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BB (LEC) FINALS 10/12/18 2.

INDIRECT ANTIGLOBULIN TEST (IAT)

ANTIHUMAN GLOBULIN TEST  Detects in vitro sensitization

ANTIHUMAN GLOBULIN TEST AHG REAGENT:

2 MAJOR TYPES OF BLOOD GROUP POLYSPECIFIC AHG


ANTIBODIES
 Contains anti-IgG and anti-
IgM C3b/C3d

 Large PENTAMER structure MONOSPECIFIC AHG


 Binds to corresponding antigen and
directly agglutinate RBCs  Contains only one antibody
suspended in saline specificity
 Anti-IgG
IgG  Anti-C3b/C3d
 MONOMER structure PREPARATION OF AHG REAGENT
 Some IgG antibodies are termed
nonagglutinating or incomplete Involves injecting human serum or purified
antibodies globulins into laboratory animals.
 Too small to directly agglutinate
 Human globulin behaves as foreign
sensitized RBCs
antigen
NOTES:  Laboratory animal will produce
antibodies against the human
 Adding AHG that contains anti-IgG globulins
to RBCs sensitized with IgG
antibodies allows for TWO TYPES OF ANTIBODY
hemagglutination. PRODUCTION

ANTIHUMAN GLOBULIN TEST  Polyclonal AHG production


 Monoclonal AHG production
 Also referred to as COOMB’S TEST
 Can be used to detect RBCs PREPARATION OF AHG REAGENT
sensitized with
1. POLYCLONAL AHG PRODUCTION
 IgG alloantibodies
 IgG autoantibodies  Usually prepared using RABBITS
 Complement proteins  Prepared by conventional technique
 SHEEP OR GOATS may be
2 TYPES OF AHG TEST:
used when large volumes of
1. DIRECT ANTIGLOBULIN TEST (DAT) antibody are required
 Rabbits are injected with either
 Detects in vivo sensitization complement or IgG
 Antibody produced is collected and
purified:
 Anti-IgG  Drug-induced Hemolytic
 Anti-C3b/C3d Anemia
 Monospecific antibodies are
INDIRECT COOMB’S TEST (IAT)
pooled together to form AHG
Polyspecific reagents  Detects in VITRO SENSITIZATION
 Used in the following situations:
2. MONOCLONAL AHG PRODUCTION
 Compatibility testing
 Usually prepared using MICE  Antibody screening
 Employs the use of HYBRIDOMA  Determination of RBC
TECHNOLOGY phenotype
 Mice is injected with purified  Determination of Du (weak
human globulin D)
 After a suitable immune
response, spleen cells CHECK CELLS
containing antibody-secreting
COOMB’S CELLS
lymphocytes are harvested
 Spleen cells are combined with  Group O RBCs sensitized with IgG
myeloma cells in the presence of  Added to Negative Antiglobulin
PEG Tests to validate negative AHG
 Spleen Cells + Myeloma reactions
Cells = HYBRIDOMA  After addition of O check cells,
CELLS agglutination indicates:
 Clone cells that produce only  AHG reagent was added
one type of antibody  AHG reagent was not
 Fused cells are placed in a HAT neutralized
medium  Lack of agglutination invalidates
 Hypoxanthine, result
Aminopterin, and
Thymidine FACTORS AFFECTING AHG TESTING
 Only allows the growth of
RATIO OF SERUM TO CELLS
Hybridoma cells
 ↑ ratio = ↑ sensitivity of the test
DIRECT COOMB’S TEST (DAT)
 Minimum target ratio 40:1
 Detects in VIVO SENSITIZATION  2 drops of serum
 Conditions that can result in in vivo  1 drop of 5% RCS
sensitization of RBCS:
1. ALBUMIN
 Hemolytic Disease of the
Newborn (HDN)  Makes use of 22% BSA (bovine
 Hemolytic Transfusion serum albumin)
Reactions (HTR)  Decreases incubation time from 60
 Autoimmune Hemolytic minutes to 30 minutes.
Anemia (AIHA)
2. LOW IONIC STRENGTH SOLUTIONS  Vigorous resuspension =
(LISS) false-negative

 Enhance antibody uptake by


reducing the zeta potential between
red cells
 Decreases incubation time from 30-
60 mins to 10-15 mins

3. POLYETHYLENE GLYCOL (PEG)

 Increases antibody uptake by


removing water molecules
surrounding the RBCs
 Concentrating antibody

TEMPERATURE

 Rate of reaction of antibodies is


optimal antibodies at 37°C

INCUBATION TIME

 Saline = 30-120 mins


 LISS/PEG = 10-15 mins at 37°C

WASHING OF RBCS

 Cells must be washed at least 3


times before adding AHG reagent
 Inadequate washing causes a
FALSE-NEGATIVE REACTION

SALINE FOR WASHING

 Buffered to a pH of 7.2-7.4

ADDITION OF AHG

 AHG should be added immediately


after washing

CENTRIFUGATION FOR READING

 Recommended method: 1,000 RCF


for 20 seconds
 ↑ RCF = ↑ sensitivity
 Inadequate resuspension =
false-positive

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