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Introduction to Coagulation Testing
Laura Worfolk, Ph.D.
Scientific Director, Hematology
Quest Diagnostics Nichols Institute, Chantilly, VA
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Hemostasis• Intricate system maintaining blood in fluid state
– Reacts to vascular injury to stop blood loss and seal vessel wall
• Involves platelets, clotting factors, endothelium, and inhibitory/control mechanisms– Highly developed system of checks and balances
Bleeding Thrombosis
Normal HemostasisAbsence of overt bleeding/thrombosis
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Interested Specialties
Bleeding & Thrombosis
OB/GYNFetal loss, Infertility,
Menorrhagia
NeurologyStroke
NephrologyAV Graft Occlusion
Vascular SurgeryGraft Occlusion
PAD
AnesthesiologyAnticoagulant Management
HIT
HematologyHemophilia
Thrombophilia
Primary CarePractice patterns vary
CardiologyPremature CAD
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Hemostasis Statistics
• #1 cause of death is CVD (includes heart attack & stroke)*
• ~1-2% of population w/ von Willebrand’s disease†
• ~18,000 Americans w/ hemophilia†
• ~600,000/year w/ venous thromboembolism‡
– ~½ with long-term health consequences; ~60,000 fatalities†
– ~5-8% of population w/ thrombophilia†
*WHO.†CDC
‡www.dvt.org..
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Primary Hemostasis
• Platelet role:
– Adhesion (via vWF), post injury to vessel wall
– Activation: shape changed, contents released
– Aggregation, ie, “plug formation”
– Formation of surface for coagulation reactions - “fibrin glue”
vWF, von Willebrand factor.
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Coagulation Cascade
XII XIIa
XIaXI
IX IXaVIIIa
X Xa
Va
HMWK/Prekallikrein
Prothrombin ThrombinXIII
XIIIaFibrinogen
Fibrin(soluble)
Fibrin (insoluble)
TF/VIIa
VII
TF
Injury
V
VIII
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Cascade Simplified
Activation/InjuryActivation/Injury
Intrinsic Pathway(XIIa, XIa, IXa, VIIIa)Intrinsic Pathway(XIIa, XIa, IXa, VIIIa)
Extrinsic Pathway(TF, VIIa)
Extrinsic Pathway(TF, VIIa)
Common Pathway(Xa, Va, IIa, Fibrinogen)Common Pathway(Xa, Va, IIa, Fibrinogen)
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Thrombin Regulation
• Activity and formation tightly controlled
– Antithrombin III• Inactivation of IIa and other enzymes involved in its
formation
– Protein C and protein S pathway• Inactivation of cofactors Va and VIIIa
– Tissue factor pathway inhibitor• Turns off extrinsic pathway (TF, VIIa)
Defects in regulatory mechanisms: thrombosis
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Fibrinolytic Pathway
Clot lysis vital in prevention of vessel occlusion
Plasminogen Plasmin
uPA, tPA
Fibrin Clot
Fibrin(ogen) Degradation Products
PAI-1
Alpha-2 AP
Defects: bleeding or thrombosis
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Hemostasis Balance
CoagulationCoagulation FibrinolysisFibrinolysis
Thrombin Generation(ie, Factors II – XII, cells)
Thrombin Generation(ie, Factors II – XII, cells)
Thrombin Regulation
(ie, PC/PS, AT, TFPI, cells)
Thrombin Regulation
(ie, PC/PS, AT, TFPI, cells)
Plasmin Regulation
(ie, PAI-1, cells)
Plasmin Regulation
(ie, PAI-1, cells)
HealingHealing
Plasmin Generation(ie, tPA, uPA, cells)
Plasmin Generation(ie, tPA, uPA, cells)
Cellular contribution: platelets, endothelium, monocytes
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Alteration of Balance
• Factor deficiencies• Acquired inhibitors• Anticoagulant therapy• Consumption (DIC)• Dysfibrinogenemia• Platelet defects• von Willebrand’s disease
BleedingThrombosis
Laboratory testing indicated if
DIC, Disseminated intravascular coagulation
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Alteration of Balance
Laboratory testing indicated if
Bleeding
Thrombosis
• Inhibitor deficiencies
• Acquired inhibitors (eg, lupus anticoagulant)
• DIC
• Heparin induced thrombocytopenia
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Case Study #1
• 21 y/o female with vague family history of bleeding disorder; evaluated prior to taking scuba diving lessons
• Has nose bleeds following aspirin ingestion
• Differential diagnosis?– Role of laboratory testing???
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Case #1: Lab Testing
• Screening assays
– aPTT: assesses intrinsic & common pathways
– PT: assesses extrinsic & common pathways
– Fibrinogen: hypo- or dysfibrinogenemia?
– CBC: platelet count
• von Willebrand’s disease (vWD) evaluation
– Multiple tests required to classify vWD type
• Antigenic and functional assays
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Case #1: Test ResultsTest Result
(Ref. Range)Comment
aPTT 33.7 sec(25.3 – 35.8)
Detects intrinsic/common pathway factor deficiency
Platelet count 231 K/L(130 – 400)
Rule out thrombocytopenia
Factor VIII activity*
85%(50 – 150)
Rule out FVIII deficiency
vWF antigen* 40%(50 – 150)
Consistent with vWD
*Acute phase proteins.
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Case #1: Test Results
Test Result Comment
ABO blood type AB+ Type O: levels of vWF Ag
vWF Functional Assays
Test Result (Ref Range)
Comment
Ristocetin cofactor activity
22%(50 – 150)
If abnormal activity:antigen ratio, suspect qualitative (ie, Type II) defect
Platelet aggregation
No aggregation Indicates abnormal vWF function
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Case #1: Multimer Analysis
Shown is representative gelof normal and type 1 and 2A vWF deficiencies
Patient results demonstrated absence of high and intermediate molecular weight multimers consistent with type 2A vWD
Normal
Type 2A
Type 1
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Case #1: Summary
Probable diagnosis
von Willebrand’s disease type 2A (bleeding disorder)
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Case Study #2
• 38 y/o Caucasian man admitted for evaluation of portal hypertension; history of recurrent thrombosis (>10 years)
PT, aPTT, fibrinogen: normal
• Positive family history; father and sister with venous thrombotic episodes, but no laboratory investigation
• Differential diagnosis??
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Inherited Thrombophilia Risk Factors
Condition% in
Healthy% in VTE
RR (%) of Thrombosis
APC resistance/FV Leiden mutation
5 21 3 – 7
AT deficiency 0.02–0.17 1 15 – 40
Protein C deficiency 0.3 3 5 – 12
Protein S deficiency 0.7 2 4 - 10
Prothrombin (FII) 20210GA mutation
2 6 2 – 3
Hyperhomocysteinemia 5–10 10 – 25 3 - 4
VTE, venous thromboembolism; RR, relative risk; APC, activated protein C; AT, antithrombin.
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Case #2: Lab Testing
Test Result (Ref. Range) Comment
Protein C activity 80% (70-180%) Activity assays detect qualitative or quantitative deficienciesProtein S activity 95% (70-150%)
AT III activity 110% (80-120%)
APC Resistance 1.1 (< 2.0) Positive; suggestive of FV Leiden mutation; genetic testing for confirmation
Prothrombin gene mutation
Not detected
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Case #2: Summary
Probable diagnosis
Thrombosis caused by APC resistance/factor V Leiden mutation
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Value of Thrombophilia Testing
• Testing does not affect management of acute events
• Test results may influence decisions
– How long & how intensively to treat• Prevention of recurrence
– Prophylaxis during high-risk procedures
– Need to evaluate family members
– Estimate future risk (ie, risk associated with HRT)
HRT, hormone replacement therapy.
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Case Study #3
• 40 y/o woman with iron deficiency anemia due to menorrhagia; hysterectomy delayed due to prolonged screening test
aPTT: 47.8 sec (elevated)
PT: 13.0 sec (normal)
Fibrinogen: 300 mg/dL (normal)
• No history of bleeding or bruising; no family history
• Differential diagnosis??
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Lupus Anticoagulants
• Antiphospholipid antibodies (APA) are directed against proteins bound to phospholipid membrane surfaces
• Lupus anticoagulants (LA) are a type of APA– Associated with thrombosis & recurrent fetal
demise– Characterized by prolongation of phospholipid
dependent clotting assays (ie, aPTT)
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ISTH Criteria for Lupus Anticoagulants
1. Prolongation of a phospholipid dependent clotting assay (ie, aPTT)
2. Evidence of inhibition in mixing studies
3. Evidence that inhibition is phospholipid dependent
4. Lack of specific inhibition by any one coagulation factor or other circulating inhibitor (ie, FVIII inhibitors, heparin)
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Case #3: Lab Testing
Test Result Comment
aPTT mixing studies
No correction Differentiate factor deficiency from inhibitor
Lupus Anticoagulant Testing
Test Result Comment
dRVVT screen & confirm
Positive Consistent with presence of lupus anticoagulantHexagonal phase
confirmPositive
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Case #3: Summary
Probable diagnosis: Lupus anticoagulant
LA may be asymptomatic or associated with thrombotic events or recurrent abortion. A bleeding history requires other coagulopathies be excluded. Since LA may be transient, international consensus guidelines suggest waiting at least 12 weeks before retesting to confirm antibody persistence.
J Thromb Haemost. 2006;4:295.
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Role of Laboratory Testing
• Assist in diagnosis of bleeding and thrombotic disorders; for example:
– Screen for von Willebrand’s disease in patients with menorrhagia (ACOG recommendation)
– Test for thrombophilia risk factors in patients with recurrent spontaneous abortion or thrombotic events
• Monitor anticoagulant therapy
– Oral anticoagulants, heparin, thrombin inhibitors
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• Monitor replacement therapy
– Factor levels (ie, FVIII, vWF)
• Pre-op screening
• Risk assessment
Role of Laboratory Testing
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Pre-analytical Considerations
• Proper specimen handling, processing, and storage is critical for accurate and precise results
• General specimen requirements available– www.questdiagnostics.com (click on Test Menu)
– www.nicholsinstitute.com (click on lab information specimen requirements)
– Quest Diagnostics Nichols Institute Directory of Services (contact your local representative)
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• Laboratories performing routine, specialty, and esoteric hemostasis testing
• Consultative services available @ Quest Diagnostics Nichols Institute
• Quest Diagnostics Interpretive Guide: http://www.questdiagnostics.com/hcp/intguide/hcp_ig_main.html
Resources
Mervyn Sahud, MDSan Juan Capistrano, CA949-728-4794
Jeffrey Dlott, MDChantilly, VA703-802-6900, x7259
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Case-Oriented Symposium on Bleeding & Thrombosis• October 11-12, 2007, Renaissance Hotel,
Washington DC; topics:– Pediatric hemostasis issues– Thrombophilia– Platelet disorders– Thrombotic thrombocytopenia purpura– FVIII Inhibitors– Point of Care testing– New technologies & more
For more information on this CME approved symposium, go to: http://www.nicholsinstitute.com/Coagulation/Default.htm.
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References• ACOG committee opinion. von Willebrand’s disease
in gynecologic practice. Int J Gynaecol Obstet. 2002;76:336.
• Brandt JT, et al. Laboratory identification of lupus anticoagulants: Results of the Second International Workshop for Identification of Lupus Anticoagulants. On behalf of the Subcommittee on Lupus Anticoagulants/ Antiphospholipid Antibodies of the ISTH. Thromb Haemost. 1995;74:1597.
• Miyakis et al. International consensus statement on an update of the classification criteria for definite APS. J Thrombo Haemost. 2006;4:295.
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References• Press et al. Clinical utility of FV Leiden testing for the
diagnosis and management of thromboembolic disorders. Arch Pathol Lab Med. 2002;126:1304.
• Sadler et al. Update on the pathophysiology & classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thrombo Haemost. 2006;10:2103.
• Thrombophilia: Laboratory support of risk assessment and diagnosis. Available at: http://www.questdiagnostics.com/hcp/intguide/jsp/showintguidepage.jsp?fn=CF_Thrombophilia/CF_Thrombophilia.htm. Accessed March 21, 2007.
Thank you.