Robert Gosselin

1
von Willebrands Disease

• Robert Gosselin
• MT (ASCP), CLS

2

• von Willebrand disease (VWD) evidence-based
  diagnosis and management guidelines, the National
  Heart, Lung, and Blood Institute (NHLBI) Expert
  Panel report (USA) (Hemophilia 2008 14171-232)
• von Willebrand Factor Assays Guideline Second
  Edition (H51-2)
• (in progress)

3

• 1924 Erik vonWillebrand evaluated a 5 year old
  girl with bleeding diathesis and ultimately 66
  family members in Finland and noted
• Mucocutaneous bleeding
• Autosomal inheritance
• Prolonged bleeding times
• Normal clotting times
• Not corrected with blood transfusions

4

• In 1950 plasma protein defect causing prolonged
  bleeding time
• Deemed von Willebrand factor
• As cryoprecipitate and F8 concentrates were
  produced, it was noted that they complexed
  together

5

• VWF produced in two areas
• endothelium synthesized and stored in
  Weibel-Palade bodies
• megakaryocytes stored in alpha granules
• Assembled from identical subunits to form linear
  strings
• multimers formed in the Golgi complex

6

• In circulation, VWF travels as a complex with F8
• Upon injury, VWF undergoes conformational change
  and adheres to the subendothelium under high
  shear conditions
• Platelets adhere via GPIb-IX
• Half-life is 12 hours

7

• VWF multimers
• Degradation by ADAMTS13
• A Disintegrin-like And Metalloprotease
  domain with ThromboSpondin type 1 motif, member
  13
• Deficiency of ADAMTS13 have been associated
  with TTP

8
VWF functions
Hemophilia 2008 14177
9
von Willebrands Disease

• Plasma protein defect of vonWillebrands factor
  (vWF)
• Quantitative defect
• Partial deficiency type 1
• Total deficiency type 3
• Qualitative defect (type II)
• Functional aberration
• Short arm of chromosome 12

10
von Willebrands Classification

• Type 1-autosomal dominant
• Type 2-autosomal dominant
• 2A (some autosomal recessive)
• 2B
• 2M
• 2N (autosomal recessive)
• Type 3 (autosomal recessive)
• Pseudo-type VWD-autosomal dominant
• Acquired VWD

11
vWF Protein-Gene/mRNA/Functional domains
12
von Willebrands Disease

• 1 of population with ? vWF
• 125/1,000,000 with significant bleeding
• In UK, 6294 registered vWD patients, but only 10
  received treatment in calendar year 2001
• Type I accounts for 75 of all vWD Dx
• Mild to severe
• Hx
• Easy bruising
• Nose bleeds
• Oral cavity bleeding
• Menorrhagia
• Bleeding variability
• Mild to severe

13
Terminology

• VWD von Willebrands disease
• VWF - mature von Willebrands protein
• VWFRCo - ristocetin cofactor activity, measures
  vWF protein activity
• VWFAg - VWF antigen
• VWFCB - collagen binding assay, another
  component of vWF function
• FVIIIC - factor VIII activity
• VWFFVIIIB - vWF-factor VIII binding assay
• RIPA - ristocetin induced platelet aggregation
• VWFpp - vWF propeptide

Hemophilia 2008 14177
14
Laboratory Testing for vWD and PLT defects
15

• Basic (recommended first tier) testing
• Screening tests
• PT and aPTT
• PFA (Siemens) or Plateletworks (Helena Labs)
• NOT bleeding time
• VWFRCo measures activity
• Ristocetin driven
• Ristocetin independent
• VWFAg measure protein
• VWFRCo/VWFAg ratio
• F8 to r/o type 2N

May miss some VWD subtypes
16

• Second tier (subsequent)
• Multimer analysis in suspected type 2
• Ristocetin aggregation
• Low dose (0.6mg/ml)
• VWFCBA in suspected type 2
• VWFpp in suspected type 1 variants
• Other
• DDAVP (desmopressin) challenge
• IV or nasal
• Molecular analysis
• Antibodies
• Acquired

17

• Factors affecting VWF levels
• Exercise
• Stress
• Psychological
• Physiological
• Prolonged tourniquet time
• Diet??
• Other
• Pregnancy, oral contraceptives, HRT
• Trauma
• Inflammatory process

18

• Other factors influencing VWF levels
• Sample type
• 3.2 sodium citrate preferred
• ABO blood group VWFAg mean (range)
• Type O 75 (36-157)
• Type A 106 (48-234)
• Type B 117 (57-241)
• Type AB 123 (64-238)
• Using filters to achieve PPP

Hemophilia 2008 14177
19
Conjugated Anti-human VWF antibody
VWF ()
Chromogenic tag ?
Wash
Wash
Microwell containing anti-VWF



Color



?
?
?









Amount of color proportional to amount of
antibody present
Incubate
Incubate
ELISA and LIA based testing
Reagent beads coated with anti-vWF
Patient vWF ?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Instrument readingchanges in optical density
secondary to aggregates
?
?
Incubate
Testing well
20
Results compared to Std curve
Platelet agglutination and aggregation testing
Patients plasma Ristocetin
Ristocetin 1.2 mg/mL (l) 0.6 mg/mL (r)
Patients platelets plasma Ristocetin
Results compared to normal population
Zhou and Schmaier Am J Clin Pathol
2005123172-183
21
VWFRCo

• Platelet poor plasma
• Stabilized platelets
• Commercially prepared
• May be combined with ristocetin
• Ristocetin
• Initially used as antibiotic
• Peptide from Amycolatopsis lurida
• Thought to mimic shear effect of shear stress

22
Problems associated with VWFRCo

• Variability
• Between lots of ristocetin
• Between reagent methods
• Imprecision not ideal (up to 15)
• Low level sensitivity
• Automated methods lower limit 15-20

23
VWF activityRistocetin independent

• Platelet poor plasma
• Latex bead coated with monoclonal antibody
  (anti-GPIb)
• Recognizes functional domain of VWF
• LIA and ELISA method
• May reduce variability associated with VWFRCo

24
IL VWFAct versus Siemens VWFRCO
25
IL VWFAct versus Siemens VWFRCo
26
VWFRCo vs RIPA

• VWFRCo
• Measures vWF activity
• Exogenous PLTs
• Exogenous ristocetin
• Platelet agglutination
• ELISA methods (VWFAct)
• LIA methods (VWFAct)

• RIPA
• Measures interaction between vWF and platelet GPs
• Patients plasma
• Patients platelets
• Exogenous ristocetin
• Platelet aggregation

vWFRCo measures plasma vWF only, while RIPA
measures both plasma vWF and vWF interaction with
platelets
27
VWFAg

• Most labs use either ELISA or LIA testing
• ELISA testinghours
• LIA testingminutes
• Traditional method is gel electrophoresis
• Less variability than VWFRCo
• Possible prozone effects with automated methods
  (LIA)
• Test may be used for other indications
• Vasculitis Rx

28
VWFCB

• ELISA method
• Plate coated with type I or type III collagen
• Equine-bovine source
• Better correlation with HMW vWF
• Better reproducibility than vWFRCo
• Improved detection at lower levels
• No FDA approved kit in US
• Bummer!!!

29
Collagen binding assay
Ratio vWFAg to vWFCB
Favaloro, Am J Clin Pathol 2000608-618
Type 1 VWD
Type 2 VWD
30
Multimeric analysis of vWF-type II
Favaloro, Thromb Haemost 2007 98346-358
31
vWF propeptide (vWFpp)

• Stored in a granules or Weibel-Palade bodies
• After secretion into plasma, t½ 2-3 hrs
• vs mature vWF t½ 10 hrs
• Plasma concentrations 1µg/mL
• vs mature vWF 10 µg/mL
• Studies suggest
• Increased vWFpp to vWFAg ratio with reduced
  vWFAg true genetic defect with ? vWF survival
• ? vWF survival would also suggest alternative Rx
  strategies

32
Pseudo-VWD

• Similar to type 2
• Decreased PLT count
• Gain in function
• Aggregation with low dose ristocetin
• Must perform VWFPB
• Increased with VWD2B
• Normal with psuedo-VWD
• Abnormal GPIb receptor

33
From M Ledford-Kramer, Editor Clot-Ed
34
CLSI Guideline H51-2

• Recommendations for
• Sample type, processing
• Testing
• Calibration and results reporting
• Same calibrator for VWFAct and VWFAg
• Different calibrator for therapeutics
• Units
• Ratios
• Implementation/validation proposals??
• Implementing new method
• Lot-to-lot

35
Type I vWD

• Hx significant for mucocutaneous bleeds
• vWFRCo, vWFCB, vWFAg are lt50 IU/ml
• vWFRCo/vWFAg ratio gt0.7
• Normal PLT count
• Normal multimeric analysis
• Factor VIII activity normal/?
• Decreased RIPA
• PFA C.T. usually ?, BT slightly better than coin
• Propeptide survival??

36
Type 2A vWD

• Qual variant with ? PLT-dependent function with
  absence of HMW multimers
• Two groups of mutations
• Group 1 defective intracellular transport, ?
  retention of large multimers in ER
• Group 2 enhanced susceptibility to proteolysis
  of vWF, ? of large multimers
• Missense, deletions, insertions
• Clinical Hx (personal or familial) mucosal
  bleeding

37
Type 2A vWD

• Laboratory finding
• Normal PLT count
• Decreased vWFRCo
• Decreased vWFCB
• Normal/? factor VIII activity
• vWFAg usually normal/ ?
• Ratio of vWFRCo or vWFCBA to vWFAg is lt0.7
• RIPA is reduced
• PFA C.T. usually ?
• Absence of high molecular weight multimers

38
Type 2B vWD

• Qual variant with gain of function
• Increased affinity for for platelet GpIb
• 4 mutations (nucleotide substitutions) within the
  vWFA1 domain account for 90 of type IIb
• Similar laboratory parameters as pseudo-type vWD
• Clinical Hx (personal or familial) mucosal
  bleeding

39
Type 2B vWD

• Laboratory finding
• Normal to slightly ? PLT count
• Decreased (usually) vWFRCo
• Decreased (usually) vWFCBA
• Normal/? factor VIII activity
• vWFAg usually normal/ ?
• Ratio of vWFRCo or vWFCBA to vWFAg is lt0.7
• RIPA is ?
• low dose of ristocetin (0.75 mg/mL)
• PFA---normal to ?
• Absence of high molecular weight multimers

40
Pseudo-type vWD

• Mutations in GpIb-IX complex on platelet surface
• Variable clinical bleeding
• Similar laboratory parameters as type 2B
• Variable thrombocytopenia
• Enhanced RIPA
• Decreased HMW multimers

41
Differential Lab Dx Type IIb versus Psuedo type
vWD

• Normal PLTs to type IIb sample
• Enhanced RIPA
• Normal Cryoprecipitate to pseudo-type vWD
• Spontaneous aggregation

VWFPB assay preferred/definitive
42
Type 2M vWD

• Qual variant with ? PLT-dependent function
  without absence of HMW multimers
• Missense, small frame delections
• Clinical Hx (personal or familial) mucosal
  bleeding

43
Type 2M vWD

• Laboratory finding
• Normal PLT count
• Decreased vWFRCo
• Decreased vWFCBA
• Normal/? factor VIII activity
• vWFAg usually normal/ ?
• Ratio of vWFRCo or vWFCBA to vWFAg is lt0.7
• RIPA is reduced
• PFA C.T. usually ?
• Presence of high molecular weight multimers
• increased amounts may be seen

44
Type 2N vWD

• A qualitative defect resulting in decreased
  vWFFVIII binding
• Autosomal recessive
• Heterozygotes with normal factor VIII
• Bleeding Hx related to surgery or trauma

45
Type 2N vWD

• Laboratory finding
• Normal PLT count with prolonged aPTT
• Normal vWFRCo
• Normal vWFCBA
• ? factor VIII activity (usually between 5-30
  IU/ml)
• Decreased vWFFVIIIC binding
• vWFAg normal
• Ratio of vWFRCo or vWFCBA to vWFAg is gt0.8
• RIPA is normal
• PFA C.T. normal
• Multimer pattern is normal

46
Type III vWD

• A virtual absence of vWF protein
• Presentation early in life
• Multiple mutations, including frame shifts and
  nonsense mutations, as well as deletions

47
Type III vWD

• Laboratory finding
• Prolonged aPTT
• Absence of vWFRCo
• Absence of vWFCBA
• Markedly ? factor VIII activity
• Absence of vWFAg
• RIPA is markedly abnormal
• PFA C.T. markedly abnormal
• No detectable multimer pattern