IMMUNOHEMATOLOGY LECTURE

Topic: Antihuman globulin test

● Introductory terms

Moreschii ● 1908
- Described the principle of the test using rabbit
anti-goat serum to agglutinate rabbit RBCs
sensitized with low non-agglutinating doses of
goat anti-rabbit RBC serum.

Robin Coombs, Robert Race, Arthur Mourant ● 1945

- Described the use of antiglobulin test to
detect weak non-agglutinating Rh ab.
● 1946
- Described the use of AHG to detect in vivo
sensitization of RBCs in babies with HDN
- Described kell blood group using antiglobulin
test

Kohler & Milstein - Devised the monoclonal antibody technique

that is used in production of high-titer ab for
AHG reagents specific to IgG and fragments
of C3d

AHG - Antihuman globulin; coombs’ serum,

- Serum from rabbit or other animal previously
immunized with purified human globulin.
- Crosslinks immune complexes to form visible
agglutination

Direct Antihuman globulin Test (DAT) - Demonstrate in vivo sensitization of RBCs

with antibodies or complement, IgG and C3d
in particular.

Indirect Antihuman globulin Test (IAT) - Detects the existence of atypical antibodies in
a patient's serum that can coat antigens in
type 'O' RBCs in vitro.
- Principle used in:
- Weak D test
- Red cell phenotyping
- Antibody screening and identification
- crossmatching

Coombs’ cell - Check cells; sensitized type ‘O’ cells that is

used to detect presence of AHG in
mixture/solution

AHG reagent

● Types
○ Polyspecific Antihuman globulin reagents

1. Rabbit polyclonal Contains anti-IgG and anti-C3d

2. Rabbit/murine monoclonal blend Contains a blend of rabbit polyclonal anti human IgG
and anti-C3d

A murine monoclonal IgM antibody

○ Monospecific Antihuman globulin reagent

Anti-IgG (rabbit polyclonal) Contains Anti-IgG with no anti complement activity

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Anti-IgG (Gamma-clone AHG) Murine monoclonal IgM ab secreted by hybridoma
cell lines

Anticomplement A murine monoclonal ab to C3d. Anti-C3d, causes

- Anti-C3d agglutination of RBCs coated with C3d and/or C3b
complement components.

AHG complexes

1. Antigen-Antibody complex [Ag:Ab]

2. Antigen-C3 complex [Ag:C3]

3. Antigen-drug metabolite [Ag: drug metabolite]

complex

AHG Test

A. Direct Antihuman globulin Test (DAT)

● In vivo sensitization; test for Ab or complement bound to Red cells

● Sample choice: EDTA blood

- EDTA prevents in vitro activation of the complement pathway.
- Prevents detection of complement-dependent antibodies when plasma is used.

● For positive result to occur there needs to be:

○ 100 to 500 IgG molecules per RBC
○ 400 to 1,100 molecules of C3d per RBC

● Medical significance
○ Diagnosing Hemolytic conditions
Hemolytic Disease of the Fetus and Newborn Maternal antibody coating fetal RBCs

Hemolytic Transfusion Reaction Recipient antibody coating donor RBCs (Patient

antibody reacting with donor cells coated with IgG)

Autoimmune Hemolytic Anemia Autoantibody coating individual’s RBCs

● Warm type (WAIHA) = IgG Autoantibody reacting to individual’s RBC in vivo

● Cold type (CAS) = IgM
- IgM autoagglutinin binds to RBCs in
peripheral circulation (32C)
- IgM binds complement as RBCs return to
warmer parts of circulation, and will
dissociate leaving RBCs coated with only the
complement.

Drug Induced Hemolytic Anemia - Type 1 (hapten dependent ab) - penicillin

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- Type 2 (autoantibody) - associated with Kidd
blood group & alpha-methyldopa produced
autoantibody
- Type 3 (durg-dependent ab) - e.g., Quinidine

Paroxysmal Cold Hemoglobinuria - IgG autoantibody reacts with RBCs in colder

parts of the body causing the complement to
bind irreversibly to red cells.
- Complement will elute at warmer
temperature
- Has Biphasic Ab that can react to cold
temperature (sensitization) and warm
temperature (lysis).

➡️
● Result interpretation
○ (+) = Agglutination Patient has Hemolytic disease
■ (+) Polyspecific reagent
● RBC is coated with either IgG or complement molecule
■ Monospecific anti-IgG and anti-C3d reagent
● Will determine the specific type of protein (IgG or C3d) sensitizing the cells
Anti-IgG Anti-C3d Type of AIHA

[+] [+] WAIHA/ Mixed-type AIHA (cold

and warm)

[+] [-] WAIHA

[-] [+]* CAS;PCH, WAIHA

*(+) for Anti-C3d and (-) for anti IgG means that AIHA leans toward cold reaction (optimal reaction at cold
incubation)

● Procedure
❌ Incubation

✅ Washing (3x)

✅ AHG

✅ Check cells (if AHG is negative, otherwise

don’t add)
○ Incubation - is not included because it is assumed that sensitization/complex formation already
occurred
○ (remember) incubation is done to sensitized ag to blood cells
○ Centrifugation after AHG addition will bring complexes closer together.

Scenario 1
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● Unbound or free IgG antibodies have to be removed to prevent neutralization of AHG.

Scenario 2

● The result of Coombs’ check cell addition will only be a 2+ reaction and will NEVER be a 4+
reaction because there is also the presence of red cells covered with antigen that will not react
with AHG.

➡️
● Reasons for negative result with check cells
1. No AHG no agglutination with check cells
2. Expired AHG reagent
3. Neutralized AHG or diluted AHG (further explained in false negative result with AHG)

B. Indirect Antihuman globulin Test (IAT)

● In vitro sensitization; Detection of Atypical antibodies in the patient’s serum

● Sample choice: Serum/Plasma

● Medical significance

Antibody detection - Compatibility testing & Ab screening (e.g.,

major & minor crossmatch)

Antibody identification - Ab panel

RBC phenotype - RBC antigen

- Atypical ab screening and
determination

Weak D test

Antibody titration - Rh antibody titer

● For positive result to occur there needs to be:

○ 100 to 200 IgG or C3d molecules on the cell surface.

● Procedure
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✅ Incubation = 30 mins @ RT or 10-15 mins
with LISS

✅ Washing (3x)

✅ AHG

✅ Check cells (if AHG is negative, otherwise

don’t add)
● Incubation to perform in vitro sensitization
● LISS - is added to shorten incubation time

Scenario 1
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Scenario 2

● The result of Coombs’ check cell addition will only be a 2+ reaction and will NEVER be a 4+
reaction because there is also the presence of red cells covered with antigen that will not react
with AHG.

Factors Affecting AHG test

1. Ratio of Serum to Cells

● [40:1] is the serum to RCS ratio used to prevent zonal phenomenon to occur, thus preventing
false negative.
● Increase drops of serum = increases detection rate of weak Antibodies

2. Reaction medium

Potentiator* Mechanism of action

AHG reagent Crosslinks immune complexes (IgG and C3d

sensitized RBC)

Low Ionic Strength Saline (LISS) Increases rate of antibody uptake, shortening
incubation time to 5-15 mins

Polyethylene glycol (PEG) Removes water molecule by in the environment thus

increasing ab concentration; enhances IgG ab

Does not react with IgM

Proteolytic enzymes (papain, ficin, trypsin) Removes negative charges from RBCs which
reduces zeta potential. Denatures RBC antigens
(e.g., Duffy, MNSs) making them undetectable.

Albumin Does not enhance warm ab (IgG)

*potentiators - reduces zeta potential
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3. Temperature
● IgG = optimally reacts at 37C (also optimum temp for complement activation)

4. Incubation time
● Extended incubation time can cause elution of Ab from Ag
● LISS and PEG = shortens incubation time to 5-15 mins

5. Washing
● Inadequate washing = traces of free IgG ab that can neutralize AHG (false negative reaction)
● Saline that is not buffered should always be freshly prepared.
● Buffered saline = pH: 7.2 to 7.4
● Decrease in pH causes enhanced elution process which might cause neutralization of AHG.
● Monoclonal ab = narrow pH range for optimum reactivity

6. Centrifugation for reading

● Inadequate resuspension (dislodging) can cause false positive result
● Too Vigorous resuspension can cause false negative result.

- False positive results -

1. RBC are agglutinated before washing potent cold reactive ab of patient origin
step & addition of AHG

2. Use of dirty glassware RBC can bind to dirt

3. Over Centrifugation Cell button is packed too tight.

- Possible reasons for false negative result with AHG -

1. Inadequate Washing Trace unbound IgG ab due to inadequate washing

will neutralize the AHG

2. Delayed adding of AHG Elution (paghiwalay) of IgG ab bound to antigen on

the surface of RBCs can occur. The eluted antibody
will neutralize the AHG.

3. Diluted AHG Trace NSS due to failure of removing all NSS after
the last washing will dilute the AHG.

4. Weak reacting AHG Improper storage, Expired AHG, contaminated with

human serum

5. Failure to add AHG Technical error

6. Inappropriate RCS conc.