Clinical Applications Of Molecular Diagnostics In The Coagulation Laboratory

The increased demand for thrombophilia testing is explained in part by the relatively common occurrence of venous thromboembolism (VTE): VTE is the third most common cardiovascular disease in the United States and accounts for 100,000 deaths annually (72). Subsets of these patients have a hereditary or acquired predisposition for thrombosis referred to generally as thrombophilia. Thrombophilia could be an important cause of unprovoked idiopathic or recurrent VTE, thrombosis in patients <50 yr of age, thrombosis at unusual sites, family history of VTE, and complications of pregnancy. Up to 70% of patients with thrombophilia might have one or more of the major inherited coagulation defects. In this context, molecular genetic testing for hereditary defects associated with thrombophilia has become readily available.

FVL mutation is the most common hereditary throm-bophilia. In patients with recurrent venous thromboembolism,

40-50% are heterozygous for the Factor V Leiden (15,68). In a prospective study of a large cohort of apparently healthy men, the FVL mutation was associated with an increased risk of venous thrombosis, whereas no increased risk was seen for myocardial infarction or stroke (73). Of note, although testing for the FVL mutation might determine the risk of thrombosis in a patient, this analysis, in turn, is limited because of the low predictive value of the test (74). Furthermore, although the homozygous state for FVL might be associated with an 80-fold increased risk for thrombosis (15), this in itself does not represent a disease state (74). In contrast, the homozygous states for protein C and S deficiencies are usually associated with a severe clinical condition such as neonatal purpura fulminans (20). Compared to antithrombin, protein C, and protein S deficiencies, FVL has a higher prevalence among the white population, but a smaller risk associated with thrombosis (75).

A positive genetic test has variable implications depending on the selected population. Screening for the FVL mutation could have important clinical benefits in selected patients, such as patients with central venous catheter-associated thrombosis (76) or in renal transplant recipients (77). On the other hand, the benefits, if any, of screening for the FVL mutation in other populations is not known. Although the combined effect of FVL and use of oral contraceptives might have a multiplicative effect on the risk of VTE (78), there is no consensus on routine FVL screening for all women considering use of oral contraceptives (2). In a recent consensus conference organized by the College of American Pathologists, screening for the FVL in women before use of oral contraceptives was not generally recommended (68).

In some patients, combined genetic defects might be present. A positive FVL test along with another hereditary defect(s) appears to compound the risk of thrombosis (79,80). Individuals with coexistent FVL and prothrombin mutations (79) and FVL and protein C deficiency (80) are at an even higher risk of thrombosis than with a single gene defect. As a result, testing for the FVL and prothrombin 20210 mutations along with antithrombin, protein C, and protein S deficiencies are all recommended for hereditary thrombophilia work-up, because a combined defect might affect clinical decision-making.

The presence of the FVL mutation could also indicate that other family members might be at risk of developing venous thrombosis (68); therefore, FVL testing has been extended to family members of patients who experienced VTE and tested positive for the FVL. Although testing for the FVL mutation in this population has the potential to identify asymptomatic carriers who might benefit from thromboprophylaxis during periods of high risk, the reportedly low rate of VTE among family members with the FVL mutation did not justify the use of prophylactic anticoagulation (81).

The prothrombin gene mutation G20210A is the second most common hereditary thrombophilic defect, with a prevalence in the general population as high as 1-4% (42,82). The pro-thrombin mutation is a well-established risk factor for venous thrombosis (83); its role in arterial thrombosis remains controversial (84). Similar to the FVL, the prothrombin mutation has a higher prevalence than antithrombin, protein C, and protein S deficiencies, but a lower risk associated with thrombosis.

Table 2

College of American Pathologists Consensus Recommendations for Factor V Leiden and Prothrombin 20210A Testing

History of recurrent VTE VTE before the age of 50 yr Unprovoked VTE at any age VTE at unusual sites

VTE patients at any age with a positive family history (first-degree relative with VTE before the age of 50) VTE secondary to pregnancy, oral contraceptives, or hormone replacement therapy

Routine screening of the general population or as an initial screening test during pregnancy or before use of oral contraceptives is not recommended (64). However, in select patient populations, testing for the prothrombin mutation is advised (64).

The third most commonly performed genetic test for thrombophilia is the methylenetetrahydrofolate reductase (MTHFR) mutation analysis. Based on prospective studies, there appears to be a weak association between hyperhomocys-teinemia and arterial thrombosis; the association with venous thrombosis is controversial (52). Whereas homozygosity for the MTHFR mutation is associated with elevated levels of homocysteine, the mutation itself is not associated with cardiovascular disease (85). Homocysteine levels are recommended for patients with documented atherosclerotic disease; however, given the lack of evidence of homozygos-ity being an independent risk factor for arterial and venous thrombosis, genotyping for either the C677T or A1298C mutations are not generally recommended (52).

Molecular diagnostics in the area of coagulation is not exempt from the ongoing controversy and debate surrounding testing in thrombophilic disorders. The impact of a positive PCR test for Factor V Leiden mutation on therapeutic decisionmaking (life-long anticoagulation?) as well as the potential financial consequences (reduced insurability) are reasons for much concern surrounding the appropriate use of these tests. Based mostly on retrospective studies or multiple anecdotal studies, the College of American Pathologists consensus conference recommended molecular testing for FVL and pro-thrombin in selected patients (Table 2).

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