Embed Size (px)
Citation preview
vWD—Disorder of Primary Hemostasis
} Most common of the congenital bleeding disorders } 1-‐2 % of the general popula@on } Symptoma@c in only about 1/10,000
} 1926 – Erik von Willebrand à 5 y-‐o-‐f and her family who lived on the Åland Islands – Hereditär pseudohemofili, 1926
} Ini@ally described as “pseudohemophilia”
1
vWD—Disorder of Primary Hemostasis
} Clinical manifesta@ons } Mucocutaneous bleeding of varying severity in males and females 1. Ecchymoses 2. Epistaxis 3. Gastrointes@nal bleeding 4. Menorrhagia
} Defec@ve platelet adhesion } Reduced FVIII levels
2
vWF • Large mul@meric protein – ranges from 600 kD to >20 million kD
– Synthesized by endothelial cells and megakaryocytes • Endothelial cells source of plasma vWF
• Gene for vWF is located on chromosome 12p • 178 kB, 52 exons
3 Hoffman: Adapted from Ginsburg D, Bowie EJW: Molecular geneGcs of von Willebrand disease. Blood 79:2507, 1992.)
Synthesis of vWF
} vWF synthesized in endothelial cells and megakaryocytes 1. Stored in Weibel-‐Palade bodies of
endothelial cells 2. Stored in α-‐granules of platelets
Steps in synthesis of vWF 1. First synthesized as a pre-‐
pro-‐vWF monomer 2. DimerizaGon occurs in ER 3. Pre-‐pro-‐vWF monomers
linked together at the carboxyl terminal end
4. Dimeric molecules pass to the Golgi apparatus
5. Dimers mulGmerize 6. Propep@de is cleaved off
à mature subunit 4
N-Terminal Multimerization
C-Terminal Dimerization
High Molecular
Weight Multimer
ER Golgi
vWF Release
Valentijn K M et al. Blood 2011;117:5033-5043
5
Func@on of vWF } vWF serves two important biologic func@ons
1. Serves as a carrier protein for plasma FVIII a. VWF protects Factor VIII in circula@on b. VWF co-‐localizes FVIII at sites of vascular injury
2. Serves as a ligand that binds to the gpIb receptor on platelets to ini@ate platelet adhesion to the damaged endothelium a. VWF binds to extravascular collagen b. Platelets adhere to the bound vWF c. Adherent platelets become ac@vated
6
Platelets
Clotting factors Vessel wall
VWF
Func@on of vWF
7
Elsevier
8 Elsevier
Classifica@on of vWD • vWD – extremely heterogenous, complex disorder with > 20 dis@nct
subtypes
• Types of vWD
1. Quan@ta@ve Defects
• Type 1 – Par@al quan@ta@ve deficiency – Autosomal dominant
• Type 3 – Complete absence/severely decreased – Autosomal recessive
2. Qualita@ve Defects
• Type 2 – 2A – 2B – 2M – 2N – Autosomal recessive
9
Subgroups
Type I vWD
} Most common type of vWD } 80% of pa@ents with vWD fall into this category
} Caused by heterozygous muta@on leading to a par@al quan@ta@ve deficiency of vWF } Gene@c abnormality in ONE of the vWF alleles } Accounts for a 50% reduc@on in vWF } Mild secondary deficiency in FVIII
} Endothelial cells and platelets contain normal, but reduced levels of vWF } DDAVP can induce the release the stored vWF
} Bleeding symptoms range from asymptoma@c to mild
10
Type I vWD } Lab findings } Normal to decreased
1. FVIII (aPTT) 2. vWF:Ac@vity (Ristoce@n Cofactor) 3. vWF:An@gen
4. Prolonged BT • (PFA-‐100—Col/EPI, Col/ADP)
5. Propor@onal decrease of ALL vWF mul@mers
11
Type 3 vWD
} Most severe form of the disease
} Results from the homozygous muta@on leading to a deficiency of vWF with absent or profound deficiency in levels of plasma vWF
} Autosomal recessive
} vWF levels are <5% } FVIII is markedly cleared from the plasma with levels below 5-‐10% } FVIII is not as severely depressed as in severe Hemophilia A } Spontaneous bleeding } Severe mucocutaneous bleeding } Soj @ssue/musculoskeletal bleeding
} 1-‐5% of case } Prevalence increases in regions of consanguineous marriages
12
Type 2A vWD
• Muta@ons commonly occur in the A2 region
• Presence of only the smaller vWF mul@mers in plasma à reduced binding to platelets • LOSS platelet-‐dependent funcGon
• Two proposed mechanisms: ▫ Abnormal assembly and secre@on of
large vWF mul@mers ▫ Increased suscep@bility of vWF to
proteolysis in circula@on
• Pa@ents exhibit moderate to severe mucocutaneous bleeding
13
Type 2B • Muta@on in the A1 domain of the vWF
gene • Absence of the high-‐molecular-‐weight
mul@mers – Caused by “gain of func7on” muta@on in
vWF à increased affinity to bind to the gpIb platelet receptor
– Spontaneous binding of vWF to platelets – Large mul@mers are synthesized but
rapidly cleared due to increased binding to platelets
– Thrombocytopenia
• DDAVP contraindicated à would cause increased thrombocytopenia as platelets would be hyper-‐reacGve to the released vWF
14
Type 2M vWD
• Muta@ons in Exon 28 in A1 domain
• Defect leads to decreased or absent binding of vWF to platelet gpIb receptor
• Decreased platelet dependent func@on
• Normal mul@mer profile
• Plasma binding to FVIII is normal
15
FVIII GPIb collagen RGDSGPIIb/IIIacollagen
D1 D2 D‘D3 A1 A2 A3 D4 B1B2 B3 C1 C2N C
Type 2N vWD
• Also referred to as “autosomal hemophilia” or the Normandy variant • Caused by muta@ons in the FVIII binding region of vWF
• Markedly decreased affinity for binding to FVIII – Rapid turnover of the unbound FVIII à reduced levels • Lab findings
1. Decreased FVIII 2. Normal vWF an@gen and ac@vity 3. Normal bleeding Gmes (PFA-‐100) 4. Platelet binding to vWF is normal 5. Similar to “mild” hemophilia
• GeneGc counseling and treatment is different from hemophilia
16
FVIII GPIb collagen RGDSGPIIb/IIIacollagen
D1 D2 D‘D3 A1 A2 A3 D4 B1B2 B3 C1 C2N C
Pseudo-‐von Willebrand Disease – (Platelet type vWD)
} NO gene@c defect of the vWF molecule – vWF molecule is NORMAL
} “Gain in func@on” muta@on in the platelet gpIb receptor } Increased affinity of platelets for vWF } Enhanced clearance of vWF and platelets from circula@on
} Defect is in the platelet à standard approaches to trea@ng vWD are not helpful
} Lab findings 1. Loss of high molecular weight mul@mers 2. Platelet count is low** 3. Platelet aggrega@on with low dose ristoce@n (RIPA)
17
Acquired vWD
• Qualita@ve, structural, or func@onal disorder of vWF not inherited and is associated with an increased risk of bleeding
• Associated with – Autoimmune clearance – lymphoprolifera@ve, MGUS, SLE, hypothyroidism
• Autoan@bodies à increased clearance of vWF from plasma
– Fluid shear stress-‐induced proteolysis – aor@c stenosis, LVAD
18
Aspect Acquired vWD Congenital vWD Personal History Late onset bleeding Early onset bleeding
Family History Nega@ve Posi@ve
AVWS associated disorder
Posi@ve Nega@ve
Laboratory associated disorder
Inhibitor to vWF Gene@c muta@on
Treatment • Remission ajer IVIg • Short lived response
ajer vWF-‐containing product
vWF-‐containing product
Assays for vWD
• Platelet Func@on Screen (BT) • vWF an@gen assay • vWF ac@vity assay • FVIII:C
• Mul@mer Analysis
19
Assays for vWD
• vWF:An@gen – Immunoassay that measures the concentra@on of vWF protein in plasma
• Actual protein responsible for binding to FVIII and gp Ib/IX/V complex – Detects all forms of vWF (func@onal and nonfunc@onal forms) – Cannot discriminate between mul@mer size
20
Patient vWF å
Testing well
Reagent beads coated with anti-vWF
å
åå
å
å
å
å
åå
å
å
Incubate
LIA based tesGng
Instrument reading—changes in optical density secondary to aggregates
Assays for vWD
• vWF:Ac@vity – Ristoce@n cofactor assay (gold standard)
• Measures the ability of vWF (pa@ent) to induce agglu@na@on of normal fixed platelets in the presence of Ristoce@n
• Mix paGent’s plasma + normal donor platelets + ristoceGn à platelet aggluGnaGon reacGon occurs on platelet aggregometer
21
Assays for vWD
• vWF:Ac@vity – Latex par@cle enhanced immunoturbidimetric assay
• Specific anG-‐vWF monoclonal anGbody adsorbed onto latex reagent directed against the platelet binding site of vWF (gp Ib receptor)
• Reacts with vWF in the pa@ent’s plasma • Degree of agglu@na@on is directly propor@onal to ac@vity of vWF in
pa@ent's plasma – Mix paGent’s plasma + latex beads coated with an anG-‐vWF
monoclonal anGbody è aggluGnaGon of parGcles
22
Assays for vWD
• FVIII ▫ Circula@ng level of FVIII ▫ Clot-‐based assay that measures the ability of plasma FVIII to shorten
the clopng @me in FVIII-‐deficient plasma
• Mul@mer Analysis ▫ QualitaGve assay (electrophoresis) to depict the variable
concentraGons of different-‐sized vWF mulGmer
23
Preanaly@cal Variables
– Race • Higher levels seen in African/African-‐Americans
– Age • Levels increase with age
– ABO Blood Groups
24
ABO Type N Mean Mean +/-‐ 2SD
O 456 74.8 35.6 – 157.0
A 240 105.9 48.0 – 233.9
B 196 116.9 56.8 – 241.0
AB 109 123.3 63.8 – 238.2
Preanaly@cal Variables
– Condi@ons with elevated vWF levels • Age • Acute and chronic inflamma@on • Diabetes • Malignancy • Pregnancy or OCT • Stress, exercise • Hyperthyroidism
– Condi@on with reduced vWF levels • Hypothyroidism • Type O blood
25
Treatment of von Willebrand Disease
1. DDAVP (deamino-‐8-‐arginine vasopressin) • Synthe@c analogue of the natural pituitary hormone • Releases intracellular vWF from endothelial cells à raises plasma levels of
vWF • Treatment of choice in Type I
• Variable response in 2A and 2M • Ineffec@ve in Type 3 • Contraindicated in 2B
• Can be given as an inhaler—S@mate
26
Treatment of von Willebrand Disease
2. Humate-‐P – contains FVIII and vWF (large mul@mers) – FDA-‐approved – Alphanate, Koate
3. ε-‐Aminocaproic Acid (EACA) and Tranexamic Acid
– Fibrinolysis inhibitors
4. Cryoprecipitate – Source of fibrinogen, factor VIII and VWF – Only plasma frac@on that consistently contains VWF mul@mers – USE WITH CAUTION!!!
27
LAB
28
Current Instrumentation
Methods of Endpoint Detec@on
• Mechanical
Ø Electromechanical
(Impedance) Method
Ø Magne@c, Steel Ball Method
• Op@cal
– Photo-‐op@cal
– Nephelometric
– Chromogenic
– Immunologic
• Electrochemical
• Automa@on approaches to clot detec@on: – SEE
• Turbidometric • Nephelometric
– FEEL • Mechanical • Viscosity based
Electromechanical Method. • When coagulation process takes
place, the concentration of clotting factors (charges) and inorganic ions will change along the time and the measured impedance or conductance w i l l b e a l s o c h a n g e d correspondingly at the same time.
• During the reaction, one probe moves in and out of the solution at constant intervals. The electrical circuit between the two probes is not maintained as the moving probe rises in and out of the solution.
• When a clot (fibrin) is formed in the solution, the fibrin strands maintain electrical contact between the two probes when the moving probe leaves the solution, which stops the timer.
Magne@c, Steel Ball Method Mechanica l c lot detec@on invo lves monitoring the movement of a steel ball within the test solu@on using a magne@c sensor As clot forma@on occurs, the movement of the ball changes, which is detected by the sensor
• The sample is introduced to a cuvete that has a small steel ball inside
• The cuvete con@nuously moves when tes@ng begins
• The clot ini@a@ng reagents are added to the sample
• The fibrin strands begin to form and atach to the moving ball
• An electrical circuit is either opened or closed when the ball moves away from the magnet because of the fibrin strands
• Clot @me is recorded.
Photo-‐op@cal Method • Detec@on of clot forma@on measured by a change in OD of
a test sample is the basis of photo-‐op@cal instrumenta@on, which is also known as turbidometric methodology:
• When a light source of a specified wavelength is passed through a test solu@on (plasma), a certain amount of light is detected by a photodetector or photocell located on the other side of the solu@on.
• The amount of light detected is dependent on the color and clarity of the plasma sample and is considered to be the baseline light transmission value.
• When soluble fibrinogen begins to polymerize into a fibrin clot, forma@on of fibrin strands causes light to scater, allowing less light to fall on the photodetector (i.e., the plasma becomes more opaque, decreasing the amount of light detected.
• When the amount of light reaching the photodetector decreases to an exact point from the baseline value as predetermined by the instrument, this change in OD triggers the @mer to stop, indica@ng clot forma@on.
Op@cal Clot Detec@on (Turbidimetry)
• Sample is added to a cuvete • A light source is directed through the
cuvete • Ini@al absorbance of transmited light is
measured • Clot ini@a@ng reagents are added by the
automated instrumenta@on • The plasma becomes more opaque
when clopng is ini@ated, decreasing the light transmited through the cuvete
• The change in transmited light is used to calculate the result
Nephelometric Clot Detec@on Nephelometry uses a light -‐emipng diode at a high wavelength (usually >600 nm) to detect varia@ons in light scater as an@gen-‐an@body complexes are formed. When the light rays encounter insoluble complexes such as fibrin strands, they are scatered in both forward (l80-‐degree) and side (90-‐degree) angles
• Sample is added to a sample cuvete • The op@cally clear cuvete passes in front of light
source • The clot ini@a@ng reagents are added • Light is scatered as the fibrin strands form • The light scaters at different angles and is measured
by detectors • A clot curve is generated by consecu@ve readings
un@l clot comple@on.
36
37
