Prelim Notes For Ih 1119 Lecture PDF

ANTIHUMAN GLOBULIN
TEST
Compiled by: Fortune Lapira – Torrecampo, RMT, MPH
DEFINITION
• Antihuman: antibodies against human antibodies
• Globulin: all antibody molecules are globulins
Therefore: Antihuman Globulin is antibody directed

against the Fc portion of human antibodies and/or
complement components
fortune lapira - torrecampo, RMT, MPH 2

HISTORY
• The AHG test is also called the Coombs’ test, in
honor of one of the investigators who first
developed the test for laboratory use
(Coombs,1945), although the principle was
actually described much earlier (in 1908) by
Moreschi

RELEVANCE OF AHG
• Some very clinically significant unexpected antibodies
(Kidd, Duffy, etc.) attach to red cell antigen or activate
complement, but do NOT cause agglutination at
o
immediate spin or 37 phase.
• Yet, these antibodies are capable of causing severe

hemolytic transfusion reactions or hemolytic disease of
the newborn.
RELEVANCE OF AHG
• AHG techniques enable the detection of these antibodies or C’
components that otherwise went undetected. AHG enables detection
of both in vitro (Indirect Antiglobulin Test) and in vivo (Direct
Antiglobulin Test) antibody attachment.
AHG testing is the primary method of detecting all Antibodies except

those of the ABO System.

Reagent Definition
Rabbit polyclonal has anti-IgG, -C3d; rabbit/murine
monoclonal blend has rabbit polyclonal anti-IgG and
Polyspecific (rabbit polyclonal, rabbit/murine murine monoclonal anti-C3b, -C3d; murine monoclonal
monoclonal blend, murine monoclonal) has monoclonal anti-IgG,
-C3b and -C3d
Rabbit polyclonal has only anti-IgG; IgG heavy chains

Monospecific anti-IgG (rabbit polyclonal; only has antibody to gamma chains; monoclonal has
IgG heavy chains; monoclonal IgG) murine anti-IgG
Contains only antibodies against designated

Anti-C3d and anti-C3b or Anti-C4b, - Complement components with no anti-immunoglobulin
C4d, -C3d (rabbit polyclonal) activity
Anti-C3d or anti-C3b, -C3d (murine Contains only antibodies against designated

Complement components with no anti-immunoglobulin
monoclonal)
activity
Table 12.1, page 260, AABB Technical Manual, 13th Edition

REAGENT PREPARATION
• Polyclonal or monoclonal sources
• Polyclonal - Animals hyperimmunized with human globulins; bled for

antisera to obtain high titered, high avidity specificity to human
• Monoclonal - Hybridoma cells from mice; collection of culture or

ascites fluid yields antisera
• mice hyperimmunized with human globulins
• prepare cell suspension from spleens; fuse with immortalized myeloma cells
• screen hybridoma clones for desirable specificities and affinities
• maintain cultures of clones in vivo or in vitro
• Product is highly regulated for potency

REAGENT PREPARATION
• Polyspecific AHG
• Abs to human IgG, and
• Abs to human C3d (C3b breaks down to C3c and C3d)
• Advantage is that polyspecific AHG may detect complement-
dependent Abs on RBCs (anti-Jka)
• Disadvantage - more nuisance positives

REAGENT PREPARATION
• Monospecific AHG
• Abs to human IgG only or human C3d or C3b only
• Fewer nuisance positives; may miss an important Ab

Anti-IgG
• Reagents labeled “anti-IgG” contain no anticomplement activity
• Major component of anti-IgG is antibody to human gamma heavy

chains, but unless labeled as “heavy-chain-specific,” these reagents
may exhibit some reactivity with light chains, which are common to
all immunoglobulin classes
• An anti-IgG reagent not designated “heavy-chain-specific” must be

considered theoretically capable of reacting with light chains of IgA or
IgM

Anti-IgG
• A positive DAT with such an anti-IgG does not definitively prove the
presence of IgG, although it is quite rare to have an in-vivo coating
with IgA or IgM in the absence of IgG
• Many workers prefer anti-IgG over polyspecific AHG in antibody

detection and compatibility tests because anti-IgG AHG does not react
with complement bound to red cells by cold-reactive antibodies that
are not clinically significant

Anti-C3b, C3d
• Anti-C3b, -C3d reagents prepared by animal immunization contain no
activity against human immunoglobulins and are used in situations
described for anti-C3d
• This type of anti-C3d characteristically reacts with C3b and possibly

other epitopes present on C3-coated red cells
•
• Murine monoclonal anti-C3b, -C3d reagent is a blend of hybridoma-
derived antibodies

PRINCIPLE OF ANTIGLOBULIN TEST
• All antibody molecules are globulins
• Animals injected with human globulins produce antibody to the

foreign protein
• After the animal serum is adsorbed to remove unwanted agglutinins,

it will react specifically with human globulins and can be called AHG
serum

• AHG sera with varying specificities can be produced, notably, anti-IgG
and antibodies to several complement components
• Hybridoma techniques are used for the manufacture of most AHG

• Anti-IgG combines mainly with the Fc portion of the sensitizing
antibody molecules, not with any epitopes native to the red cell
• The two Fab sites of the AHG molecule form a bridge between
adjacent antibody coated cells to produce visible agglutination
• Cells that have no globulin attached will not be agglutinated.

• The strength of the observed agglutination is usually proportional to the
amount of bound globulin
• AHG will react with human antibodies and complement molecules that are
bound to red cells or are present, free, in serum
• Unbound globulins may react with AHG, causing false-negative antiglobulin

tests
• Unless the red cells are washed free of unbound proteins before addition of
AHG serum, the unbound globulins may neutralize AHG and cause a false-
negative result

DIRECT ANTIGLOBULIN TEST (DAT)

24
DIRECT ANTIGLOBULIN TEST
• Detects in vivo sensitization of RBCs
• Procedure
• Wash unknown RBCs 3X with saline (removes unbound
globulins)
• Add AHG (binds to IgG or C3d, if any, that is bound to RBCs;
forms agglutinates)
• Centrifuge (accelerates agglutination)
• Grade and record agglutination as 0 to 4+
• Add Ab-coated RBCs (check cells) to all negatives, spin, read and
record (checks that AHG was added and was functioning)
COOMBS’ 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.

COOMBS’ 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.

PREPARATION OF COOMBS’ CONTROL CELLS
• Indication for use:
• Coomb’s control cells are used when the antiglobulin test is NEGATIVE to
confirm that washing has been adequate and the antiglobulin reagent is
reactive. These control cells can be prepared in the transfusion service
• Principle:
• A group “O”, Rh positive donor sample is mixed with a 1:10 dilution of reagent
with anti-D and allowed to incubate after the red cells are sensitized. They are
washed with saline and suspended to a 5% red cell suspension. These
sensitized red cells are then used to confirm the anti-IgG activity in the AHG
reagent.

• Reagents:
• Freshly drawn group O, Rh positive cells
• Anti-D reagent
• 0.85% saline solution

Procedure:
1. Select a group “O”, Rh positive, freshly drawn donor sample. The sample
must be negative for hepatitis, VDRL, and antibody screen
2. Centrifuge tube and remove all plasma
3. Dilute the reagent and anti-D 1:10 dilution with normal saline (0.5ml anti-D
with 4.5 ml of normal saline)
4. Mix equal volumes of “O” Rh positive red cells with 1:10 dilution of anti-D
(200ul of PRBC “O” cells + 200uL of diluents anti-D). Incubate at 37 ̊C for 30
minutes mixing several times during incubation.
5. Centrifuge the tube to remove all supernatant anti-D. Wash the red cells 6x
with large volumes of saline

Procedure:
6. Suspend the sensitized cells in saline to a 5% red cell suspension
7. Add two drops of anti-human globulin reagent to 2 drops of
sensitized cells. Centrifuge. It should give a 2+ to 3+ reaction
8. Label the prepared red cell suspension as Coombs’ control cells.
Include the date and the initials of the person who prepared it.
9. Store at 1-6 ̊C when not in use
10. Record all information on the Coombs’ control cells log sheet

• INTERPRETATION:
• Coombs’ control cells should give a 2+ to 3+ reaction when tested with AHG

If the Check Cells agglutinate, it indicates
three (3) things:
• The test was adequately washed prior to addition of the

AHG reagent.
• AHG reagent was added to the test tube.
• The AHG reagent that was added was in an ACTIVE form.

What agglutinated Check Cells DOES NOT
mean!
• That the test was performed correctly!!
Why? Because…
• Patient Serum could have been left out of tube
• The WRONG Patient Serum could have been added
• The WRONG test cells could be used
• Incubation time or temperature may be incorrect
• Enhancement media (LISS) may not have been added
• The tube could be mis-labeled
• The results could be mis-read
• The results could be recorded incorrectly
DAT: CLINICAL CONDITIONS
• Alloimmune hemolysis
• Hemolytic disease of the newborn (HDN) (maternal IgG crosses

placenta and binds to infant RBCs; may be up to a 4+ reaction)
• Hemolytic transfusion reaction (recipient Ab coats donor RBCs;

usually about a 1+ reaction)
• Autoimmune hemolytic anemia (AIHA) (autoAbs coat own RBCs;

reaction strength variable)

• Hemolytic disease of the newborn (also known as HDN or
erythroblastosis fetalis)
• 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)
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)

DRUG-INDUCED IMMUNE-MEDIATED HEMOLYSIS
• Methyldopa (IgG mediated type II hypersensitivity)
• Penicillin (high dose)
• Quinidine (IgM mediated activation of classical

complement pathway and Membrane attack complex)

DAT: INTERPRETATION
• Usually do initial DAT using polyspecific AHG and then more

detailed testing, if necessary, with monospecific AHG
• With a positive DAT, consider:
• Evidence of in vivo hemolysis?
• Recently transfused?
• Unexpected allo- or autoAbs?
• Medications?
• Positive DATs with no clinical significance may be detected in
up to 2% of population
Indirect antiglobulin test

INDIRECT ANTIGLOBULIN TEST (IAT)
•Detection of in vitro sensitization of RBCs

•Procedure
• Same as DAT, except:
• Step 1 entails incubating RBCs (reagent or unknown)
with antisera (reagent or unknown) allowing time for
in vitro attachment of Abs to RBCs

IAT: APPLICATIONS
• When the sample is a sera:

• Detection of Abs in recipient sera that may be incompatible with
donor RBC Ags (compatibility test - in this case, the RBCs may
also be considered “unknown”)
• Detection of unexpected allo- or autoAbs in unknown sera
(antibody screen)
• Identification of unexpected allo- or autoAbs in unknown sera
(antibody identification)
• Titration of Ab in unknown sera or amniotic fluid

IAT: APPLICATIONS
• When the sample is a RBC:

• Determination of RBC phenotype (detection
of Ags) using known antisera (weak D testing)

Indirect Antihuman globulin Test (IAT)
Indications
• The IAT is done to determine the presence of sensitization of
red cells with IgG and/or complement in vitro in the following
conditions.
1.Compatibility testing.
2.Screening and detection of unexpected antibodies in
serum.
3.Determination of red cells phenotype K, Lea, Fya Fyb, Jka, Jkb
and sub-group of Rh etc by using known sera.
Set of Problems…
1. Even 10 mL of AHG can be neutralized by a tiny amount of free serum

protein.
2. A technologist can forget to add AHG to a test tube.
3. A couple of drops of residual saline can dilute the AHG reagent below
detectable levels.
• Normally people do NOT produce unexpected Antibodies. Therefore

the test should normally be negative.
HOW DO YOU KNOW A NEGATIVE AHG TEST IS A TRUE NEGATIVE?
TEST SENSITIVITY
• DAT detects about 150 to 500 IgG or C3d

molecules/cell
• IAT detects about 100 to 200 IgG or C3d

molecules/cell

FACTORS AFFECTING SENSITIVITY
• Ratio of serum to cells (increasing ratio by adding more serum may increase
sensitivity)
• Temperature (37oC is optimal)
• Incubation time
• in saline (30 to 60 min)
• in LISS (10 to 15 min)
• Washing must be thorough (else, neutralization of AHG) and rapid (else, elution of
bound Abs)
• Centrifugation (1000 RCF for 20 seconds)

REACTION MEDIA
• 22% albumin
• decreases zeta potential by buffering
• allows Ab-coated cells to come closer together
• Low Ionic Strength Solution (LISS)

• decreases zeta potential
• serum/cells very important; increase amount of serum with caution
• Polyethylene glycol (PEG)

• removes water, concentrating Ab
• use monospecific AHG with anti-IgG (else, false positives)
• do not read aggl. microscopically (false positives)

ABO (ISBT 001) and H (ISBT 018)
BLOOD GROUP SYSTEMS
Why is it important?
• pretransfusion testing
• expected antibodies
• transfusion reactions
fortune lapira - torrecampo, rmt, mph

ABO SYSTEM
• Most important blood system in
• transfusion
• organ transplantation
• Found on:
• red cells, platelets, and many circulating proteins
• Histo – blood group antigens:

• endothelium, kidney, heart, bowel, pancreas, and lungs
Soluble antigens are detected in secretions and all body fluids EXCEPT CEREBROSPINAL
FLUID. ABO blood group system antigens, which are intrinsic to the red cell membrane,
exist as either glycolipid or glycoprotein molecules, whereas the soluble forms are
primarily glycoproteins.
ABO SYSTEM
• Transfusion of ABO incompatible blood is associated with:

• Acute intravascular hemolysis
• Renal failure
• death

ABO SYSTEM
• Transplantation of ABO incompatible organ is associated with:

• Acute humoral rejection

ABO antigens

BIOCHEMISTRY
• ABO antigens: carbohydrate antigens
• On RBC: expressed on type 2 chain oligosaccharides

ABO IN DEVELOPMENT AND AGING
• ABO antigens: detected on red cells of embryos as early as 5 – 6 weeks of
gestation
• Adult levels of ABO expression: 2-4 years of age

ABO INHERITANCE
“An individual inherits one ABO gene from each parent
and that these two antigens determine which ABO
antigens are present on the RBC membrane” -
Bernstein 1924
• Codominant in expression
• Genotypes: AO, BO, OO, AA, BB, AB
• Phenotypes: “A”, “AB”, “B”, “O”

Location
• ABH antigens on the red blood

cell membrane
• controlled by H gene
• ABH antigens in secretions

• indirectly controlled by Se gene

ABO Antigen Genetics
• H gene
• H and h alleles (h is an amorph)
• Se gene
• Se and se alleles (se is an amorph)
• ABO genes
• A, B and O alleles

H Antigen
• The H gene codes for an enzyme that adds the sugar fucose
to the terminal sugar of a precursor substance (PS)
• The precursor substance (proteins and lipids) is formed on an

oligosaccharide chain (the basic structure)

H antigen
• H antigen immunodominant sugar: L-fucose
• H enzyme: fucosyltransferase
• A and B genes code for enzymes that add an immunodominant sugar to

the H antigen
• “A” antigen immunodominant sugar: N-acetylgalactosamine
• “B” antigen immunodominant sugar: D-galactose

RBC Precursor Structure
RBC
Glucose
Galactose
Precursor
Substance (stays
the same) N-acetylglucosamine
Galactose

Formation of the H antigen
RBC
Glucose
Galactose
H antigen
N-acetylglucosamine
Galactose
Fucose fortune lapira - torrecampo, rmt, mph

A and B Antigen
• The “A” gene
• codes for an enzyme (transferase) that adds
N-acetylgalactosamine to the terminal sugar of the H antigen
- enzyme: N-acetylgalactosaminyltransferase
• The “B” gene

• codes for an enzyme that adds D-galactose to the terminal sugar of the H
antigen
• enzyme: D-galactosyltransferase

Formation of the A antigen
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
N-acetylgalactosamine
fortune lapira - torrecampo, rmt, mph Fucose
Formation of the B antigen
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Fucose Galactose fortune lapira - torrecampo, rmt, mph

“AB” AND “O” INDIVIDUALS
• “AB”: Both A and B structures are synthesized
• “O”: Neither A nor B are synthesized as a result of mutation in the ABO

gene

Genetics
• The H antigen
• found on the RBC when you have the Hh or HH genotype
• NOT from the hh genotype
• The A antigen
• found on the RBC when you have the Hh, HH, and A/A, A/O, or A/B genotypes
• The B antigen
• found on the RBC when you have the Hh, HH, and B/B, B/O, or A/B genotypes
H antigen
• Certain blood types possess more H antigen than others:
O>A2>B>A2B>A1>A1B
Greatest amount Least amount
of H of H

ABO Antigens in Secretions
• Secretions
• include body fluids like plasma, saliva, synovial fluid, etc
• Blood Group Substances are soluble antigens (A, B, and H)

that can be found in the secretions
• controlled by the H and Se genes

ABH ANTIGENS ON RBCS SECRETOR ABH GLYCOPROTEIN SUBSTANCES
L-fucose D - galactose L-fucose D - galactose
N-acetylglucosamine N-acetylglucosamine
D - galactose D - galactose
Glucose N-acetylgalactosmine
Protein
Ceramide backbone
Secretor Status
• The secretor gene

• consists of 2 alleles (Se and se)
• responsible for the expression of the H antigen on glycoprotein structures
located in body secretions
• If the Se allele is inherited as SeSe or Sese, the person is called a

“secretor”
• 80% of the population are secretors

Secretors
• Secretors
• express soluble forms of the H antigen in secretions that can then be converted to A
or B antigens (by the transferases)
• Individuals who inherit the sese gene are called “nonsecretors”

• se allele is an amorph (nothing expressed)
• sese individuals do not convert antigen precursors to H antigen and has neither
soluble H antigen nor soluble A or B antigens in body fluids

Secretor Status Summary
• The Se gene codes for the presence of the H antigen in secretions,

therefore the presence of A and/or B antigens in the secretions is
contingent on the inheritance of the Se gene and the H gene
A antigen
Se gene
H antigen in and/or
(SeSe or Sese) secretions
B antigen
se gene (sese) No antigens secreted in saliva or

other body fluids

ABH Antigens on Red Cells A, B, and H Soluble substances
RBC antigens can be glycolipids, Secreted substances are glycoproteins
glycoproteins, or glycosphingolipids
RBC antigens are only synthesized on type 2 Secreted substances are primarily
precursor chains synthesized on type 1 precursor chains
Type 2 chain refers to a beta1 4 linkage Type 1 chain refers to a beta 1 3 linkage
in which the number one carbon of the in which the number one carbon of the
galactose is attached to the number three galactose is attached to the number three
carbon of the N-acetylglucosamine sugar of carbon of the N-acetylglucosamine sugar
the precursor substance of the precursor substance
Enzyme produced by the H gene (α-2-L- Enzyme produced by the Se gene (α-2-L-
fucosyltransferase)acts primarily on type 2 fucosyltransferase) preferentially acts on
chains, which are prevalent on the RBC type 1 chains in secretory tissues
membrane
BIOLOGICAL ROLE OF ABH ANTIGENS
• Linked ABO types with higher incidence of many diseases: autoimmune,

neoplastic, and infectious disorders

ABO Subgroups
• Have less antigen
• Result of less effective enzymes
• Subgroups of A are more common

Subgroups of A
Two different A antigens: described by von Dungern in

1911

Subgroups of A
• The 2 principal subgroups of A are: A1 and A2

• Both react strongly with reagent anti-A
• To distinguish A1 from A2 red cells, the lectin Dolichos
biflorus is used (anti-A1)
• 80% of group A or AB individuals are subgroup A1
• 20% are A2 and A2B

A1 and A2 Subgroups
Anti-A Anti-A1 Anti-H ABO antibodies in # of antigen sites

antisera antisera lectin serum per RBC
A1 4+ 4+ 0 Anti-B 900 x103
A2 4+ 0 3+ Anti-B & anti-A1 250 x103
*Adapted from Flynn, J. (1998). Essentials of Immunohematology

A2 Phenotype
Why is the A2 phenotype important?
Production an anti-A1
May cause discrepancies when a crossmatch is done

(incompatibility)

A1vs A2 Phenotypes
• Quantitative differences:
• More antigenic sites on A1 than A2
• Qualitative differences between A1 and A2 antigens:

• 1-8% of A2 individuals produce anti-A1
• 25% - 35% of A2B individuals produce anti-A1.

Other A subgroups
• There are other additional subgroups of A

• Aint (intermediate), A3, Ax, Am, Aend, Ael, Abantu
• A3 red cells cause mixed field agglutination when polyclonal anti-A or

anti-A,B is used
• May contain anti-A1

B Subgroups
•Very rare and are less frequent than A subgroups

•B subgroups demonstrate variations in the strength of
the reaction using anti-B and anti-A,B
•Examples are: B3, Bx, Bm, Bel

B Subgroups

B(A) PHENOTYPE
ACQUIRED B PHENOTYPE
cis- AB PHENOTYPE
NULL AND WEAK PHENOTYPES

B(A) phenotype
• Autosomal dominant phenotype
• Weak A expression on group B red cells
• Due to synthesis of A antigen by the B gene enzyme

B(A) phenotype
• Serological reaction:
• red cells react strongly with anti-B and weakly with anti-A (<2+)
• possess a strong anti-A reactive with both A1 and A2 red cells

B(A) phenotype
FORWARD TYPING REVERSE TYPING
ANTI-A ANTI-B A1 CELLS B CELLS
PATIENT 1+ 4+ 4+ 0

• Acquired enzymatic modification of group A1 red
cells in vivo
• Reflects enzymatic deacetylation of group A

antigen to form a B-like antigen on RBCs
• Acquired, TRANSIENT serologic discrepancy

encountered in group A individuals
• Occurs in type A individuals with:

• Colon cancer, intestinal obstruction, gram negative sepsis
• Bacteria deacetylate group A sugar (GalNAc); remaining

galactosamine crossreacts with reagent anti-B

•Serological reaction:
• Strong agglutination with anti-A
• Weak agglutination with monoclonal anti-B (2+ or
less)
• Contains strong anti-B in the serum

ACQUIRED B TYPING RESULT
Forward Reverse
Anti-A Anti-B Interp A1 cells B cells Interp
4+ 1+ “AB” 0 4+ “A”

TO RESOLVE PATIENT’S TRUE RED CELL TYPE:
Reaction with anti-B is negative, if:

• Acidify serum
• Acetic anhydride treatment
• Auto incubation

cis-AB PHENOTYPE
•A and B antigens are synthesized by the

same enzyme and are inherited as a
single, autosomal dominant allele

ABO antibodies
Landsteiner’s Rule:
“Normal, healthy individuals
possess ABO antibodies to
the ABO antigen absent from
their RBCs”

Nature of antibodies
• Non-red blood cell stimulated (previously discussed)
• ABO antibodies
• Red blood cell stimulated

• Antibodies formed as a result of transfusion, etc
• Usually IgG
• Active at 37°C
• Can occur in group O (may occur in group A or B)

ABO antibodies
• Not present at birth, or if present, are of maternal origin
• Endogenous synthesis of anti-A and anti-B: as early as age 3 to 6

months
• Anti-A and Anti-B reach adult levels: within 5 to 10 years
• Declines slightly with advancing age

ABO Antibodies
• High titer: react strongly (4+)
• Newborns may passively acquire maternal antibodies (IgG crosses

placenta)
• Reverse grouping (with serum) should not be performed on
newborns or cord blood

ABO antibodies
• group A serum contains anti-B

• group B serum contains anti-A
• group AB serum contains no antibodies
• group O serum contains anti-A, anti-B, and anti-A,B

Anti- A and Anti-B
• Predominant isotype found in A and B individuals: IgM
• Predominant isotype in group “O” serum: IgG
• Agglutinates at room temperature (20-24 °C) or cooler
• Activate complement at 37 °C

Anti-A
• Group O and B individuals contain anti-A in their serum
• Anti-A can be separated into different components: anti-A and anti-

A1
• Anti-A1 only agglutinates the A1 antigen, not the A2 antigen
• There is no anti-A2
Anti-A1
• Naturally occurring antibody found in the sera of:
• A2
• A2B
• Weak A subtypes
• O (mixture of Anti-A and Anti-A1)
• Implicated in:
• transfusion reactions
• solid organ rejection
Anti-A1
• Usually IgM
• Cause ABO discrepancies during routine testing and can lead to

incompatible crossmatches
• Considered clinically significant if reactive at 37 °C

Anti-A1
• What should be given to A2 patients with an anti-A1 reactive at 37
°C?
• Group O or A2 red cells
• What should be given to A2B patients with an anti-A1 reactive at 37

°C?
• Group O, A2, or A2B red cells
Anti-A,B
• Found in the serum of group O individuals
• Reacts with A, B, and AB cells
• Predominantly IgG, with small portions being IgM
• Anti-A,B is one antibody, it is not a mixture of anti-A and anti-B antibodies
• Saliva containing secreted A or B substances can inhibit the activity of Anti-A, B

against both A and B red cells
Anti-H
• Usually benign
• Naturally occurring antibody in the sera of:

• A1 and A1B nonsecretors
• Reacts more strongly with

• O followed by A2, B, A2B, A1 and A1B
• Autoantibody in most individuals

Laboratory Testing:
ABO typing
The Use of Lectins for Antigen Confirmation
• Dolichos biflorus = anti-A1

• Ulex europaeus = anti-H

ABO Blood Groups
ABO Antigen Antigen Missing Antibody Present

Group Present
A A B anti-B
B B A anti-A
O None A and B anti-A, anti-B, anti-

A,B
AB A and B None None

Forward & Reverse Typing
Reaction of cells Reaction of serum

tested with: tested with:
anti-A anti-B A cells B cells ABO group
1 0 0 + + O
2 + 0 0 + A
3 0 + + 0 B
4 + + 0 0 AB

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ANTIHUMAN GLOBULIN

Therefore: Antihuman Globulin is antibody directed

fortune lapira - torrecampo, RMT, MPH 2

fortune lapira - torrecampo, RMT, MPH 3

• Yet, these antibodies are capable of causing severe

AHG testing is the primary method of detecting all Antibodies except

fortune lapira - torrecampo, RMT, MPH 6

Rabbit polyclonal has only anti-IgG; IgG heavy chains

Contains only antibodies against designated

Anti-C3d or anti-C3b, -C3d (murine Contains only antibodies against designated

Table 12.1, page 260, AABB Technical Manual, 13th Edition

• Polyclonal - Animals hyperimmunized with human globulins; bled for

• Monoclonal - Hybridoma cells from mice; collection of culture or

• Product is highly regulated for potency

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• Major component of anti-IgG is antibody to human gamma heavy

• An anti-IgG reagent not designated “heavy-chain-specific” must be

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• Many workers prefer anti-IgG over polyspecific AHG in antibody

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• This type of anti-C3d characteristically reacts with C3b and possibly

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• Animals injected with human globulins produce antibody to the

• After the animal serum is adsorbed to remove unwanted agglutinins,

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• Hybridoma techniques are used for the manufacture of most AHG

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• Cells that have no globulin attached will not be agglutinated.

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• Unbound globulins may react with AHG, causing false-negative antiglobulin

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• To show that test cells were properly washed and that no

• In a negative antiglobulin test the anti-human globulin should remain

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• Agglutination of the IgG sensitized cells after mixing and centrifuging

• Failure of the IgG sensitized cells to agglutinate indicates that the

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• The test was adequately washed prior to addition of the

• AHG reagent was added to the test tube.

• The AHG reagent that was added was in an ACTIVE form.

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• Hemolytic disease of the newborn (HDN) (maternal IgG crosses

• Hemolytic transfusion reaction (recipient Ab coats donor RBCs;

• Autoimmune hemolytic anemia (AIHA) (autoAbs coat own RBCs;

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• Rhesus D hemolytic disease of the newborn (also known as Rh

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• Penicillin (high dose)

• Quinidine (IgM mediated activation of classical

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• Usually do initial DAT using polyspecific AHG and then more

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•Detection of in vitro sensitization of RBCs

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• When the sample is a sera:

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