1. Coagulation Assays – Part 2
Larry Smith, PhD
Director, Coagulation/Hemostasis
Assistant Attending

2. 2
Thrombophilia Testing
• Increased tendency to VTE
• Not a disease per se but may be associated with
▫ Disease  cancer
▫ Drug exposure  HRT, OCT
▫ Modifiable conditions  pregnancy, immobilization
▫ Non-modifiable conditions  age, gender
• Affects 1-2 individuals/1000 in the general population
▫ In the US
 ~2,000,000/year
 ~500,000 deaths
 Many survive with complications

3. 3
Virchow’s Triad
Post-operative state
Casting/splinting
Sedentary state
Leukostasis syndrome (AML)
Congenital heart disease
Stasis
Thrombosis
Vascular
Injury
Arterial
Changes in Blood
Composition
Central line, Sepsis
Inherited thrombophilia
Trauma, APA
Acquired thrombophilia
Chemotherapy/toxins
Hyperhomocysteinemia
Rudolph Virchow

4. 4
Risk Factors
• Multiple risk factors—multi-factorial process
▫ Hereditary
▫ Acquired
• Multi-hit hypothesis
▫ Individual hereditary or acquired risk factors has a relatively small
individual effect
▫ Risk for thrombosis is greatly increased when two or more risk factors
combine
• Classification of Thrombophilia
▫ Congenital
▫ Acquired
 Physiologic factors
 Environmental factors

5. 5
Thrombophilic Risk Factors
Congenital Risk Factors
•
•
•
Protein C
Protein S
AT
•
•
•
•
FVL
PG20210
FVIII
Homocysteine
•
•
(acquired also)
Non-modifiable
Inhibitory
Prothrombotic
Mechanism

6. 6
Acquired Risk Factors
 Acquired risk factors
 Pregnancy
 Malignancy
 Surgery
 Immobilization
 Hormone therapy (HRT, OCT)
 Trauma
 Obesity
Modifiable
 Antiphospholipid antibodies
 Physiologic risk factors
 Gender (hormonal changes)
 Age –Increases ~1%/year of age
 Childhood = 1/100,000
 40 years = 1/1000
 75 years = 1/100
Non-modifiable
 Identify a population at risk but have low predictive value for individual

7. 7
Role of the Laboratory and Physician
• Laboratory
• Provide reliable assays to identify these risk factors
• Identify preanalytical variables that can affect the accuracy
of those assays
• Physician
• Assess the potential benefit to each patient before
performing a battery of expensive tests

8. 8
Who should be tested
• Patients presenting with
▫ Venous thrombotic event before 40-50 years of age
▫ Unprovoked or Recurrent thrombosis at any age
▫ Thrombosis at unusual site
▫ Positive family history of thrombosis
▫ Unexplained abnormal laboratory test (PT, aPTT)
▫ Short or prolonged
•
Age of first episode
▫ 0-12 years
▫ 13-45 years
▫ 45-60 years
▫ 60+ years
Rare
Highly probable
Probable
Possible
Congenital
Risk Factors

9. 9
Testing: When and Why
• Optimal time for testing
▫ Asymptomatic
▫ Not on anticoagulant therapy
▫ Anytime for molecular testing
• Why do we test
▫ Pathologic basis for the thrombotic event
▫ Duration and intensity of therapy
▫ Prophylaxis for high risk patients
▫ To alert the patient's immediate family members to the
presence of possible inherited risk factors

10. 10
Types of Assays
• Functional Assays
 Clot-based assays
 Good screening assays
 Based on a functioning cascade
 Affected by preanalytical variables
▫ May require additional testing
 Chromogenic assays
 Measure the activity of a specific enzyme rather than
general biologic function
 Not affected by most preanalytical variables

11. 11
Types of Assays
• Antigenic assays
 LIA- or ELISA-based technologies
• Confirmatory assays
▫ DNA-based assays

12. 12
Types of Deficiencies
• Type I Deficiency
 Decrease in both FUNCTION and AMOUNT of protein
present
 True deficiency
 Functional assay
• Type II Deficiency
 Identify the total AMOUNT of protein only
 Do NOT measure the function of the protein
 To identify a dysproteinemia
 Functional + Antigenic assays

13. 13
Antithrombin Deficiency
• Single chain glycoprotein
▫
▫
Synthesized in the liver
SERPIN
• Inherited deficiencies
▫
▫
▫
Associated with increased risk of VTE
0.02 – 0.2% in general
1-2% of patients with VTE
• Acquired deficiencies
▫
Decreased synthesis

▫
Drug-induced decreased synthesis


▫
LD
L-asparaginase
Heparin
Increased clearance



Active thrombosis
DIC
Nephropathies

14. 14
Antithrombin Deficiency
Type
Type I
Type II
Interpretation
• Parallel reduction in functional and immunologic AT –
Quantitative
50% of normal
• Antigen = Activity
Qualitative
• Greater reduction in functional assay in comparison to
immunologic assay
• Antigen > Activity
T2-HBS  mutation in heparin binding domain
T2-RS  mutation in reactive site of AT
T2-PL  mutation in both heparin binding domain and reactive site of AT

15. 15
Antithrombin Assays
• Two different designs of the AT assay
1. AT is added to the test system as a reagent to ensure 100%
levels of AT are present
 Better reflection of the absolute drug concentration
2. The second relies on the AT in the patient’s plasma***
• Better reflection of the functional AT status of the patient
Inversely proportional to the AT activity concentration in the plasma sample

16. 16
Protein C Deficiency
• 1960 – Seeger described it
anticoagulant role
• 1980 – Griffin et al associated PC
deficiency with VTE
• VKD serine protease that is activated
by IIa to aPC
▫ Inhibits Va and VIIIa  shuts off
thrombin generation
▫ Exhibits
 Anti-inflammatory activities
 Anti-apoptotic activities
▫ Inhibits PAI-1  enhanced
fibrinolysis

17. 17
Protein C Deficiency
• Congenital Deficiency
• Heterozygous PC deficiency:
•
•
•
~0.2% of the general population
~3% of patients with VTE
~85% of PC mutations are type 1
• Acquired Deficiency
▫ Decreased synthesis
 LD
 VKD/VKA
▫ Drug-induced decreased synthesis
 L-asparaginase
▫ Increased clearance





Acute thrombosis
Acute medical illness
DIC
SCD
Trauma
More common that the
congenital deficiency

18. 18
Protein C Assays
• No single test for PC is 100% sensitive and specific for abnormalities
▫ Functional assays
Clot-based Assay (functional)
Chromogenic Assay (functional)
aPTT assay to measures the anticoagulant
effect of aPC  due to its ability to inhibit
FV and FVIII
aPC cleaves a substrate  release of
chromophore generating a color change
Subject to a number of preanalytical
variables
Subject to fewer preanalytical variables
 Detects most functional defects but
not all
 PF3 binding defect
 May be affected by OAT
 FVIII
 FVL
▫




Misleadingly low levels
Antigenic assay
DTI
Heparin
Lupus Anticoagulant
OAT
False normal results

19. 19
PC Assays
• Dilute patient’s plasma (1:10) in PC-deficient plasma
▫ Clot-based assay
▫ Chromogenic-based assay

20. 20
Protein S Deficiency
• Described in 1984, Comp
• TOTAL PS circulates in 2 forms:
▫ Bound PS—60%
 C4B-BP—nonfunctional
▫ Free PS—40%-functional
• Serves as a cofactor for PC
▫ Binds aPC to the
phospholipid surface
▫ VKD glycoprotein synthesized in
the liver
Total Protein S
C4B-BP
Bound
PS
Free
PS

21. 21
Protein S Deficiency
• Congenital Deficiency
• Heterozygous PS deficiency:
▫ ~2% in general population
▫ ~3-6% in recurrent thrombosis or family history
• Acquired Deficiency
▫ Decreased synthesis
 LD
 VKD/VKA
▫ Drug-induced decreased synthesis
 L-asparaginase
 OCT
 HRT
▫ Increased clearance




Acute thrombosis
DIC
Acute medical illness
Trauma
More common that the
congenital deficiency

22. 22
Protein S Assays
Patient plasma diluted 1:10 in PSdeficient plasma
Three types of assays
1. Clot-based functional PS assay—”activity” assay
 Based on aPC inactivation of FVa and FVIIIa
 Plasma + PNP(PS free)+ aPC + Bovine FVa + Add CaCL2
2.
3.
Clot
Antigenic-based Free PS assay
 Immunologic assay  measures “free” (functional) portion of PS
 Plasma + PNP(PS free)+ aPC + Bovine FVa + Add CaCL2
Clot
Antigenic-based Total PS assay
 Immunologic assay that measures PS bound to C4BBP + free PS
Type
PS (Activity)
PS (Free)
PS Total
C4bBP
I
Decreased
Decreased
Decreased
Normal
II
Decreased
Normal
Normal
Normal
III
Decreased
Decreased
Normal
Elevated

23. 2323
Interpretation of PS Deficiency
• Activity assay may be misleading low
▫ FVL
▫ Elevated FVIIII
▫ VKA/VKD
▫ LD – not universal (there are extra-hepatic sites of PS synthesis)
▫ SCD
▫ Type II deficiencies are very rare
 May be helpful if there is a high index of suspicion
▫ Pregnancy and some women on OCT
▫ HIV infection
▫ Acute phase response
Type III
• Marlar et al. (2012) AJCP. 137; 173-175
▫ Found increased number of falsely low PS activity when PS activity is used as
first assay
• Marlar et al. (2011) Am J Hem. 86;418-421
▫ Free PS assay  considered by many to be more reliable than activity
▫ Diagnoses 95-99% of PS deficiencies

24. 24
aPC-Resistance—Screening assay
• aPC-resistance
▫ Dahlbäck et al in 1993
http://www.wardelab.com/arc_2.html
•
 Noted a blunted response in
aPTT’s of a group of patients
with thrombophilia when
aPC was added
▫ Shuts-off thrombin
generation
 Via inhibition FVa and
FVIIIa
▫ Ratio of 2 aPTT’s
__(aPTT plus APC)__
(aPTT minus APC)
•
•
Normal : adding aPC prolongs aPTT
FVL: adding aPC does NOT prolong aPTT

25. 25
aPC-Resistance—Screening assay
•
• ~95% of aPC Resistance is caused
by a defect in the Factor V
molecule
• “Screening assay” for FVL
mutation
▫ Substitution of adenine for
guanine at 1691 – G1961A
▫ Changes arginine to glutamine
at 506 – R506Q
• Sensitivity and specificity
approach 100% with modified
assay
▫ Uses FV-deficient normal
plasma + patient plasma
http://www.wardelab.com/arc_2.html
a. Screening assay is affected by
 Lupus anticoagulant
 DTI’s
b. High FVIII levels may lower
APC ratio
(pregnancy/inflammatory
states)
c. DNA-based assay confirms
FVL

26. 26
Factor V Leiden—Confirmatory Assay for FVL Mutation
• Mutation later described in 1994 by Bertina et al
• Caused by single point mutation in the FV gene
▫ A single nucleotide substitution of adenine for guanine at
nucleotide 1691 of the FV gene  replacement of Arg (R) with
Gln (Q) at position 506 in FV protein
• Higher risk for thrombosis
• Venous thrombosis most common manifestation

27. 27
PG20210 Mutation
 Poort et al, 1996
 Single nucleotide substitution
G20210A in the 3’ UT regions of the
prothrombin gene
 G A substitution at nucleotide
20210 in prothrombin gene
 Results in elevated levels of
prothrombin (~30% increase)
 No screening test available
 Occurs primarily in Caucasians--~3%
in general population
 2-5-fold increased risk of VTE

28. 28
Lupus Anticoagulant/APAs
Y
or
Prothrombin
Y Y Y
• Lupus Anticoagulant
▫ Auto-antibodies directed
against phospholipid-binding
proteins
▫ Targets
 β2GPI—thrombosis
 Prothrombin—bleeding
 PC, PS, Annexin V—
thrombosis
2 GPI
Y
 Prolonged clotting time in vitro
 Thrombosis in vivo
ANTIBODY-MEDIATED THROMBOSIS
Y
• Paradox
▫ LA is a riddle wrapped in a
mystery inside an enigma
Antibody:
Y
Membrane
lupus anticoagulant
anticardiolipin
antiphosphatidylserine
anti 2GPI
anti Annexin V

29. 29
ISTH Criteria for Lupus Anticoagulant Testing
 The ISTH has defined the minimum diagnostic criteria for lupus
anticoagulants to include
1. A prolonged clotting time in a screening assay such as the aPTT
2. Mixing studies indicating the presence of an inhibitor
3. Confirmatory studies demonstrating phospholipid dependence of the
inhibitor
a. Screen – decreased amount of phospholipids  prolonged
clotting time
b. Confirm—increased amount of phospholipids  shortened
clotting time
4. No evidence of other inhibitor-based coagulopathies
 Specific factor assays if the confirmatory step is negative or there is
evidence of a specific factor inhibitor

30. 30
ISTH Criteria for Lupus Anticoagulant Testing
• Updated ISTH guidelines (2009) ISTH
▫ Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG. Update of
the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7:
1737–40
▫ Choice of tests
1. Two tests based on different principles
2. dRVVT should be the first test considered
3. Seconds test should be a sensitive aPTT (low
phospholipids and silica as activator)
4. LA should be considered as positive if one of the two tests
gives a positive result

31. 31
dRVVT Screen (Normal plasma)
X
dRVVT
Xa
Prothrombin
Xa
Phospholipid
(PF3)
Va
Ca2+
Thrombin
Fibrinogen
Fibrin

32. 32
dRVVT Screen (Lupus Anticoagulant)
X
dRVVT
Xa
Prothrombin
Xa
Va
Low
Phospholipid
Content
Ca2+
Thrombin
Fibrinogen
Fibrin

33. 33
dRVVT Confirm (LA)
X
dRVVT
Xa
Prothrombin
Xa
Va
High
Phospholipid
Content
Ca2+
Thrombin
Fibrinogen
Fibrin

34. 34
Detection of LA
 dRVVT*
 SCT*
 HEX
 Kaolin CT
 dPT
Clot-based assays
• Assays
SCT
DRVVT
dPT
Why do we see so few LA’s
on the extrinsic side???

35. 35
Summary
• Broad menu of assays that can potentially be performed as part of a
hypercoagulable workup
• These assays help to identify risk factors that may contribute to thrombosis
• Clot-based assays should be interpreted with caution
• All of the assays have their advantages and disadvantages
▫ Chromogenic PC and Free PS assays are the assays of choice to screen for these
deficiencies
▫ ISTH Subcommittee on Thrombosis and WHO recommend Free PS to screen for
PS deficiency
Ballard RB, Marques, MB. Pathology Consultation on the Laboratory Evaluation of Thrombophilia:
When, How, and Why. Am J Clin Pathol 2012;137-553-560

36. 36
The End

37. Homocysteine
 McCully suggested an association between elevated
levels of homocysteine in plasma and arterial disease
 Most common congenital form due to:
1. (C677T)* in MTHFR gene
2. B-cystathionine synthase gene
 Acquired form due to deficiencies in
Folate, B-12, B-6
 Genetic testing* is controversial
 Homocysteine levels may provide
more information
 Normal values increase with age
 Higher in males
37

38. 38
Elevated Factor VIII (congenital)
Independent risk factor for venous thrombosis
•
•
•
•
•
FVIII activity >150%
Results in 5-6-fold higher risk for DVT, especially recurrent DVT
Associated with ischemic heart disease
Elevated FVIII persistent over time
Clusters in families—suggests a genetic component
• Mechanism of action for VTE
▫ Enhanced thrombin generation
▫ Induction of aPC-resistance state
Elevated Factors VII, IX, X, XI, XII
–
Also identified as risk factors for venous thrombosis
•
2-fold increase in risk