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Ain’t Nobody Got Time for

DATs…Lindsay Peters MLS(ASCP)SBB

Reference Laboratory Supervisor

Mississippi Valley Regional Blood Center

OBJECTIVES

Define and describe the antiglobulin test.

Identify the reagents used in the antiglobulin test.

Compare direct and indirect antiglobulin testing.

Describe DAT performance.

Discuss uses of the DAT.

Investigate positive DAT results.

Explore the causes of a positive DAT.

Antiglobulin Test

Definition: Antiglobulin Test

Determines if the RBC is coated by IgG/complement or both.

Described by Coombs, Mourant and Race in 1945.

What is detected?

RBC bound IgG/C3

How is it detected?

AHG Reagent

Polyspecific/ Monospecific

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Also known as…

What Reagents are Used?

Polyspecific

Anti-IgG + Anti-C3

Screening tool

Monospecific

Anti-IgG

Anti-C3

Globulin coated reagent cells (Check Cells)

Where do the Reagents Come From?

Polyclonal

Rabbits, Sheep, Goats

Immunized with “antigen”

High titer, high avidity antibodies to the human antibody

Monoclonal

Immunization of laboratory animals

Spleen cells harvested and fused with myeloma cells

High titer antibodies with well defined specificity

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Antiglobulin Test- What can we do with it?

Direct Testing (DAT)

IN VIVO sensitization

NO incubation

Applications:

HDN

AIHA

HTR/DHTR

Indirect Testing (IAT)

IN VITRO sensitization

Incubate of aby and

ag

Applications:

Aby detection

Aby ID

Crossmatch

Antigen typing

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DAT- How is the Test Performed?

Patient Cells Washed

Antiglobulin Added

CentrifugeRead

agglutination

If Negative, Add Check

Cells

Can detect: (sensitivity)

100-500 molecules of IgG attached to the red cell

400-1100 molecules of C3d attached to the red cell

Is it always significant? (specificity)

Up to 0.1% of Blood Donors

1-15% of hospital patients

Most have no signs of clinical hemolysis

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False Positives

Improper sample

Bacterial contamination

Over centrifugation

False Negatives

Inadequate WASHING

Cell suspension too weak /too heavy

Under centrifugation

Delay/ Interruption in testing

Indicate Hemolysis

Immune response to transfused blood

Potentially serious drug interaction

Suspected Hemolysis

Neonates

Suspected Transfusion Reaction

Performed as part of an antibody investigation

Evaluation of possible Drug Hemolysis

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Which type of DAT do I use?

Polyspecific

• SCREENING TOOL

Anti-IgG

• Detect Bound IgG Antibody

Anti-C3

• Detect Bound C3 component

• C3 attached by an antibody

• ABO, Le, Jk, P

The DAT is positive, now what?

1st- What do I do Next?

Elution

Aby Screen

2nd- Is it causing cell destruction?

Other Labs to Evaluate for Hemolysis

Clinical Symptoms

3rd-Why is it positive?

What is an Elution and How Do I use It?

Elution

• Disrupt bonds between antibody and RBC

• Collect antibody to test

• Physical or Chemical

Eluate

• Test against panel of reagent cells

• Can determine if there is a specificity

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Lui Freeze/ Thaw

Used mostly for ABO HDN

Heat (56C)

Used mostly for IgM antibodies, ABO HDN

Acid

IgG Antibdies

Most commonly used

Chemical (Ether, Digitonin)

Time Consuming

Dangerous

What causes the positive DAT?

Hemolytic Disease of the Newborn

Maternal aby crosses the placenta and attaches to baby’s RBCs

Aby MUST be IgG

Ag must be well developed on Baby’s red cells

Anemia, Jaundice, Enlargement of Liver and Spleen, Edema

DAT on Cord cells

Must WASH!!

IgG only

Eluate?

Considered Unnecessary

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What causes the positive DAT?

Hemolytic Disease of the Newborn

Most Common:

ABO

O mom’s higher levels

of IgG

Usually not severe

UV Light

Most Severe: Anti-D

Monitor and intervene

Treatment (after birth)

Transfusion UV Light

What causes the positive DAT?

AutoImmune Hemolytic Anemia

Cannot be diagnosed with Blood Bank Testing ALONE

Warm AIHA

Cold Agglutinin Syndrome/ Cold Agglutinin Disease

Mixed-type AIHA

Paroxysmal cold hemlgbinuria

Alloimmune

Drug Induced

Determine Treatment

AIHA- How do we know which one?

DAT Ig Eluate Plasma Specificity

WAIHA

IgG

IgG+C3

C3

IgG Pos IAT PosBroad

Rh

CAS/CAD C3 IgM Neg RT or colder I

MixedIgG+ C3

C3IgG, IgM Pos

IAT + RT or

colder??

PCH C3 IgG Neg

Pos

Biphasic

Hemolysin

P

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WAIHA, What do we do?

Pan Reactive plasma and eluate

May show Rh specificity – most commonly –e

Ficin Enhanced, PeG Enhanced

Antibody Panel

Cell 1 Cell 2 Cell 3 Cell 4 Cell 5 Cell 6 Cell 7 Cell 8 Cell 9 Cell 10 Auto

Plasma 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+

Eluate 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+

Plasma-Ficin 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+

WAIHA- How Do We Deal?

Adsorption

Autologous

Allogenic

Time Consuming

Requires more sample

Complex interpretation

What causes a positive DAT?

Transfusion Reaction

Patient has antibody

Donor unit is positive for antigen

Aby + Ag (In vivo)= positive DAT

Acute vs. Delayed

STEP 1

RECOGNIZE THAT A TRANSFUSION REACTION IS OCCURING

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Transfusion Reactions- What do we need to do?

Pre- transfusion sample:

ABO/RH

Visual Check for hemolysis/ icteric

Antibody Screen (ABID)

DAT

Post- transfusion sample:

ABO/RH

Visual check for hemolysis/ icteric

DAT

Other testing as indicated

Pre Post

Clerical OK OK

ABO/RH O pos O pos

Aby Scr Neg Neg

Visual OK Hemolysis +

DAT Neg POS

Transfusion Reaction- How do we find the aby?

Plasma

Eluate

•Pre-transfusion

•Post-transfusion

•Enhancements

•Remove aby bound to donor cells

•Concentrate

•Test like plasma

Medications

Positive DAT with and without Hemolysis

MANY Drugs

Severe Hemolysis is RARE

1 in 1 million

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4 Different Mechanisms

Drug Dependent Antibodies

Reactive with Drug-Treated Cells

Reactive with Untreated Cells in the Presence of the Drug

Drug Independent Antibodies

Nonimmunologic Protein Adsorption

How do Medications cause a positive DAT?

Drug Dependent

Aby Reactive w/Drug Treated

RBC

• IgG (+C3)

• DAT +++

• Plasma antibody/ Antibody eluted- ONLY reacts with drug treated cells

Drug Dependent Aby Reactive w/Untreated

RBCs in Presence of

Drug

• C3 (+IgG)

• Antibody IgM, IgG or both

How do Medications cause a positive DAT?

DrugIndependent

Antibody

• Indistinguishable from AUTOANTIBODY

Non immunologic

Protein Adsorption

(NIPA)

• RARELY hemolytic

• Positive DAT on RBCs that have been in the presence of the drug but eluate is non-reactive with RBCs in the presence of the drug

• Proteins “stick” to the RBC membrane

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How do we know what the cause is?