Biomarkers to Enhance Accuracy and Precision of Prediciton of Short Term and Long Term Outcome ICH

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  STUDY PROTOCOL Open Access Biomarkers to enhance accuracy andprecision of prediction of short-term andlong-term outcome after spontaneousintracerebral haemorrhage: a studyprotocol for a prospective cohort study A. Kumar 1 , P. Kumar 1 , S. Misra 1 , R. Sagar 1 , P. Kathuria 1 , D. Vibha 1 , S. Vivekanandhan 1 , A. Garg 1 , B. Kaul 2 , S. Raghvan 2 ,S. P. Gorthi 3 , S. Dabla 4 , C. S. Aggarwal 5 and Kameshwar Prasad 1* Abstract Background:  Several studies reported prognostic value of biomarker in intracerebral hemorrhagic (ICH) but theyare either preliminary observation or inadequately powered to analyse independent contribution of biomarkersover and above clinical and neuroimaging data. Objective:  To examine whether the biomarker can significantly add to the predictive accuracy of prognosis of ICH. Method/design:  In a multi-centric prospective cohort study, 1020 patients with ICH within 72 hours of onset arebeing recruited. After obtaining written informed consent from patients/proxy, venous blood sample (10 ml) isbeing collected and analysed for C-reactive protein (CRP) level, S100B, Glial fibrillary acidic protein (GFAP), Troponin,change in leukocyte count and Copeptin levels. The patients are telephonically followed using stroke scales (BarthelIndex and modified Rankin Scale) at 3, 6, 12 months and 2 years after the recruitment. Discussion:  This protocol will aim at predicting the short term or long term prognosis with the use of clinical,neuroimaging and biomarkers in order to help clinician to stratify patients for early referral or intervention. Keywords:  Biomarkers, Intracerebral hemorrhage, Prediction, Outcome, Multicentric study Background Stroke has emerged as the second most common causeof mortality worldwide and a major public health prob-lem. Intracerebral haemorrhage (ICH) accounts for ap-proximately 20-30 % of acute strokes in India and is stillassociated with a mortality of up to 35 – 50 % [23]. Thereare several fold higher incidence rates of ICH (61/100,000) in Asian countries [11], including India than inwestern countries. It is more common in men than inwomen [14]. Overall, the prognosis for ICH is poor:37-47 % of patients die within the first year after theevent and a substantial proportion of the survivors areleft with serious neurological deficits. About 25 to 30 %of patients deteriorate within first 24 hours in hospitalbecause of hematoma growth [12]. Thus, there is an ur-gent need for a simple diagnostic test which can help inthe hospital management of ICH patients.Several prospective studies have reported that in-creased levels of acute inflammatory markers, such asC-reactive protein (CRP), and white blood cell (WBC)count are associated with increased risk of death or dis-ability. Greater changes in leukocyte count over the first72 hours after admission predicted both short term andlong term worse and functional outcomes after ICH [1].Blood glucose (BG) represents a novel prognosticmarker in acute ICH, playing a major role in the patho-genesis of the acute inflammatory response in ICHpatients. The prognostic role of these inflammatory  * Correspondence:; 1 Department of Neurology, Neurosciences Centre, All India Institute of Medical Sciences, New Delhi, IndiaFull list of author information is available at the end of the article © 2015 Kumar et al.  Open Access  This article is distributed under the terms of the Creative Commons Attribution 4.0International License ( ), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the srcinal author(s) and the source, provide alink to the Creative Commons license, and indicate if changes were made. The Creative Commons Public DomainDedication waiver ( ) applies to the data made available in thisarticle, unless otherwise stated. Kumar  et al. BMC Neurology   (2015) 15:136 DOI 10.1186/s12883-015-0384-3  markers after ICH is less clear. Early prediction of out-come in patients with ICH is important and biomarkersmight allow the individualization of care by stratifyingrisk of reperfusion haemorrhage, predicting relative volumeof penumbral tissue, and providing additional prognosticinformation. In this study we propose to investigate therole of CRP level, serum glial fibrillary acidic protein,troponin, change in leukocyte count, S100B, copeptinlevels as independent predictors of the neurologicaloutcome in patients with primary intracerebralhaemorrhage. Copeptin Copeptin has emerged as a new diagnostic and prognos-tic biomarker in various diseases, but its prognostic value in ICH is still unknown. Its level is high in patientswith ICH. One study suggested that copeptin levels werehigher in patients who died in 30 days than in 30 dayssurvivors. Its levels were also higher in patients with anunfavourable clinical outcome at 90 days in ICH [26].Increase in level of plasma copeptin is an independentprognostic marker of 1-year mortality, 1-year unfavor-able outcome and early neurological deterioration [25]and associated with mortality and outcome in patientswith ICH [26]. Copeptin is a new prognostic marker inpatients with ICH, wrote Zweifel and colleagues, Univer-sity Hospital, Department of Neurosurgery and also sug-gested that  “ if this finding can be confirmed in largerstudies, it might serve as an additional valuable tool forrisk stratification and decision making in ICH patients. ” In this study we will assess the level of copeptin andidentify its relationship with the prediction of outcomein ICH patients. Troponin Cardiac troponin level is being used as a test of choicefor the detection of myocardial injury. One study sug-gested that in surgically treated ICH, elevated cardiactroponin levels are predictor of mortality and should beconsidered in managing the decisions of ICH [13].Higher level of troponin on admission is a significantrisk factor for in-hospital mortality in haemorrhagic pa-tients [3]. Elevated level of cardiac troponin has been as-sociated with adverse prognosis in patients with acuteneurological diseases. Only few studies have been con-ducted to know the relation between cTnT and progno-sis of ICH, but results are not conclusive [17,21]. In this study, we will identify the relationship between cTnTand outcome in patients with spontaneous ICH. S100B S100B is a member of calcium-mediated low molecularweight glial protein (approximately 10 kDa). Various com-binations of subunits ( α  and  β ) make up the S100 proteinfamily, which diverge into the hetero- and homodimerforms of ( α  and  β ) subunits. S100B is comprised of the( α - α ,  α -  β ,  β - β ). The highly specific form is  α -  β ,  β - β  forms,to nervous tissue, and is found in abundance in the cerebralastroglial compartment, peripheral Schwann cells, andextraneuronally in melanocytes, adipocytes, and chondro-cytes [9]. The concentration of S100B is 40-fold higher inCSF than in serum. Serum S100B levels after injury accur-ately predicts neurological function at discharge after supra-tentorial ICH in the first 24 h [18]. We hypothesize thatS100B could be useful as a biomarker for ICH patients. Leukocyte count After the spontaneous ICH, increase in peripheral leuko-cytes is an important marker of response of the immunesystem and causes the activation of the inflammatory cascade [2]. This is the most routinely used biomarkerto know the amount of inflammation mounted with72 hours after the intracerebral haemorrhage. Measure-ment of leukocyte count after the spontaneous intracere-bral haemorrhage may accurately reflect the extent of neuroinflammation. It independently predicts poor func-tional outcomes in terms of discharge disposition [1].We hypothesize that change in leukocyte count could beuseful as a biomarker for ICH patients. C - reactive protein C-reactive protein (CRP) is an inflammatory protein andits level rises in response to inflammation in an acutephase of inflammatory reactions. It binds with phospho-choline expressed on the surface of dead or dying cellsin order to activate the complement system. It also has arole in complex modulatory functions. It may directly participate in enhancing inflammation in cerebral vesselsand brain injury through activation of complement cas-cade, initiation of leukocyte chemotaxis and expressionof adhesion molecules through a positive feedbackmechanism [15]. It also induces apoptosis through acaspase-dependent mechanism [4]. Data are limitedregarding the role of CRP in the pathophysiologicalmechanisms and predictive outcome after sICH. Higherlevels of CRP are significantly associated with 30-day death after sICH [7] and an independent predictor of poor outcome in intracerebral haemorrhage [8]. Wehypothesize that it could be an important predictor of outcome in ICH patients. Glial fibrillary acidic protein (GFAP) Glial fibrillary acidic protein (GFAP) is a member of cytoskeleton protein family. It is expressed by numerouscell types of the central nervous system including epen-dymal cells and astrocyte cells. It helps to maintainastrocyte mechanical strength, as well as the shape of the cells. It is evident that GAP is considered to be Kumar  et al. BMC Neurology   (2015) 15:136 Page 2 of 6  important sensitive and specific marker for the rapidastrocyte response to injury and disease. It is alsoinvolved in promotion of normal blood brain barrier.One study indicated that serum GFAP may function as areliable biomarker for intracerebral haemorrhage inacute stroke. Increased GFAP levels are associated withthe blood brain barrier injury [20]. GFAP may be usefulas a surrogate marker and may be helpful for manage-ment of hemorrhagic stroke [12]. We hypothesize thatGFAP is immediately detectable in serum in acute phaseof ICH and could be useful as a biomarker for ICHpatients.A prediction model based on single or multiple bio-markers may help in clinical management, if it can pre-dict haematoma growth or long-term outcome. Atpresent, there is no single biomarker or panel of bio-markers that has the level of accuracy or precision to beuseful in clinical practices. A combination of clinical,neuroimaging and biomarkers may be able to achievethis objective. The main objectives of the present study are (i) to determine whether any of the biomarkersS100B, Copeptin, CRP, Leukocyte count, Troponin, Glialfibrillary acidic protein are independent predictors of theneurological outcome in patients with primary intracere-bral haemorrhage, and (ii) whether any of them (singly or in combination) improve the predictive accuracy of clinically important outcomes. Methods/design Ethical considerations Study Protocol has been approved from Institute EthicsCommittee. Design of study Prospective cohort study. Inclusion and Exclusion criteria Patients will be eligible if they meet all the inclusion criteriaand none of the exclusion criteria. Inclusion criteria Patients will be judged eligible if they have all of the fol-lowing: (1) Sudden onset of focal neurological deficit orimpairment of consciousness; (2) Age greater than18 years; (3) CT scan showing parenchymal haematomain brain; (4) Admission within 72 hours after the onsetof the qualifying event; (5) Accessibility for follow-up by telephone (landline or mobile). Exclusion criteria Patients meeting the inclusion criteria will be consideredineligible for the study if they have any one of the fol-lowing: (1) Suspicion or documented history of a bleed-ing disorder; (2) History of recent head trauma; (3)History of ingestion of anticoagulant drugs within sevendays of onset of stroke; (4) A documented A-V malfor-mation; aneurysm or cerebral neoplasm as the under-lying cause of primary supratentorial intracerebralhaemorrhage; (5) Pre-morbid organ failure or disability leading to dependence on others for activities of daily living; (6) Unwillingness to provide written informedconsent (by self or next of kin); (7) Hemorrhagic trans-formation of cerebral infarct; (8) Concurrent major renalor hepatic disease; (9) Pregnancy. Centre ’   s eligibility criteria (1) Be a Department of Scientific and Industrial Re-search (DSIR) certified or a medical college or govern-ment hospital; (2) Admitting at least 5 patients withstroke per month; (3) Have access to CT scan; computerand internet; (4) Have access to a laptop and refrigeratorwith  − 80 °C. Consent and Recruitment Witnessed written informed consent will be requestedfrom all eligible patients or their next of kin (in case of patients with aphasia or impaired consciousness). Liter-ate subjects or their next of kin will be requested to givetheir written consent by one of the physician investiga-tors with a nurse witnessing the consent. Subjects ortheir next of kin who are not literate will have the con-tents of the consent form read out and explained by theinvestigator and their left thumb impression will betaken according to the current practice with a nurse as awitness. All questions from the subjects or their next of kin will be answered by the investigator.Potentially eligible patients will be recruited from theemergency services and ward of the participating centre.All potentially eligible patients will undergo emergency head CT for diagnosis of primary intracerebral haemor-rhage. All patients with primary intracerebral haemor-rhage will be admitted and their eligibility will beassessed by the investigators. Patients suspected to haveaneurysm or arteriovenous malformation will be ex-cluded unless digital subtraction angiography. A logbookof all patients screened, those eligible will be maintained.This log will be used to estimate the proportion of allpotentially eligible patients who enter the study and willbe used to assess the generalizability of study results. Baseline assessment Standardized forms will be used to record patienthistory, general and neurological examination. Clinical,laboratory and radiological findings to be recorded atbaseline will include: Kumar  et al. BMC Neurology   (2015) 15:136 Page 3 of 6   A. Clinical assessment  Age, Sex, History of hypertension, diabetes, smoking andfamily history of stroke or coronary artery disease, Treat-ment history for hypertension and diabetes, if known tohave one or both of these, Time of onset of the stroke,Blood pressure at the time of recruitment, Glasgow Coma Scale (GCS) score at the time of recruitment,Barthel index (BI) score at the time of recruitment,National Institute of Health Stroke Scale (NIHSS) andICH Score. B. Radiological assessment  Site and volume of haematoma and any midline shift asdetermined on CT scan, Intraventricular extension of the haematoma as determined by CT scan, CT scans willbe uploaded on the day of recruitment using software tobe installed by AIIMS neuro-radiology department. Thescans will be viewed for quality on the same day, and if not satisfactory, repeat CT will be done within hours. C. Biomarker Assay and Sample transportation After the written informed consent of patient/relatives venous blood sample (10 ml) will be collected in a spe-cific serum vacutainer and EDTA coated vial. For serumcollection it will be left standing at room temperaturefor 30 min until clotted. It will then undergo centrifuga-tion at 3000 rpm for 15 min, after which the serum willbe separated into two equal aliquots in serum containing vials. Both aliquots will be stored at  − 80 °C. EDTA tubeswill be centrifuged for plasma separation and storedat  − 80 °C until analysis. It will be packed within a monthat participating centres and transferred to PrincipalCentre (AIIMS) for the biomarker analysis. Prior to ship-ment the serum samples will be removed from the  − 80 °Crefrigerator and will be placed inside one of two identicalreusable thermal insulated pouches. A single frozen icepack, gel pack and dry ice will be placed inside one of thetwo insulated pouches and sealed. Samples will be trans-ferred by using standard courier service to the AIIMS by priority overnight delivery. Sample will be used to assay the biomarker levels using available methods as men-tioned in Table 1. Sample Size As this study involves multiple variables (11 clinical, 03neuroimaging and 06 biomarkers), we plan to use prog-nostic modelling. According to Harrell et al. [16] a stableand replicable model will require 20 events per variable.As the outcome events in ICH over six months has afrequency of approximate 50 % [24], and 23 variables areplanned to be examined, we need 920 patients withcomplete follow up. Adjusting this for 10 % loss to fol-low up, yields a sample size of 1020. Patient management All subjects will receive standard medical therapy con-sisting of maintenance of adequate airway, fluids andelectrolyte balance, and good pulmonary and cardiacfunction. Nasogastric feeding will be instituted in uncon-scious patients. Patients who experience a seizure at any time since onset of the stroke will be given a loadingdose of an anticonvulsant intravenously (usually pheny-toin 15 – 20 mg/kg body weight i.v. push) followed by amaintenance dose (usually phenytoin 5 mg/kg dividedover a period of three time a day i.v. push) until recovery of consciousness when oral administration of the samedose will be continued for six months. The dose and thedrug will be adjusted to minimize the side-effects. Severehypertension (defined as systolic BP more than 200 mgHg and diastolic BP more than 120 mm Hg) will betreated with diuretics and/or ACE inhibitors so as tokeep systolic BP between 160 – 200 mm Hg and diastolicBP between 100 – 110 mm Hg. Corticosteroids, glycerolor antithrombotics will not be used. Hyperventilationwill be used to control intracranial pressure especially when signs of brain herniation develop. The medicalmanagement of all patients will be carried out by a teamof neurologists and neuroanaesthetists in the intensivecare unit settings according to National guidelines [22].Patients meeting the criteria according to the Nationalguideline will be operated. All interventions adminis-tered to the patients will be recorded and documentedfor trial purposes. In-hospital follow-up All subjects will be assessed daily by the neurologist in- vestigators with the help of neurology residents. CT scanwill be repeated between 24 to 48 hours to detecthaematoma growth. Serum chemistry and blood gaseswill be monitored daily for unconscious patients. Otherinvestigations will be done when clinically indicated. Award physiotherapist will see each subject twice a weekand implement an appropriate physiotherapy plan.Nurses will provide nursing care to all subjects regularly.All subjects will be observed in the hospital for a mini-mum period of 30 days post-inclusion with the Table 1  List of Biomarkers to be assayed S.No Name of Biomarker Source Method of Assessment1 S100B Plasma ELISA [6]2 Copeptin Plasma ELISA [10]3 CRP Serum Immunoturbidimetricassay [7]4 Glial fibrillary acidic protein Serum ELISA [19]5 Leukocyte count Blood Automated cellcounters [5]6 Troponin Serum ELISA [21] Kumar  et al. BMC Neurology   (2015) 15:136 Page 4 of 6
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