What laboratory workup is recommended for von Willebrand disease?

Laboratory Workup for von Willebrand Disease

The initial laboratory workup for von Willebrand disease should include three essential plasma tests: VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and factor VIII coagulant activity (FVIII), preceded by basic hemostasis screening with CBC, PT, and aPTT. 1

Initial Hemostasis Screening Tests

Before ordering VWD-specific assays, obtain basic coagulation studies to exclude other bleeding disorders: 1

• Complete blood count (CBC) - to assess for thrombocytopenia or thrombocytosis
• Prothrombin time (PT) - to evaluate coagulation factor deficiency
• Activated partial thromboplastin time (aPTT) - to screen for factor deficiencies

These tests do not diagnose VWD but help identify alternative causes of bleeding such as coagulation factor deficiencies or platelet count abnormalities. 1

Core VWD Diagnostic Panel (Grade B Recommendations)

All three of the following tests must be ordered simultaneously for initial VWD evaluation: 1

• VWF antigen (VWF:Ag) - measures the quantity of VWF protein
• VWF ristocetin cofactor activity (VWF:RCo) - assesses VWF functional activity for platelet binding
• Factor VIII coagulant activity (FVIII) - evaluates FVIII levels, which are often reduced in VWD

Diagnostic Thresholds

A VWF level <30 IU/dL is the recommended cutoff for definitive VWD diagnosis. 1 This threshold accounts for the high frequency of blood type O (associated with naturally lower VWF levels), bleeding symptoms in normal individuals, and the absence of VWF gene abnormalities in many with mildly low VWF levels. 1

However, VWD diagnosis remains possible with VWF:RCo levels of 30-50 IU/dL when supported by strong clinical or family bleeding history. 1

Critical Ratio Calculation

Calculate the VWF:RCo/VWF:Ag ratio to distinguish Type 1 from Type 2 VWD variants. 1 A ratio <0.5-0.7 suggests Type 2 VWD (qualitative defect), while a ratio >0.5-0.7 with proportionally decreased values suggests Type 1 VWD (quantitative defect). 1

Specialized Testing (Reflexive or Secondary)

VWF multimer analysis should only be performed if initial testing shows abnormal results, not as a screening test. 1 Indications for multimer analysis include: 1

• Abnormally low VWF:RCo
• VWF:RCo/VWF:Ag ratio <0.5-0.7
• Clinical suspicion of Type 2 VWD despite borderline initial results

Multimer analysis is technically complex, qualitatively interpreted, and used specifically for VWD subtype determination. 1

Critical Pre-analytical Considerations

Laboratory results are highly vulnerable to pre-analytical variables that can falsely elevate or decrease VWF levels: 1

Patient Factors to Control

• Avoid stress during phlebotomy - struggling, crying, or anxiety can falsely elevate VWF and FVIII levels 1
• No recent exercise - physical activity elevates VWF 1
• Document inflammatory conditions - acute/chronic inflammation, pregnancy, or estrogen/oral contraceptives increase VWF 1
• Record ABO blood type - Type O individuals have VWF levels 25% lower than other blood groups 1
• Atraumatic blood draw - minimize tissue factor exposure 1

Sample Handling Requirements

Blood samples for VWF assays must be transported at room temperature to prevent cryoprecipitation. 1 Specific handling protocols include: 1

• Transport samples at room temperature (never refrigerated)
• Separate plasma from blood cells promptly at room temperature
• Centrifuge thoroughly to remove all platelets
• If assaying within 2 hours, keep at room temperature
• For storage/transport to reference labs, freeze promptly at ≤-40°C
• Thaw frozen samples at 37°C and keep at room temperature <2 hours before assay

FVIII activity decreases 10-20% in frozen-thawed plasma compared to fresh plasma, and more with suboptimal handling. 1

Tests NOT Recommended for Routine Screening

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

Laboratory Quality Considerations

• Calibrators should reference WHO plasma standards 1
• VWF assays have high coefficients of variation (10-30%), especially VWF:RCo 1
• Interlaboratory variability is substantial 1
• Results should be reported as IU/dL when WHO-linked calibrators are used 1

Common Pitfalls to Avoid

1. Ordering only one or two of the three core tests - all three (VWF:Ag, VWF:RCo, FVIII) are required for proper diagnosis and classification 1
2. Ordering multimer analysis as initial screening - this is expensive, complex, and should be reserved for cases with abnormal initial results 1
3. Testing during acute illness or pregnancy - these conditions artificially elevate VWF levels and may mask the diagnosis 1
4. Improper sample handling - refrigeration or delayed processing can invalidate results 1
5. Failing to repeat testing - VWF levels can vary, and repeat testing may be necessary for borderline cases 1