AHG testing

What is anti human globulin used for?

To detect non-agglutinating antibodies in serum, also used to detect rbcs coated with antibody and/or complement

What does the direct antiglobulin test detect?

In vivo coating of rbcs with Ab and/or C’

What does the indirect antiglobulin test detect?

In vitro reaction of serum and reagent rbcs

What are antibodies/complement made of?

globulins (proteins)

Which IgG subclass is the best at crossing the placenta?

IgG1

Which IgG subclass is the least likely to activate complement?

IgG3

What are the steps to make antiglobulin?

• Stimulate animal to produce antibody to foreign protein

• Adsorb the serum to produce reagents with certain specificities

What does polyspecific AHG reagent contain?

Antibody to human IgG and complement (C3d,C3b,C4d,C4b)

What antibodies does polyspecific AHG have little reactivity to?

IgM and IgA

What is the most important antibody that AHG should detect?

IgG

What does monospecific AHG detect?

Either IgG or complement

What does monospecific AHG to IgG detect?

Primarily gamma chains, may react with light chains. Will not detect cold reactive antibodies that fix complement

What does monospecific AHG to complement detect?

Anti C3d, C3b. Should have no anti IgG activity. Not used in routine testing but is for investigation of positive DATs

What are the two mechanisms of complement binding IN VIVO?

• Ag-Ab complex binding complement

• immune complexes activating complement

What are some common complement binding antibodies?

• anti-A

• anti-B

• Anti- Jka

• Anti- Lea

What will AHG react with?

Human globulin molecules attached to rbcs and/or free in the plasma

What must be done to cell before adding AHG?

They must be thoroughly washed to prevent neutralization of the AHG reagent

What must be done to all negative AHG reaction?

Must be observed microscopically to confirm no agglutination

What must be added to all negative tubes?

Check cells that are coated with IgG to assure the activity of AHG reagent

Summarize the DAT procedure.

• prepare a 3-5% suspension of cells

• add 2 drops of suspension to tube and wash with saline to remove unbound globulin

• add 2 drops of AHG to cell button, centrifuge and read

• add 1 drop check cells to all negative tests

What is DAT used to evaluate?

• transfusion reactions

• hemolytic disease of the newborn

• autoimmune hemolytic anemia

What is a transfusion reaction?

Where the recipient has created an antibody that attached to the donor cells

What is hemolytic disease of the newborn?

All cord blood samples contain maternal antibody, some recognize the baby as foreign and attach

What is autoimmune hemolytic anemia?

Patient has antibody directed against their own antigens. Can be drug induced

Summarize the IAT procedure.

• add 2 drops of patient serum to tube

• Add 1 drop of reagent screening cells

• Centrifuge and read

• add enhancement media, incubate

• centrifuge and read

• wash

• add 2 drops of AHG, centrifuge and read

• add check cells

What does it mean if the check cells are positive?

AHG test is a true negative

What does is mean if the check cells are negative?

Invalid test, start over

Was has been done to all reagent screening cells?

Has been antigen typed so “all” antigens on it’s surface are known

What does the IAT cover?

Multiple phase of reaction

• At room temp-cold antibodies (IgM)

• 37 degrees- detect warm antibodies (IgG)

How does AHG detect cells that are non-agglutinated by sensitized?

Serves as bridge

What factors affect IAT testing?

• temperature: affects attachment of antibody to rbc

• Ionic strength

• serum to cell ratio

How temperature affect antibody attachment?

• decrease ab binding

• damage the ab and/or rbcs

How does ionic strength affect IAT testing?

Shielding affect that hinders Ag-Ab binding

What antibodies are clinically significant?

IgG “warm” antibodies

What reagents can minimize the effect of ionic strength?

• low ionic strength saline

• albumin

• polybrene

• Polyethylene glycol

What are the incubation times for saline/albumin and LISS/PEG?

• Saline/albumin: 15-30 minutes

• LISS/PEG: 10-15 minutes

What are some application of IAT?

• pre-natal testing

• pre-transfusing testing

• compatibility testing

• antibody identification and titers

• RBC phenotyping

What are some sources of false negatives with DAT and IAT testing?

• inadequate washing of rbcs

• delay of testing

• inactive AHG reagent

• improper centrifugation

• improper strength cell suspension

• prozone

• IgG cryoprecipitate

• saline pH

• check cells

What are some sources of false positives with DAT and IAT testing?

• agglutinated rbcs

• containers

• dirty glassware

• over centrifugation

• AHG reagent

What are some cause of false negatives with DAT testing?

• low number of attached Abs

• Complement binding

What are some causes of false positives with DAT?

• use of a serum sample (fibrinogen interference)

• gel separator tubes

• samples from IV lines

• septicemia

What are some causes of false negatives in IAT testing?

• sample storage

• plasma samples

• temperature and incubation times

• cell suspension

What are some sources of false positives with IAT testing?

• cells coated with IgG (positive DAT)