Thrombophilia Failure of the Inherent Anticoagulation Defense System Mervyn A. Sahud, M.D., A.B.I.M.-Hem. Medical Director, Coagulation, Quest Diagnostics

Thrombophilia Failure of the Inherent Anticoagulation

Defense System

Mervyn A. Sahud, M.D., A.B.I.M.-Hem.Medical Director, Coagulation, Quest Diagnostics Nichols Institute

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Outline• Thrombophilia (impact on DVT and PE)

• Risk factors

• Coagulation cascade

• Thrombophilia testing– Antithrombin– Protein C– Protein S– Activated protein C resistance (APCR)– Prothrombin (factor II) mutation– Lupus anticoagulant– Factor VIII excess

• Case studies• Recommended work-ups

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• Thrombophilia (impact on DVT and PE)

• Risk factors




Outline

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Deep Vein Thrombosis — A Leg Up on Blood Clots

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Valve Cusp Thrombus (Autopsy Specimen)

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DVT only

DVT & PE

Die from PE

• 2 million Americans affected with DVT each year

• 600,000 of these develop pulmonary embolism (PE)

• 60,000 of those with DVT and PE die each year

Thrombophilia

DVT, deep vein thrombosis.Rosendall FR. Lancet. 1999:353:1167-1173.

Anderson FA Jr, et al. Arch Intern Med. 1991;151:933-938.

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• 5% - 8% of population affected by genetic defects leading to thrombosis predisposition

• 25% suffer chronic swelling, skin ulceration, and impaired mobility secondary to “venous hypertension”

Thrombophilia

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DVT…What’s New?• 50% (soon to be 100%) of “unprovoked” DVT

cases associated with hereditary thrombophilia

• 60% of DVT cases in pregnant women associated with factor V Leiden mutation (A1698G)

• DVT often associated with multiple genetic and acquired risk factors

• DVT responds to prolonged anticoagulant therapy (eg, low molecular weight heparin)

Bucciarelli P, et al. Arterioscler Thromb Vasc Biol. 1999;19:1026-1033.

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DVT…What’s New?• New anticoagulants available for non-responsive

patients and those who experience side-effects from standard anticoagulants

• Family members request DVT screening prior to being placed in a “high-risk” situation

• Elevated D-dimer level often indicates thrombosis

• Normal D-dimer level has strong NPV

NPV, negative predictive value..

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What Else is New?• Pulmonary embolism

– 4 million patients present to U.S. emergency departments with shortness of breath each year

– Shortness of breath = heart failure or pulmonary embolism (PE)

– 60% of patients who die in the hospital have PE

– PE diagnosis missed in 70% of hospital cases

– 10% of patients with acute PE die within first 60 minutes

Clagett GP. Chest. 1998;114(Suppl 5):531S-560S.

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Thrombophilia Is Often Multigenic

Multiple risk factors raises the risk of thrombosis

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• Thrombophilia (Impact on DVT and PE)

• Risk factors




Outline

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Clinical DVT Risk Factors

• Age >60 y

• Extensive surgery*

• Marked immobility, pre- or postoperative

• Major orthopedic surgery (eg, hip, knee)

• Fracture of pelvis, femur, or tibia

*Risk of postoperative thrombosis increases as patient’s age and surgery duration increases; risk also increases with presence of varicose veins and obesity.

• Surgery for malignant disease

• Postoperative sepsis

• Major medical illness– Heart failure

– Inflammatory bowel disease

– Sepsis

– Myocardial infarction

– Stroke

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Frequency of Biological Risk Factors

Factor VIII excess (30%)

APCR, Factor V mutation (20%)

Prothrombin (factor II) mutation (15%)

Hyperhomocysteinemia (10%)

Protein C (10%)

Protein S (10%)

Unknown (5%)Antithrombin (4%)

LA (3%) Other (<1%)

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Synergistic Effects of Thrombotic Risk Factors

Factor V Leiden Factor IIa Thrombotic Risk

Normal Normal 1.0

Abnormal Normal 2.8

Normal Abnormal 2.4

Abnormal Abnormal 22.8aProthrombin 20210GA mutation.

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Factor V Leiden MTHFR Thrombotic Risk

Normal Normal 1.0

Abnormal Normal 2.8

Normal Abnormal 1.8

Abnormal Abnormal 16.5

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Factor V Leiden

Factor IIa MTHFR Thrombotic Risk

Normal Normal Normal 1.0

Abnormal Normal Normal 2.8

Normal Normal Abnormal 1.8

Normal Abnormal Normal 2.4

Abnormal Abnormal Abnormal 56.5aProthrombin 20210GA mutation.

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Risk of Recurrent Venous Thromboembolism

Risk of recurrent venous thromboembolism (VTE) in individuals with a thrombophilic defect relative to risk in those with a first episode of VTE and no thrombophilic defect.

Weitz J, et al. Hematology Am Soc Hematol Edu Program. 2004;424-438.

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Age of Thrombosis Onset in Patients with Common Thrombotic Risk Factors

Risk FactorAge of Thrombosis Onset

Homozygote Heterozygote

Protein C deficiency Birth 26 years

Protein S deficiency Birth 31 years

APCR* 28 years 63 years

Antithrombin Embryonic death 33 yearsAPCR, activated protein C resistance.*May be responsible for venous thrombosis in individuals older than 55-60 years.

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Relative Frequency of Various Thrombosis Types

Type of Thrombosis

Patient with APCR Patient with Protein C or S Deficiency

DVT and/or PE 82% 76%

Abdominal 6% 11%

Stroke/MI 4% 6%

Other 8% 7%

Total 100% 100%

Salomon O, et al. Arterioscler Thromb Vasc Biol. 1999;19:511-518.

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• Risk factors




Outline

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Coagulation Cascade

Crowther MA, Kelton JG. Ann Intern Med. 2003;138:128-134.

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Coagulation Cascade

Factor VIIa binds to tissue factor (TF) at sites of vascular injury, causing a cascade that ultimately leads to factor IIa (thrombin) generation. Thrombin activates platelets; converts fibrinogen to fibrin; leads to generation of additional thrombin through an autocatalytic loop; converts factor XIII to factor XIIIa, which stabilizes the thrombus; inhibits fibrinolysis; and acts as an anticoagulant by activating protein C. The principal inhibitors of coagulation are activated protein C (which, in concert with protein S, inactivates factors Va and VIIIa), antithrombin (which forms an inactive covalent complex with thrombin and factors Xa, IXa, and XIa), and tissue factor pathway inhibitor (which bonds with and inactivates the tissue factor-factor VIIa complex).

Crowther MA, Kelton JG. Ann Intern Med. 2003;138:128-134.

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• Risk factors




Outline

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How to test?

When to test? What to test?

Why test?

Whom to test?

Thrombophilia Testing

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Antithrombin Deficiency

Genetic deficiency

• Rare

– <1% of general population

– ~1% - 8% of those with VTE

• Heterozygotes

– VTE by ~30 years of age

– 5- to 50-fold increased risk of VTE

– High risk of VTE during pregnancy and postpartum

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Antithrombin Deficiency

Acquired deficiency• Causes

– Liver disease– Malnutrition – Inflammatory bowel disease – Prematurity– DIC– Transfusion reactions – Chemotherapy – Heparin therapy

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AT

IIa

AT

Inactive Complexes

IIa

IIa

AT

Xa

AT

IXa

AT

XIa

AT

XIIa

AT

Heparin

Role of antithrombin

• Direct inhibitor of IIa

• Inhibits other factors leading to indirect inhibition of IIa formation

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Therapeutic Monitoring with Antithrombin III

Normal antithrombin III

Antithrombin III deficient

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Factors Affecting Test Results

Antithrombin Protein C and S

Heparin Long-term therapy results

False in results from clot-based assays (>1.0 IU/mL)

OAC or vitamin K deficiency

No effect Vitamin K dependent proteins; OAC will levels

FVIII No effect High levels will falsely results from clot-based assays

Circulating inhibitors

No effect May falsely results from clot-based assays

OAC, oral anticoagulant.

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Factors Affecting Test Results

Antithrombin Protein C Protein S

Pregnancy No effect

Oral contraceptives or no change No effect

Hormone replacement therapy

or no change No effect

Liver disease, DIC, active thrombosis, recent surgery

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Protein C Deficiency – Genetic Mechanism

Autosomal inheritance

• Complete penetrance (dominant mutation)– Heterozygotes– Primary symptom: venous thrombosis

• Mild or no penetrance (recessive mutation)– Homozygotes: symptomatic (purpura fulminans)– Heterozygotes: asymptomatic

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Protein C Deficiency• Genetic deficiency

– Rare– Heterozygotes

• <1% of population

• ~1-11% of patients with VTE

• 30% will have an event during lifetime

• ~50% experience VTE by age 40

• Acquired deficiencies– Warfarin, vitamin K deficiency, liver disease,

DIC, renal insufficiency

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Which Protein C Functional Assay?

Chromogenic• Pro

– Fewer interfering substances

• Con

– Measures cleavage of synthetic substrate

– Rare mutations not detected by this method

Clotting

• Pro

– Measures cleavage of natural substrates (factor Va or VIIIa)

• Con

– Many interfering substancesHigh factor VIII levels

Lupus anticoagulants

Heparin >1.0 IU

Direct thrombin inhibitors

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Functional Protein C Assay

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120 140 160

Chromogenic Assay

Clot

Ass

ay2 patients missed by clot assay

8 patients missed by chromogenic assay

36Normal adult reference range: 70%

Protein C Testing Algorithm

No further testing

Protein C Clot Assay

Decreased(<70%)

Borderline(70% - 75%)

Normal or

Protein CAntigen

Repeat ClotAssay

Decreased(<70%)

Increased(70%)

Borderline(70% - 75%)

Chromogenic Assay

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Therapeutic Monitoring with Protein C

Normal protein C

Protein C deficient

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Protein S DeficiencyGenetic deficiency

• Homozygous (rare) – Purpura fulminans

• Heterozygous – < 1% of general population – ~1% to 3% of VTE population– ~50% experience first VTE by 45 years of age

Acquired deficiency• Pregnancy and oral contraceptive/HRT use• Inflammation and acute thrombosis

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Protein CfPS

PS

TM

TM

TM

IIa

PC

APC

Va or VIIIa

VIIIai or Vai

Free PS

C4b-B

P

IIa

fPS

Inactive Cofactors

Role ofprotein C & S

• Inhibition of IIa formation

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Therapeutic Monitoring with Protein S

Normal protein S

Protein S deficient

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APC Resistance in Patient with Thrombosis

APC, when added to plasma from a middle-aged man with recurrent thrombosis, did not result in a prolongation of the clotting time (APC resistance). In contrast, when added to normal plasma (control), APC prolongs the clotting time in a dose-dependent manner.

Dahlback B. J Thromb Haemost. 2003;1:3-9.

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APC Resistance – Genetic Mechanism

• Molecular cause Mutation of APC cleavage site within Factor Va

• Mutation site Amino acid #506 (arginine) mutates to a glutamine

Amino acid: Arg506→ GinNucleotide: CGA→ CAA

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APC Resistance – Genetic Mechanism

• Molecular function– APC cleaves bond between arginine and

glycine

– If Arg506 residue is mutated

Bond not cleaved

Factor Va not inactivated by APC

Continued generation of thrombin and clot formation

Thrombosis

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Acquired APC Resistance

Associated with:

• Increased plasma levels of FVIIIa

• Presence of antiphospholipid antibodies

• 3rd generation oral contraceptives, relative to 2nd generation oral contraceptives

• Pregnancy

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Incidence of APC Resistance in “Normals”

Ethnicity Percent (%)

Caucasian 4

African American 0.8

Asian American 0.6

Hispanic/Latino 2.3

Native American 0.2

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Prothrombin (Factor II) 20210GA Mutation

A, adenine; G, guanine. Zoller B, Svensson PJ, He X, et al. J Clin Invest. 1994;94:2521-2524.

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Lupus Anticoagulant Screening

• Screening tests– LA sensitive aPTT reagent– Kaolin clotting time (KCT)– Dilute russell viper venom time (dRVVT)– Dilute prothrombin time (dPT)

• Minimum of 2 screening tests

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Factor VIII Excess

O’Donnell J, et al. Thromb Haemost. 1997;77:825-828.

Factor VIII Level Relative Risk

>150% (>1.5 IU/mL) 3.0

>200% (>2.0 IU/mL) 5.5

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Factor VIII Excess vs Other Thrombophilia Markers

AbnormalityFrequency (%)

No Historya With Historya

APCR 14-20 20-50

Prothrombin mutation 4-8 10-20

Hyperhomocysteinemia 5-10 NA

Protein C deficiency

Protein S deficiency

Antithrombin deficiency

APCR/factor VIII mutation

1-4

1-4

1-3

25

3-12

3-12

1-6

NA

O’Donnell J, et al. Thromb Haemost. 1997;77:825-828.

aHistory of thromobophilia.

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• Risk factors




Outline

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Case Study #1• 61 year-old white male with multiple myeloma

(in remission)

• Receiving no treatment

• Flies to Singapore for vacation

• Experiences discomfort in right leg, no swelling, no rubor

• 2 days later, sudden dyspnea, left pleuritic chest pain

• Hospitalized with DVT/PE

• Returns home 2 weeks later on coumadin

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Case Study #1: Laboratory WorkupTest ResultHemoglobin 12.4 g/dLPlatelet count 177,000/LWBC 3,100/LINR 2.2D-dimer <0.79 g/mL FEUAPCR 1.3 ratioAntithrombin 87%Protein C <41%Protein S 22%

Factor V Leiden HomozygousFollow-up duplex ultrasound Residual thrombus in iliac

system and IVC

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Case Study #2

• 37 y/o, G1P1 W/F attempting to get pregnant for the 2nd time

• 1st childbirth at age 21 y without incident

• Family history of protein S deficiency– Mother on long-term coumadin– Sister had DVT/PE while on birth control

• Lab results at 12 weeks– Protein S 47% (normal, 60-120%)– Protein C 141% (normal, 60-130%)– Antithrombin III 78% (normal, 60-120%)

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Case Study #2 (cont’d)

• 2 weeks later, concern raised about need for prophylactic anticoagulant

• Lab results– C4 binding protein 212 mg/L (normal, 75-140)– Total protein S 81%– Free protein S 53%– Functional protein S43%

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• Physician discusses drug therapy– Coumadin – Unfractionated heparin – Low molecular weight heparin– Arixtra (fondaparinux)

• Decision made to use graded elastic stockings and aspirin

• Lab results at 20 weeks– Protein S 33%– Protein C 162%

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• Lab results at 36 weeks– Protein S 21%

• Enoxaparin initiated: 40 mg s.c. daily

• Delivery of baby uneventful

• Lab results 4 weeks postpartum– Protein S 31%

• Patient told she probably has hereditary protein S deficiency.

• Restarts enoxaparin for 4 weeks, daily 40 ng s.c.

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Months postpartum Protein S (%)

3 74

6 81

12 80

Protein S deficiency unlikely

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Case Study #3• 39 year-old, physically active, white male bicyclist

develops rapidly progressive drawing pain in right calf after 40-mile event

• History of DVT in left lower extremity after laparoscopic knee surgery at age 21

• Mother on coumadin

• Father died mysteriously of unknown causes at age 51 years

• Sister has had recurrent miscarriages

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Test Result

PTa 10.4 sec. (normal, 10 – 12)

aPTTa 33.0 sec. (normal, <36 sec.)

Factor V Leiden R506Q heterozygous

Prothrombin gene G20210A heterozygous

MTHFR Heterozygous

Lupus anticoagulant screen Negative

Protein Cb 31% (normal, 62 – 124)

Protein Sb 19% (normal, 58 – 112)

Antithrombin IIIb 77% (normal, 62 – 114)

MRI, vena cava Thrombus to vena cava; duplicate cava; old thrombosis noted

aTested prior to initiation of anticoagulant therapy.bTested after initiation of coumadin therapy; INR 3.1.

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Case Study #3 Conclusion

Long-term anticoagulation indicated

Schulman S, et al. N Engl J Med. 1997;336:393-398.

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Case Study #4• Active 49 year-old white male experienced “charley horse” of right leg for >6 days

• Pain, swelling, and redness progressed to lower thigh area

• 16-year history of smoking

• Episode of superficial phlebitis following IV insertion during routine colonoscopy

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• Thrombophilic workup negative for– Factor V G1691A– Prothrombin G20210A– Lupus anticoagulant– Protein C and S– Antithrombin

• Ultrasound duplex assessment demonstrated DVT involving both lower extremities

• Patient hospitalized for IV heparin therapy followed by coumadin therapy

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Case Study #4 (cont’d) Lab Results at Admission

Test Result

Platelet count 515,000/L (normal, 147,000 – 390,000)

Prothrombin time 9.9 secs (normal, 10.3 – 12.7)

aPTT 20.7 secs (normal, 23.1 – 34.2)

Fibrinogen 584 mg/dL (normal, 200 – 400)

D-dimer 4000-8000 ng/mL (normal, <250)

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Test Result

D-dimer 500 – 1000 ng/mL (normal, <250)

Thrombin-antithrombin 11.3 g/L (normal, <4.0)

Soluble tissue factor (CD142) 1114 pg/mL (normal, <250)

Prothrombin fragment 1.2 7.6 nM/L (normal, 0.32 – 2.45)


Lab Results 30 d Post Discontinuation of Coumadin Therapy

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Case Study #4 Conclusions1. Finding of unprovoked DVT and negative

thrombophilic workup requires closer look for possible malignancy even without referable symptoms

2. Development of superficial thrombophlebitis after brief IV insertion and subsequent development of unprovoked bilateral DVT is significant

3. High level of fibrinogen, elevated platelet counts, and shortened prothrombin time and aPTT are suggestive of hypercoagulability

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Case Study #4 Conclusions4. Elevated thrombin generation markers 14

days post coumadin therapy are turning point in this patient’s workup & eventual diagnosis

5. High level of thrombin generation markers and presence of soluble tissue factor suggest antithrombin or Xa inhibitors (eg, low molecular weight heparins) would be more appropriate therapy

6. CT scan of chest reveals 3 cm mass, and bronchoscopic washing demonstrated malignant cells, ie, bronchogenic carcinoma

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Case Study #5

• 19 year-old ballet dancer strains left calf and ankle

• Recently recovered from infectious mononucleosis

• Negative ultrasound duplex exam

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Test Result

D-dimer 497 µg/mL (normal, <0.79)

Fibrin monomer +/-

PT 11.4 sec (normal, 12)

aPTT 31.7 sec (normal, <34)

HAMA Negative

Rheumatoid factor Negative

Fibrinogen 232 mg %

ANA Negative

Repeat ultrasound duplex (post 7 d)

No DVT


69Medical Devices Agency 2002 Evaluation Report.

• Patient recovers, but D-dimer still elevated (241 µg/mL)

• False-positive identified

1. Demonstrated high heterophile antibody titer in patient serum

2. Incubated serum w/ heterophile blocking antibody, then repeated D-dimer test

3. Repeat D-dimer test normal


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Heterophile antibodies may contribute to false-positive D-dimer results in various methods

Medical Devices Agency 2002 Evaluation Report.

FEU, fibrinogen equivalent units; cut-off values are either those recommended by the manufacturer or 95% VTE sensitivity.

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• Risk factors




Outline

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Hereditary Thrombophilia Evaluation

Who• Age 18-45 years • Family history of HTD• No other cause of HTD

When• Asymptomatic• Usually not receiving therapy, but may be on stable oral

anticoagulant therapy

How• Work up in stages• Most common factors first

HTD, hereditary thrombophilia evaluation.

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Cost-Effective Hypercoagulation Workup

Below Age 45• APCR• Protein C• Protein S• Antithrombin

If APCR is abnormal or borderline normal, perform factor V Leiden mutation analysis.

If aPTT is abnormal, complete LA workup.

If homocysteine is abnormal, perform MTHFR mutation analysis.

• Prothrombin mutation• Homocysteine• LA (aPTT) and

ACA/APS

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Cost-Effective Hypercoagulation Workup

Over Age 45• APCR• Homocysteine• Prothrombin mutation• LA (aPTT) and ACA/APS

If APCR is abnormal or borderline normal, perform factor V Leiden mutation analysis.

If aPTT is abnormal, complete LA workup.

If homocysteine is abnormal, perform MTHFR mutation analysis.

Thrombophilia Quiz

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Thrombophilia Quiz

Which of the following statement(s) concerningactivated protein C resistance is/are true?

a. Activated protein C resistance is the most commonly inherited thrombophilic condition.

b. Factor V Leiden mutation is reported in 3% to 7% of the Caucasian population.

c. Approximately 20% to 60% of patients with venous thromboembolism have the Factor V Leiden mutation.

d. All of these

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Thrombophilia Quiz

Which of the following statements concerningprothrombin mutation are true?

a. A mutated 20210 allele is a risk factor for venous and arterial thrombosis.

b. The increased thrombotic risk in patients with a mutated 20210 allele appears to be caused by excessive activation of the coagulation pathway.

c. In patients with a history of venous thrombosis, there is a 9- to 11-fold increase in the presence of the mutant 20210 allele.

d. All of these

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Thrombophilia Quiz

All of the following statements concerningantithrombin are true except:

a. Patients with homozygous antithrombin deficiency usually present in the neonatal period with purpura fulminans.

b. Antithrombin is a major regulator of coagulation and inhibits activated Factor II, IX, X, XI, and XII.

c. Thrombosis is recurrent in approximately 60% of antithrombin deficient patients.

d. There are multiple amino acid substitutions responsible for congenital deficiencies of antithrombin.

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Thrombophilia Quiz

Of patients who experience venous thrombosis,deficiencies of protein C, protein S, andantithrombin are collectively present in:

a. Approximately 15% of patients

b. Approximately 30% of patients

c. Approximately 50% of patients

d. Approximately 70% of patients

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Thrombophilia Quiz

Patients with deficiency of protein C, protein S,or antithrombin usually present with:

a. Arterial thrombosis

b. Venous thrombosis

c. Recurrent arterial thromboembolic disease

d. All of these

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Thrombophilia Quiz

Which of the following statement(s) concerningmethylene tetrahydrofolate reductase (MTHFR)is/are true?

a. MTHFR and cystathionine β synthase are the primary enzymes responsible for methionine metabolism.

b. At least 7 mutations of MTHFR have been reported to increase plasma homocysteine levels.

c. It is estimated that up to ½ of the North American population is heterozygous for a MTHFR mutation.

d. All of these

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Thank You

Documents

Thrombophilia Failure of the Inherent Anticoagulation Defense System Mervyn A. Sahud, M.D., A.B.I.M.-Hem. Medical Director, Coagulation, Quest Diagnostics