It’s medicine’s equivalent of being a claims adjuster: mastering detailed, painstaking work that no one else wants to do. Not that it’s drama-free: Think of how E.G. Robinson undid Barbara Stanwyck and Fred MacMurray in “Double Indemnity.”

In pathology, several surprises are emerging from the literature. From the July 2006 issue (vol. 32, issue 5) of Seminars in Thrombosis and Hemostasis, which was devoted to lab issues in vWD, comes a fresh picture of how to screen, confirm, and subclassify this maddening disease.

That’s good news for pathologists. In Dr. Hayes’ view, coagulation in general is short-changed in medical school and residency training, and the field—including von Willebrand testing—is rife with myths. “There’s an audience out there who’s highly receptive to what you have to offer,” says Dr. Hayes, a member of the CAP Coagulation Resource Committee.

“I’ve found, over time, that once you make connections with physicians and you’re able to show that you add value, they welcome the conversations,” he continues. At Maine Medical Center, it’s not unusual for clinicians to cold fax to the laboratory their patients’ histories and physicals, along with their own office notes, before and after a von Willebrand workup—a practice many laboratorians can only dream about. “They’ve seen how that helps them take better care of their patients,” says Dr. Hayes.

Wayne Chandler, MD, vice chair for clinical operations, Department of Laboratory Medicine, University of Washington, Seattle, likes to start by asking, Why do most clinicians order von Willebrand workups? “This may seem like an obvious question, but sometimes I think you have to ask that question directly to decide what you’re going to try to tell them,” says Dr. Chandler, who’s also a member of the Coagulation Resource Committee.

With vWD, there seems to be no shortage of tests and information that pathologists can share. Levels of von Willebrand factor in patients vary from day to day. The protein is unstable, and factor levels can be stimulated by an acute phase response, including inflammation, infection, cancer, and pregnancy. Factor levels also vary by blood type, with type O having lower levels and types A, B, and AB having somewhat higher levels.

Little wonder von Willebrand workups have been intricate and nuanced affairs. The Seminars issue is an effort to streamline and improve the diagnosis of vWD, pulling together experts from all over the world. Along the way, it also took aim at some standard practices that may one day find themselves put out to pasture.

Screening

The traditional screening test, the bleeding time, has poor sensitivity. So when the PFA-100 test emerged some 10 years ago as a possible replacement, it caught on quickly. “Many laboratories have brought that test onboard with the hope it would be the definitive screening test,” says Mark T. Cunningham, MD, associate professor of pathology and medical director of laboratory hematology, University of Kansas Medical Center, Kansas City. As evidence of that hope, the CAP launched a proficiency testing Survey for PFA-100, with 600 to 700 labs now enrolled.

Yet PFA-100 is falling short. A review article in Seminars (pages 537–545) authored by Emmanuel Favaloro, PhD, of the Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales, Australia, shows that PFA-100 has considerable limitations. “The overall sensitivity for von Willebrand disease is 85 to 90 percent, which isn’t bad—it’s better than the bleeding time,” says Dr. Cunningham. “But it’s not as good as you’d like for an ideal screening test.”

It appears that the PFA-100 test misses some cases of type 1 vWD, with sensitivity ranging from 50 to 100 percent, depending on the study. Despite this, some experts recommend using the test in selected clinical scenarios.

The first would be if a rapid result is needed—in the case of an urgent surgical procedure, for example. The PFA has a quick turnaround time—less than 10 minutes—and it’s easy to do, according to Dr. Cunningham.

Another scenario would be if the physician simply wants to exclude an unlikely diagnosis. “But if the clinical suspicion is high, some of the experts are recommending that the testing go directly to specific von Willebrand factor assays, bypassing the PFA,” Dr. Cunningham says.

The PFA test may also help determine therapy. Many patients can be treated with desmopressin, which releases vWF from endothelial cells. A PFA test performed shortly after a patient receives a test dose of the drug will indicate whether the patient is responding. “We’ll take someone who has an abnormal PFA, and if you give them the drug and they respond, the PFA test will normalize,” Dr. Cunningham says.

The PFA is not useful for monitoring response to factor replacement therapy. “For some reason, it doesn’t normalize the PFA,” says Dr. Cunningham. “You would have to do specific von Willebrand factor assays to monitor that therapy, like the ristocetin cofactor or a direct von Willebrand factor antigen test.”

Confirmatory testing

The ristocetin cofactor assay has been the test of choice. Recent studies, however—including those by the College as well as NEQAS, the United Kingdom’s external QC group—show this assay has very poor precision. “The interlaboratory coefficient of variations are high—they range from 30 to 50 percent,” says Dr. Cunningham. This could be causing some diagnostic errors, he suggests. A study by the NEQAS group published in Seminars (Kitchen S, et al. 492–498), in which normal plasma was submitted to multiple clinical laboratories for testing, reported a seven percent rate of false-positive diagnoses of vWD. “The reason for those errors isn’t entirely clear,” says Dr. Cunningham, “but one possibility is that it’s related to the poor precision of the ristocetin cofactor test. A test with that high of a coefficient of variation could produce artificially low numbers in someone who really doesn’t have von Willebrand disease.” The CAP’s PT program, which also showed significant variability in the assay (CVs between 23 and 30 percent) was described in another Seminars paper (Hayes TE, et al. 499–504).

Dr. Chandler suggests the problem may be somewhat intrinsic to the test. Patient plasma is added to donor platelets (either formalin fixed or washed formalin fixed, or, occasionally, fresh); ristocetin is then added, and observers see how fast the platelets aggregate. The aggregation tracings have a fair amount of variation, depending on how the slope is determined as well as the instrument, software, and platelet source. “A lot of people like the assay, they’ve done it for years, but the data from proficiency testing is consistent across Australia, the U.S., and Europe: that it has a high imprecision.” Another group, the North American Specialized Coagulation Laboratory Association, or NASCOLA (Dr. Chandler is a member), compared manual versus automated methods for this test and found very little difference between the two. “It seems to be a problem with the assay itself—it’s just not that precise,” he says.

Another development is the relatively new collagen-binding activity test, which is quite popular in Australia, gaining popularity in the United Kingdom, and rarely used in the United States.

The test, available commercially in an ELISA format, measures the ability of vWF to bind to collagen coated onto microtiter plates; this vWF-collagen interaction is critical for normal platelet adhesion to damaged endothelium. The assay is more sensitive than the ristocetin cofactor assay in diagnosing certain types of vWD—specifically types 2A and 2B, which are characterized by dysfunctional proteins. It can also detect some type 2Ms. The increased sensitivity may be related to the fact that the collagen-binding assay seems to be quite sensitive to decreased concentrations of high-molecular-weight forms of vWF, which characteristically are deficient in types 2A and 2B.

The assay does not detect all types of 2M disease. (A research assay, the glycoprotein 1B binding assay, might address one subset of 2M patients.) “So this is not going to be a replacement assay for the ristocetin cofactor assay. It’s going to be a complementary assay,” says Dr. Cunningham.

Ristocetin-induced platelet aggregation, or RIPA, is also used to help classify type 2B. The test determines the smallest amount of ristocetin at which platelet aggregation will be stimulated. In normal patients, this will happen in the range of 1 to 1.5 mg/dL of ristocetin; in patients with type 2B disease, aggregation will occur roughly in the range of 0.4 to 0.6 mg/dL.

Subclassification

How accurate are laboratories’ diagnoses of vWD? Recent studies are a bit unsettling.

Diagnosing the most common subtype, type 1, is based on the level of vWF and the vWF activity-to-antigen ratio. This requires an activity test—either the ristocetin cofactor test or the collagen-binding assay—and an antigenic test to measure protein quantity. The ratio value is the main feature distinguishing type 1, which is a partial quantitative deficiency, from type 2, which is a qualitative deficiency.

Two recent external quality assurance studies, one from Australia (the Royal College of Pathologists of Australasia) and one from the Netherlands (the ECAT Foundation), have shown that the subclassification error rate for type 1 vWD is anywhere between 13 and 40 percent, says Dr. Cunningham. Those errors may be related to the poor precision of the activity tests. “The antigenic tests are not all that accurate either,” Dr. Cunningham reports. Some may have coefficients of variation ranging from 20 to 25 percent.

The Australian group also looked at errors in identifying type 2 disease. In one study, known type 2 samples were misidentified as type 1 or type 3 20 percent of the time. “It’s not entirely clear why these errors are happening,” Dr. Cunningham says. “That’s the next step, to figure out what’s causing it. But it does highlight the problem, which wasn’t clear before.”

The College has not yet established PT programs for either confirmatory testing or subclassification. “What we’ve done previously is send normal plasma or diluted plasma specimens, and that doesn’t get to the accuracy of the final diagnosis. It gets at how good the tests are in terms of their precision and accuracy,” says Dr. Cunningham. “But to get at the diagnostic error rate, it would be valuable to do things that other proficiency testing organizations have done over the last five or six years, and that is send out known plasmas derived from patients, and asking labs to make a diagnosis.”

Multimer analysis

One of the Seminars papers (Budde U, et al. 514–521) raised questions about the accuracy of multimer analysis.

The multimer test, which looks at the molecular weight distribution of all vWF molecules in plasma, has historically been used to subclassify type 2 vWD into types 2A, 2B, 2M, and 2N. Types 2A and 2B exhibit an abnormality in their multimer patterns; specifically, the high-molecular-weight forms are absent or deficient. “Once you diagnose a type 2 von Willebrand disease by looking at the activity-antigen ratio, then you look at the multimer analysis and ask, Are there decreased amounts of high-molecular-weight multimers? And if the answer is yes, then you can separate it into type 2A or 2B. If it’s normal, it’s type 2M,” says Dr. Cunningham.

“Now, type 2N is a different story,” he continues. (Of course it is.) And 2B, naturally, requires an additional test, the RIPA, in which ristocetin is added to a patient’s plasma; hyper-responsiveness to low concentrations of ristocetin indicates a gain of function mutation in vWF receptor.

It appears that some labs are misclassifying known type 1 samples as either type 2A or 2B, based in part on multimer analysis; specifically, they identified abnormal patterns of high-molecular-weight multimers that didn’t actually exist in the samples. Says Dr. Cunningham: “That might be another quality assurance problem we’re only starting to detect.” He professes little surprise that there may be a problem, noting that QA issues may arise if labs fail to do an adequate electrophoretic transfer procedure, since it takes longer for the high-molecular-weight multimers to transfer to the blotting media. He offers this up as a theory, not fact. “We know that people are making false diagnoses because of multimer analyses that are in error. The question is, Why are they making those errors?”

Genetic testing

Genetic testing for vWD is not in high demand. Seminars ran a paper (James P, et al. 546–552) advocating genetic testing for the subtype 2N, which is characterized by mutations in the factor VIII binding region. An alternative test for diagnosing type 2N is a factor VIII binding activity assay for vWF. “If we had to do that, I don’t know where I’d go to get [one],” Dr. Cunningham says. “I’ve actually looked at a few reference laboratories, and I don’t see it offered.” Type 2N is relatively uncommon. It presents like hemophilia A, except it lacks a sex-linked inheritance pattern.

In some type 3 patients, whose disease is marked by a complete deficiency of vWF, genetic testing might also be valuable. Of the variety of mutations that can cause type 3, one is a deletion mutation, which puts patients at risk for life-threatening anaphylactic reactions to factor replacement therapy.

Certainly all this is enough to keep pathologists’ heads spinning. So how are clinicians supposed to keep up?

Von Willebrand disease remains one of the most common inherited coagulation disorders, and treatment depends on accurate subtyping. Desmopressin is contraindicated in types 2B and type 3, for example, so if a patient is falsely diagnosed with those subtypes, they’ll be denied a drug that likely will work well for them. And failing to make any diagnosis means clinicians will lose an opportunity to treat and prevent bleeding episodes.

Physicians may find it hard to give up certain tests—as anyone past the age of 15 knows, it can be tough to walk away from certain relationships, even if they’re no good. Assays such as the ristocetin cofactor test have been used for years to make diagnoses. Even though it’s becoming clear that they may not be as useful as previously thought, “Those sorts of assays are difficult to give up,” says Dr. Chandler. “You get used to them.”

A good example is bleeding time, he says. “It’s been around for a long time, and lots of labs still do it, although the papers show it’s not a good pre-op screen for bleeding. A number of labs, including ours, have stopped doing bleeding times—we haven’t done them in some of our hospitals for three or four years now. Other places still crank out lots of them.”

The single smartest move labs can make may be to remind their colleagues that a single test or workup for vWD may be insufficient, since borderline normal results during an acute phase response may mask a true deficiency.

“One way to sort of crudely evaluate this is to look for other acute phase factors, such as CRP or elevated fibrinogen,” Dr. Chandler suggests. A patient who’s borderline normal with a fibrinogen of 600 mg/dL “makes you wonder” what will happen when the fibrinogen returns to a lower level. “We will often call the physician and say, ‘Today we have to tell you there’s no evidence of von Willebrand disease because the level’s normal. But we’re concerned the patient may be acute phasing, and you probably ought to repeat in a month.’” Patients may also be transiently low, he says, though this situation may be less common.

“Just doing it once and seeing borderline normal is not good enough if the patient has a good clinical history. Now of course, that’s the part the lab has no control over,” Dr. Chandler continues, “and it’s the really tricky part, because from our own data, just in our own labs, we’re working up lots of older individuals who probably don’t have a prior history of bleeding and therefore are unlikely to have genetic von Willebrand disease.” That means the lab has to look for acquired disease, which is linked to several factors: very high platelet counts associated with essential thrombocytopenia, for example, or a stenotic valve, either the patient’s own valve or a replacement valve. And occasionally a patient may spontaneously develop an antibody to vWF.

Even relatively simple results can befuddle, however. Case in point: physicians who see a result showing a high level of vWF—due to an acute phase response—and tell the patient he or she has vWD. When they request a further workup, “We have to tell them, ‘This is a deficiency disease.’ It’s not that they’re dumb,” Dr. Chandler says. “They’re just looking at the report for an asterisk, an abnormality.”

Since most clinicians aren’t well versed in hematology, it only makes sense for labs to provide a relatively simple interpretive comment alongside numerical results. Dr. Chandler likens it to the remarks accompanying AP results. “They should have a sentence that says, ‘No evidence of von Willebrand disease,’ or, ‘Reduced von Willebrand factor, possible increased risk of bleeding,’ or, ‘Low von Willebrand factor consistent with possible type 1 von Willebrand disease,’” he says. “Clinicians shouldn’t be pulling out a book, looking up tables and trying to figure out what the numbers mean. I don’t think the average family practice doctor can be expected to know this.”

Occasionally pathologists will need to reach for the phone, Dr. Chandler says. “It’s not like you’re calling 20 guys a day. It’s that you’re calling somebody once a week or so to say, ‘You may want to have this person come back in again, because we’re betting they have von Willebrand disease—they’re right on the cutoff, and it looks like there’s a good chance they’re having an acute phase response.’”

At Maine Medical Center, Dr. Hayes and his colleagues do very little screening, because most of their patients have been seen by hematologists rather than primary care physicians. “We tend to launch right into a complete evaluation,” he says. “We use the hematologists as kind of our screening tool.”

He and his colleagues will typically do the entire factor VIII complex, as well as two measures of vWF: the ristocetin cofactor and the vWF antigen. Occasionally the lab will get requests for just one of those tests. Dr. Hayes discourages this, since an incomplete panel can cause labs to under- or overdiagnose as well as mis-type vWD.

At Maine, “We also, for better or for worse, do a bleeding time.” This test may soon fall by the wayside because it adds little diagnostic value, “at least in our hands. It’s kind of a holdover from previous years.” The lab also does PTs, PTTs, and fibrinogens, which are a relatively inexpensive way to monitor specimen integrity as well as identify non-von Willebrand coagulopathies. The vast majority of samples will also undergo a platelet aggregation assay.

This menu evolved over time, during which Dr. Hayes often took the clinical histories himself. Now, anytime a patient comes in for a platelet aggregation assay, someone from his small crew of coagulation specialists will take the history. Without that information, Dr. Hayes is loath to interpret results.

He professes amazement at the number of patients who, despite having seen a hematologist, arrive for testing while on medications that can influence test results. This includes a substantial number of patients who take thyroid replacement—acquired vWD can occur in patients who are either hypothyroid or who have subtherapeutic replacement of thyroid hormone.

Dr. Hayes often adds another step to the workup: He and the chief coagulation technologist often sign out cases together. Since vWF can be released by anxiety, fear, stress, exercise, and the like, it’s helpful to know if, for example, a child in for a workup was especially anxious or combative. “It’s hard to interpret these assays if you don’t have a good understanding of all the variables. Pathologists have to be more proactive.”

He likes to joke that “coag” is short for “quagmire,” not “coagulation.” He himself has only learned to become comfortable with von Willebrand testing as a result of his confabs with colleagues. Telling a surgeon a patient is unlikely to bleed, for example, without following up on that prediction, is pointless. “I need that feedback from physicians. That’s helped me over the years to have a better handle on which patients I need to worry about and which ones I can worry about less. Unfortunately, that’s not something that comes overnight. It takes experience and a good working relationship with the medical staff.”