AHG Testing

Question 1

Anti-D added to Rh-negative cells

Answer 1

Reaction is negative
QC of anti-D reagent

Question 2

Indirect AHG test detects

Answer 2

Antibody in patient serum

Question 3

Antiglobulin Testing History

Answer 3

Coombs, Mourant, and Race
Developed test to detect non-agglutinating antibodies in serum

Question 4

Two Types of AHG Testing

Answer 4

Direct Antiglobulin Test (DAT)
Indirect Antiglobulin Test (IAT)

Question 5

Direct Antiglobulin Test (DAT)

Answer 5

In vivo coating of RBCs with AB and or complement

Question 6

Indirect Antiglobulin Test (IAT)

Answer 6

In vitro reactions of serum and reagent RBCs

Question 7

Antibody/complement

Answer 7

Globulins (proteins)
- IgM
- IgA
- IgG

Question 8

IgG subclasses

Answer 8

IgG 1 is best at crossing placenta
IgG 4 is the least likely to activate complement

Question 9

Steps for Antiglobulin Testing Reagent

Answer 9

Step 1
- Stimulate animal to produce antibody to a foreign protein
Step 2
- Adsorb serum to produce reagents with certain specificities: Polyspecific, Monospecific

Question 10

Polyspecific AHG

Answer 10

Anti-IgG and anti C (use every day in lab)

Question 11

Monospecific AHG

Answer 11

anti-IgG
anti-C; c3d or c3b (specialty reagent issue in specific instance)

Question 12

Polyspecific Information

Answer 12

Contains antibody to human IgG and component of Complement (C3d, C3b, C4d, and C4b)
Has little or no reactivity with IgM or IgA

Question 13

Monospecific AHG Anti-IgG Info

Answer 13

Contains antibody to human IgG
- primarily gamma chain
May react with light chains
Will NOT detect cold reactive cold reactive antibodies that fix complement

Question 14

Monospecific AHG Anti-C3d, C3b

Answer 14

Contains antibody to human complement component
Has no anti-IgG activity
Not used in routine testing
Used in investigation of positive DATs

Question 15

Complement’s Role in Reactions

Answer 15

2 Mechanisms of Complement in vivo
- Ag-Ab complex binds complement
- Immune complexes activate complement
Complement binding may or may not cause cell lysis

Question 16

Common complement binding antibodies

Answer 16

Anti-A
Anti-B
Anti-Jka (most common to fall below detection levels)
Anti-Lea

Question 17

AHG Reagents

Answer 17

React with human globulin molecules attached to RBCs and/or free in plasma

Question 18

AHG Antibody

Answer 18

Fab part of AHG reacts with Fc part of antibody molecule

Question 19

DAT Info

Answer 19

Cells must be thoroughly washed
- Prevent neutralization of the AHG reagent
All negative reactions must be observed microscopically
Check Cells must be added to all negative tubes
- Assures activity of AHG reagent

Question 20

Summary of DAT Procedure

Answer 20

Prepare 2-5% suspension of cells
Add 2 drops of suspension to tube and wash with saline
- removes unbound globulin
Add 2 drops of AHG to cell button, centrifuge, and read
Add 1 drop Check Cells to all negative tests

Question 21

Transfusion Reactions

Answer 21

Recipient has created an antibody that attached to the donor cells

Question 22

Hemolytic Disease of the Newborn

Answer 22

All cord blood samples contain maternal antibody
- some recognize the baby as foreign

Question 23

Autoimmune Hemolytic Anemia

Answer 23

Patient has antibody directed against their own antigen
Can be drug induced

Question 24

DAT Application

Answer 24

Transfusion Reactions
Hemolytic Disease of Newborn
Autoimmune Hemolytic Anemia

Question 25

Summary of IAT Procedure

Answer 25

Add 2 drops of patient serum to tube Add 1 drop of reagent screening cells Centrifuge and read (RT/IS phase) Add enhancement media; incubate (PEG/LISS) Centrifuge and read (37 C for 10-30 minutes) LISS only Wash to remove unbound globulin Add 2 drops of AHG reagent Centrifuge and read Check microscopically all negative samples Add Check Cells (should be positive)

Question 26

IAT Phases

Answer 26

Covers Multiple Phases of REaction - RT detects cold antibodies (IgM) - 37 C enhanced & incubated detect warm antibodies (IgG) - AHG reagent serves as a bridge (detects non-agglutinated but sensitized cells)

Question 27

IAT Factors Affecting Testing

Answer 27

Temperature (attachment of antibody) RT (IS) - IgM, +/- clinical significance 37 C - IgG, clinically significant Ionic Strength - shielding that hinders Ag-Ab binding Reagents to minimize effect (LISS/PEG/Albumin) Portion of Serum to Cells (2 drops serum:1 drop of cells) Time of Incubation

Question 28

IAT Testing Applications

Answer 28

Pre-natal testing Pre-transfusion testing Compatibility testing (crossmatch) Antibody identification and titers RBC phenotyping

Question 29

Sources of Error in DAT and IAT Testing

Answer 29

False Negatives False Positives

Question 30

False Negatives in DAT & IAT Testing

Answer 30

Inadequate washing of RBCs Delay of Testing - IgG decreased Inactive (or absent) AHG reagent Improper Centrifugation Improper strength of cell suspension Prozone IgG cryoprecipitate Saline pH Check Cells

Question 31

False Positives in DAT & IAT Testing

Answer 31

Agglutinated RBCs Containers (causes spontaneous agglutination) Dirty glassware (spontaneous agglutination) Over centrifugation AHG reagent

Question 32

False Negatives for DAT only

Answer 32

Low number of attached Abs Complement binding

Question 33

False Positives for DAT only

Answer 33

Use of serum sample Gel separator tubes Samples from IV lines (not appropriately flushed) Septicemia (blood has additional immunoglobulins)

Question 34

False Negatives for IAT only

Answer 34

Sample storage Plasma samples Temperature and Incubation Time Cell Suspension

Question 35

False Positives for IAT only

Answer 35

Cells coated with IgG (positive DAT)

Question 36

Why are cells washed prior to adding AHG reagent?

Answer 36

Remove any unbound antibody and avoid neutralizing the AHG reagent

Question 37

What is being detected by AHG reagent in the DAT test?

Answer 37

Detects antibody bound to patient RBCs

Question 38

What is being detected by AHG reagent in the IAT test?

Answer 38

Detects antibody from patient serum that is bound to reagent RBCs

Question 39

What are the consequences of inadequate washing of cells?

Answer 39

Cause a false negative due to neutralization of AHG Indicated by negative reactions of check cells

Question 40

If antiglobulin test is positive with polyspecific AHG reagent, what does that indicate?

Answer 40

IgG and/or complement is attached to the RBCs

Question 41

What are possible causes of a false positive in IAT?

Answer 41

Rouleaux Positive DAT

Question 42

What are possible causes of a false negative in DAT?

Answer 42

Inadequate washing of the RBCs AHG reagent not added

Question 43

What is the purpose of using EDTA sample for DAT?

Answer 43

Anticoagulation of sample with EDTA prevents IN VITRO complement activation

Question 44

EDTA, Heparin Testing

Answer 44

Within 2 days

Question 45

Clotted Whole Blood, Citrate, Oxalate Testing

Answer 45

Within 14 days

Question 46

ACD, CPD Testing

Answer 46

Within 28 Days

Question 47

CPDA-1 Testing

Answer 47

Within 35 days