at 37C.
 IgM reacts optimally at RT (20-24C) or below (4C)
 Incubation time
 Choose incubation time which favors maximal binding of
antibody to antigen.
 Enhancement agents increase antibody uptake onto cells thereby
decreasing incubation time.
Variables Affecting Second Stage
 Immunoglobulin Class
 IgG is a small monomer with the ability to sensitize RBCs but
NOT agglutinate.
 IgM is huge pentamer, very easily agglutinates RBCs
 Antigen sites may be present in low numbers on surface
resulting in no agglutination.
 Electrostatic Repulsion Forces
 Lattice formation depends on overcoming electrical charges
(repulsion forces) of RBCs.
 Zeta potential is the term used to describe the electrical forces
which keep RBCs separated, use methods to decrease.
Zeta Potential
 IgG unable to overcome zeta potential to cause direct agglutination.
 I could not copy the original picture.
 Visit http://tinyurl.com/4f9d24y , EXCELLENT materials
Principle of the Antiglobulin Test
 Most important test in the blood bank after ABO/D typing.
 Small IgG antibodies can sensitize but rarely cause
agglutination.
 Cannot overcome zeta potential
 If undetected (false negative) will cause destruction of RBCs.
 1945 Coombs, Mourant and Race described test for non-
agglutinating coating antibodies now called the anti-human
globulin (AHG or Coomb’s) test.
 You MUST know the principle of the test backwards,
forwards and sideways.
Principle of the Antiglobulin Test
 A reagent antibody to IgG (anti-IgG) and/or complement is
produced (AHG serum) and is added to washed cells after
incubation with serum/plasma or antisera.
 If cells are sensitized (coated) with IgG during incubation the
anti-IgG will attach to the Fc portion of the IgG coating the
cell causing agglutination.
 You must MEMORIZE the principle of this test and be
able to draw a diagram for future exams.
Coomb’s Test
Importance of Coomb’s Test
 Prior to 1945 could only detect IgM class antibodies, only a
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few blood groups had been identified.
ABO antibodies and antigens were detected but hemolysis of
ABO compatible transfused cells continued to occur.
This test provided the explanation that IgG class antibodies
were responsible, could sensitize but not be detected in
direct agglutination tests.
Determined that IgG was capable of destroying transfused
RBCs and also crossed the placenta to destroy fetal RBCs.
IT IS NOW A REQUIRED TEST
Preparation of AHG Serum
 Two methods.
 Animal Inoculation
 Purified human IgG injected into suitable animal, usually
rabbits, and rabbit would make anti-IgG.
 Animal bled, unwanted antibodies removed.
 AHG will react with IgG bound to cells or free in
serum/plasma.
Preparation of AHG Serum
 Monoclonal Reagent Preparation
 Animal (mouse most popular) injected with appropriate antigen
(IgG) and produces anti-IgG
 Make hybridoma
 Fuse antibody secreting cell (mouse spleen cell) with myeloma cell which
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produces massive quantities of antibody.
The myeloma cell produces antibody without specificity.
Fusion with mouse spleen cell “programs” the myeloma cell to produce
antibody with one specificity.
Cell is immortal.
Grow in culture.
 Harvest the antibody containing fluid from hybridoma.
Monoclonal Antibody Production
Types of AHG
 THREE types
 Polyspecific
 Monospecific anti-IgG
 Monospecific anti-Ce
Polyspecific AHG
 Consists of a POOL of anti-human IgG, anti-C3b and anti-C3d.
 May be produced by hybridomas, rabbits or a mixture.
 May be used for routine compatibility testing, antibody identification
and DAT.
 Most important function is to detect IgG antibodies coating the cells.
 The importance of the presence of anti-complement in AHG serum is
very controversial for routine compatibility testing.
 Antibodies which are detectable only by their ability to bind
complement are rare, so it's not as useful for compatibility testing as
many false positive reactions occur.
Anti-IgG (monospecific)
 Major component is antibody to human gamma chains (anti-IgG)
contains no anti-complement, is heavy chain specific.
 Used as alternative to polyspecific AHG in routine blood
bank procedures.
 Advantage of utilizing anti-IgG is that it eliminates the false
positives frequently obtained when using polyspecific AHG
due to complement and non-specific cold reacting antibodies
yet will detect clinically significant alloantibodies.
Anti-Complement (Monospecific)
 Contains anti-complement antibodies to detect complement coating
the cells.
 Used for classifying the coating proteins on RBCs to
determine if IgG, complement, or both are present.
 Primary importance is for classification of autoimmune
hemolytic anemias and drug induced hemolysis of RBCs.
Antiglobulin Techniques
 TWO applications of the antiglobulin test
 Direct antiglobulin test (DAT)
 Indirect antiglobulin test (IAT)
Direct Antiglobulin Test (DAT)
 Used to demonstrate or detect in-vivo coating of RBCs with
globulin, particularly IgG and/or C3.
 One drop of a patient's cell suspension is washed 3 times to
remove all contaminating proteins.
 AHG is added directly to the washed cells, centrifuged and
observed for agglutination, which is indicative of IgG antibodies
coating the cells, the AHG serum will bind to the IgG on the
cells forming lattices = agglutination.
 Primary use is detecting maternal antibody coating fetal
cells.
 Also used in detecting autoantibody coating on patient cells.
Direct Antiglobulin Test
Indirect Antiglobulin Test (IAT)
 Detects in-vitro sensitization with antibody complement or both.
 Patient serum (or special types of anti-serums) are added to RBCs and
incubated at 37 C (body temperature) for a specified time.
 May be read after incubation because some IgG antibodies may coat the
RBCs heavily enough after incubation to cause agglutination.
 The RBCs are washed free of contaminating protein and AHG serum is
added, if the cells have been coated with IgG during the incubation
phase the anti-antibody in the AHG will form lattices which resulting in
agglutination.
Indirect Antiglobulin Test
Antiglobulin Techniques
 The crossmatch procedure may require the IAT,
 patient serum is incubated with donor cells, washed and tested
with AHG serum.
 Agglutination indicates that the patient has antibody to an
antigen on the donor cells
 The unit cannot be given to the patient (it is incompatible).
 When a patient has an antibody identified in their serum it is
critical to find donor units which lack the antigen, typing the
donor may require the IAT.
False Negative Results in the DAT and
IAT. MEMORIZE!
 Failure to wash red blood cells adequately.
 Testing interrupted or delayed during the washing
procedure.
 Loss of activity in AHG reagents
 Failure to add AHG.
 Improper centrifugation
 Under centrifugation
 Over centrifugation
 Incorrect concentration of red blood cells in test system.
 Prozone reactions
False Negative Results in the DAT and
IAT. MEMORIZE!
 Negative DAT may not necessarily mean absence of coating
globulins.
 Poly and anti-IgG will detect approximately 200 molecules of
IgG per cell.
 Presence of weak antibody may go undetected.
 If a negative reaction is obtained, IgG coated reagent RBCs
(check cells), are added to the test system, agglutination
ensures that the AHG serum is active.
 In the DAT complement coating may not be apparent as
agglutination upon immediate reading.
False Negatives in the IAT
 IAT and DAT are different.
 Red cells and serum lose reactivity if improperly stored.
 Occasional examples of anti-Jka and anti-Jkb may be detected
only in the presence of active complement.
 Temperature and incubation time affect attachment of
antibody or complement to the red blood cells.
 Optimal proportion of serum to cells should be achieved.
False Positive Results in the DAT and
IAT. MEMORIZE!
 Red cells may be agglutinated before they are washed.
 Saline stored in glass bottles may contain colloidal silica
particles that can leach from the container into the saline.
 Improperly cleansed glassware.
 Overcentrifugation
 Improperly prepared AHG reagents.
DAT - additional considerations
 Complement components, primarily C4, may nonspecifically
bind to red blood cells from clotted blood samples kept at
4C.
 False positive DAT results may occur with blood collected
into tubes containing silicone gel.
 Blood samples collected from intravenous fluid lines used to
administer solutions.
 Septicemia in a patient or bacterial contamination of stored
specimens.
IAT - Additional Considerations
 Red cells coated with IgG cannot be tested with antisera that
react only by IAT, such as weak D or anti-Fya.
 Procedures are available for removing IgG from in vivo
coated red blood cells
Role of Complement in the Antiglobulin
Reactions
 Complement-binding antibodies attach complement to the
cell surface when they react with red cell antigens.
 Some blood group antibodies bind complement to the red cell
membrane.
 Clinical significance of complement binding antibodies is
variable.
 Immune complexes present in plasma activate complement
components that may adsorb to red blood cells in a nonspecific manner.
 Complement coated red cells may or may not proceed to
hemolysis.
References
 Indian Immunohematology Initiative
http://www.indianinitiative.org/lectures/
 Life’s Blood – EXCELLENT
http://tinyurl.com/4ar2q56
 Merck Manual
http://www.merckmanuals.com/professional/sec11/ch131/ch131a.html
 Monocolonal Antibody Productions
http://www.accessexcellence.org/RC/VL/GG/monoclonal.php
 Monoclonal Cell Culture Picture
http://tinyurl.com/4mnyrpa
End of Unit 4