lecture 5

what is Antiglobulin Test (AGT) used for?

• used for detecting IgG antibody(ies) and / or C' bound to RBCs.

  • Binding of IgG Ab or C' can occur in-vivo or in-vitro

• also known as Coombs' Test

Why does IgG not cause immediate visible hemagglutination like IgM in the Antiglobulin Test (AGT)?

• Due to its size (small) , IgG usually does NOT cause immediate "visible" hemagglutination like IgM.

  • BUT when IgG is attached to the RBC (sensitization), the AGT provides a "bridging molecule" / "bridge RBCs together" to help make hemagglutination visible

What is the role of the Anti-Human Globulin (AHG) reagent in the AGT?

• is an Ab solution made from immunized animals (e.g., rabbit)

• it makes human IgG or C' Abs that bind to human immunoglobulins found attached to human RBCs.

types of Antiglobulin / Coombs' Tests (2)

1. Direct Antiglobulin Test (DAT) OR Direct Coombs' Test (DCT)

   • Measures in-vivo sensitization of the RBCs; sensitization occurs inside the body

2. Indirect Antiglobulin Test (IAT) OR Indirect Coombs' Test (ICT)

   • Measures in-vitro sensitization of the RBCs; sensitization occurs outside the body

Direct Antiglobulin Test (DAT)/ Direct Coombs' Test (DCT)

1. Detection of in-vivo sensitization of RBCs

2. Detects both IgG and / or C

DAT/DCT Clinical Conditions

• HDFN (Hemolytic Disease of the Fetus / Newborn) - maternal IgG crosses placenta barrier & binds to infant RBCs (up to 4+ reaction)

• HTR (Hemolytic Transfusion Reaction) - transfused recipient Ab binds to donor RBCs

• AIHA (Autoimmune Hemolytic Anemia) [Cold or Warm] - medical disease condition or drug-induced phenomenon that creates allo- Abs or auto-Abs towards RBCs

  • IgM = Cold; IgG = Warm

• all of the above clinical conditions fall into the Class / Type II Hypersensitivity category

DAT/DCT Test Procedure

• to ALL negative test results: add reagent Coomb’s Check Cells (CCCs)/ Check Cells (CCs)

  • CCs are RBCs coated w/ IgG Ab and are used to verify:

    • 1) washing was adequately performed

    • 2) that AHG was added

    • 3) that the AHG reagent is working

  • CC rxn positive (at least 2+) = valid

  • CC rxn negative = invalid, MUST repeat

DAT/DCT interpretation

• When the polyspecific AHG test result is a positive reaction, repeat the DAT / DCT using monospecific AHG reagents (individual IgG reagent and individual C' reagent).

• A positive DAT / DCT test result, you should consider:

  • Evidence of in-vivo hemolysis or sequestration of RBCs (intra- or extra-vascular, respectively)

  • Recently transfused, unexpected IgG allo-Ab

  • Medical condition, IgG auto-Ab or C'

  • Medication (IgG or C')

• Positive DATs / DCTs with no clinical significance may be detected in up to 2% of the population

Factors that affect DAT/DCT test

• body conditions

  • Ab titer: primary cause for Ab production

  • whether Ab is binding C'

• Saline washing of the RBCs (critical).

• Addition of AHG reagent (proper amount or inadvertent omission).

• AHG reagent integrity is maintained.

• Centrifugation speed is optimal

DAT/DCT Panel Patterns

Indirect Antiglobulin Test/ Coomb’s Test (IAT/ICT)

• used in Antibody Screen Test (ABS) & Antibody Identification (Ab ID) Test

• Detects unexpected IgG allo- / auto- Abs in recipient sera that

• Identification of unexpected allo Abs in unknown sera

• Titration of IgG Abs

why do the IAT/ICT ?

Promotes

• safe and effective RBC transfusions

• RBC longevity to maintain oxygen-carrying capacity to vital organs

• normal body functions without compensations like:

  • Tachycardia: rapid heartbeat

  • Hyperventilation: elevated breathing

Help Avoids

• Transfusion of incompatible RBCs

• Decreased survival of donor RBCs

• Hemolytic Transfusion Reactions: causing morbidity

Factors that Affect The IAT/ICT Test

• Serum:Cell Ratio (SC ratio)

• Reaction media

• Temperature

• Incubation time

• Washing of RBCs

• Addition of AHG reagent (proper amount or inadvertent omission)

• Centrifugation

types of Coombs Reagent (2)

Polyspecific AHG

• Reagent is clear

• Contains Abs to human IgG AND human C3d

• Advantage: may detect C'-binding Abs on RBC (ex: Jka)

• Disadvantage: may detect C' on cells that may not be clinically significant (nuisance reactions)

Monospecific AHG

• IgG = green color

  • contains Abs to human IgG only

• C3d = clear

  • small vial; contains Abs to human C3d only

• Fewer nuisance positives

• Very specific in targeting either IgG or C3d

polyclonal or monoclonal?

polyclonal : Animal hyper-immunized with human globulins, bled for antisera to obtain high titer, high avidity specificity to human globulin.

• rabbit

monoclonal : Use hybridoma cells from mice; collection of culture or ascites fluid yields antisera

• mice

Coombs Control Red Cell Reagent aka Check Cells

• These are reagent RBCs coated with IgG that are added to all negative AHG test results.

• Checks for neutralization of AHG by free globulin molecules or inadvertent omission of Coombs' reagent

Key Concepts of BB testing

test “knowns” against “unknowns”

key concepts of AHG testing

For the DAT test, we test the patient's RBCs to see if Ab(s) is / are coating the RBCs.

For the IAT test, we test the patient's serum / plasma to see if the patient has any Ab(s).