CLS 306 - ANTIGLOBULIN TEST / ANTIHUMAN GLOBULIN (AHG) TEST

Antiglobulin Test / Antihuman Globulin (AHG) Test

Antiglobulin Test Overview

• The Antiglobulin Test (AGT) detects IgG antibody(ies) and/or complement (C') bound to RBCs.
• Binding of IgG Ab or C' can occur in-vivo or in-vitro.
• Also known as the Coombs' Test, named after Dr. Robert R. A. Coombs, who developed it in 1945.
• IgG, due to its size, doesn't usually cause immediate visible hemagglutination like IgM.
• When IgG is attached to the RBC (sensitization), the AGT provides a bridging molecule to help make hemagglutination visible.
• The Anti-Human Globulin (AHG) reagent is an antibody solution from immunized animals (e.g., rabbit). These antibodies bind to human IgG or complement (C') attached to human RBCs.

Types of Antiglobulin / Coombs' Tests

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 / Coombs' Test (DAT / DCT)

• Detection of in-vivo sensitization of RBCs
• Detects both IgG and/or C'

Clinical Conditions Detected by DAT / DCT

• HDFN (Hemolytic Disease of the Fetus / Newborn) - Maternal IgG crosses the 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, i.e., IgM = Cold; IgG = Warm
• These clinical conditions fall into the Class / Type II Hypersensitivity category.

DAT / DCT Test Procedure

• Wash unknown (patient) RBCs at least 3x with saline (removes unbound globulins).
• Add polyspecific AHG reagent (detects IgG and/or C3d bound to patient RBCs). Mix.
• Centrifuge (accelerates agglutination) & read.
• Grade (0 to 4+) and record agglutination.
• To all negative test results, add reagent Coombs' Check Cells (CCCs) / Check Cells (CCs). Mix.
  • CCs are RBCs coated with IgG Ab and are used to verify:
    1. Washing was adequately performed
    2. That AHG was added
    3. That the AHG reagent is working
• Spin, read, and record reaction.
• If CC reaction is positive (≈ 2+), test is valid.
• If CC reaction is negative, test is invalid and must be repeated.

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.

DAT / DCT - Factors That Affect the Test

• What is going on inside the body, e.g., Ab titer, primary cause for Ab production, binding C', etc.
• 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 / Coombs' Test (IAT / ICT)

• Used in the Antibody Screen (ABS) Test and the Antibody Identification (Ab ID) Test.
• Detects unexpected IgG allo- / auto- Abs in recipient sera that may be incompatible with donor RBC ags.
  • 0.5 - 2% of the general patient population have irregular RBC Abs.
  • 6 - 35% of various multiply-transfused populations have irregular Abs.
• Identification of unexpected allo Abs in unknown sera.
• Titration of IgG Abs.

Why Do the IAT / ICT?

• Promotes
  • Safe and Effective RBC transfusion.
  • RBC longevity to maintain oxygen-carrying capacity to vital organs.
  • Normal body functions without compensations, e.g., tachycardia, hyperventilation, etc.
    • tachy-rapid; cardia- heartbeat; hyper-elevated; ventil-breathing; ation-connected with a process or action
• Avoids
  • Transfusion of incompatible RBCs.
  • Decreased survival of donor RBCs.
  • Clinical effects of Hemolytic Transfusion Reactions (causing morbidity).

IAT / ICT Test Procedure

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

Antiglobulin Reagents

• Coombs' Serum
  • Serum from a rabbit or other animal previously immunized with purified human globulin to prepare antibodies directed against IgG and complement, used in the direct and indirect Coombs' tests. Also called antihuman globulin.

Coombs' Reagent Types

• Polyspecific AHG
  • Reagent is clear
  • Contains Abs to human IgG AND human C3d
  • Advantage – may detect C'-binding Abs on RBC (e.g., 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

Reagent Manufacturing

• Polyclonal OR Monoclonal
  • POLYCLONAL - Animal hyper-immunized with human globulins, bled for antisera to obtain high titer, high avidity specificity to human globulin.
  • MONOCLONAL - Use hybridoma cells from mice; collection of culture or ascites fluid yields antisera.
• Product is regulated (by FDA) for its potency

Coombs' Control Red Cell Reagent aka, Check Cells (CC)

• 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.
• Other acronyms / symbols used to refer to this reagent:
  1. CCC = Coombs' Control Cells
  2. CC = Check Cells
  3. √ = Check Cells

Key Concepts of BB Testing - Overview

• In blood banking, we test “knowns” against “unknowns,” i.e.,
  • Known: Reagent antisera + patient RBCs = Unknown
  • Known: Reagent RBCs + patient serum = Unknown

Key Concepts of the AHG Testing

• For the DAT test, we test the patient's RBCs to see if Ab(s) is / are coating the RBCs.
  • Known: Reagent AHG antisera + patient RBCs = Unknown
• For the IAT test, we test the patient's serum / plasma to see if the patient has any Ab(s).
  • Known: Reagent Screening Cells + patient serum = Unknown
  • After incubation & washing of screening cells, add Reagent AHG antisera (known)

Comparison of the DAT vs IAT Tests

False Positive AGT

1. Improper specimen (refrigerated, clotted sample = > C' attachment)
2. Dirty glassware, i.e., glass bits inside tube; usually caused by broken tubes in box
3. Autoagglutinable cells
4. Bacterial contamination of cells or saline
5. Cells with positive DAT used for IAT, i.e., auto control
6. Over-centrifugation & over-reading
7. Polyagglutinable cells
8. Preservative-dependent Ab in LISS reagents
9. Contaminating Ab in AHG reagent
10. Saline contaminated by heavy metals or colloidal silica

False Negative AGT

1. Inadequate or improper washing of cells
2. AHG reagent not added
3. Neutralization of AHG reagent due to inadequate washing
4. Deterioration of AHG reagent
5. Serum nonreactive due to deterioration of Ab or C'
6. Inadequate incubation conditions in the IAT
7. Cell suspension either too weak or too heavy
8. Under-centrifuged or over-centrifuged
9. In test tube: testing error, CLS may shake tube too hard
10. Poor reading technique
11. Patient serum not added in IAT