Thrombophilia- Lab Assessment CH 14 & CH 9&19 (algorithmic) Flashcards
1
Q
What are some common things to
know about thrombophilia?
A
- hereditary thrombophilias only marginally increase the risk of VTE and recurrence
- there is no evidence that a negative lab thrombophilia panel is associated with lower risk of recurrence
- clinical risk factors typically outweigh genetic ones in management
- recurrent VTE is common after the initial unprovoaked VTE
2
Q
What recurrence risk of VTE is
used to weight the benefits vs. risks of anticoagulation?
A
- recurrence risk of >5% in the first year would outweigh the risk of long term anticoagulation
3
Q
What are the 2 main lab findings
that could portend a clinically significant increased
risk of recurrent VTE if anticoagulation is stopped ?
A
- antiphospholipid antibodies
- antithrombin III deficiency
4
Q
What is associated with VTE formation
in family members ?
A
- unprovoked VTE at a young age (<45 years)
- assocaited with increased risk of VTE in family members
- the risk is further increased if it occurs in multiple family members
Note: Factor V Leiden and PT gene mutations are rather weak predictors
5
Q
What is the relationship between
estrogen supplementation and thrombosis ?
A
- dose dependent increased risk of thrombosis
- thought to be related to altered levels of clotting factors
- synergistic effect with hereditary causes, particularly Factor V leiden
6
Q
What is key to know about Antithrombin III ?
A
- also called Heparin cofactor I
- synthesized in the liver
- half life is 2.8-4.8 days
- Inhibits
- thrombin (factor II)
- IXa, Xa, XIa, and XIIa
- accelerated 1000x by Heparin
7
Q
What is the inheritance pattern of
Antithrombin III ?
A
- autosomal dominant with variable penetrance
- Type I deficiency
- quantitative
- homozygous deficiency is lethal
- Type II deficiency
- qualitative
- Type I deficiency
8
Q
What are the three types of
type II defects in Antithrombin III ?
A
- Type II reactive site (RS), defect in the thrombin binding site
- Type II heparin binding site (HBS)
- most common
- least thrombophilic mutation
- exception is the homozygous type that develops thrombosis early on in life
- Type II PE
- produces a conformational change that affects binding to heparin and thrombin
9
Q
How is testing for antithrombin III deficiency performed ?
A
- first line testing
- functional assay - Heparin cofactor activity
- chromogenic assay
- detects all types of antithrombin III defects
- performed by adding heparin to patient plasma which will bind to patient’s antithrombin III
- then exogenous thrombin and/or factor Xa is added
- once the antithrombin III activity is exceded there is light emittance
- functional assay - Heparin cofactor activity
10
Q
What is a pitfall in treatment with heparin
derivatives when evaluating Antithrombin III ?
A
- derivative of heparin reduce antithrombin III levels
- other anticoagulants may cause increases in levels
- lead to false positive
- direct factor Xa inhibitors may lead to an overestimation of ATIII IF using a Xa based assay
- direct thrombin inhibitors may lead to overestimation of ATIII in thrombin based assays
11
Q
What are causes of acquired antithrombin III
deficiencies ?
A
- acute thrombosis
- transiently reduces Antithrombin III
- cirrhosis
- leads to reduced synthesis of both procoagulant and anticoagulant proteins
- nephrotic syndrome
- reduces ATIII levels in the absence of clotting
- protein losing enteropathies
- ECMO
- due to heparin, consumption, and dilutional defects
- may lead to heparin resistance
- heparin therapy
- reduces ATIII by 30%
- likely due to increased clearance of heparin:ATIII complexes
- burns- levels correlate with the extent of thermal injury
12
Q
How does aspariginase therapy (frequently seen in
treatment for B-ALL) affect ATIII levels ?
A
- asparaginase impairs the synthesis of proteins that contain asparagine
- ATIII is one of them
13
Q
What is the odds ratio and clinical
presentation of ATIII deficiency ?
A
- first VTE OR is 16
- at risk of recurrent VTEs if they stop anticoagulation
- low levels of ATIII
- lead to heparin resistance, where you need higher levels to reach anticoagulation
14
Q
Which medications do not require
antithrombin III levels in order to work ?
A
- direct Xa inhibitors and direct thrombin inhibitors
- their actions are not affected by low ATIII levels
15
Q
Can people with ATIII deficiency develop
arterial thrombosis ?
A
- arterial thrombosis has been documented but is uncomon and may not actually be associated with this
16
Q
What is the mechanism of action of
Protein C ?
A
- synthesized in the liver and is vitamin K dependent
- Protein C when activated is a serine protease
- primary function is to prevent clot extension into areas of intact endothelium
- thrombomodulin
- found on intact endothelium
- thrombomodulin binds thrombin and alters it’s function
- promotes cleavage of protein C into a functional protein C
17
Q
Where is protein C located and
what is it’s function ?
A
- localized to the area of thrombomodulin
- connected by endothelial cell C protein
- activated C
- cleaves factor Va and VIIIa
- thus slowing clot formation
18
Q
What are the protein C deficiencies?
A
- Type I deficiency (most common type)
- quantitative
- decreased protein with full function
- Type II deficiency
- qualitative
- dysfunctional
- Protein C deficiency is autosomal dominant
- rare homozygotes may present with neonatal purpura fulminans
Note: acquired protein C deficiency is much more common
19
Q
What testing is performed to evaluate for
Protein C deficiency ?
A
- functional assays detect most types of Protein C deficiency
- 2 types of assays: clot based and chromogenic
- both use venom from the southern copperhead snake which activates protein C
- clot based tests (use either a PTT or Russell viper venom clotting time) suffer from lots of interference
- DOACs or lupus anticoagulants can cause false increases in protein C
- elevations of VIII and factor V leiden can falsely decrease protein C activity
- clot based tests (use either a PTT or Russell viper venom clotting time) suffer from lots of interference
- chromogenic assays are preferred due to the interferences
20
Q
How does the chromogenic assay work
for the Protein C functional assay ?
A
- southern copperhead snake venom is added to the patient’s plasma
- this activates protein C
- a chromogenic protein C substrate is added and when cleaved emits a light detected by spectrophotometry
21
Q
How do protein C levels vary in
certain normal conditions ?
A
- levels are significantly lower in neonates
- levels increase throughout childhood and may not reach final levels until age 30
- increase in normal pregnancy and postpartum
- in contrast to protein S which decreases
22
Q
If low levels of protein C are found
on the chromogenic assay, what subsequent
test is performed ?
A
- immunoassay to determine levels
23
Q
What can cause an
acquired protein C deficiency ?
A
- acute thrombosis
- cirrhosis cause reduced production
- DIC, with severe purpura fulminans in meningococcal disease
- Vitamin K or Warfarin
- Nephrotic syndrome
- L-asparaginase therapy
24
Q
What types of VTE can
occur in protein C deficiency ?
A
- cerebral venous thrombosis
- splanchnic vein thrombosis
25
What are some important facts
about protein S ?
* synthesized in the liver, vitamin K dependent
* does not have direct enzymatic activity
* is a cofactor for protein C
* approximately 70% is bound in a 1:1 ratio to the complement regulatory protein C4b-binding protein
* 30% free protein S is responsible for most of the activity
* 3 types of deficiencies
* Type I- quantitative
* Type II- qualitative
* Type III- decreased free protein S antigen but normal total protein levels
26
How is protein S deficiency inherited ?
* autosomal dominant
* rare homozygotes get neonatal purpura fulminans
27
How is testing for protein S deficiency conducted?
* functional assay NOT recommended up front
* technically difficult
* functional protein S deficiencies are rare (type II)
* clot based assay technique
* DOACs and Lupus anticoagulants falsely increase protein S levels
* increased Factor VIIIa and Factor V leiden falsely decrease protein S levels
* also SUPER sensitive to pre-analytical variables (~10% of cases)
* preferred test is the quantitative test
28
How is the quantitative protein S test performed ?
* preferred initial evaluation
* both total protein S levels and free protein S levels are important
* total protein S levels should not be ordered in isolation because there is poor correlation with thrombosis
* IMP
* free protein S levels of \< 33% correlate with VTE
* many patients have mutations in protein S
29
What are causes of acquired protein S
deficiency ?
* acute thrombosis
* cirrhosis
* pregnancy
* oral contraceptives
* DIC
* Vitamin K deficiency or Warfarin therapy
* nephrotic syndrome
* newborns (healthy) have 15-30% of adult levels of total protein S
* but C4b binding is also less so free protein levels are about the same
30
What are the most common presentations
of patients with protein S deficiency ?
* DVT and PE
* splanchnic vein thrombosis
31
What is the cause of Factor V Leiden?
* caused by a single nucleotide polymorphism in the factor V gene that changes arginine 506 to glutamine
* arginine 506 is the normal cleavage site for activated protein C
* mutation here leads to slower inactivation of Va
* thus there is more thrombin generation
32
What is the testing for Factor V leiden ?
* genetic testing for the mutation is standard
* can do a functional assay but not necessary because most cases are caused by the single mutation
33
What ethnicity is factor V Leiden most common ?
* European ancestry
* 4-5% of white individuals
* many are heterozygous for the mutation
* most patients never experience a VTE and of those that do fatal VTE is very rare
34
What clinical manifestations are seen
with Factor V leiden ?
* DVT and PE
* cerebral vein thrombosis
* portal/hepatic vein thrombosis
35
What is the prothrombotic mechanism
of the prothrombin mutation ?
what is the mutation ?
* mutation: G20210
* there is an increase in the prothrombin plasma levels
* increases PT mRNA and ultimately protein
* leads to increased thrombin formation
* enhanced tendency to clot
* test for by genetics
36
Why is testing for plasma PT
levels not recommended ?
* becuase the levels fluctuate throughout the day normally
37
Are patients with the PT gene mutation
at risk for recurrent VTE ?
* No
38
Which coagulation test do Lupus
anticoagulants affect more ?
* PTT more often affected
* PT less often affected because there is a higher amount of excess phospholipid in the test
39
What is the source of extra phospholipid
for determining lupus anticoagulantS?
* frozen washed platelets
* aka platelet neutralization procedure (PNP)
* purified hexagonal phase array phospholipids
* LA-insensitive phospholipid
Note: all of these are clot based assays
40
What factors can confound
LA testing ?
* anticoagulant medication
* factor deficiency
* specific factor inhibitor
41
What is the association between
increased factor VIII levels and VTE ?
* appear to increase VTE risk in a dose-dependent manner
* no definite cutoff that defines the disorder for how high the levels need to be
* independent risk factor for first unprovoaked DVT
42
What are common things that can
elevate factor VIII?
* aging
* acute phsyiologic stress (epinephrine)
* liver disease
* pregnancy
* possibly warfarin
43
Where is factor VIII produced ?
* made in specialized hepatic sinusoids
* IMP:
* most other factors are made in the hepatocytes themselves
* most factor VIII circulates noncovalently bound to von Willebrand factor (vWF)
* lots of variability in levels in different people
* 50% of variability may be related to blood type
* type O blood has lower levels of FVIII and vWF (due to faster clearance)
44
What are acquired causes of elevated
factor VIII ?
* acute thrombosis
* levels may remain high for up to 6 months
* Warfarin therapy
* cirrhosis
* normal aging
* pregnancy
* African American race
45
please see pg. 254 for a list of
minimal or null increase in thrombophilia
46
When should testing for protein C
deficiency occur ?
* delay testing for 4 weeks after discontinuing Warfarin
* delay testing for several days after factor Xa inhibitors
Note: elevated protein C levels are of no significance
47
How does heparin affect the clot-based
functional assay for Protein C activity ?
* it will overestimate protein C activity
* also seen with direct thrombin inhibitors or factor Xa anticoagulants
* a specific factor inhibitor
* or lupus anticoagulant
48
What is the type I protein S
deficiency?
* accounts for 80% of inherited forms
* concordant reductions of total protein S antigen, free protein S antigen and protein S activity
49
What is the type II protein S
deficiency ?
* due to protein S mutations that lead to a dysfunctional protein
* decreased protein S activity
* normal free protein
* normal total protein S antigen
50
What is type III protein S
deficiency ?
* debateable if this even exists
* low activity
* normal total S protein
* low free protein S antigen
Note:
* attributed to an increased binding affinity of protein S for C4bBP
* but it could just be seen due to age related increases in C4bBP
IMP: acquired protein S deficiency is much more common
51
Why do protein S activity and free protein S
antigen decrease with some combined
hormone OCPs and/or pregnancy ?
* usually do to increased hepatic synthesis of C4b binding protein
52
Where is antithrombin produced
and what is it's function ?
* produced in the liver
* inactivates factor Xa and thrombin (factor IIa)
* accelarated by Heparin
* Type I mutation
* hyposynthesis
* antigen=activity
* Type II mutation
* dysnfunctional protein
* antigen \> activity
* there are 3 subtypes
53
How is antithrombin affected by
hemodilution ?
* hemodilution from cardiopulmonary bypass reduces the response of antithrombin to heparin
54
How is antithrombin measured ?
* chromogenic assay
* in the presence of heparin (heparin cofactor activity)
* or absence of heparin (progressive antithrombin activity)
* use of bovine thrombin or human factor Xa is needed
* because they are not inactivated by Heparin cofactor II, which is another plasma protein with inhibitor activity
55
What are the advantages of the
heparin cofactor activity antithrombin assay ?
* sensitive for all types of antithrombin deficiency
* superior precision and accuracy
* lack of interference from all types of coagulation factor activities, lupus anticoagulants, or heparin
Because it is a measure of optical density things that interfere include:
* high concentrations of hemoglobin, bilirubin, and triglycerides
56
What has homocysteine been implicated as a
risk factor for?
* implicated as a risk factor for venous thrombosis, coronary artery disease, MI and stroke
* no mitigation in thrombosis risk by treating elevated homocysteine levels with vitamin B12 and B6
