BB AHG p1

AHG test

This is a vital blood test used to detect antibodies,

specifically IgG.

Coombs’ Test

AHG is aka?

AHG test

aids in diagnosing immune mediated hemolytic

anemias, hemolytic disease of the fetus and newborn

and transfusion reactions by identifying these immune

components on RBCs

AHG test

Based on the binding of Antihuman globulins from

immunized nonhuman species to human globulins (IgG or

complement)

● Detects globulins either free in serum or attached in RBC

antigens.

● Essential in transfusion medicine to ensure patient safety.

Direct AHG test

● A test that directly examines a patient's RBCs for

pre-existing antibodies or antibody or complement

binding.

● crucial for investigating AIHA or transfusion reactions.

Indirect antiglobulin test

● A test that screens a patient's serum for free antibodies

capable of binding to RBCs.

● Essential for antibody screening in blood banking,

prenatal testing for maternal antibodies and ensuring

blood compatibility before transfusion.

IgM antibodies

● Pentameric structure

● Since IgM antibodies can directly agglutinate RBCs in

saline because of its large pentameric structure

IgG antibodies

● Monomer structure

● Too small to directly agglutinate RBCs, hence, they are also

called as non-agglutinating/incomplete antibodies.

hemagglutination.

When RBCs are coated with IgG antibodies, adding AHG

that targets IgG will cause the RBCs to clump together.

AHG reagent

essentially acts as an antibody against

other antibodies making Ag-Ab reactions visible that

would otherwise be too small to see.

anticomplement component

If complement is involved, -x- can be added to AHG reagent (polyspecific

AHG)

To detect the presence of IgG or complement-sensitized RBCs.

PURPOSE OF AHG TEST

1945:

Coombs and associates introduced Antiglobulin test

to detect weak and non-agglutinating Rh Antibodies.

1946:

Coombs and coworkers described using AHG to

detect in vivo RBC sensitization in infants with hemolytic

disease of the newborn (HDN) which became DIrect

Antiglobulin Test - DAT

1947:

Coombs and Mourant identified the anti-gamma

globulin fraction as the component responsible for

detecting Rh Antibodies.

1951:

Dacie suggested another Antibody activity

influencing the AGT reaction.

1957:

Dacie and colleagues determined that:

○ Warm antibodies react due to anti-gamma globulin activity

○ Cold antibodies react due to anti-non gamma globulin activity specifically beta globulin and complement specificity (C3 & C4)

1908:

Moreschi first described the AGT principle using rabbit anti-goat serum to agglutinate rabbit RBCs sensitized with goat anti-rabbit RBC serum.

Indirect Antiglobulin Test (IAT)

Detects in vitro sensitization (e.g., antibody screening, crossmatching)

Direct Antiglobulin Test (DAT)

Detects in vivo sensitization (e.g., HDN, autoimmune hemolytic anemia)

DAT

In vivo antibody coating the RBC (Y shape). And these antibodies are directed against antigens on the surface of other RBCs. May cause Autoimmune hemolytic anemia (AIHA)

WASHING

step is to remove any unbound or free antibodies, ensuring that the only antibodies present are the antibodies that are attached to the RBC and are

detected. (DAT)

AHG reagent

target the human IgG antibodies that are attached on the RBCs surface (star shape).

Agglutination

What happens if IgG Abs are coating the RBC? (DAT) this also signifies a positive rxn

IAT

Antibodies present in serum of the patient + the reagent RBC. The two are subjected to incubation

INCUBATION AND WASHING

2 steps involved in IAT

POLYSPECIFIC AHG

● Contains Abs to human IgG and C3d complement.

May include;

○ Anti-C3b

○ Anti-C4b

○ Anti-C4d

● Facilitates agglutination when RBCs are sensitized with IgG, C3b, or both.

● Commercial x has minimal or no reactivity against IgA and IgM heavy chains.

● May contain Abs to kappa and lambda light chains, which could react with IgG or IgM molecules.

MONOSPECIFIC AHG

● Contains only 1 Ab specificity, either:

○ Anti-IgG

○ Ab to specific complement components (e.g., anti-C3b, anti-C3d)

○ Anti-IgG

○ Anti-C3b-C3d

what are the common licensed monospecific AHG reagents:

ANTI-IgG REAGENTS

● Specific for the Fc fragment of the gamma heavy chain of IgG.

● Does not contain anticomplement activity

● If not labeled “gamma heavy chain-specific” (it may react with IgM, IgA, and IgG due to anti-light chain specificity)

○ Because it could contain antibodies that could recognize the common light chains found in all these immunoglobulins.

ANTICOMPLEMENT REAGENTS

● Include anti-C3b and anti-C3d

● Specific to complement components, with no reactivity against human Igs.

● Monospecific anticomplement reagents are often a blend of monoclonal anti-C3b and anti-C3d.

Rabbit

Leporine

Mouse

Murine

Rabbit polyclonal (Polyspecific)

Contains anti-IgG and anti-C3d (may contain other anticomplement and other anti-Ig Abs)

Rabbit/ Murine Monoclonal Blend (Polyspecific)

Contains a blend of rabbit polyclonal antihuman IgG and murine monoclonal anti-C3b and anti-C3d

Murine monoclonal

Contains murine monoclonal anti-IgG, anti-C3b, and anti-C3d

Rabbit Polyclonal (Monospecific ant-IgG)

Contains anti-IgG with no anticomplement activity

IgG heavy-chain specific (Monospecific ant-IgG)

Contains only antibodies reactive against human gamma chains

Monoclonal IgG (Monospecific anti-IgG)

Contains murine monoclonal anti-IgG

Rabbit Polyclonal Anti-C3d and Anti-C3b (Anticomplement)

contains only Abs reactive against the designated complement

Rabbit Polyclonal Anti-C3d, anti-C4b, anti-C4d (Anticomplement)

Components with no anti-immunoglobulin activity

Murine Monoclonal Anti-C3d

Contains only Abs reactive against the designated complement (Murine)

Murine Monoclonal Anti-C3b, anti-C3d

Component with no anti-Ig activity (murine)

CLASSIC METHOD OF AHG PRODUCTION

Human serum or purified globulin is injected into laboratory animals (e.g., rabbits, sheep, goats)

CONVENTIONAL METHOD (polyclonal Antihuman Globulin)

● Pooled donor Ag allows for a broader spectrum of reactivity, but the source of antibody is limited to the lifespan of the inoculated animal.

●-x- may be manufactured by combining polyclonal anti-IgG with either polyclonal or monoclonal anticomplement components.

Polyclonal Abs

• recognize multiple epitopes on an Ag

• Provide high avidity but may have variability in potency

Monoclonal Abs (Hybridoma technology)

• derived from a single plasma cell clone

• Recognize only one epitope, ensuring high specificity and consistency

Rabbits

most commonly used in production animals

Sheep or goats

what animal is used for large-scale production (sofer luoy:()

Block titration

is performed to determine the optimal Ab dilution to prevent prozone effects (false negatives).

Anti-C3 pools

are tested using C3b-and C3d-coated RBCs. Ensuring clinically significant detection of RBC-bound C3d while minimizing false positives.

Polyclonal reagents

may have varying levels of anti-C3b and anti-C3d, which can be avoided with monoclonal antibodies

by Kohler & Milstein

the monoclonal antibody technique was developed by

MONOCLONAL AHG PRODUCTION

widely used for producing high-titer AHG reagents with well defined specificities to IgG & C3 fragments

Monospecific anti-IgG

Detects only IgG-coated RBCs.

Monospecific anti-C3b or anti-C3d

Detects only complement-coated RBCs, essential in cases where

antibodies fix complement but do not directly agglutinate red blood cells.

buffers, stabilizers, and bacteriostatic agents

what do AHG reagents contain to maintain stability?

Anti-IgG

• AHG reagents are crucial for detecting IgG Abs that do not directly clump RBCs

IgG1 and IgG3 subtypes

most clinically important RBC Abs

anti-light-chain activity

AHG might also contain activity against IgA and IgM, and the presence of this allows detection of all immunoglobulin classes

• Kidd blood group system (Anti-Jka, Anti-Jkb)

• Lutheran blood group system (Anti-Lub)

Abs that frequently bind complement

• Duffy (anti-fya)

• Kell (Anti-K)

• MNS (anti-S, anti-s)

Abs that have variable binding

Rh blood group system (anti-D, anti-E, and anti-c)

Abs that rarely binds to complement

Complement activation

what results in the breakdown of C3 and C4 into smaller components?

C3b and C4b

what complement binds to RBCs

c3a C4a

what complement remains in plasma?

Anti-C3d

what is essential in detecting complement-coated RBCs especially in warm and cold AIHA

Polyspecific AHG (IAT)

detects both IgG and complement C3b/C3d components on RBCs

Monospecific anti-IgG (IAT)

detects only IgG coated RBCs

• HDN

• HTR Autoimmune

• Drug-induced Hemolytic Anemia

clinical conditions associated with (+) DAT