Mutations add up to thrombophilic risk

Testing for FVL and prothrombin G20210A should also be considered in women with a first VTE related to pregnancy, the puerperium, or oral contraceptive use; women with events related to hormone replacement therapy; and those with unexplained pregnancy loss during the second or third trimester. A good interview in a woman requesting oral contraceptives or HRT is important, Dr. Press notes. Testing is advised if justification is found in the personal or family history.

The conference created a separate category for controversial applications of testing, chiefly persons over age 50 who have a first provoked VTE in the absence of cancer or an intravascular device.

"A minority believe you should test for Leiden in virtually everybody with a venous thrombotic event," Dr. Press says. "That was not our consensus. Personally, I think we should be testing only people with thrombophilic predispositions." Accordingly, testing was not recommended as a general population screen or as a routine screening test during pregnancy or before prescribing oral contraceptives.

Test methods include functional assays for activated protein C(APC) resistance (some FDA-approved), which are either chromogenic or based on clotting time, and DNA-based tests that directly detect the Leiden mutation. Appropriately validated DNA-based methods are extremely accurate and precise. "It was the conclusion of the group that first-generation APC resistance functional assays, in which plasma is not prediluted before addition of APC, have variable sensitivity and specificity that precludes their routine clinical use," Dr. Press says.

Second-generation APC resistance assays, with dilution of patient plasma into FV-deficient plasma, look about equivalent to DNA-based tests, at least in some laboratories. "If you are going to test for Leiden, use a second-generation functional test or a DNA-based mutation detection method," Dr. Press sums up.

An initial DNA method is preferred to screen patients with a strong lupus inhibitor and family members of patients known to have FVL. And confirmatory DNA-based testing should be done in patients with "borderline" APC resistance values, as defined in each laboratory. DNA confirmatory testing is also recommended in patients with positive APC tests to distinguish heterozygotes from homozygotes.

"I don't think this varies much from what people are doing," Dr. Press says. "In my experience, most practitioners are either using a DNA test initially or a functional assay followed by DNA confirmation."

Among DNA-based methods, each laboratory must decide which method best suits its situation. Many in-house assays for FVL have been developed. "As with any other in-house test," Dr. Press says, "the laboratory needs to validate the assay." Commercial DNA-based methods for Leiden detection are under development.

In his laboratory, Dr. Press uses the Magna Pure instrument to prepare DNA, followed by real-time PCR analysis for Leiden and G20210A mutations on the Light-Cycler using allele-specific hybridization probes for amplification and detection.

At the Cleveland Clinic, Ilka R. Warshawsky, MD, PhD, associate staff in the Department of Clinical Pathology, also tests for FVL and prothrombin G20210A by DNA extraction on the Magna Pure and real-time PCR on the Light-Cycler. "We use the Magna Pure to extract DNA from 200 µL of blood," Dr. Warshawsky says. Blood and reagents are placed into cartridges on the Magna Pure and bar-coded patient information is input. Preparation takes about 40 minutes. After extraction, the instrument pipettes DNA samples and MasterMix (Roche's proprietary reagent mix containing primers and probes specific for the mutation, taq polymerase, nucleotide precursors, etc.) into capillaries in a carousel. Capillaries are centrifuged to move the reaction solution to the bottom of the capillaries, and the carousel is manually carried into the PCR room and put into the Light-Cycler, along with the disc containing bar-coded information.

"We do runs of 28 samples on preset days each week," says Dr. Warshawsky. (Four slots are used for controls.) "We haven't gotten any complaints about turnaround time." Over the past eight months, 77 to 91 percent of FVL samples were normal, nine to 21 percent were heterozygous for FVL, and zero to two percent were homozygous. Testing for prothrombin G20210A was introduced in July. Of 500 samples tested since then, 12 were heterozygous for the mutation.

Light-Cycler assay can detect new mutations in the region where probes hybridize if they have atypical melting curves, triggering sequencing. "We have found one new prothrombin mutation," Dr. Warshawsky says, "which has the same melting curve as another reported mutation. That would be
a concern."

Dr. Warshawsky now tests for MTHFR C677T, a much lower-volume assay, by PCR and restriction enzyme digestion. But she plans to set it up on Magna Pure and Light-Cycler.

Dr. McGlennen also tests for FVL, prothrombin G20210A, and MTHFR C677T. He considers the contribution of MTHFR C677T controversial, calling it a "risk modifier"—a mutation that by itself does not increase thrombotic risk, but one that can augment risk in combination with other mutations. In addition, he probes for another mutation in the factor V gene, HR2, which also results in a factor V protein resistant to inactivation.

Many CAPconference recommendations for testing flow from a patient's personal and family history, Dr. McGlennen points out. "Given that a patient has a series of blood clots under age 50," he says, "we really do need to have some family history or other evidence to conclude that testing is worthwhile." In this sense, he asserts, "Testing for thrombophilia has reinvigorated taking a good family history. Clinicians must go beyond simply asking, Has anybody in your family clotted?"

While acknowledging that the standard of care is to do a thrombophilia panel only in patients whose personal or family history suggests thrombophilia, Dr. McGlennen says, "I have added a handful of additional scenarios in which identifying a thrombophilic tendency is helpful." For example, a person who has surgery is immobilized for a time and may develop a blood clot. When that patient resumes walking, the clot can migrate, generating a pulmonary embolism.

"Our job is to find those individuals before they get into bed," Dr. McGlennen says, "so we can prevent clot formation by anticoagulation." As a result, he says, "I am looking at a transition from thrombophilia panels being ordered primarily by internists or hematologists, to ordering by surgeons who are deciding what to do with post-operative patients and gynecologists making decisions about oral contraceptives or HRT."

Even so, he agrees that generalized screening cannot be justified.

As for methodology, Dr. McGlennen aims to find the technology platforms that allow them to test inexpensively. "My goal is to make gene-based testing for thrombophilia as common as hematology-based coagulation assays," he says. He uses an in-house DNA microarray bearing probes for the four mutations for which he tests. "We have shown that we can do those four reactions for about the same cost as doing one test," he says. "And we could probably expand to one or two additional markers for minimal incremental cost." Although testing by microarray is unusual, Dr. McGlennen says, "All commercial microarray manufacturers are working assiduously to come up with their own thrombophilia panels."

He focuses the commentary in his lab report on how gene-based defects create increased risk. "I try to leave the clinician with a sense of numerical proportionality of risk relative to the general population," he says. "And I may include one sentence stating the recommendation about therapy." Physicians know the risk of long-term anticoagulation. Before they consider initiating this therapy, they need to see a multiple of risk—however arbitrary—that justifies it.

Dr. Triplett, too, stresses the role of history-taking in decisions about whether to test for thrombophilia. "For our baseline evaluation," he says, "we would, if possible, get a history in more than one generation and in both men and women." Women who carry the FVL gene are the principal group at increased risk of VTE. "Women are challenged as men are not," he says. Women in their reproductive years take oral contraceptives, pregnancy predisposes a woman with FVL to deep-vein thrombosis and other thromboembolic events, and many women take hormone replacement therapy later in life.

Perhaps Dr. Triplett's heightened awareness of FVL's impact on women is due to the first FVL patient he saw—a female college freshman who had just started on oral contraceptives. She had a massive DVT and was admitted to the hospital with nonfatal pulmonary embolism. "That kind of scenario tells you that educating people about the relative risks of FVL, especially its increased risk to women, is very important," Dr. Triplett says. He believes that any clinician evaluating a woman for an OC prescription should ask, Have you or any members of your family, especially women, had
a thrombosis?

Of population screening, he says simply, "We really do not want to do that."

As director of the Midwest Hemostasis and Thrombosis Laboratory, Dr. Triplett offers testing for FVL, prothrombin G20210A, and MTHFR C677T. "In most laboratories, these three tests are performed by molecular testing," he says. In his laboratory, gene-based testing is done using the Invader system from Third Wave Technologies (Madison, Wis.), which he finds easy to use.

Dr. Marchant, who coordinated the section in the CAP conference report on testing for deficiencies of proteins C and S and antithrombin, says that these tests are reserved for persons who have had an episode of venous thrombosis under age 50 with no apparent inciting factors, such as malignancy, immobilization, surgery, or obesity—VTE that occurs "out of the blue." The local coroner recently called Dr. Marchant about a 22-year-old man who died suddenly of pulmonary embolism; on autopsy a large metastatic testicular tumor was found. She recommended against testing for thrombophilic mutations because of the documented tumor.

Functional assays, rather than gene-based tests, are used for initial testing for proteins C and S and antithrombin deficiencies, because, unlike FVL and prothrombin G20210A, these deficiencies are due to multiple mutations. Protein C deficiency, for instance, can be due to any of more than 160 mutations. "Any one genetic test is unlikely to detect all those mutations," Dr. Marchant says.

It is best to measure protein C remote from an acute thrombotic event, when the patient has been off oral anticoagulation for a month. However, many thrombosis patients come from a distance and can be tested only in the hospital. A practical recommendation is to measure protein C when you can. "If it is normal, that can exclude a deficiency," Dr. Marchant says. An abnormal result in the setting of an acute event or during oral anticoagulant therapy is uninterpretable.

Recommended screening tests for protein C deficiency include chromogenic (amidolytic) assays that are widely available for automated coagulation instruments and have good sensitivity and reproducibility. Because these assays measure activity, they detect deficiencies due to gene deletions and point mutations.

A clotting-based assay is also widely available. "I don't recommend this as strongly for general testing," Dr. Marchant says, "since it is subject to multiple interferences, including heparin, high levels of factor VIII or FVL, and lupus anticoagulant." The clotting assay does measure the ability of protein C to bind to protein S or factor V, which could be missed with a chromogenic assay unless the mutation is at the active site. "However," Dr. Marchant says, "most protein C mutations are not missed by chromogenic assays."

Testing for protein S deficiency "remains problematic," she says. There is no direct functional assay, because protein S is not an enzyme, but a cofactor for protein C activity. A clotting assay is available, but it is subject to the same interferences as for protein C. Dr. Marchant uses the clotting assay and screens the sample for all possible interfering substances if the initial clottable protein S result is decreased—heparin, lupus anticoagulant, and elevated factor VIII or FVL. "That's a lot of testing just to make sure your result is valid," she says.

Another complication with protein S is that it is present in plasma both in the free state and bound to a complement regulatory protein, C4bBP. Only free protein S is active. Persons with high C4bBP activity may have low plasma protein S activity. An antigenic assay that measures total and free protein S with polyclonal antiserum, polyethylene glycol precipitation, and centrifugation is "notoriously inaccurate," Dr. Marchant says. "In the conference, we recommended that probably the best screening assay is one of the new antigenic assays that use a monoclonal antibody to detect free protein S, which are not subject to as many interferences." Her laboratory is switching to a monoclonal assay.

Because the activity of both protein C and protein S is vitamin K-dependent, protein C or S cannot be measured accurately in patients on anticoagulant therapy. Waiting one month after cessation of therapy is recommended. Also, antithrombin levels are decreased in patients on heparin therapy. Unfortunately, Dr. Marchant says, "As a reference laboratory, we get many samples with no information on whether a patient is on an anticoagulant. In these cases, results can be difficult to interpret." For this reason, she often does a pro-time, aPTT, or anti-Xa assay to determine whether a patient is on coumadin or heparin.

Antithrombin assays, in contrast, are fairly straightforward. Chromogenic or amidolytic assays are most widely used and have fairly good sensitivity and precision, though patients on heparin may have decreased antithrombin activity.

With one set of tests (proteins S and C and antithrombin) being done in the coagulation laboratory and the others (FVL, prothrombin G20210A, and MTHFR C677T) in the molecular laboratory, how are results coordinated? Typically, they are not.

"Unless a clinician requests a clinical pathology consult, which they rarely do, individual test results are sent back with individual interpretations," Dr. Press says. "No one in the laboratory puts the whole picture together." At Oregon Health Sciences University, many patients getting thrombophilia workups are seen in the thrombophilia clinic by hematologists, who can usually integrate results themselves.

However, Dr. Press says, about three-fourths of his testing is done on cases from community hospitals. He offers an interpretive report with molecular test results. "But I am completely in the dark about protein S and C and antithrombin results," he says, "as well as about the patient's clinical status. As in most of pathology, the appropriate clinical information usually doesn't accompany the specimen. So we are often interpreting these tests in the dark. I worry a bit about that and I think many of my colleagues do
as well."

Midwest Hemostasis and Thrombosis Laboratory does a full spectrum of testing for patients who bleed or have thromboembolic disorders. In other places, Dr. Triplett says, "Even among those with a substantial degree of sophistication in a coagulation laboratory, many offer only antithrombin and protein C and S. They may be sending genetic tests to a reference laboratory." They would need sufficient volume to justify incorporating gene-based tests into their own laboratory, and they would need a coagulation specialist who is comfortable with those mutations. If not, it might be wise to refer patients and families with thrombophilic mutations to another hospital.

Therapeutic implications of thrombophilia assays are uncertain. "One of the things we said up front in the recommendations," Dr. Press says, "is that there is really no evidence that any of these test results are going to impact therapy." It is not clear that you would treat a patient who is positive for FVL or prothrombin G20210A longer or more intensively. However, some test results may affect prophylaxis in family members, especially female family members. "We believe that the knowledge of carrying the Leiden mutation may, and perhaps should, impact decisions such as choice of contraception and perhaps use of prophylactic anticoagulation during and/or after pregnancy," Dr. Press says.

Dr. Marchant calls therapeutic decisions in patients with thrombophilic mutations, especially those with a combination of mutations, "an area of great clinical controversy."

Amid all this uncertainty, Dr. McGlennen says, "We have a remarkable opportunity." But what can laboratorians do to get primary care physicians and surgeons to think about gene-based testing for thrombophilia? Coagulation laboratories, which primarily work up patients with a tendency to bleed, are always in the minds of clinicians doing a workup. "Along come gene-based tests to identify individuals who have a tendency to form abnormal clots, which appear to be orders of magnitude more common than bleeding problems," Dr. McGlennen says. "Now we have gene-based markers to look for more common diseases and a conventional laboratory algorithm that is well ensconced in routine practice but typically only for rare events. What I am suggesting is that we have an opportunity to look at a common thing commonly with DNA-based testing for FVL and prothrombin mutations," he says. "This is a challenge that I am working on."

William Check is a medical writer in Wilmette, Ill.