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  Looking for trouble in all the right places


CAP Today




January 2008
Feature Story

Karen Titus

Dot Adcock, MD, practically emits sunbeams when she talks about coagulation. It’s a subject that has caused many clinicians, and even some pathologists, to start muttering darkly, but Dr. Adcock, medical director of coagulation at Esoterix Laboratory Services, Englewood, Colo., says things like, “Gosh, it seems I’ve always been interested in coagulation” and “It’s great fun for me.”

Don’t be fooled. Dr. Adcock has a dark side. It’s the side that worries constantly about what can go wrong in coagulation testing, the side that goes looking for trouble when all appears well. Yet somehow, Dr. Adcock strikes a balance between sunny-side up and sunny-side down—think Doris Day in the role of Captain Ahab.

Dr. Adcock launched her coagulation career when trouble came looking for her, in the form of a furious anesthesiologist. At the time, Dr. Adcock was a resident on the clinical pathology service at the veterans hospital in Denver. A patient undergoing surgery was bleeding significantly, and the anesthesiologist and surgeon couldn’t figure out why. “They decided it wasn’t anything that they were doing wrong, that it was a coagulopathy,” Dr. Adcock recalls. “I’ll never forget: The anesthesiologist came downstairs and said, ‘The patient’s bleeding, it’s heparin rebound, and it’s your fault.’”

She didn’t know what heparin rebound was, or if that was why the patient was bleeding. (It wasn’t, as it turned out.) It wasn’t even clear to her if the anesthesiologist knew what heparin rebound was. What was clear was his anger at the lab. That’s when Dr. Adcock came to a decision: “I was never going to not know what they were throwing at me again.”

Given her prominent role at one of the nation’s largest laboratories for esoteric testing, Dr. Adcock appears to have made good on her vow. But, she maintains, her outlook is more than just a personal creed, or at least it should be; she’d like it to be the norm in coagulation laboratories.

She doesn’t see that happening, however. Instead, she sees a fault line emerging. It’s not that coagulation doesn’t intrigue physicians; nevertheless, she says she’s seeing fewer of them delve into it with deep understanding. “As the testing becomes more complex, as it has over the years, I think there’s a greater disparity between physicians’—and even other pathologists’—comfort level with the difficult degree of testing that is now available.”

This plays out endlessly in the coagulation lab, says Dr. Adcock, even with tests that might seem routine. “Even with our bread-and-butter special coagulation tests, you have to always be thinking, What else could this be? Could I be going down the wrong track?”

When labs are asked to perform a factor VIII inhibitor, she says, “I would guess 95 percent of the time they’re going to be right”—the patient is tested for factor VIII inhibitor and does indeed have a factor VIII inhibitor. But it takes a seasoned, and suspicious, expert to identify those other patients.

If the laboratory does not have a good understanding of what scenarios may mimic a factor VIII inhibitor, it may report out a low factor VIII activity with an inhibitor titer that is, in fact, spurious.

What are these imposters? One is a lupus anticoagulant—a far different clinical scenario for the patient, Dr. Adcock notes, since it is not typically a cause of bleeding but rather predisposes toward thrombosis.

The other mimicker—and one that is poorly appreciated, says Dr. Adcock—can occur if the sample is EDTA plasma, rather than citrated plasma. This is an important difference that’s impossible to see. “Plasma collected into different anticoagulant tubes and serum samples all look the same.”

“But one of the things that a knowledgeable coagulation laboratory should do is begin working on the puzzle as soon as it gets the sample,” she says. Step one: Take nothing for granted. “So when we get a sample that has a low factor VIII activity, we begin to try to figure out if it is in fact one of these mimickers. And you’d be surprised how often we see this.”

Lupus anticoagulants can mimic a factor VIII deficiency with inhibitor since the antibody can interfere with the phospholipid needed in the factor VIII activity assay and the antibody can also give a false-positive in the inhibitor assay.

Dr. Adcock suggests looking at the mixing studies and at other intrinsic factors, in addition to the factor VIII activity. With a true factor VIII inhibitor, an incubated plasma mixing study will show prolongation, so “if we have a low factor VIII activity with what looks like a positive inhibitor titer, and we don’t see prolongation on an incubated mixing study, we begin to get suspicious. We’re always suspicious, as a matter of fact. So we might also look at a factor IX or a factor XI activity, and if that’s also low, then that would give us a clue to non-specific inhibition as may occur with a lupus anticoagulant. It can be very difficult, though, because a strong factor VIII inhibitor can do the same thing,” she says.

That’s why her lab will also perform a chromogenic factor VIII activity assay, which measures the activity of factor VIII using a method that is essentially not phospholipid dependent. With a true factor VIII inhibitor, the chromogenic VIII activity would be low, but with a spurious inhibitor due, for instance, to the presence of a lupus anticoagulant, the chromogenic VIII activity would be normal.

With an EDTA sample, says Dr. Adcock, clues start emerging with the running of a prothrombin time and a partial thromboplastin time. A true factor VIII typically would generate a normal PT and a prolonged PTT; with an EDTA sample, both measurements are prolonged. And neither shows corrections with a plasma mixing study.

“I guess this actually does get pretty complex,” she muses. So does testing for von Willebrand disease. From the get-go it requires multiple assays for an appropriate diagnosis. If a clinician orders just one assay to evaluate for von Willebrand disease, alarm bells should start ringing, and this is when Dr. Adcock might call the physician. Her lab recommends evaluating a panel of assays in the investigation for von Willebrand disease including factor VIII activity and von Willebrand factor antigen, and when measuring von Willebrand factor activity, the laboratory likes to evaluate both ristocetin agglutination and collagen binding. The lab then considers the results as a whole to make sure all the pieces fit. If not, it will pull another aliquot for testing to make sure something hasn’t gone wrong.

What can go wrong? “A million things,” says Dr. Adcock, who’s apparently exaggerating only slightly. For starters, von Willebrand factor and factor VIII are both acute phase reactant proteins and are also highly subject to preanalytical conditions that can interfere with results. Both are cryoprecipitable, so failing either to thaw the sample adequately or to mix it adequately once it’s thawed may result in protein loss. Or, if the sample is thawed in a warm water bath and left sitting too long, or thawed at too high a temperature, both factors will be degraded. In fact, factor VIII can degrade if the sample is left sitting too long at room temperature on the phlebotomists’ table or the medical technologists’ bench.

If the whole blood sample is refrigerated, both factors will be lost as well. Ditto if the sample is put on ice prior to its being spun. “We instruct our clients very carefully how to submit the samples. By evaluating the panel of assays and in particular using the two von Willebrand factor activity assays, it can give a really clear picture, we believe, of what’s going on with the patient,” Dr. Adcock says. “Because the ristocetin activity assay is not a very robust assay, and it’s not very reproducible. And we may see low results, which many times are spurious.” In a study published in Seminars in Thrombosis and Hemostasis (Adcock DM, et al. 2006;32:472–479), Dr. Adcock and her colleagues evaluated nearly 500 cases and found that the ristocetin cofactor assay was commonly 10 percent to 20 percent lower than the antigen or the collagen-binding assays.

The study highlights another role for coagulation experts—unearthing, and addressing, problems that can occur in the lab to better ensure quality testing. “We work very closely with CLSI generating guidelines and standards for laboratory testing,” Dr. Adcock says. “Many of us in coagulation are very involved in generating these documents, as we should be.”

That’s teaching pathologists. What about others? Like most coagulation gurus, Dr. Adcock gives plenty of credit to her medical technologists, most of whom have decades of experience solving coagulation puzzles. Though they initially learned the complicated workups by following algorithms, the steps now come naturally, Dr. Adcock says.

And when those technologists retire? Dr. Adcock’s lab is not alone in facing an aging technologist population. Her solution is to bring younger technologists onboard regularly, then educate them endlessly. That includes pairing newer arrivals with veterans. The lab also does regular case-of-the-week reviews to deconstruct unusual problems. In addition, the lab relies on what Dr. Adcock calls medical validation. “When a panel of tests are ordered, we never send out a single result in isolation—results are reviewed and sent out as a conglomerate,” she says. “And the techs are instructed to look at all those results together. When they see something they don’t understand, they bring it to me or to a senior tech for explanation and resolution.”

“We purposefully try to spread the knowledge,” she says. “It’s the only way we can keep doing what we’re doing.” As for educating her clinical colleagues, Dr. Adcock hedges a bit. “Coagulation sometimes gets kind of, well, quirky,” she says, sounding amused yet slightly guarded, the way parents might when explaining that their son enjoys, say, wearing a princess costume to preschool. “There are nuances that are evident only in this field, and I think it can be difficult to teach them.”

Instead of expecting clinicians to know all the things that can go wrong with a test, she says it’s up to the pathologist or hematologist to keep clinical colleagues informed. That means working as a consultant—perhaps a dirty word in some industries, but definitely not in the coag lab. “You have to be able to speak to the test,” she says. “You need to understand the basis of it, and help clinicians interpret it and then know what to do with it.”

It also means answering the phone. “We try to hold all our staff meetings or technical meetings in my office, so that I can be available to pick up the phone when a doctor calls with questions.” Dr. Adcock says the response to such a simple act is overwhelming. “When they find out there’s a readily available resource out there to help them, they’re thrilled,” she says.

Coagulation labs also need to let physicians know what new tests might be coming down the pike. Dr. Adcock points to companion diagnostics—laboratory testing linked to therapeutics. This includes warfarin pharmacogenomics testing, for starters.

Close to 50 percent of the variability to warfarin response is due to the presence of polymorphisms, which occur in two regions: the drug’s target, and the way the drug is metabolized, Dr. Adcock says. Warfarin’s target is the vitamin K epoxide cycle, which can be affected by a polymorphism in the VKORC1 enzyme complex. The rate of metabolism can be affected by a genetic variation in the cytochrome P450 system, called CYP2C9. The polymorphisms are quite common, Dr. Adcock noted in a fall ASCP teleconference: CYP variants occur in one-third of the Caucasian population, while VKORC1 variants pop up in one-half of that same population.

Less known, perhaps, is the role of companion diagnostics in antiplatelet therapy. There are undoubtedly polymorphisms that relate to how a patient responds to these therapies, says Dr. Adcock, whether aspirin or clopidogrel. Unlike warfarin pharmacogenomics, genetic testing related to antiplatelet therapies is not performed. “At this point, however, we can use platelet-function studies or measure platelet metabolites such as 11-dehydrothromboxane B2 to begin to get an idea whether a patient is responding to antiplatelet therapy,” Dr. Adcock suggests.

Coagulation problems are an ideal target for personalized medicine, given the endless combinations and interactions of procoagulant and anticoagulant factors. This leads Dr. Adcock to another area of interest: thrombin generation. Her lab offers this global assay (restricted, for now, to use in clinical trials) that looks at a patient’s ability to generate thrombin by assessing the balance of their procoagulant factors (such as factors VIII and IX), and anticoagulant factors (such as proteins C and S). It can also be used to dose replacement therapies such as to determine whether a hemophiliac has received an adequate amount of factor VIII; or, on the flip side, if a patient requiring blood thinner has been adequately anticoagulated.

Dr. Adcock offers the example of two severely hemophiliac patients. Patient A may bleed to a greater degree than patient B, even though they both have factor VIII levels of less than one percent, because of different baselines in their thrombin generation—one could be heterozygous for factor V Leiden, for example. The thrombin generation test epitomizes personalized medicine. “It looks at each individual patient’s propensity for thrombin generation, and based on this, one can evaluate if the patient has a bleeding or clotting potential, or ultimately it can also be used in treating the patient so that a physician can administer just enough factor concentrate, or just enough anticoagulant.

“This is very exciting,” Dr. Adcock says. “But it’s not quite ready to introduce.” While it’s a global assay, it must be optimized for the indication for which it is used, she cautions, be it measuring bleeding potential, evaluating for a hyper- or hypocoagulable state, or measuring response to factor concentrate or anticoagulation therapy. “You have to use different activators of coagulation at different concentrations to evaluate that particular aspect you’re interested in. So it’s a very, very complex assay. It’s got a lot of potential, but it’s got to be used and introduced properly. That, again, is another area where you can really go wrong,” she says.

Even assays as common as PTTs can be problematic, too, she says, if their use and limitations are not properly understood. New antithrombotics, such as direct thrombin inhibitors, are commonly monitored with PTTs. “Which is a travesty,” says Dr. Adcock, who considers the test inappropriate for monitoring heparin therapy as well as direct thrombin inhibitors.

It’s just another example of What Can Go Wrong. “One of the biggest problems we need to alert our laboratorians to is that when using the PTT to monitor direct thrombin inhibitors, you will see a plateau effect, where the elevation of the PTT flattens out. You don’t get continuous prolongation of the APTT, despite increasing concentration of direct thrombin inhibitor in the blood. So the PTT may be—may be, and I use that loosely—sufficient to monitor a therapeutic range, but once you become supratherapeutic, it’s still going to look like the PTT is therapeutic.” Instead, she says labs need to offer testing that’s more directed toward the antithrombotic, whether that be an anti-factor Xa assay, anti-factor IIa, an ecarin clotting time, or the thrombin generation assay. “You don’t want to miss an overdose,” she says bluntly.

Again with the looking for trouble. When her cell phone rings (as it did partway through her interview with CAP TODAY), those within earshot will hear a few choice bars of “Fanfare for the Common Man.” Not bad, but perhaps “Suspicious Minds” would be a better fit. Though Dr. Adcock wants her lab to produce results that are right, she and her colleagues spend just as much time asking what might be wrong. It’s the nature of coagulation. And suspicious minds may be some of the best tools at their disposal.

Karen Titus is CAP TODAY contributing editor and co-managing editor.