HEM 2133

Immunohaematology IImmunohaematology I

Lesson 9: Antihuman Globulin

Reaction

History of Antiglobulin Testing

Discovered by Coombs, Mourant and Race in 1945

Found that red blood cells (RBC) can become sensitized without visible agglutinationsensitized without visible agglutination

Preceded by two important discoveries by Race and Wiener

Race found what he believed to be anti-D components in some patients sera that contained detectable anti-D

These anti-D components seemed to combine

with D-positive red cells but did not cause

direct agglutination of those D-positive red

cells incomplete antibodiescells incomplete antibodies

Coombs visualized red cells coated with

human, incomplete antibodies being linked

together by other antibodies that were

directed against human antibodies

This simple hypothesis was then tested by

Coombs, Mourant and Race as the principle of

the antiglobulin test

The routine application of the antiglobulin test The routine application of the antiglobulin test

to immunohematology have allowed major

discoveries in human blood groups, immune

and acquired hemolytic anemias and

antiglobulin reagents

Principle of the Antiglobulin Test

The antiglobulin test depends on the following

basic premises:

Antibodies are globulins

Animals injected with human globulins Animals injected with human globulins

produce anti-human globulins. These anti-

human antibodies include antibodies to

complement components and IgG

The anti-human antibodies bind to the Fc

portion of sensitizing antibodies and form

bridges between antibody-coated red cells,

resulting in visible agglutinationresulting in visible agglutination

Anti-human antibodies will bind to human

antibodies that are both bound to red cells

(sensitizing) and unbound. Anti-human

antibodies will react first with any unbound

human antibodies

Depending on the amount of unbound human

antibody present for binding with the anti-

human globulins (AHG), AHGs may not be

available to form bridges between sensitized available to form bridges between sensitized

red cells

Principle of the Antiglobulin Test

IgM pentameric (large structure) when

bound to RBCs antigens, agglutination will

occur (usually at room temperature)

Principle of the Antiglobulin Test

When IgG antibodies attach to their

appropriate red cell antigens may fail to

cause agglutination

Will remain firmly bound even when the cells Will remain firmly bound even when the cells

are thoroughly washed in saline

These bound antibody may be detected by the

addition of anti-human globulin (AHG) act as

a bridge molecule to help IgG to agglutinate

RBCs

Principles of the Antiglobulin Test

Principles of the Antiglobulin Test

Injecting an animal with human globulin (IgG)

and complement proteins

This stimulates the animal to produce

antibodies to these foreign proteinsantibodies to these foreign proteins

AHG will react with human globulin molecules

whether bound to RBCs or free in serum

AHG will agglutinate with washed RBCs coated

with human globulin

Principles of the Antiglobulin Test

If the red cells in the test are sensitized with

IgG or complement, the AHG reagent will

crosslink and cause agglutination

Anti-IgG attaches to the Fc portion of the IgG

molecule that is bound to the red cellmolecule that is bound to the red cell

Anti-C3 attaches to C3 molecules bound to the

red cell

Agglutination = positive antiglobulin test

No agglutination = negative antiglobulin test

Immunoglobulin

Positive Antiglobulin Test

Negative Antiglobulin Test

Reagents

1. Monoclonal (single clone of cells-specificity)

2. Polyclonal (human source: mixture of cells-

contains multiple antibodies)

Assist in identifying antigen present on Assist in identifying antigen present on

patients RBC that may cause and reaction in

vivo

Polyspecific AHG

Abs to human IgG and Abs to human C3d (anti-

C3d to detect Ab bound with complements

and those rare Abs)and those rare Abs)

Advantage is that polyspecific AHG may detect

complement-dependent Abs on RBCs

Monospecific AHG

Abs to human IgG or human C3d only

May miss an important antibody

The AHG serum that is routinely used by most

labs for pretransfusion IATs is monospecific

anti-IgG

When doing DATs to detect in vivo When doing DATs to detect in vivo

sensitization with IgG or C3, blood banks must

use polyspecific AHG containing anti-IgG, anti-

C3b and anti-C3d

Zeta Potential

An expression of the difference in electrostatic

potential at the surface of the red cell and the

ionic cloud of positive cations that are

attracted to the negative charges on the attracted to the negative charges on the

surface

The net negative charge surrounding red cells

is part of the force that repels red cells from

each other

Reduce the zeta potential facilitate the RBCs

to agglutinate by IgG molecules

zeta potential zeta potential

Agglutination

Enhancement Media

Low Ionic Strength Solution (LISS)

Decrease the ionic strength of a reaction medium

Thus, reduce the zeta potential

Leads to an increase in the attraction between

positively charged Ab molecules and negatively positively charged Ab molecules and negatively

charged red cells

Increase the rate of antibody uptake incubation

time of 5-15 mins; instead of 30-60 mins

Albumin

Enhances antibody detection test by reducing

the zeta potential

Allow Ab-sensitized cells to become closer Allow Ab-sensitized cells to become closer

together

Do not promote the antibody uptake stage of

agglutination

Enzymes

Can enhance or suppress the reaction of

certain blood group antigens and antibodies

There are certain enzymes can be used:

Ficin (Fish)Ficin (Fish)

Trypsin (pig stomach)

Papain (papaya)

Bromelin (pineapple)

Action of Enzyme

The treatment of red cells with enzyme

results in the release of sialic acid from the

membrane of RBC

Subsequent decrease in the negative charge Subsequent decrease in the negative charge

of the cells which lead to reduction in the

zeta potential

The use of proteolytic enzymes in in vitro test

systems facilitates antibody identification

Blood group antigen-antibody reactions are

differently affected by enzyme treatmentdifferently affected by enzyme treatment

Enzymes cleave certain proteins from the

surface of the red blood cells, destroying some

antigens while enhancing others

Abs reactivity which can be enhanced by

enzyme treatment are Rh, Kidd, P1, Lewis and

I Ags

Ags that can be destroyed or depressed by Ags that can be destroyed or depressed by

enzyme treatment are Fya, Fyb, M, N and S

Reagent Uses

Bovine serum albumin

(BSA)

Rh antibodies enhanced

at 37C

Low ionic strength saline

(LISS)

Increases antibody

uptake, decreases

incubation time, cost-incubation time, cost-

efficient

Proteolytic enzymes

(papain, ficin, trypsin,

bromelin)

Kidd, Rh, Lewis

antibodies enhanced,

generally warm and cold

autoantibodies

enhanced