Role of laboratory testing for venous thromboembolism risk assessment

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Role of laboratory testing for venous
thromboembolism risk assessment

• Charles Eby M.D

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Why order a thrombophilia risk factor laboratory
evaluation?

• To provide an explanation for VTE
• To identify risk factors that can be modified
• To predict risk of VTE in asymptomatic kin
• To assess risk for recurrent VTE
• To identify risk factors for pregnancy
  complications

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Risk Factors for Venous Thromboembolism

Vessel Wall Trauma Thrombophilic Conditions in
blood Stasis
Virchows Triad 1856
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venous thromboembolism acquired risk factors

• temporary
• Major trauma
• Orthopedic surgery
• Major surgery
• Immobilized leg
• Acute medical illness
• Central venous catheter
• Pregnancy/OCP/HRT
• Prolonged travel

• persistent
• Cancer and cancer therapy
• Myeloproliferative disorders
• PNH
• Phospholipid antibodies/Lupus Anticoagulant
• Previous VTE

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VTE incidence increases with age
Anderson Arch Int Med 1991, 151 933-938
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Protein C pathway
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TFPI
TAFI
plasmin
thrombin
TAFIa
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VTE congenital risk factors 2006

• coagulation regulation
• partial deficiencies antithrombin, protein C,
  protein S
• coagulation factors
• factor V Leiden , GgtA 20210 prothrombin mutation
• factor XI IX antigens, and VIII activity,
  fibrinogen
• miscellaneous
• dysfibrinogenemia, plasminogen deficiency,diminish
  ed fibrinolysis, hyperhomocysteinemia (MTHFR
  CgtT677)
• Hypothetical
• TFPI, TAFI, protein Z, heparin cofactor II

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the original thrombophilia trio partial
deficiencies of regulators of thrombin generation

• Antithrombin protein C
  protein S
• Inheritance autosomal dominant, incomplete
  penetrance
• Prevalence 16001 -50002 1 2003-6004 1
  800-40005
• VTE
• Prevalence 1-3 1-3 1-3
  
• Relative risk? 100
  10 10
• 1 AT activity 2 AT antigen 3 PC antigen 4 PC
  activity 5 fPS and total PS

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PC, PS, AT Deficiency Classifications

• Protein C
• Type I (85) quant. defect
• activity antigen
• Type II qual. defect actltant

• Antithrombin
• Type I (80) quant. defect activity antigen
• Type II qual. defect actlt ant
• Progressive AT activity
• Abnormal
• RS reactive site
• PE pleotropic
• Normal
• HBS heparin binding site defect minimal
  VTE risk

• Protein S
• Type I (70)quant. defect PSact, fPS, totPS
  reduced
• Type II qual. defect PS act reduced, free and
  total antigen normal
• Type III decreased PS act and fPS, normal total
  PS

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Confirmation of Protein C and S heterozygotes is
particularly challenging
Thromb Haemst 70 281-5
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2.6
0.15
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Clot-based activity assays maximize sensitivity
PC,PS, AT deficiencies

• PC activity ex-vivo activation of PC with
  southern copperhead venom PT or aPTT reagent
• PS activity aPC plus PT or aPTT reagent
• AT activity FXa or IIa progressive AT assay
• BUT
• Poor specificity due to multiple pre-analytical
  and analytical variables heparin, warfarin, DTI,
  FVL,LA, liver dz, reactive increases of FVIII,
  fibrinogen, FDP

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Compromise design more robust assays at the
expense of sensitivity

• Chromogenic protein C activity
• Chromogenic heparin cofactor antithrombin
  activity
• Monoclonal antibodies specific for free PS
• or
• Clot-based PS activity using dilute test plasma
  aPC bovine FV

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2001 College of American Pathology coagulation
test proficiency

• Specimen 70 NPP, 30 buffer
• Test range median CV
• Antithrombin 46-121 66 15
• Protein S total 26-61 54
  16
• Protein S free 32-70 49
  20
• Protein S act 20-86 45
  21
• Protein C antigen 30-78 56 19
• Protein C act 34-100 58
  16

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Blockbusters of the 1990s

• Act. Protein C resistance
• gt90 factor V Leiden
• Arg506gln eliminates aPC cleavage site in factor
  Va
• 5 carriers, U.S. whites
• 2.3 carriers Hispanics
• 1.5 carriers U.S. blacks
• 20 spont.VTE are FVL
• Relative risk
• FVL heterozygous 7
• FVL homozygous 80

• Prothrombin G20210A
• 30 higher FII activity
• 2 carriers, U.S. whites
• 6 spont. VTE are PG 20210
• Relative risk
• PG heterozygous 2.8
• PG homozygous ?

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Modified APCr test is a sensitive screening test
for FVL
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Why order a thrombophilia risk factor laboratory
evaluation?

• To provide an explanation for VTE
• To identify risk factors that can be modified
• To predict risk of VTE in asymptomatic kin
• To assess risk for recurrent VTE
• To identify risk factors for pregnancy
  complications

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Typical estimates of prevalence of thrombophilia
risk factors in VTE patients
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Why order a thrombophilia risk factor laboratory
evaluation?

• To provide an explanation for VTE
• To identify risk factors that can be modified
• To predict risk of VTE in asymptomatic kin
• To assess risk for recurrent VTE
• To identify risk factors for pregnancy
  complications

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What tests should be included in a thrombophilia
evaluation?

• Antithrombin activity
  (chromogenic)
• Protein C activity
  (chromogenic)
• Protein S free antigen/ activity
• Factor V Leiden first aPC screen,
  if , genotype to confirm
• Prothrombin G20210A genotype
• Homocysteine fasting
• Phospholipid antibody IgGIgM
  anticardiolipin
• IgGIgM anti B2GP1 soon
• Lupus Anticoagulant

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What tests do the experts order?

• ASH education session 2002 Too many tests, too
  much conflicting data
• Frits Rosendaal, John Heit, Ken Bauer
• Test ordering patterns
• Rosendaal caveat no testing done for first VTE
  unless exceptional (very young, very large,
  unusual location, positive family hx)

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Who is likely to be at increased risk for
recurrent VTE?

• Patients with
• Weak temporary risk factors
• Spontaneous DVT/ PE
• Onset lt 50 years old
• Recurrent events
• Definitive positive family history in 10
  relatives

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Recurrence rate is minimal for transient VTE risk
factors
Transient risk group Post op DVT, no hx
of Previous VTE, cancer
11/66
0/43
Levine MN Thrombosis Haemostasis 746061995
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Do positive thrombophilia tests predict increased
risk of recurrent VTE?

• Based on recent prospective studies
• Factor V Leiden (/-) NO
• Prothrombin 20210 (/-) NO
• Anticardiolipin IgG YES
• Lupus anticoagulant YES
• AT, PC,PS, multiple risks excluded/too rare

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AIM 2003 138128-134
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Candidates for prolonged, OAT

• One VTE and
• Reversible risk factor 3 mo.
• Phospholipid antibody syndrome gt12 mo
• Active cancer LMWH gt3-6 mo
• AT, protein C, or protein S deficiency, FVIIIgt
  90th percentile, FVL, PG 20210, homocysteine
  gt6-12 mo
• FVL homozygote or 2 inherited risk factors gt 12
  mo
• Combined inherited risk factors (FVL and PG
  20210)
• Two or more VTEs
• Indefinite oral anticoagulation
• Target INR 2.5 0.5 (2-3)
• 7th AACP consensus conference Chest
  2004126(suppl 3)

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Risk of VTE recurrence based on D-dimer cut-off
of 250 ng/ml
N 610 13 recurred Sensitivity
80 Specificity 36 NPV 92 PPV 16
Eichinger, JAMA 2003, 290 1071-4.
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D-dimer cut-off 500 ng/ml
N 282 New VTE 12
NPV 93 PPV 16
Palareti, Circulation 1083132003
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n130 New VTE 15
NPV 96 PPV 27
Palareti, Circulation 1083132003
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Why order a thrombophilia risk factor laboratory
evaluation?

• To provide an explanation for VTE
• To identify risk factors that can be modified
• To predict risk of VTE in asymptomatic kin
• To assess risk for recurrent VTE
• To identify risk factors for pregnancy
  complications

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Testing asymptomatic kin for thrombophilia risk
factors case 1

• 33 white male, objectively confirmed spontaneous
  DVT
• 30 year old brother with spontaneous DVT one
  month earlier
• Their 58 year old father had spontaneous PE at
  28, recurrence after stopping warfarin, on
  chronic OAT

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• Thrombophilia testing repeatedly low
  antithrombin activities (50-60)
• both sons have two children, lt 10 years old
• Should the 3 daughters and one son be screened
  for AT deficiency?
• If deficient, what kind of prophylaxis should be
  recommended?

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Case 2

• 52 white male
• First spontaneous DVT 2000, OAT x 6 months
• Superficial phlebitis 2002
• Second spontaneous DVT 2003
• Factor V Leiden heterozygous
• 2 sons (20s) and one daughter (18)
• Should his children be screened for FVL?

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Prospective study of VTE risk in FVL carriers
247 probands 470 asymp carriers Mean age 43 Ave.
F/U 3.3 yr
Ann Intern Med 353222001
VTE incidence .58 (n 9) 4 spont (incidence
.28) 1 post-op (7 d. LMWH) 1 HRT (2.9/yr
.8-15.3) 3 OCP (1.8/yr .4-5.2) 0 pregnancy
(9/17 LMWH)
Screening 200 daughters /sisters of FVL
probands will potentially prevent 2 OCP related
VTEs
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Case 3

• A 55 year old woman is diagnosed with a DVT 2
  weeks after removal of a cast for treatment of an
  ankle fracture.
• Thrombophilia testing is performed, and she is PC
  heterozygous. Family history otherwise negative
  for VTEs.
• Should her 25 year old daughter who is taking
  OCPs be screened?

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conclusions

• Testing for thrombophilic risk factors, both
  inherited and acquired, is recommended for
  selected patients at high risk for recurrent VTE
• Borderline low test results for antithrombin,
  protein C, and protein S should be viewed with
  great skepticism due to analytical impression and
  biologic variability
• Screening asymptomatic first degree relatives
  should be tempered by the identified risk factor
  in the affected proband, and by the
  thrombophilic severity of the kindred.

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Extra Slides
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PCPS
TFPI
Pro Z
AT
TAFI
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VTEs in asymptomatic carriers of AT, Protein C
and S deficiencies
Unselected probands 10 and 20 relatives
Annual incidence 1.5
Blood 9437021999
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protein C and antithrombin activities during
acute DVT
Antithrombin
Protein C
Reiter Thrombosis Haemostasis 745961995
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Thrombophilia testing what else can go wrong?

• Phospholipid antibody testing
• Phospholipid antibody syndrome diagnosis based on
  one test result. Confirm persistence
• Minimally cardiolipin antibodies, isolated IgA
• Commercial labs with long phospholipid antibody
  panels
• No gold standard method for L.A. gt 2 screening
  tests should be performed, and screen confirmed

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protein S function during acute DVT
Total protein S
Free protein S
Thrombosis and Haemostasis 745961995
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VTE risk in thrombophilic FVL kindreds
Retrospective 182 10 and 20 relatives 12
propositi (AT,PC,PS neg)
93
10 20 carriers (n 92)
annual VTE incidence carriers .58 non-carriers
.17
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first degree carriers (n 36)
25 VTE in FVL spontaneous
Brit J Haem 1109392000
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VTE risk in unselected FVL kindreds
467 10 relatives 118 propositi retrospective
FVL .10
50 VTE Spontaneous 30 OCP/preg 20 surgery
FVL .45
VTE in 5/235 FVLpreg All peripartum
VTE in OCPFVL 3.3 relative risk, .48 annual
incidence
Ann Intern Med 128151998
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Thrombophilia testing What can go wrong?

• Misinterpreting temporary decreases in protein C,
  protein S, and antithrombin as inherited
  deficiencies
• Acute thrombosis
• Heparin therapy
• Warfarin therapy
• Protein S and concurrent pregnancy/OCP use
• Poor test precision
• Elevated homocysteine due to
• Non-fasting
• Ex-vivo release from red cells

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Thrombophilia testing what can go wrong?
continued

• Activated protein C resistance (aPCr)
• Rarely false positive if pt. plasma is prediluted
  with factor V depleted plasma
• FVL molecular tests
• Remote chance of aPCr mutation at alt. site
• Prothrombin G20210A
• genetic testing is specific and sensitive

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Do global tests of hemostasis predict recurrent
VTE?

• Fibrinolysis NO
• Euglobulin lysis time pre/post venous occlusion
• Thrombin generation NO
• Prothrombin fragment F12
• Thrombin and fibrinolysis Maybe
• D-dimer levels