Transcript Slide 1

von Willebrands
Disease
Robert Gosselin
MT (ASCP), CLS
von Willebrand disease (VWD):
evidence-based diagnosis and
management guidelines, the National
Heart, Lung, and Blood Institute
(NHLBI) Expert Panel report (USA)
(Hemophilia 2008; 14:171-232)
von Willebrand Factor Assays:
Guideline – Second Edition (H51-2)
(in progress)
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
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
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
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
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
VWF functions
Hemophilia 2008; 14:177
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
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
vWF ProteinGene/mRNA/Functional domains
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
•
•
•
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Easy bruising
Nose bleeds
Oral cavity bleeding
Menorrhagia
Bleeding variability
– Mild to severe
Terminology
• VWD – von Willebrand’s disease
• VWF - mature von Willebrands protein
• VWF:RCo - ristocetin cofactor activity, measures
vWF protein activity **
• VWF:Ag - VWF antigen
• VWF:CB - collagen binding assay, another
component of vWF function
• FVIII:C - factor VIII activity
• VWF:FVIIIB - vWF-factor VIII binding assay
• RIPA - ristocetin induced platelet aggregation
• VWFpp - vWF propeptide
Hemophilia 2008; 14:177
Laboratory Testing for
vWD and PLT defects
Basic (recommended first tier) testing
– Screening tests
• PT and aPTT
• PFA (Siemens)* or Plateletworks (Helena Labs)*
• NOT bleeding time
– VWF:RCo – measures activity
• Ristocetin driven
• Ristocetin independent
– VWF:Ag – measure protein
• VWF:RCo/VWF:Ag ratio
– F8 – to r/o type 2N
* May miss some VWD subtypes
Second tier (subsequent)
– Multimer analysis – in suspected type 2
– Ristocetin aggregation
• Low dose (0.6mg/ml)
– VWF:CBA – in suspected type 2
– VWFpp – in suspected type 1 variants
– Other
• DDAVP (desmopressin) challenge
– IV or nasal
• Molecular analysis
• Antibodies
– Acquired
Factors affecting VWF levels
– Exercise
– Stress
• Psychological
• Physiological
– Prolonged tourniquet time
– Diet??
– Other
• Pregnancy, oral contraceptives, HRT
• Trauma
• Inflammatory process
Other factors influencing VWF levels
– Sample type
• 3.2% sodium citrate preferred
– ABO blood group VWF:Ag mean (range)**
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•
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•
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; 14:177
VWF (+)
Conjugated Anti-human VWF antibody ¤
Chromogenic tag 
Wash
Microwell
containing
anti-VWF
+
Wash
¤ ¤
¤
++ +
+ +
Incubate

 
¤
+ ¤
+ ¤
+
Incubate
Color
Amount of color
proportional to
amount of antibody
present
ELISA and LIA based testing
Reagent beads coated with
anti-vWF
Patient
vWF 


 



Testing well








Incubate
Instrument reading—
changes in optical density
secondary to aggregates
Results
compared
to Std curve
Platelet
agglutination
and aggregation
testing
Patient’s plasma + Ristocetin
Ristocetin 1.2 mg/mL (l) & 0.6 mg/mL (r)
Patient’s platelets &
plasma + Ristocetin
Zhou and Schmaier Am J Clin Pathol 2005;123:172-183
Results
compared to
normal
population
VWF:RCo
• 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
Problems associated with
VWF:RCo
Variability
– Between lots of ristocetin
– Between reagent methods
Imprecision not ideal (up to 15%)
Low level sensitivity
– Automated methods lower limit 15-20%
VWF activity
Ristocetin 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
VWF:RCo
D i f fe r e n c e ,
VWF:ACT %NHP %
N H P
IL VWF:Act versus Siemens
VWF:RCO
200
30
180
20
160
10
140
0
120
-1 0
100
y = 0.6981x + 14.094
VWF:Activity R2 = 0.7309
0
20
40
60
80
100
120
140
160
180
- 280
0
- 360
0
- 440
0
- 520
0
- 6 00
-7 0
0
50
100
150
VWF:ACT % NHP
M e a n V W F :A c t
200
250
IL VWF:Act versus Siemens
VWF:RCo
70
TOP VWF:Activity, %
60
50
40
30
20
10
0
0
5
10
15
20
25
30
Reported VWF:Activity, %
35
40
45
VWF:RCo vs RIPA
VWF:RCo
Measures vWF activity
Exogenous PLTs
Exogenous ristocetin
Platelet agglutination
ELISA methods (VWF:Act)
LIA methods (VWF:Act)
RIPA
Measures interaction between
vWF and platelet GPs
Patients plasma
Patients platelets
Exogenous ristocetin
Platelet aggregation
vWF:RCo measures plasma vWF only, while RIPA measures both
plasma vWF and vWF interaction with platelets
VWF:Ag
• Most labs use either ELISA or LIA testing
– ELISA testing…hours
– LIA testing…minutes
• Traditional method is gel electrophoresis
• Less variability than VWF:RCo
• Possible prozone effects with automated
methods (LIA)
• Test may be used for other indications
– Vasculitis Rx
VWF:CB
• ELISA method
– Plate coated with type I or type III collagen
– Equine-bovine source
– Better correlation with HMW vWF
– Better reproducibility than vWF:RCo
• Improved detection at lower levels
– No FDA approved kit in US
• Bummer!!!
Collagen
binding
assay
Ratio: vWF:Ag
to vWF:CB
Favaloro, Am J Clin
Pathol 2000;608-618
Type 1 VWD
Type 2 VWD
Multimeric analysis of vWF-type II
Favaloro, Thromb Haemost 2007; 98:346-358
vWF propeptide
(vWF:pp)
• Stored in α 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 vWF:pp to vWF:Ag ratio with reduced
vWF:Ag true genetic defect with  vWF survival
–  vWF survival would also suggest alternative Rx
strategies
Pseudo-VWD
• Similar to type 2
– Decreased PLT count
– “Gain in function”
• Aggregation with low dose ristocetin
– Must perform VWF:PB
• Increased with VWD:2B
• Normal with psuedo-VWD
– Abnormal GPIb receptor
From M Ledford-Kramer, Editor Clot-Ed
CLSI Guideline H51-2
• Recommendations for
– Sample type, processing
– Testing
– Calibration and results reporting
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Same calibrator for VWF:Act and VWF:Ag
Different calibrator for therapeutics
Units
Ratios
– Implementation/validation proposals??
• Implementing new method
• Lot-to-lot
Type I vWD
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Hx significant for mucocutaneous bleeds
vWF:RCo, vWF:CB, vWF:Ag are <50 IU/ml
vWF:RCo/vWF:Ag ratio >0.7
Normal PLT count
Normal multimeric analysis
Factor VIII activity normal/
Decreased RIPA
PFA C.T. usually , BT slightly better than coin
• Propeptide survival??
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
Type 2A vWD
• Laboratory finding
– Normal PLT count
– Decreased vWF:RCo
– Decreased vWF:CB
– Normal/ factor VIII activity
– vWF:Ag usually normal/ 
• Ratio of vWF:RCo or vWF:CBA to vWF:Ag is <0.7
– RIPA is reduced
• PFA C.T. usually 
– Absence of high molecular weight multimers
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 pseudotype vWD
• Clinical Hx (personal or familial) mucosal
bleeding
Type 2B vWD
• Laboratory finding
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–
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Normal to slightly  PLT count
Decreased (usually) vWF:RCo
Decreased (usually) vWF:CBA
Normal/ factor VIII activity
vWF:Ag usually normal/ 
• Ratio of vWF:RCo or vWF:CBA to vWF:Ag is <0.7
– RIPA is 
• low dose of ristocetin (0.75 mg/mL)
• PFA---normal to 
– Absence of high molecular weight multimers
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
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
VWF:PB assay preferred/definitive
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
Type 2M vWD
• Laboratory finding
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–
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Normal PLT count
Decreased vWF:RCo
Decreased vWF:CBA
Normal/ factor VIII activity
vWF:Ag usually normal/ 
• Ratio of vWF:RCo or vWF:CBA to vWF:Ag is <0.7
– RIPA is reduced
• PFA C.T. usually 
– Presence of high molecular weight multimers
• increased amounts may be seen
Type 2N vWD
• A qualitative defect resulting in
decreased vWF:FVIII binding
• Autosomal recessive
• Heterozygotes with normal factor VIII
• Bleeding Hx related to surgery or
trauma
Type 2N vWD
• Laboratory finding
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–
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Normal PLT count with prolonged aPTT
Normal vWF:RCo
Normal vWF:CBA
 factor VIII activity (usually between 5-30 IU/ml)
• Decreased vWF:FVIIIC binding
– vWF:Ag normal
• Ratio of vWF:RCo or vWF:CBA to vWF:Ag is >0.8
– RIPA is normal
• PFA C.T. normal
– Multimer pattern is normal
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
Type III vWD
• Laboratory finding
– Prolonged aPTT
• Absence of vWF:RCo
– Absence of vWF:CBA
– Markedly  factor VIII activity
– Absence of vWF:Ag
– RIPA is markedly abnormal
• PFA C.T. markedly abnormal
– No detectable multimer pattern