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Blood Bank

Leukocyte/Platelet Testing

The evaluation of leukocytes and platelets in the Clinical Blood Bank extends beyond standard red cell immunohaematology. While red cell testing focuses primarily on the ABO and Rh systems to prevent hemolytic reactions, leukocyte and platelet testing focuses on Human Leukocyte Antigens (HLA) and Human Platelet Antigens (HPA). These testing methodologies are critical for managing platelet refractoriness, diagnosing alloimmune thrombocytopenias, and ensuring solid organ or hematopoietic stem cell graft survival. The testing spectrum ranges from functional serologic assays, such as cytotoxicity, to precise molecular genotyping

Cytotoxicity (Leukocyte/HLA Testing)

Cytotoxicity testing serves as the functional cornerstone for detecting antibodies against HLA antigens. While molecular methods have largely superseded serology for the identification (typing) of HLA antigens, Complement-Dependent Cytotoxicity (CDC) remains a vital physiological assay. It demonstrates not just the presence of an antibody, but its biological capability to activate complement and destroy tissue

The Complement-Dependent Cytotoxicity (CDC) Assay

The CDC assay, often termed the Terasaki lymphocytotoxicity test, relies on the principle of dye exclusion to assess cell membrane integrity. The test requires the isolation of pure lymphocytes (T-cells and B-cells) from peripheral blood. T-cells are used to detect antibodies against HLA Class I antigens (A, B, C), while B-cells are used to detect antibodies against both HLA Class I and Class II antigens (DR, DQ, DP). The physiological process involves incubating patient serum with donor lymphocytes. If specific antibodies bind to the HLA antigens on the cell surface, they effect a conformational change allowing the binding of exogenous rabbit complement

  • Principle of Lysis: The complement cascade is activated via the classical pathway, resulting in the formation of the Membrane Attack Complex (MAC). The MAC inserts into the lipid bilayer of the lymphocyte, creating a pore that leads to osmotic lysis
  • Visualization: A supravital dye (Eosin Y or Trypan Blue) is added. Viable cells with intact membranes exclude the dye and appear bright and refractile. Dead (lysed) cells take up the dye, appearing large, dark, and non-refractile. A reaction is scored based on the percentage of dead cells in the well
  • Clinical Utility: This method is used for HLA antibody screening (Panel Reactive Antibody or PRA) and the HLA Crossmatch. A positive CDC crossmatch is a contraindication for renal transplant as it predicts hyperacute rejection

Molecular Correlations in HLA Testing

While CDC is a serologic functional assay, it is limited by its inability to detect non-complement fixing antibodies and its requirement for viable cells. Consequently, molecular testing has become the standard for HLA typing:

  • PCR-SSP (Sequence-Specific Primers): Uses primers designed to amplify specific HLA alleles. If the allele is present, amplification occurs; if absent, no product is formed. This is a rapid method often used for deceased donor typing
  • PCR-SSO (Sequence-Specific Oligonucleotides): DNA is amplified and then hybridized with probes bound to microbeads (Luminex technology). This allows for high-throughput typing
  • SBT and NGS (Sequencing Based Typing / Next-Generation Sequencing): These methods determine the exact nucleotide sequence of the HLA gene, providing the highest resolution typing necessary for Hematopoietic Stem Cell Transplantation (HSCT)

Platelet Serologic & Molecular Testing

Platelet testing is distinct because platelets express both shared tissue antigens (HLA Class I) and lineage-specific antigens (HPA). The laboratory approach must differentiate between these two, as the clinical management for HLA-mediated refractoriness differs from HPA-mediated disorders like Neonatal Alloimmune Thrombocytopenia (NAIT) or Post-Transfusion Purpura (PTP). Unlike RBCs, platelets do not agglutinate visibly in a tube; therefore, high-sensitivity binding assays are required

Solid Phase Red Cell Adherence (SPRCA)

Solid phase technology is the most common screening method for platelet antibodies due to its adaptability to automation. It utilizes a “capture” technique

  • Methodology: Donor platelets are fixed to the bottom of microtiter wells. Patient serum is added, and if anti-platelet antibodies are present, they bind to the fixed platelets. After washing, anti-IgG coated indicator red blood cells are added
  • Interpretation: A positive reaction is indicated by the adherence of the indicator RBCs across the surface of the well (a “carpet” or monolayer). A negative reaction occurs when the indicator RBCs fail to attach and settle to the bottom in a compact button. This test detects both HLA and HPA antibodies but does not distinguish between them

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA assays provide higher specificity and are often used to differentiate the source of the immune response

  • Glycoprotein-Specific Assays: Modern ELISA kits (such as the “Pak” assays) utilize purified platelet glycoproteins (GPIIb/IIIa, GPIb/IX) or solubilized HLA antigens immobilized in the wells. This allows the laboratory scientist to determine if the patient has antibodies solely against HLA, solely against HPA, or both
  • Clinical Significance: Differentiating these antibodies is crucial. If a patient has only HLA antibodies, they require HLA-matched platelets. If they have HPA antibodies, they require HPA-antigen-negative units

Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA)

Considered the reference standard for HPA antibody identification, MAIPA avoids the interference of HLA antibodies that often clouds other tests

  • Mechanism: This is a “double antibody” or sandwich technique. A mouse monoclonal antibody captures a specific platelet glycoprotein from a cell lysate. The patient’s serum is allowed to react with this captured complex. Because the assay isolates the specific glycoprotein (e.g., exclusively GPIIb/IIIa), any reactivity detected can be definitively attributed to an HPA antibody, effectively ignoring any HLA antibodies present in the serum

Molecular Genotyping of Platelet Antigens

Serology is limited if the patient is severely thrombocytopenic (making autologous phenotyping impossible) or if reagents are unavailable for low-frequency antigens. Molecular genotyping is the definitive method for determining a patient’s or donor’s HPA type

  • HPA Genotyping: Polymerase Chain Reaction (PCR) techniques are used to identify the specific single nucleotide polymorphisms (SNPs) that define HPA alleles (e.g., differentiating HPA-1a from HPA-1b). This is essential in the workup of NAIT to determine fetal risk (by genotyping the father) and for screening blood donors to create an inventory of rare HPA-negative platelet units