When can you use an antibody to find another antibody?

When can you use an antibody to find another antibody?

The Antiglobulin TestBasic Principle, Procedures, and Applications

The Acquired Immune Response

From our Immunology Review we know: The body makes antibodies in response to foreign

antigen. These antibodies coat the foreign object leading to:

Clearance of the foreign antigen by the RES Lysis of the foreign object via complement activation

IgG is the predominant antibody produced in most responses

Incomplete antibody Usually not detected at room temperature/immediate spin phase

of testing

The Antiglobulin Test

The purpose of the antiglobulin test is to detect cells that have become coated with antibodies &/or complement.

The test is also known as the Coombs test.

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Anti-Human Globulin

The main reagent used in the antiglobulin test is anti-human globulin (AHG). Also called Coombs serum.

Anti-human globulin (AHG) is an IgG antibody directed against human immunoglobulins or complement components.

AHG Production

AHG Production

The globulins that AHG may be directed against include: IgG IgM IgA C3

AHG Contents

Polyspecific AHG reagent contains antibodies to both IgG and C3.

Monospecific AHG contains antibodies to either IgG or C3.

AHG Action

AHG combines with the Fc portion of a sensitizing antibody.

This completes the antigen-antibody bridge, allowing agglutination to occur.

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When used to detect clinically significant antibodies, AHG reagent MUST contain

anti-IgG.

ANYTIME YOU ARE USING AHG REAGENT, AND GET A

NEGATIVE TEST RESULT (by tube)---

YOU MUST ADD

COOMBS CONTROL CELLS

AND GET POSITIVE RESULTS!!!

Control

Coombs Control Cells

Rh positive cells coated with anti-D antibodies or cells coated with the C3 portion of complement.

Coombs Control Cells will react with the antibody in the AHG reagent.

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Coombs Control Cells will prove that…

Coombs reagent was added. Coombs reagent was active. The wash step was adequate to

remove any unbound globulins. The most common error made

when performing the antiglobulin test is inadequate washing.

Procedures

Direct Antiglobulin Test (DAT) Detects antibody (or complement)

sensitizing red cells In vivo sensitization Uses patient’s cells

Procedures

Indirect Antiglobulin Test (IAT) Antibody is free Uses incubation at 37oC to force red cell

sensitization in vitro. May be used to detect antigens or antibodies.

Both DAT and IAT utilize Anti-Human Globulin reagent (AHG).

The Direct Antiglobulin Test

Procedure

Steps to the DAT Procedure (tube method)

1. One drop of patient’s red cells are washed with 0.9% NaCl a minimum of 3 times to remove plasma that may contain unbound antibodies.

2. AHG reagent is added.3. Tube is centrifuged.4. If IgG or C3 is coating the cells, agglutination will occur

(positive test). This will depend on the type of AHG reagent used i.e. if C3 is coating

the cell, and monospecific anti-IgG AHG reagent is used, there will be NO agglutination.

If neither is present there will be no agglutination (negative test).

5. Each negative test is validated (controlled) through the addition of Coombs Control Cells (also called check cells).

Pt ID

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DAT Procedure (tube method)

Washed cell button

The Direct Antiglobulin Test

Applications

What causes a cell to become coated with antibodies in vivo?

Hemolytic Transfusion Reaction Hemolytic Disease of the Fetus and Newborn Drugs Disease

Indirect Antiglobulin Test

Procedure

Steps to the IAT Procedure(tube method)

1. One or two drops of serum (may contain an antibody) are added to a test tube.

2. One drop of red cells (antigen source) is added to the tube.

3. The tube is incubated at 37oC. The length of incubation is dependant on the medium.

4. Following incubation, the cells are washed with saline a minimum of 3 times, to remove any unbound antibody.

5. Following the final wash, two drops of AHG reagent are added to the dry cell button. The tube is centrifuged and results are read. The tube may be read microscopically, depending on the test medium.

6. Coombs control cells are added to each negative test. The tubes are centrifuged and results read.

Indirect Antiglobulin Test Tube Method

37C inc√

Indirect Antiglobulin Test

Antibodies sensitize the red cells during incubation at 37oC.

Following the wash step, AHG reagent is added.

AHG reagent completes the “bridge” between red cells, allowing for visible agglutination.

Indirect Antiglobulin Test

Applications

Indirect Antiglobulin Test

Looking for in vitro cell sensitization. Uses incubation at 37oC to allow antibody to

sensitize red cell. Uses AHG reagent to complete the “bridging”

between red cells. Visible agglutination as a positive endpoint.

Enhancement reagents may be added during incubation phase to increase sensitization and agglutination.

Applications Using Patient’s Serum

Antibody screen Detects antibodies in patient’s serum Uses reagent red cells as a source of known

antigen

Antibody panel Identifies antibodies Uses reagent red cells as a source of known

antigen

Applications Using Patient’s Serum

Antiglobulin crossmatch Determines patient’s compatibility with donor Uses donor red cells (antigens) and patient’s

serum (antibodies) Usually performed only when a patient has an

antibody or a history of antibodies

Applications Using Patient’s Cells

Antigen Typing Weak D test

Both use commercial anti-serum which contains antibodies, versus the patient’s cells (antigen).

Auto control – Patient’s plasma vs. patient’s cells

NOTE:

If the patient has a positive DAT, the results of any IAT using the patient’s cells will be invalid. Cells are already coated

with antibody before the incubation step!

Factors affecting the IAT

Serum/Cell ratio Incubation temperature pH Length of incubation Test environment (enhancement

media)

POTENTIATORS

Some incomplete antibodies will not react in a saline environment.

Potentiators are reagents that adjust the test environment. Reduce the zeta potential Promote agglutination Enhance antibody uptake

22% Albumin Polyethylene glycol

(PEG) : a low ionic strength medium. Removes water from the test system, thereby concentrating any antibody present.

LISS Enzymes

Sources of Error in the Antiglobulin Test

Adequate wash Centrifugation Problems with reagents/saline Problems reading reactions