American Association for Clinical Chemistry
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March 2008 Clinical Laboratory News: Stroke

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March 2008: Volume 34, Number 3


Stroke
A Look at Emerging Biochemical Markers 

By Robert H. Christenson, PhD, DABCC, FACB and Svetlana Dambinova, DSc, PhD

 

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The third leading cause of death in the U.S., stroke is outranked only by “diseases of the heart” and all forms of cancer. According to 2004 American Heart Association (AHA) data, stroke killed more than 150,000 people in the U.S. Stroke is also the most important cause of serious, long-term disability, with 30–50% of stroke survivors suffering permanent disability. Approximately 700,000 Americans suffer stroke each year, of which 500,000 are first attacks. Data also show that stroke disproportionally affects women. Women account for 61% of stroke deaths, and about 22,000 more women than men die of the condition each year. Moreover, the statistics indicate that race influences death rates for both sexes. For Caucasian males, the rate is 48.1 deaths per 100,000 people compared to 73.9 for African American males. The rate is 47.4 for white females and 64.9 for African American females per 100,000.

 

Not surprisingly, the cost of stroke to the healthcare system is enormous and growing. Estimates put emergency department visits for assessment of stroke symptoms at 3 million per year and hospital discharges at about 1 million. In 2006, the total estimated annual cost associated with stroke, both direct and indirect, was nearly $57.9 billon. No doubt, the burden stroke places on the healthcare system will increase as the U.S. population ages.

 

Although the role of the laboratory is relatively modest at present, promising biochemical markers and strategies are emerging that may one day contribute to the determination of long-term cerebrovascular risk and acute assessment of suspected stroke patients. Here we provide a look at what is known about those markers and some basic background on the different types of stroke, with a focus on risk assessment and diagnosis of ischemic stroke.

 

What is Stroke?

 

Stroke is caused by an acute disruption of blood supply to the brain. There are two broad categories of stroke: ischemic stroke, in which there is a blockage of arteries supplying blood to the brain with resulting ischemia, and hemorrhagic stroke, in which a blood vessel ruptures, causing bleeding in the brain. Hemorrhagic stroke can be further subcategorized according to where the bleeding occurs: subdural indicates that the bleeding involves the surface of the brain and epidural indicates bleeding in the brain’s interior.

The majority of strokes, about 88%, are of the ischemic type. The symptoms of hemorrhagic and ischemic stroke are frequently different, as are the outcomes. Thirty-day mortality for hemorrhagic stroke is about threefold higher at 37–38%, compared to 8–12% for ischemic stroke. Treatment options that improve outcomes for hemorrhagic stroke patients are very limited. However, if management is initiated quickly (<3 hours), studies show that outcomes for patients with ischemic stroke can be dramatically improved.

Ischemic stroke is frequently caused by thrombus formation in the cerebrovascular system. With the variable nature of thrombus, as well as the physiological response to acute blockage, ischemic stroke consists of a continuum of disease ranging from transient symptoms to large areas of cell injury. At one end of the continuum is transient ischemic attack (TIA) or mini-stroke, ischemic strokes that last less than an hour. Importantly, thrombolytic therapy with recombinant tissue plasminogen activator (rtPA), the so called “clot-busting drug,” can be an effective treatment for ischemic stroke. Timing of therapy is critical, however, and unfortunately, fewer than 4% of patients receive life-saving thrombolytic therapy within the critical treatment window. On the other hand, thrombolytic therapy is strictly contraindicated in hemorrhagic stroke, as it would likely worsen intracranial bleeding.

 

Treating a Brain Attack

 

Analogous to “heart attack” in acute coronary syndrome (ACS), healthcare professionals are increasingly applying the term “brain attack” to describe stroke. This terminology is quite appropriate because both “attacks” represent a medical emergency. In addition, stroke and ACS share many of the same risk factors, including hypertension, atherosclerosis, and family history.

Today, clinicians view ischemic stroke as a continuum of disease impacting the brain. As stated above, ischemic strokes are very responsive to treatment with rtPA; however, the benefit of rtPA is extremely dependent on the timing of therapy. Treatment must be initiated within 2–3 hours after the first symptoms in order to maximize the benefit, making rapid diagnosis and initiation of management critical.

 

Clinical Need for Stroke Biomarkers

 

As with other diseases or conditions, lab tests would be a valuable tool for screening, diagnosis, and prognosis of stroke patients, as well as for guiding management, determining the efficacy of treatment, or monitoring treatment. Although stroke biomarkers continue to generate great interest and substantial research, there is currently no biochemical marker or strategy in routine use for any of these applications.

 

Forty years ago, creatine kinase measurements were shown to be associated with meningitis, head trauma, and stroke (1). Then in 1996, researchers found that levels of the neuroexcitatory amino acid, glutamate, in plasma and cerebrospinal fluid had significant relation to infarct size and neurological deficit in ischemic stroke (2). As research continues, scientists have identified the following attributes of an ideal stroke biomarker: rapid release from injured neurons or glial cells; being measurable in blood soon after any acute event; ability to discriminate between ischemic and hemorrhagic stroke; and sensitivity to early ischemia. The marker’s release pattern would also be indicative of disease progression and would not be elevated in other conditions with similar clinical presentation. For effective diagnosis of ischemic stroke, a rapid assay for the marker and a local strategy for minimizing the time from order to result, such as point-of-care testing, would need to be in place because timing of therapy is so critical.

 

Although several promising biomarker candidates have been identified, some researchers have proposed that a panel approach is most appropriate because stroke is complex and involves a number of different physiological systems (3). Whether a single biomarker or a multiple biomarker strategy emerges, it is clear that any biomarker must add value to the clinical signs and symptoms, existing risk factors, and imaging technologies that clinicians currently use.

To date, feasibility studies of biomarker panels have suffered from assay reliability, with the biomarkers weakly correlating with damage size as defined by radiographic gold standards. This area of stroke diagnostics may prove ideal, however, for establishing a complementary role between imaging and laboratory medicine tests, an emerging area of medicine.

 

Candidates for Stroke Risk Assessment

 

Situations conducive to unstable plaque are the predominant risk factors for stroke. Consequently, there are many risk factors shared by stroke and ACS. A marker that has received attention as a potential candidate for assessing risk of future adverse events, including stroke, is Lp-PLA2. Produced by macrophages, Lp-PLA2 is a calcium-independent serine lipase associated with LDL. The biomarker is expressed in greater concentrations in atherosclerotic lesions and participates in oxidative modification of LDL, hydrolyzing oxidized phosphatidylcholines, lysophosphatidylcholine, and oxidized free fatty acid. In this way, Lp-PLA2 is involved with production of potent proinflammatory products that contribute to formation of atherosclerotic plaques.

In a case cohort study called Atherosclerosis Risk in Communities (ARIC), investigators tested 12,773 healthy middle-aged individuals for Lp-PLA2 blood levels with the PLAC test (Diadexus Diagnostics, South San Francisco, Calif.) (4). The investigators examined the relationship between Lp-PLA2, CRP, traditional risk factors, and stroke outcome over 6 years. Individuals in the highest tertile were at significantly higher risk of ischemic stroke compared to patients in the lowest tertile. Further, the researchers found that patients with values in the highest tertile of both Lp-PLA2 and systolic blood pressure (>130 mmHg) were 6.4-fold (95% CI: 2.75–14.86) more likely to have ischemic stroke. Subsequently, the FDA cleared the PLAC test for Lp-PLA2 determinations in human plasma in conjunction with clinical evaluation and patient risk assessment and as an aid in predicting risk for ischemic stroke associated with atherosclerosis.

 

Another novel biomarker that appears promising in the more immediate risk assessment of stroke is measurement of products of the N-methyl-D-aspartate neuroreceptors (NMDAR). NMDAR is an excitatory receptor in the brain that plays an essential role in calcium homeostasis and signal transduction across neural cell membranes. These receptors are very susceptible to perturbations caused by vascular occlusion, either from thrombus formation or embolus, that disrupt the function of NMDARs and lead to a breakdown in cell membrane integrity, as well as damage to neurons, glia, and endothelial cells. This cell injury cascade compromises the blood-brain barrier, causing increased permeability. Further, serine proteases that are activated by thrombus formation cleave brain-specific NMDA-receptor into fragments (NR2). Release of these NR2 fragments into cerebrospinal fluid and subsequently into the bloodstream initiates an immune response that results in the formation of NMDA-receptor autoantibodies that are measurable in blood.

In a retrospective analysis of a blinded, multicenter clinical trial, researchers examined the prognostic ability of NMDAR antibodies (NR2Ab) for neurological deficits using the Gold Dot-1 test (CIS Biotech, Atlanta, Ga.) in high-risk adult patients who underwent coronary artery or valve replacement surgery with cardiopulmonary bypass (CPB). The study also looked at S100B, a calcium-binding protein that is synthesized in abundance in astrocytes and is believed to modulate long-term synaptic plasticity, and the inflammatory protein CRP (5). Ninety-six percent (24/25) of patients with increased preoperative NR2Ab concentrations had neurological complications within 48 hours post-CPB, versus only 5.4% (20/373) of patients without NR2Ab elevated concentrations, resulting in a 17.9-fold increase (95% CI, 11.6–27.6) in postoperative neurological complications for patients with high levels of NR2 antibodies. Preoperative serum S100B and CRP did not predict neurological complications from CPB. Further, decreased mini-mental status examination scores for orientation, attention, and recall are frequently associated with neurological adverse events early after CPB. Preoperative serum concentrations of NR2Ab, but not S100B or CRP, were predictive of severe neurological adverse events after CPB. Overall, patients with elevated preoperative NR2Ab values were nearly 18-fold more likely to experience a postoperative neurological event.

In an unpublished study conducted at the Imperial College (London, U.K.), researchers evaluated the profile of NR2 peptide in patients undergoing endarterectomy and carotid artery stenting in relationship to hemodynamic and embolic events detected using transcranial Doppler. They found that the NR2 peptide indicated acute cerebral ischemia within 6 hours of the procedure and was associated with adverse neurological events after carotid revascularization. Transcranial Doppler findings suggest that the elevated NR2 peptide levels may be due to an increase in microembolization and cerebral hypoperfusion.

While none of these biomarkers has yet to be widely adapted, it seems possible that the development of algorithms for assessing the long-term and more immediate risk of stroke in selected populations, analogous to the Framingham Risk Score for estimating cardiovascular events, may be valuable. Available data suggest that biomarkers may contribute unique information to such algorithms, and researchers will undoubtedly explore such avenues in the future.

 

Ischemic Stroke: Current Guidance

 

The AHA and the American Stroke Association Stroke Council have recently published guidelines for ischemic stroke diagnosis and management (6). As with virtually all diagnostic problems, clinicians are advised to take a careful history and perform a physical exam with attention to the “ABCs”: airway, breathing, and circulation. Clinicians should also perform a neurological examination and assess the patient using clinical stroke scale scores (6). With regard to initiating therapy, the single most important component from the history is the time of stroke onset. It is also important for clinicians to obtain the circumstances around development of neurological symptoms because there may be a number of other causes for the patient’s symptoms termed “stroke mimics” (Table 1, below).

 

 

Table 1
Stroke Mimics and Clinical Features
Disorder
Clinical Features
Conversion disorder Lack of cranial nerve findings, neurological findings in a nonvascular distribution, inconsistent examination
Hypertensive Headache, delirium, significant hypertension, encephalopathy cerebral edema
Hypoglycemia History of diabetes, serum glucose low, decreased level of consciousness
Complicated migraine History of similar events, preceding aura, headache
Seizures History of seizures, witnessed seizure activity, postictal period
Adapted from reference 7.

 

The guidelines further recommend that the physical exam include assessment of any seizure activity, carotid disease or congestive heart failure, hepatic function, coagulopathy, or platelet disorders. Overall, the goal is to expeditiously determine patient eligibility for intervention, identify any potential complications, and decide on a treatment plan within 60 minutes of when the patient presents.

 

Included in the guidelines are recommendations for a number of lab tests to evaluate patients for systemic conditions that mimic ischemic stroke and therefore may influence the diagnosis and therapeutic options (Table 2, below). Blood glucose is included to determine if hyper- or hypoglycemia is a factor. Platelet count, prothrombin time (PT)/international normalized ratio (INR) and activated partial thromboplastin time (APTT) are included as aids for assessing potential bleeding. Although these laboratory procedures are listed as important, if thrombolytic therapy is being strongly considered, initiation of the drug therapy should not be delayed while the lab completes the PT/INR, APTT, or platelet count unless a bleeding abnormality or thrombocytopenia is suspected. Similarly if the patient is on warfarin or heparin, or if the anticoagulation status is unknown, therapy should not be initiated before the results of the tests are available.

 

Table 2
Diagnostic Studies Needed Immediately for Evaluation of Suspected Ischemic Stroke

All Patients

  • Non-contrast brain CT or brain MRI
  • Blood glucose
  • Serum electrolyte and renal function tests
  • Electrocardiogram
  • Markers of cardiac ischemia
  • Complete blood count, including platelet count
  • Prothrombin time with International Normalized Ratio
  • Activated partial thromboplastin time
  • Oxygen saturation

Selected Patients

  • Hepatic function tests
  • Toxicology screen including ethyl alcohol concentration
  • Pregnancy test
  • Arterial blood gas testing
  • Lumbar puncture testing
  • Electroencephalogram

Note: rtPA therapy should not be delayed unless: 1. there is clinical suspicion of a bleeding abnormality or thrombocytopenia; 2. the patient has received heparin or warfarin; or 3. the use of anticoagulants is unknown.

Adapted from reference 7.

Clearly, a team effort is needed for assessing stroke in order to meet the 60-minute decision time. In fact, the guidelines assign a Class IA recommendation to developing a collaborative plan for the emergency evaluation of patients with suspected stroke that includes stakeholders from laboratory medicine, diagnostic radiology, and clinical departments (7).

Candidates for Ischemic Stroke Diagnosis

Specific stroke biomarkers are currently not part of the standard diagnosis and management guidelines for acute ischemic stroke. Clearly, any new stroke diagnostic test or strategy must demonstrate added value in order to be useful to clinicians. This point has important implications for designing trials that show how any new test or algorithm is to be used and how the results will be analyzed to demonstrate an independent contribution to the diagnostic process. If the analysis shows that addition of the biomarker(s) increases (positive likelihood ratio) or decreases (negative likelihood ratio) the post test odds substantially, then the marker is of potential value. A few biomarkers are showing such promise for TIA and stroke.

In one study, researchers measured autoantibodies to NR2A/2B in a cohort of 360 neurological patients within 3 hours of their cerebrovascular event (8): 105 from TIA/stroke patients and 255 from controls, including patients with controlled hypertension/atherosclerosis and sex- and age-matched healthy individuals (7). The controls were matched for age and sex and were at low risk for ischemic stroke. The results of the test showed a clear separation of the groups, with lower values for hemorrhagic stroke compared to the ischemic stroke cohort. CIS Biotech is working to develop this marker for the acute evaluation of stroke.

Another marker that shows promise is matrix metalloproteinases (MMPs), which are involved in tissue destruction produced by the inflammatory response that occurs after ischemic stroke. MMPs comprise a family of zinc-endopeptidases whose expression can be up-regulated or down-regulated after cerebral ischemia. MMPs are thought to play an important role in tissue remodeling and may contribute to blood-brain barrier breakdown, as well as hemorrhagic stroke transformation in ischemic stroke patients after treatment with rtPA (9). Collectively, MMPs are capable of degrading many extracellular matrix proteins and may be active earlier than 3 hours after an acute cerebrovascular event.

In one study, researchers measured an array of MMPs in 24 patients with cerebral artery occlusion who received rtPA (9). The study demonstrated that the neuroinflammatory response produces high concentrations of MMP-9 and MMP-13, which were involved in extension of the stroke lesion and suggests an early role for these markers after brain injury. It is important to note that the study was not designed to examine performance characteristics in a clinical setting, so the results must be considered preliminary.

Other researchers have explored the feasibility of a diagnostic panel of biochemical markers of cerebral ischemia. In one study, serial blood samples were obtained from 222 patients, 65 with suspected ischemic stroke and 157 control subjects, presenting to an academic medical center emergency department (10). The researchers analyzed 26 markers believed to play a role in the ischemic cascade and created a 3-variable logistic regression model to predict the clinical diagnosis of stroke, defined as persistent neurological symptoms of cerebral ischemia lasting >24 hours. Of the 26 markers analyzed, univariate logistic analysis revealed that four were highly correlated with stroke (P<0.001): a marker of glial activation (S100b), two markers of inflammation (MMP-9 and vascular cell adhesion molecule), and one marker of thrombosis (von Willebrand factor). When the outcome level was set to a cutoff of P=0.1, the logistic model provided a sensitivity and specificity of 90% for predicting stroke. While this study design must be considered exploratory because it was not possible to adjust for important covariates such as age and race, the investigation does illustrate that a panel of biomarkers might be helpful in diagnosing ischemic stroke patients.

Another approach examined more than 50 protein biomarkers in healthy donors and patients diagnosed with either hemorrhagic or ischemic stroke (11). After the initial comparison, the top five markers were S-100b, MMP-9, von Willebrand factor, plus monocyte chemotactic protein-1, and B-type neurotrophic growth factor. Use of these markers provided clinical sensitivity and specificity values of 92% and 93%, respectively. However, the study cohort—normal individuals and patients with ischemic and hemorrhagic stroke—did not allow a true indication of performance that could be expected in clinical use. The multimarker approach used in this study has evolved to include D-Dimer, but updated clinical performance data remains unavailable in a peer-reviewed form.

Improving Patient Outcomes

Cerebrovascular disorders are an emerging segment in non-infectious disease testing, where the real value of any test will be determined by whether it improves diagnostic certainty in the earliest stages of brain injury. Appropriate validation of known tests and strategies for discovery of novel brain markers offers promise. Academics in laboratory medicine and professionals in industry must work together to produce a user-friendly format and put these assays to the test for assuring consistency and quality. Improving tools for the diagnosis and prognosis of stroke is a great opportunity for the laboratory medicine field.

For now, the appropriate response to a brain attack is emergency action, both by the person it strikes and the healthcare team, which should include laboratorians. A rapid blood test that improved the timeliness and accuracy of diagnosis would represent a substantial advance in getting appropriate treatment to patients who could benefit from rtPA. Also, markers that add independent information for quantifying risk of an acute ischemic stroke would be an important stride forward.

References

  1. Dubo H, Park DC, Pennington RJ, Kalbag RM, Walton JN, Serum-creatine-kinase in cases of stroke, head injury, and meningitis. Lancet 1967;2:743–8.
  2. Castillo J, Dávalos A, Naveiro J, Noya M. Neuroexcitatory amino acids and their relation to infarct size and neurological deficit in ischemic stroke. Stroke 1996;27:1060–5.
  3. Dambinova SA, Khounteev GA, Skorometz AA. Multiple panel of biomarkers for TIA/Stroke evaluation. Stroke 2002;33: 1181–1182.
  4. Ballantyne CM, Hoogeveen RC, Bang H, Coresh J, Folsom AR, Chambless LE, Myerson M, Wu KK, Sharrett AR, Boerwinkle E. Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk for incident ischemic stroke in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study. Arch Intern Med 2005;165:2479–84.
  5. Bokesch PM, Izykenova GA, Justice JB, Easley K, Dambinova SA. Predicting adverse neurological outcome before cardiac surgery using blood biomarkers. Stroke 2006;37:1432–1436.
  6. www.ninds.nih.gov/doctors/NIH_Stroke _Scale_Booklet.pdf
  7. Adams HP Jr, del Zoppo G, Alberts MJ, Bhatt DL, Brass L, Furlan A et al. Guidelines for the early management of adults with ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: the American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists. Stroke 2007;38:1655-711.
  8. Dambinova SA, Khounteev GA, Izykenova GA, Zavolokov IG, Ilyukhina AY, Skoromets AA. Blood test detecting autoantibodies to N-methyl-D-aspartate neuroreceptors for evaluation of patients with transient ischemic attack and stroke. Clin Chem 2003;49:1752–62.
  9. Rosell A, Alvarez-Sabín J, Arenillas JF, Rovira A, Delgado P, Fernández-Cadenas I, Penalba A, Molina CA, Montaner J. A matrix metalloproteinase protein array reveals a strong relation between MMP-9 and MMP-13 with diffusion-weighted image lesion increase in human stroke. Stroke 2005;36:1415–20.
  10. Lynch JR, Blessing R, White WD, Grocott HP, Newman MF, Laskowitz DT. Novel diagnostic test for acute stroke. Stroke 2004;35:57–63.
  11. Reynolds MA, Kirchick HJ, Dahlen JR, Anderberg JM, McPherson PH, Nakamura KK, Laskowitz DT, Valkirs GE, Buechler KF. Early biomarkers of stroke. Clin Chem 2003;49:1733–9.

Christenson.jpgRobert H. Christenson, PhD, is Director of the Core Laboratories at the University of Maryland Medical Center, and Professor of Pathology and Professor of Medical and Research Technology at the University of Maryland School of Medicine, Baltimore, Md.

 

 

Daminbrova

Svetlana A. Dambinova, DSc, PhD, is currently Visiting Professor at Emory University, Atlanta, Ga. She also retains her professorship at Pavlov Medical University, St. Petersburg, Russia. Dr. Dambinova consults for Siemens, Roche, CIS Biotech and Grace Laboratories, LLC.