This document provides information about the Coombs test, which is used to detect antibody or complement coating of red blood cells. It describes the history and principles of the test, as well as the direct and indirect Coombs test procedures. The direct Coombs test detects in vivo coating of red blood cells and is used to diagnose conditions like hemolytic disease of the newborn. The indirect Coombs test detects in vitro coating of red cells and is used for compatibility testing and antibody screening. Factors affecting the tests and causes of false positive and negative results are also outlined.

1. COOMBS TEST
MR. MANOJ MEHTA

2. HISTORY
• 1908,
• Moreschii, described the principle of the test (the use of
rabbit anti-goat serum to agglutinate rabbit RBCs that were
sensitized with low non-agglutinating doses of goat anti-rabbit
RBC serum.)
• 1945 ,
• R R coombs and associates described the use of the
antigolubulin test for the detection of weak and non
agglutinating Rh antibodies in the serum .
• 1946,
• coombs and coworker describe the use of AHG to detect in
vivo sensitization of the RBCs of babies suffering from HDN
• Kell blood grouping was described by using antiglobulin test

3. INTRODUCTION
• It It is based on the principle of AHG obtained from
immunized non human species bind to human
globulin IgG or complement either free or attached to
Ag of RBCs
• The use of AHG serum to detect sensitization of red cells
in vitro can be:
• One stage technique , the direct anti globulin test
(DAT).
• Two stage technique , the indirect antiglobulin test
(IAT).

4. PRINCIPLE
 Normal human red blood cells, in presence of antibody
directed towards the antigen they possess, may fail to
agglutinate when centrifuged and become sensitized.
This may be due to the particular nature of the antigen and
antibody involved.
 Sensitization of RBC’s may be with IgG or complement.
 In order for agglutination to occur an additional of anti-
antibody or anti-complements, which reacts with the
Fc portion of the IgG antibody, or with the C3b or C3d
component of complement alternatively.

5. • This will form a "bridge" between the antibodies or
complement coating the red cells, causing
agglutination.
• The coating (sensitization) of red cells can occur in vivo
or in vitro following incubation at 37°C with serum
containing antibody.
PRINCIPLE

6. ANTI- HUMANGLOBULINREAGENT
• a kind of antibody that react with the human globulin
• AHG combines with the Fc portion of a sensitizing antibody.
• This completes the antigen-antibody bridge, allowing agglutination
to occur.
• Can be of two types
• Polyspecific Anti-human Globulin: blend of Anti-IgG and Anti-C3b, -C3d
• Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone

7. PREPARATIONOFAHG
• Polyclonal AHG :-
• mixture of Ab from different plasma cell clone
• Can recognize different antigenic determinants(epitope
• Or same epitope by different affinities
• Monoclonal AHG:-
• Detect only one epitope on the antigen
• They will consist of only one antibody subtype where a
secondary antibody is required for the detection , an
antibody against the correct subclass should be chosen

8. Preparation of polyclonal AHG
Preparation of purified IgG AND C3b containing serum form many
donors
Injection of serum to lab animal
After potency and specificity is determine by blood sample from
animal an meet the required criteria animal are bleed
Pooling of anti –IgG
Purification of AHG is done
Content in determine I e its potency affinity and specificity
AHG is diluted to get required concentration with required potency

9. MONOCLONALAHG
Immunization of animal (mice) with purified human globulin
Spleen cells contains Ab-secreting lymphocytes are fused with
myeloma cell( hybridization)
The resulting hybridoma are screened for antibodies with required
specificity
Ab secreting clones are then passes to tissue culture or back to new
mice (ascites)
AHG is obtained an diluted to required concentration

10. COOMB’S CELLS
 To show that test cells were properly washed and that no
neutralization or reagent deterioration has occurred,
antibody-coated cells are used as a positive indicator.
 In a negative antiglobulin test the anti-human globulin
should remain active and this can be demonstrated by the
addition of IgG sensitized cells.
 Agglutination of the IgG sensitized cells after mixing and
centrifuging confirms that the anti-human globulin was
added to the test, that the test cells were properly washed
and all free globulin molecules were removed and that the
anti-human globulin was active.
 Failure of the IgG sensitized cells to agglutinate indicates
that the original negative antiglobulin test result is not valid
and testing must be repeated.

11. PREPARATION SENSITIZED CELL
Washgroup o positive RBCs 3 times in normal saline
Make a 50% cell suspension
Dilute the incomplete anti-D reagent 1:5 in saline
Add 1 volume cell suspension to 10 voumes diluted anti-d
Incubate at 37c for 20 mins
Wash RBCs 4 times with normal saline
Make 5% suspension of red cells
Add 1 drop of 5% cells to ! Drop of AHG reagent an spin
Agglutination of cell showing 1+ or 2+ show presence of sensitizes
cell

12. DIRECt COOMBS TEST (DCT)

13. • The direct antiglobulin test (DAT) detects sensitized red
cells with IgG and/or complement components C3b and
C3d in vivo.
• In vivo coating of red cells with IgG and/or complement
may occur in any immune mechanism is attacking the
patient's own RBC's.
• These mechanism could be:
• Autoimmunity
• Alloimmunity
• Or a drug-induced immune-mediated mechanism.

14. PRINCIPLE OF DAT
• Sensitization of RBCs occur in vivo
• AHG reagent is added to the washed RBCs and incubated to
observe agglutination
Sensitized RBCs addition of AHG agglutination

15. METHOD OF DCT
• Tube method :-
• Gel technique method:-
• Based on column technology
• RBCs are filtered through a column containing a gel which contained in
strips of micro well
• Gel medium contain anti IgG and anti C3b
• Sensitized RBCs binds anti-glubulin and gets trapped in gel column
• Unsensitized cell pellet at the bottom of column
• Flow cytometry method :-
• Immunofluorescences technique is utilized
• Can detect and measure low level of cell bound IgG

16. PROCEDURE OF TUBE METHOD

17. RESULT INTERPRETATION
• Alloimmune hemolysis :-
• Hemolytic transfusion reaction :-
• The patient has an antibody in the plasma that is directed against an
antigen on the donor red cells.
• The patient’s antibody coats the donor cells.
• In an acute (immediate) hemolytic transfusion reaction,
complement is activated.
• The donor cells are lysed (intravascular hemolysis).
• The DAT may be positive due to IgG or complement.
• In a delayed hemolytic transfusion reaction, the antibody
coated cells are removed via phagocytosis.(extravascular lysis)
• A drop in hemoglobin usually occurs 2-10 days following transfusion.
• The DAT is usually positive due to IgG.

18. HEMOLYTICDISEASEOFTHENEWBORN(ALSOKNOWN
ASHDNORERYTHROBLASTOSISFETALIS)
• Occurs in :-
• Rhesus D hemolytic disease of the newborn (also known as Rh disease)
• ABO hemolytic disease of the newborn (the indirect Coombs test may only be
weakly positive)
• Anti-Kell hemolytic disease of the newborn
• Rhesus c, E hemolytic disease of the newborn
• Other blood group incompatibility (RhC, Rhe, Kidd, Duffy, MN, P and others)
• Occurs by
• Mother must have been stimulated to make an IgG antibody.
• Antibody must cross the placenta.
• Fetus must be antigen positive.
• Fetal cells become coated with maternal antibody & are cleared by
the fetal RES. In HDFN, the infant’s cells are coated with maternal IgG
antibody, resulting in a positive DAT in the infant.
• The DAT is THE diagnostic test for HDFN.

19. AUTOIMMUNE HEMOLYSIS
• Warm antibody autoimmune hemolytic anemia:-
• Idiopathic
• Systemic lupus erythematosus
• Evans' syndrome (antiplatelet antibodies and hemolytic
antibodies)
• Cold antibody autoimmune hemolytic anemia:-
• Idiopathic cold hemagglutinin syndrome
• Infectious mononucleosis
• Paroxysmal cold hemoglobinuria (rare)

20. DRUG INDUCE HEMOLYSIS
• Mechanism of hemolysis
• Drug Adsorption
• Drug attaches to RBC.
• Antibody directed at drug only
• DCT positive due to IgG
• Immune Complex
• Antibody directed at a “neoantigen” having determinants on both the
drug and the red cell membrane.
• Antibody activates complement
• DCT positive due to complement
• Some drugs showing positive DCT
• Methyldopa (IgG mediated type II hypersensitivity)
• Penicillin (high dose)
• Quinidine (IgM mediated activation of classical complement pathway
and Membrane attack complex)

21. INDIRECT COOMBSTEST(ICT)
• The ICT is performed to determine in-vitro sensitization of RBCs
• Serum containing incomplete Ab in incubated with RBCc to
sensitized and than AHG is added to observe agglutination
• Application :-
• Detection of incomplete (nonagglutinating) antibodies to
• potential donor RBCs (compatibility testing) or to screening cells
(antibody screen) in serum
• 2. Determination of RBC phenotype using known antisera
• (e.g., Kell typing, weak D testing)
• 3. Titration of incomplete antibodies

22. PROCEDURE OF TUBE ICT

24. FACTORS AFFECTING THE IAT
• Serum/Cell ratio:- increasing the ratio increases the sensitivity so
minimum 40:1 is selected’
• Reaction medium or use of potentiaters
• Incubation temperature:- 37⁰c is selected as majaority of reaction occurs
at this temperature
• Length of incubation:- saline 30-60n, LISS 10-15
• Washing of RBC:- at least three time washing is required to remove
unbound AB before adding AHG . Should be done as soon as possible
• Saline use to wash :- should of pH 7.2-7.4 , stored long time in plastic
container can decrease the pH causing elution of Ab from RBCs
• Addition of AHG:-
• Centrifugation for reading

25. POTENTIATORS
• Some incomplete
antibodies will not react in
a saline environment.
• Potentiators are reagents
that adjust the test
environment.
• Reduce the zeta potential
• Promote agglutination
• Enhance antibody uptake

26. • 22% bovine solution :-
• High molecular weight protein
• Reduces the zeta potential by dispersing some of the cations
surrounding each negatively charged red cell.
• Increases the dielectric constant, defined as a measure of ability to
dissipate a charge.
• Polyethylene glycol :-
• a low ionic strength medium.
• Removes water from the test system, thereby concentrating any
antibody present.
• Antibody uptake is also increased.
• PEG can cause cellular aggregation, therefore, tests using PEG can not be
centrifuged and evaluated following a 37oC incubation. Testing should proceed
immediately to the wash phase, with a minimum of 4 washes performed.
• PEG is not the potentiator of choice when the patient has elevated proteins,
such as in multiple myeloma. In these cases, LISS is the preferred
enhancement.
POTENTIATORS

27. • Improper sample(refrigerated , clotted ) may causes
invitro complement attachment
• Autoagglutinable cells
• Bacterial contamination of cells or saline used in washing
• Cells with positive direct AhG test used for the ICT
• Saline contaminated by heavy metals or colloidal silica
• Dirty glassware
• Over centrifugation and over reading
• Polyagglutinable cells
• Preservative –dependent antibody in LISS reagent
• Contaminating antibodies in the AHG reagent
• Centrifugation of test with polyethylene gylcol prior to
washing
FALSE POSITIVE RESULTS

28. FALSE NEGATIVE RESULTS
• Inadequate or improper washing of cells
• AHG reagent nonreactive because of deterioration or
neutralization
• AHG reagent not added
• Serum not added in the indirect test
• Serum nonreactive because of deterioration of
complement
• Inadequate incubation conditions in the IAT
• Cell suspension either too weak or too heavy
• Undercentrifuged or overcentrifuged
• Poor reading technique
• Low pH of saline

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