Testing

We have a fantastic toolkit for detecting antigen-antibody interactions! From simple direct agglutination (great for IgM like ABO) to the essential Antiglobulin Test (for detecting IgG/Complement in DAT and IAT), and newer technologies like Solid Phase and Gel that offer standardization and sensitivity. Choosing the right test depends on what you’re looking for – antigen or antibody, IgM or IgG, in vivo or in vitro sensitization! Understanding the principle behind each test is crucial for performing it correctly and interpreting the results accurately

Let’s see how we actually detect those antigen-antibody interactions. Think of it like this: We know antibodies are super specific (the “lock and key” idea), but most of the time, just having an antibody bind to a red cell antigen doesn’t produce an obvious signal. We need clever methods to visualize that binding event

Overarching Principles of Testing

Specificity is Key: All our tests rely on the highly specific nature of antigen-antibody binding. We use known antibodies (reagents) to detect unknown antigens on cells, or known antigens (on reagent cells) to detect unknown antibodies in patient plasma/serum
Need for a Visible Endpoint: Since we can’t see individual antibodies binding, we need a way to make the reaction visible. The most common endpoint in blood banking is agglutination (clumping of red cells), but hemolysis (red cell destruction) is also a crucial positive indicator. Newer methods use adherence patterns
Two Stages of Agglutination: Visualizing agglutination usually involves two steps:
- Stage 1: Sensitization: The antibody in the plasma/serum binds to the corresponding antigen on the red blood cell surface. This is an invisible step! Factors influencing this include temperature, incubation time, pH, and ionic strength
- Stage 2: Lattice Formation: Antibody molecules cross-link multiple red blood cells together, forming visible clumps (agglutinates). This depends on factors like the distance between cells (zeta potential), antibody class (IgM is much better at this than IgG), and centrifugation to bring cells closer

Common Testing Methods and Their Principles

Let’s look at the main techniques we use:

Hemagglutination Methods (Direct Agglutination)

Principle: Directly visualizing the clumping of red blood cells caused by antibodies cross-linking antigens on adjacent cells. This works best with antibodies that are large enough or present in high enough concentration to bridge the gap between red cells suspended in saline
Method (Classic Tube Testing)
1. Combine known (reagent) or unknown (patient/donor) red cells with known (reagent) or unknown (patient) serum/plasma in a test tube
2. Centrifuge briefly to encourage lattice formation (Immediate Spin phase)
3. Gently resuspend the cell button and observe for clumping (agglutination)
4. Incubation at different temperatures (Room Temp, 37°C) can be added before centrifugation, depending on the antibody suspected
Interpretation
- Positive: Visible clumping of red cells (graded 1+ to 4+). Hemolysis is also considered a positive result (indicates complement activation leading to cell lysis)
- Negative: Smooth, homogenous suspension of red cells after resuspension
When is it Used?
- ABO Typing (Forward & Reverse): Uses potent IgM anti-A and anti-B reagents (forward) and patient plasma against known A1 and B cells (reverse). Usually detected at Immediate Spin
- Detecting potent cold-reacting IgM antibodies: Often react at Immediate Spin or Room Temperature phases
- Immediate Spin Crossmatch: A rapid check for ABO incompatibility
Key Antibody Class: Primarily detects IgM antibodies, which are large pentamers and excellent agglutinators. Some high-titer IgG might cause weak direct agglutination

Antiglobulin Testing (The Coombs Test) - Detecting “Invisible” Binding

Principle: This is CRITICAL! Many clinically significant antibodies (especially IgG) bind to red cells (sensitization) but are too small to cause direct agglutination in saline. The Antiglobulin Test uses a special reagent – Anti-Human Globulin (AHG) – which contains antibodies against human antibodies (anti-IgG) and/or complement components (anti-C3d). If IgG or C3d is bound to the red cells, the AHG reagent will bind to them and act as a “bridge” to cross-link the sensitized cells, causing visible agglutination
There are TWO main types of Antiglobulin Tests
- Direct Antiglobulin Test (DAT)
  - Principle: Detects in vivo sensitization – meaning, it checks if a patient’s red blood cells circulating in their body are already coated with IgG antibodies and/or complement
  - Method
    1. Take a sample of the patient’s red blood cells
    2. Wash the cells thoroughly (at least 3 times) with saline. This is VITAL to remove any unbound plasma proteins that could neutralize the AHG reagent
    3. Add AHG reagent (polyspecific or monospecific)
    4. Centrifuge briefly
    5. Resuspend and examine for agglutination
  - Interpretation
    - Positive: Agglutination occurs. This means the patient’s red cells were coated with IgG and/or C3d in their body
    - Negative: No agglutination
  - Applications: Investigating suspected Hemolytic Disease of the Fetus and Newborn (HDFN), Autoimmune Hemolytic Anemia (AIHA), or Hemolytic Transfusion Reactions (HTR)
- Indirect Antiglobulin Test (IAT)
  - Principle: Detects in vitro sensitization – meaning, it checks if unexpected antibodies are present in the patient’s serum or plasma that are capable of binding to specific red cell antigens under laboratory conditions
  - Method
    1. Combine patient’s serum/plasma with known reagent red blood cells (expressing specific antigens)
    2. Incubate at 37°C (optimal for IgG binding). Often use enhancement media (like LISS or PEG) during incubation to speed up sensitization
    3. Wash the cells thoroughly (at least 3 times) to remove unbound antibodies from the serum. Again, VITAL!
    4. Add AHG reagent
    5. Centrifuge briefly
    6. Resuspend and examine for agglutination
    7. Add Check Cells (Coombs Control Cells) If the test is negative, add IgG-coated check cells. The test must then become positive (agglutinate). This confirms the AHG reagent was added, active, and the washing steps were adequate. If check cells don’t agglutinate, the negative result is invalid!
  - Interpretation
    - Positive: Agglutination occurs. This means the patient’s serum/plasma contains an antibody that reacted with an antigen on the reagent red cells
    - Negative: No agglutination (and check cells work)
  - Applications: Antibody Screening (detecting presence of unexpected antibodies), Antibody Identification (determining specificity of antibodies), Crossmatching (checking compatibility between patient serum and donor cells), Weak D testing, and Antigen Typing (using known antisera to detect antigens on patient/donor cells)
AHG Reagents
- Polyspecific: Contains both anti-IgG and anti-C3d (and sometimes anti-C3b). Used for routine screening (DAT & IAT)
- Monospecific: Contains only anti-IgG or only anti-C3d. Used in DATs to determine what is coating the cells (IgG, complement, or both)

Enhancement Media in IAT

Principle: Used during the IAT incubation step to increase the rate and sensitivity of IgG antibody binding to red cell antigens
Methods
- Low Ionic Strength Saline (LISS): Reduces the “ionic cloud” around red cells, allowing antibodies to approach antigens more easily and quickly. Shortens incubation time (usually 10-15 min)
- Polyethylene Glycol (PEG): Removes water molecules, effectively concentrating antibodies near the red cells. Very sensitive for detecting weak IgG antibodies. Requires careful technique (no reading after 37°C incubation before washing, as PEG can cause non-specific aggregation)
- Bovine Serum Albumin (BSA): Older method, thought to reduce zeta potential slightly. Less effective than LISS or PEG
- Enzymes (e.g., Ficin, Papain): Proteolytic enzymes that cleave certain proteins from the RBC surface. This can enhance reactivity of some antibodies (like Rh, Kidd) by removing obstructions or reducing negative charge, but it destroys other antigens (like Duffy, MNSs). Used in specialized antibody ID panels

Solid Phase Red Cell Adherence (SPRCA) Assays

Principle: Instead of looking for agglutination in solution, these methods immobilize either antigen or antibody onto the surface of microplate wells. Detection relies on the adherence of red cells to the well surface
Method (Antibody Screen Example - Antigen Capture)
1. Microplate wells are coated with purified red cell antigens or membranes
2. Patient serum/plasma is added and incubated (allows antibody binding to immobilized antigen)
3. Wells are washed to remove unbound antibody
4. Indicator Red Cells (reagent cells coated with anti-IgG) are added
5. Plate is centrifuged
Interpretation
- Positive: Indicator cells bind to the antibody captured on the well surface, forming a diffuse layer covering the well bottom (adherence)
- Negative: No antibody was captured, so indicator cells pellet into a tight button at the bottom of the well after centrifugation
Applications: Antibody screening, antibody identification, crossmatching. Often used in automated platforms. Can also be reversed (antibody capture) for antigen typing or DAT

Gel Technology (Column Agglutination)

Principle: Uses microtubes (“cards”) containing a dextran-acrylamide gel matrix. The gel acts as a sieve. Agglutinated red cells get trapped in the gel during centrifugation, while unagglutinated cells pass through and form a pellet at the bottom. AHG reagent can be incorporated into the gel for antiglobulin tests
Method (IAT Example)
1. Microtube contains gel + Anti-IgG
2. Add reagent red cells and patient serum/plasma to the reaction chamber above the gel
3. Incubate (if necessary, e.g., for IAT)
4. Centrifuge the card. Red cells pass through the gel
Interpretation
- Positive: Agglutinated cells are trapped in the gel matrix, forming a line at the top or dispersed throughout the gel (depending on reaction strength). Clear supernatant
- Negative: Unagglutinated cells pass freely through the gel and form a distinct pellet at the bottom of the microtube
Applications: ABO/Rh typing, antibody screening, antibody identification, DAT, crossmatching. Very popular due to clear endpoints, stable reactions, and standardization

Key Terms

Agglutination: The visible clumping of particles (e.g., red blood cells) caused by cross-linking by antibodies
Hemolysis: The rupture or destruction of red blood cells, releasing hemoglobin. In testing, often indicates complement activation by an antibody
Sensitization: The binding of antibody or complement to antigens on the red blood cell surface (Stage 1 of agglutination, often invisible)
Lattice Formation: The cross-linking of sensitized red blood cells by antibodies to form visible clumps (Stage 2 of agglutination)
Anti-Human Globulin (AHG) / Coombs Reagent: Reagent containing antibodies directed against human IgG and/or complement C3d, used to detect sensitization of red blood cells
Direct Antiglobulin Test (DAT): Detects in vivo sensitization of patient red blood cells with IgG or complement
Indirect Antiglobulin Test (IAT): Detects antibodies in patient serum/plasma capable of sensitizing reagent red blood cells in vitro
Enhancement Media (LISS, PEG): Solutions used in IAT to increase the rate and sensitivity of IgG antibody binding to red cells
Check Cells (Coombs Control Cells): IgG-coated red blood cells added to all negative AHG tests to validate the result (confirm AHG was active and washing was adequate)
Solid Phase Red Cell Adherence (SPRCA): Testing method using microplate wells coated with antigen or antibody; detects adherence patterns of red cells
Gel Technology (Column Agglutination): Testing method using microtubes containing a gel matrix that traps agglutinated cells during centrifugation

Principles

Complement