AHG Testing Flashcards
Anti-D added to Rh-negative cells
Reaction is negative
QC of anti-D reagent
Indirect AHG test detects
Antibody in patient serum
Antiglobulin Testing History
Coombs, Mourant, and Race
Developed test to detect non-agglutinating antibodies in serum
Two Types of AHG Testing
Direct Antiglobulin Test (DAT)
Indirect Antiglobulin Test (IAT)
Direct Antiglobulin Test (DAT)
In vivo coating of RBCs with AB and or complement
Indirect Antiglobulin Test (IAT)
In vitro reactions of serum and reagent RBCs
Antibody/complement
Globulins (proteins)
- IgM
- IgA
- IgG
IgG subclasses
IgG 1 is best at crossing placenta
IgG 4 is the least likely to activate complement
Steps for Antiglobulin Testing Reagent
Step 1
- Stimulate animal to produce antibody to a foreign protein
Step 2
- Adsorb serum to produce reagents with certain specificities: Polyspecific, Monospecific
Polyspecific AHG
Anti-IgG and anti C (use every day in lab)
Monospecific AHG
anti-IgG
anti-C; c3d or c3b (specialty reagent issue in specific instance)
Polyspecific Information
Contains antibody to human IgG and component of Complement (C3d, C3b, C4d, and C4b)
Has little or no reactivity with IgM or IgA
Monospecific AHG Anti-IgG Info
Contains antibody to human IgG
- primarily gamma chain
May react with light chains
Will NOT detect cold reactive cold reactive antibodies that fix complement
Monospecific AHG Anti-C3d, C3b
Contains antibody to human complement component
Has no anti-IgG activity
Not used in routine testing
Used in investigation of positive DATs
Complement’s Role in Reactions
2 Mechanisms of Complement in vivo
- Ag-Ab complex binds complement
- Immune complexes activate complement
Complement binding may or may not cause cell lysis
Common complement binding antibodies
Anti-A
Anti-B
Anti-Jka (most common to fall below detection levels)
Anti-Lea
AHG Reagents
React with human globulin molecules attached to RBCs and/or free in plasma
AHG Antibody
Fab part of AHG reacts with Fc part of antibody molecule
DAT Info
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
Summary of DAT Procedure
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
Transfusion Reactions
Recipient has created an antibody that attached to the donor cells
Hemolytic Disease of the Newborn
All cord blood samples contain maternal antibody
- some recognize the baby as foreign
Autoimmune Hemolytic Anemia
Patient has antibody directed against their own antigen
Can be drug induced
DAT Application
Transfusion Reactions
Hemolytic Disease of Newborn
Autoimmune Hemolytic Anemia
Summary of IAT Procedure
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)
IAT Phases
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)
IAT Factors Affecting Testing
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
IAT Testing Applications
Pre-natal testing
Pre-transfusion testing
Compatibility testing (crossmatch)
Antibody identification and titers
RBC phenotyping
Sources of Error in DAT and IAT Testing
False Negatives
False Positives
False Negatives in DAT & IAT Testing
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
False Positives in DAT & IAT Testing
Agglutinated RBCs
Containers (causes spontaneous agglutination)
Dirty glassware (spontaneous agglutination)
Over centrifugation
AHG reagent
False Negatives for DAT only
Low number of attached Abs
Complement binding
False Positives for DAT only
Use of serum sample
Gel separator tubes
Samples from IV lines (not appropriately flushed)
Septicemia (blood has additional immunoglobulins)
False Negatives for IAT only
Sample storage
Plasma samples
Temperature and Incubation Time
Cell Suspension
False Positives for IAT only
Cells coated with IgG (positive DAT)
Why are cells washed prior to adding AHG reagent?
Remove any unbound antibody and avoid neutralizing the AHG reagent
What is being detected by AHG reagent in the DAT test?
Detects antibody bound to patient RBCs
What is being detected by AHG reagent in the IAT test?
Detects antibody from patient serum that is bound to reagent RBCs
What are the consequences of inadequate washing of cells?
Cause a false negative due to neutralization of AHG
Indicated by negative reactions of check cells
If antiglobulin test is positive with polyspecific AHG reagent, what does that indicate?
IgG and/or complement is attached to the RBCs
What are possible causes of a false positive in IAT?
Rouleaux
Positive DAT
What are possible causes of a false negative in DAT?
Inadequate washing of the RBCs
AHG reagent not added
What is the purpose of using EDTA sample for DAT?
Anticoagulation of sample with EDTA prevents IN VITRO complement activation
EDTA, Heparin Testing
Within 2 days
Clotted Whole Blood, Citrate, Oxalate Testing
Within 14 days
ACD, CPD Testing
Within 28 Days
CPDA-1 Testing
Within 35 days
