For complex coag disorders,
  plots thicken

November 2005
Feature Story

William Check, PhD

“When you hear hoofbeats, think horses, not zebras.”

In medicine, that’s usually good advice. Yet a good laboratorian needs to be able to carry out daily testing for routine questions while at the same time knowing when to suspect an uncommon disorder and which tests to perform when that possibility arises. No where is this more important than in the coagulation laboratory.

At a symposium in September at the CAP ’05 annual meeting, two speakers discussed new information about complex coagulation disorders. Timothy E. Hayes, MD, DVM, chief of clinical pathology at Maine Medical Center, Portland, presented a new scheme for classifying and diagnosing von Willebrand’s disease that is being proposed in the literature. And Mark T. Cunningham, MD, director of the hematology laboratory at the University of Kansas Medical Center, Kansas City, described a new biochemical understanding of thrombotic thrombocytopenic purpura.

A third speaker, Elizabeth M. Van Cott, MD, director of the coagulation laboratory at Massachusetts General Hospital and assistant professor of pathology at Harvard Medical School, discussed something that is becoming increasingly common: the use of newer anticoagulants, occasioned at least in part by the not-uncommon complication, heparin-induced thrombocytopenia. As newer anticoagulants enter clinical practice, the type and volume of monitoring assays ordered could change, Dr. Van Cott said in an interview with CAP TODAY. “Adoption of newer anticoagulants, some of which don’t require monitoring, could reduce the volume seen in a routine coagulation laboratory,” she says. “You might see fewer PTTs [activated partial thromboplastin times] if heparin use decreases and fewer PTs [prothrombin times] if clinicians use less Coumadin.” On the other hand, increased use of newer anticoagulants will expand the workload in special coagulation laboratories that offer chromogenic factor X or anti-factor Xa assays.

Von Willebrand’s disease, or vWD, named after the early 20th century Finnish internist who studied the first cases, is an inherited bleeding disorder due to abnormalities of von Willebrand factor, or vWF, a multimeric glycoprotein. When vascular endothelium is injured, vWF promotes hemostasis by linking collagen fibrils in the damaged endothelial tissue matrix to platelets in the circulation, promoting clot formation. If a defect causes loss of vWF protein or reduces its function, variable degrees of bleeding tendency can result. Reduction in vWF activity also affects secondary hemostasis, since vWF acts as a protective carrier protein for factor VIII.

Many types of assays have been proposed to diagnose vWD. In the symposium, Dr. Hayes listed nine separate categories: bleeding time, PTT, factor VIII activity, an immunologic assay for vWF antigen, an activity test for vWF function (such as ristocetin cofactor, collagen-binding assays, and ELISA/flow cytometry assays), Platelet Function Analyzer (PFA-100), vWF multimers, and molecular assays. No single test is ideal. Measuring bleeding time does not correlate well with bleeding symptoms. Decreased vWF levels may or may not prolong the PTT. In mild vWD, factor VIII levels are often in the low-normal range. Measuring vWF multimers is difficult to do and is performed only in specialty laboratories. On the positive side, measurement of vWF antigen is a simple and useful test. An activity assay, usually ristocetin cofactor, is also helpful.

At least as problematic as testing for vWD is classifying its various manifestations. The primary system in use until about 1994 evolved to the occasional use of multimer patterns alone for subgroup classification. Unfortunately, Dr. Hayes said, this does not always correlate well with clinical behavior or type of genetic lesion.

More recent classification systems have been based on the axiom that all vWD is caused by mutations at the vWF locus. Therefore, cases of “acquired vWD,” due to such conditions as malignancy, lupus, or drugs (for example, ciprofloxacin, valproic acid), are not included, Dr. Hayes noted. In practice, three types of true vWD are distinguished: type 1, in which there is a partial quantitative deficiency of vWF; type 2 (which has several subclasses), in which there is a qualitative deficiency of vWF; and type 3, in which there is a complete quantitative deficiency of vWF.

Two facts are central to diagnostic testing: vWD types 2 and 3, which together account for about one-fourth of all cases, are fairly easy to diagnose, while vWD type 1, which makes up about 75 percent of all cases, is often difficult to diagnose and is frequently overdiagnosed. “The majority of those felt to have vWD type 1 present a significant diagnostic and therapeutic challenge,” Dr. Hayes said. He described a newly proposed but not yet widely accepted framework for addressing this challenge.

While some people with vWD type1 have very low levels of vWF (<15 percent), experience repeated and serious bleeding, and appear to have autosomal dominant inheritance, most patients thought to have vWD type 1 do not meet these three criteria. Rather, they have vWF levels between 15 percent and 50 percent of normal, have only mild bleeding, and come from families in which some family members don’t even experience mild bleeding.

(Aspects of heredity are especially problematic in vWD type 1. While the cornerstone of the classification is that it must be an inherited bleeding disorder, Dr. Hayes told CAPTODAY that “a lot of genetic databases have proven much more fruitful in shedding light on type 2 rather than type 1.” The genetic situation, he says, “is very complex and there is a lot that we still don’t understand about type 1.” Current classifications of vWD are still primarily phenotypic. “We have not found a primary genetic defect that can explain the majority of type 1 disease as it is currently defined,” Dr. Hayes says. “Except for persons with type 2 and 3, we don’t have a good idea of why their vWF is in the low range.”)

Dr. Hayes listed four reasons why vWD type 1 is difficult to diagnose. His analysis was taken from a paper titled “Von Willebrand Disease Type 1: A Diagnosis in Search of a Disease” (Sadler JE. Blood. 2003; 101:2089–2093).

There is a broad distribution of normal vWF levels: The normal range is 50 percent to 200 percent of the mean. About 30 percent of this variation is accounted for by ABO blood type. Within type O, there is considerable overlap in vWF values between obligate heterozygotes and normals, with many normals in the vWD range and many heterozygotes in the normal range.

There is a high prevalence in the normal population of mild bleeding symptoms. Surveys of healthy controls show excessive nosebleeds, gum bleeding, and menorrhagia in five percent to 39 percent, seven percent to 51 percent, and 23 percent to 44 percent of people, respectively. At the lowest estimate, 25 percent of males and 46 percent of females have at least one bleeding symptom. These common symptoms may be the basis for (mis)diagnosing “a bleeding diathesis consistent with VWD,” Dr. Hayes says.

Most diagnoses of vWD type 1 are false-positives. Given a prevalence of bleeding in the normal population of 25 percent, and defining vWD type 1 as the lowest 2.5 percent of the mean vWF value (two standard deviations from the mean), then 0.6 percent of the population will have both low vWF and bleeding by chance. This is equivalent to the published prevalence of vWD type 1.

There is a weak relationship between vWF level and bleeding. For instance, in an Italian study, there was a striking lack of concordance between low vWF levels and the occurrence of mild bleeding symptoms. A low vWF level did not predict surgical bleeding; factor VIII was more important.

In summary, Dr. Hayes quoted Sadler’s conclusion: “Rendering a diagnosis of vWD type 1 is often difficult to confirm and may be more confusing rather than helpful.”

Rather than thinking of vWD as a binary “yes/no” diagnosis, Sadler proposed an approach that views bleeding risk as a continuum, which is related to vWF level. He suggested reserving a “diagnosis” of vWD type 1 for individuals with vWF levels of less than 15 percent, frequent bleeding, and dominant inheritance. For these people, appropriate lifestyle changes and prophylactic interventions are justified. “Using this exceptionally low level of vWF to define type 1 disease would reduce misdiagnosis (overdiagnosis),” Dr. Hayes says. Those with vWF levels between 15 percent and 50 percent would be told they have “low von Willebrand factor,” which would be regarded as a risk factor for bleeding and treated empirically.

At the present time, Dr. Hayes told CAP TODAY, “I don’t know how much exposure Sadler’s concept has gotten. It is not mainstream yet; it is working its way through the system. People are mulling it over.”

Dr. Hayes does like Sadler’s concept. “It is not black and white. This has been a gray area for a fairly long time, and Sadler’s scheme offers a more practical and manageable approach to these patients.” It is too early to tell how clinicians and specialty societies are going to react to it, he adds.

An abnormality in vWF activity also underlies the new understanding of the etiology of thrombotic thrombocytopenic purpura, or TTP, an inherited or acquired disorder occurring in about four individuals per million. Acquired cases, which make up 90 percent of TTP, can be due to autoimmunity, malignancy, pregnancy, or a variety of drugs such as the antithrombotic drugs clopidogrel (Plavix) and ticlopidine (Ticlid); about half of acquired cases are idiopathic. TTP is characterized by microvascular thrombosis localized to the kidney and central nervous system and affecting principally small vessels—arterioles and capillaries. Clots are platelet-rich on biopsy and lack fibrin staining. Purpura lesions on the skin often occur, due to hemorrhages consequent to platelet depletion.

Dr. Cunningham said TTP is rare—most hospitals will see one or two cases per year at most. Yet he believes that physicians need to be knowledgeable about TTP because, when it does present, it can be rapidly fatal and treatment is effective. “Recognizing the clinical presentation and making an accurate diagnosis is life-saving,” he says. Most helpful in diagnosis are two universal features: thrombocytopenia and microangiopathic hemolytic anemia. Schistocytes are increased to e4 per field under 100x oil objective. Lactic dehydrogenase is elevated seven to 10 times normal.

Underlying the inherited form of TTP and many cases of the acquired form is altered vWF homeostasis (Moake JL. Semin Hematol. 2004;41: 4–14). VWF is made by endothelial cells as an ultra-large (UL) multimer that is proteolyzed to smaller, normally active multimers by an enzyme with the clumsy name “a disintegrin and metalloprotease with thrombospondin-like domains,” or ADAMTS13 for short. When ADAMTS13 is nonfunctional, UL multimers are not cleaved and TTP results. Inherited cases of TTP are caused by mutations in the gene that codes for ADAMTS13. Most acquired cases are thought to be due to autoantibodies that neutralize the enzyme’s function.

As described for vWD, cleaved vWF multimers promote clot formation by causing platelet aggregation in damaged vessels. Uncleaved UL multimers also promote platelet aggregation, but in a damaging manner. Like an eager but uncoordinated child who only wants to help, uncleaved UL multimers do considerable harm. “Uncleaved multimers are hyperfunctional,” Dr. Cunningham explains. “They spontaneously induce platelets to clump under high shear forces, usually in the arterial circulation.” Platelet aggregates catalyzed by uncleaved UL multimers consist of three-dimensional clumps, or plugs, of platelets, as opposed to the physiologic platelet monolayers caused by cleaved multimers.

Laboratory testing for TTP focuses on measuring ADAMTS13 enzyme activity or autoantibody titer. Many enzyme assays are available—ELISA, electrophoresis, platelet agglutination. In all assays, patient plasma is added to a standardized solution of multimers and the products are evaluated, for instance, by electrophoresis (for example, Gerritsen HE, et al. Thromb Haemost. 1999;82:1386–1389). Samples must be collected in citrate tubes to avoid EDTA chelation of zinc and calcium, cofactors for the enzyme.

In six studies, a range of sensitivities has been reported for activity assays, ranging from 15 percent to 100 percent, depending on the population. Sensitivity is 100 percent for inherited TTP and for acquired TTP due to drugs. For TTP secondary to pregnancy or autoimmune disease, sensitivity is about 50 percent. Specificity for TTP is 100 percent at less than five percent enzyme activity. “If you suspect TTP from historical or clinical criteria, a test result of less than five percent defines the disease,” Dr. Cunningham said. A normal level does not rule out TTP. Enzyme activity levels also have prognostic significance: Less than five percent enzyme activity predicts an 83 percent response rate to therapy, while people with a level greater than five percent have only a 33 percent response. (“Response” is defined as life or death following therapy.) Therapy for inherited TTP is plasma infusion. For acquired cases, plasma exchange is required. In drug-induced TTP, symptoms resolve if the drug is removed.

Sensitivity of the autoantibody assay is 79 percent for idiopathic TTP, 100 percent for drug-induced TTP, and about 45 percent for TTP due to autoimmunity or pregnancy.

Dr. Cunningham noted several disadvantages of both types of laboratory tests for TTP. They have limited availability and are done only in specialty reference laboratories because of their high complexity. They also have a long turnaround time—four to 10 days—so they can’t be used to determine initial treatment. Therapy is based on clinical criteria and the presence of the two key features—microangiopathic hemolysis and thrombocytopenia. “In our center, TTP is often diagnosed and treated on an outpatient basis before enzyme levels come back,” Dr. Cunningham said. Treatment must be started as soon as possible because the condition can progress swiftly and early treatment is necessary to prevent neurological symptoms and renal failure.

“The diagnosis of TTP is still largely a clinicopathologic diagnosis,” Dr. Cunningham concluded. Even so, he said, “The discovery of this enzyme has advanced our understanding of TTP in terms of its etiology and pathophysiology.”

Dr. Van Cott elucidated the relation between the third clotting disorder, heparin-induced thrombocytopenia, or HIT, and newer anticoagulants. In normal hemostasis, platelet adhesion and aggregation are followed by formation of a fibrin clot. Coumadin and heparin both inhibit formation or extension of the fibrin clot, heparin by enhancing the activity of antithrombin III 1,000-fold and Coumadin by interfering with post-translational modifications that are required for activity of vitamin K-dependent clotting factors. Development of many new anticoagulants has been stimulated by the drawbacks of these drugs, such as imperfect prophylaxis of deep vein thrombosis by heparin, osteoporosis when heparin is used for extended lengths of time, and bleeding risk with both heparin and Coumadin. Another important inducement to look for improved anticoagulants has been HIT, which Dr. Van Cott called “the most significant adverse effect of heparin after bleeding.” HIT is a drug reaction in which antibodies are formed to the heparin-platelet factor 4 (PF4) complex. Binding of the antibody to the complex causes platelet aggregation, leading to venous and arterial thrombosis.

Dr. Van Cott cited a recent meta-analysis of 15 studies that concluded that 2.6 percent of patients exposed to heparin (most undergoing orthopedic surgery) got HIT, as defined by a decrease in platelets to less than 50 percent or to less than 100 x 109/L and a positive HIT assay (Martel N, et al. Blood. 2005; 106:2710–2715). For low-molecular-weight heparin, the corresponding figure was 0.2 percent. Depending on the study, 25 percent or more of patients who have such low platelet counts will have thrombosis within 30 days, Dr. Van Cott told CAP TODAY. The number of patients exposed to heparin is rather large, she says, including not only those undergoing orthopedic procedures and deep vein thrombosis prophylaxis or treatment, but also patients having heparin flushes or heparin-coated catheters and many cardiac patients.

Commonly used laboratory assays for HIT are ELISAs that measure the antibody against the heparin-PF4 complex. Dr. Van Cott presented data on the consequences of HIT from the first 90 patients with the condition seen at Massachusetts General who were ELISA-positive. These patients were seen over an 18-month period during 1996 and the first half of 1997, when the coagulation laboratory started doing the Diagnostica Stago HITELISA assay. In 69 percent of patients thrombocytopenia without thrombosis was present, and in 30 percent of patients both thrombosis and thrombocytopenia occurred. In one percent, thrombosis occurred without thrombocytopenia. Mortality in patients with HIT and thrombosis was 25 percent, while 32 percent experienced pulmonary embolism. Many other sequelae were also seen, such as stroke and amputation. Length of stay was significantly increased. Many had “massive” clots, Dr. Van Cott observed.

Among 205 patients in the surgical ICU at Massachusetts General who had an HIT ELISA based on clinical suspicion, 19 were positive. Of these 19 patients, 32 percent died, much higher than the seven percent mortality rate among controls. Heparin flushes were the sole source of heparin that induced HIT in 12 of the 19 patients.

Dr. Van Cott presented results of a study that she and her colleagues did to evaluate whether repeat ELISA testing is useful. They found that 13 of 30 (43 percent) patients who had a high-titer negative HIT assay, defined as 66.7 percent of the threshold positive value, turned positive over the next three days or so (Refaai MA. Am J Clin Pathol. 2003;119:61–65). The laboratory now reports high-titer negative results with the comment, “Negative but borderline; suggest repeat in three days.”

Therapy for HIT is to discontinue all heparin and low-molecular-weight heparin, or LMWH. However, this leaves the patient in danger from existing thrombosis or the risk of future thrombosis. Enter the newer anticoagulants—the direct thrombin inhibitors recombinant hirudin (r-hirudin), bivalirudin, and argatroban and the synthetic heparin derivative fondaparinux, a pentasaccharide comprising the active site of heparin specifically for antithrombin III. Only r-hirudin and argatroban are FDA-approved for treating HIT, and only argatroban is approved for HIT thrombosis prophylaxis. However, Dr. Van Cott’s experience is that clinicians are also using bivalirudin and fondaparinux in HIT cases.

Some of these newer anticoagulants have additional indications. Argatroban and bivalirudin are approved for use in percutaneous coronary intervention/angioplasty, and fondaparinux is approved for preventing venous thrombosis in knee and hip surgery and to treat inpatients with deep vein thrombosis or pulmonary embolism. While these drugs have clinical advantages over heparin, they carry a familiar drawback—expense. Fondaparinux and LMWH are more expensive than heparin and approximately equivalent to each other in price. Most expensive are the three direct thrombin inhibitors.

All four newer anticoagulants have a more predictable dose-response pattern than heparin, so laboratory monitoring is more convenient and less crucial. For argatroban and r-hirudin, PTT is used. Bivalirudin requires no monitoring with angioplasty, although the ACT can be used. Like LMWH, fondaparinux does not require monitoring, but anti-factor Xa assays can be used.

Monitoring anticoagulation during a transition to Coumadin is more difficult with r-hirudin or argatroban than with heparin. Normally a patient with thrombosis is started on heparin and Coumadin is begun simultaneously. However, Coumadin takes about five days to exert its effect. During this period, Coumadin’s impact is tracked by calculating the INR (international normalized ratio) from the PT value. When the INR has been in the Coumadin therapeutic range for two days, heparin is stopped. However, if argatroban or r-hirudin is used instead of heparin, this simple scheme does not work, because both of these drugs can prolong the PT/INR. An INR of two might no longer signal a therapeutic Coumadin level. “In this situation you have to be fancier,” Dr. Van Cott says. One alternative is to use a chromogenic factor X assay, targeting the 20 percent to 40 percent range. A second alternative that is useful for r-hirudin, which interferes less than argatroban, is to decrease r-hirudin 50 percent when the INR reaches two and stop it when the INR reaches 2.5. The manufacturer suggests reducing hirudin such that the PTT is 1.5 times baseline, and stopping it when the INR reaches two. For argatroban, a conversion graph is provided in the package insert. For both hirudin and argatroban, their effect on the PT varies among the different PT reagents and is affected by the ISI of the reagent used in the PT assay. Fondaparinux is easier to overlap with Coumadin because it does not prolong the PT as much.

In practice, more monitoring is ordered for newer anticoagulants than is strictly required, all the speakers agreed in followup interviews, though that may change as physicians become more comfortable using them. Monitoring for low-molecular-weight heparins (enoxaparin, dalteparin) set the pattern, according to Dr. Van Cott. “In our hospital,” she says, “clinicians were initially ordering anti-factor Xa for LMWH even when it was not needed. Now they mostly shy away from it.” Monitoring for LMWH is done only for special indications, such as obesity, renal disease, pregnancy, or pediatric cases. In the same vein, clinicians are ordering anti-factor Xa assays for fondaparinux, even though it is not required. Dr. Van Cott attributes this to clinicians’ not being familiar with fondaparinux yet. She expects that monitoring will decline with time.

For r-hirudin and argatroban she sees orders for PTTs, “though these tend to be much more stable, so we don’t see as many PTTs, since there are not as many out-of-range PTTs as with heparin.”

Laboratory interaction with clinicians is more frequent when a newer anticoagulant is used than with heparin and Coumadin. While the laboratory does provide PTs/INRs for Coumadin and PTTs for heparin, Dr. Van Cott says, “it is not often the laboratory will need to be consulted, unless the patient is more complicated.” One such special situation is a patient who has lupus anticoagulant. In that case, the laboratory would try to provide an anti-factor Xa assay for heparin. “Probably not many laboratories do anti-factor Xa in-house,” Dr. Van Cott says. “Most places have to send out.” In the presence of lupus anticoagulant a chromogenic factor X assay is suggested for Coumadin. Again, this assay is not readily available. In both cases, a specialized coagulation laboratory can provide the assay.

Monitoring needs will change as patterns of anticoagulant use change. Much heparin use is being substituted by LMWH, Dr. Van Cott finds. “This is one of the newer anticoagulants that has really been making headway,” she says. One area where heparin is “almost essential,” she says, is cardio pul monary bypass surgery. “Alternative anticoagulants have not been well established for bypass surgery,” she says. In addition, “For patients with renal dysfunction, clinicians are wary of LMWH because of its increased dependence on renal clearance,” Dr. Van Cott says. “Aside from these settings, it is kind of open territory for LMWH.”

However, LMWH is contraindicated for heparin-induced thrombocytopenia, so the newest anticoagulants are used there. In fact, Dr. Van Cott finds that right now the newest anticoagulants are “pretty much limited to HIT patients.”

Dr. Hayes agrees that heparin is being used less. “We’ve seen a dramatic increase in our pharmacy bill for all the newer anticoagulants, including LMWH, all the direct thrombin inhibitors, and fondaparinux,” he says. “Our pharmacy bills are skyrocketing.”

With regard to monitoring, he says they still do a fair amount of PTT work. “What we are finding is that we are doing much less anti-factor Xa monitoring for heparin,” he says. “I think that is because clinicians are using newer anticoagulants for those challenging cases that previously needed monitoring with anti-factor Xa. We clearly are monitoring people on LMWH more than we need to.” Dr. Hayes calls such monitoring “a perceived comfort factor for many clinicians.” In his hospital, clinicians are mostly using PTT to monitor direct thrombin inhibitors. “This has all been protocolized with order sets and clinical pathways,” he says.

With traditional anticoagulant drugs, there is still significant interaction between the laboratory and clinicians, says Dr. Cunningham, “particularly with Coumadin and unfractionated heparin and low-molecular-weight heparin.” For instance, he says, “the laboratory needs to be able to provide test capability in terms of INR for patients on Coumadin.” In addition to patients with DVT or PE, he observes that many people are on Coumadin for atrial fibrillation. In fact, he says, “Atrial fibrillation probably represents the bulk of patients that we see on Coumadin.” It’s also important for the laboratory to communicate proper therapeutic target ranges for Coumadin. The same considerations apply for patients being treated with heparin or LMWH, he notes, for instance, those who have just had a myocardial infarction.

Dr. Cunningham sees two potential impacts of the newer anticoagulants as they are introduced into clinical use. “One thing we are seeing now is that these new drugs have an impact on coagulation tests we perform to evaluate patients with idiopathic bleeding disorders,” he says. For instance, argatroban and r-hirudin influence all coagulation tests that depend on thrombin generation—PT, aPTT, the lupus anticoagulant test, and standard factor assays such as factor VIII for hemophilia A and factor IX for hemophilia B. Coagulation specialists need to be aware of these interactions to avoid false results. He, too, emphasizes a point Dr. Van Cott made: the use of different monitoring assays with the newer anticoagulants.

“We need to be cognizant of the impact of newer anticoagulants on standard tests and of the different ways needed to monitor these new drugs,” he says, “and to implement them in the laboratory and make them known to our clinical colleagues.”