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.