In a healthy 30‑year‑old woman with frequent spontaneous bruising, what is the recommended von Willebrand factor testing panel and subsequent platelet function testing?

Von Willebrand Factor Testing Panel and Platelet Function Testing

Initial VWF Testing Panel

The initial laboratory evaluation for von Willebrand disease must include three mandatory tests ordered simultaneously: VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and factor VIII coagulant activity (FVIII). 1, 2

Core Testing Components

• VWF:Ag measures the total quantity of von Willebrand factor protein present in plasma, providing essential information about whether the defect is quantitative 1, 3

• VWF:RCo assesses the functional capacity of VWF to bind to platelet glycoprotein Ib, which is critical for platelet adhesion at sites of vascular injury 1, 4

• FVIII activity evaluates factor VIII levels, which are carried and stabilized by VWF in circulation, and helps distinguish VWD from hemophilia A 1, 2

• Normal reference ranges for all three tests are 50-200 IU/dL, with VWF:RCo <30 IU/dL indicating definitive VWD diagnosis 2

Newer VWF Activity Assays

The traditional VWF:RCo assay is being replaced by more accurate automated alternatives, though VWF:RCo remains the reference standard 5:

• VWF:GPIbR uses recombinant wild-type GPIb fragments with ristocetin, offering improved precision and lower detection limits (down to 1-10 IU/dL) 5

• VWF:GPIbM employs gain-of-function mutant GPIb fragments that bind VWF spontaneously without ristocetin, eliminating false-positive results in certain populations 5

• VWF:Ab measures binding of VWF A1 domain to monoclonal antibodies, but cannot replace VWF:RCo as it misses certain type 2M mutations (e.g., p.G1324A) and has limited sensitivity to loss of high molecular weight multimers 5

• Chemiluminescence-based procedures demonstrate the lowest inter-laboratory variability, best low-level VWF detection (down to <1 U/dL), and fewest diagnostic errors compared to other methodologies 6

Interpretation of Initial Results

Diagnostic Thresholds

• VWF:RCo <30 IU/dL establishes definitive VWD diagnosis 2

• VWF:RCo 30-50 IU/dL with supportive clinical or family history indicates likely VWD 2

• VWF:RCo/VWF:Ag ratio <0.5-0.7 suggests type 2 (qualitative) VWD rather than type 1 (quantitative deficiency), requiring specialized subtyping tests 1, 2

Key Considerations Affecting Results

• ABO blood type O is associated with 25% lower VWF levels compared to non-O blood types 1

• Physiological elevators including stress, recent exercise, inflammation, pregnancy, and oral contraceptives can falsely elevate VWF and FVIII levels 1, 2

• VWF:RCo assay variability has a coefficient of variation of 10-30%, which may necessitate repeat testing 2

Specialized Subtyping Tests

VWF multimer analysis should not be ordered for initial screening but reserved for reflex testing when initial results are abnormal or the VWF:RCo/VWF:Ag ratio is <0.7. 1, 2

Additional Specialized Assays

• VWF collagen binding (VWF:CB) measures VWF binding to collagen and shows substantially lower inter-laboratory variability and error rates compared to VWF:RCo and VWF:GPIbM 6, 7

• Ristocetin-induced platelet aggregation (RIPA) using low-dose ristocetin helps identify type 2B VWD, which shows enhanced platelet aggregation 1, 3

• FVIII-VWF binding assay is necessary to diagnose type 2N VWD, where VWF cannot properly bind and stabilize factor VIII 3, 7

• VWF propeptide measurement can help distinguish increased VWF clearance (type Vicenza) from decreased synthesis 3, 7

• Platelet VWF measurement reveals defective VWF synthesis patterns: severe type 1 VWD shows reduced platelet and plasma VWF, type Vicenza shows normal platelet VWF despite low plasma levels, and type 2A can show either reduced or normal platelet VWF depending on subtype 8

Platelet Function Testing

Limited Role in VWD Screening

Bleeding time and platelet function analyzer (PFA-100) tests are not recommended for routine VWD screening due to conflicting data regarding sensitivity and specificity. 1, 2

• These tests lack adequate sensitivity and specificity for VWD diagnosis and should not be relied upon as screening tools 1

• PFA-100 may be abnormal in VWD but can also be normal in mild cases, and abnormal results are not specific to VWD 1

When Platelet Function Testing Is Indicated

Platelet function testing becomes relevant when:

• Initial hemostasis tests (CBC, PT, PTT) suggest a platelet disorder rather than VWD 1

• Thrombocytopenia is present on CBC, requiring evaluation for platelet consumption or production defects 2

• Clinical presentation suggests inherited platelet function disorders (e.g., Glanzmann thrombasthenia, Bernard-Soulier syndrome) rather than VWD 4

Critical Pre-Analytical Requirements

Proper sample handling is essential to avoid false results that lead to misdiagnosis. 1, 2

Blood Collection and Processing

• Perform atraumatic blood draw to minimize platelet activation 2

• Minimize patient stress and avoid testing during acute illness, inflammation, or pregnancy 1, 2

• Transport samples to the laboratory at room temperature (never refrigerate or freeze whole blood) 1, 2

• Separate plasma from blood cells promptly at room temperature 1, 2

• If testing will be delayed beyond 2 hours, freeze plasma at or below -40°C 1, 2

Common Diagnostic Pitfalls

Critical Errors to Avoid

• Relying on a single test for VWD diagnosis—all three initial tests (VWF:Ag, VWF:RCo, FVIII) are mandatory 2

• Ordering VWF multimer analysis as initial screening—this complex test should only be performed after abnormal initial results 1, 2

• Improper sample handling significantly affects test results, potentially causing false negatives or positives 1, 2

• Ignoring ABO blood type when interpreting borderline results, as type O individuals have physiologically lower VWF levels 1

• Testing during physiological stress states (pregnancy, inflammation, acute illness) can mask VWD diagnosis 1, 2

• Misinterpreting laboratory results accounts for approximately 40% of all errors in type 2 VWD diagnosis 6

When to Refer to Hemostasis Specialist

Immediate referral is indicated for 2:

• Abnormal initial VWD assay results requiring subtyping
• Strong bleeding history with repeatedly normal test results
• Suspected acquired von Willebrand syndrome (AVWS) in patients with aortic stenosis, lymphoproliferative disorders, or monoclonal gammopathies
• Guidance on repeat testing interpretation or access to specialized assays including multimer analysis