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Friday, September 3, 2010


Okay, so I admit, I come from a time when we still did tilt tube testing, I still believe every coagulation tech should know what a clot looks like in a tube, how is forms and how and if it lasts.  You never know when it could come in handy.  So molecular testing in coagulation, not even a glimmer in anyone’s eye when I started in the laboratory.

Now, we even have guidelines for molecular testing in coagulation- 

CLSI document:  H21-45 Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis!  It discusses what samples are acceptable, (including buccal smears) what additives may inhibit PCR reactions (like heparin), extraction for DNA samples (up to 922 days with acceptable yields!), DNA isolation, and storage

Molecular genetic markers:

One of the most widely utilized molecular testing is in regard to the detection of the molecular basis of activated protein C (APC) resistance.  This was discovered in 1994 by Bertina.  Prior to this, many patients thrombophilic states could not be linked to a disorder and were describes as idiopathic.  One of the advantages of molecular testing is that patients can be tested while they are on anticoagulation. The range of molecular genetic markers, linked with a clearly documented increased risk of thrombophilia are   include mutations of factor V Leiden 506R/Q, of protrombin 20210G/A, MTHFR 677C/T in homozygous form, mutation of PAI-1 4G/5G, mutations of different coagulation inhibitors and finally a range of polymorphisms with still not precisely defined increased risk for thrombophilia (F XIII Val34leu, platelets glycopeproteins, endothelial protein C receptor and thrombomodulin 

It is known that VTE is a multifactorial disorder and can impact the incidence by combining genetic risk factors and acquired or environmental conditions such as pregnancy, oral contraceptive use, estrogen therapy, malignancy, stroke with extremity paresis, trauma, surgery, or immobility. The more stimulus that you have the greater risk increases of a clot. Known genetic causes are present in approximately 25% of unselected venous thrombosis cases and up to 63% of familial cases. Factor V R506Q (Leiden), (FVL) causing activated protein C (APC) resistance,  is the most common genetic risk factor for venous thrombosis. Protein C is a circulating vitamin K-dependent zymogen, which is activated to APC, the active enzyme, by the thrombin-thrombomodulin complex. APC functions as a natural anticoagulant byinactivating (via proteolysis) procoagulant factors Va and VIIIa in the presence of protein S. Factor V Leiden appears to account for 90-95% of cases of APC resistance. Two rare mutations in the factor V gene have been described and are of dubious clinical significance.

Factor V-Cambridge (R306T) is not strongly associated with venous thrombosis in controlled epidemiologic studies. Factor V-Hong Kong (R306C) has been found in 1-2% of Chinese patients but does not appear to be associated with APC resistance.The R2 allele (H1299R, or A4070G) of the factor V gene, associated with a haplotype known as HR2, is present in 10% of the general population, and early studies indicate that it increases the risk of venous thrombosis in individuals heterozygous for factor V Leiden an additional 3-fold beyond their already 7-fold increased risk. 

FVL  is present in 5% of Caucasian Americans, it is rarer in Hispanic-Americans, rarer still in African-Americans, and virtually absent in Africans and Asians.  It is believed to produce a relative risk of venous thrombosis of about 7-fold in the heterozygous state and about 80-fold in the homozygous state. It is  found in 11-20% of individuals of all ages presenting with their first episode of venous thrombosis.  If the patient is under 50 years of age, up to 40% present with this FVL mutation is involved,   The environmental factor most extensively discussed in this context is oral contraceptive use in women, which produces a 30-fold increase in thrombotic risk when the factor V Leiden mutation is also present. 

The following recommendations regarding FVL can be accessed at:
Consensus Statement on Factor V Leiden Mutation Testing Page 5/14/2007

Issue 1: Which methodology should be used: Factor V Leiden DNA testing or functional activated protein C (APC) resistance testing?

Recommendation 1 

When appropriate clinical care requires testing for the factor V Leiden allele, either direct DNA-based genotyping or a factor V Leiden-specific functional assay is recommended. Patients who test positive by a functional assay should then be further studied with the DNA test for confirmation and to distinguish heterozygotes from homozygotes. Patients on heparin therapy or with known lupus anticoagulant should proceed directly to molecular testing if the modified functional assay is not used. When relatives of individuals known to have factor V Leiden are tested, the DNA method is recommended. 

Issue 2: Who should be tested?

Recommendation 2

Opinions and practices regarding factor V Leiden testing vary. Some physicians advocate testing of all patients with venous thrombosis except when active malignancy is present. Others exclude testing in patients over age 60 in the absence of a family history of thrombosis or a previous thrombotic event. There is growing consensus that testing should be performed in at least the following circumstances (these are the same general recommendations for testing for any thrombophilia):


- Age <50, any venous thrombosis.

- Venous thrombosis in unusual sites (such as hepatic, mesenteric, and cerebral veins).

- Recurrent venous thrombosis.

- Venous thrombosis and a strong family history of thrombotic disease.

- Venous thrombosis in pregnant women or women taking oral contraceptives.

- Relatives of individuals with venous thrombosis under age 50.

- Myocardial infarction in female smokers under age 50.

Testing may also be considered in the following situations:

- Venous thrombosis, age >50, except when active malignancy is present.

- Relatives of individuals known to have factor V Leiden. Knowledge that they have factor V Leiden may influence management of pregnancy and may be a factor in decision-making regarding oral contraceptive use.

- Women with recurrent pregnancy loss or unexplained severe preeclampsia, placental abruption,intrauterine fetal growth retardation, or stillbirth. Knowledge of factor V Leiden carrier status mayinfluence management of future pregnancies.

Random screening of the general population for factor V Leiden is not recommended. Routine testing is not recommended for patients with a personal or family history of arterial thrombotic disorders (e.g., acute coronary syndromes or stroke) except for the special situation of myocardial infarction in young female smokers. Testing may be worthwhile for young patients (<50 years of age) who develop acute arterial thrombosis in the absence of other risk factors for atherosclerotic arterial occlusive disease. 

Issue 3: Should testing be offered to individuals with environmental risk factors?

Recommendation 3

Factor V Leiden testing is recommended in women with venous thromboembolism during pregnancy or oral contraceptive use. In contrast to general screening before administration of oral contraceptives, targeted testing of women with a personal or family history of venous thrombosis is advisable. Routine screening for factor V Leiden in asymptomatic women contemplating or using oral contraceptives is not recommended, except for those with a personal history of thromboembolism or other medical risk factors. Those women with a family history of thromboembolism, APC resistance, or documented factor V Leiden mutation should be counseled about their risks and options and considered for testing depending on the overall clinical situation. Women with a history of recurrent late-trimester fetal loss should also be considered for testing. Whether or not the woman smokes would not alter these recommendations. Screening of asymptomatic individuals with other recognized environmental risk factors such as surgery, trauma, paralysis, and malignancy is not necessary or recommended, since all such individuals should receive appropriate medical prophylaxis for thrombosis regardless of carrier status. 

Issue 4: Should patients found to be positive for factor V Leiden or APC resistance be tested for any of the other heritable thrombophilic risk factors?

Recommendation 4

Patients testing positive for factor V Leiden or APC resistance should be considered for molecular genetic testing for the most common other thrombophilias with overlapping phenotype for which testing is easy and readily available. At present, only the prothrombin 20210A variant fits these criteria. It is present in 1-2% of the general population, its involvement in venous thromboembolism is well-established, and the DNA test is as simple as that for factor V Leiden (with which it can even be multiplexed). Protein S, protein C, and antithrombin III deficiencies are too genetically heterogeneous for routine molecular genetic testing, but testing by functional coagulation assays may be considered, especially if there is a strong family history of venous thrombosis. Hyperhomocysteinemia should be considered and tested (in most cases by measuring plasma homocysteine levels) as another potential risk factor in those found to be positive for factor V Leiden. Patients with classic homocystinuria are at extremely elevated risk of thromboembolism and should probably be tested for other available thrombophilic risk factors. 

Issue 5: Should testing for other heritable thrombophilic factors be performed simultaneously with factor V Leiden testing?

Recommendation 5

Physicians ordering factor V Leiden on a venous thrombosis patient for any of the indications recommended here should also consider the utility of functional, biochemical, and molecular screening for other heritable thrombophilic factors, especially prothrombin 20210A and plasma homocysteine levels. 

Issue 6: Are there any other factor V mutations in addition to factor V Leiden which should be tested?

Recommendation 6

The factor V Leiden (R506Q) mutation is currently the only molecular analysis of the factor V gene indicated in the routine workup of thrombotic risk. 

Issue 7: What are the recommended methodologies and quality assurance standards for performing these tests?

Recommendation 7

The factor V Leiden mutation test should be performed using any of the accepted technical approaches as long as they have been properly validated by the laboratory, while adhering to current ACMG/CAP quality assurance guidelines for molecular genetic testing. 

Issue 8: What are the appropriate pre- and postanalytic procedures to be followed in factor V Leiden testing?

Recommendation 8

Individuals being tested should be made aware that this is a genetic test, that test results have implications about risk in other family members,and that there may be attendant issues of confidentiality and possible insurance discrimination. The laboratory's report should state explicitly the relative risk implications for factor V Leiden heterozygotes and homozygotes,the risk that other relatives may have the mutation, and the recommendation, if indicated, for testing for other inherited hypercoagulabilities. It is important for individuals testing positive for factor V Leiden to understand the risk implications and genetic implications of their result. Patients should be counseled about these implications by their physician or genetic counselor. 

FUTURE PROJECTS: Molecular Genetics of Coagulation Disorders from:

The NIH has a Program Project to explore the molecular basis for selected disorders of coagulation and thrombosis and the role of hemostatic balance in vascular disease pathogenesis. The individual projects in this proposal emphasize the use of new technologies to provide improved biologic insight and develop new treatments for hemorrhage, thrombosis and related cardiovascular disorders. The three individual projects contained in this PPG will: (1) continue a whole genome ENU mutagenesis analysis in the mouse to identify genetic modifiers of factor V Leiden, while also taking advantage of natural murine strain variation to identify additional thrombosis modifiers, as well as modifier genes for thrombotic thrombocytopenic purpura (TTP); (2) continue to explore the critical factors that limit factor VIII expression. The current proposal focuses on the molecular mechanisms responsible for the oxidative stress that results from misfolded FVIII in the ER lumen; and (3) explore the role of bacterial streptokinase (SK) and its interaction with plasminogen in the pathogenesis of Group A streptococcal infection; high throughput chemical screening will be used to develop specific SK inhibitors as a potential new class of antibiotics for this important human infection. The PPG will continue to support 4 cores: (A) the Mouse Coagulation Laboratory, (B) the Genetics Core, (C) the Administrative Core, and (D) the Morphology Core. The PPG will aim to increase interaction and collaboration between individual project participants, as well as among the large number of other laboratories at the University of Michigan already engaged in research on coagulation, thrombosis and vascular disease. We anticipate that the overall program resulting from the combined efforts of all participants will significantly exceed the sum of the individual parts. Relevance to public health: Abnormalities in the control of blood clotting are a critical factor in a number of diseases, including heart attack and stroke (the leadings causes of death in the US), as well as several important infectious diseases. This Project will identify key genes in this system that should provide valuable new diagnostic tools as well as suggest novel approaches to treatment.

Donna Castellone

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Donna Castellone,  MS, MT(ASCP)SH

Donna Castellone,

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