COOMB’S TEST

BY DR. SOMYA MISHRA

• Coombs test is a laboratory test done to detect antibodies coated on
RBC surface.
• Moreschi described the use of antiglobulin reactions as an aid to RBC
agglutination, in 1908.
• In 1945, Coombs et al developed antiglobulin test and its role in
identification of maternal IgG on fetal RBCs in 1946, in Hemolytic
Disease of Newborn
Antibodies on RBC surface can be of two types :
• IgG
• IgM
After coating these antibodies will lead to destruction of RBCs by
various pathways.
• Warm antibodies : commonly IgG type, agglutinates RBCs at zero
degrees, may/ maynot fix complement, primarily RBC loss by spenic
mediated destruction

• Cold antibodies : commonly IgM type, agglutinates RBCs at 37

degrees, fix complement, immediate intravascular RBC destruction,
hepatic mediated

• Mixed antibodies consisting of both warm and cold type antibodies,

responsible for more troublesome disease.
Zeta potential of RBCs

• There is a negative charge due to sialoglycoprotein coat leading to

RBC repulsion
• The minimum separation attaiable between two RBCs is 18nm
• Other factirs also involved in this zetavpotenyial like water
• IgM molecules, large pentameric structure, 30nm distance between
adjacent binding sites and can cause agglutination, complete
antibodies, critical no. 25-50 per RBC
• IgG molecules, smaller, 12nm distance between adjacent binding
sites, hence, cannot cause agglutinatiom alone, incomplete
antibodies, critical no. 7000-20000 per RBC
For serologic detection of IgG antibodies, enhancement of
agglutination by IgG antibodies by :
• Enzyme treatment
• Centrifugation
• Addition of albumin, PVP (polyvinylpyrrolidone), dextran
• Coombs test
• Polyspecific Antiglobulin Sera : Contains antibodies to human IgG, C3
and C4 components of the complement
• Monospecific Antiglobulin Sera : Maybe against any one of the human
IgG, IgM, IgD, IgA or complement C3 or C4.
Direct Coombs Test ( Direct antiglobulin test)
Principle :
RBCs with surface bound immunoglobulin and/or complement and
antisera with reactivity to human IgG and/or complement cross link to
agglutinate patients RBCs
Indications
• Autoimmune haemolytic anemia
• Hemolytic disease of newborn
• Drug induced red cell sensitisation
• Hemolytic transfusion reaction
 Method
• Take 1ml of blood sample collected in EDTA vial and same amount of
positive and negative control samples
• Centrifuge for 10 minutes
• Remove supernatant plasma
• Take 10 times normal saline in the
centrifuged sample and vortex
(washing)
• Centrifuge for 10 minutes
• Washed two more times
• After this we can do either card test
or tube test.
Tube Test
• 50 microliter of RBC button is taken after
discarding the supernatant after washing
• Add 100 microliter of Anti Human Globulin
• Vortex and then, centrifuge for 1 minute
• Mix gently
• Let it stand for 2-3 minutes
• Examine the bottom of the tube
• Make wet mount
• Analyse
Interpretation : Gross
Interpretation : Microscopic
NEGATIVE POSITIVE POSITIVE
Card Test
• 10 microliter of RBC button is taken after
discarding the supernatant after washing

• Add 1 ml of LISS (Low Ionic Strength

Solution- increases the rate and degree of
antibody uptake by red cells and reduces the
incubation time significantly) [1:100 ratio]
• Vortex
• Take 50 microliter of this solution and put in
cylinder of card
• Then, this card is centrifuged for 10 minutes
with proper balance
• Analysis
Interpretation
• The microtubes of a card contain a solid phase matrix and the
antiglobulin reagent to which patients red cells are added
• During centrifugation, unagglutinated cells pass to the tip of the tube,
but agglutinates fail to pass through gel which acts as sieve
Grading of gel card test
• HTR : Hemolytic transfusion reaction
• AIHA : Autoimmune haemolytic anemia
• CAD : Cold agglutinin disease
• DI-IHA : Drug induced immune haemolytic anemia
• PCH : Paroxysmal cold Hemoglobinuria
False positive DAT
• With a silica gel derived from glass
• Rarely on healthy individuals
False negative DAT
• Failure to wash cells properly (antisera will be neutralised by
immunoglobulins or complements in the sera)
• Excessive agitation: may break up agglutinates
• Non potent anti sera
• Antisera lacking the antibody corresponding to the immunoglobulin
coating the RBCs
• Presence of antibodies that is readily dissociable
Indirect Antiglobin Test
• Principle : Patient’s serum/plasma containing immunoglobulins are
attached to reagent RBCs and is detected with antiglobulin sera,
crosslinking RBCs together and producing agglutination
Indications
• Compatibility testing
• Detection of red cell antigens using specific antibodies reacting only
in antiglobulin test eg. Fya, Fyb, Jka, K, Jkb etc.
• Screening and identification of unexpected antibodies in serum
Method
• Take two drops of patients serum and same amount of positive and
negative control samples
• Add 1 drop of 3% red cell suspension
• Mix thoroughly and incubate for 50 minutes at 37 degrees
• Wash these cells with normal saline 3 times
• after discarding supernatant, add 2 drops of antiglobulin serum
• Vortex and centrifuge for 15 seconds
• Re-mix the cells gently and observe for agglutination
• Analyse
Interpretation
• Agglutination of reagent RBCs is suggestive of immunoglobulin
presence in patients serum
False negative IAT
• Inadequate washing of cells
• Delay / interruption of washing procedure
• Failure to add antihuman globulin
• Under centrifugation
• Too heavy/ weak red cell suspension
• Prozone effect
• Insufficient incubation
• Reagent that has lost potency
Prozone effect
False positive IAT
• Agglutination of red cells before adding Antihuman globulin
• Over centrifugation
• Dirty glassware
• Cold autoantibodies in sample
• Non specific agglutination