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Blood Bank

Column Agglutination

Column Agglutination Technology (CAT), often called the Gel Test™ (which is actually a specific brand name, but frequently used generically) is a really popular and visually elegant alternative to traditional tube testing for many routine and special blood bank procedures!

Think of it as a sophisticated sieve in a tiny tube. Instead of looking for clumps floating in liquid, we see if clumps get trapped in a special gel or bead matrix during centrifugation

The Core Purpose: Detecting Agglutination in a Column

CAT provides a standardized and stable method for performing many common immunohematology tests, including:

  • ABO Forward and Reverse Grouping
  • Rh(D) Typing (including Weak D testing)
  • Antibody Screening
  • Antibody Identification
  • Direct Antiglobulin Test (DAT)
  • Compatibility Testing (Crossmatching)
  • Red Blood Cell Antigen Typing

The Principle: Sieving Through Gel/Beads

The magic happens inside specially designed microtubes (or “columns”) embedded in plastic cards (often 6 microtubes per card)

  • The Microtube: The top part is a reaction chamber. Below this, the microtube contains a matrix of either Sephadex gel or glass beads suspended in a buffered solution. This solution often contains pre-dispensed reagents depending on the test card type (e.g., Anti-A, Anti-B, Anti-D, AHG)
  • The Process
    1. Patient/donor red cells and/or plasma/serum are added to the reaction chamber at the top of the microtube, according to the specific test protocol
    2. The card is incubated (if required, e.g., for IAT methods at 37°C)
    3. The card is then centrifuged in a special centrifuge designed to hold the cards
  • The Separation: During centrifugation:
    • Unagglutinated Red Blood Cells: Are small enough to pass through the spaces in the gel/bead matrix and form a compact pellet at the bottom of the microtube
    • Agglutinated Red Blood Cells: Form larger clumps that are too big to pass through the matrix. They get trapped within the gel/beads at various levels depending on the size and strength of the agglutinates

Types of Gel Cards

Different cards are pre-filled for specific tests:

  • Neutral/Saline Cards: Contain only the buffered gel/bead matrix. Used for ABO reverse grouping, autocontrols, or situations where no specific reagent is needed in the column itself
  • Antisera Cards: Contain specific blood grouping antisera (like Anti-A, Anti-B, Anti-D) within the gel/matrix. Used for forward ABO/Rh typing
  • AHG Cards (Anti-Human Globulin): Contain Anti-IgG (and sometimes Anti-C3d or polyspecific AHG) within the gel/matrix. These are crucial for:
    • Indirect Antiglobulin Tests (IAT): Antibody screen, antibody ID, Weak D, crossmatch. Plasma/serum and cells are incubated before being added to the AHG card for centrifugation
    • Direct Antiglobulin Test (DAT): Patient’s washed red cells are added directly to the AHG card and centrifuged

Reading and Interpreting Results

This is where CAT really shines due to its clarity and stability:

  • Negative Reaction (0): All red blood cells pass through the matrix and form a sharp, compact pellet at the very bottom of the microtube. There are no agglutinates trapped in the column
  • Positive Reaction (1+ to 4+): Agglutinated cells are trapped within the gel/bead matrix. The strength of the reaction correlates with how high up the cells are trapped:
    • 4+: A solid band of agglutinated cells trapped at the top of the gel/bead column
    • 3+: Agglutinated cells primarily in the upper half of the column
    • 2+: Agglutinated cells dispersed throughout the length of the column
    • 1+: Agglutinated cells mainly in the lower half of the column, with some cells possibly reaching the bottom
  • Mixed Field Reaction: Characterized by a layer of agglutinated cells trapped at the top or dispersed in the column and a pellet of unagglutinated cells at the bottom. Often seen post-transfusion or with certain weak subgroups
  • Hemolysis: Hemolysis (due to complement activation, etc.) occurring before or during centrifugation is also considered a positive result if it’s antibody-mediated. The supernatant above the gel/beads may appear pink or red, and there may be fewer intact cells to pellet or get trapped

(Visualizing this helps! Imagine a clear tube filled with gel/beads. Negative = red dot at bottom. Positive = red line/smear trapped in the gel, higher up = stronger reaction.)

Advantages of Column Agglutination Technology

  • Standardization: Reduces variability associated with tube testing techniques (e.g., inconsistent shaking/resuspension)
  • Objectivity & Stability: Results are stable for hours (sometimes days), easy to read, photograph, or review. Grading is generally more consistent between technologists
  • Sensitivity: Often considered as sensitive as, or slightly more sensitive than, traditional LISS tube testing for detecting weak IgG antibodies
  • Reduced Sample Volume: Can often use smaller amounts of sample/reagents
  • Automation: Easily incorporated into semi-automated and fully automated blood bank testing platforms
  • Safety: Reduced aerosol production compared to tube methods

Disadvantages and Considerations

  • Cost: Cards and specialized centrifuges/incubators can be more expensive than basic tube testing supplies
  • Specialized Equipment: Requires dedicated centrifuges and sometimes incubators designed for the cards
  • Hemolysis Detection: Detecting in vitro hemolysis within the opaque gel column itself can sometimes be harder than seeing it in the supernatant of a tube, though hemolysis above the column is visible
  • Fibrin/Particulate Interference: Fibrin strands or other debris in a sample can sometimes get trapped nonspecifically at the top of the gel, potentially mimicking a weak positive reaction. Clean samples are important
  • Training: Requires familiarity with the specific reading and grading criteria

Key Terms

  • Column Agglutination Technology (CAT) / Gel Test™: Method using microtubes containing a gel or glass bead matrix to trap agglutinated red blood cells during centrifugation
  • Microtube/Column: The individual reaction vessel within the card containing the gel/bead matrix
  • Gel Matrix / Glass Beads: The sieving medium within the microtube that separates agglutinated from unagglutinated cells
  • Agglutinates: Clumps of red blood cells formed by antibody binding
  • Pellet: Compact button of unagglutinated red blood cells at the bottom of the microtube (negative result)
  • Trapped Cells: Agglutinated cells caught within the gel/bead matrix (positive result)
  • Mixed Field: Reaction pattern showing both trapped agglutinates and a pellet of free cells at the bottom
  • AHG Card: Gel card containing Anti-Human Globulin (Anti-IgG +/- Anti-C3d) pre-dispensed in the matrix, used for IAT and DAT