People in the bleeding disorders community are no strangers to blood tests. They give blood samples routinely at comprehensive treatment center visits, prior to surgery, before dental procedures and virtually anytime the sample is needed to verify factor protein levels or measure other blood components. When evaluated as part of research studies at national laboratories, personal blood samples can serve a higher purpose: helping the entire bleeding disorders population.
Of the many laboratories housed at the Centers for Disease Control and Prevention (CDC) headquarters in Atlanta, two are dedicated to bleeding disorders research. The CDC’s Division of Blood Disorders (DBD), part of the National Center on Birth Defects and Developmental Disabilities, houses a clinical coagulation or hemostasis protein laboratory, and a cell and molecular laboratory. Both work together to conduct basic research on bleeding disorders, standardize testing methods, provide diagnostic tools for emerging diseases and maintain a national reference laboratory. Blood samples from consumers provide the basic material from which key findings are made.
Clinical Coagulation Lab
Connie Miller, PhD, has directed the Clinical Coagulation Laboratory for the past nine years. She oversees a team of researchers that analyzes blood serum and plasma, focusing on hemostatic proteins—the key components of blood clotting. Assays, highly specialized lab tests that measure the amount or activity of a specific substance such as factor VIII protein, are performed there. A relatively recent innovation called high-throughput screening—fast analysis of active compounds, antibodies and genes using computer technology—means millions of tests at the biochemical level can produce mounds of data in a short time period.
At the National Hemophilia Foundation’s (NHF’s) 59th Annual Meeting in Orlando, Florida, in November 2007, Miller presented findings from her laboratory on the diagnosis of women’s bleeding disorders. “One of the areas we’ve focused on is variability in the von Willebrand factor (VWF) test results,” she says. Von Willebrand disease (VWD) results from a qualitative defect (abnormal structure or function) or quantitative defect (decreased amount) of VWF.
Variables the lab has noted include changes in VWF levels during a woman’s menstrual cycle, race and ethnicity differences, and ABO blood group variations. “We’ve found that African-American women have higher levels of VWF,” says Miller. Since African-American women with VWD may have VWF levels within normal range, these women could go undiagnosed. “That’s a significant diagnostic issue,” Miller says. In addition, it is known that people with type AB blood have 60% to 70% higher levels of VWF than those with type O blood. However, Miller says there is no consensus yet on whether patients should be diagnosed solely on the basis of blood groups or ethnicity.
Since testing women for VWF can be tricky—in Type I VWD, the mildest form, the VWF level can fall within the normal range, and there are several VWD subtypes—Miller says questions abound as to which women to test, when to test and what test to use. “For example, should all women with menorrhagia (prolonged, heavy bleeding during menstruation) be screened for VWD, or does it depend on other symptoms?” she asks. Besides being a logistical nightmare—the CDC could not handle such a heavy volume of tests all at once—needlessly subjecting women to the panel of tests would be controversial.
In addition to fluctuating during menstruation, VWF levels change during pregnancy and postpartum. They can also be affected by other conditions, such as diabetes, says Miller.
Miller is leaning toward testing teens. “Testing adolescents would be the most fruitful because they would not have yet developed the secondary disorders that we see in women who’ve been having menorrhagia for a long time.”
Because the average time between onset of symptoms and diagnosis of VWD is 16 years, Miller feels early testing is vital. “There should be education on what’s normal and what’s not. Our projection is that this may need to be addressed from a public health perspective.”
One of the main functions of the CDC labs is surveillance, testing samples for disease-causing agents before they become public health problems. The need for surveillance in the bleeding disorders community was a direct result of two back-to-back crises that struck the community—the spread of HIV and hepatitis C in contaminated blood products in the 1980s and 1990s.
“The community came together after HIV and hepatitis C were continuing to be transmitted in products and said, ‘If you’re going to consider us the canaries in the coal mine for blood safety in the country, we want a surveillance system set up that will really protect us so that thousands of cases don’t occur before this is discovered,’” says J. Michael Soucie, PhD, an epidemiologist and associate director of science at the DBD.
That outcry led to the establishment of the Universal Data Collection (UDC) project, which, among other things, monitors blood safety for people with bleeding disorders who are taking factor products. “This is an active surveillance system, wherein we get specimens from patients, do the testing and monitor the outcomes,” says Soucie. Prior to the UDC project, a passive reporting system was in place for occurrences such as new cases of hepatitis. “We were relying on somebody else to do the testing and report these new cases to us.”
Routinely collecting blood specimens from patients who volunteer for the UDC project means that testing is proactive. An initial blood sample is used to measure baseline data and can then be compared with samples gathered at the patient’s annual comprehensive treatment center visit. The samples are all routinely tested for hepatitis A, B and C and HIV; some of each sample is saved to investigate other possible blood safety threats in the future. For example, stored specimens were tested for West Nile virus to make sure that it was not being transmitted in factor products. If even a handful of patients suddenly test positive for an indicator that had previously been negative, an investigation can be initiated immediately, says Soucie.
Inhibitor Pilot Study
Contrary to what one might expect, though, virulent viruses are not the most pressing threat to the bleeding disorders community. Inhibitors are. “The biggest blood safety issue acknowledged in the world for the hemophilia population is inhibitors,” says Soucie. He observes that inhibitors would not develop at all if the underlying bleeding disorder were not treated with factor products. “So something about the way we’re treating them—what we’re treating them with—has to enter into it.”
The impetus for the CDC’s Inhibitor Pilot Study was a meeting with the US Food and Drug Administration (FDA) in 2005. Officials told the CDC that studies of new products typically involved so few patients that the FDA could not determine the causal relationship between inhibitor development and the product used. “When one or two people developed inhibitors, did it mean that the product was worse for all patients? Nobody could answer that question,” says Soucie.
The inhibitor study is being conducted at nine adult and four pediatric hemophilia treatment centers (HTCs) around the country. So far, more than 500 patients have enrolled. A challenging aspect of the study is that patients are required to keep detailed treatment records.
“What we’re doing is collecting not only information on the product—what it is, how much they’re using and how often—but also what they’re using it for,” says Soucie. “Is it for prophylaxis? To treat a bleed? Before surgery? All these things have been identified as potential risk factors for developing an inhibitor.” And so has the product itself.
By taking a blood sample when a patient switches to a new product, the CDC hopes to be at the forefront of prevention. “We’ll be able to judge whether there were any changes,” says Miller. “If we do that in large enough numbers, we’ll pick up new occurrences of inhibitors much more rapidly than by simply collecting data.”
The advantages of one lab overseeing all the testing are manifold. The CDC can standardize the way blood is drawn and prepared. It can test samples when patients first enroll, a year later when they return for their annual comprehensive visit and again when they change factor products. In addition, it has the resources to collect data prospectively—before patients develop inhibitors—and compare it if and when the patients develop inhibitors.
Miller says a distinct line of demarcation is already being seen in regard to titers—the antibody level measured in Bethesda units, or BUs, indicating the strength of the body’s response in producing an inhibitor. “With people who don’t have a history of inhibitors, the results seem to show that they fall below an inhibitor titer of 0.5 Bethesda units. We are helping establish a cut-off point.”
While Miller’s lab is analyzing blood components, Craig Hooper’s team is analyzing the DNA in genes, looking for mutations. Gene mutations, which are alterations in how the basic building-block proteins are instructed to convey information, come in many forms. These include single base-pair alterations; insertions, or the addition of extra DNA; deletions, or missing DNA; and inversions, in which entire sections of DNA are reversed. Mutations in the factor VIII and IX genes can determine the severity of hemophilia A and B in an individual. Large deletions or inversions in the gene can cause severe hemophilia.
“For the inhibitor project, we are sequencing the whole factor VIII and FIX genes,” says Hooper, PhD, director of the CDC’s Laboratory of Molecular and Clinical Hemostasis. That is a formidable feat, considering the large size of the FVIII gene. “We are looking for all mutations, including inversions. We sequence those two genes using high-throughput methods that are accurate, efficient and cost-effective.”
Understanding the genotype, or genetic constitution, of each person will also provide necessary information. The inhibitor pilot study is breaking new ground by being the first to report results on such a large scale in the United States. “It’s very complex to do this. It’s very expensive and time-intensive,” says Miller. “But it’s something we’ve wanted to do for the community for a long time.”
Prior to her appointment at the CDC, Miller was employed at an HTC. “I understand that there has not been a testing mechanism available to most families that wanted to find out what the mutation is and test other family members,” she says. Once the patient’s gene is scanned, a report of the results is sent back to the patients’ treatment center so that if other family members decide to undergo genetic testing, another lab can pick up where the CDC left off.
By looking so closely at each participant’s genes, the research team expects to find mutations that cause inhibitors, which could help determine patient eligibility in gene therapy trials. “We need to know whether these people have a particular mutation that might lead to inhibitors,” says Miller. On the other hand, she says, the results may reveal that patients have mutations that are more correctable.
Preliminary results are already revealing important information on mutations. “We are finding a large number of mutations that have not been reported before,” says Miller. These new mutations came to light when the CDC lab compared data from the US population with mutations among the European population. And the results will also reveal information on women who are carriers, helping determine why some experience bleeding symptoms and others do not.
The DBD’s laboratories have national and international reach. “As a reference lab, we sometimes get specimens sent in from sites within the US that are having trouble making a diagnosis,” says Miller. The lab also collaborates on international studies, standardizing test methods and improving quantification of factors.
The only way to answer many commonly asked consumer questions—How many patients have hemophilia? Is the disease in different races the same? What is the best method of preventing or treating joint disease?—is by participating in CDC research, says Roshni Kulkarni, MD, former director of the DBD. “Sharing health information through participating in the UDC helps answer these questions. It also helps the CDC develop prevention messages as well as educate both consumers and healthcare providers.”
Kulkarni is quick to remind consumers of the confidentiality of the data. “No identifiers, such as the patient’s name, address and date of birth, are sent to the CDC. These studies are voluntary, and it is up to patients to decide to participate upon consent,” she says. More than 80 percent of patients with hemophilia seen at federally funded HTCs are currently enrolled in the UDC surveillance study.
The CDC continues to solicit volunteers from the bleeding disorders community for its own sake and for the community at large. The more specimens on hand, the more data it has at its fingertips. If a new or emerging infection comes along, the stockpile of specimens is readily available for further testing, says Soucie.
“In the future, if we need to go back to those specimens and see if there was an infectious agent, we’ll have the material on hand to do the rapid testing,” says Miller. A rapid assessment of risk can then be made and steps taken to minimize any impact on the bleeding disorders community, Soucie says.
“We don’t want another HIV crisis to happen and not be able to do something rapidly to deal with it,” Miller adds.
“So we truly are a surveillance lab,” says Miller. “We are looking for problems that complicate bleeding disorders.”