ANTIGLOBLULIN TEST

KAREN MAE A. MARGAJA,

RMT

DEFFINITION OF TERMS
ALLOANTIBODIES
= an antibody produced following introduction of an alloantigen into the
system of an individual of a dif. Genes but same species
AUTOANTIBODIES
= an antibody produced by an organism in response to a constituent of its
own tissues.
POLYCLONAL
= antibodies derived from more than one antibody producing parent cell
MONOCLONAL
= antibodies derived from a single ancestral antibody-producing parent cell.
SENSITIZED
= RBC antigens bound to antibodies either in vitro or in vivo.
AGGLUTINATION
= clumping together of RBCs or any particle resulting from interaction of
antibody and its corresponding antigen.
ADSORPTION
= Adsorption is a procedure used by blood bankers to bind antibodies to red
blood cells in order to remove them from the plasma and better analyze the
antibodies that might remain behind.

DEFFINITION OF TERMS

COMPLEMENT
= Complementis a system of
plasmaproteinsthat can be activated
directly by pathogens or indirectly by
pathogen-bound antibody, leading to a
cascade of reactions that occurs on the
surface of pathogens and generates active
components with various effector functions.
POTENCY
= EFFECTIVITY (ex. The potency of the
harvested pooled anti-IgG)

History of COOMBS TEST

discovered by ROBIN COOMBS and
associated in 1945.
BEFORE ONLY IgM ANTIBODIES were
detected
AHG permitted the detection of non
agglutinating IgG antibodies.
Led to the discovery and characterization
of many new blood group systems.
Kell, Rh, P, LUTHERAN, ETC.

2 MAJOR TYPES OF BLOOD GROUP

ANTIBODIES

IgM = PENTAMERIC = AGGLUTINATING

IgG = MONOMERIC= NONAGGLUTINATING =
TOO SMALL TO AGGLUTINATE SENSITIZED
RBCs DIRECTLY.
ADDITION OF AHG TO THE RBCs SENSITIZED
W/ IgGs and/or complement, ALLOWS
HEMAGGLUTINATION OF THESE SENSITIZED
CELLS.

ANTIGLOBULIN TEST
A.K.A COOMBS TEST
DETECT IgG AND/OR COMPLEMENT
SENSITIZED RBCs.

BASIC PRINCIPLE:
AHGs OBTAINED FROM IMMUNIZED
NONHUMAN SPECIES BIND TO
HUMAN GLOBULINS SUCH AS
IgG/COMPLEMENT, EITHER FREE IN
SERUM OR ATTACHED TO ANTIGENS
ON RBCs.

AHG

AHG PRODUCTION

IN THE HUMAN BLOOD.

SO WHAT DOES AHG DO?

BINDS IgG

ANTICOMPLEMENT

2 types of Coombs test

DIRECT COOMBS TEST (DAT)
THE USE OF AHG TO DETECT IN VIVO
SENSITIZATION OF RED CELLS IS A ONE-STAGE
TECHNIQUE.

INDIRECT COOMBS TEST (IAT)

THE USE OF AHG TO DETECT IN VITRO
SENSITIZATION OF RED CELLS IN A TWO-STAGE
TECHNIQUE.

ANTIHUMAN GLOBULIN
REAGENTS
2 KINDS:
1. POLYSPECIFIC
2. MONOSPECIFIC
THESE REAGENTS ARE DEFINED BY
THE FOOD AND DRUG
ADMINISTRATION (FDA) CENTER FOR
BIOLOGICS EVALUATION AND
RESEARCH (CBER).

POLYSPECIFIC AHG
Contains anti-IgG and anti-C3d
Other anti-complement antibodies are
anti-C3b, anti-C4b, or anti-C4d.
commercially prepared polyspecific AHG:
> contains little, if any, activity against IgA
and IgM heavy chains.
> may contain antibody activity to kappa
and lambda light chains = reacting w/ IgA
or IgM.

MONOSPECIFIC ANTIHUMAN
G
CONTAIN ONLY ONE ANTIBODY
SPECIFICITY.
EITHER ANTIBODY TO SPECIFIC
COMPLEMENT COMPONENTS : C3b
or C3d or Anti-IgG
Licensed monospecific AHG
Reagents in common use are AntiIgG and Anti-c3b-CBd

ANTI-IgG
NO ANTI-COMPLEMENT ACTIVITY
CONTAIN ANTIBODY SPECIFIC FOR
THE FC FRAGMENT OF THE GAMMA
HEAVY CHAIN OF THE IgG MOLECULE.
IF NOT LABELLED GHC= MAY
CONTAIN ANTI-LIGHT CHAIN=
REACTIVE TO IgM and IgA.

ANTI-COMPLEMENT
ANTI-C3b-C3d
SPECIFIC/ REACTIVE AGAINST THE
DESIGNATED COMPLEMENT
COMPONENTS ONLY.
CONTAIN NO ACTIVITY AGAINST
HUMAN IMMUNOGLOBULINS.

GENERAL PRINCIPLE OF ANTIHUMAN GLOBULIN

REAGENTS

IgG ANTIBODIES ARE HETEROGENEOUS

THUS:
POLYCLONAL= ANTIBODIES ARE FROM A
MIXTURE OF ANTIBODIES FROM
DIFFERENT PLASMA CELL CLONES.
= RECOGNIZE DIFFERENT ANTIGEN
DETERMINANTS (EPITOPES) OR
= THE SAME PORTION OF THE ANTIGEN
BUT W/ DIFFERENT AFFINITIES.
ESSENTIAL IN PRODUCTION REAGENTS FOR
ROUTINE USE.

MONOCLONAL ANTIBODIES
BY HYBRIDOMA TECHNIQUE.
DERIVED FROM ONE CLONE OF
PLASMA CELLS AND RECOGNIZE A
SINGLE EPITOPE.
USED TO PRODUCE AHG AND HAS
PROVED PARTICULARLY USEFUL IN
PRODUCING HIGH TITER ANTIBODIES
W/ WELL-DEFINED SPECIFICITIES TO
IgG AND TO THE FRAGMENTS OF C3.

POLYSPECIFIC VS.
MONOSPECIFIC
THE MOST IMPORTANT FUNCTION OF
POLYSPEFIC AHG IS THE DETECTION OF
THE IgG ANTIBODIES.
ONE MUST BALANCE THE ADVANTAGE OF
DETECTING CLINICALLY SIGNIFICANT
COMPLEMENT-ONLY ANTIBODIES W/ THE
DISAVANTAGES RESULTING FROM USING
ANTIGLOBULIN SERUM CONTAINING ANTICOMPLEMENT ACTIVITY.

ANTIHUMAN GLOBULIN REAGENTS

AND THE DIRECT ANTIGLOBULIN TEST
DAT = IN VIVO SENSITIZATION OF RBCs W/
IgG AND OR COMPLEMENT COMPONENTS.
DAT IS HELPFUL IN DETECTING WARM OR
COLD AUTOIMMUNE HEMOLYTIC ANEMIAS.
W-AIHA= ASSOCIATED W/ BOTH IgG AND
C3d COATING THE RBCs.
C-AIHA= ONLY C3d MAY BE DETECTABLE
ON THE RBC MEMBRANES.
DAT = 1ST POLYSPECIFIC = IF POSTIVE =
MONOSPECIFIC.

Wash 3 times

READ/ INTERPRET

DAT APPLICATIONS

HDN
HTR
AIHA
DRUG INDUCED

DAT APPLICATIONS

PATIENT SAMPLE
PREPARATION

DONOR SAMPLE
PREPARATION

HECK FOR HEMOLYSIS

 FACTORS AFFECTING THE ANTIGLOBULIN

TEST
1. RATIO OF SERUM TO CELLS
2. REACTION MEDIUM
3. TEMPERATURE
4. INCUBATION TIME
5. WASHING OF CELLS
6. SALINE FOR WASHING
7. ADDITION OF ANTIHUMAN GLOBULIN
8. CENTRIFUGATION FOR READING