PRELIM: IMMUNOHEMATOLOGY

JC
Title
❖ Title

ANTIGLOBULIN TEST
▪ IgM: binds corresponding antigen and directly agglutinates rbc
suspended in saline because of its large pentameric structure.
o Most efficient at agglutination reaction
o large structure
▪ IgG: called non-agglutinating or incomplete antibody due to its single
monomer structure
o too small to directly agglutinate the sensitized rbc

PURPOSE OF AHG TEST:

▪ to detect the igG antibodies and in addition to igG; the complement
proteins attached to red cells but doesnt produce visible agglutination. ▪ Henry’s (source)
▪ From the word: “Anti” - against and “globulin” - antibody; anti antibody ▪ Direct Antiglobulin test: patient’s sample is sensitized in vivo, no need
or anti igG that forms/serves as a link or bridge to sensitize rbc to form for incubation step.
visible agglutination. ▪ Indirect Antiglobulin test: Patient sample is antibody + red cell antigen
na reagent or red cell yung patient sample + antibody reagent.
OBJECTIVE o Need ng incubation timekasi dapat magkaroon muna
❖ State the principle of the antiglobulin test. sensitization before ma add ang AHG reagent and upon addition
❖ Differentiate monoclonal from polyclonal and monospecific from of reagent magkakaroon ng formation of latticework with visible
polyspecific AHG reagents. agglutinates.
❖ Describe the preparation of monoclonal and polyclonal AHG
PRINCIPLE OF AHG TEST:
reagents.
❖ Compare and contrast the IAT and DAT. ▪ Antihuman globulins obtained from immunized nonhuman species bind
❖ Include an explanation of (1) principle, (2) applications, and (3) RBC to human globulins such as IgG or complement, either free in serum or
sensitization. attached to antigens on RBCs.
❖ List the factors that affect the antiglobulin test. ▪ The test uses a reagent which has been prepared by injecting animals
(usually rabbit), sheep or goats may be used for large antibodies.
HISTORY ▪ The rabbit is injected with human antibody molecules (Human igG and
complement proteins -> these 2 are recognized as foreign -> na
1. 1945, Coombs, Mourant & Race: use of antiglobulin test for
produce na antibodies ng rabbits ang gagamitin na sa AHG reagent -> sa
detection of weak and nonagglutinating Rh antibodies in serum
test system ma add yung reagent that is specific sa igG antibody and
2. 1946, Coombs & coworkers: use of AHG to detect in-vivo
c3b antibodies and it then magkakaroon ng reaction)
sensitization of the RBCs of babies suffering from HDN
▪ It is essential na yung red cells ay ma wash with saline to remove any
3. 1947, Coombs & Mourant: demonstrated that antibody activity
unbound molecules before addition of AHG reagent, if the red cells are
that detected Rh antibodies was associated with the anti-gamma
inadequately wash, yung unbound antibodies or complement na present
globulin fraction in the reagent
sa test, sila yung mag bbind sa AHG reagent that will inhibit the reaction
4. 1952, Dacie: observed that different reaction patterns were
of antibody or complement attached to the red cells called
obtained when dilutions of AHG were used to test cells sensitized
neutralization (blocks the antibodies).
with warm (IgG, 37C) as compared with cold antibodies (IgM, <22C)
5. 1957, Dacie & coworkers: showed that reactivity of AHG to cells AHG TEST CAN BE USED TO DETECT RBCS SENSITIZED
sensitized with warm antibodies resulted from anti-gamma globulin WITH:
activity, whereas anti-nongamma globulin activity by cold antibodies ✓ igG alloantibodies: formed during tranfusion when exposed to foreign
antigens
AHG TEST ✓ igG autoantibodies: formed against self antigens sa autoimmune
▪ also called the Coombs' test diseases
▪ first developed by Coombs but principle was o Presence of autoantibodies will cause problems to the test
described by Moreschi procedures, usually (+) DAT results.
▪ involves red cells coated with incomplete o DAT: performed to see if may transfusion reaction ang nag
antibodies (IgG), which are agglutinated by occur.
anti-IgG in antiglobulin serum thru linking of the o (+) DAT: it indicated na may transfusion reaction or HDN or
IgG molecules on neighboring red cells hemolytic anemia ang nag occur meaning hindi compatible ang
▪ AHG/Coombs Reagent: green blood ang naisalin sa patient. Mahirap hanapan ng compatible
▪ Named after the discoverer Robin Coombs na blood.
▪ Principle of test was described by Carlo ✓ Complement components: during blood transfusion, foreign blood
Moreschi cells enter the body and in the presence of allantibodies to antigenic
polymorphisms on the recipients tissue, the complement system can be
activated which can result in adverse transfusion reactions.

▪ All of these components can be detected by AHG test.

TWO AHG TEST:

1. Direct Antiglobulin Test (DAT): test is used detect antibodies or

complement bound to RBCs in vivo
2. Indirect Antiglobulin Test (IAT): test is used to detect the reaction
of antibody and RBCs in vitro after an appropriate incubation phase

AHG REAGENTS AND PROCEDURES

▪ Anti igG component of the AHG reagent serves as the link/bridge para
magkaroon ng visible agglutination. 1. Polyspecific Reagents: produced by hyperimmunizing animals,
usually rabbits, with purified Ig or complement to produce high-
titered, high-avidity IgG antibodies
2. Monospecific Reagents
 1. Polyspecific AHG Reagent: contains both anti-IgG and anti-C3d
antibodies and detects both IgG and C3d molecules on red cells PREPARATION OF AHG
o Kaya c3b and c3b ang complement na involved kasi during ▪ AHG production involves injecting human serum or purified globulin into
complement activation yung c3 is split into c3a and c3b, laboratory animals such as rabbits.
yung c3b ang nag bbind to rbc membrain and yung c3a it ▪ The human globulin behaves as foreign antigen, the rabbit's immune
passe into the fluid phase, and then yung c3b is further response is triggered, and an antibody to human globulin is produced.
cleaved to c3c and c3d, yung c3c during incubation yung
number ng determinant sites ay nag ffa, rapidly and then
yung c3d nag bbind din sa rbc membrane.

2. Monospecific AHG Reagent: reagents prepared by separating the

specificities of the polyspecific AHG reagents into individual sources
of anti-IgG and anti-C3d/anti-C3b
o Ginagamit ito sa further investigation or testing ng (+) DAT
result, this is to identify kung igG or complement ang nag
ccause ng reaction, called differential DAT.
✓ Anti-IgG: contain antibodies specific for the Fc fragment of the
gamma heavy chain of the IgG molecule
✓ Anti-Complement: are reactive against the designated
complement components only and contain no activity against
human immunoglobulins

SUMMARY OF AHG REAGENTS:

❖ ANTIHUMAN GLOBULIN REAGENTS: TWO METHOD OF AHG TEST:

DIRECT ANTIGLOBULIN TEST (DAT)

▪ One-stage procedure
▪ Ordered to detect IgG or complement proteins bound to patient cells,
which is a consequence of certain clinical events, including AHA, HDFN,
drug-related mechanism, or an antibody reaction to tranfused red cells
▪ A positive DAT (agglutination) is an important indicator of potential
immune-mediated red cell destruction in the body
▪ Negative DAT (no agglutination)
▪ Specimen: collected in EDTA
▪ DIFFERENTIAL DAT: test that uses monospecific anti-IgG and
monospecific anti-C3d/anti-C3b reagents to determine the cause of a
positive DAT with polyspecific antiglobulin reagents

▪ Polyclonal: made from several different B cell clones that secretes

antibodies of different specificity.
▪ Monoclonal: made from a single B cell clone that secretes antibody of
the same specifity and recognize a single epitope.
o Derived from hybridroma technique.

CLINICAL EXAMPLES CAUSING A POSITIVE DAT

Clinical condition Caused by Source of igG

Transfusion reaction Donor cells with igG Recipient (patient)
antibody
HDFN (Hemolytic Fetal red cells coated Maternal antibody
Disease of the fetus and with igG crossing the placenta
newborn)
Autoimmune hemolytic igG or C3 on patient red Patient autoantibody
anemia cells
Drug related igG-drug complex Immune complex formed
mechanisms attached to cells with drug
 FACTORS AFFECTIC ANTIGLOBULIN TEST
INDIRECT ANTIGLOBULIN TEST (IAT) 1. Ratio of Serum to Cells: increasing the ratio of serum to cells
▪ Two-stage procedure; this incudes the incubation step. increases sensitivity of defense system
▪ Incubation step will allow time for the antibody na ma attach sa rbc. 2. Reaction Medium: discussion niyo raw last meeting yung
▪ Designed to detect in vitro sensitization of red cells enhancement media: common dito -> albumin(rarely used),
▪ Used in the following situations: LISS(advantage: decrease of incubation time (from 30-60 mins to
✓ Detection of incomplete (nonagglutinating) antibodies to potential 10-15 mins nalang) by reducing the zeta potential surrounding the
donor RBCs (compatibility testing) or to screening cells (antibody rbc; mechanism of LISS: reduction of zeta potential, PEG
screen) in serum (Polyethylene glycol) this removes water molecule surround the rbc,
✓ Determination of RBC phenotype using known antisera thereby effectively concentrating antibodies.
✓ Titration of incomplete antibodies 3. Temperature: 37C
4. Incubation Time: saline (30-100 mins); LISS (reduced to 10-15
mins)
5. Washing of RBCs
6. Saline for Washing: fresh with suggested open expiration of 30 days
and buffered to pH 7.2-7.4. Saline when they are spoiled for long
periods in plastic containers it has been shown to decrease in pH
which may increase the rate of antibody elution during washing
process resulting false negative results.
7. Addition of AHG: add immediately after washing, to minimize the
chance na ma remove ang antibodies sa red cells
8. Centrifugation for Reading: recommended for evaluation for AHG:
1000 rcf for 15-20 seconds

SOURCE OF ERROR IN ANTIGLOBULIN TESTING

❖ COMMON SOURCE OF FALSE POSITIVE ERROR IN ANTIGLOBULIN
TESTING
False positive Possible explanation
Red cells re agglutinated before Potent cold negative antibody of
APPLICATIONS OF DIRECT ANTIGLOBULIN TEST IN THE
IMMUNOHEMATOLOGY LABORATORY washing step and addition of patient origin
antihuman globulin reagent.
Use of dirty glassware Particles or contaminants
Procedure Purpose
Improper Centrifugation— Red cell button packed to tightly on
Antibody screening Detects antibodies with specificity to red cell antigens
overcentrifugation centrifugation that nonspecific
Antibody identification Identifies specificity of red cell antibodies
clumping cannot be dispersed
Crossmatch Determines serologic compatibility between donor
and patient before transfusion ▪ Tignan niyo raw sa table ng harmening kasi madami raw don
Antigen typinf Identifies a specific red cell antigen in a patient or
donor ❖ COMMON SOURCE OF FALSE NEGATIVE ERROR IN ANTIGLOBULIN
TESTING
COMPARISON OF DIRECT AND INDIRECT False negative Possible explanation
ANTIGLOBULIN TEST PROCEDURES Failure to wash cells adequately during Unbound human serum globulins
the test procedure before the addition neutralize AHG reagent
Direct antiglobulin test Indirect antiglobulin test of AHG reagent
Detects igG and complement coated Detects igG and complement coated Testing is interrupted or delayed; AHG Bound IgG or complement molecules
red cells red cells reagent is not added immediately after may detach from the coated red cells
igG attachment to red cell has occurred igG attachment and red cells occurred washing
within the patient’s body during thr incubation step Failure to identify weak positive Technical error in testing
One stage procedure Two stage procedure reactions
Patient’s red cells are tested with Test required an incubation step before Loss of reagent activity Improper reagent storage, bacterial
antiglobulin reagent without an the addition of antiglobulin reagent contamination, or contamination with
incubation step human serum
Failure to AHG reagent Technical error in testing
Test for certain clinical condition: Used as a reactiom phase of several
HDFN, Hemolytic transfusion reaction test in immuhematology: antibody Improper centrifugation: Conditions for promoting agglutination
and autoimmune hemolytics anemia screen and antibody indentification undercentrifugation are not optimal
panel Inappropriate red cell concentration: Concentration of red cells influences
red cell suspensions fall outside the the asglutination reaction
optimal 2%-5%

MODIFIED & AUTOMATED ANTIGLOBULIN

TEST TECHNIQUES

SOLID PHASE TECHNOLOGY

▪ May be used for performing antiglobulin tests
▪ One of the test reactants (either antigen or antibody) is bound to a solid
phase support (usually a microtiter well) before the test is started

GEL TEST
▪ Detects RBC antigen-antibody reactions by means of a chamber filled
with polyacrylamide gel
▪ The gel acts as a trap; free unagglutinated RBCs form pellets in the
bottom of the tube, whereas agglutinated agglutinated RBCs are trapped
in the tube for hours
▪ NEGATIVE reactions appear as pellets in the bottom of the microtube;
▪ POSITIVE reactions are fixed in the gel

▪ Need daw ito i memorize sabi ni maam cutie kahit ayaw mu

▪ Memorize mo lahat ng table ma konsensya ka kasi tinype ko yan
hehe
 AHG METHODOLOGIES
1. Saline Tube testing
2. LISS Tube testing
3. PEG Tube testing
4. Gel
5. Solid Phase

QUALITY CONTROL OF AHG TEST

▪ To standardize antiglobulin sera and to confirm true negative antiglobulin
reactions, two types of QC RBCs are normally used:
✓ those coated with IgG
✓ those coated with C3b and/or C3d
▪ To sensitize RBCs with IgG, Rh antibodies are usually used
▪ RBCs coated with C3b are prepared by incubation of whole blopd in LISS
or with human anti-Le(a) or anti-I
▪ C3d-coates RBCs are prepared by incubating C3b-coated with fresh
serum or trypsin to split C3b→C3d
▪ IgG or complement-coated control cells should give a 1+ to 2+ reaction
when tested with anti-IgG or anti-C3b+C3d

Book: henry’s,
harmening
Lecture Notes
PPT