ahg Flashcards by Ruth Joy De Guzman

obtained
from immunized nonhuman species bind to human
globulins such as IgG or complement

antiglobulin test

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AHG reagent contains

IgG and complement proteins

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Important step in AHG testing

washing the RBC

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Unwashed rbc may lead to

false negative result

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Mechanism of AHG testing

detection of the unknown, unidentified or weakly reactive antibodies

detection of weak and
nonagglutinating Rh antibodies in serum.

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principle of AHG testing

sensitization of RBC

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Agglutination reaction of IgM

large aggregation, large
particles

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two types of AHG testing

direct and indirect type of AHG

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IgG antibodies are termed

non-agglutinating

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In vitro sensitization of RBC: it is a two-stage
process/ two stage technique are referred to as

indirect antiglobulin test

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In vivo sensitization of RBC: a one-stage
procedure which is referred as

direct antiglobulin tes

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RBCs are sensitized with

IgG alloantibodies, IgG autoantibodies, complement components

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Reacts with Fc Region of the Gamma heavy chain of
the IgG molecule

Polyspecific AHG

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If positive with polyspecific AHG

use monospecific AHG
to determine which components reacts with the red cell

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License monospecific AHG

Anti-IgG and Anti-C3b to C3d

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involves injecting
human serum or purified globulin into laboratory
animals, such as rabbits.

classic method of AHG

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Production of POLYSPECIFIC AHG may occur in two ways:

Polyclonal production:
Monoclonal production

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are a mixture of antibodies
from different plasma cell clones

polyclonal antibodies

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are produced by identical
plasma cells

monoclonal antibodies

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Produced ex vivo using tissue-culture
techniques

monoclonal production / ab

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Usually produced in live animals

polyclonal production

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Monoclonal production also called as

hybridoma technology

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usually prepared in rabbit

POLYCLONAL AHG PRODUCTION

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Usually mice is used in monoclonal antibody production/
hybridoma technique

MONOCLONAL AHG PRODUCTION

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produced by immunizing one colony of rabbits with human immunoglobulin (IgG) antigen and another colony with human C3 antigen.

Conventional polyspecific antiglobulin reagents

Both colonies of animals are hyperimmunized to produce antibodies

high-titer, high-avidity IgG antibodies

performed by reacting dilutions of each antibody against cells sensitized with different amounts of IgG

block titration for anti-IgG pools

excess IgG antibody may lead to

prozoning

can be used to prepare monospecific antibody as well as polyspecific antibody

MONOCLONAL AHG PRODUCTION

discovered a technique to produce antibody arising from a single B cell

George Kohler and Cesar Milstein

Are screened for antibodies with the required specificity and avidity

monoclonal antibody

Useful in producing high-titer antibodies with well defined specificities to IgG and to the fragments of C3.

MONOCLONAL AHG PRODUCTION

After fusing of spleen cells and myeloma cells, we need to suspend it on a media complemented with

PEG

After the fusion, the cells are placed now in a selective culture media

HAT or hypoxanthine-aminopterin-thymidine medium

Enzyme lacks in myeloma cells

HGPRT Hypoxanthine-guanine phosphoribosyl transferase

is prepared by a production process similar to that described for polyspecific AHG

monospecific AHG

Clinical significance in DAT

- Hemolytic disease of the newborn (HDN) - Hemolytic transfusion reaction (HTR) - Autoimmune and drug-induced AIHA.

not a required test in routine pretransfusion protocols

DAT

Sensitization For DAT

at least 100 to 500 IgG molecules per RBCs and 400 to 1100 molecules of C3d per RBCs.

does not require the incubation phase because of the antigen-antibody complexes formed in vivo

DAT

Interpreting the significance of a positive DAT requires

knowledge in patient's diagnosis, drug therapy, recent transfusion history,

performed to determine in-vitro sensitization of RBCs and is used in the following situations

IAT

Requires the incubation phase because of the antigenantibody complexes formed in vitro

IAT

sensitization for IAT

there must be between 100 and 200 IgG or C3 molecules on the cell to obtain a positive reaction.

* Sensitization can be influenced by several factors:

o Ratio of serum to cells o The use of enhancement medium o Temperature o Incubation time o Washing of RBCs o Addition of AHG reagent

RATIO OF SERUM TO CELLS:

40:1 or 2 drops of serum and 1 drop of a 5 percent red cell suspension

ration of Detecting weak antibodies

4 drops of serum with 1 drop of 3% RCS)

incubation time for 22% bovine serum albumin

30 mins

Works by reducing the zeta potential, it allows the RBC to approach each other

Tend to lower the Zeta potential of that particular RBC,

LISS

a water-soluble linear polymer and is used as an additive to increase antibody uptake. Its action is to remove water, thereby effectively concentrating antibody.

PEG

Temperature

optimum temp - 37 C

saline with decreased pH due to long prolonged storage periods

increase rate of antibody elution during washing process.

Why should AHG should be added immediately after washing

to minimize the chance of antibody eluting from the cell and subsequently neutralizing the AHG reagent.

FALSE-POSITIVE RESULTS

* Overcentrifugation and overreading * dirty glassware * cells with a positive DAT will yield a positive IAT

FALSE-NEGATIVE RESULTS

* Inadequate or improper washing of cells * Undercentrifuge or overcentrifuge * Serum:cell ratio are not ideal * Cell suspension either too weak or too heavy * Low pH of saline * Inadequate incubation conditions in the IAT * Poor reading technique

MODIFIED AND AUTOMATED AHG TEST TECHNIQUES

* Low Ionic Polybrene technique * Enzyme-Linked Antiglobulin Test * Gel Technique * Solid Phase technique

An RBC suspension is added to a microtiter well and washed with saline.

ENZYME-LINKED ANTIGLOBULIN

A rouleaux-forming reagent is added to allow the sensitized cells to approach each other to permit crosslinking by the attached antibody.

LOW IONIC POLYBRENE TECHNIQUE

The patient’s serum or plasma is added to each well in the screen cell set along with LISS.

SOLID PHASE TECHNIQUE

added to reverse the rouleaux; however, if agglutination is present, it will remain.

high ionic strength solution

In solid phase technique what needs to be added after washing

indicator cell

If no sensitization occurred

indicator cells form a pellet in the bottom of the well.

If sensitization occurred

forming a diffuse pattern in the well

gel used in gel technique

dextran acrylamide gel

Does not contain any specific reagent and acts only by its property of trapping agglutinates

Neutral

use a specific reagent incorporated into the gel and are useful for antigen determination.

Specific gel tests

a valuable application of the gel test and may be used for the IAT or the DAT

AHG test

how much percentage pf LISS is needed

0.8%