Blood Bank: AHG

IgG or C’ bound to RBCs

The antiglobulin test (AGT/AHG/Coomb’s) is a test used for detecting (—2—). Binding of these components can occur either in vivo or in vitro

provides a bridging molecule

IgG does not usually cause immediate visible agglutination, so when IgG is attached to an RBC, AGT provides a (——) to make hemagglutination visible

Direct antiglobulin test

Measures the in vivo sensitization of RBCs

Indirect antiglobulin test

Measures in vitro sensitization of the RBCs

(pt RBC)-Ab + AHG → Agglutination

What reaction is happening in DAT?

(Class II hypersensitivity)

• HDFN

• HTR

• AIHA

Clinical conditions tested with DAT (3)

1. Wash pt RBCs 3x with saline

2. Add polyspecific AHG

3. Centrifuge

4. Grade and record agglutination

Describe the DAT procedure (4)

Coomb’s check cells

To all negative DAT results, add (——), where at least a 2+ reaction is valid and a negative indicates the test must be repeated

• Adequate washing

• AHG was added

• AHG is working

What does the addition of check cells ensure? (3)

• In vivo sensitization

• Recent transfusion w/ allo IgG

• Medical condition w/ IgG, auto Ab, or C’

• Medication w/ IgG or C’

(+ result w/ no clinical significance in 2% of population)

With a positive DAT result, you should consider… (4)

• Inadequate washing leaves residual serum proteins that can neutralize AHG

• Addition of AHG reagent (amt/omission/contamination)

• AHG reagent integrity

Factors that affect the DAT test (3)

• IgM Ab present

• CHD

• PCH

• C’ binding IgG

What does it mean when DAT is only positive for C3d? (4)

Indirect Antiglobulin Test (IAT)

This technique is used in the Antibody Screen Test and the Antibody Identification Test

1. Known phenotype RBCs + Pt serum → in vitro sensitization

2. RBC-Ab + AHG → Agglutination

What are the reactions involved in IAT?

1. Incubate pt sera with reagent RBCs

2. Wash at least 3x

3. Add AHG and mix

4. Centrifuge

5. Grade and record agglutination

6. Add check cells to negative reactions

Describe the IAT procedure (6)

• SC ratio

• Reaction media

• Temperature

• Incubation time

• Washing

• Adding AHG (amt/omission)

• Centrifugation

Factors that affect the IAT test (7)

• Advantage: may detect C’ binding Abs on RBCs (i.e., Jka)

• Disadvantage: may detect C’ normally found on cells (nuisance rxns)

Advantage and disadvantage of using polyspecific AHG reagent

• Polyspecific = clear

• Mono IgG = green

• Mono C3d = clear

What are the colors of the AHG reagents? (3)

Polyclonal reagent

Reagent manufactured by an animal being hyper immunized with human globulins, bled for antisera to obtain high titer, high avidity specific to human globulin

Monoclonal reagent

Reagent manufactured by hybridoma cells from mice; collection of culture or ascites fluid yields antisera

1. Improper specimen (refrigerated, clotted → C’ bind)

2. Dirty glassware

3. Autoagglutinable cells

4. Bacterial contamination

5. DAT (+) cells used for IAT (i.e., autocontrol)

6. Over-centrifugation/reading

7. Polyagglutinable cells

8. Preservative-dependent Ab in LISS

9. Contaminating Ab in AHG

10. Saline contaminated by heavy metals or colloidal silica

Things that cause false positive results in AHG test (10)

1. Inadequate washing

2. AHG not added

3. Neutralization of AHG (washing)

4. Deterioration of AHG reagent

5. Deterioration of Ab or C’ in serum

6. Inadequate incubation in IAT

7. Cell suspension too heavy/weak

8. Under/over-centrifuged

9. Testing error (i.e., shake tube too hard)

10. Poor reading technique

11. Pt serum not added in IAT

Things that cause false negative results in AHG test (11)