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Thrombophilia: Laboratory Support of Diagnosis and Management.

Thrombophilia: Laboratory Support of Diagnosis and Management

Clinical Focus

Thrombophilia

Laboratory Support of Diagnosis and Management

  

Contents:

Clinical Background  - Table 1 - Table 2 - Table 3

Individuals Suitable for Testing

Test Availability

Test Selection - Figure

Test Interpretation - Table 4 - Table 5 - Table 6

Sample Collection Considerations

References

Appendix 1

Appendix 2
 

Clinical Background [return to contents]

Thrombophilia is characterized by hypercoagulability and an increased propensity for thrombosis. Almost 2 million Americans succumb annually to a thromboembolic event,1 with venous thrombosis the third most common cardiovascular disease after ischemic heart disease and stroke. Venous thrombosis affects 1 to 2 in 1000 individuals every year and is associated with life-threatening conditions such as pulmonary embolism (PE).1 Though less clearly delineated, hypercoagulability is also believed to play a role in the pathogenesis of arterial thrombosis.2

Conditions associated with an increased risk of venous thrombosis can be either inherited or acquired (Tables 1 and 2). One or more predisposing factors are identifiable in 80% of individuals with a first episode of thrombosis, and an inherited cause of thrombophilia can be identified in approximately 30% to 35% of individuals with a first thrombotic episode.3 Manifestations include deep vein thrombosis (DVT) of the lower limbs, PE, superficial thrombophlebitis, mesenteric or cerebral vein thrombosis, fetal loss (spontaneous abortion or stillbirth), preeclampsia, and neonatal purpura fulminans.

Table 1. Inherited Conditions Associated with Venous Thrombosis [return to contents]
Condition

Frequency (%) in Healthy Individuals

Frequency (%)

in Patients

with Venous

Thrombosisa

Relative Risk

(%) of

Thrombosis

Activated protein C resistance/factor V mutationb,c,4,5

5

21

3-7

Antithrombin deficiency6

0.02-0.17

1

15-40

Factor VIII excessd,7

11

25

6

Hyperhomocysteinemiae,8,9

5-10

10-25

3-4

Protein C deficiency6

0.3

3

5-12

Protein S deficiency6

0.7

2

4-10

Prothrombin (factor II) 20210G>A mutationb,10,11

2

6

2-3

Other rare inherited conditions that may be associated with inherited venous thrombosis include sticky platelet syndrome, heparin cofactor II deficiency, factor XII deficiency, and dys- and hyperfibrinogenemia.

Deficiency of antithrombin, protein C, and protein S may also be acquired.

a Data is for heterozygotes.

b Affects primarily Caucasian population.

c Homozygosity of the factor V HR2 allele increases the risk of venous thrombosis 3- to 4-fold in the presence of the factor V Leiden mutation (no increase if factor V Leiden mutation is absent).

d Can also be acquired.

e Dietary deficiency of folate and/or vitamins B6 and B12.

Table 2. Acquired Conditions Associated with Venous Thrombosis [return to contents]

Antiphospholipid syndrome (most common cause)

Autoimmune disorders (eg, systemic lupus erythematosus)

Combined oral contraceptives

Elevated factor IX, XI

Endocrine disorders (eg, diabetes mellitus, Cushing syndrome)

Heparin-induced thrombocytopenia (HIT)a

Hormone replacement therapy

Liver disease

Malignancyb

Myeloproliferative disorders (eg, polycythemia vera, chronic myelogenous leukemia)

Nephrotic syndrome

Obesity

Paroxysmal nocturnal hemoglobinuria

Pregnancy and puerperium

Thrombotic thrombocytopenic purpura

a Should be considered in any individual who has received heparin within the 30 days preceding a thrombotic episode and has a decrease in platelet count to <100,000/µL or more than 50% of baseline.

b A thrombotic event can precede the diagnosis of malignancy by months to years.

Individuals at high risk for venous thrombosis include those with a personal or family history of thrombosis, inherited coagulation disorders, homocystinuria, paroxysmal nocturnal hemoglobinuria, essential thrombocythemia, polycythemia vera, recurrent spontaneous abortion and stillbirth, and malignancy. Additional risk factors include surgery, trauma, physical inactivity (bed confinement or paralysis), warfarin induced skin necrosis, diabetes, hyperlipidemia, vasculitis, thrombocytopenia, sepsis, congestive heart failure, and use of purified prothrombin complex concentrates. Other factors that may be associated with increased thrombotic risk include plasminogen deficiency and elevations of plasminogen activator inhibitor-1, lipoprotein (a), D-dimer, and thrombin-activatable fibrinolysis inhibitor. 

The risk of thrombosis increases with the number of defects or risk factors present; ie, individuals with multiple conditions associated with thrombosis are at greater risk than those with only one condition.1 Risk factors for venous thrombophilia are generally not associated with risk of arterial thrombosis, with the exception of hyperlipidemia, antiphospholipid syndrome (Appendix 1), elevated homocysteine, protein S deficiency, and dysfibrinogenemia.2

The identification of thrombotic risk factors and diagnosis of thrombophilia contributes to patient management in multiple ways (Table 3). Such diagnosis is based on personal and family history of thrombosis (especially during adolescence and young adult years), clinical manifestations, and laboratory testing.

Table 3. Value of Thrombophilia Diagnosis [return to contents]
Identify pathologic basis of thrombotic event
Aid in selection of appropriate therapy
Determine duration and intensity of treatment
Determine need for prophylaxis
Estimate future thrombotic risk
Determine degree of risk associated with oral contraceptives and estrogen replacement therapy
Determine need for evaluation of family members

Clear guidelines how to best manage individuals with a family or personal history of documented risk factors and who have not experienced a thrombotic episode have not been established. The decision for prophylactic therapy should be based on an individual’s clinical history.12 Screening general populations for inherited disorders associated with venous thrombosis is not recommended; however, the clinical utility of global screening assays in high-risk populations is being evaluated.

Patients with acute thrombosis are treated with intravenous heparin or oral anticoagulants such as warfarin (Coumadin®). Prophylactic treatment is provided to diagnosed patients when in high-risk situations, eg, surgery, prolonged immobilization, and pregnancy and puerperium. Lifelong prophylactic therapy may be considered for those with recurrent thrombotic episodes, high-risk disorders, or with multiple-risk factors and may include plasma transfusions (eg, antithrombin concentrates), oral anticoagulants, low dose aspirin, and heparin.12 Heparin is of limited benefit post thrombosis in patients with antithrombin deficiency, however, and heparin selection for pregnant women should be individualized due to risk of bone fracture.13 Low molecular weight heparin (LMWH) may be a better option for those at risk of osteoporosis since LMWH does not cause bone thinning. Individuals with hyperhomocysteinemia may be treated with vitamin supplementation (folic acid, cobalamin, pyridoxine).

Individuals Suitable for Testing [return to contents]

  • Symptomatic individuals

  • Individuals with a personal or family history of unprovoked thrombosis, thrombosis at a young age (<45 years), or thrombophilia-associated mutations

  • High-risk individuals predisposed by surgery, trauma, immobility, pregnancy, oral contraceptives, etc.

Note: high-risk pregnant women include those with a personal or family history of thrombosis, previous neural tube defect affected fetus, recurrent spontaneous abortions, severe early onset preeclampsia, cesarean section, obesity, advanced maternal age, higher parity, and prolonged immobilization.13

Test Availability [return to contents]

Tests available to assist in diagnosis and management of thrombophilia disorders are listed in Appendix 2. Additionally, Quest Diagnostics offers panels that include multiple tests, thereby simplifying the test ordering process. Refer to the Quest Diagnostics Directory of Services for information on these panels, which are typically named according to the medical condition.

Test Selection [return to contents]

Diagnosis

A venous thrombosis laboratory work-up for high-risk or symptomatic individuals begins with a personal and family history. Test selection may vary for each individual based on his/her history as well as a particular defect’s prevalence in specific populations. For example, venous thrombosis in a pediatric patient suggests the likelihood of an inherited disorder; in an individual with SLE, antiphospholipid syndrome should be considered; and in an older individual, malignancy. Testing for multiple etiologies is recommended since venous thrombosis is a polyfactorial disorder, and presence of multiple etiologies increases the risk for thrombosis.14,15 Generally accepted testing guidelines suggest the use of first and second line testing in the thrombophilia diagnosis (Figure).14,17

Figure. Individual with Documented Thrombotic Episode or at High Risk

First line testing for an individual with venous thrombosis typically includes a CBC with smear and aPTT; activated protein C resistance (APCR); functional (activity) assays for antithrombin, proteins C and S, and factor VIII; prothrombin 20210G>A mutation detection; homocysteine; and anticardiolipin and antiphospholipid antibodies (see Appendix 1). APCR and prothrombin 20210G>A mutation detection need not be performed initially in non-Caucasian individuals since these disorders are primarily observed in Caucasians. Likewise, if a first thrombotic event occurs after the age of 50, testing for protein C, S, and antithrombin deficiency may be postponed as hypercoagulability due to these disorders usually manifests as thrombosis earlier than the fifth decade. Testing for heparin-induced thrombocytopenia should be considered for any individual who has received heparin within the 30 days preceding a thrombotic episode and has a decrease in platelet count to <100,000/μL or more than 50% of baseline. Additional testing directed toward diagnosis of other causes of acquired thrombophilia such as systemic lupus erythematosus, liver disease, nephrotic syndrome, polycythemia vera (JAK2 mutations), chronic myelogenous leukemia (BCR-ABL1 gene rearrangement), diabetes mellitus, Cushing syndrome, etc. may be indicated (see Table 2).

Positive functional assays can be confirmed by genetic testing in some cases or by demonstration of the abnormality in another family member. For example, a borderline or positive APCR test can be confirmed with factor V (Leiden) mutation analysis. Such analysis differentiates homozygous and heterozygous states, providing additional prognostic information. Factor V HR2 allele mutation analysis provides even more prognostic information in factor V (Leiden) carriers. Homocysteine elevations may be due to an acquired nutritional deficiency (vitamin B12, B6, or folate). Acquired causes for antithrombin, protein C, and protein S deficiencies can be ruled out by liver function testing, a disseminated intravascular coagulation screen (D-dimer, fibrin degradation product, PT, aPTT, fibrinogen, platelet count), and a proteinuria test (urine albumin).18 Decreased antithrombin and protein C and S activities (function) can be further characterized as a deficiency [type I or III (protein S only)] or dysproteinemia (type II) by using antigenic assays; however, such characterization will not affect treatment decisions.

If all of the aforementioned testing is negative, the patient may have a rare disorder that can be identified by testing for factors IX and XI, lipoprotein (a) [Lp(a)], plasminogen activity (function), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (TPA); evaluation for dysfibrinogenemia may also be helpful (Figure). Testing for rare disorders is only recommended for individuals with a strong personal and family history of thrombosis and negative first line tests or in whom clinical suspicion is high. Since all thrombophilia etiologies are not yet known, it is possible for all of these tests to be negative.

Management

Since individuals with variations in the CYP2C9 and VKORC1 genes may require lower warfarin doses, mutation analysis should be performed to assist in selecting the initial dosage and to prevent over anticoagulating the patient. Warfarin therapy can be monitored using the prothrombin time test, reported as INR, except in 1) some patients with a strong lupus anticoagulant and 2) patients receiving direct thrombin inhibitors. For these patients, monitoring with chromogenic factor X is preferred.

When injectible anticoagulants are used, patients can be monitored using a Xa inhibition assay. Quest Diagnostics offers 3 such tests, 1 for each class of injectibles (LMWH, unfractionated heparin, or synthetic LMWH [fondaparinux])). See Appendix 2 under Heparin and Fondaparinux for test details.

Test Interpretation [return to contents]

Acute thrombosis, anticoagulant therapy, drug therapy, and certain medical conditions can affect the results and interpretation of tests used to diagnose causes of thrombophilia (Tables 4 and 5). Additional interpretive information, specific to each test, is provided below.

Table 4. Confounding Effects of Anticoagulant Therapy and Acute Thrombosis on Testing Used in the Diagnosis of Thrombophilia [return to contents]
Test

Confounding Effect

Warfarin Heparin Acute Thrombosis

Antithrombin

None (rarely increases)a

Decreases

May decreaseb

Antiphospholipid antibodies

None

Potential false
positive

None

APCR/Factor V Leidenc

Nonsensical results

Noned

None

aPTT

May increase

Increases

Increases

Homocysteine

None

None

None

Lupus anticoagulant

None when confirmatory testing is performed

None when confirmatory testing
is performedd

None

Protein C

Decreases

Noned

May decreaseb

Protein S

Decreases

Noned

May decreaseb

Prothrombin gene mutation

None

None

None

Reptilase time

None

None

Increases

Thrombin time

Increases

Increases

Increases

APCR, activated protein C resistance; aPTT, activated partial thromboplastin time.

a Can be increased to normal range in individuals with heterozygous deficiency.

b Results are inaccurate during acute thrombosis; however, a normal level during an acute thrombotic event essentially excludes deficiency of these proteins.

c Mutation analysis not affected.

d Heparin up to 1 U/mL does not affect results.

Table 5. Conditions That Affect Testing for Inherited Causes of Thrombophilia [return to contents]

Condition Effect

Lupus anticoagulant

Decreases APCR, protein S, and intrinsic clotting factors

Deficiency of vitamin B12, B6, or folate

Methotrexate, phenytoin, theophylline

Hypothyroidism, malignancy, menopause

Increases homocysteine

Oral contraceptives

Estrogen replacement

Decreases antithrombin and protein C and S

Pregnancy and puerperium

Decreases APCR and protein C and S

Increases homocysteine

Acute phase reaction, inflammation, infection

Increased factor VIII

Decreases protein S

Kidney disease/nephrotic syndrome

Decreases antithrombin and protein S

DIC, liver disease, sepsis, l-asparaginase therapy

Decreases antithrombin and protein C and S

Surgery, recent thrombosis

Decreases antithrombin and protein C and S

Increases homocysteine

Vitamin K deficiency

Decreases protein C and protein S

APCR; activated protein C resistance, DIC; disseminated intravascular coagulation.

AccuType® Warfarin

The cytochrome P450 enzyme CYP2C9 participates in the metabolism of a number of important drugs, including warfarin. Individuals carrying variants in the CYP2C9 gene (CYP2C9*2, CYP2C9*3, CYP2C9*5, and CYP2C9*6) have reduced metabolism of warfarin, and those with 2 copies of variant alleles are at high risk of life-threatening side effects. Presence of the -1639G>A mutation in the VKORC1 gene leads to a decrease in the level of vitamin K-dependent clotting factors and increased sensitivity to warfarin. VKORC1 and CYP2C9 polymorphisms, together with clinical factors, account for 50% to 60% of the variability in an individual’s response to warfarin21-23; individuals with these polymorphisms may require lower warfarin doses.

Activated Partial Thromboplastin Time (aPTT)

The aPTT will be prolonged if there is deficiency or inhibition of factors of the intrinsic pathway including high molecular weight kininogen (HMWK), prekallikrein, factors V, VIII, IX, X, XI and XII, prothrombin, and fibrinogen. Prolongation is also seen in individuals with lupus anticoagulant.

Activated Protein C Resistance (APCR)

A decreased ratio of clotting times obtained with and without exogenous activated protein C is suggestive of activated protein C (APC) resistance and increased risk of deep vein thrombosis. Assay sensitivity and specificity approach 100%, even in the presence of anticoagulants and heparin.24,25 In most cases, APCR is caused by a factor V (Leiden) mutation. In <5% of cases, APCR is found without a corresponding factor V (Leiden) mutation, perhaps indicative of an unknown mutation. Such cases are also associated with increased venous thrombosis risk.26

Antithrombin

Decreased levels of antithrombin are associated with an increased risk of both arterial and venous thrombosis and are seen in individuals with hereditary antithrombin deficiency, nephrotic syndrome, colitis, liver disease, active thrombosis, disseminated intravascular coagulation (DIC), those receiving l-asparaginase therapy or oral contraceptives, and individuals who are pregnant or have undergone surgery. Levels are also decreased in individuals receiving heparin. Levels in neonates are approximately half of the adult level, which is reached by 6 months of age. Low levels in both the activity and antigen assays indicate type I deficiency, whereas low activity levels in the presence of normal antigen levels indicate type II deficiency (dysproteinemia). Increased levels may be due to oral anticoagulants or heparin cofactor II.

C4 Binding Protein

Approximately 65% of protein S circulates in plasma bound to C4 binding protein. Increased levels of C4 binding protein may cause decreased levels of free protein S, and subsequent increased risk of thrombosis, and are associated with inflammation, pregnancy, diabetes mellitus, SLE, AIDS, allograft rejection, estrogen and progesterone administration, and smoking.

Cardiolipin Antibodies

Anticardiolipin antibodies of the IgA, IgG, and IgM isotype are associated with the antiphospholipid syndrome and, when >40 GPL units, increase the risk for venous thrombosis 5- to 8-fold. IgG antibodies appear to be more predictive of disease activity, while IgM antibody occurs more often in drug-induced disorders and infectious disease (eg, syphilis). Higher antibody titers are generally correlated with greater thrombotic risk (see Appendix 1).

D-Dimer

Elevated levels are associated with myocardial infarction, deep vein thrombosis, pulmonary embolism, DIC and other coagulation disorders, surgery, trauma, sickle cell disease, liver disease, severe infection, sepsis, inflammation, malignancy, pregnancy, and hyperfibrinolysis. When clinical probability is low, a negative result (normal level) essentially rules out DVT.27

dRVVT Screen with Reflex to dRVVT Confirm and dRVVT 1:1 Mix

The dRVVT screen and dRVVT confirm are intended for the detection of a lupus anticoagulant. The direct activation of factor X in the test system bypasses the intrinsic pathway of coagulation, thereby excluding interference from deficiencies and/or inhibitors of factors VIII, IX, XI, and XII. An uncorrected dRVVT in the mixing study rules out factor deficiencies, specifically those induced by warfarin therapy. A falsely prolonged dRVVT test may occur when heparin is >1.0 IU/mL. A false-negative dRVVT test may be due to platelet contamination of the plasma. Samples with moderate or severe icterus or lipemia are contraindicated.

Factor V HR2 Allele DNA Mutation Analysis

The HR2 allele is associated with APCR and increased risk of venous thrombosis in individuals also heterozygous for the factor V (Leiden) mutation. Such co-inheritance increases the risk of venous thromboembolism 3- to 4-fold when compared with factor V (Leiden) alone. An individual heterozygous positive for the HR2 allele and negative for factor V (Leiden) is not at increased risk of thrombosis compared to factor V (Leiden) alone. However, homozygosity for factor V HR2 is associated with increased risk of thrombosis even in the absence of a factor V (Leiden) mutation.

Factor V (Leiden) Mutation Analysis

The 1691G>A factor V Leiden mutation results in the laboratory finding of APCR. Factor V (Leiden) confers an approximately 7-fold increase in venous thromboembolic events in heterozygous individuals and an 80-fold increase in homozygous subjects.28 When a heterozygous mutation is coupled with oral contraceptive use, the risk increases synergistically to 30-fold.29 The mutation is also associated with arterial thrombosis (especially in smokers), complications of pregnancy (including fetal loss),30 and increased levels of factor VIII. Although this test is highly specific, identification of a mutation may occur in the absence of APCR in rare cases. A negative result does not rule out APCR or an increased risk of venous thrombosis.

Factor VIII

Factor VIII is an acute phase reactant and increased levels are found during periods of stress, postoperatively, and in inflammatory conditions. Elevated levels are also found at birth and during pregnancy. Increased levels are associated with increased risk for venous thrombosis,31 whereas decreased levels are associated with hemophilia A.

Fibrin Monomer

The presence of soluble fibrin monomer complexes in plasma indicates intravascular thrombin generation. It can be used to support diagnosis of DIC in the context of other laboratory and clinical findings.

Fibrinogen

Increased levels are associated with acute phase reactions, pregnancy, and an increased risk of thrombosis. Low fibrinogen activity levels are associated with afibrinogenemia, hypofibrinogenemia, or dysfibrinogenemia (which may be associated with thrombophilia in rare instances), as well as with DIC, systemic fibrinolysis, pancreatitis, severe hepatic dysfunction, and l-asparaginase or valproate treatment. Individuals with afibrinogenemia or hypofibrinogenemia will have decreased activity and antigen levels. Individuals with dysfibrinogenemia will typically have decreased activity levels and normal or decreased antigen levels.

Fibrinogen Degradation Products (FDP)

FDP result from the breakdown of fibrinogen, as well as fibrin, by plasmin. Normally, the fibrinolytic process is localized to fibrin, however, during conditions such as DIC, fibrinolysis spreads and becomes systemic. Elevated levels of FDP are seen in many clinical states (eg, DVT and PE); thus measurement of FDP is useful for their diagnosis. Persistent elevations indicate that abnormal fibrinolysis and fibrinogenolysis are occurring.

Fondaparinux Sodium (Xa Inhibition)

Fondaparinux is a synthetic pentasaccharide administered subcutaneously and used to prevent or treat thromboembolic conditions. Measurement is used to monitor therapeutic levels. The therapeutic range is 1.20-1.26 μg/mL, and the prophylactic range is 0.39-0.50 μg/mL. These ranges are applicable to samples collected approximately 3 hours after administration of the drug.

β2-Glycoprotein I Antibodies

β2-Glycoprotein I antibodies of the IgA, IgG, and IgM isotype are associated with the antiphospholipid syndrome, and their presence is more specific but less sensitive than cardiolipin antibodies for the diagnosis of antiphospholipid syndrome. Individuals who are positive for cardiolipin antibodies and negative for β2-glycoprotein I antibodies are more likely to have an infection (varicella, rubella, adenovirus, HIV) or drug exposure (amoxicillin, chlorpromazine, hydralazine) than antiphospholipid syndrome (Appendix 1).

Heparin Anti-Xa (Low Molecular Weight Heparin)

LMWH are prepared by the chemical or enzymatic degradation of unfractionated heparin, and are used in the prevention and treatment of thromboembolic conditions. Measurement of LMWH in plasma is used to monitor therapeutic levels. The therapeutic and prophylactic ranges for samples collected 4 hours after subcutaneous administration are shown in Table 6.

Table 6. Therapeutic and Prophylactic Ranges for Low Molecular Weight Heparin
[return to contents]
Medication Target Range, U/mL
Dalteparin therapy, once daily >1.05

Enoxaparin therapy

Twice daily

Once daily

 

0.6-1.0

 >1.0

Tinzaparin therapy, once daily >0.85

PCI, percutaneous coronary intervention.

Heparin Anti-Xa (Unfractionated Heparin)

Unfractionated heparin is used for the prevention and treatment of thromboembolic conditions and measurement is used to monitor therapeutic levels. When administered as an intravenous infusion, the therapeutic range is 0.30 to 0.70 IU/mL.

Homocysteine

Levels are increased in the following: cardiovascular disease, vitamin B12 and folate deficiencies, chronic renal disease, homocystinuria, hypothyroidism, selected malignancies, individuals whose diet is rich in methionine (high meat intake), cigarette smokers, and in individuals treated with corticosteroids, methotrexate, cyclosporin, vitamin B6 antagonists (isoniazid, azauridine, penicillamine, procarbazine), anticonvulsants (phenytoin, carbamazepine), and S-adenosyl-methionine. When coupled with the factor V (Leiden) mutation, venous thrombosis risk increases synergistically.32 Falsely increased levels may occur if serum or plasma is not separated from the red cells within 1 hour of collection.

Homocysteine is decreased in pregnancy (except in some women carrying a fetus with a neural tube defect), individuals less than 15 years of age, and individuals taking oral contraceptives or hormone replacement therapy.

Human Platelet Antigen 1 (HPA-1) Genotype
The HPA-1b platelet antigen polymorphism is associated with increased platelet thrombogenecity, neonatal alloimmune thrombocytopenia, and post-transfusion purpura.

Lipoprotein (a) [Lp(a)]

Increased levels of Lp(a) are observed in patients with coronary artery disease, stroke, cerebrovascular and peripheral vascular disease, and venous thrombosis. Substantial increases are secondarily (not genetically related) observed in nephrotic syndrome and end-stage renal disease. Decreased Lp(a) levels may be seen in several rare disorders (lecithin:cholesterol acyltransferase [LCAT] deficiency, lipoprotein lipase [LPL] deficiency, liver disease). Normal levels in the African American population may be 2 to 3 times the values in Caucasian and Asian populations. Native Americans and Mexican Americans have lower normal levels (no lower than one half) relative to the Caucasian and Asian populations.

Mixing/Correction Study

Test results are consistent with an intrinsic factor deficiency when a prolonged aPTT is normalized after mixing patient plasma with normal plasma and the normalized result does not reverse after incubation of the mixed sample. A prolonged aPTT that normalizes immediately after mixing with normal plasma, and does not reverse after incubation, indicates an intrinsic or common pathway factor deficiency. A prolonged aPTT that remains prolonged after mixing with normal plasma, or an aPTT that normalizes immediately after mixing with normal plasma but prolongs after incubation, is indicative of an inhibitor (eg, factor-specific inhibitor, lupus anticoagulant, or heparin). A prolonged PT that is corrected in the mixing study, along with a normal aPTT, suggests a factor VII deficiency. Test results are consistent with a single or multiple deficiency of factors II, V, or X (common pathway) when a prolonged PT is normalized after mixing studies and when a prolonged aPTT normalizes after mixing studies and remains normalized after incubation.

Phosphatidylserine, Phosphatidylinositol, Phosphatidylcholine, Phosphatidylethanolamine, Phosphatidylglycerol, and Phosphatidic Acid Antibodies

These antibodies may be associated with thrombosis and/or recurrent pregnancy loss, clinical features of antiphospholipid syndrome (see Appendix 1).

Plasminogen Activator Inhibitor-1 (PAI-1)

Increased levels of PAI-1 antigen and PAI-1 activity are associated with decreased fibrinolytic activity and increased risk for venous thrombosis and coronary artery disease. Interpretation of increased levels is confounded by circadian variation (morning values being about 2-fold higher than afternoon values), increases associated with the acute phase response (eg, following myocardial infarction, major surgery, severe trauma, or sepsis), as well as increases associated with normal pregnancy and disorders such as endotoxemia, liver disease, obesity, hyperinsulinemia, hypertriglyceridemia, and malignancy. Severely decreased or undetectable levels may be associated with bleeding problems. The antigenic, but not the activity test can help distinguish between a constitutional deficiency of PAI-1 and a dysproteinemia.

Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism

The 4G allele is associated with increased PAI-1 antigen and activity levels. Similar to PAI-1 antigen and activity levels, data regarding utility of the 4G/5G polymorphism in predicting venous thrombosis is conflicting.33 It may be more useful when co-inherited with another thrombophilia marker. When co-inherited, the 4G allele may further increase the risk of thrombophilia. For example, Visanji et al found the risk for venous thrombosis increased approximately 2-fold in patients with at least 1 copy of the 4G allele (4G/4G or 4G/5G genotypes) relative to that in patients with the 5G/5G genotype.34 All patients were heterozygous for factor V (Leiden) mutation and had experienced at least 1 venous thromboembolic event. Furthermore, Zoller et al reported an approximate 4-fold increase in the risk of pulmonary embolism among subjects with hereditary protein S deficiency who were homozygous for the 4G allele.35

Plasminogen

Decreased levels are associated with liver disease, DIC, thrombolytic agents, primary fibrinolysis, tissue plasminogen activator and, rarely, with venous thrombosis and pulmonary embolism (homozygous state only). Increased levels are associated with trauma, infection, acute myocardial infarction, surgery, and chronic inflammation. A functional assay is usually the preferred assay because the presence of a normal amount of antigen does not exclude a qualitative defect of the protein. The antigen level is most often obtained to assess a quantitative abnormality of the protein.

Protein C

Decreases are associated with venous thrombosis, recurrent superficial thrombophlebitis, neonatal purpura fulminans, arterial thrombosis (rarely), oral anticoagulant-induced skin necrosis, DIC, infection, acute illness such as the flu or a gastrointestinal disorder, malignancy, liver disease, vitamin K deficiency, surgery, and L-asparaginase therapy. Low values may be obtained in individuals on oral anticoagulant therapy. Heparin levels up to 1 IU/mL do not interfere with test results for antigen and activity assays. Protein C levels are significantly decreased in neonates; adult levels are reached only after 10 years of age. Low levels in both activity and antigen assays are suggestive of type I deficiency, whereas low activity levels in the presence of normal antigen levels indicate type II deficiency (dysproteinemia). A rare hereditary deficiency can be confirmed by demonstration of deficiency in another family member.

Elevations in protein C are not considered clinically relevant.

Protein S

Decreases are associated with increased risk for venous, and possibly arterial, thrombosis; oral anticoagulant-induced skin necrosis; neonatal purpura fulminans; DIC; acute phase reactions; oral anticoagulants; vitamin K deficiency; liver disease; surgery; L-asparaginase therapy; oral contraceptives; estrogen replacement therapy; pregnancy; nephrotic syndrome; infections (HIV, varicella); Crohn’s disease; and ulcerative colitis. Levels are significantly decreased in neonates; however, adult levels are reached by 6 months of age. Low levels in both activity and antigen (free and total) assays are suggestive of type I deficiency, whereas low activity in the presence of normal total antigen levels indicate type II deficiency (dysproteinemia). Type III deficiency is characterized by low levels in the activity and free antigen assays, but normal levels in the total antigen assay. A rare hereditary deficiency can be confirmed by demonstration of deficiency in another family member. Protein S levels are not affected by heparin (up to 1 IU/mL) or factor VIII (up to 250%).

Prothrombin (Factor II) Mutations

The prothrombin 20210G>A mutation is associated with increased prothrombin levels, increased risk for venous thrombosis,11 increased risk for obstetric complications (eg, preeclampsia, abruptio placenta, fetal growth retardation, and stillbirth),30 and, possibly, premature coronary heart disease. Venous thrombosis risk increases synergistically in the presence of oral contraceptive use.36 The combination may also lead to cerebral sinus and spinal vein thromboses.

Another prothrombin mutation, 19911A>G, is associated with a slight elevation in plasma prothrombin levels and increased venous thrombotic risk. The 19911G>A mutation may exacerbate the venous thrombotic risk linked to factor V (Leiden) or prothrombin 20210G>A mutations.19,20

Prothrombin Fragment 1.2

Prothrombin fragment 1.2 is the amino terminus fragment of prothrombin released when prothrombin is converted to thrombin. Elevated levels are associated with an increased risk of thrombosis and found in patients with trauma, eclampsia, pre-eclampsia, DIC, DVT, and PE. Levels are also increased in individuals with antithrombin deficiency.

Prothrombin Time (PT)

Prolonged PT results suggest a potential bleeding disorder that may be caused by a deficiency in factor II, V, VII, or X. Prolonged results are also associated with very low fibrinogen levels, factor-specific inhibitors, or a lupus anticoagulant (minimal increase). When patients taking anticoagulants such as warfarin have a prolonged result, they are at risk of a bleeding episode, and a dose adjustment should be considered. Results are interpreted based on the international normalized ratio (INR).

PTT-LA with Reflex to Hexagonal Phase Confirmation

Lupus anticoagulants are non-specific antibodies that extend the clotting time of phospholipid-dependent clotting assays, and lupus anticoagulant antibodies bind to hexagonal phase phospholipids. A reduction of the aPTT after the addition of hexagonal phase phospholipid is considered confirmation of the presence of a lupus anticoagulant and an increased risk of thrombosis. The sensitivity and specificity of this test are 96% and >95%, respectively. False-positives may be seen with specific factor inhibitors (factors VIII and V) and direct thrombin inhibitors. While false-negatives are rare, if clinical suspicion of LA is high, dRVVT Screen with Reflex to dRVVT Confirm and dRVVT 1:1 Mix assay is suggested.

Reptilase Clotting Time

Unlike thrombin, reptilase is not affected by the presence of heparin or hirudin; thus, a prolonged thrombin time in an individual with a normal reptilase time is consistent with contamination or the presence of heparin. Reptilase clotting time is also prolonged in individuals with a-, hypo-, and dysfibrinogenemias.

Serotonin Release Assay (SRA)

A value >20% is considered positive and strongly suggests heparin-induced thrombocytopenia.

Thrombin-Antithrombin (TAT) Complex

Elevated TAT complex is a risk factor for thrombosis and found in individuals with DIC, malignancies, and those receiving heparin and fibrinolysis therapy.

Thrombin Clotting Time

Prolonged clotting times may indicate abnormal fibrinogen levels (elevated or decreased), dysfibrinogenemia, the presence of heparin, hirudin, paraproteins, uremia, or increased levels of fibrin degradation products. Normalization of clotting time after mixing indicates hypo- or dysfibrinogenemia, whereas continued prolongation indicates the presence of heparin, hirudin, paraproteins, uremia, or increased levels of fibrin degradation products.

Tissue Plasminogen Activator (TPA)

TPA converts plasminogen to plasmin, which in turn degrades fibrin to soluble degradation products. TPA is inhibited by plasminogen activator inhibitor-1. Elevated TPA levels are associated with an increased risk of atherosclerosis, myocardial infarction, stroke, and recurrent venous thrombosis.

von Willebrand Factor Protease (ADAMTS-13) Activity with Reflex to Protease Inhibitor

von Willebrand factor is released into circulation as high molecular weight multimeric forms that are broken down into smaller, less active multimers by von Willebrand factor cleaving protease (ADAMTS-13). Deficiency of von Willebrand factor cleaving protease can result in persistence of supranormal high molecular weight multimers, which are associated with enhanced platelet aggregation and thrombotic thrombocytopenic purpura.37

Sample Collection Considerations [return to contents]

Anticoagulants may interfere with some test results (Table 4). When clinically indicated, replace an oral anticoagulant with heparin for 7 to 10 days, then stop the heparin and draw the sample 12 to 24 hours later.6

Certain medicines and medical conditions may also affect some test results (Table 5).

Platelets significantly decrease the sensitivity of antiphospholipid antibody testing; thus, the specimen must be double centrifuged (15 minutes at 2000 g, followed by centrifugation of the supernatant for 10 minutes at 2500 g) to remove platelets prior to freezing.38 The final platelet count must be <10,000 platelets/μL of plasma (note that 1 μL=1 mm3).

References [return to contents]

  1. Heit JA. Venous thromboembolism: disease burden, outcomes and risk factors. Thromb Haemost. 2005;3:1611-1617.

  2. Prandoni P, Bilora F, Narchiori A, et al. An association between atherosclerosis and venous thrombosis. N Engl J Med. 2003;348:1435-1441.

  3. Cohen AT, Alikhan R, Arcelus J, et al. Assessment of venous thromboembolism risk and the benefits of thromboprophylaxis in medical patients. Thromb Haemost. 2005;94:750-759.

  4. Ridker PM, Miletich JP, Hennekens CH, et al. Ethnic distribution of factor V Leiden in 4047 men and women. Implications for venous thromboembolism screening. JAMA. 1997;277:1305-1307.

  5. Castoldi E, Rosing J. Factor V Leiden: a disorder of factor V anticoagulant function. Curr Opin Hematol. 2004;11:176-181.

  6. De Stefano V, Finazzi G, Mannucci PM. Inherited thrombophilia: pathogenesis, clinical syndromes, and management. Blood. 1996;87:3531-3544.

  7. Schambeck CM, Hinney K, Haubitz B, et al. Familial clustering of high factor VIII levels in patients with venous thromboembolism. Arterioscler Thromb Vasc Biol. 2001;21:289-292.

  8. den Heijer M, Koster T, Blom HK, et al. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med. 1996;334:759-762.

  9. Martinelli I, Battaglioli T, Pedotti P, et al. Hyperhomocysteinemia in cerebral vein thrombosis. Blood. 2003;102:1363-1366.

  10. Rosendaal FR, Doggen CJ, Zivelin A, et al. Geographic distribution of the 20210 G to A prothrombin variant. Thromb Haemost. 1998;79:706-708.

  11. Poort SR, Rosendaal FR, Reitsma PH, et al. A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88:3698-3703.

  12. Gallus A. Management options for thrombophilias. Semin Thromb Hemost. 2005;31:118-126.

  13. Girling J, de Swiet M. Inherited thrombophilia and pregnancy. Curr Opin Obstet Gynecol. 1998;10:135-144.

  14. Franchini M, Veneri D. Inherited thrombophilia: an update. Clin Lab. 2005;51:357-365.

  15. Van Cott EM, Laposata M, Prins M. Laboratory evaluation of hypercoagulability with venous or arterial thrombosis. Arch Pathol Lab Med. 2002;126:1281-1295.

  16. Committee on Practice Bulletins–Gynecology, American College of Obstetricians and Gynecologists. ACOG Practice Bulletin No. 84: prevention of deep vein thrombosis and pulmonary embolism. Obstet Gynecol. 2007;110:429-440.

  17. Carrier M, Le Gal G, Wells PS, et al. Systematic review: the Trousseau syndrome revisited: should we screen extensively for cancer in patients with venous thromboembolism? Ann Intern Med. 2008;149:323-333.

  18. Van Cott EM, Laposata M. Laboratory evaluation of hypercoagulable states. Hematol Oncol Clin North Am. 1998;12:1141-1166.

  19. Martinelli I, Battaglioili T, Tosetto A, et al. Prothrombin A19911G polymorphism and the risk of venous thromboembolism. J Thromb Haemost. 2006;4:2582-2586.

  20. Perez-Ceballos E, Corral J, Alberca I, et al. Prothrombin A19911G and G20210A polymorphisms’ role in thrombosis. Br J Haematol. 2002;118:610-614.

  21. Caldwell MD, Berg RL, Zhang KQ, et al. Evaluation of genetic factors for warfarin dose prediction. Clin Med. 2007;5:8-16.

  22. Gage BF, Eby C, Milligan PE, et al. Use of pharmacogenetics and clinical factors to predict the maintenance dose of warfarin. Thromb Haemost. 2004;91:87-94.

  23. Sconce EA, Khan TI, Wynne HA, et al. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. Blood. 2005;106:2329-2333.

  24. Wilmer M, Stocker C, Buhler B, et al. Improved distinction of factor V wild-type and factor V Leiden using a novel prothrombin-based activated protein C resistance assay. Am J Clin Path. 2004;122:836-842.

  25. Gessoni G, Valverde S, Manoni F. Comparison between two functional assays to detect factor V Leiden. Int J Lab Hematol. 2010;32:e188-e189.

  26. de Visser MC, Rosendaal FR, Bertina RM. A reduced sensitivity for activated protein C in the absence of factor V Leiden increases the risk of venous thrombosis. Blood. 1999;93:1271-1276.

  27. Wells PS, Anderson DR, Rodger M, et al. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. N Engl J Med. 2003;349:1227-1235.

  28. Rosendaal FR, Koster T, Vandenbroucke JP, et al. High risk of thrombosis in patients homozygous for factor V Leiden. Blood. 1995;85:1504-1508.

  29. Vandenbroucke JP, Koster T, Briet E, et al. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet. 1994;344:1453-1457.

  30. Kupferminc MJ, Eldor A, Steinman N, et al. Increased frequency of genetic thrombophilia in women with complications of pregnancy. N Engl J Med. 1999;340:9-13.

  31. Triado I, Mateo J, Soria JM, et al. The ABO blood group genotype and factor VIII levels as independent risk factors for venous thromboembolism. Thromb Haemost. 2005;93:468-474.

  32. Ridker PM, Hennekens CH, Selhub J, et al. Interrelation of hyperhomocyst(e)inemia, factor V Leiden, and risk of future venous thromboembolism. Circulation. 1997;95:1777-1782.

  33. Francis CW. Plasminogen activator inhibitor-1 levels and polymorphisms: association with venous thromboembolism. Arch Pathol Lab Med. 2002;126:1401-1404.

  34. Visanji JM, Seargent J, Tahri D, et al. Influence of the �675 4G/5G dimorphism of the plasminogen activator inhibitor 1 promoter on thrombotic risk in patients with factor V Leiden. Br J Haematol. 2000;110:135-138.

  35. Zoller B, Garcia de Frutos P, Dahlback B. A common 4G allele in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene as a risk factor for pulmonary embolism and arterial thrombosis in hereditary protein S deficiency. Thromb Haemost. 1998;79:802-807.

  36. Martinelli I, Taioli E, Bucciarelli P, et al. Interaction between the G20210A mutation of the prothrombin gene and oral contraceptive use in deep vein thrombosis. Arterioscler Thromb Vasc Biol. 1999;19:700-703.

  37. Arya M, Anvari B, Romo GM, et al. Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood. 2002;99:3971-3977.

  38. Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009;7:1737-1740.

Appendix 1. Antiphospholipid Syndrome

Antiphospholipid syndrome, the most common cause of acquired thrombophilia, is characterized by the presence of antiphospholipid antibodies such as lupus anticoagulants (LA), anticardiolipin antibodies (ACA) (IgG and/or IgM), and β2-glycoprotein I antibody (IgG and/or IgM).1 The syndrome is associated with both arterial and venous thrombosis, systemic lupus erythematosus (SLE), connective tissue and autoimmune disorders, malignancy, HIV infection, drug ingestion, and obstetric, hematologic, neurologic, dermatologic, and cardiac complications (Appendix 1 Table).

Appendix 1 Table. Complications Associated with Antiphospholipid Syndrome
Obstetric – Maternal Hematologic Dermatologic
Deep vein thrombosis Thrombocytopenia Skin necrosis
Chorea Hemolytic anemia Ulceration
Postpartum syndrome Evans syndrome Livedo reticularis
Eclampsia/preeclampsia Low complement 3 & 4 Digital gangrene
Obstetric – Fetal Neurologic Cardiac
Intrauterine fetal death Chorea Coronary artery disease
Recurrent spontaneous abortion Stroke Libman-Sacks endocarditis
Fetal growth retardation Multi-infarct dementia Arterial graft occlusion
Prematurity TIA/CVA  
Neonatal thrombosis Transverse myelitis
Migraine
  

Because no single test is 100% sensitive or specific for LA, the diagnosis usually begins with demonstration of prolonged phospholipid-dependent clotting times using 2 different methods (eg, dRVVT and PTT-LA).1,2 A prolonged dRVVT screen is automatically followed by a phospholipid-rich confirmatory test (at an additional charge and CPT code). Similarly, a positive dRVVT confirm test is reflexed to a 1:1 mixing study; an uncorrected dRVVT rules out factor deficiencies, specifically those induced by warfarin therapy. When the PTT-LA is prolonged, LA is confirmed using hexagonal phase neutralization (hexagonal phase confirmation test, STACLOT® LA). The Lupus Anticoagulant Evaluation with Reflex panel (test code 7079) includes all these tests.

Although highly sensitive, ACA testing is not specific for thrombotic risk. Transient LA or ACA may be due to infectious diseases (varicella, rubella, adenovirus, HIV) or drug exposure (amoxicillin, chlorpromazine, hydralazine). Since transient LA or ACA are not associated with clinical complications, 2 positive tests (titer >40 GPL or MPL units or >99th percentile), more than 3 months apart, are recommended for diagnosis of clinically significant antiphospholipid antibodies.1,3

The diagnosis of antiphospholipid syndrome may be improved by using the β2-glycoprotein I antibody assay. β2-glycoprotein I antibodies (titer >99th percentile on 2 occasions more than 3 months apart)1 are associated with the antiphospholipid syndrome, and their presence is more specific but less sensitive than cardiolipin antibodies for the diagnosis of antiphospholipid syndrome. Individuals who are positive for cardiolipin antibodies and negative for β2-glycoprotein I antibodies are more likely to have an infection (varicella, rubella, adenovirus, HIV) or drug exposure (amoxicillin, chlorpromazine, hydralazine) than antiphospholipid syndrome.

Phosphatidylserine, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid antibodies are not included in the international consensus APS classification criteria1; however, they are sometimes used to identify patients at risk of developing APS clinical symptoms when such patients test negative for first-line tests. For example, phosphatidylserine antibodies are associated with stroke and both phosphatidylserine and phosphatidylinositol antibodies are associated with pregnancy morbidity.4,5 Annexin V antibody, which is linked to recurrent pregnancy loss, can also be used as a second-line test.6

References

  1. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006;4:295-306.

  2. Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009;7:1737-1740.

  3. Van Cott EM, Laposata M. Laboratory evaluation of hypercoagulable states. Hematol Oncol Clin North Am. 1998;12:1141-1166.

  4. Kahles T, Humpich M, Steinmetz H, et al. Phosphatidylserine IgG and beta-2-glycoprotein I IgA antibodies may be a risk factor of ischaemic stroke. Rheumatology. 2005;44:1161-1165.

  5. Ulcova-Gallova Z, Krauz V, Novakova P, et al. Anti-phospholipid antibodies against phosphatidylinositol, and phosphatidylserine are more significant in reproductive failure than antibodies against cardiolipin only. Am J Reprod Immunol. 2005;54:112-117.

  6. Bizzaro N, Tonutti E, Villalta D, et al. Prevalence and clinical correlation of anti-phospholipid-binding protein antibodies in anticardiolipin-negative patients with systemic lupus erythematosus and women with unexplained recurrent miscarriages. Arch Pathol Lab Med. 2005;129:61-68.

Appendix 2. Tests Used in the Diagnosis and Management of Thrombophilia

The Appendix 2 Table contains a comprehensive list of tests used in the diagnosis and management of thrombophilia.

Appendix 2 Table. Tests Used in the Diagnosis and Management of Thrombophilia

19911A>G Mutation Analysisa

Test Code

Method

Description

16533(X)
 

PCR

The 19911A>G prothrombin gene mutation is detected by PCR followed by single nucleotide primer extension and capillary electrophoresis.

AccuType Warfarina

Test Code

Method

Description

16160(X)
 

PCR
 

PCR of the promoter region of VKORC1 and CYP2C9 exons 3, 5, and 7 is followed by single nucleotide primer extension and detection of fluorescent extension products on an automated DNA sequencer.

Activated Partial Thromboplastin Time (aPTT)

Test Code

Method

Description

763
 

Photo-optical clot detection
 

An ellagic acid phospholipid reagent is mixed with patient plasma, and calcium chloride is then added to initiate clot formation. The time elapsed for formation of a fibrin clot is measured.

Activated Protein C Resistance

Test Code

Method

Description

22
 

Photo-optical clot detection
 

The patient sample is incubated with and without exogenous activated protein C (APC). Activated factor V (FVa) is broken down by APC, reducing conversion of prothrombin to thrombin and extending the clotting time. If factor V mutation is present, FVa breakdown is inhibited, leading to a shorter clotting time. Results are expressed as the ratio of clotting times obtained with and without exogenous APC.

Antithrombin III Activity

Test Code

Method

Description

216
 

Chromogenic
 

Antithrombin in the patient’s sample binds to thrombin; excess thrombin cleaves a synthetic thrombin substrate, the amount of which is inversely proportional to the amount of antithrombin activity in the plasma.

Antithrombin III Antigen

Test Code

Method

Description

5158(X)

Nephelometry
 

Antithrombin antigen is measured in a fixed rate time nephelometry assay.

BCR-ABL1 Gene Rearrangement, Quantitative PCRa

Test Code

Method

Description

91065
 

Real-time, reverse transcription PCR

BCR-ABL1 P210 transcripts (e13a2 and e14a2) and P190 transcript (e1a2) are amplified. If there is no amplification of fused mRNA (BCR-ABL1/ABL1 ratio = 0), the result is reported as negative. Positive results are reported as BCR-ABL1/ABL1 % values; P210 transcript levels are also standardized to the international scale.

C4 Binding Protein

Test Code

Method

Description

15914
 

Radial immunodiffusion
 

The patient’s sample is diffused through an agarose gel matrix containing sheep anti-human C4 binding protein antibodies. Antigen-antibody complexes are detected as a visible precipitating ring. The C4 binding protein concentration is calculated based on the ring diameter.

Cardiolipin Antibody IgA, IgG, IgM

Test Code

Method

Description

7352

EIA

The level of antibodies directed against various plasma proteins that bind to phospholipid surfaces (eg, damaged endothelial membranes, monocytes, tumor cells, etc) is measured. The method is highly sensitive but not specific for β2-glycoprotein antibodies.

Cytochrome P450 2C9 Genotypea

Test Code

Method

Description

11294(X)
 

PCR
 

Single nucleotide primer extension and detection of fluorescent extension products are utilized to detect the 2 most common variants in the CYP2C9 gene, CYP2C9*2 and CYP2C9*3.

D-Dimer, Quantitative

Test Code

Method

Description

8659
 

Immunoturbidimetric
 

The increase in light absorption caused by the agglutination of D-dimer antibody-coated latex particles with endogenous D-dimer is measured.

dRVVT Screen with Reflex to dRVVT Confirm and dRVVT 1:1 Mixc

Test Code

Method

Description

15780(X)
 

Photo-optical clot
detection, calculation
 

The time elapsed for clot formation following Russell viper venom activation of factor X is determined. When the dRVVT is prolonged, another dRVVT test is performed (at an additional charge [additional CPT code]) after adding excess phospholipid in the presence of a heparin neutralizer. The ratio of the 2 dRVVT tests is calculated, and the result is reported as “positive” or “negative.�

Factor V HR2 Allele DNA Mutation Analysisb,e

Test Code

Method

Description

17902(X)
 

PCR, oligonucleotide ligation, fluorescent detection

The HR2 allele (4070A>G) in the factor V gene is detected by amplification of the gene region with PCR, followed by oligonucleotide ligation and hybridization to color-coded microspheres. Results are reported as no mutation detected, heterozygous positive, or homozygous positive.

Factor V HR2 Allele DNA Mutation Analysise

Test Code

Method

Description

10905
 

Invader® assay

The HR2 allele (4070A>G) in the factor V gene is detected by allele-specific hybridization, signal amplification, and fluorescent detection. Results are reported as negative, heterozygous positive, or homozygous positive.

Factor V (Leiden) Mutation Analysise

Test Code

Method

Description

17900

PCR, HyBeacon® probes

The 1691G>A factor V Leiden mutation is detected by amplification of the gene region by PCR in the presence of a HyBeacon probe specific for the mutated sequence. The melting temperature (Tm) of the probe-target complex is then determined using melting curve analysis. Presence of the factor V (Leiden) mutation results in a higher Tm than that obtained with the wild-type sequence.

Factor VIII Activity, Chromogenic

Test Code

Method

Description

16049
 

Chromogenic
 

This assay provides increased sensitivity (relative to the factor VIII clotting assay) at levels >150% and is insensitive to the effects of direct thrombin inhibitors and heparin. It is, therefore, the preferred assay when a lupus anticoagulant is present.

Factor VIII in the patient sample is activated by mixing with thrombin and activated factor IX. Activated factor VIII then accelerates the factor IXa-mediated conversion of factor X to Xa. Factor Xa activity, which is proportional to factor VIII activity, is measured by the change in a chromogenic substrate.

Factor VIII Activity, Clotting

Test Code

Method

Description

347
 

Photometric clot detection
 

Patient plasma is mixed with factor VIII-deficient normal plasma. The aPTT clotting time of the mixed plasma is compared to that of reference plasma. Results are reported as percent of normal factor VIII activity.

Fibrin Monomer

Test Code

Method

Description

11074
 

Hemagglutination
 

Patient plasma is mixed with human erythrocytes coated with purified fibrin monomer. The presence of soluble fibrin monomer complexes in plasma results in agglutination.

Fibrinogen Activity, Clauss

Test Code

Method

Description

461
 

Photo-optical clot
detection
 

Fibrinogen in the patient sample is converted to fibrin in the presence of excess thrombin. The clotting time obtained is inversely proportional to the fibrinogen level (activity).

Fibrinogen Degradation Products (FDP)

Test Code

Method

Description

458(X)
 

Agglutination
 

In the presence of corresponding antigens, latex particles coated with monoclonal anti-FDP antibodies agglutinate to form macroscopic clumps.

Fibrinogen Antigen, Nephelometry

Test Code

Method

Description

37801
 

Nephelometry
 

Patient plasma is reacted with highly specific antiserum to form insoluble antigen-antibody complexes, which are detected in solution as turbidity.

Fondaparinux Sodium (Xa Inhibition)e

Test Code

Method

Description

16103

Chromogenic

The level of fondaparinux in plasma is measured by the inhibition of activated factor X (Xa) activity in the presence of excess antithrombin.

β2-Glycoprotein I Antibodies (IgA, IgG, IgM)

Test Code

Method

Description

30340(X)

EIA

This enzyme immunoassay semi-quantitatively measures antibodies directed against β2-glycoprotein I (β2-GPI, apolipoprotein H). Individual tests for IgA, IgG, and IgM are available (separate order codes).

Heparin Anti-Xa (Low Molecular Weight Heparin)

Test Code

Method

Description

30292
 

Chromogenic
 

The level of low molecular weight heparin in plasma is measured by the inhibition of activated factor X (Xa) activity in the presence of excess antithrombin.

Heparin Anti-Xa (Unfractionated Heparin)

Test Code

Method

Description

404(X)
 

Chromogenic
 

The level of unfractionated heparin in plasma is measured by inhibition of activated factor X (Xa) activity in the presence of excess antithrombin.

Homocysteine (Cardiovascular)

Test Code

Method

Description

31789
 

Competitive immunoassay
 

The level of total homocysteine (ie, protein-bound, oxidized, and free, reduced homocysteine) is measured in a competitive immunochemiluminometric assay.

Human Platelet Antigen 1 Genotype

Test Code

Method

Description

10707(X)
 

DNA-based capture/binding
assay

This assay uses polymerase chain reaction (PCR) amplification targeting the HPA-1 polymorphism region of the GPIIIa gene followed by binding of the amplicons to allele-specific probes, which are then detected colorimetrically. Genotype is expressed as the ratio of HPA-1b to HPA-1a absorbance relative to the cutoff.

JAK2 Mutation (V617F) Analysis, Quantitative, Plasma-based, Leumeta® a,b

Test Code

Method

Description

16175

Real-time, reverse
transcription PCR,
sequencing

PCR amplification targets a 269 bp region on the JAK2 gene, encompassing the V617F point mutation region on chromosome 9p24. Results are reported as pg JAK2 V617F per μL plasma.

JAK2 Exons 12 and 13 Mutations, Qualitative, Leumeta® a

Test Code

Method

Description

16536(X)

Reverse transcription
PCR, sequencing

PCR amplification targets the V167F mutation region on chromosome 9p24 and the exon 12 and 13 regions of the JAK2 gene.

JAK2 V617F Mutation, Qualitative PCR, Leumeta® with Reflex to Exons 12,13a,c

Test Code

Method

Description

16539(X)

Reverse transcription
PCR, sequencing

PCR and sequencing are performed as described above to detect the V617F mutation; if negative, an additional PCR and sequencing will be performed to detect JAK2 mutations in exons 12 and 13.

JAK2 V617F, QL, Leumeta® w/rfl to Exons 12,13 and rfl to MPL W515, S505a,c

Test Code

Method

Description

16538(X)

Reverse transcription
PCR, sequencing

This assay uses PCR and sequencing to detect the V617F mutation; if negative, presence of JAK2 mutations in exons 12 and 13 will be sought; if these are also negative, then MPL mutations will be sought.

Lipoprotein (a)

Test Code

Method

Description

34604

Immunoturbidimetric

Agglutination occurs between lipoprotein (a) in the patient sample and anti-lipoprotein (a) antibodies adsorbed to latex particles, and is measured photometrically.

Mixing Study

Test Code

Method

Description

8922(X)
 

Clot detection
 

A PT and/or aPTT is measured after mixing patient plasma and normal plasma to determine the cause (eg, factor deficiency, factor inhibitor, and/or nonspecific inhibitor) of a prolonged PT and/or aPTT test result.

Phosphatidic Acid Antibodies (IgG, IgA, IgM)d,f

Test Code

Method

Description

1771

EIA

The presence of IgG, IgA, and IgM antibodies to phosphatidic acid is detected in an enzyme immunoassay. Individual tests for IgA, IgG, and IgM antibodies are available (separate order codes).

Phosphatidylcholine Antibodies (IgG, IgA, IgM)d

Test Code

Method

Description

30037

EIA

The presence of IgG, IgA, and IgM antibodies to phosphatidylcholine is detected in an enzyme immunoassay. Individual tests for IgA, IgG, and IgM antibodies are available (separate order codes).

Phosphatidylethanolamine Antibodies (IgG, IgA, IgM)d

Test Code

Method

Description

16619(X)

EIA

The presence of IgG, IgA, and IgM antibodies to phosphatidylethanolamine is detected in an enzyme immunoassay. Individual tests for IgA, IgG, and IgM antibodies are available (separate order codes).

Phosphatidylglycerol Antibodies (IgG, IgA, IgM)d,f

Test Code

Method

Description

18926

EIA

The presence of IgG, IgA, and IgM antibodies to phosphatidylglycerol is detected in an enzyme immunoassay. Individual tests for IgA, IgG, and IgM antibodies are available (separate order codes).

Phosphatidylinositol Antibodies (IgG, IgA, IgM)d

Test Code

Method

Description

10480(X)

EIA

The presence of IgG, IgA, and IgM antibodies to phosphatidylinositol is detected in an enzyme immunoassay. Individual tests for IgA, IgG, and IgM antibodies are available (separate order codes).

Phosphatidylserine Antibodies (IgG, IgA, IgM)

Test Code

Method

Description

10062
 

EIA
 

The presence of IgG, IgA, and IgM antibodies to phosphatidylserine is detected in an enzyme immunoassay. Individual tests for IgA, IgG, and IgM antibodies are available (separate order codes).

Plasminogen Activator Inhibitor (PAI-1) Antigen

Test Code

Method

Description

36555(X)

EIA
 

The concentration of plasminogen activator inhibitor-1 (antigen) is measured in an enzyme immunoassay.

Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5Ga,b

Test Code

Method

Description

11368
 

PCR
 

This test employs PCR amplification of a 150-bp fragment of the PAI-1 gene, followed by single nucleotide primer extension and capillary electrophoresis.

Plasminogen Activity

Test Code

Method

Description

4458(X)
 

Chromogenic
 

Plasminogen in the patient sample forms a complex with streptokinase. Hydrolysis of this complex produces a color, the intensity of which is directly proportional to the percent of normal plasminogen activity.

Plasminogen, Antigenic

Test Code

Method

Description

5164(X)
 

Nephelometry
 

Specific antiserum to plasminogen forms antigen-antibody complexes that are detected in solution as turbidity.

Protein C Activity

Test Code

Method

Description

1777

Clot detection

The Protac® glycoprotein from snake venom is used to activate protein C in the patient sample, and CaCl2 is added to initiate clotting. The amount of time required for clot formation is proportional to the amount of activated protein C in the sample.

Protein C Antigen

Test Code

Method

Description

4948(X)
 

EIA
 

Protein C antigen is measured via an enzyme immunoassay.

Protein S Activity

Test Code

Method

Description

1779
 

Clot detection
 

Protein S in the patient sample enhances the anticoagulant action of activated protein C, resulting in a prolonged clotting time. The increase in clotting time is directly proportional to the percent of normal protein S activity.

Protein S Antigen, Total

Test Code

Method

Description

5165
 

Immunoturbidimetric
 

The level of total protein S (bound and free) is measured in a microlatex particle-mediated immunoassay.

Protein S Antigen, Free

Test Code

Method

Description

10170
 

Immunoturbidimetric
 

The increase in light absorption caused by the agglutination of free protein S antibody-coated latex particles with endogenous free protein S is measured.

Prothrombin (Factor II) 20210G>A Mutation Analysisb,e

Test Code

Method

Description

17909

PCR, HyBeacon probes

The 20210G>A mutation is detected by amplification of the gene region with PCR, followed by oligonucleotide ligation and hybridization to color-coded microspheres. Reaction products are detected by fluorescent detection of the microspheres. Results are reported as no mutation detected, heterozygous, or homozygous positive.

Prothrombin Fragment 1.2

Test Code

Method

Description

37674(X)
 

EIA
 

Prothrombin fragment 1.2 antibodies are used to measure the level of prothrombin fragment 1.2.

Prothrombin Time with INR

Test Code

Method

Description

8847
 

Clot detection
 

Thromboplastin and calcium chloride are mixed with patient plasma, and the time to clot formation is measured photometrically. Results are reported in seconds and INR units.

PTT-LA with Reflex to Hexagonal Phase Confirmationc

Test Code

Method

Description

17408
 

Clot-based PTT
 

A sensitive PTT with dilute phospholipid is used. Prolonged PTT-LA test results are confirmed (at an additional charge [additional CPT code]) with hexagonal phase neutralization (hexagonal phase confirmation test, STACLOT® LA).

Reptilase Clotting Time

Test Code

Method

Description

37700
 

Clot detection
 

Reptilase, an enzyme derived from the venom of Bothrops atrox, cleaves fibrinopeptide A from fibrinogen resulting in the production of fibrin and subsequent clot formation. Clotting time is determined by measuring the increase in viscosity after reptilase is added to test plasma.

Serotonin Release Assay, Fondaparinuxe

Test Code

Method

Description

16285

Radiobinding 14C

serotonin radiolabel

Donor platelets are incubated with 14C-serotonin.

Porcine heparin (0.1, 0.5, and 100 U) is then added along with patient serum. The presence of antibodies to the heparin-PF4 complex in the sera of patients with HIT causes the release of serotonin with 0.1 and 0.5 U heparin doses; serotonin release is suppressed with the 100 U dose. Results are reported as percent serotonin released.

Serotonin Release Assay, LMWHe

Test Code

Method

16284

 

Radiobinding 14C

serotonin radiolabel

Serotonin Release Assay, Unfractionated Heparine

Test Code

Method

14627

Radiobinding 14C

serotonin radiolabel

Thrombin-Antithrombin (TAT) Complex

Test Code

Method

Description

10162(X)
 

ELISA
 

Thrombin is inhibited by antithrombin and the resulting inactive proteinase/inhibitor complex is measured quantitatively by ELISA.

Thrombin Clotting Time

Test Code

Method

Description

883(X)
 

Clot detection
 

Thrombin is mixed with patient plasma and clotting time is determined photometrically.

Tissue Plasminogen Activator (TPA), EIA

Test Code

Method

Description

29816
 

EIA

This EIA utilizes goat anti-TPA IgG to determine the level of TPA.

von Willebrand Factor Protease Activity with Reflex to Protease Inhibitorc,d

Test Code

Method

Description

14532

ELISA

von Willebrand factor cleaving protease (VWF-CP), also known as ADAMTS-13, cleaves a recombinant substrate to form a cleavage product, the amount of which is proportional to the amount of ADAMTS-13 in the patient’s sample. The presence of inhibitors is determined (at an additional charge [additional CPT code]) using the same method by mixing a heat-inactivated patient sample with pooled normal plasma (PNP) and calculating the residual activity (activity of mixture vs activity of PNP).

EIA, enzyme immunoassay; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; INR, international normalized ratio.
a This test was developed and its performance characteristics have been determined by Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical performance of the test.
b Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche Molecular Systems, Inc.
c Reflex tests are performed at an additional charge and are associated with an additional CPT code(s).
d This test is performed using a kit that has not been approved or cleared by the FDA. The analytical performance characteristics of this test have been determined by Quest Diagnostics Nichols Institute. This test should not be used for diagnosis without confirmation by other medically established means.
e This test was developed and its performance characteristics have been determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Drug Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.
f Test codes 1771 and 1775 are specific to the performing laboratory. When ordering through your regional business unit, please indicate that the test code is specific for Quest Diagnostics Nichols Institute, Valencia, CA.

 

Content reviewed 02/2013

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* The tests listed by specialist are a select group of tests offered. For a complete list of Quest Diagnostics tests, please refer to our Directory of Services.