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Blood Bank

Platelet Testing

Platelet testing in the immunohematology laboratory differs significantly from routine red blood cell testing. While RBC testing is universal for transfusion candidates, platelet serology is typically a reflex investigation triggered by specific clinical complications, most notably refractory thrombocytopenia (failure to increment) or suspected alloimmune disorders. The physiological basis for these tests rests on the detection of antibodies against two distinct categories of antigens found on the platelet membrane surface: Human Leukocyte Antigens (HLA) and Human Platelet Antigens (HPA)

Physiological Basis: Platelet Antigens

To understand platelet immunology, one must distinguish between the ubiquitous tissue antigens and the lineage-specific antigens expressed on the thrombocyte surface

HLA Class I Antigens

  • Expression: Platelets express HLA Class I antigens (A, B, and C loci), but they do not express HLA Class II antigens. The density of HLA antigens on platelets is variable and can be influenced by clinical conditions
  • Source: The HLA antigens on platelets are primarily intrinsic (integral to the membrane), though historically there was debate regarding how much was adsorbed from plasma. Current consensus focuses on the integral expression
  • Clinical Impact: HLA antibodies are the most common cause of immune-mediated platelet refractoriness. Because HLA antigens are highly polymorphic, multiparous women and multiply transfused patients frequently develop alloantibodies against them

Human Platelet Antigens (HPA)

  • Structure: HPAs are located on specific platelet membrane glycoproteins that function as receptors for adhesion and aggregation (e.g., GPIIb/IIIa, GPIb/IX). These are distinct from HLA
  • Terminology: They are named numerically (HPA-1, HPA-2, etc.) with alleles usually designated’a’ (high frequency) and’b’ (low frequency). For example, HPA-1a is the most clinically significant
  • Clinical Impact: Antibodies against HPA are responsible for specific clinical syndromes such as Neonatal Alloimmune Thrombocytopenia (NAIT) and Post-Transfusion Purpura (PTP). Unlike HLA antibodies, HPA antibodies can cause destruction of platelets even in the absence of broad sensitization

Pathophysiology: Immune Platelet Destruction

When a patient possesses IgG antibodies against specific platelet antigens, the pathophysiology of destruction mimics that of extravascular hemolysis in red cells. The antibody binds to the specific antigen on the donor platelet surface. These sensitized platelets are then recognized by the Fc receptors of macrophages in the reticuloendothelial system (RES), primarily in the spleen. This results in the rapid clearance of transfused platelets from circulation, preventing the patient from achieving hemostasis

Refractoriness

Refractoriness is defined as the repeated failure to achieve the desired rise in platelet count after transfusion. It is critical to differentiate between “Non-Immune” and “Immune” causes before ordering serological testing:

  • Non-Immune Causes: Sepsis, fever, splenomegaly (sequestration), DIC, and bleeding are the most common causes of low increments. These consume platelets faster than they can be replenished
  • Immune Causes: Alloimmunization to HLA (most common) or HPA. This requires laboratory investigation and antigen-matched products

Corrected Count Increment (CCI)

To determine if the physiology of the transfusion failure is immune-mediated, a CCI is calculated. This standardizes the increment based on the patient’s body size and the dose of platelets transfused

  • Formula: \(CCI = \frac{(\text{Post-count} - \text{Pre-count}) \times \text{Body Surface Area (m}^2)}{\text{Number of Platelets Transfused (} \times 10^{11})}\)
  • Interpretation: A CCI of less than 5,000 to 7,500 (depending on the lab) occurring after two sequential transfusions indicates immune refractoriness. This triggers the need for platelet antibody screening

Serological Testing Methodologies

Because platelet antigens can be elusive and antibodies may be low-titer, specialized assays are required. Unlike RBC agglutination, platelet reactions are rarely visualized directly as “clumping” in a tube

Solid Phase Red Cell Adherence (SPRCA)

This is a widely used commercial method (e.g., Capture-P) for screening and crossmatching

  • Mechanism: Platelets are immobilized as a monolayer on the bottom of microplate wells. Patient serum is added. If antibodies attach to the platelets, they are detected by adding anti-IgG-coated indicator red blood cells
  • Positive Result: The indicator RBCs adhere to the antibody-coated platelets spread across the bottom of the well (a “carpet” of cells)
  • Negative Result: The indicator RBCs do not attach and pellet to the bottom of the well in a tight button

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is highly specific and often used to distinguish between HLA and HPA antibodies

  • Mechanism: Purified platelet glycoproteins or solubilized HLA antigens are bound to the well. Patient serum is added, followed by an enzyme-labeled anti-human globulin
  • Pak Assays: Specific kits (like GTI Pak) can separate HLA antibodies from HPA antibodies, helping determine if the patient needs HLA-matched platelets or HPA-negative platelets

Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA)

Historically considered the “gold standard” for differentiating platelet antibodies, though labor-intensive

  • Mechanism: A “sandwich” technique where a monoclonal antibody captures a specific platelet glycoprotein (like GPIIb/IIIa) from a lysate. This ensures that any human antibody detected is reacting strictly with that glycoprotein (HPA) and not an HLA antigen. This effectively eliminates interference from HLA antibodies when looking for HPA specificity

Clinical Syndromes & Testing Strategies

Neonatal Alloimmune Thrombocytopenia (NAIT)

NAIT is the platelet equivalent of Hemolytic Disease of the Fetus and Newborn (HDFN)

  • Pathophysiology: The mother lacks a specific platelet antigen (most commonly HPA-1a) which the fetus has inherited from the father. The mother produces IgG anti-HPA-1a, which crosses the placenta and destroys fetal platelets. Unlike HDFN (Rh), this can affect the first pregnancy
  • Testing: Requires genotyping the father (to see if he is homozygous or heterozygous for the antigen) and screening the mother’s serum for the specific HPA antibody. The mother’s platelets are phenotyped/genotyped to confirm she is antigen-negative

Post-Transfusion Purpura (PTP)

A rare but severe complication occurring 5–12 days post-transfusion

  • Pathophysiology: A patient (usually HPA-1a negative) is sensitized via transfusion or pregnancy. Upon re-exposure to antigen-positive platelets, the patient destroys the transfused platelets. Uniquely, the immune response also destroys the patient’s own antigen-negative platelets via a “bystander” mechanism or cross-reactivity, leading to profound thrombocytopenia
  • Testing: Demonstration of a high-titer HPA antibody (usually anti-HPA-1a) in a patient with severe thrombocytopenia following recent transfusion

Drug-Induced Immune Thrombocytopenia

Certain drugs (historically quinine/quinidine, currently heparin) can induce antibody formation

  • Heparin-Induced Thrombocytopenia (HIT): The antibody targets a complex of Heparin and Platelet Factor 4 (PF4). This causes platelet activation and consumption, leading to thrombosis rather than bleeding
  • Testing: Testing for HIT involves ELISA for PF4-heparin antibodies or functional assays like the Serotonin Release Assay (SRA)

Selection of Compatible Platelets

When testing confirms immune refractoriness, random donor platelets are ineffective. The laboratory must provide compatible products:

  • HLA-Matched Platelets: For patients with HLA antibodies. Donors are selected from a registry of HLA-typed apheresis donors to match the recipient’s A and B antigens. The “Match Grade” (A through D) predicts the likelihood of a successful increment
  • Crossmatched Platelets: If HLA-matched units are unavailable or ineffective, local inventory units can be crossmatched using SPRCA methods to find a unit that does not react with the patient’s serum
  • Antigen-Negative Units: For HPA antibodies (NAIT or PTP), donors must be screened (genotyped) to be negative for the specific antigen (e.g., HPA-1a negative)