Immunohematology: Blood Bank Technologies & Automation

Column Agglutination Technology (CAT) - Gel Method

  • Developed by Yves Lapierre at DiaMed AG.

  • Hemagglutination test with controlled centrifugation of RBCs through dextran-acrylamide gel containing predispensed reagents.

  • Large agglutinates are trapped at the top of the gel during centrifugation, while unagglutinated RBCs form a pellet at the bottom.

Agglutination Reaction Grading:

  • 4+: Solid band of agglutinated RBCs at the top.

  • 3+: Most agglutinated RBCs remain near the top with few staggered below.

  • 2+: RBC agglutinates distributed throughout the upper and lower halves.

  • 1+: RBC agglutinates predominantly in the lower half.

  • 0: RBCs form a well-delineated pellet at the bottom.

  • Hemolysis: Reddish color in gel area.

Advantages:

  • Standardization of procedures.

  • No wash step or need for antiglobulin control cells.

  • Decreased sample volume requirement (50ul of blood).

  • Enhanced sensitivity and specificity.

  • Improved productivity.

Disadvantages:

  • Sample restrictions and need for special equipment.

  • Hemolyzed or icteric samples may be difficult to interpret.

  • Lipemic samples may clog the column.

  • Rouleaux may produce false positives.

  • Special incubators, centrifuges, and pipettes are needed.

Gel Technology Applications

  • Approved by the FDA for:

    • ABO forward and reverse grouping.

    • Rh typing.

    • Direct Antiglobulin Test (DAT).

    • Antibody Screen and Identification (AHG reagent in the gel).

    • Compatibility testing (AHG & potentiator in the gel).

Solid Phase Technology

  • Examples: Immucor (Solid-phase red cell adherence - SPICA), Bio-Rad (Solid-phase protein A), GTi Diagnostics (Solid-phase ELISA).

  • First-generation tests: Target antigen added by the user.

  • Second-generation tests: Target antigen bound to microplate wells by the manufacturer.

  • FDA approved for antibody screening, identification, Weak D Testing, IgG autologous control, and compatibility testing.

  • Positive tests show adherence of indicator RBCs to part or all of the well bottom.

Advantages:

  • Standardized.

  • Stable endpoints.

  • No predilution of reagents required.

  • Possible to test hemolyzed, lipemic, or icteric samples.

  • Long shelf life for test plates.

Disadvantages:

  • Need for specialized equipment.

Solid-Phase Protein A Technology

  • Protein A: Component of Staphylococcus aureus with high affinity for the Fc portion of most immunoglobulin classes.

  • Solidscreen II: Assay using antiglobulin testing to detect red cell antibodies, perform compatibility tests, detect IgG on patients' red blood cells, and type red cells for weak D and partial D antigens.

  • IgG antibodies are captured in microwells coated with protein A.

Solid-Phase Enzyme-Linked Immunosorbent Assay (ELISA)

  • GTi Diagnostics developed an ELISA assay to test for antibodies to platelet glycoproteins.

  • PAK*1 assay detects antibodies to platelet antigens on glycoprotein IIb/IIIa.

  • PAK*2 assay detects antibodies to antigens on platelet glycoproteins IIb/IIIa, Ia/IIa, Ib/IX, GP1V and HLA.

  • GTi Diagnostics developed ELISA assays to screen for and identify HLA antibodies (Class I and Class II).

Applications:

  • Used by GTi Diagnostics: MACE (MACE1 and MACE2) and PAK (PAK1, PAK2-LE, PAK12, PAK12G and PAKPLUS*) for screening and identifying platelet antibodies.

Principle:

  • Patient's serum/plasma added to microwells coated with platelet glycoproteins, allowing antibody binding.

  • Unbound antibodies washed away.

  • Alkaline phosphatase-labeled AHG reagent added and incubated.

  • Unbound AHG reagent washed away, and substrate PNPP added.

  • Reaction stopped with sodium hydroxide solution.

  • Optical density measured in a spectrophotometer.