By Renee DiIulio

 Proteomics will soon enable speedy, sensitive, specific diagnosis of ischemic stroke. This will result in rapid treatment and improved outcomes for patients.

“The future, for the general physician, lies in genomics, or analyses that identify the conditions a patient is at risk of developing in the course of his or her lifetime. The acute care physician’s future is in proteomics, analyses that determine which proteins are in the patient’s bloodstream,” predicts Norman Paradis, MD, medical director, Emergency Department, University of Colorado Hospital, and associate professor at the University of Colorado Heath Sciences Center (Denver). “Since many proteins should not be present in the blood at all or should be present only at certain levels, tests that provide this information will help determine diagnoses and treatment courses.”

This is good news for stroke victims, whose prognoses improve with faster treatment. Paradis, also a spokesperson for the National Stroke Association (NSA) of Englewood, Colo, believes that the new generation of imaging studies and biomarker analyses currently in development have the potential to revolutionize stroke care, providing physicians with diagnostic tools that will offer them the assurances currently lacking in today’s stroke diagnostic techniques. Confidence will allow them to comfortably prescribe potentially life-improving treatment that can also be life threatening if used when not actually needed.

Norman Paradis, MD

Brain Attack
Stroke results from the sudden interruption of blood flow to the brain, leading some to refer to it as a “brain attack.” Also called cerebrovascular accident or stroke syndrome, strokes are currently classified as either hemorrhagic or ischemic in origin.

Ischemic stroke is by far the more common of the two, accounting for somewhere between 80% and 88% of all strokes. Ischemic stroke results when blood flow to the brain is blocked, either by a clot or a narrowed artery. Clots may develop directly within the cerebral arteries, a condition known as a thrombotic stroke, or they may travel from elsewhere in the body, typically the heart, to become lodged in the vessels of the brain, referred to as an embolic stroke.

The less common but equally symptomatic hemorrhagic stroke occurs when a vessel in the brain bursts, bleeding into the brain tissue and/or surrounding areas. There are two types of hemorrhagic stroke: intracerebral hemorrhage, which results from the rupture of an artery in the brain itself; and subarachnoid hemorrhage, in which the location of the burst vessel causes blood to be spilled into the space around the brain. There are typically two causes of such strokes: aneurysms, which result in ruptures from the ballooning of a weakened portion of a blood vessel, and arteriovenous malformations (AVMs), which are clusters of abnormally formed blood vessels, any of which can be the source of the break.

Rapid Cell Death
The consequences of a stroke are determined by its length and location within the brain. Some people will experience relief of their symptoms almost immediately, while others will live with the effects for the rest of their lives.

The symptoms of transient ischemic attacks (TIAs), or mini-strokes, disappear within minutes or hours, leaving no permanent damage. Though the patient is typically fully recovered within 24 hours, the occurrence of TIAs can indicate that a more serious attack may manifest later and preventive measures should be taken.

A stroke or brain attack can result in reduced blood flow to portions of the brain causing an associated loss of function. Imaging can help to rule out other reasons for the damage.

More serious strokes, either ischemic or hemorrhagic, may result in the loss of cognitive and physical functions dependent on the area of the brain affected. According to the NSA, “Within the last decade, researchers have learned exactly why brain cells die during stroke. Most strokes culminate in a core area of cell death (infarction) in which blood flow is so drastically reduced that the cells usually cannot recover. This threshold seems to occur when cerebral blood flow is 20% of normal or less. Without neuroprotective agents, nerve cells facing 80% to 100% ischemia will be irreversibly damaged within a few minutes. Surrounding the ischemic core is another area of tissue called the ischemic penumbra or transitional zone in which cerebral blood flow is between 20% and 50% of normal. Cells in this area are endangered, but not yet irreversibly damaged.”

When brain cells die, the abilities controlled by these cells are lost. This can include speech, movement, and memory. Patients may experience sudden weakness, loss of sensation, and/or difficulty speaking, seeing, or walking.

According to the NSA, the five most common stroke symptoms include: sudden numbness or weakness of the face, arm, or leg, particularly on one side of the body; sudden confusion, trouble speaking or understanding; sudden trouble seeing in one or both eyes; sudden trouble walking, dizziness, loss of balance, or coordination; and/or a sudden severe headache with no known cause.

It is critical for patients to recognize the symptoms of stroke and seek immediate medical care and for doctors to quickly diagnose the disorder and administer treatment.

The ischemic cascade, or chain reaction of cell death resulting from a stroke, endangers more cells and therefore more functions. Because of the rapid progression, the window for interventional treatment for an ischemic stroke is roughly 6 hours. Hemorrhagic strokes also require immediate care. It is therefore important for patients to recognize symptoms and seek immediate medical attention and for doctors to quickly diagnose the disorder and administer treatment.

Stroke Stats

• The World Health Organization estimates that in 2001 there were more than 20.5 million strokes worldwide; 5.5 million of these were fatal.

• Stroke is the third leading cause of death in the United States, behind heart disease and cancer.

• Stroke is the leading cause of serious, long-term disability in the United States.

• Each year, about 700,000 people suffer a stroke in the United States. About 500,000 of these are first attacks.

• On average, someone in the United States suffers a stroke every 45 seconds; every 3.1 minutes someone dies of a stroke.

• Stroke killed 283,000 people in 2000 and accounted for about 1 of 14 deaths in the United States.

• About 47% of stroke deaths occur out of a hospital.

• About 4.7 million stroke survivors (2.3 million men, 2.4 million women) are alive today.

• Within 30 days, 7.6% of ischemic strokes and 37.5% of hemorrhagic strokes result in death.

• After a heart attack, 8% of men and 11% of women will have a stroke within 6 years.

• Within a year, 14% of people who have a stroke or transient ischemic attack will have another.

• Within a year, 22% of men and 25% of women who have an initial stroke die.
—From “Stroke Statistics,” the Internet Stroke Center, [removed][/removed].

• For 2004 the estimated direct and indirect cost of stroke is $53.6 billion.
—From “About Stroke,” American Stroke Association,

• From 1991 to 2001, the death rate from stroke declined 3.4%, but the actual number of stroke deaths rose 7.7%.

• In 1999, more than 1.1 million American adults reported difficulty with functional limitations, activities of daily living, etc., resulting from stroke.

• There are 981,000 hospital discharges every year; less than 4% of patients received life-saving thrombolytics.

—From “New Dimensions in Diagnosis, Stroke Diagnostic Program,” Biosite Inc 2004.

Slow Patient Response
One of the largest problems in stroke care today, however, is patient response. The NSA cites studies that have found that 42% of stroke patients wait as long as 24 hours before presenting themselves to the emergency room, with the average wait being 13 hours—well beyond the current treatment window.

Paradis notes that people do not have the same reaction to stroke symptoms as they do to those of a heart attack. “A heart attack is crushing pain, but stroke symptoms are more subtle. It might just be numbness of the arm, as if it’s fallen asleep. People expect the symptom to wear off and so wait before coming to the hospital,” he says.

However, the longer the delay in patient presentation, the more damage can occur and the less recovery can be achieved. New clot-busting medications and neuroprotective drugs work on ischemic strokes when administered as quickly as possible; interventional treatments for hemorrhagic strokes also work when employed rapidly and are actually best when used as preventive measures.

Short Treatment Window
By delivering treatment quickly, doctors aim to stop the ischemic cascade and resulting cell death, thus preserving brain cells and corresponding functions. Hemorrhagic strokes are best treated with surgical intervention or endovascular procedures, the aim of which is to introduce an obstruction that will prevent the rupture and/or bleeding that has resulted. Thrombolytic or fibrinolytic drugs do this by dissolving the obstructing clot of a thrombotic stroke and restoring circulation; neuroprotective drugs minimize the effect of the ischemic cascade itself.

The new neuroprotective drugs show great promise, but currently none are available commercially. Several different types are in clinical trials for acute ischemic stroke, including glutamate antagonists, calcium antagonists, opiate antagonists, and antioxidants.

Other novel treatments in development for ischemic stroke include:
    • Desmoteplase, or vampire bat plasminogen activator, a genetically engineered version of a protein found in the saliva of vampire bats. It dissolves clots with less risk of hemorrhage than current clot busters and may therefore expand the treatment window. Paion GmbH (Aachen, Germany) successfully completed phase II clinical trials in February.
    • The Concentric Merci Retrieval System by Concentric Medical Inc of Mountain View, Calif. This is a small, nickel and titanium corkscrew device that pulls the clot from the vessel. Currently in phase II clinical trials, this procedure is also expected to expand the treatment window.

While these developments are being closely watched by the medical community, they are not available for use outside of the trials. The only treatment currently approved by the Food and Drug Administration (FDA) is tissue plasminogen activator (t-PA), and while its introduction has also been revolutionary, its short interventional period and associated risks have prevented it from being widely used.

According to the American Stroke Association, a division of the American Heart Association (AHA) in Dallas, only 3% to 5% of patients reach the hospital in time for its application. Recent studies published on the Web site of the National Institute of Neurological Disorders and Stroke (NINDS), part of the National Institutes of Health in Bethesda, Md, indicate that improved recovery may be achieved if administered within 4 hours, but by 6 hours, no benefits are seen.

Because the treatment carries a high risk of hemorrhage, doctors are leery of using the drug outside the treatment window. “The doctor’s oath is to do no harm,” says Paradis. “Therefore, they don’t want to prescribe a drug that may result in hemorrhaging and death.”

Imaging can provide valuable information regarding the activity in the brain.

Wide Range of Diagnostic Tests
With such high stakes, a diagnosis of stroke, and particularly the type of stroke, is critical. However, current diagnostic methods do not provide the surety many would like, though they do rule out other conditions.

The AHA “Guidelines for the Early Management of Patients with Ischemic Stroke” states, “In one series of 821 consecutive patients initially diagnosed with stroke, 13% were later determined to have other conditions. Several conditions mimic stroke. Frequent alternative diagnoses include unrecognized seizures, confusional states, syncope, and toxic or metabolic disorders, including hypoglycemia, brain tumors, and subdural hematoma. These stroke mimics are commonly, but not always, associated with global rather than focal neurological symptoms and are usually readily detected with standard laboratory tests.”

Therefore, the guidelines suggest that the first goal in a diagnostic evaluation of ischemic stroke is to rule out other systemic or neurological illnesses, including hemorrhagic stroke. “Differentiation of ischemic or hemorrhagic stroke is especially important because of the marked difference in the management of these conditions. Because clinical findings overlap, a brain imaging study is mandatory to distinguish ischemic stroke from hemorrhage or other structural brain lesions that may imitate stroke.”

The second goal is to determine advisability for acute treatment with thrombolytic agents. The third goal is to screen for acute medical or neurological complications of stroke. And the final goal is to provide historical data or other information that can be used to establish the “vascular distribution of the stroke and to provide clues about its likely pathophysiology and etiology.” The data is used to determine treatment and a course of action for prevention of recurrent stroke.

Daniel Laskowitz, MD

The treating physician will typically review the events bringing the patient in, obtain a medical history, complete a physical and neurological examination, run laboratory tests, perform medical imaging, and use whatever other diagnostic tests he deems necessary, including electrical and blood flow tests.

Brain imaging, whether computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET), can provide valuable information regarding the activity in the brain. Paradis notes that current CT scans provide a null set in that they don’t positively identify stroke. Daniel Laskowitz, MD, associate professor of medicine (neurology), Duke University Medical Center (Durham, NC), agrees. “CT is everywhere and is sensitive for hemorrhage or a mass lesion but is not sensitive for ruling in stroke.”

It does provide information regarding the location and extent of damage to the brain, and new generation imaging shows even more promise, with the ability to more clearly identify blood flow. “Perfusion MRI is more sensitive for acute ischemic stroke but is not always readily available and is not fast,” says Laskowitz.

The guidelines also review the blood tests that should be routinely performed both to identify stroke mimics and help determine treatment. These include blood glucose, electrolytes, complete blood count with platelet count, prothrombin time, activated partial thromboplastin time, and renal and hepatic function studies. Because of the small treatment window, results of the prothrombin and partial thromboplastin times should not delay treatment unless there is suspicion of a bleeding abnormality.

Other tests that should be run if the physician has related questions include a toxicology screen, blood alcohol level, pregnancy test, arterial blood gas levels (to test for hypoxia), and a cerebrospinal fluid exam (in cases of suspected subarachnoid hemorrhage).

It is also suggested that these tests be available at all times as treatment cannot be delayed to wait for technicians or equipment.

Specific and Sensitive Diagnostics on the Horizon
None of these tests, however, offers a rapid, positive diagnosis for stroke. But as Paradis has predicted, proteomics stand to alter the landscape. Biosite Inc of San Diego is currently developing a biomarker assay panel that will provide a rapid, specific, sensitive diagnostic tool for stroke. The company expects to submit the test to the FDA in fourth-quarter 2004 with a United States launch to follow in fourth-quarter 2005. The product is expected to be launched in Europe in fourth-quarter 2004.

Ken Buechler, PhD

According to Ken Buechler, PhD, Biosite cofounder and vice president of research and development, the product has been in development for about 21/2 years. In order to establish a sample base, the company started its research with the collection of very characterized samples from stroke patients who had clinical histories. It then identified roughly 50 markers important to stroke. Using more than 1,000 samples, it has narrowed the protein targets to develop a panel that utilizes six markers, which are currently being held in confidentiality.

Laskowitz, who has conducted parallel research academically and collaborated with Biosite, has worked with three markers. “Stroke is complicated so it was necessary to pick multiple markers that provide complementary information. You want to use it as a screening test so you need a high sensitivity that will capture everyone who has the condition but will also avoid false-negatives. Because the doctor is in an emergency room setting and treatment carries risk, he wants to be absolutely sure before he gives meds; therefore, you also want high specificity,” he says.

Initial studies on the Biosite product have produced such results. Testing the panel at times from onset ranging from 3 to 48 hours, the sensitivity (at 90% specificity) was found to be 97.1%, and specificity (at 90% sensitivity) was 98.7%. A comparison to CT scans has found the markers to have three times better sensitivity for ischemic stroke. In addition, it delivers these results in 15 minutes and has been designed as a point-of-care diagnostic test.

Looking into his crystal ball once more, Paradis sees the value of the new biomarkers in their ability to be used as advanced screening. “The emergency medical services team can utilize the test in the field so that patients are identified before coming into the hospital. Once there, new imaging techniques will be able to identify the location and extent of the stroke, and a treatment course will be decided upon quickly.” The result will mean better prognoses and outcomes for patients, making their futures brighter as well.

1) Ken Buechler, PhD, vice president, Research and Development, and cofounder, Biosite Inc (San Diego); Contact: Nicole Palmer, senior corporate communications specialist, (858) 597-4815 x3686; [email protected].
2) Daniel Laskowitz, MD, associate professor of medicine (neurology), Duke University Medical Center (Durham, NC); Contact: Tracey Koepke, senior medical writer, public information officer, Medical Center News Office, Duke University Health System, (919) 684-4148; (919) 660-1301; [email protected].
3) Norman Paradis, MD, medical director, Emergency Department, University of Colorado Hospital (Denver); associate professor, University of Colorado Health Sciences Center (Denver); spokesperson for the National Stroke Association (Englewood, Colo) (303) 372-5901; contact: Barbara Haskel, assistant, (303) 372-4065; [email protected].

Renee DiIulio is a contributing writer for Clinical Lab Products.