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LAB
NEWS Chairman: Peter Jatlow, M.D. Editors: Edward L. Snyder, M.D.; Petrie M. Rainey, M.D., Ph.D. Production Assistant: Terri M. Fiondella Contributors: Peter Marone, MT (ASCP), MBA; Petrie Rainey, M.D., Ph.D.; Henry M. Rinder, M.D.; Brian Smith, M.D. FACTOR V LEIDEN IN HYPERCOAGULABLE STATESThe diagnostic work-up for hypercoagulable (thrombophilic) patients has changed significantly over the past few years. Previously, fewer than 10-15% of patients with thrombophilia were found to have a congenital cause of recurrent thromboses, specifically deficiencies of the naturally occurring anticoagulants Protein C, Protein S, or Antithrombin III. Recently, Dahlback and colleagues have identified a novel mechanism in familial thrombophilia that is characterized by a poor plasma anticoagulant response after addition of excess activated Protein C, so-called "APC-resistance." When excess activated Protein C is incubated with normal citrate-anticoagulated plasma, followed by initiation of clotting with calcium and thromboplastin, the time to clotting (usually measured by the PTT) is significantly prolonged when compared to normal plasma treated without excess activated Protein C. Dahlback and other investigators found that plasma from a significant proportion of patients with venous thrombosis (20-80% depending on age and number of thrombotic episodes) failed to significantly prolong the PTT with addition of excess APC. These patients were termed "APC-resistant." It was subsequently determined that most of these abnormal functional assays could be corrected by addition of normal factor V, the co-factor substrate on which Protein C acts. Subsequently, the underlying molecular defect responsible for APC-resistance was defined as a guanidine to adenine substitution in position 1691 of the factor V gene, termed the "factor V Leiden" mutation. This results in a factor V molecule with a glutamine substituted for arginine 506 which is at a critical cleavage site for APC.Studies have now determined that the prevalence of carriers (heterozygotes) of the Factor V Leiden mutation is between 3-5% in the general population. That percentage increases to near 20-25% in patients presenting with their first deep vein thrombosis and is probably >50% in probands with strong family histories of venous thrombosis. Therefore, this particular genetic mutation is by far the most common cause of a tendency towards venous thrombosis. The significance of these findings has not been lost on cardiologists, vascular surgeons, and hematologists who treat and follow hypercoagulable patients. The YNHH laboratory has now developed both functional and molecular assays to diagnose APC-resistance. The functional APC-resistance test has been recently described in detail in Lab News. The molecular assays involve polymerase chain reaction (PCR) amplification of the factor V region containing the mutation, followed by restriction endonuclease digestion of the amplified product. The G to A substitution results in loss of a restriction site to Mnl 1 endonuclease and appearance of a unique band on gel electrophoresis. In patients suspected of a hypercoagulable state, the functional APC ratio test, which is less expensive than the mutational analysis, should be performed first. Patients who are not receiving anticoagulants and who have a normal baseline PTT should be initially screened with this functional assay for APC-resistance. In the literature and in our studies at YNHH, the negative predictive value of the functional study has consistently been >95%. Specifically, a normal APC-resistance ratio means that the patient is very unlikely to have the factor V Leiden mutation and that patient does not require additional genetic analysis. The general work-up for hypercoagulability in such patients should then proceed with measurement of Proteins C and S and Antithrombin III. Published clinical studies and our own work have found that the positive predictive value of the functional APC-resistance ratio is between 45-60%; thus, patients with borderline or low APC ratios suggestive of APC resistance often do not have the factor V Leiden mutation. Even in highly rigorous studies of probands with strong histories of familial thrombophilia, 20% of individuals with functional APC-resistance lacked the Leiden mutation. It is unclear why APC resistance is present in these subjects; it is also unclear whether such patients have a relative increased risk of thrombosis or not. There are other critical cleavage sites on factor V; however, a second mutation in factor V has not yet been described. Other causes of false-positive functional assays exist. Patients who are studied at the time of an acute thrombosis may have decreased factor levels because of active clot formation and thus have borderline or low functional APC resistance when their acutely drawn plasma is studied. Since such patients are usually immediately placed on anticoagulants, genetic analysis of factor V Leiden is the only other option (see below). Pregnant women in the second and third trimesters have a high incidence of functional APC resistance, but very few of these women have the factor V Leiden mutation; moreover, when these women have been followed for weeks to months post-partum, it was determined that their functional APC ratios normalized. Thus, genetic analysis of factor V Leiden is probably necessary in all patients with borderline or low functional APC ratios. There are also some instances in which the functional APC-resistance assay cannot be used as a screen, and analysis for the factor V Leiden mutation itself is appropriate as the initial work-up. Prolongation of the PTT by APC in the functional assay can be adversely affected by reduced levels of Protein S, and factors II, V, VIII, IX, and X. Although newer functional assays are being developed to improve specificity in these situations, at present, patients on oral anticoagulants cannot be diagnosed with the functional assay. Similarly, those patients with a baseline prolongation of the PTT, either because they are on heparin or because of the presence of an anti-phospholipid antibody, cannot be tested with the functional APC-resistance assay. Thus, patients who are being treated with anticoagulants (oral or intravenous) should be screened with the genetic assay for factor V Leiden, instead of the functional assay. Should any asymptomatic patients be screened? The answer to this question remains uncertain. If a proband is identified as homozygous for factor V Leiden, most clinicians would agree that the family members should be screened for factor V Leiden. Heterozygotes for factor V Leiden are probably at increased risk for venous thrombosis with major surgery, prolonged immobilization, and perhaps in association with use of oral contraceptives. Because of the high frequency of this mutation in the general population, prospective studies, some of which are ongoing, will be needed to determine any additional role for these assays in screening potentially high-risk subpopulations. The mutational analysis for factor V Leiden requires one tube (lavender top) of EDTA-anticoagulated blood which should be kept on ice and sent to the Immunology Laboratory at Yale-New Haven Hospital between Monday and Friday. Direct questions can be referred to the Immunology Laboratory at 785-2440, or the Laboratory Medicine Resident on call at 340-3411. References
Henry M. Rinder, MD |
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