von Willebrand Assays

Overview of von Willebrand Assays

Definition: A panel of laboratory tests used to assess the quantity and function of von Willebrand factor (vWF), a protein essential for normal blood clotting
Purpose:
- Diagnosis of von Willebrand Disease (vWD): Confirms the diagnosis and helps classify the type of vWD
- Differentiation of vWD from other Bleeding Disorders: Helps distinguish vWD from hemophilia and other platelet function disorders
- Evaluation of Unexplained Bleeding: Used to investigate patients with a personal or family history of mucocutaneous bleeding
- Monitoring Treatment: Used to monitor the response to treatment for vWD (e.g., desmopressin, vWF concentrates)
Key Assays:
- vWF Antigen (vWF:Ag)
- vWF Activity (vWF:RCo)
- Factor VIII Activity (FVIII:C)
- vWF Multimeric Analysis
- Platelet Function Analyzer (PFA-100) - though not specific to vWF, it can assist in diagnosis

Components of von Willebrand Assays

Specimen Collection:
- Collection Tube: Sodium citrate (light blue top) tube with a 3.2% or 3.8% sodium citrate concentration
- Blood-to-Anticoagulant Ratio: The correct ratio is critical for accurate coagulation testing:
  - 9:1 ratio of blood to anticoagulant
- Processing:
  - Centrifuge the sodium citrate tube to obtain platelet-poor plasma (PPP)
  - PPP should have a platelet count < 10 x 10^9/L
  - Test should be performed within 4 hours of collection or the plasma should be frozen if testing will be performed later. Specifics will vary from lab to lab
Reagents:
- vWF:Ag Reagent: Contains antibodies specific for vWF
- vWF:RCo Reagent: Contains ristocetin, a substance that induces vWF-dependent platelet agglutination
- Factor VIII Reagent: Contains purified factor VIII

Individual Assays

von Willebrand Factor Antigen (vWF:Ag):
- Principle: Measures the amount of vWF protein in the plasma, regardless of its function
- Methods:
  - Immunoturbidimetry: Measures the turbidity caused by the formation of antigen-antibody complexes
  - Nephelometry: Measures the light scattered by the antigen-antibody complexes
  - ELISA (Enzyme-Linked Immunosorbent Assay): Uses enzyme-labeled antibodies to detect vWF
- Procedure:
  1. Add anti-vWF antibodies
  2. Read for what test is used whether it is measuring the antibody or test light to determine the concentration with the reference range
- Reporting:
  - Reported as a percentage of normal or in IU/dL
  - Reference Range: Varies depending on the laboratory and the assay used (e.g., 50-200%)
- Interpretation:
  - Decreased vWF:Ag: Suggests a quantitative deficiency of vWF (seen in type 1 and type 3 vWD)
  - Normal vWF:Ag: Does not rule out vWD; may be seen in qualitative vWF defects (type 2 vWD)
von Willebrand Factor Activity (vWF:RCo):
- Principle: Measures the ability of vWF to bind to platelets in the presence of ristocetin, a cofactor that promotes vWF-dependent platelet agglutination
- Method:
  - Ristocetin-Induced Platelet Agglutination (RIPA):
    - Measures the rate of platelet agglutination after the addition of ristocetin and vWF-containing plasma
    - The degree of agglutination is proportional to the vWF activity
  - Automated vWF Activity Assays:
    - Use modified latex particles coated with GPIbα (the vWF receptor on platelets)
    - Measure the binding of vWF to the latex particles in the presence of ristocetin
    - Automated latex agglutination test can also be called vWF:Glycoprotein 1b Binding Assay (vWF:GPIbM)
- Procedure:
  1. Test is ran through an analyzer
  2. The agglutination is read
- Reporting:
  - Reported as a percentage of normal or in IU/dL
  - Reference Range: Varies depending on the laboratory and the assay used (e.g., 50-200%)
- Interpretation:
  - Decreased vWF:RCo: Suggests a functional defect in vWF (seen in most types of vWD)
  - Increased vWF:RCo: Seen in type 2B vWD (vWF has increased affinity for platelets)
Factor VIII Activity (FVIII:C):
- Principle: Measures the activity of factor VIII, a coagulation protein that is bound to and stabilized by vWF
- Methods:
  - Chromogenic Assay: Measures the amount of activated Factor X (Factor Xa) produced in the presence of Factor VIII and other coagulation factors
  - Clotting Assay: Measures the ability of plasma to correct the aPTT of Factor VIII-deficient plasma
- Procedure:
  - Follow Factor Assay procedure
- Reporting:
  - Reported as a percentage of normal or in IU/dL
  - Reference Range: Varies depending on the laboratory and the assay used (e.g., 50-150%)
- Interpretation:
  - Decreased FVIII:C: Suggests vWD type 2N (decreased binding of vWF to FVIII) or hemophilia A (Factor VIII deficiency)
vWF Multimeric Analysis:
- Principle: Evaluates the distribution of vWF multimers, which are polymers of vWF subunits
- Method:
  - Gel Electrophoresis: vWF multimers are separated by electrophoresis on an agarose gel
  - Immunoblotting: The separated multimers are transferred to a membrane and detected with an antibody specific for vWF
  - Visualization: The multimers are visualized using a staining or detection method
- Interpretation:
  - Normal Multimer Pattern: A full range of vWF multimers is present
  - Abnormal Multimer Pattern: Decreased high-molecular-weight multimers (HMW multimers) are seen in type 2A vWD
- Clinical Significance: Helps differentiate type 2A vWD from other subtypes of vWD

PFA-100 (Platelet Function Analyzer)

Principle: Measures platelet function under high shear stress by aspirating blood through a small aperture coated with collagen and epinephrine or collagen and ADP
Procedure:
- A citrated whole blood sample is aspirated through a small aperture coated with collagen and either epinephrine or ADP
- The time it takes for the aperture to become occluded by a platelet plug is measured (closure time)
Results:
- Closure Time (CT):
  - Epinephrine Closure Time (Epi-CT): Measures platelet function in response to collagen and epinephrine
  - ADP Closure Time (ADP-CT): Measures platelet function in response to collagen and ADP
Interpretation:
- Prolonged Closure Time: Suggests platelet dysfunction
  - Prolonged Epi-CT and ADP-CT: May be seen in vWD, aspirin use, or other platelet function disorders
- Normal Closure Time: Does not rule out vWD or other platelet function disorders
Limitations:
- Affected by Hematocrit: Must be performed on samples with a hematocrit within the specified range
- Affected by Platelet count: Must be performed on samples with a platelet count within the specified range
- Not Specific for vWD: Prolonged closure time can be seen in other platelet function disorders

Interpreting von Willebrand Assay Results

The following table summarizes the typical patterns of results seen in different types of vWD:

vWD Type	vWF:Ag	vWF:RCo	FVIII:C	Multimers	PFA-100
Type 1	Low	Low	Low (may be)	Normal	Prolonged
Type 2A	Normal or Low	Low	Normal	Decreased HMW multimers	Prolonged
Type 2B	Normal or Low	Increased	Normal	Normal	Prolonged
Type 2M	Normal	Low	Normal	Normal	Prolonged
Type 2N	Normal	Normal	Low	Normal	Normal or Prolonged
Type 3	Very Low	Very Low	Very Low	Absent	Prolonged

Factors Affecting von Willebrand Assay Results

Pre-Analytical Variables:
- Improper Collection Technique: Tissue thromboplastin contamination or hemolysis
- Incorrect Blood-to-Anticoagulant Ratio: Underfilling or overfilling the collection tube
- Clotted Sample: Invalidates the results
- Delayed Testing: vWF and FVIII can degrade over time
- Improper Storage: Incorrect storage temperatures can affect results
Analytical Variables:
- Instrument Malfunction: Ensure proper calibration and maintenance of the coagulation analyzer
- Reagent Problems: Use fresh, properly stored reagents and follow the manufacturer’s instructions
- Interfering Substances: Lipemia or icterus can interfere with optical clot detection
- Assay Sensitivity: Different assays have different sensitivities for detecting vWF abnormalities
Patient-Related Variables:
- Blood Type: vWF levels are typically lower in individuals with blood type O
- Age: vWF levels increase with age
- Pregnancy: vWF levels increase during pregnancy
- Estrogen Therapy: Increases vWF levels
- Acute Phase Reactants: vWF is an acute phase reactant, so levels can increase during inflammation or infection

Troubleshooting Erroneous Results

If the vWF 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
- Consider repeating the vWF assays after a period of stability (to account for normal fluctuations in vWF levels)
- Evaluate for potential interfering substances or medications
- Consult with a pathologist or coagulation expert

Reflex Testing

If the initial vWF assays are abnormal, further testing may be performed to:
- Characterize the type of vWD (e.g., multimer analysis, FVIII binding assay)
- Rule out other causes of bleeding (e.g., platelet function disorders)
- Perform genetic testing to identify specific vWF gene mutations

Key Terms

von Willebrand Factor (vWF): A protein that mediates platelet adhesion and carries factor VIII
vWF Antigen (vWF:Ag): Measures the amount of vWF protein
vWF Activity (vWF:RCo): Measures the ability of vWF to bind to platelets
Factor VIII (FVIII:C): A coagulation protein that is carried by vWF
Ristocetin: An antibiotic that induces vWF-dependent platelet agglutination
Multimer Analysis: Evaluates the distribution of vWF multimers
Platelet Function Analyzer (PFA): A test that measures platelet function under high shear stress
Desmopressin (DDAVP): A synthetic analog of vasopressin that stimulates the release of vWF and factor VIII
Type 1 vWD: Partial quantitative deficiency of vWF
Type 2 vWD: Qualitative defects in vWF function
Type 3 vWD: Severe quantitative deficiency of vWF

Inhibitor Assays

Platelet Function