**Laboratory Testing and the Thrombophilia Workup**

Thomas L. Ortel, M.D., Ph.D.

From the Division of Hematology, Department of Medicine, and the
Department of Pathology, Duke University Medical Center, Durham, NC

Supported, in part, by grants from the National Institutes of Health
(HL077878; HL072289) and the Centers for Disease Control and Prevention
(U18 DD00014).

Correspondence to: Thomas L. Ortel, M.D., Ph.D.

Division of Hematology, Department of Medicine

Box 3422, Duke University Medical Center

Durham, NC 27710

Tel: 919 684-5350

Fax: 919 681-6160

e-mail:

**Introduction**

The clinical laboratory plays several essential roles in the diagnosis
and management of patients with venous thromboembolism. First, certain
laboratory tests can be helpful in the initial assessment of a patient
presenting with symptoms and clinical manifestations suggestive of
venous thromboembolism. Second, certain clinical laboratory tests play
essential roles in therapeutic monitoring of several of the
anticoagulant agents that are used to treat patients with venous
thromboembolism. Lastly, the clinical laboratory plays an essential role
in the identification of patients who might have specific inherited
and/or acquired risk factors for venous thromboembolism. Finding one or
more thrombophilic risk factors may have an impact on how long a patient
would need to stay on therapy after presenting with a new thromboembolic
event. This chapter will focus primarily on the use of the clinical
laboratory to identify which patients might have an increased risk for
an initial, or recurrent, venous thromboembolic event.

**What clinical laboratory tests are useful when a patient first
presents with symptoms suggestive of a blood clot?**

Routine initial laboratory studies that should be obtained on a patient
who presents with signs and/or symptoms suggestive of venous
thromboembolism should include a complete blood count, baseline
coagulation studies with a prothrombin time (PT) and activated partial
thromboplastin time (aPTT), and a serum creatinine. These tests
establish normal baseline parameters for the patient, which is necessary
to ensure correct antithrombotic therapy dosing.

One blood test that is particularly useful in the acute setting is the
D-dimer, which is elevated in patients who present with a new blood
clot. If the D-dimer level is *normal*, particularly in the setting of a
low clinical suspicion for venous thromboembolism, it is extremely
unlikely that the patient has had an event . An elevated D-dimer level
is less helpful, however, given the frequency of false-positive results,
particularly in certain patient populations .

For patients who present with an acute pulmonary embolism, cardiac
biomarkers can be useful for risk stratification . Elevated circulating
levels of cardiac troponins and brain natriuretic peptide reflect right
ventricular dysfunction in patients with acute pulmonary embolism. These
tests are particularly useful for their negative predictive value when
not elevated, by identifying patients with pulmonary embolism who are at
low-risk for in-hospital death . However, these biomarkers have not yet
been incorporated into formal guidelines for therapeutic decision-making
(*e.g*., anticoagulants vs. fibrinolytic therapy).

In general, a comprehensive laboratory workup for an inherited
thrombophilic state is not indicated when a patient initially presents
with a new thromboembolic event. In fact, several of the analytical
methods can be adversely affected by the acute thromboembolism and/or
the initial therapy used to treat the patient (for example, a low
functional protein S level can occur in the setting of acute
thrombosis). Furthermore, identification of an underlying thrombophilic
disorder generally does not have an impact on the early management of a
patient with a new thromboembolic event. An exception to this would be
the patient who presents with a new venous thromboembolism who also has
a prolonged activated partial thromboplastin time (aPTT) result. The
aPTT can be prolonged in patients with lupus anticoagulants, which can
interfere with the ability to monitor heparin therapy.

**Who should be considered for thrombophilia testing?**

Individuals who should be considered for evaluation for a
hypercoagulable state include the following groups: \[1\] patients with
spontaneous, or 'idiopathic', venous thromboembolism, particularly if 50
years of age or younger; \[2\] patients with thromboembolism that is
unusually extensive, or located in blood vessels not usually affected by
blood clots; \[3\] patients with recurrent thromboembolic events; and
\[4\] patients who have a strong family history for venous
thromboembolism . The potential clinical significance of positive test
results in these different populations is discussed further below.

**What blood tests should be done?**

A complete evaluation for an increased risk for venous thromboembolism
includes the laboratory tests listed in Table 1. These thrombophilic
disorders can be grouped into three broad classes . First, thrombophilic
states associated with *decreased* levels or function of the natural
anticoagulant proteins include deficiency states of antithrombin,
protein C, and protein S. These deficiency states are relatively
uncommon, but many patients with these disorders will have experienced a
venous thrombotic event by age 60 years . The second group of
thrombophilic states is associated with *increased* levels or function
of specific coagulation factors (*e.g*., factor V Leiden, prothrombin
G20210A polymorphism, elevated factor VIII levels). These disorders are
much more common than the deficiency states, but generally have a lower
risk for thrombosis .

The remaining thrombophilic disorders include the antiphospholipid
antibody syndrome, elevated homocysteine levels, and less common
disorders such as fibrinolytic defects, selected dysfibrinogenemias, and
elevated lipoprotein(a) levels . Testing for antiphospholipid antibodies
includes functional assays for lupus anticoagulants as well as serologic
assays for anticardiolipin antibodies and anti-~2~-glycoprotein I
antibodies. The presence of an antiphospholipid antibody in a patient
with a thromboembolic event needs to be reproduced on one additional
occasion, at least twelve weeks apart, in order to meet criteria for the
antiphospholipid syndrome . It is unclear if patients with a transient
antiphospholipid antibody (*i.e*., an antibody that is present on
initial testing, but not on repeat testing) have the same risk for
recurrent thromboembolism as patients with a persistent antiphospholipid
antibody.

When evaluating a patient for an inherited thrombophilic state, the
ethnicity and race of the individual being studied needs to be taken
into consideration . For example, factor V Leiden and the prothrombin
G20210A polymorphism are much less commonly identified in African
Americans with venous thromboembolism than Caucasians, even though the
frequencies of venous thromboembolism in these two groups are similar .
Notably, these two polymorphisms are essentially absent from the
indigenous peoples of Africa and Asia . Less information is available
for other patient populations or the other hypercoagulable states.

Evaluation for a thrombophilic disorder should also consider that more
than one inherited disorder might exist in an individual patient . In
addition, acquired prothrombotic risk factors can modulate the risk for
thrombosis in a patient with an inherited disorder (for example, the
increased risk of venous thrombosis in oral contraceptive users who are
carriers of the factor V Leiden polymorphism).

Testing for thrombophilia in children with 'spontaneous' venous
thromboembolism requires a different strategy than for adults. In these
patients, more than one thrombophilic state is frequently present, which
is associated with a substantially increased risk for recurrent
thromboembolism . Pediatric patients with elevated levels of factor VIII
and/or D-dimers are also at an increased risk for recurrent venous
thromboembolism .

**How do the results of these laboratory tests affect the clinical
management of patients with venous thromboembolism?**

As noted above, the presence of a thrombophilic disorder has relatively
little impact on the initial management of patients with venous
thromboembolism. Most patients with venous thromboembolism will be
initially treated with unfractionated heparin or a low-molecular weight
heparin. A subset of patients will receive thrombolytic therapy, but
this decision will depend on clinical manifestations and not the
presence of an inherited hypercoagulable state. Patients are
subsequently converted to oral anticoagulation with a vitamin
K-antagonist (e.g., warfarin) with a target international normalized
ratio (INR) of 2 to 3. The target INR is not adjusted for patients with
venous thrombosis and an inherited thrombophilic disorder . Although
earlier, retrospective studies suggested that patients with
antiphospholipid antibody syndrome might need to be maintained at a
higher target INR (2.5 to 3.5), more recent prospective studies have
demonstrated that the majority of these patients can be effectively
anticoagulated with a target INR of 2 to 3 .

The primary impact of identifying one or more inherited prothrombotic
risk factors is on the duration of anticoagulant therapy. For patients
with a thromboembolic event in the setting of a transient risk factor,
such as recent surgery, it is generally agreed that a limited course of
anticoagulant therapy is indicated (*e.g*., 3 to 6 months) . For
patients with an idiopathic thromboembolic event, the American College
of Chest Physicians guidelines recommend 6 to 12 months of therapy, with
consideration for a more extended course in certain circumstances.
Variables that would potentially impact on the decision to recommend an
extended course of therapy (*i.e*., greater than 12 months) include the
presence of one or more inherited risk factors, the continued presence
of acquired risk factors (*e.g*., malignancy), the severity of the
clinical event, other co-morbid conditions, and bleeding risk. The
presence (or absence) of an inherited hypercoagulable state may
contribute to the decision of how long to treat an individual patient,
but this decision must be individualized with careful consideration of
the risks and benefits of continued warfarin therapy.

Identification of an inherited hypercoagulable state in selected
patients may also impact in other ways. For example, identification of
an inherited risk factor may provide an 'explanation' for why a patient
sustained a thromboembolic event. It can also identify a specific
disorder that needs to be considered when evaluating family members,
whether symptomatic or not, as discussed further below.

**Should asymptomatic individuals be screened for thrombophilic
states?**

Several authorities have recommended screening for thrombophilic states
in asymptomatic first-degree relatives of an affected individual with a
known inherited thrombophilia . Screening for hypercoagulable states may
also be cost-effective in selected patients in high-risk situations in
the absence of a known familial disorder (*e.g*., selective screening of
women prior to using hormone replacement therapy) . Indiscriminate
testing for thrombophilic disorders is generally inappropriate, however,
and should be avoided .

There is relatively little evidence-based medicine to guide how the
results of thrombophilic testing should be used in clinical practice.
For example, the presence of "molecular hypercoagulability" places
surgical patients in a "high risk" category for venous thromboembolism,
which generally leads to more aggressive recommendations for
thromboprophylaxis with certain surgical procedures . Prospective
studies documenting the utility of this strategy for specific inherited
hypercoagulable states are lacking. Inherited hypercoagulable states are
also associated with a significant risk for venous thromboembolism
during pregnancy . Although little information is available concerning
the efficacy of prophylactic interventions in pregnant patients with a
thrombophilic risk factor but no prior personal history of
thromboembolism, recommendations for thromboprophylaxis vary depending
on the thrombophilic defects identified .

Individuals who test positive for a thrombophilic disorder need
counseling in regards to: \[1\] the risk of thrombosis for themselves
and their family members; \[2\] signs and symptoms suggestive of a
venous thromboembolic event which need medical evaluation; and \[3\] the
risks and benefits of antithrombotic prophylaxis in various clinical
settings (*e.g*., during pregnancy, postoperative) . Testing should not
be performed in the absence of appropriate counseling, to avoid
confusion, false reassurance, and unnecessary anxiety . Unfortunately,
one survey found that more than half of patients diagnosed with factor V
Leiden did not feel that their healthcare provider understood the
disorder .

**Future directions**

Prospective clinical studies are urgently needed to identify the role(s)
of thrombophilia testing in individuals with venous thromboembolism.
Specific areas that need to be addressed include: \[1\] impact of
specific thrombophilic disorders on anticoagulant therapy management of
patients with venous thrombosis; and \[2\] identification of optimal
prophylactic strategies for asymptomatic individuals with an inherited
thrombophilic risk factor during high-risk situations.

**Table 1. Thrombophilia Workup.**

-   Testing for Deficiencies of Natural Anticoagulant Proteins

```{=html}
<!-- -->
```
-   Antithrombin functional assay.

-   Protein C functional assay.

-   Protein S functional assay, with antigenic testing for free and
    total protein S if decreased.

```{=html}
<!-- -->
```
-   Testing for Disorders Associated with Increased Levels or Function
    of Coagulation Proteins

```{=html}
<!-- -->
```
-   Activated protein C resistance, with factor V Leiden analysis if
    positive.

-   Prothrombin G20210A genotype analysis.

-   Factor VIII assay.

```{=html}
<!-- -->
```
-   Testing for Other Disorders Associated with an Increased Thrombotic
    Risk.

    -   Antiphospholipid antibody testing.

        -   Lupus anticoagulant testing.

        -   Anticardiolipin antibody testing.

        -   Anti-~2~-glycoprotein I antibody testing.

```{=html}
<!-- -->
```
-   Homocysteine level.

-   Dysfibrinogenemias.

-   Fibrinolytic defects.

-   Lipoprotein (a).