Biochemical markers in the diagnosis of acute ischaemic stroke

M arkers of Strokeas published in CLI November 2004analyteof the monthWith the development of effective therapeutic interventions for ischaemic stroke, the rapiddiagnosis of cerebral ischaemia has become a priority in the management of the disease. In thisbrief review, we discuss the role of biochemical markers in the pathogenesis of stroke, as wellas recent data demonstrating that the measurement of biochemical markers in the blood is analternate approach to sensitive but costly neuroimaging techniques for the early diagnosis ofischaemic stroke.Biochemical markers in thediagnosis of acute ischaemic strokeby James Floyd and Dr. Daniel LaskowitzTreatment for stroke is dependent on whether thestroke is ischaemic or haemorrhagic, and thereforediagnosis within the first six hours after onset ofsymptoms is essential if patients with ischaemicstroke are to benefit from t-PA therapy. Standardneuroimaging techniques, such as computedtomography (CT) and magnetic resonance imaging(MRI), and models based on clinical data have allbeen evaluated in the diagnosis of stroke. However,they are insufficiently accurate to serve as reliablediagnostic tests. Although newer, more sensitiveimaging modalities are being developed and validated,they are time-consuming and are typicallyavailable only in academic centres and institutionswith stroke units. Measurement of biochemicalmarkers in the blood is an alternative approach.Mechanisms of injury from cerebral ischaemiaAcute cerebral ischaemia is caused by the occlusionof a cerebral blood vessel, resulting in decreasedblood flow to the brain. As blood flow falls belowthreshold levels, normal neuronal function beginsto fail and a host of molecular and cellular changesleads to cerebral infarction.One of the first events to occur is energy failurefrom reduced ATP synthesis. The subsequentimpairment of ion homeostasis and the presence ofexcess extracellular glutamate result in neuronaldepolarisation, triggering an influx of calciumthrough voltage-gated channels. Intracellular calciumoverload promotes numerous enzyme-dependentprocesses that lead to or exacerbate neuronal celldeath, including the production of reactive oxygenspecies. Free radicals are generated in the oxygendeprivedenvironment, damaging proteins, DNA,and lipids. Conversion from aerobic respiration toanaerobic glycolysis results in lactic acidosis, whichfurther contributes to tissue damage by promotingintracellular oedema and impairing mitochondrialrespiration [1].After these early mechanisms of injury have beeninitiated, apoptosis and inflammation mediate furthertissue injury after ischaemia. Apoptosis, or programmedcell death, occurs primarily within theischaemic penumbra but also within the ischaemiccore. Although apoptosis occurs for many days aftercerebral ischaemia, upregulation of caspases, theenzymatic mediators of this process, is evidentwithin hours of the initial insult. The inflammatoryresponse is characterised by the migration ofleukoctyes - first neutrophils and then macrophages- into the brain, where they exert their damagingeffects by releasing toxic substances, modulating thevasoreactivity of cerebral vessels, and through theirphagocytic activity. This is preceded by the productionof many different cytokines by glial cells, leukocytes,and endothelial cells. Cytokines attract leukocytesand stimulate the production of adhesionmolecules on leukocytes and endothelial cells, facilitatingmigration across the blood-brain barrier [2].Biochemical markers in cerebral ischaemiaMany biochemical markers that correlate with cerebralischaemia have been identified [Table 1].Although much of the data on these markers derivefrom animal models, this article will discuss onlyhuman studies, and primarily those that involveblood measurements.Neuron-specific enolase (NSE) is an enzyme foundin the cytoplasm of neurons and neuroendocrinecells that has been studied extensively as a marker ofneuronal injury. Levels of this enzyme are elevatedin the blood and CSF of patients presenting withischaemic stroke, and they correlate with infarctsize. Glial acidic fibrillary protein (GFAP) andS100β are two markers of glial activation that havebeen evaluated in the setting of cerebral ischaemia.Serum levels of both proteins are elevated as early aswithin 8 hours after symptom onset, correlatingwith infarct size and functional outcome, as well aswith each other.The profile of various inflammatory mediators inthe blood during cerebral ischaemia has been studiedextensively in the clinical setting [2, 3]. Inhumans, blood levels of the cytokines interleukin-1receptor antagonist (IL-1ra), interleukin-6 (IL-6),and tumour necrosis factor-α (TNF-α) become elevatedafter ischaemic stroke, and for IL-6 thesechanges occur within a few hours of symptomonset. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that attracts mononuclear leukoctyes,is elevated in the CSF of stroke patients presentingwithin 24 hours of symptom onset, but notin the blood. In a study of circulating levels of solubleselectin- and immunoglobulin-type adhesionmolecules in patients with ischaemic stroke, vascularcell adhesion molecule-1 (VCAM-1) was foundto be elevated within 4 hours of symptom onset andendothelial leukocyte adhesion molecule-1 (ELAM-1) within 8 hours. Elevated levels of the acute phasereactant C-reactive protein (CRP) have been shownin multiple prospective cohort studies to correlatewith an increased incidence of stroke. However, thisbiochemical marker has not yet been evaluated inan acute setting.Different components of the coagulation systemhave also been evaluated in ischaemic strokepatients. Levels of D-dimer, a marker of plasminactivity and fibrinolysis, are elevated within hoursof onset of cerebral ischaemia and remain so formonths, reflecting both acute and chronic coagulationabnormalities. The coagulation factorvonWillebrand factor (vWF) has been evaluated inpatients four weeks after the occurrence of transientischaemic attack (TIA) or ischaemic stroke and as apredictive marker in a prospective cohort study.Levels of vWF are elevated after cerebral ischaemia,Table 1. Some biochemical markers associated with cerebralischaemia.

M arkers of Strokeas published in CLI November 2004imaging modalities may be unavailable, such a test could contribute to a physician'sconfidence in administering thrombolytic therapy or assist in the decisionto transport patients with suspected cerebral ischaemia to a hospital with astroke unit for further evaluation. Without such a test, the diagnosis of strokeremains one that is made solely on clinical grounds in most hospitals.Table 2. Biochemical markers significantly correlated with the presence of ischaemic stroke inpatients presenting within six hours, and from 6 to 24 hours after onset of symptoms [4].The study of biochemical markers involved in the ischaemic cascade has providedimportant information regarding the pathogenesis of stroke and has identifiedpotential targets for therapeutic intervention. However, these surrogatemarkers for cerebral ischaemia are useful as a diagnostic modality only ifpatients with ischaemic stroke can be identified with high sensitivity and specificity.A recent report of a diagnostic panel of biochemical markers [5] suggeststhat this novel approach may serve as a useful diagnostic test for ischaemicstroke during the acute period.and higher levels at baseline are associated with an increased incidence of stroke.Various growth factors have been implicated in facilitating recovery after stroke,some by stimulating angiogenesis in regions of infarction. In post-mortemanalyses of brain specimens, increased expression of transforming growth factor-β(TGF- β) and platelet-derived growth factor (PDGF) were found ininfarcted areas and border zones. The measurement of vascular endothelialgrowth factor (VEGF) in the blood of ischaemic stroke patients revealed elevatedlevels on the day of admission, persisting for two weeks. Conversely, apoptosisleads to continued necrosis after cerebral ischaemia, particularly in theischaemic penumbra. Immunochemical staining of brain specimens frompatients with symptomatic cerebral ischaemia demonstrated upregulation ofcaspase-3 in selectively vulnerable areas of the brain.The acute time-windowAlthough the number of studies of biochemical markers in ischaemic strokepatients has increased dramatically over the past few years, very few have includedmeasurements made during the first few hours after symptom onset - a criticalperiod for therapeutic decision-making. Recently, plasma levels of 26 differentbiochemical markers have been evaluated in patients presenting acutely withcerebral ischaemia, and compared with healthy controls [4]. These markers -many of which were examined in relation to ischaemic stroke for the first time -included inflammatory mediators, growth factors, and markers of glial activation,cellular injury, myelin breakdown, thrombosis, and apoptosis. The clinicaloutcome of ischaemic stroke was defined as a neurological deficit lasting longerthan 24 hours resulting from cerebral ischaemia, confirmed either radiographicallyor through the exclusion of other aetiologies.Significant correlations between many of the biochemical markers and the presenceof ischaemic stroke were demonstrated in patients with blood drawn bothbefore 6 hours and between 6 and 24 hours after symptom onset [Table 2].Although many of the individual correlations were excellent - as with previousstudies examining biochemical markers - no single marker possessed the requisitepredictive capacity for it to serve as a clinically useful diagnostic test.A panel approach for the diagnosis of ischaemic strokeTo optimise diagnostic accuracy, the biochemical markers that best correlatedwith ischaemic stroke were entered into a predictive multivariate model and athree-marker panel was developed that included matrix metalloproteinase-9(MMP-9), vascular cell adhesion molecule (VCAM), and vWF [4]. This panelwas able to distinguish ischaemic stroke patients presenting within 6 hours ofsymptom onset from controls with a sensitivity and specificity of over 90%[Figure 1]. Another panel, which included S100β rather than MMP-9, was ableto distinguish stroke patients presenting between 6 and 24 hours after symptomonset from controls with a similar sensitivity and specificity. Abnormalities inthese markers reflect glial activation and derangements in the coagulation systemand inflammatory processes - events that occur early in the ischaemic cascade.A point-of-care test that measures biochemical markers of cerebral ischaemiacould be cost- and time-saving in comparison with expensive neuroimagingtechniques currently being investigated. In the community setting, where theseFigure 1. Received operating characteristics (ROC) curve demonstrating predictive capacityof biomarker panel: sensitivity vs 1-specificity [4].References1. Barber PA, Demchuk AM, Hirt L, et al. Biochemistry of ischemic stroke. Adv Neurol2003; 92: 151 - 164.2. Pantoni L, Sarti C and Inzitari D. Cytokines and cell adhesion molecules in cerebralischaemia: experimental bases and therapeutic perspectives. Arterioscler Throm VascBiol 1998; 18: 503 - 513.3. Price CJS, Warburton EA and Menon DK. Human cellular inflamation in the pathologyof acute ischemia. J Neurol Neurosurg Psychiatry 2003; 74: 1476 - 1484.4. Lynch JR, Blessing R, White WD, et al. Novel diagnostic test for acute stroke. Stroke2004; 35: 57 - 63.5. Reynolds MA, Kirchick HJ, Dahlen JR, Anderberg JM, McPherson PH, Nakamura KK,Laskowitz DT, Valkirs GE and Buechler KF. Early Biomarkers of Stroke. ClinicalChemistry 2003; 49: 1733 - 1739.For a full list of references contact the corresponding author.The authorsJames S. Floyd, BSDuke University School of MedicineDaniel T. Laskowitz, MD, MHSAssociate ProfessorDepartments of Medicine (Neurology) and AnesthesiologyDuke University Medical CenterDurham, NC 27707U.S.A.Corresponding authorDaniel T. Laskowitz. MD, MHSBox 2900, Duke University Medical CenterDurham, NC 27710, USA.Fax +1 919 684 6514Email: danl@neuro.duke.edu