Which laboratory tests should be ordered to evaluate an adult with unexplained bleeding, bruising, mucosal hemorrhage, or a personal or family history of abnormal clotting?

Laboratory Testing for Clotting Disorders

First-Line Testing Panel

For any adult with unexplained bleeding, bruising, mucosal hemorrhage, or personal/family history of abnormal clotting, order the following first-line tests simultaneously: complete blood count with platelets and peripheral smear, PT/INR, aPTT, fibrinogen level (Clauss method), VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and Factor VIII coagulant activity. 1, 2

Core Coagulation Tests

• Complete blood count (CBC) with platelets and peripheral blood smear evaluates for thrombocytopenia, abnormal platelet morphology, and evidence of chronic blood loss 2, 3

• Prothrombin time (PT/INR) assesses the extrinsic coagulation pathway (factors II, V, VII, X) and should be reported as both clotting time and INR 2, 4, 3

• Activated partial thromboplastin time (aPTT) evaluates the intrinsic coagulation pathway (factors VIII, IX, XI, XII) and detects hemophilia states when prolonged in isolation 2, 4, 3

• Fibrinogen level (Clauss method) identifies fibrinogen disorders and is essential for detecting disseminated intravascular coagulation 2, 3

Von Willebrand Disease Testing

• VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and Factor VIII coagulant activity must be ordered together as a panel—84% of bleeding evaluations include these tests in first-line workup 5, 1, 2

• These three tests establish VWD diagnosis, type, and severity, with normal reference ranges of 50-200 IU/dL for all three 1, 6

• A VWF:RCo/VWF:Ag ratio below 0.5-0.7 suggests Type 2 (qualitative) VWD rather than Type 1 (quantitative deficiency) 1, 6, 2

Additional First-Line Tests

• ABO blood group should be obtained because type O patients have 25-30% lower VWF levels than type AB 5, 1, 2

• Iron studies (ferritin, serum iron, TIBC) evaluate for chronic blood loss as a cause or consequence of bleeding 5, 2

Second-Line Testing (When First-Line Tests Are Normal But Bleeding History Is Convincing)

Coagulation Factor Assays

• Intrinsic pathway factors (FVIII, FIX, FXI) should be tested if aPTT is normal but bleeding persists 5, 2

• Extrinsic pathway factors (FII, FV, FVII, FX) should be tested if PT is normal but bleeding continues 5, 2

• Factor XIII activity is frequently overlooked but clinically important—it causes delayed bleeding with normal PT/aPTT 5, 2

Platelet Function Testing

• Light transmission aggregometry (LTA) with multiple agonists (ADP, collagen, epinephrine, ristocetin, arachidonic acid) is the gold standard for detecting inherited platelet function defects 5, 2

• Platelet flow cytometry evaluates platelet surface glycoproteins and activation markers 5, 2

• PFA-100/200 serves as an alternative platelet function screen, though it has conflicting data regarding sensitivity and specificity and should not be used for routine screening 5, 1, 2

Specialized VWD Testing

• VWF multimer analysis should be performed if the VWF:RCo/VWF:Ag ratio is below 0.5-0.7 or if initial VWD testing shows abnormal results—this distinguishes Type 2A, 2B, and 2M subtypes 5, 1, 6, 2

• VWF collagen binding (VWF:CB) and ristocetin-induced platelet aggregation (RIPA) may be necessary for subtyping 1, 6

• Newer automated assays (VWF:GPIbR, VWF:GPIbM) provide improved analytical precision compared to traditional VWF:RCo 6

Critical Pre-Analytical Considerations

• Atraumatic blood draw is essential—traumatic venipuncture activates coagulation and falsely elevates VWF levels 1, 6

• Avoid testing during acute illness, pregnancy, stress, or recent exercise—these conditions falsely elevate VWF and FVIII levels by 2-3 fold 1, 6

• Sample handling: Transport at room temperature, separate plasma promptly, and freeze at ≤-40°C if testing will be delayed beyond 2 hours 1, 6

• Exclude aspirin and NSAID use for at least 7-10 days before platelet function testing, as these medications confound assessment of inherited platelet function defects 5

Interpretation Algorithm

If PT/INR prolonged alone:

• Suspect vitamin K deficiency, warfarin effect, liver disease, or factor VII deficiency 4, 3

If aPTT prolonged alone with bleeding history:

• Suspect hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency), or factor XI deficiency 4, 3

If both PT and aPTT prolonged:

• Suspect vitamin K deficiency, liver disease, disseminated intravascular coagulation, or combined factor deficiencies 4, 3

If VWF:RCo <30 IU/dL:

• Definitive diagnosis of VWD 1, 6

If VWF:RCo 30-50 IU/dL with supportive clinical/family history:

• Likely VWD—repeat testing may be necessary due to assay variability (coefficient of variation 10-30%) 1, 6

If all tests normal but bleeding history convincing:

• Proceed to second-line testing including factor assays, platelet function testing, and consider bleeding disorder of unknown cause (BDUC) 5, 2

Common Pitfalls to Avoid

• Do not rely on a single test—no single laboratory test can screen for all forms of bleeding disorders 1, 2

• Do not order VWF multimer analysis as initial screening—this complex test should be reserved for reflex testing when initial results are abnormal 1, 6

• Do not use bleeding time or PFA-100 as routine screening tests—current evidence does not support their use due to poor sensitivity and specificity 1, 2

• Do not ignore ABO blood type—failure to consider blood type O (which has 25-30% lower VWF levels) leads to misdiagnosis 1, 6

• Do not test during physiological VWF elevations—pregnancy, oral contraceptives, inflammation, and stress can mask underlying VWD 1, 6

When to Refer to Hemostasis Specialist

• Abnormal initial VWD assay results requiring subtyping 6, 2

• Strong bleeding history with repeatedly normal tests (possible BDUC) 5, 6

• Suspected acquired von Willebrand syndrome (AVWS) in patients with aortic stenosis, lymphoproliferative disorders, or monoclonal gammopathies 6

• Need for guidance on repeat testing interpretation or access to specialized assays 6, 2