CLS 3311Advanced Clinical

Immunohematology

Antihuman Globulin Testing

(AHG)

Antihuman Globulin

Definition:• Antihuman: antibodies against human antigens

• Globulin: all antibody molecules are globulins

Therefore: Antihuman Globulin is antibody directed against the Fc portion of human

antibodies and/or complement components.

Antihuman Globulin• In the past AHG was made by injecting rabbits (or

sheep, etc.) with human globulin and complement (C’). The rabbit would then make antibody to the human globulin and C’ components.

• The antihuman antibody (polyclonal) would then be harvested, fractionated and purified for use in the blood bank as anti-A, anti-B, etc. Each lot # was quite unique.

• Today we use, almost exclusively, Monoclonal AHG produced using mouse hybridoma cells to make very specific AHG. Figure 4.1, page 75 Harmening

Antihuman Globulin Reagents

1. Polyspecific:

2. Monospecific:

• Table 4.1, page73 Harmening

1. Contains anti-IgG and anti-C3d (Complement)

2. Contains only one specificity: either anti-IgG or anti-C3d. NOT both, only one.

• AHG Reagents Include:– Anti-IgG (Anti-IgG)– Anti-C’ (Anti-C3/C4)– Anti-IgG & C’ (Polyspecific AHG)

• These Anti-Human Globulins must be diluted to achieve optimum reactivity. – In doing this anti-IgM is diluted

BELOW detectable level.– The presence of IgM on the RBC is

NOT detected using AHG reagents.

Reagent Definition

Polyspecific (rabbit polyclonal, rabbit/murine monoclonal blend,

murine monoclonal)

Rabbit polyclonal has anti-IgG, -C3d; rabbit/murine monoclonal blend has rabbit polyclonal anti-IgG and murine monoclonal anti-C3b, -C3d; murine monoclonal has monoclonal anti-IgG, -C3b and -C3d

Monospecific anti-IgG (rabbit poly- clonal; IgG heavy chains; monoclonal IgG)

Rabbit polyclonal has only anti-IgG; IgG heavy chains only has antibody to gamma chains; monoclonal has murine anti-IgG

Anti-C3d and anti-C3b or Anti-C4b, -C4d, -C3d (rabbit polyclonal)

Contains only antibodies against designated Complement components with no anti-immunoglobulin activity

Anti-C3d or anti-C3b, -C3d (murine monoclonal)

Contains only antibodies against designated Complement components with no anti-immunoglobulin activity

Table 12.1, page 260, AABB Technical Manual, 13th Edition

AHG Technique’s: What is the relevance?

• Some very clinically significant unexpected antibodies (Kidd, Duffy, etc.) attach to red cell antigen or activate complement to do so, but do NOT cause agglutination at immediate spin or 37o phase.

• Yet, these antibodies are capable of causing severe hemolytic transfusion reactions or hemolytic disease of the newborn.

Relevance of AHG• AHG techniques enable the detection of these

antibodies or C’ components that otherwise went undetected. AHG enables detection of both in vitro (Indirect Antiglobulin Test) and in vivo (Direct Antiglobulin Test) antibody attachment.

AHG testing is the primary method of detecting all Antibodies except

those of the ABO System.

Expected Vs. Unexpected Antibodies

Expected Antibodies• If an ABO antigen is missing from an individuals red

blood cell membrane then it is EXPECTED that the individual will produce an antibody to that antigen. These have also been called “naturally occurring”.

• Example: Group A individual does NOT have B antigens on their red blood cell membrane. They will normally produce anti-B antibodies.

Unexpected Antibodies• In ALL other blood group systems if the antigen is

missing from the red cell, the individual is NOT expected to produce an antibody against it, normally.

• When these antibodies are produced they are termed UNEXPECTED antibodies.

• EXAMPLE: An individual lacking the “D” antigen on their RBC membrane is NOT expected to have anti-D antibodies in their plasma, normally. It requires exposure to the D antigen from a foreign RBC either by transfusion or pregnancy.

• CONCLUSION: Only ABO antibodies are expected to be produced in the absence of foreign RBC Ag.

• All other blood group systems require, with a few exceptions, exposure to a foreign red cell antigen (transfusion or pregnancy) to stimulate production of an unexpected antibody.

Indirect Antiglobulin Test (IAT)

• Another name is “Antibody Screen”• Patient serum or plasma containing

unexpected Antibodies is incubated with RBC’s possessing the corresponding antigen (Screen Cells).

• The unexpected IgG class Antibody will bind to the corresponding RBC Antigen during incubation.

OR …• The Patients unexpected IgM or IgG class

antibody will bind C’ to the RBC membrane with the corresponding antigen during incubation.

• The RBC’s are then washed with saline to remove all UNBOUND serum proteins including IgG & C’.

• An Anti-Human Globulin reagent is added to the washed, dry RBC button.

If unexpected antibodies (or C’) are present on the red cell membrane AHG will bind and cross link

the red blood cells causing agglutination.

Set of Problems…1. Even 10 mL’s of AHG can be neutralized by a tiny

amount of free serum protein.

2. A technologist can forget to add AHG to a test tube.3. A couple of drops of residual saline can dilute the

AHG reagent below detectable levels.

• Normally people do NOT produce unexpected Antibodies. Therefore the test should normally be negative.

HOW DO YOU KNOW A NEGATIVE AHG TEST IS A TRUE NEGATIVE?

Coomb’s Control or Check Cells

• Addition of IgG coated red cells to all negative AHG tests is required by AABB Standards for Blood Banks and Transfusion Services for antibody screen and crossmatch tests.

• Negative AHG test should contain “FREE” AHG reagent still capable of binding to IgG coated RBC’s

• If IgG coated RBC’s are added to “negative” test and centrifuged, then the “free” AHG should bind to them and cause agglutination.

If the Check Cells agglutinate, it indicates three

(3) things:

• The test was adequately washed prior to addition of the AHG reagent.

• AHG reagent was added to the test tube.

• The AHG reagent that was added was in an ACTIVE form.

What agglutinated Check Cells DOES NOT

mean!• That the test was performed correctly!!

Why? Because…– Patient Serum could have been left out of tube

– The WRONG Patient Serum could have been added

– The WRONG test cells could be used

– Incubation time or temperature may be incorrect

– Enhancement media (LISS) may not have been added

– The tube could be mis-labeled

– The results could be mis-read

– The results could be recorded incorrectly

Purpose of the IAT

• The purpose of the Indirect Antiglobulin test is to detect unexpected antibodies in patient or donor serum.

• Screening test only, not for antibody identification.• RBC’S used are called SCREEN CELLS

– Reason: they’re used to screen for unexpected antibodies present in the donor or recipient serum.

– Use a set of cells - Usually 2 or 3 cells, each cell from a different source/donor so each has a unique phenotype

Purpose of the IAT

What type of cells are used as the “Screen Cells”?– Group O cells – Why?

– The cells MUST have antigens that most commonly stimulate production of unexpected antibodies (Kell, Kidd, Duffy and Rh).

– SOURCE OF CELLS: Individual donors with specific phenotypes

DIRECT ANTIGLOBULIN TEST (DAT)

• DAT tests for in vivo sensitized red blood cells– Attachment of antibody or complement in vivo

Problem:• Clotted samples still contain serum with Ca++

present so that C’can be activated non-specifically after collection causing false positive results.

• Solution: Perform test on EDTA sample. EDTA chelates calcium stopping C’ activation. The blood in the EDTA sample represents in vivo C’ activity.

DIRECT ANTIGLOBULIN TEST1. Collect sample in EDTA tube

– Make 3-4% RBC suspension and wash 3-4 times – Cord blood needs additional washing.

2. Take 1 drop of 3-4% RBC suspension and centrifuge to get dry button

3. Add AHG to the dry button– If IgG or C’ attached to the RBC’s in vivo, the

AHG will cause agglutination (Autoimmune process, HDN)

– NORMAL RESULT: Negative– Again, how do we know that the Negative result is

a TRUE Negative?

Application of AHG testing include detection of:

1. Hemolytic Anemia's

2. Transfusion Reactions

3. Hemolytic Disease of the Newborn

4. Antibody Screening of donors and recipients

5. Final step in Antibody Identification

I II III Auto

IS 0 0 0 0

37 LISS 0 0 0 0

AHG 0 0 0 0

CkCells + + + +

InterpretNo Unexpected

Antibodies Detected

This represents a general layout for an Antibody Screen report and suggested interpretation of an IAT without reactivity. Can I report it out as negative? Are we able to detect all unexpected antibodies with this system?

I II III Auto

IS 0 0 0 0

37 LISS 1+ 0 0 0

AHG 2+ 0 0 0

CkCells + + +

InterpretUnexpected antibody of

Class IgG.

Why do we suspect that this unexpected antibody is IgG class? Why not IgM? Is it clinically significant?

I II III Auto

IS 0 0 0 0

37 LISS 0 0 0 0

AHG 0 0 1+ 0

CkCells + + +

Interpret

Must know what AHG reagent used. If anti-IgG, then unexpected IgG class

antibody;

If anti-IgG, anti-C3, then coating IgG or Complement Dependent antibody

detected.

I II III Auto

IS 1+ 0 0 0

37 LISS 2+ 0 0 0

AHG 4+ 0 0 0

CkCells + + +

Interpret

1. Unexpected antibody detected, IgG class – Strong reacting2. Unexpected antibodies, may be multiple (IgM and/or IgG) detected.

I II III Auto

IS 1+ 0 0 0

37 LISS 0 1+ 0 0

AHG 0 2+ 0 0

CkCells + + +

InterpretUnexpected antibodies

detected, one of IgM class and one of IgG class

I II III Auto

IS 0 0 0 0

37 LISS 2+ W+ 0 0

AHG 4+ 1+ 0 0

CkCells + +

Interpret

May indicate Multiple antibodies or Single antibody of class IgG

showing dosage.

I II III Auto

IS 0 0 0 0

37 LISS 0 0 0 0

AHG 0 0 0 1+

CkCells + + +

Interpret

Positive auto control, should be followed up with DAT using EDTA sample to determine if

coating is in vivo.