4. Antihuman Globulin (AHG)

Question 1

AHG: Principle

Answer 1

• Human serum (globulin) is injected into rabbit
• Acts as Ag- stimulated rabbit to make Ab
• Anti-human globulin (AHG) harvested from rabbit - will bind to any human globulin (Ab) bound to red blood cells - will form lattice agglutination - complete ABSC

Question 2

Overview of Antihuman Globulin Test

Answer 2

Test to detect globulin (antibody and/or complement) coating red blood cells

Question 3

Coating of RBCs with globulin can occur:

Answer 3

In vivo (within the body)
In vitro (in the laboratory)

Question 4

Two routine BB antiglobulin tests

Answer 4

1) Direct Antiglobulin Test (DAT)

2) Indirect Antiglobulin Test (IAT)

Question 5

AHG Test, Coombs Test: History

Answer 5

1) Direct - detects IgG or complement coating patient cells (DAT = Direct Antiglobulin Test)
2) Indirect - detects IgG or complement in patient serum or plasma that is capable of coating reagent (or donor) red blood cells (IAT = Indirect Antiglobulin Test or ABS/ABSC = Antibody Screen/ing)

Question 6

Polyspecific Reagents

Answer 6

Polyspecific AHG reagent = broad spectrum

• Contains anti-IgG and anti-C3bC3d
• Will detect IgG Ab and/or C3bC3d bound to RBC

USES: DAT, IAT, can be neutralized

Question 7

Monospecific Reagents

Answer 7

Anti-IgG - heavy chain specific

• No complement activity
• Mixture of 4 IgG subclasses
• Will detect only IgG Antibody bound to RBC

USES: DAT, IAT, can be neutralized

Question 8

Anti-C3bC3d

Answer 8

• No anti-IgG activity
• Will detect only C3b or C3d bound to RBC Anti-C3bC3d vs. Anti-C3d

USES: DAT, not used for IAT, can be neutralized

Question 9

Control cells: Negative result using polyspecific AHG reagent or Anti-IgG AHG reagent

Answer 9

How do we know the reagent was not neutralized?
Test all negative tubes with Coombs control cells - agglutination confirms NEG result with PS AHG or Anti-IgG (these are known as “check” cells)

Question 10

Control cells: Negative result using anti-C3bC3d

Answer 10

How do we know if the reagent was not neutralized?`

Test all negative tubes with complement control cells - agglutination confirms NEG result with anti-C3bC3d

Question 11

Direct Antiglobulin Test: Indications (looking for in-vivo Ag-Ab reaction)

Answer 11

• Hemolytic Disease of Fetus and Newborn (HDFN)
• Transfusion reaction
• Allo vs. Autoantibody
• Evaluation of Hemolytic Anemia

Question 12

Direct Antiglobulin Test: less complicated than the IAT

Answer 12

• No need to try to attach antibody to red cells

- RBCs are already sensitized in the patient’s specimen (if positive)

Question 13

Direct Antiglobulin Test: Whole blood collected in an EDTA (purple top) tube

Answer 13

• EDTA whole blood is used to reduce interference of the DAT caused by complement
• The EDTA binds calcium, prevents coagulation cascade
  e. g. Red top tubes (clotted)= rich source of complement

Question 14

DAT Procedure: Polyspeciific AHG Reagent

Answer 14

Two drops Polyspecific AHG reagent
One drop 2-5%  suspension washed RBC
Spin and read immediately (IgG)
5 min RT incubation, spin and read 
immediately (C3)
BOTH readings negative = negative 
interpretation
Confirm with 1 drop of Coombs control 
cells, spin and read (pos)
One and/or the other reading(s) 
positive = use monospecific reagents to 
determine cause of reactivity
DAT

Question 15

DAT Procedure: Anti-IgG Readent

Answer 15

Two drops Anti-IgG reagent

One drop 2-5% suspension washed RBC

Spin and read immediately

Negative = negative interpretation

Confirm with 1 drop of Coombs control
cells, spin and read (pos)

Question 16

DAT Procedure: Anti-C3bC3d

Answer 16

Two drops Anti-C3bC3d reagent

One drop 2-5% suspension washed RBC

Spin and read immediately; if neg
5 min RT incubation, spin and read
immediately

BOTH readings negative = negative
interpretation

Confirm with 1 drop of Complement
control cells, spin and read (pos)

Question 17

IAT Test: Indications

Answer 17

•Looking for “in vitro” Ag-Ab reaction
•The IAT is used to detect red cell antibodies in patient serum
•AKA the “antibody screen” or ABSC
•Part of the type and screen and type and crossmatch procedure
•Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both
•Most clinically significant alloantibodies are IgG antibodies that react best at 37C and are formed as a result of: Previous exposure via transfusion or pregnancy or transplant
e.g. of clinically significant include: antibodies to Rh, Kell, Kidd, and Duffy antigens

Question 18

IAT: IgM antibodies

Answer 18

• IgM antibodies are usually not clinically significant (except for ABO antibodies) but are a source of in vitro serologic difficulty that may delay transfusion
  e. g. of IgM antibodies include antibodies to the Lewis, M, N, I, and P1 red cell antigens (will come back to LMNOP’s)
• IgM antibodies react best at cold temperatures (4C) and are usually naturally occurring in that they do not require a sensitizing event

Question 19

IAT: Looking for “in-vitro” reaction

Answer 19

Testing for presence of antibody that could cause a transfusion reaction

Antibody Screening Test

If positive, identify
antibody(ies) present

Patient should receive Ag
NEG RBCs

Testing for compatibility of patient plasma with donor cells (crossmatch)

Question 20

IAT: Indications – Phenotyping for presence of antigen on RBCs

Answer 20

Some phenotyping antisera require an IAT test to detect presence of antigen on RBCs

Question 21

IAT Procedure for Antibody Detection (Screening): Immediate Spin (IS) Testing

Answer 21

If 3-cell antibody screening:
Label 3 tubes I, II, III with patient ID
Two drops patient plasma (or serum)
One drop 2-5%  suspension reagent RBC 
(I in 1st tube, II in 2nd tube, III in 3rd tube)
Spin and read immediately (IgM)
Record reaction

Question 22

IAT Procedure for Antibody Detection (Screening): AHG Phase of Testing

Answer 22

Wash tubes 3x isotonic saline
Dry pack
Add 2 drops of AHG reagent (Anti-PS 
AHG or Anti-IgG)
Spin and read immediately (IgG)
Record reaction
If neg, add Coombs control cells (1 dr) 
to each neg tube, spin, read, record

Question 23

Sources of Error: False POSITIVES

Answer 23

BOTH DAT & IAT: 
- Saline stored in glass 
bottles (not done now)
- Improperly cleaned 
glassware
- Over-centrifugation
- Improperly prepared AHG 
reagents

DAT:
- Clotted specimens stored 
at 4oC 
- Clotted specimens in 
tubes with silicone gel
- Specimens collected from 
IV lines
- Polyagglutination

IAT: 
- When Phenotyping 
RBC coated "in vivo" - 
Pos DAT
- Requires: Chloroquine 
treatment – cells