SEED Coagulation
Sysmex Educational Enhancement and Development
April 2013
Laboratory evaluation of thrombophilia
What is thrombophilia?
The most simplistic definition of thrombophilia is an in­­
creased tendency to form blood clots in either the veins or
arteries. The term thrombophilia should not be considered
to be a disease but rather a risk factor for thrombosis. The
thrombophilias can therefore also be defined as a group of
inherited or acquired disorders that increase a person’s risk
of thrombosis. Thrombosis will manifest when the interac­
tion of genetic and environmental risk factors collectively
tip the haemostatic balance towards clotting. This predis­
position towards thrombosis is also referred to as hyper­
coagulability or a hypercoagulable state.
What is the difference between
thrombophilia and thrombosis?
Thrombosis refers to the actual presence of a pathological
blood clot which causes occlusion of a blood vessel. This
can occur in either the venous or arterial side of the circu­
lation. The term thrombophilia refers to the increased risk
or tendency to thrombosis. However, the simple fact that a
person has a thrombophilia and a greater chance of devel­
oping abnormal clotting than a person without thrombo­
philia does not mean that the former individual will ever
have a thrombosis. Fortunately, not all people with throm­
bophilia will have a thrombotic episode in their lifetime,
whereas unfortunately many patients who do experience
thrombotic events (such as deep vein thrombosis) may
not have any detectable thrombophilia at all.
Thrombophilias can be inherited or acquired, or both. The
haemostatic system is a highly regulated, fine-tuned inter­
action of multiple processes, involving the blood vessel wall,
principally the endothelium, platelets and the non-cellular
blood constituents. The non-cellular blood constituents
are comprised of both procoagulant proteins (the clotting
factors) and anticoagulant proteins which ensure that clotting
is confined only to sites of vascular injury and that clot for­
mation does not cause total vessel occlusion. Deficiencies
of these so-called natural anticoagulants, namely protein C
(PC), protein S (PS) and antithrombin (AT, sometimes still
referred to as ATIII) are classic inherited causes of throm­
bophilia. Other much more common inherited causes of
throm­bophilia are factor V Leiden (FVL) and the prothrom­
bin 20210A mutation (Tab. 1).
1. Procoagulant protein a) Factor V Leiden abnormality
abnormalities (activated protein C resistance)
b) Prothrombin 20210A mutation
c) Increased levels of clotting factors
(FII, FVIII, FIX, FXI) and fibrinogen
d) Some dysfibrinogenaemia variants
(rare)
e) Factor XIII polymorphism (rare)
2. Anticoagulant protein a) Antithrombin deficiency
abnormalities b) Protein C deficiency
c) Protein S deficiency
3. Other a) Elevated homocysteine levels
Tab. 1 Inherited causes of thrombophilia
Acquired causes of thrombophilia are plentiful. The most
well described acquired cause of thrombophilia, for which
diagnostic laboratory tests exist, is the so-called antiphos­
pholipid syndrome. These antiphospholipid antibodies are
procoagulant and are also commonly referred to as lupus
anticoagulant. The remaining acquired thrombophilias, as
listed in Tab. 2, are not associated with any specific coag­
ulation abnormality for which a definitive laboratory test
exists. As thrombophilia, in its broadest sense, simply means
a tendency to thrombosis, it includes conditions such as
disseminated intravascular coagulopathy, where clot for­
mation in the microvasculature is widespread. This edition
will however be restricted to those conditions that present
with localised occlusive blood clots.
SEED Coagulation – Laboratory evaluation of thrombophilia
Sysmex Educational Enhancement and Development | April 2013
1. Autoimmune a) Antiphospholipid antibody
conditions syndrome
2. Oestrogen a) Pregnancy and postpartum state
b) Oral contraceptive use
c) Hormone replacement therapy
3. Malignancy a) Myeloproliferative disorders
b) Paroxysmal nocturnal haemo-
globinuria
c) Solid tumours
4. Drugs a) Heparin induced thrombocyto- effect of thrombin is limited to the site of vascular injury and,
penia
b) Certain chemotherapeutic agents
5. Other a) Nephrotic syndrome
b) Sickle cell disease
c) HIV infection
d) Tuberculosis
Tab. 2 Acquired causes of thrombophilia
There is some overlap between the concept of an acquired
thrombophilia and a risk factor for thrombosis. Anything
that will swing the haemostatic balance in favour of clotting
is a risk factor and likewise would be deemed to be a throm­
bophilia. Conventionally however, the term thrombophilia is
usually restricted to those conditions that have an identifia­
ble coagulation abnormality that can be confirmed by means
of laboratory investigations. Common risk factors, without
identifiable specific laboratory abnormalities, include sur­
gery and any period of prolonged immobilisation including
long distance travel. Here the primary procoagulant influ­
ence is venous stasis. In the case of surgery, the release of
procoagulant material into the circulation plays a role, which
is most pronounced in orthopaedic surgery involving exten­
sive manipulation, e.g. hip and knee replacements. The more
risk factors a patient has, the greater the chance that he or
she will develop a thrombosis. Furthermore, most patients
with an inherited abnormality, with the exception of severe
AT, PC or PS deficiency, will generally remain asymptomatic
until exposed to an environmental risk factor or thrombo­
philic state such as pregnancy or surgery.
It has been estimated that about 5% of the world’s popula­
tion has an underlying thrombophilia. Furthermore, about
50% of people that present with an idiopathic thrombosis
are shown to have an underlying identifiable thrombophilic
state. Most conditions listed in Tab.1 and 2 predispose to
2
venous thrombosis, where blood constituent aberrations
play a much bigger role. Antiphospholipid antibodies and
elevated homocysteine levels are associated with both arte­
rial and venous thrombosis as these can induce endothelial
damage, which is the main stimulus for arterial thrombosis.
The natural inhibitors of coagulation
The natural inhibitors of coagulation (Fig. 1) ensure that the
together with the fibrinolytic system, ensure that blood ves­
sels are protected from occlusion. The first inhibitor to act
is tissue factor pathway inhibitor (TFPI). It is synthesised by
endothelial cells and is found in both plasma and platelets.
TFPI becomes concentrated at sites of tissue damage due
to release from activated platelets as they aggregate and
form a primary platelet plug. TFPI inhibits TF/FVIIa complex
thereby blocking off the extrinsic arm of the coagulation
cascade. As absence of TFPI is incompatible with life, defi­
ciency states are not a recognised cause of clinical throm­
bosis.
Antithrombin (AT) is a circulating inhibitor which inacti­
vates serine proteases, most notably thrombin as well as
the activated clotting factors FXa, FIXa and FXIa. Inhibition
takes place by way of a direct binding to the active site on
these molecules. This action is markedly enhanced by the
presence of heparin.
Protein C (PC) and Protein S (PS) are inhibitors of FVa and
FVIIIa, the cofactors of coagulation. Both PC and PS are
vitamin K dependent proteins. When thrombin comes into
contact with intact endothelium, it binds to the surface
receptor thrombomodulin. This complex activates PC, which
is brought into close proximity to the thrombin: thrombo­
modulin complex through its own binding to endothelial
protein C receptor (EPCR), also an endothelial cell surface
receptor. Activated PC, using PS as a co-factor to localise
to the activated platelet surface where the active clotting
enzyme complexes are assembled, in turn inhibits FVa and
FVIIIa. In doing so, it impedes further thrombin generation
by blocking the ‘intrinsic’ arm of the coagulation cascade.
Activated PC also enhances fibrinolysis by inhibiting tissue
plasminogen activator inhibitor (t-PAI).
SEED Coagulation – Laboratory evaluation of thrombophilia
Sysmex Educational Enhancement and Development | April 2013
XIa XI
AT IX IXa (VIIIa/Ca++/PL)
X Xa
TF VII/a PC/PS
(Va/Ca++/PL)
TF/VIIa
TFPI
II thrombin
Fibrinogen fibrin
XIIIa XIII
Fibrin polymer
Fig. 1 A schematic representation of the sites of coagulation cascade
inhibition of the natural anticoagulants. TFPI = tissue factor pathway
inhibitor; AT = antithrombin; PC = protein C; PS = protein S; TF/VIIa =
Tissue factor/activated factor VII complex, II = prothrombin.
Roman numerals represent the respective clotting factors. Roman
numerals followed by ‘a’ (e.g. Xa) represent the respective activated
clotting factors.
Assessment for the presence of thrombophilia
a) Clinical assessment
Evaluation of a possible underlying thrombophilia begins
with a detailed history. Important questions would in­­
clude an exploration of all possible risk factors including
any past personal or family history of thrombosis, any
co-existing disease, clinical events (e.g. previous surgery)
and medication use. A comprehensive physical examina­
tion is equally important. As the assessment for throm­
bophilia is generally only applicable if a patient currently
presents with or has a history of repeated past throm­
bosis, it is imperative to objectively confirm that throm­
boembolism has occurred before embarking on extensive
laboratory testing.
b) Screening for occult cancer
Although cancer is a well-documented risk factor for
thrombosis, extensive testing for occult cancer in
pa­­­tients presenting with idiopathic thromboembolism
is not warranted. Doing so has not been proven to in­­
crease patient survival. Any testing undertaken should
be restricted to age-specific screening.
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c) Laboratory testing
The nature of laboratory testing to be undertaken will
depend on the clinical situation. If the patient has had
an acute thrombosis, then baseline coagulation testing
is required prior to the commencement of anticoagula­
tion treatment in order to monitor efficacy. The latter
will include an activated partial thromboplastin time
(APTT) and prothrombin time (PT). A baseline complete
blood count (CBC) is also recommended as heparin
therapy may induce thrombocytopenia.
As there is no global screening assay for thrombophilia,
testing of individual parameters is required. Specialised
coagulation testing consists of a battery of tests for both
inherited and acquired conditions. However, as such test­
ing is costly and may not be universally available, referral
to a specialised facility may be required. Consequently
it is prudent to start with testing for those conditions
where the thrombotic association is well established.
Furthermore, it is critical that testing is performed under
optimal conditions as multiple factors may influence
the results which could lead to erroneous result inter­
pretation (Tab. 3).
I. Factor V Leiden
The presence of a single point mutation results in glu­
tamine instead of arginine being present in the clotting
factor V– called factor V Leiden (FVL). This mutation
occurs at position 506, which is the site where activated
PC would ordinarily cleave and inactive FVa. The pres­
ence of this mutation on chromosome 1 renders the
FVa molecule resistant to inactivation – hence the term
activated protein C resistance (APCR). As FVa remains
active longer than it ordinarily would in the absence of
FVL, it continues to support thrombin generation and
hence increases the risk of thrombosis. FVL is by far
the commonest cause of inherited causes of thrombosis
as approximately 5% of Caucasians are carriers but is
absent in blacks. In this regard, it would be wasteful to
test for FVL in blacks as the findings will be negative.
The risk of thrombosis in heterozygous (single gene af­­­
fected) carriers is about 4 to 8-fold increased with ho­­
mozygotes being at much greater risk (~80-fold). The
risk is compounded in pregnant women and those taking
oestrogen containing oral contraceptives. FVL is almost
exclusively associated with venous thromboembolism.
SEED Coagulation – Laboratory evaluation of thrombophilia
Sysmex Educational Enhancement and Development | April 2013
The APCR assay is a clot based assay which is designed
to detect functional APC resistance. In essence it is
comprised of two APTT tests; one in which the reagent
additionally contains activated protein C (APC), and one
without. The principle of the assay is that the presence
of FVL will not demonstrate the expected prolongation
of the APTT clotting time by the addition of APC. Con­
sequently the APCR ratio (APTT with APC/APTT without
APC) will be smaller. Although FVL is the commonest
cause of APCR other causes exist. Other rarer mutations
in the FV molecule have since been described. A re­­duced
APCR ratio is also sometimes observed if FVIII is mark­
edly elevated, in the presence of lupus anticoag­ulant
(LAC) as well as being physiological in pregnancy.
In order to make the functional APCR assay more spe­
cific for FVL, the test is commonly performed on patient
plasma that has been diluted with FV deficient plasma.
In so doing, the only variable influencing the final result
is the patient’s own FV. Positive results can then be
selectively confirmed by DNA testing – most commonly
by means of the polymerase chain reaction (PCR).
II. Prothombin 20210A mutation
The prothrombin 20210 mutation is a point mutation
in the 3’ untranslated region of the prothrombin gene
which increases the concentration of prothrombin
in the plasma. Like FVL, it is relatively common with
a prevalence of ~2% and is restricted to people of
Cau­casian ancestry. The risk of venous thrombosis
is increased about 3-fold in heterozygote carriers and
is multiplied several fold in pregnancy. DNA based
tests are required for prothrombin 20210A detection.
III. Protein C
Protein C deficiency is inherited in an autosomal domi­
nant fashion. The prevalence has been estimated to be
about 0.5% of the general population. The defects can
manifest either as a quantitative abnormality (reduced
production or unstable protein) or qualitative where the
protein itself has abnormal function (e.g. defective bind­
ing to its target sites on FV or FVIII). The severity will
depend on the nature of the mutation and whether one
or both gene copies are abnormal. Although the haemo­
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static defect is highly variable from patient to patient,
on average PC deficiency confers about an 8–10 fold
increased risk of venous thrombosis. Arterial thrombo­
sis is rarely described.
PC is a vitamin K dependent protein with a very short
half-life. In this regard, it declines rapidly shortly after
commencing warfarin therapy (vitamin K antagonist).
Testing for PC once patients have commenced warfarin
therapy (even if the INR is still sub-therapeutic) will
result in reduced levels, and hence may lead to a false
diagnosis of PC deficiency. Testing must therefore be
delayed until warfarin therapy has been discontinued.
Laboratory tests for PC tend to be functional assays.
Although both clot-based and chromogenic assays are
available, the latter are considered to be more reliable.
The principle of the assay is illustrated in Fig. 2.
PC activator
Protein C activated PC
activated PC
Substrate- Substrate +
colour colour
Reagent
Measured and
Patient
sample
converted to PC
value in %
Fig. 2 Principle of chromogenic PC assay. The reagent contains PC
activator, which activates any PC present in the sample plasma. This
in turn acts on a colour substrate resulting in release of the chromo-
phore. The degree of colour change is directly proportional to the PC
activity in the plasma. The colour change is measured and converted
into a PC value expressed as a percentage.
IV. Protein S
Protein S is a vitamin K dependent naturally occurring
anticoagulant that acts as a co-factor for PC. It facili­
tates the co-localisation of the PC/PS complex onto the
activated platelet surface in close proximity to the clot­
ting factor enzyme complexes, thereby bringing PC in
SEED Coagulation – Laboratory evaluation of thrombophilia
Sysmex Educational Enhancement and Development | April 2013
contact with its substrate, FVa and FVIIIa. About 60%
of total PS is bound to a protein called C4B binding
protein. Only free PS is able to support PC activity.
PS deficiency is inherited in an autosomal dominant
fashion. The abnormality may manifest as a reduced
antigen level of functionally normal PS, or as a func­
tional abnormality, both of which will lead to reduced
FVa and FVIIIa degradation, thereby increasing the risk
of thrombosis. About 1:500 individuals have mild PS
deficiency. Severe deficiency is rare although the exact
prevalence is not known.
Tests for PS include quantification of free and total PS,
either by means of antigen detection or by functional
analysis. Antigen testing is generally performed by ELISA
methods. With the exception of haemostasis referral
centres, most laboratories restrict their testing to func­
tional free PS as this provides the most meaningful clini­
cal information. The functional PS assay is a clot based
assay. Most commercial assays include a step whereby
the plasma sample is diluted in PS deficient plasma,
thereby minimising the influences of factors other than
the patient’s own PS on the result outcome.
As for PC, warfarin will induce a functional deficiency
of PS. Testing must therefore be performed prior to
initiation of anticoagulation, or done once treatment
has been discontinued. It must also be noted that PS
is physiologically low in pregnancy. Testing must there­
fore be postponed until at least 6 weeks post-partum.
V. Antithrombin
Antithrombin is the most potent inhibitor of coagula­
tion and exerts its action by inhibiting the activated
clotting factors FIIa, FXa, FXIa, FIXa by cleaving off a
part of the molecule. Its action is dramatically enhanced
by the presence of heparin.
Congenital antithrombin deficiency is uncommon with
prevalence rates of between 1 in 500 and 1 in 5000 being
reported. The risk of thrombosis is highly variable (5 to
50-fold) depending on the nature of the genetic defect.
The deficiency is either classified as type I (quantitative)
or type II (qualitative). Most cases are heterozygous.
Homozygosity for AT deficiency is rare as almost all
5
cases lead to foetal death. Deficiency is associated
with venous thromboembolism and pregnancy loss
but arterial events are unusual.
The nature of the functional abnormalities is highly
variable. Consequently not all variants are equally well
detected by the various AT assays available. This may
account for the wide variation in published prevalence
rates. Both antigen and functional assays exist. The appro­
priate first test for AT deficiency should be a functional
assay. There is no need to perform an antigen test, either
as a screening test or as a secondary assay if the activity
is normal. If the antigen level is normal it does not rule
out a type II deficiency and type II is much more common.
A significant number of cases of AT deficiency would be
missed if only antigen assays were used for screening
purposes.
AT activity is measured using a chromogenic assay. The
assay assesses the ability of AT, in the presence of hep­
arin, to cleave its target substrate. As the activated clot­
ting factors are inherently unstable, the assay uses an
artificial substrate as a substitute. The degree of colour
change observed is indirectly proportional to the amount
of AT present. The principle of the assay is shown in Fig. 3.
Heparin
AT + Thrombin excess + substrate-colour
[AT-Thrombin] + residual Thrombin = substate + colour
This is the unknown amount of
AT in the patient sample
Colour change
is measured
Reagent
Fig. 3 Principle of chromogenic functional AT assay. The reagent
contains excess thrombin (or FXa), heparin and a chromogenic
substrate. The AT in the plasma sample, binds heparin and inhibits
the thrombin. The residual thrombin is then free to cleave the
chromo­genic substrate. The lower the AT concentration, the less it
is able to inhibit the excess thrombin hence more thrombin (or FXa)
is available to act on and release the chromophore from the arti­­­-
ficial substrate. The colour change is measured and converted into
an AT value expressed as a percentage.
SEED Coagulation – Laboratory evaluation of thrombophilia
Sysmex Educational Enhancement and Development | April 2013
Acquired causes of AT deficiency include disseminated
intravascular coagulopathy, pregnancy associated micro­
angiopathies, liver disease, severe sepsis, nephrotic syn­
drome as well as being physiological in neonates and
infants.
Variable Influence
Warfarin therapy PC and PS will both be low.
Vitamin K deficiency PC and PS will both be low.
Pregnancy Physiological PS deficiency and APC
resistance.
Age <12 months Haemostatic system is immature, PC, PS
and AT significantly lower than adult levels.
Liver disease PC, PS and AT produced in liver. All may be
low.
Acute thrombosis Consumption of PC, PS and AT during acute
event.
Heparin therapy Prolongs all clot-based tests.
Extent of influence depends on individual
assay.
(Some reagents include heparin inhibitors
and therefore the concentration at which
heparin would give false results varies).
Haemolysed sample May interfere with chromogenic assays.
Extent of influence is assay specific.
The free plasma HGB will be included in the
total measurement of “colour” and give
false results.
Assays where activity of measurement
parameter is directly proportional to colour
change, e.g. PC, will be falsely high.
Assays where activity of measurement
parameter is indirectly proportional to
colour change, e.g. AT, will be falsely low.
Icteric (jaundiced) sample As above. Free bilirubin will contribute to
measured ‘colour’.
Tab. 3 Precautions for interpretation of coagulation test results
6
VI. Lupus Anticoagulant
Lupus anticoagulant (LAC) represents a family of several
different antibodies which may lead to thrombosis and/
or miscarriage.
Precaution
May falsely diagnose PC or PS deficiency.
May falsely diagnose PC or PS deficiency.
May falsely diagnose PC or PS deficiency.
May falsely diagnose PC, PS or AT deficiency.
Always interpret results with age specific reference ranges.
May falsely interpret low results as an inherited deficiency.
May falsely interpret low results as an inherited deficiency.
Best to postpone testing for suspected thrombophilia to a
later stage.
May give false impression of APC resistance and low PS.
LAC tests may be uninterpretable.
Results may be falsely high (PC) or falsely low (AT).
Most cases of haemolysis are due to traumatic collection
or delayed analysis. In this case a repeat sample should be
requested as other variables may be affected.
If the patient is actively haemolysing, and testing is required,
results must be interpreted with caution. The package insert
or analyser assay reference guide will state the concentration
above which results may be affected.
Plasma HGB can be measured and the sample diluted to
reduce the HGB concentration to below the threshold. The
sample can then be analysed and the result multiplied by the
dilution factor to obtain an estimate.
As above.
Plasma bilirubin can be measured, sample diluted, analysed
and result multiplied by dilution factor to obtain estimate.
SEED Coagulation – Laboratory evaluation of thrombophilia
Sysmex Educational Enhancement and Development | April 2013
Conclusion
The presence of the inherited thrombophilias would not
alter the acute management of the thrombotic episode and
therefore testing is best delayed until 6 months after the
acute event and only once anticoagulant treatment has
been discontinued for a couple of weeks. The likelihood
of finding an abnormality will depend on concomitant risk
factors, ethnicity, prevalence rate and relative risk of throm­
bosis associated with each abnormality. Although relative
risks are not well established, they range from AT deficiency
(the most severe) to PC/PS deficiency (intermediate) to
FVL/prothrombin 20210A mutation being least severe.
Tests for thrombophilia are not indicated in all patients,
especially if the cause of venous thromboembolism is obvi­
ous. If however a patient has an unexplained thrombosis
or a recurrent episode then testing may be helpful. Identifica­
tion of a thrombophilia would enable the clinician to ascer­
tain risk of recurrence and thereby determine duration of
treatment and make provision for prophylaxis during any
future high risk situations (e.g. surgery, immobilisation or
pregnan­­cy). Screening for the specific defect may also be
offered to first degree relatives as they may be carriers that
are still asymptomatic, and may benefit from counselling
and pos­sible primary prophylaxis during high risk situations.
LAC should be tested for in all patients as this may cause
a prolongation of the APTT and therefore interfere with hep­
arin monitoring. Likewise testing for AT deficiency is war­
ranted at the outset of treatment as effective heparin (and
LMWH) anticoagulation is dependent on the presence of
adequate levels of AT.
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7
Take home message
■■ There is no solid evidence that inherited abnormalities
are linked to arterial thrombosis – investigation of arterial
thrombosis should therefore be restricted to LAC testing.
Some centres include screens for homocysteine levels.
■■ Routine thrombophilia testing is not indicated in all
patients presenting with venous thrombosis – it should
be done on an individual basis.
■■ Patients with recurrent pregnancy loss, with or without
thrombotic episodes, should be tested for AT and LAC.
■■ If a decision to perform thrombophilia testing has been
taken, then a full battery of tests should be performed
as combine defects, which confer a higher risk, are well
described.
■■ Thrombophilia screening should include tests for LAC,
AT, PC, PS, APCR (FVL) and the prothrombin 20210A
mutation. Testing for FVL and prothrombin 20210A
should only be done in individuals of Caucasian descent
as these mutations are absent in blacks. Some centres
include screens for FVIII and homocysteine levels.
■■ Results must be interpreted in relation to age specific
reference ranges. PC, PS and AT values are much lower
in infants only reaching adult levels at about 6 months.
■■ Be aware of interfering variables when interpreting
results.
■■ Always exclude acquired causes before making a diagnosis
of an inherited thrombophilic abnormality.
References
[1] Pruthi RK and Heit JA. (2009): Laboratory evaluation of throm-
bophilia. Practical Hemostasis and Thrombosis, 2nded, editors Key,
Makris, O’Shaughnessy and Lilicrap, Chapter 3, pages 17–24.
[2] Tripodi A and Mannucci PM. (2001): Laboratory investigation
of thrombophilia. Clinical Chemistry 47(9): 1597-1606.
Compiled by
Dr Marion Münster