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Immunohematology > AHG Testing > Flashcards

AHG Testing Flashcards

1
Q

Anti-D added to Rh-negative cells

A

Reaction is negative
QC of anti-D reagent

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2
Q

Indirect AHG test detects

A

Antibody in patient serum

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3
Q

Antiglobulin Testing History

A

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

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4
Q

Two Types of AHG Testing

A

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

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5
Q

Direct Antiglobulin Test (DAT)

A

In vivo coating of RBCs with AB and or complement

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6
Q

Indirect Antiglobulin Test (IAT)

A

In vitro reactions of serum and reagent RBCs

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7
Q

Antibody/complement

A

Globulins (proteins)
- IgM
- IgA
- IgG

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8
Q

IgG subclasses

A

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

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9
Q

Steps for Antiglobulin Testing Reagent

A

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

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10
Q

Polyspecific AHG

A

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

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11
Q

Monospecific AHG

A

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

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12
Q

Polyspecific Information

A

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

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13
Q

Monospecific AHG Anti-IgG Info

A

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

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14
Q

Monospecific AHG Anti-C3d, C3b

A

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

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15
Q

Complement’s Role in Reactions

A

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

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16
Q

Common complement binding antibodies

A

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

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17
Q

AHG Reagents

A

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

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18
Q

AHG Antibody

A

Fab part of AHG reacts with Fc part of antibody molecule

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19
Q

DAT Info

A

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

20
Q

Summary of DAT Procedure

A

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

21
Q

Transfusion Reactions

A

Recipient has created an antibody that attached to the donor cells

22
Q

Hemolytic Disease of the Newborn

A

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

23
Q

Autoimmune Hemolytic Anemia

A

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

24
Q

DAT Application

A

Transfusion Reactions
Hemolytic Disease of Newborn
Autoimmune Hemolytic Anemia

25
Q

Summary of IAT Procedure

A

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)

26
Q

IAT Phases

A

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)

27
Q

IAT Factors Affecting Testing

A

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

28
Q

IAT Testing Applications

A

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

29
Q

Sources of Error in DAT and IAT Testing

A

False Negatives
False Positives

30
Q

False Negatives in DAT & IAT Testing

A

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

31
Q

False Positives in DAT & IAT Testing

A

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

32
Q

False Negatives for DAT only

A

Low number of attached Abs
Complement binding

33
Q

False Positives for DAT only

A

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

34
Q

False Negatives for IAT only

A

Sample storage
Plasma samples
Temperature and Incubation Time
Cell Suspension

35
Q

False Positives for IAT only

A

Cells coated with IgG (positive DAT)

36
Q

Why are cells washed prior to adding AHG reagent?

A

Remove any unbound antibody and avoid neutralizing the AHG reagent

37
Q

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

A

Detects antibody bound to patient RBCs

38
Q

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

A

Detects antibody from patient serum that is bound to reagent RBCs

39
Q

What are the consequences of inadequate washing of cells?

A

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

40
Q

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

A

IgG and/or complement is attached to the RBCs

41
Q

What are possible causes of a false positive in IAT?

A

Rouleaux
Positive DAT

42
Q

What are possible causes of a false negative in DAT?

A

Inadequate washing of the RBCs
AHG reagent not added

43
Q

What is the purpose of using EDTA sample for DAT?

A

Anticoagulation of sample with EDTA prevents IN VITRO complement activation

44
Q

EDTA, Heparin Testing

A

Within 2 days

45
Q

Clotted Whole Blood, Citrate, Oxalate Testing

A

Within 14 days

46
Q

ACD, CPD Testing

A

Within 28 Days

47
Q

CPDA-1 Testing

A

Within 35 days

Immunohematology (17 decks)

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