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Thrombosis andTh b hiliThrombophilia

Kathleen Winner MS,MT(ASCP)SHSr. Clinical Hemostasis SpecialistBeckman Coulter, Inc.

Objectives• Describe thrombotic disease states

• Define the biological basis for hypercoagulability and thrombosis

• Discuss acquired and inherited thrombophilia

• Identify laboratory tests for the diagnosis and treatment of thrombophilia

• Review the 2009 ISTH Guidelines for the identification of Lupus Anticoagulant

Introduction

History

• Throughout history a key medical problem has been the control of blood loss

Present

• Today the greater problem is not the risk of people bleeding to death but rather the risk of “clotting to death”to death

Thrombotic Disease

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The Extent of Thrombotic Disease Annually in the U.S.

• 1.5 million MIs‐ Mortality of 30% (450,000)

• 500 000 CVAs• 500,000 CVAs‐ Mortality of 30% (150,000)

• 2 million DVTs

• 200,000 deaths from PE

CVA

Venous Thromboembolism

• Third most common cardiovascular disease

• Significant morbidity and mortality

• VTE includes:– Deep Venous Thrombosis (DVT)– Pulmonary Embolism (PE)

Deep Vein Thrombosis

• Blood clot of lower leg or thigh

• Approximately 1 per 1,000 pp y ppeople affected by DVT

• Hospitalization for 5 to 7 days

• 50% of patients with DVT are asymptomatic

Deep Vein Thrombosis

Pulmonary Embolism

• Dislodged blood clot entering the pulmonary circulation

• Accounts for 5-10% of all hospital deathshospital deaths

• Estimated 60,000 die of PE each year

• 80% of patients die within the first 2 hours CT Scan of Pulmonary Embolism Basic Coagulation

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Coagulation BasicsThe process of blood clotting and then the subsequent dissolution of the clot is termed hemostasis.

ClotFormation

ClotDissolution

Thrombosis

• If the clotting system is activated

• Or the fibrinolytic system in inhibited

• A hypercoagulable state exists that leads to abnormal clot formation

Losing that balanceThrombogenesis

• Arterial thrombi– platelet aggregates bound together

by fibrin strands (white clots)

• Venous thrombi– Consist mainly of fibrin and RBCs

(red clots)

Arterial thrombus

Virchow’s Triad

Stasis

Thrombosis

Hypercoag-ulability

VascularInjury

ArterialRudolph Virchow

Vascular Injury

• Trauma

• Surgical manipulation

• Prior thrombosis

• Atherosclerosis

VascularInjury

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Stasis• Immobility

– Post-op state, debility, coma

– “Economy Class Syndrome”

• Pressure– catheter, tumor obstruction

• Increased viscosity• Increased viscosity– Polycythemia– Dehydration– EPO Stasis

Blood Hypercoagulability

• Increased procoagulants

• Decrease in inhibitors

I i d fib i l i• Impaired fibrinolysis

Hypercoag-ulability

Blood Hypercoagulability

Thrombophilia

Thrombophilia

• Tendency to develop thrombosis

• Can be acquired or inherited or both

• Manifested as venous thromboembolism (VTE)

• Multi-hit theory

Venous Thrombosis

Acquired Thrombophilia

• Antiphospholipid antibody syndrome

• Pregnancy

• Oral contraceptives

• Malignancy

• Heparin-induced thrombocytopenia with thrombosis

• Hormone replacement therapy

• Surgery

• Trauma

• Advancing Age

• Nephrotic syndrome

• Elevated Factor VIII

• Hyperviscosity

• Myeloproliferative disorders

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Inherited Thrombophilia

• Resistance to activated protein C (Factor V Leiden)

• Antithrombin deficiency

• Protein C, Protein S deficiency

• Prothrombin gene mutation 20210A

• Elevated Factor VIII

• Dysfibrinogenemia

• Hyperhomocysteinemia

Sword of Damocles

Unexplained30-45%

Protein S3-6%

Protein C3-7% Antithrombin

2-5%

Homocysteine12-15%

Genetic Risk Factors in Patients with Venous Thromboembolism

Activated Protein CResistance

30-50%

ProthrombinMutation

10-15%

Laboratory Investigation

Laboratory Investigation of Thrombophilia

• Discovery of antithrombin deficiency in 1965

– First identification of a heritable cause for thrombophilia

• Deficiencies of Protein C and S in the 1980s

• Comprise only 5%-10% of thrombosis patients

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Activated Protein C Resistance

In 1993 Björn Dahlbäck found patients with resistance to activated protein Cactivated protein C– Autosomal dominant trait

– 30-50% of all cases of VTE

– Highly prevalent in the general population

Protein C and S

Factor V Leiden

In 1994 scientists at the University of Leiden identified a single mutation in the gene for factor V

– Point mutation of arginine (R) at position 506 in factor V molecule with glutamine (Q)

– denoted FV:Q506 or Factor V Leiden

Proved to be the most common hereditary cause of thrombophilia

Rembrandt’s birthplace in Leiden

Holland

Coagulation

FVIIIa FVIIIi

Coagulation inhibition

+

APC

PS

Insoluble Fibrin

FVa FVi

Factor V Leiden

F. VaF.Xa F.Va

Leiden mutation

F.Xa

ActivatedProtein C

Normal-protein C inhibits activity of F.Va-F.Xa complex

Protein S

ActivatedProtein C

F.V Leiden mutation prevents protein C binding

Protein S

Why test for APCr ?

• Heterozygotes carry a 3-7 fold increased risk of thrombosis

• Homozygotes carry a 50-100 fold increased risk of thrombosis

F.V Leiden and Risk Factors for DVTCondition Relative Risk Incidence/year

Normal 1.0 0.008

Prothrombin gene mutation

2.8 0.022u a o

Oral contracep. 4 0.03

F.V Leiden heterozygotes 7 0.06

Oral contracep.+F.V Leiden heterozygotes

35 0.30

F.V Leiden homozygotes 80 0.5-1.0

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Laboratory Diagnosis of APCR

• APTT with APC added to CaClCaCl2

• APTT without APC

• Ratio of two APTT assays Ratio less than 2.0 indicates APC

resistance

Copperhead

Limitations of APCR assay

• Heparin or patients OAT

• Interference from inhibitors (lupus)• Interference from inhibitors (lupus)

• Genotype (homozygote vs. heterozygote)

• Confirmation required

Modified APCR Assay (APCR-V)

• Prediulte with Factor V deficient plasma

• Polybrene

• Perform APTT with and without APC

• Calculate ratio

Dilution with Factor V Reagent

• Selectivity for Factor V:Q506 is increased– (sensitivity and specificity close to 100%)

• Normalizes the Vit.K dependent proteins– testing of patients on OAT

• Neutralizes heparin– testing of patients on heparin

– Can easily be performed on coag analyzers

Antithrombin

• Major inhibitor of blood coagulation

• Inhibits thrombin (F IIa),FXa and IXa, XIa and XIIa

• Heparin binds to AT

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Antithrombin Deficiencies

Congenital deficiency• Heterozygotes have AT levels of

40-60%• Homozygotes incompatible with• Homozygotes incompatible with

life• Found in 0.3% of the population

Acquired deficiency• Liver disease• DIC

Blood clot

Clinical Symptoms

• Recurrent venous thromboembolic episodes– DVT and PE

• Symptoms appear between ages 10-25

• Precipitated by trauma, surgery, pregnancy, or infections.

Angiogram of left humeral artery

Laboratory Testing

• Functional assays are recommended by WHO/ISTH– detect quantitative and qualitative deficiencies– Chromogenic– Clottingg

• Measure neutralization of thrombin or F Xa* in the presence of heparin.

*Factor Xa is preferred because it is not affected by heparin cofactor II

Chromogenic Measurement Principle

AT HEP AT HEP

Chromo SubstrateXa

AT HEP XaEXCESS

AT HEP

Xa XaRESIDUAL

Protein C & S

• Naturally occurring anticoagulants

• Vitamin K dependent proteins

• Serve as inhibitors of coagulation– inactivation of factors VIIIa and Va

Inherited deficiency

• Autosomal dominant inheritance

• Neonatal purpura fulminans (h )(homozygote)

• 10-16% of unexplained venous thrombosis before age 50

• Skin necrosis induced by warfarinEmbolus

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Acquired Deficiency

• Warfarin oral anticoagulant therapy

• Liver diseasee d sease

• Consumption of PC and PS-DIC-Surgery-DVT

Disease ClassificationsProtein C

Type I - total reduction of PC antigen and yp gactivity- quantitative disorder

Type II - normal antigen but reduced activity (functionally abnormal molecule) qualitative disorder

Assays for Protein C

• Functional assays - measure the anticoagulant activity of APC

-Clot-based assays-Chromogenic assays

• Antigenic assays - measure concentration

-Not used to screen patients because it leaves Type II undetected

Assays for Protein C

• Chromogenic Assays detect abnormalities of PC activation

• Clot-based Assaysdetect abnormalities of PC activation and defects in binding

Protein S

Protein S functions as a cofactor for APC

O l th f ti fOnly the free native form of Protein S has APC cofactor activity.

H N COOH

PS C4BP

H2N COOH

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PS distribution in plasma

25 µg/mL

15 µg/mL 10 µg/mL

Approximately 60% of PS in plasma is linked to C4BP while the remaining 40% circulates in the free form.

Protein S DeficiencyProtein S deficiency may be hereditary or acquired.

• Acquired deficiency may be observed during:– pregnancy– oral anticoagulant therapyoral anticoagulant therapy– oral contraceptive use– in liver disease– in new-born infants– after surgical interventions– inflammation

Protein S DeficiencyPS deficiency should be classified, according to ISTH SSC recommendations (Munich 1992)

Classification Total PS Ag Free PS Ag PS Activity

Type I Low Low Low

Type II Normal Normal Low

Type III Normal Low Low

Protein S Assays

The laboratory diagnosis of Protein S abnormalities may require both:

• quantitative (immunological)

• qualitative (functional)

Protein SParameter HemosIL Product Main FeaturesProtein S HemosIL ProS - Easy to use clotting assay

- Only two components: PT and PS Def Plasma

- No PC preactivation required

- APC co-lyophilized with PT reagent

- No interference from elevated FVIII

Very moderate interference from- Very moderate interference from Lupus and FV Leiden

HemosIL Free Protein S Antigen - Easy to use Latex assay

- C4-binding protein to capture Free Protein S

- sensitive to Type I and III PS deficiencies (Type II is very rare)

- Together with HemosIL ProS offer a complete panel for Protein S testing

Prothrombin Gene Mutation

• Mutation on the prothrombin gene (G20210A)

• Increased prothrombin activity

• Coinheritance with Factor V Leiden

• Second most common genetic risk factor for VTE

• DNA analysis is required

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Elevated Factor VIII

• Fivefold increased risk of DVT

• Increases the risk of recurrent VTE

• Heritability has been demonstrated

• Acute phase reactant

• High levels are best measured by a chromogenic method

Blood clot

Hyperhomocysteinemia

• Premature vascular disease– Thromboembolic disease at an early age

• Arterial and venous thromboembolismArterial and venous thromboembolism– M.I.

– DVT

• Graded risk factor– 40% for every 5 mol/L

M.I.

Hyperhomocysteinemia

• Congenital – Deficiency of metabolic enzymes

(MTHFR)

– 11% of the population are p phomozygous

• Acquired– Poor dietary intake of folic acid and

B12

• Effectively treated by dietary supplementation

Homocysteine Lupus Anticoagulant

Antiphospholipid Syndrome

• Also known as “lupus anticoagulant”

• Acquired biologic abnormality• Acquired biologic abnormality

• Anticoagulant in vitro (abnormal APTT)

• Excessive clotting in vivo

Elements of theImmune System

How does the Lupus Anticoagulant work?• Etiology of LA is

unclear• Not a congenital

diseaseT i t• Transient

• LA is one of the most commonly acquired, pre-disposing causes of thrombosis– LA positive 8.5-10%

of DVT (30,000 DVT/yr) SLE Workshop by Dr. Michael D. Lockshin, MD, Professor of Medicine,

Weill Medical College of Cornell University, Attending Rheumatologist, Hospital for Special Surgery (January 2000)

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Clinical Presentation

• Thrombotic events in 30% of patients– Two thirds venousTwo thirds venous

– One third arterial

• Young persons with MI, or CVA

• Multiple fetal losses.

ISTH Subcommittee Criteria - 1995

Update of the Guidelines for Lupus Anticoagulant Detection

Source: Official Communication of the Scientific and Standardization Committee on Lupus Anticoagulant / Phospholipid-dependent antibodies

Journal of Thrombosis and Haemostasis, 2009; 7: 1737-1740

Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG,

“A test is as good as a sample”

is never truer than in the

performance of an LA test.

SSC Guidelines for LA – 2009Blood Collection

• Blood collection before the start of any anticoagulant drug or a sufficient period after its discontinuation

Fresh venous blood in 0 109 M sodium

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• Fresh venous blood in 0.109 M sodium citrate, 9:1

• Platelet poor plasma: double centrifugation– Initial centrifugation: 2000g, 15 minutes, room temperature

– Transfer plasma to a non-activating plastic centrifuge tube using a plastic pipette (DO NOT INCLUDE PLATELETS!)

– Second centrifugation: >2500g, 10 minutes

NOTE: Plasma filtration is not recommended

SSC Guidelines for LA – 2009Sample handling

• Freeze as quickly as possible following venipuncture to prevent loss of coagulation factors

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• Freeze the plasma in a freezer at - 70 °C or below

• Before analysis frozen plasma must be thawed by total immersion of sample content in a water bath at 37°C for 5 minutes to avoid formation of cryoprecipitate and then mixed thoroughly before testing

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SSC Guidelines for LA – 2009Choice of the test

• Recommended:

Use 2 different tests with different assay principles.principles.

• The use of more than 2 different assays increases the risks of false positive and is discouraged

SSC Guidelines for LA – 2009Choice of the test (cont.)

• Use 2 different tests with different assay principles.

Recommended:1) dRVVT should be the first test considered)

2) APTT with Silica as an activator

low concentration of PL

• Confirmation with high phospholipid test Bilayer or hexagonal (II) phase PL Note: Freeze/thawed platelets are not recommended as a PL source

LA should be considered as positive if one LA should be considered as positive if one of the two tests gives a positive resultof the two tests gives a positive result

SSC Guidelines for LA – 2009Choice of the test (cont)• Test Not Recommended

– Kaolin based assays• problems with coagulometers and poorer reproducibility compared with other

tests available

– Ellagic Acid based assaysEllagic Acid based assays• Insensitivity to LA

– Diluted Prothrombin Time• variability in thomboplastin reagents

– Ecarin or Textarin based assays• no standardized commercial assays available Treatment

Anticoagulants

• Heparin– Unfractionated

– Low Molecular Weight

• Oral Anticoagulants Warfarin

• Direct thrombin inhibitors Hirudin

Argatroban Medicinal leech, Hirudo medicinalis

Prevention

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Know Your Risk

• Identify inherited risk factors– Family history

– Previous episodes

– Laboratory screening

• Remove or diminish acquired factors– Contraceptive choice

– Lifestyle changes

Prevention of ECS

• Book exit,bulkhead or aisle seat

• Get up once per hour

• Remain hydratede a yd a ed

• Exercise calf muscles while seated

• Avoid constrictive clothing (“knee-highs”)

• Consult physician if at risk– Anticoagulant

– Compression hose

Prevention

Prevention

Conclusion

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Conclusion

Future efforts should be aimed at developing cost-effective screening tests able to assess globally the increased tendency towardglobally the increased tendency toward thrombosis in patients with thrombophilia so that preventive measures may be taken.

Conclusion

If patients who are at risk for developing pathologic blood clots can be identified early, appropriate measures may be undertaken to decrease the incidence and severity of thrombotic events.incidence and severity of thrombotic events.

But ……

Conclusion

A little red wine

A piece of chocolate

Doesn’t hurt either!