Thrombophilia test- when and what to do? Thrombophilia is not a disease itself but a manifestation of several conditions leading to increased tendency for thrombosis. Since long it has been a grey area in thrombophilia with a lot of who, when, why and what regarding testing for this condition. As a result, the tests are being done very indiscriminately without knowing what to do with the result, whether positive or negative. Many family-screening are also commonly done simultaneously in many cases unnecessarily. The inherited thrombophilias are mutation of factor V Leiden (FVL) or prothrombin gene and deficiency of protein C (PC), protein S (PS) or antithrombin (AT). The acquired conditions are anti-phospholipid syndrome (APS), paroxysmal nocturnal hemoglobinuria (PNH) and myeloproliferative neoplasms (MPN). Various malignancies, drugs (e.g., oral contraceptives, chemotherapy), pregnancy, trauma and surgery also increase the risk of thrombosis. In clinical point of view immobility and central venous access devises (CVAD) also add fuel to the flame (Virchow’s triad). The excerpts from the compilation of various literatures including recent guidelines are as follows. Testing for “heritable thrombophilia” should be done if thrombosis occurs at a younger age (<50yrs), with no/weak provocation and preferably with family history of thrombosis in first degree relatives in young age. Tests if at all needed should be done after at least 3 months of anticoagulation, only after stopping of anticoagulation for specified duration for respective drugs and tests should not be done during an acute event. Genetic testing to predict a first episode of venous thrombosis is not recommended. For thrombosis at unusual venous sites (splanchnic veins or cortical venous sinuses) or arteries, PNH and MPN should be ruled out as per CBC and other ancillary features. JAK2 mutation may also present with thrombosis even before MPN develops. Apart from this, tests for APS should also be done in above cases and additionally in retinal vein occlusion, stroke, or myocardial infarction in young. Neonatal purpura fulminans is a matter of special concern; protein C/S should be tested at urgent basis as specific treatment can be offered. Women contemplating pregnancy or willing for oral contraceptives should be addressed as per personal or family history of thrombosis/thrombophilia. Finally, an algorithmic approach should be adopted for individualizing each case of thrombosis to avoid indiscriminate testing and to avoid unnecessary anxiety of patients and family members with a proper vision to what to do with the result.

1. TOPIC : Thrombophilia testing- when and what to do?
SPEAKER : Dr. Pritish Chandra Patra

2. Hypercoagulable states
• Who should be tested ?
• When to test ?
• Why test ?
• What to test ?
• How to treat ?
• How long to treat ?

3. Circulatory stasis
Virchow’s triad
• Immobility
• Central lines
• PICC lines
• Inherited
• Factor V Leiden (FVL) mutation (F5 G1691A)
• Prothrombin gene mutation (F2 G20210A)
• Protein C (PC) deficiency
• Protein S (PS) deficiency
• Antithrombin (AT) deficiency
• Rare- elevated F VIII, dysfibrinogenemia
• Acquired
• Antiphospholipid syndrome (APS)
• Paroxysmal nocturnal hemoglobinuria (PNH)
• Myeloproliferative neoplasms (MPN)
• JAK2 mutation in the absence of an MPN phenotype
• Malignancy
• Drugs- OCP, chemotherapy
• Pregnancy
• Trauma, Surgery
Virchow’s triad

4. Interactions of multiple factors
• Pregnancy
• Puerperium
• Cancer
• Inflammatory conditions
• Obesity

5. Case 1
• 26 yr female
• on estrogen based OCP
• presents with
• new onset left lower limb DVT
• No prior history of clots
• No family history of clots
• No surgery
• No long-distance car travel
• 19 yr female
• College student
• presents with
• new onset unprovoked left
lower limb DVT
• No prior history of clots
• No surgery
• No long-distance car travel
• Mother had DVT in her 30’s
• Uncle had DVT in his 40’s
Case 2
• 65 yr old man
• Pain abdomen  Portal vein
thrombosis
• Hb-13, TLC- 8000, Plt- 6.5L
• Past h/o- migraine- 1yr, burning of
fingers- 1 yr
Case 3

6. Prevalence of inherited thrombophilia
Name Prevalence (%)
Factor V Leiden 12-40
Prothrombin gene mutation 6-18
Deficiency of AT, Prot- C, Prot- S 5-15
Risk of thrombosis
Risk factor Relative risk
General population 1
Factor V Leiden- Heterozygous 5
Prothrombin gene- Heterozygous 5
Protein C 10
Protein S 10
AT 15-20
Factor V Leiden- Homozygous 60-80
OCP 4-5
Hetero FVL + OCP 30-35
Pregnancy & Post Partum 5-10
Croles FN et al. Risk of venous thrombosis in antithrombin deficiency: systematic review and bayesian meta-analysis. Semin Thromb Hemost. 2018;44(4):315–26.
Gindele R et al. Clinical and laboratory characteristics of antithrombin deficiencies: a large cohort study from a single diagnostic center. Thromb Res. 2017;160:119–28.

7. Excess procoagulant
• ? increase the risk of thrombosis
• A meta-analysis of 12 GWAS for VTE (Arachchillage et al, 2019)
• linked to thrombosis
• Variants in F2, F5, F11, and FGG (fibrinogen gamma chain)
• Elevation of VWF and FVIII
• Relevance in routine clinical practice- not clear
• MEGA case-control study of VTE (Rietveld et al, 2019)
• FVIII: Odds Ratio- 16.0 (CI: 9.7–26.3)
• Interaction of heritable and acquired influences on FVIII activity
• Variability in levels over time
• ? Role in management- in thrombosis or asymptomatic family members
Routine testing of coagulation factors to assess the risk of thrombosis- not recommended
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.
O

8. Deficiency of natural anticoagulants
• AT deficiency: 15 x risk
• Heterozygous PC or PS deficiency: 5-7 x risk
• PC, PS deficiency >> Risk of thrombus >> FVL, PTG mutation
• PC, PS deficiency << Prevalence << FVL, PTG mutation
• Testing: Plasma activity >> molecular testing
• Multiple different genetic variants
• Acquired causes of deficiencies should always be considered
• Test only if it will impact clinical management
• Test if
• VTE at <50 years of age
• spontaneous or weak provoking factors
• 1st degree relative at young age
• Test for deficiencies of physiological anticoagulants- only after 3 months of anticoagulation for acute thrombosis
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.
P
• Warfarin
• Vit K deficiency
• CLD
• DIC
• Heparin
• Etc…

9. Jean M. Connors. Thrombophilia Testing and Venous Thrombosis. N Engl J Med 2017;377:1177-87

10. Factor V Leiden, Prothrombin gene mutation
• Most tested genetic variants predisposing to VTE
• FVL mutation
• Present in about 5% of Europeans
• Rare elsewhere
• Prothrombin gene mutation
• Present in 1%-2% of Europeans
• Rare or absent in other ethnic populations
• Result in increased risks for 1st VTE: 3-5x (Saemundsson Y et al, 2013)
Genetic testing to predict a first episode of venous thrombosis- not recommended
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.
O

11. Acquired genetic traits
• Incidence of thrombosis- 10% (Kelly RJ et al, 2015)
• Neutrophil clone size correlates best with
thrombosis risk
• Cumulative 10-year incidence of thrombosis
• Clone size >50%- 34.5%
• Clone size <50%- 5.3%
• PV, ET, PMF (15-30% incidence) (Landolfi R, 2008)
• 2/3rd arterial & 1/3rd venous
• Thrombosis often precedes disease recognition
• JAK2 mutation may precede frank MPN
• JAK2 mutation with normal CBC
• Thromboses in PNH and MPN- can occur anywhere in the venous or arterial systems
• Usually in unusual sites-
• Splanchnic Vein Thrombosis (SVT)- portal vein (PVT), mesenteric vein (MVT), splenic vein & hepatic vein/
Budd–Chiari syndrome (BCS)
• Cerebral Venous Sinus thrombosis (CVST)
• Thrombosis at unusual site-
• Abnormal CBC (cytopenia, abnormal Red Cell indices), hemolysis- test for PNH
• Abnormal CBC s/o MPN- test for MPN panel (JAK2 V617F, JAK2 exon 12, CALR, MPL mutation)
• Normal CBC with SVT or CVST- test for JAK 2 mutation
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.
MPN
PNH
P
P
P

12. • “Triple positives”- highest thrombotic risk, risk of CAPS
• LA- most strongly associated with recurrent
thrombosis
• No difference in recurrence of thrombosis
• unprovoked OR provoked
• venous OR arterial
• VTE unprovoked
• VTE provoked by a minor risk factor
• Multiple thrombotic events with signs of organ failure s/o CAPS
• Screening for APL Ab- recommended
• Family screening- not recommended (acquired)
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.
Acquired non- genetic traits
P
O
APS

13. Recurrence of VTE & long term anticoagulation
• FVL, PTG mutation- not associated with recurrent VTE Vs controls (Méan M et al 2017)
• AT deficiency (Ensor J et al 2016)
• Recurrent VTE: 2-4x
• PC, PS deficiency (Vossen CY et al 2005)-
• Annual risk of recurrent VTE without long-term anticoagulation
• 5.1% in PC deficiency
• 6.5% in PS deficiency
• Seems to be significant but utility is limited
• In 1st episode of VTE
• Testing not recommended (per se)- to decide duration of anticoagulation
• Testing not recommended- in 1st degree relatives
• Can be done in 1st degree relatives- specific circumstances
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.
D-dimer
Heritable
thrombophilia
Long-term
anticoagulation
Low +ve O
High -ve P
• D-dimer- (Palareti et al, 2003, Boutitie et al, 2011)
• DASH score (D-dimer, Age, Sex, Hormonal therapy) (Tosetto A et al, 2012)
• Vienna prediction model (Eichinger S et al, 2010)
• HERDOO2 score (Hyperpigmentation, Edema, Redness, D-dimer, Old age, Obesity) (Rodger MA et al, 2008)
P
P
O

14. • Portal / Mesenteric /
Splenic / Hepatic
• 19% of SVT preceded the diagnosis
of PNH
• 15-30%- MPN
• PNH & MPN have specific treatment
Unusual site thrombosis- testing for
heritable thrombophilia ?
• Incidence <5%
• Test only if the thrombotic event
occurs in the absence of a clear risk
factor at a young age (<50 years)
• Other causes ruled out
• <1% of all strokes
• 85% have risk factors- OCP,
pregnancy
• Rare- APS, MPN, PNH, vasculitis,
infections, malignancy, trauma,
surgery
• In the absence of a clear risk factor,
they need long-term
anticoagulation & routine testing for
heritable thrombophilia is not
required
Retinal Vein Occlusion
(RVO)
Cerebral Venous Sinus Thrombosis
(CVST)
Splanchnic Vein Thrombosis
(SVT)
• No e/o association with
heritable thrombophilia
• APS is significantly associated with
APS LA & triple positive
• Hernández JL et al, 2020
• 10% APS Vs 4.5 % control
Thrombosis PVT BCS
AT 3.9% 2.3%
PC 5.6% 3.8%
PS 2.6% 3.0%
Thrombosis at
unusual site
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.

15. Thrombosis at unusual site- unprovoked
• Abnormal CBC (cytopenia, abnormal Red Cell indices), hemolysis- test for PNH
• Abnormal CBC s/o MPN- test for MPN panel (JAK2 V617F, JAK2 exon 12, CALR, MPL mutation)
• Normal CBC with SVT or CVST- test for JAK 2 mutation
• Testing for heritable thrombophilia- recommended- if age <45yrs in absence of other common causes
• APL Ab
• RVO (Retinal Vein Occlusion)- APL Ab
P

16. Arterial thrombosis
• FVL & Prothrombin gene mutation
• Common in Europeans- <5%
• Causative role- not established, no clinical trials
• AT, PC, PS deficiency-
• Causative role- not established
• MPN, PNH, APS-
• Arterial thrombosis- 60-70% of thrombotic events in
MPN
• In absence of other risk factors- test for MPN
• Especially in younger age
Ischemic stroke
• FVL, PTG, AT, PC and PS deficiency-
• No clear relationship- Doesn’t warrant testing
• MPN- (Landolfi R, 2008)
• Ischemic stroke- 30-40% of all thrombotic episodes
• Stroke incidence in PV-
• With phlebotomy- 4-5%
• Without phlebotomy- 10-20%
• JAK2 V617F- normal CBC- harbinger of future
MPN
• APS-
• Risk of recurrence high despite anticoagulation
• Young stroke (<50 yr)- positive for APL Ab
• 17.2% of stroke
• 11.7% of TIA
• Arterial thrombosis-
• Heritable thrombophilia- not recommended in- the association in adults is weak and does not alter the management
• APS (antiphospholipid antibodies)- recommended- in the absence of other vascular risk factors
• CBC abnormal- MPN, PNH- recommended
Myocardial infarction
O
P
P
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.

17. Pediatric VTE
• CVAD associated VTE
• Pediatric >50% & Neonatal
>90%
Neonatal VTE
• Neonatal VTE
• >90% are CVAD related
• Rest are multifactorial
• Doesn’t warrant testing
Neonatal
purpura fulminans
• Heritable thrombophilia association-
• Severe deficiency of PC, PS
• Homozygous or combined
heterozygous deficiency of PC,
PS or AT
• Homozygous AT deficiency
• FVL or PTG mutation- low-risk
• Extensive unexplained thrombosis
• APS
• Neonates & children with purpura fulminans- test urgently for Protein C & S deficiency
• Neonatal stroke- heritable thrombophilia screening is not routinely recommended
• Children or neonate- test for heritable thrombophilia
• Extensive, unprovoked, recurrent thrombus
• Family h/o VTE- 1st degree relative
• Neonates with multiple unexplained thrombosis
• Clinical evidence s/o CAPS- test for antiphospholipid antibodies
Deepa J. Arachchillage et al. Thrombophilia testing: A British Society for Haematology Guideline. Br J Haematol. 2022;198:443–458.
P
O
P
P

18. Pregnancy
• VTE in pregnancy & puerperium- 1 in 1000
• 5x Vs age matched nonpregnant females
• 20 to 80x: 1st 6 weeks post partum period
• Women with a previous unprovoked or estrogen
provoked VTE (OCP, IVF or pregnancy)
• routine thrombophilia testing- not indicated
• require thromboprophylaxis throughout pregnancy &
puerperium
• Arterial thrombosis
• Rare- 1in 4000
• Test for APL Ab
• 6 weeks after end of pregnancy & repeated after 12 weeks
Pregnancy morbidity
• Gestational hypertension and pre-eclampsia
• IUGR
• Placental abruption
• Recurrent first-trimester pregnancy loss
• Stillbirth
• ACOG- doesn’t recommend testing for heritable
thrombophilia
• Acquired thrombophilia- APS
• APS in women with h/o VTE >>> with no h/o VTE
• Triple positive- poorer outcome
Personal h/o VTE Family h/o VTE in
1o relative
Family h/o Heritable
thrombophilia
Test required Thrombo-prophylaxis
required (P+P)
P O O O P
O P O O O
O P P P P

19. • Pregnancy-
• Testing for AT deficiency- may be considered in pregnant women with a known family history of this deficiency
or evidence of heparin resistance.
• In women with a history of unprovoked VTE- testing for APL Ab should be performed outside pregnancy
• Recurrent pregnancy loss
• Testing for heritable thrombophilia- not recommended
• Testing for APS- should be done- avoid during pregnancy
P
P

20. Back to the cases
Case 1 Case 2 Case 3
• 26 yr female
• on estrogen based OCP
• new onset left lower limb DVT
• No prior/family history of clots
• No h/o surgery
• No long-distance car travel
• Her sister is planning for pregnancy
• 19 yr female
• College student
• new onset unprovoked left
lower limb DVT
• No prior history of clots
• No surgery
• No long-distance car travel
• Mother had DVT in her 30’s
• Uncle had DVT in his 40’s
• 65 yr old man
• Pain abdomen  Portal vein
thrombosis
• No CLD, Splenomegaly- 2cm
• Hb-13, TLC- 8000, Plt- 6.5L
• Past h/o- migraine- 1yr, burning of
fingers- 1 yr
To test
• BM aspiration & biopsy
• MPN mutation panel
• JAK2 exon 14 & 12, CALR, MPL
To test
• After 3 months of anticoagulation
• 2 weeks of stopping drugs
• Mutation of FVL, PTG
• Deficiency of PC, PS, AT
To test/ ?may not test  indefinite
• After 3 months of anticoagulation
• 2 weeks of stopping drugs
• Mutation of FVL, PTG
• Deficiency of PC, PS, AT

21. Summary
• Provoking factor- strong, weak, unprovoked
• Family h/o- 1st degree, young age <50, h/o HT
• Site- venous, artery, unusual site
• CBC
Case 2
Case 1 Case 3
Jean M. Connors. Thrombophilia Testing and Venous Thrombosis. N Engl J Med 2017;377:1177-87

22. Thank You