What additional laboratory tests should be performed when von Willebrand disease is suspected?

Additional Laboratory Testing for von Willebrand Disease

When von Willebrand disease is suspected, order three mandatory initial tests together: VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and factor VIII coagulant activity (FVIII). 1, 2

Initial Hemostasis Panel

Before or alongside VWD-specific testing, obtain basic coagulation studies to rule out other bleeding disorders: 3, 1

• Complete blood count (CBC) with platelet count – identifies thrombocytopenia (note: isolated low platelets may occur in Type 2B VWD) 3, 4
• Prothrombin time (PT) – screens for extrinsic pathway defects 3, 2
• Activated partial thromboplastin time (PTT) – may be prolonged if FVIII is significantly reduced, though often normal in mild VWD 3, 4
• Fibrinogen or thrombin time (TT) – optional, helps exclude fibrinogen disorders 3

If you have a strong mucocutaneous bleeding history (epistaxis, menorrhagia, easy bruising), proceed directly to the three core VWD assays without waiting for these screening tests. 1, 2

The Three Mandatory VWD-Specific Tests

These must be ordered simultaneously as a panel: 1, 2

• VWF:Ag (VWF antigen) – measures the quantity of VWF protein in plasma; normal range 50-200 IU/dL 1, 2
• VWF:RCo (VWF ristocetin cofactor activity) – assesses functional ability of VWF to bind platelets; normal range 50-200 IU/dL 1, 2
• FVIII (factor VIII coagulant activity) – evaluates FVIII levels, which are carried and protected by VWF; normal range 50-200 IU/dL 1, 2

Calculate the VWF:RCo/VWF:Ag ratio: A ratio <0.5-0.7 suggests Type 2 (qualitative) VWD rather than Type 1 (quantitative deficiency). 1, 2

Specialized Reflex Testing (Only If Initial Tests Are Abnormal)

Do not order VWF multimer analysis as part of initial screening. 3, 1 Reserve these tests for when initial results show abnormalities or when clinical suspicion remains high despite borderline results: 3

• VWF multimer analysis – distinguishes Type 2A, 2B, and 2M subtypes by analyzing the electrophoretic pattern of VWF multimers 3, 5, 6
• VWF collagen binding assay (VWF:CB) – alternative functional assay that may detect qualitative defects 5, 7
• Ristocetin-induced platelet aggregation (RIPA) – helps identify Type 2B VWD (increased sensitivity to low-dose ristocetin) 5, 8
• FVIII binding capacity (VWF:FVIIIB) – identifies Type 2N VWD (defective VWF binding to FVIII) 5, 8
• Platelet VWF studies – measures VWF content in platelets 3, 5
• VWF propeptide antigen – helps distinguish increased clearance from decreased synthesis 7, 9

Newer Automated VWF Activity Assays

Consider these alternatives to the traditional VWF:RCo assay for improved precision: 2

• VWF:GPIbR – automated assay using recombinant GPIb fragments with ristocetin; improved analytical precision with lower limit of detection (1-10 IU/dL) 2
• VWF:GPIbM – automated assay using gain-of-function mutant GPIb fragments that bind VWF without ristocetin, eliminating false-positives 2
• VWF:Ab – measures VWF A1 domain interaction with monoclonal antibodies; cannot replace VWF:RCo as it misses some Type 2M mutations 2

Critical Pre-Analytical Considerations

Timing and patient factors matter significantly: 1, 2

• Avoid testing during acute illness, pregnancy, inflammation, or within 48 hours of strenuous exercise – these falsely elevate VWF levels 1, 4
• Minimize patient stress during blood draw (stress elevates VWF) 1, 4
• Consider ABO blood type: Type O individuals have 25% lower baseline VWF levels 3, 2
• Avoid testing in patients on estrogen therapy or oral contraceptives (elevates VWF) 1, 4

Sample handling is crucial: 1, 2

• Perform atraumatic venipuncture at room temperature 2, 4
• Transport samples to laboratory at room temperature (not refrigerated) 1, 2
• Separate plasma promptly at room temperature with thorough centrifugation to remove platelets 1, 4
• If testing delayed beyond 2 hours, freeze samples at or below -40°C 1, 2

Common Pitfalls to Avoid

• Never rely on bleeding time or PFA-100 as screening tests – conflicting data on sensitivity/specificity; not recommended by current guidelines 3, 1, 4
• Never order just one or two of the three core tests – all three are necessary to establish diagnosis and suggest VWD type 1, 2
• Never order VWF multimer analysis upfront – it is complex, expensive, and should only be performed after abnormal initial results 3, 1
• Never ignore the VWF:RCo assay's high variability – coefficient of variation is 10-30%, so repeat testing may be necessary for borderline results 2, 4
• Never forget that normal tests don't exclude VWD – if bleeding history is strong and tests are normal, repeat testing or refer to a hemostasis specialist 3, 1

When to Refer to a Hemostasis Specialist

Consult or refer when: 3, 1, 2

• Initial VWD assays show abnormalities requiring subtyping 2, 4
• Strong bleeding history with repeatedly normal test results 1, 2
• VWF:RCo between 30-50 IU/dL with supportive clinical/family history 2
• Suspected acquired von Willebrand syndrome (AVWS) – associated with aortic stenosis, lymphoproliferative disorders, or monoclonal gammopathies 2
• Need for specialized assays like multimer analysis or genetic sequencing 3, 2