Diagnosis of Hemophilia and Other
Bleeding Disorders: A Laboratory Manual

Second Edition, 2010

Steve Kitchen, Angus McCraw, Marión Echenagucia on behalf of the WFH Laboratory Sciences Committee

This WFH manual is the definitive how-to guide for the accurate diagnosis of hemophilia and other bleeding disorders. Written and reviewed by the world’s leading experts in coagulation testing, this comprehensive, revised edition features new sections on two-stage clotting and chromogenic FVIII assays, von Willebrand factor collagen binding assay and multimer analysis, and the use of coagulometers.

Click here to download the complete PDF, or select individual sections below.

Also available in:

Laboratory Manual Demonstration Videos 

NOW AVAILABLE: Step-by-step video demonstrations of the three most important manual diagnostic techniques: 

  • APTT (Section 13)
  • One-stage FVIII assay (Section 23)
  • FVIII inhibitor assay (Section 28) 

Each video features: 

  • careful demonstration of correct technique
  • detailed explanations of the background theory of the assay
  • special considerations for issues of limited resources
  • plotting and interpretation of typical results
  • searchable transcripts
  • record sheets and graph paper to download
  • discussion of the important reagents

Access the videos here: 
One-stage FVIII 


  1. Laboratory Equipment
  2. Checking Pipette and Weighing Balance Calibration
  3. Laboratory Safety
  4. Sample Collection and Pre-Analytical Variables
  5. Internal Quality Control and External Quality Assessment
  6. Manual Tilt-Tube Technique
  7. Preparation and Calibration of Pooled Normal Plasma (PNP)
  8. Establishing a Normal Reference Range
  9. Reagents
  10. Platelet Count
  11. Bleeding Time
  12. Prothrombin Time (PT)
  13. Activated Partial Thromboplastin Time (APTT)
  14. Mixing Tests For Further Investigation of Abnormal PT and APTT
  15. Thrombin Clotting Time
  16. Thrombin Time in the Presence of Protamine Sulphate to Detect Presence of Heparin
  17. Reptilase Time
  18. Fibrinogen (Modifi ed Clauss Assay)
  19. Removal of Heparin from Plasma
  20. Fibrinogen Antigen Assay by Radial Immunodiffusion (RID)
  21. Factor XIII Screen
  22. Assays Based on Prothrombin Time (Factors II, V, VII, or X)
  23. Factor Assays Based on APTT (One-Stage Assay of FVIII:C, FIX, FXI, or FXII)
  24. Factor VIII:C Assays in Cryoprecipitate
  25. Two-Stage Clotting Assay for Factor VIII:C
    25.1 Production of Combined Reagent for Two-Stage Factor FVIII:C Assay
  26. Chromogenic FVIII:C Assay
  27. One-Stage Intrinsic Assay of Prekallikrein (PKK) and High Molecular Weight Kininogen (HMWK)
  28. Clotting Factor Inhibitor Screen Based on APTT
  29. Ristocetin Cofactor Activity/von Willebrand Factor Activity (VWF: RCo or VWF:Act)
    29.1 Fixed Platelet Preparation
  30. Von Willebrand Factor Antigen by Elisa (VWF:Ag)
  31. Von Willebrand Factor Collagen Binding Assay (VWF:CB)
  32. Factor VIII Binding Assay for Diagnosis of von Willebrand Disease Normandy
  33. VWF Multimer Analysis
  34. Quantitative Measurement of FVIII Inhibitors
  35. Factor IX Inhibitor Assay
  36. Factor XIII Activity Assay
  37. Lupus Anticoagulant and Anti-Phospholipid Antibodies
  38. Dilute Russell’s Viper Venom Time (DRVVT)
    38.1 Preparation of Washed Platelets for DRVVT
  39. Platelet Function Testing
    39.1 Clot Retraction
    39.2 Measurement of Platelet Aggregation
  40. Lyophilization of Plasma
  41. Evaluation and Use of Coagulometers
  42. Molecular Genetic Analysis 

Updated July 2016