Antiphospholipids

Overview of Antiphospholipid Antibody (aPL) Assays

Definition: A group of laboratory tests used to detect and measure the levels of antiphospholipid antibodies (aPL) in serum or plasma. aPLs are autoantibodies directed against phospholipids and phospholipid-binding proteins, and their presence is associated with an increased risk of thrombosis and pregnancy morbidity
Clinical Significance:
- Diagnosis of Antiphospholipid Syndrome (APS): The presence of aPLs, along with specific clinical criteria (thrombosis or pregnancy morbidity), is used to diagnose APS
- Risk Stratification: aPL titers and profiles can help assess the risk of thrombosis or pregnancy complications in individuals with APS
- Evaluation of Unexplained Thrombosis or Pregnancy Loss: aPL testing is recommended in patients with unexplained venous or arterial thrombosis, recurrent pregnancy loss, or other clinical features suggestive of APS
Key Assays: The main aPL assays include:
- Lupus Anticoagulant (LA) Testing
- Anticardiolipin (aCL) Antibody Assay
- Anti-β2 Glycoprotein I (anti-β2GPI) Antibody Assay

Key Concepts: Target Antigens and Antibody Isotypes

aPLs can target various phospholipid-binding proteins, with the most clinically relevant being:

Cardiolipin (aCL): A negatively charged phospholipid found in cell membranes
β2-Glycoprotein I (β2GPI): A plasma protein that binds to negatively charged phospholipids
Antibody Isotypes: IgG, IgM, and IgA isotypes of aCL and anti-β2GPI antibodies are measured, with IgG isotypes generally having the strongest association with thrombosis and pregnancy morbidity

Lupus Anticoagulant (LA) Testing

Principle:
- LA prolongs phospholipid-dependent clotting tests in vitro by interfering with the assembly of coagulation complexes on phospholipid surfaces
- LA is not a true anticoagulant in vivo; it is associated with an increased risk of thrombosis
Testing Algorithm:
- LA testing involves a series of tests to demonstrate:
  - Prolongation of phospholipid-dependent clotting tests (screening tests)
  - Failure to correct with mixing studies (presence of an inhibitor)
  - Correction with the addition of excess phospholipid (phospholipid dependence)
- Test Selection and Interpretation
  - Screening Test: Sensitive to the presence of LA
  - Mixing Study: In this process the addition of other test subjects will not correct issues from being outside. If it doesn’t correct, an inhibitor is present
  - Confirmatory Test: To confirm if phospholipid is present when performing a hexagonal phase
Confirmatory Tests:
- Dilute Russell’s Viper Venom Time (dRVVT)
- Silica Clotting Time (SCT)
- Hexagonal Phase Phospholipid Neutralization Assay
Interpretation:
- Prolonged Screening Test: Suggests the presence of an inhibitor
- Failure to Correct on Mixing: Confirms the presence of an inhibitor
- Phospholipid Dependence: Correction with excess phospholipid confirms that the inhibitor is a lupus anticoagulant
- Requires Persistent Positivity: To meet the laboratory criteria for APS, the LA must be present on two or more occasions, at least 12 weeks apart

Anticardiolipin (aCL) Antibody Assay

Principle:
- An ELISA-based assay that measures the levels of IgG and/or IgM antibodies that bind to cardiolipin, a negatively charged phospholipid
Procedure:
1. Microplate Wells Coated with Cardiolipin
2. Patient Sample is added to determine what happens with lipid testing.
3. An enzyme-labeled secondary antibody (e.g., anti-human IgG or anti-human IgM) is added to determine anti-cardiolipin antibodies.
4. A substrate is used to detect the anti-cardiolipin, and light passes through to help determine.
Reporting:
- Results are reported in GPL units (for IgG aCL) or MPL units (for IgM aCL)
- Reported as negative, low positive, medium positive, or high positive based on established cut-off values:
  - Negative: <20 GPL or MPL units
  - Low Positive: 20-39 GPL or MPL units
  - Medium Positive: 40-79 GPL or MPL units
  - High Positive: ≥80 GPL or MPL units
Interpretation:
- Elevated aCL Levels: Suggests the presence of aCL antibodies
- Requires Persistent Positivity: To meet the laboratory criteria for APS, the aCL antibodies must be present on two or more occasions, at least 12 weeks apart
- Clinical Significance:
  - High titers of IgG aCL antibodies are more strongly associated with thrombosis and pregnancy morbidity

Anti-β2 Glycoprotein I (anti-β2GPI) Antibody Assay

Principle:
- An ELISA-based assay that measures the levels of IgG and/or IgM antibodies that bind to β2-glycoprotein I, a plasma protein that binds to negatively charged phospholipids
Procedure:
1. Microplate wells coated with Beta-2-Glycoprotein is mixed with the plasma.
2. An enzyme-labeled secondary antibody (e.g., anti-human IgG or anti-human IgM) is added to each well to determine.
3. If positive, this will result in the enzyme causing an increased signal.
Reporting:
- Results are reported in units/mL or arbitrary units (AU/mL)
- Reported as negative, low positive, medium positive, or high positive based on established cut-off values:
  - Negative: <20 units/mL
  - Low Positive: 20-39 units/mL
  - Medium Positive: 40-79 units/mL
  - High Positive: ≥80 units/mL
Interpretation:
- Elevated anti-β2GPI Levels: Suggests the presence of anti-β2GPI antibodies
- Requires Persistent Positivity: To meet the laboratory criteria for APS, the anti-β2GPI antibodies must be present on two or more occasions, at least 12 weeks apart
- Clinical Significance:
  - High titers of IgG anti-β2GPI antibodies are more strongly associated with thrombosis and pregnancy morbidity

General Factors Affecting aPL Assay Results

Pre-Analytical Variables:
- Improper Collection Technique: May activate coagulation factors and affect results
- Incorrect Blood-to-Anticoagulant Ratio: Underfilling or overfilling the collection tube
- Clotted Sample: Invalidates the results
- Delayed Testing: aPLs can degrade over time
- Improper Storage: Incorrect storage temperatures can affect results
Analytical Variables:
- Reagent Quality: Use fresh, properly stored reagents, and follow the manufacturer’s instructions
- Instrument Calibration: Ensure proper calibration and maintenance of the instruments
- Interfering Substances: High levels of lipids, bilirubin, or paraproteins can interfere with ELISA-based assays
Patient-Related Variables:
- Medications: Warfarin, heparin, direct oral anticoagulants, and other medications can affect coagulation test results
- Acute Thrombotic Events: May consume coagulation factors and aPLs, leading to falsely negative results
- Infections: Transiently elevated aPL levels can be seen in some infections
- Autoimmune Disorders: The presence of other autoimmune antibodies (e.g., antinuclear antibodies) can affect aPL results

Troubleshooting Erroneous Results

If the aPL assay results are unexpected or inconsistent with the patient’s clinical presentation:
- Check the sample for clots or hemolysis
- Repeat the test on a fresh sample
- Ensure that the correct blood-to-anticoagulant ratio was used
- Verify the instrument and reagent quality control results
- Investigate potential interfering substances
- Review the patient’s medication list and medical history
- Ensure that the aPLs have been positive on two or more occasions, at least 12 weeks apart

Key Terms

Antiphospholipid Antibodies (aPL): Autoantibodies directed against phospholipids and phospholipid-binding proteins
Lupus Anticoagulant (LA): An antibody that interferes with phospholipid-dependent coagulation assays in vitro and is associated with thrombosis in vivo
Anticardiolipin (aCL) Antibodies: Antibodies that bind to cardiolipin, a negatively charged phospholipid
Anti-β2 Glycoprotein I (anti-β2GPI) Antibodies: Antibodies that bind to β2-glycoprotein I, a plasma protein that binds to phospholipids
Antiphospholipid Syndrome (APS): An autoimmune disorder characterized by thrombosis, pregnancy morbidity, and the presence of antiphospholipid antibodies
ELISA: Enzyme-Linked Immunosorbent Assay, a common type of immunoassay
Thrombosis: Formation of a blood clot inside a blood vessel

Assays

Protein C