JCB J Circ Biomark 2023; 12: 17-25ISSN 1849-4544 | DOI: 10.33393/jcb.2023.2502REVIEW Journal of Circulating Biomarkers - ISSN 1849-4544 - www.aboutscience.eu/jcb © 2023 The Authors. This article is published by AboutScience and licensed under Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Commercial use is not permitted and is subject to Publisher’s permissions. Full information is available at www.aboutscience.eu causative aneurysm has been obliterated (1,2). The patho- physiology of DCI is complex and multifactorial; however, it is not yet fully understood. As a result, an accurate diagnostic test and effective treatment are both lacking. Biomarkers may not only help to further elucidate the pathophysiology of DCI, but could also aid in optimizing early diagnosis and prompt treatment. Even though the present literature is brimmed with biomarker studies of DCI, accurate biomarkers of DCI have not yet been established. In 2018, an extensive systematic review proposed a panel analysis of 6 selected genetic and 15 protein biomarker can- didates for further prospective validation of DCI prediction (3). However, this review used a very broad definition of DCI, including also DCI defined solely as angiographic vaso- spasm on digital subtraction angiography or increased flow velocities on transcranial Doppler (TCD) ultrasonography. Historically, angiographic vasospasm was considered the sole cause of DCI; however, in the past decades research has Laboratory biomarkers of delayed cerebral ischemia following subarachnoid hemorrhage: A systematic review Maud A. Tjerkstra1, Homeyra Labib1, Bert A. Coert1, René Spijker2,3, Jonathan M. Coutinho4, W. Peter Vandertop1, Dagmar Verbaan1 1Amsterdam UMC, University of Amsterdam, Department of Neurosurgery, Neuroscience Amsterdam, Amsterdam, Netherlands 2Medical Library, Amsterdam UMC, University of Amsterdam, Amsterdam Public Health, Amsterdam, Netherlands 3 Cochrane Netherlands, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands 4Amsterdam UMC, University of Amsterdam, Department of Neurology, Neuroscience Amsterdam, Amsterdam, Netherlands Maud A. Tjerkstra and Homeyra Labib contributed equally. ABSTRACT Delayed cerebral ischemia (DCI) substantially contributes to disability and death in subarachnoid hemorrhage (SAH) patients; however, its pathophysiology is incompletely understood and diagnostic and therapeutic strate- gies are lacking. Biomarkers may help to elucidate the pathophysiology, optimize early diagnosis, or provide treat- ment targets. We systematically searched PubMed and Embase on October 13, 2021, for studies that evaluated at least one laboratory biomarker in patients with DCI, using the most up-to-date definition of DCI as proposed by a panel of experts in 2010. Quality of studies was assessed using the Newcastle-Ottawa Scale or Cochrane Collaboration’s risk of bias assessment tool. Biomarkers of clinical and radiological DCI were analyzed separately. Results were meta-analyzed if possible, otherwise narratively reviewed. Biomarkers were classified as signifi- cant, inconclusive, or nonsignificant. We defined validated biomarkers as those with significant results in meta- analyses, or in at least two studies using similar methodologies within the same time interval after SAH. The search yielded 209 articles with 724 different biomarkers; 166 studies evaluated 646 biomarkers of clinical DCI, of which 141 were significant and 7 were validated biomarkers (haptoglobulin 2-1 and 2-2, ADAMTS13, vWF, NLR, P-selectin, F2-isoprostane); 78 studies evaluated 165 biomarkers of radiological DCI, of which 63 were significant and 1 was a validated biomarker (LPR). Hence, this review provides a selection of seven biomarkers of clinical DCI and one biomarker of radiological DCI as most promising biomarkers of DCI. Future research should focus on determining the exact predictive, diagnostic, and therapeutic potentials of these biomarkers. Keywords: Biomarker, Delayed cerebral ischemia, Subarachnoid hemorrhage Received: October 8, 2022 Accepted: March 20, 2023 Published online: April 5, 2023 This article includes supplementary material Corresponding author: Maud Annabel Tjerkstra Department of Neurosurgery Amsterdam UMC, University of Amsterdam Meibergdreef 9 PO Box 22660, 1100 DD, Amsterdam - the Netherlands m.a.tjerkstra@amsterdamumc.nl Introduction Delayed cerebral ischemia (DCI) is an important and potentially preventable contributor to disability and death in subarachnoid hemorrhage (SAH) patients in whom the https://doi.org/10.33393/jcb.2023.2502 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:m.a.tjerkstra@amsterdamumc.nl Biomarkers of DCI after SAH18 © 2023 The Authors. Journal of Circulating Biomarkers - ISSN 1849-4544 - www.aboutscience.eu/jcb shown that DCI and vasospasms are two different entities (4). Therefore, the combined use of both vasospasm and DCI in the 2018 review may have misguided the selected panel of biomarkers. In 2010, a panel of experts proposed a strict definition of DCI, divided into a clinical and radiological definition, to be used as outcome measure and to enable more reliable meta- analyses of future clinical trials and observational studies (4). We used these definitions of DCI to systematically review the current literature on laboratory biomarkers of DCI and selected a new panel of the most promising biomarkers. The selected biomarkers may be useful for the prediction or diag- nosis of DCI, either individually or combined. Methods The Preferred Reported Items for Systematic Review and Meta-analysis (PRISMA) 2020 guidelines were followed. This review was not registered and a review protocol was not prepared. Search strategy and selection criteria PubMed and Embase were searched for relevant studies from January 1, 1985, to October 13, 2021. The search strategy can be found in the supplemental data (Supplementary material, page 2-3). Two authors (MAT and HL) independently selected eligible studies. Titles and abstracts were screened for potential eligibility when cov- ering primary studies on biomarkers of DCI in SAH patients either written in Dutch or English. Of the selected studies, full texts were screened and studies were considered eli- gible if they met the following criteria: (1) patients with SAH of whom (2) at least 95% were diagnosed with a caus- ative aneurysm, (3) measurement of at least one laboratory biomarker (4) during hospital admission, (5) using either the radiological or clinical definition of DCI as proposed in 2010 (criteria listed in Tab. I), and (6) the studies had to perform, or allow for, statistical analysis on the biomarker in question and DCI. Exclusion criteria were: (1) studies with mixed clinical and radiological DCI cohorts, in which patients with either clinical or radiological DCI could not be distinguished from the mixed cohort, (2) biomarker levels in patients with and without DCI were inconsistent (e.g., different numbers in tables/figures and text), (3) no level of statistical significance was given, nor could be calculated, (4) studies in children only, (5) the same patient popula- tion (similar years and hospital of patient inclusion) was used by more than one study and, within those studies, the same biomarker was measured and compared similarly between DCI and no DCI patients. In this case, the study with the smallest sample size, and if sample sizes were equal the most outdated study, was excluded. Lastly, we checked studies on nimodipine administration. In 1988 and 1989, two randomized controlled trials showed efficacy of nimodipine in preventing DCI, resulting in a shift in SAH management (5,6). Therefore, we excluded studies with SAH patients before 1985. SAH cohorts between 1985 and 1990 were only included if nimodipine administration was explicitly mentioned. All papers after 1990 were included, unless nimodipine was described explicitly as being not part of the treatment protocol. TABLE I - Criteria of the clinical and radiological definition of DCI Clinical DCI Defined as: • �A� decrease� in� level� of� consciousness� (described� by� a� decrease� of�the�Glasgow�Coma�Scale�score�or�an�increase�of�the�National� Institutes�of�Health�Stroke�Scale�score)�or • �Focal�neurological�impairment�(e.g.,�aphasia,�motor�impairment,� sensory�deficits,�neglect)� Excluded if: • �Vague�definitions�of�DCI,�e.g.,�“clinical�deterioration,”�“neurological� deterioration,”� “worsening� of� neurological� condition,”� without� specifically�mentioning�either�decreased�level�of�consciousness� or focal neurological impairment • �The�DCI�cohort�also�includes�patients�with�solely�signs�of�vasospasm� without�clinical�symptoms�of�DCI.�For�example,�patients�with:�  • Arterial�narrowing�on�CT-A�or�DSA  • Increased�flow�velocities�on�TCD Radiological DCI Defined as: • New�ischemia/infarction/stroke�or • Secondary�ischemia/infarction/stroke�or� • �Ischemia/infarction/stroke� due� to� DCI� (or� associated� DCI� terms,� including�delayed�ischemic/neurological�deficit,�[clinical]�vasospasm,� secondary�ischemia/infarction) Excluded if: • �Defined� as� “presence� of� ischemia/infarction/stroke”� without� defining�“new,”�“secondary,”�or�“due�to�DCI/associated�terms�of� DCI”� • �Presence�of�ischemia/infarction/stroke�determined�by�an�imaging� modality other than noncontrast CT or MRI, e.g., CT perfusion, CT-A) • �Other�nonimaging�methods�used�to� assess� ischemia/infarction/ stroke,�e.g.,�brain�tissue�oxygenation�monitoring�and�micro�dialy- sate biomarkers �CT�=�computed�tomography;�CT-A�=�CT-angiography;�DCI�=�delayed�cerebral�ischemia;�DSA�=�digital�subtraction�angiography;�MRI�=�magnetic�resonance�imaging;� TCD = transcranial Doppler. Tjerkstra et al J Circ Biomark 2023; 12: 19 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu Data extraction The same authors (MAT and HL) independently extracted baseline SAH characteristics (age, gender, World Federation of Neurological Societies [WFNS] grade and Fisher grade), the study’s definition of DCI, the number of patients with and without DCI, and biomarker-related data. If studies addressed both clinical and purely radiologi- cal DCI separately, biomarker-related data were extracted for both definitions. The biomarker-related data concerned laboratory biomarkers only (clinical and radiological biomark- ers [heart rate, blood pressure, temperature, TCD velocities, computed tomography (CT) perfusion variables etc.] were left out of consideration). Biomarker data had to concern levels of individual biomarkers or ratios of two biomarkers, that is, combinations of three or more biomarkers, such as decision tree analysis or a self-made calculated score based on multiple biomarkers, were left out of consideration. Data included the biomarker medium (blood, cerebrospi- nal fluid [CSF], microdialysate [MD], urine, or other), tim- ing of measurement(s), and all statistical analyses regarding the biomarker and the occurrence of DCI. If the biomarker medium was not mentioned, it was assumed to be blood. A biomarker measured in multiple media, for example, in blood and CSF, was considered as two separate biomarkers. Nongenetic biomarkers were stratified into biomarker groups, which were based on the multifactorial pathophysi- ology of DCI as described by a previously published system- atic review (7). Of biomarkers with multiple functions, we aimed to select the function which the authors of the study considered as DCI-related and subsequently categorized the biomarker into that specific biomarker group. Nongenetic biomarkers which could not be stratified to any biomarker group related to the pathophysiology of DCI were categorized as “other.” Within the “other” group, we aimed to cluster biomarkers with similar functions. Genetic biomarkers were categorized as a separate group. The extracted data were compared and discrepancies were discussed with a third author (DV) and solved. Quality assessment Two authors (MAT and DV) independently assessed the quality of included studies using the Newcastle-Ottawa Scale (NOS) for case control and cohort studies and the Cochrane Collaboration’s risk of bias assessment tool for random- ized trials. Studies could obtain a maximum of eight or nine points, depending on the use of a nonexposed cohort. The Cochrane Collaboration’s risk of bias assessment tool com- prises seven criteria, which can be scored as high, low, or unclear risk of bias. Discrepancies were discussed with a third opinion (WPV) and solved. Data interpretation and analysis Biomarkers of clinical DCI (primary outcome) were ana- lyzed separately from biomarkers of radiological DCI (second- ary outcome). We categorized biomarkers into significant and nonsignificant biomarkers. Biomarkers, which were studied by one study only, were considered significant if at least one measurement was significantly associated with DCI (p < 0.05) and nonsignificant if all measurements yielded nonsignificant results. If a biomarker was investigated by more than one study, the classification into significant and nonsignificant was either based on a biomarker-specific meta-analysis, if methodologically possible (similar type of analyses and tim- ing of measurement), or by descriptive comparison. Meta- analyses were performed only if all relevant studies, which investigated the biomarker in question on a specific point in time after SAH, could be included, using Review Manager 5.4. For continuous data we used the inverse variance method with random effects model to calculate weighted mean dif- ferences with 95% confidence intervals. For dichotomous data we used the Mantel-Haenszel method with random effects model to calculate estimated odds ratio with 95% con- fidence intervals. A pooled effect size was estimated for each specific timing of biomarker measurement. Descriptive com- parison of the studies was done by summarizing the results of all relevant studies in a biomarker-specific figure, which was then used to classify the biomarkers into significant, inconclusive, and nonsignificant. The biomarker-specific fig- ures display the author, year, number of patients with and without DCI, the type of analyses, and the results divided into categories of sampling timing (day 0-3, 4-8, 9-13, and >14 after SAH). The method used for classification of biomarkers into significant, inconclusive, and nonsignificant based on the biomarker-specific figures is explained in the supplemental data (Supplementary material, page 4). In order to select the most promising biomarkers of DCI, we evaluated whether significant biomarkers were validated by a second study. A validated biomarker was defined as a biomarker with significant results in at least two studies using similar methodologies within the same time interval after SAH, without any other study showing contradictory results. Biomarkers of which the meta-analysis of all relevant studies yielded a significant association were considered validated, too. Additionally, biomarkers that had only been investigated by one study were differentiated from those biomarkers that were investigated by a high-quality study. We defined high quality as a maximum (eight or nine) or maximum minus one point (seven out of eight or eight out of nine) quality assess- ment score. Data have been reported according to the PRISMA report- ing guidelines for systematic reviews and meta-analyses. Results Included studies After removing duplicates, the search yielded a total of 5,820 studies. We excluded 3,900 studies during abstract screening and 1,711 studies during full-text screening, after which 209 articles (204 cohort and 4 case-control studies, 1 randomized controlled trial) remained (Fig. 1). Baseline characteristics of all included studies are listed in Table S1 of the supplemental data (Supplementary material, page 5-12). Altogether, the 209 studies measured 724 different biomark- ers in 25,933 SAH patients (not corrected for overlapping Biomarkers of DCI after SAH20 © 2023 The Authors. Journal of Circulating Biomarkers - ISSN 1849-4544 - www.aboutscience.eu/jcb patient cohorts). A summary of the NOS quality scores is listed in Table II, and the quality scores of individual studies are displayed in the baseline characteristics table (Table S1, page 5-12). The four case-control studies scored three, four (n = 2) and seven of nine points. The randomized controlled trial scored low risk on three of seven items. Clinical DCI Biomarkers of clinical DCI were studied by 166 studies comprising 646 different biomarkers (Fig. 2) in 20,983 SAH patients of whom 5,810 (28%) developed DCI. Most biomark- ers (588 of 646, 91%) were investigated by one study and 58 (9%) by more than one study (range 2 to 14, median 3). The distribution of the biomarkers among the biomarker groups is shown in Table III. In total, 141 biomarkers (n = 88 nongenetic, n = 53 genetic) were classified as significantly associated with clinical DCI (Table S2, page 13-34). A summary of studied and significant biomarkers and specific trends observed within each bio- marker group are displayed in Table III. Of the significant bio- markers, 121 were investigated by one study and 20 by more than one study. Meta-analyses were possible for five bio- markers (haptoglobin 1-1, 2-1, and 2-2, ADAM metallopepti- dase with thrombospondin type 1 motif 13 [ADAMTS13] and Fig. 1 - PRISMA 2020 flow diagram for systematic reviews. TABLE II - Summary of the Newcastle-Ottawa Scale quality assess- ment scores of 204 cohort studies Quality score 134 studies without control group (maximum 8 points) 70 studies with control group (maximum 9 points) 1 point None None 2 points 7 studies 2 studies 3 points 24 studies 8 studies 4 points 20 studies 16 studies 5 points 35 studies 20 studies 6 points 34 studies 14 studies 7 points 10 studies 7 studies 8 points 4 studies 3 studies 9 points – None von Willebrand factor [vWF]). The meta-analyses showed that SAH patients with haptoglobin polymorphism 2-1 were less likely to develop DCI and patients with haptoglobin poly- morphism 2-2 were more likely to develop DCI (8-10). DCI patients had lower ADAMTS13 levels on day 1 and higher lev- els of vWF on day 4 after SAH, compared to patients without Tjerkstra et al J Circ Biomark 2023; 12: 21 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu Fig. 2 - Flowchart of all biomarkers, di- vided into clinical and radiological DCI. ADAMTS13 = ADAM metallopeptidase with thrombospondin type 1 motif 13; LPR = lactate/pyruvate ratio; NLR = neu- trophil/lymphocyte ratio; vWF = von Willebrand factor. TABLE III - Number of studied and significant biomarkers and specific trends observed within each biomarker group of clinical DCI Biomarker group Biomarkers of clinical DCI Specific trends observed among significant biomarkers within biomarker groupStudied Significant Nongenetic biomarkers 225 88 Brain injury 16 8 All�indicated�more�brain�injury�in�patients�with�DCI.�No�specific�trend�in�time.� Coagulation�cascade 33 15 All�indicated�increased�platelet�activity,�hypercoagulability,�or� hyperfibrinolysis.�No�specific�trend�in�time.� Cortical�spreading�depressions 2 1 Excitotoxicity�in�first�48�h�after�SAH. Endothelial injury 3 2 Both indicated more endothelial injury in DCI. Inflammation�and�immune� system 83 29 25�of�29�biomarkers�indicated�enhanced�inflammation�or�immune� response.�Most�frequently�observed�in�first�couple�of�days�following�SAH. Oxidative�stress 18 7 5�of�7�biomarkers�indicated�increased�oxidative�stress�in�patients�with� DCI.�No�specific�trend�in�time.� Vascular�tone 22 9 6�of�9�biomarkers�indicated�vasoconstriction,�2�indicated�vasodilation,�1� was�significant�with�unknown�point�estimate.� Volemic�status 10 2 Both increased and decreased diuresis and natriuresis were observed in DCI. Other 38 14 Subcategories�were:�cerebral�metabolism,�gender-specific�growth� factors, lipid metabolism, oxygen-carrying capacity, and stress response. Genetic biomarkers 421 53 Genomic MicroRNA Methylation Polymorphism RNA Single�nucleotide�polymorphism  5  51  44  13  15 293  1  0 36  3  6  7 Function�often�unclear,�some�were�involved�in�processes�of�oxidative� stress,�vascular�tone,�volemic�status,�stress�response,�inflammation�and� coagulation,�among�others. DCI�=�delayed�cerebral�ischemia;�RNA�=�ribonucleic�acid;�SAH�=�subarachnoid�hemorrhage. DCI, in two studies (11,12). The results of the remaining 53 significant biomarkers that were investigated by multiple studies that could not be meta-analyzed were summarized in biomarker-specific figures. Based on the biomarker-specific figures, three other biomarkers (neutrophil/lymphocyte ratio [NLR] and P-selectin in blood and F2-isoprostane in urine) had significant results in two or more studies using similar methodologies and timing of measurement, and were con- sidered validated biomarkers. Four studies on NLR showed a proinflammatory state on admission and preoperatively in patients with DCI compared to patients without DCI (13-16). P-selectin, a marker of endothelial activation, was found to have a significantly larger increase during DCI onset, com- pared to paired samples in the non-DCI group in two studies Biomarkers of DCI after SAH22 © 2023 The Authors. Journal of Circulating Biomarkers - ISSN 1849-4544 - www.aboutscience.eu/jcb (17,18). F2-isoprostane in urine on day 3 after SAH was sig- nificantly associated with the occurrence of DCI in two stud- ies (19,20). The quality of studies from which we found the seven validated biomarkers is listed in Table IV. Both meta- analyses and biomarker-specific figures can be found in the supplemental data (Supplementary material, Figure S1-S58, page 35-45). Among the 121 significant biomarkers investigated by one study only, 5 (n = 4 nongenetic, n = 1 genetic) biomark- ers were investigated by high-quality studies: brain natriuretic peptide (21), osteopontin (22), tenascin C (23), and neutrophil/ albumin ratio (24) in blood and endothelial nitric oxide syn- thase (SNP rs2070744 T-786C) using buccal swabs (25). Radiological DCI Biomarkers of radiological DCI were studied by 78 studies comprising 165 different biomarkers (Fig. 2) in 10,282 SAH patients of whom 2,254 (22%) developed DCI. Most biomark- ers (146 of 165, 88%) were investigated by one study and 19 (12%) by more than one study (range 2 to 8, median 2). The distribution of the biomarkers among the biomarker groups is shown in Table V. In total, 63 biomarkers (n = 39 nongenetic, n = 24 genetic) were classified as significantly associated with radiological DCI TABLE IV - Quality of relevant studies of validated biomarkers Validated biomarker Studies Quality of studies Clinical DCI ADAMTS13 Li,�2017�(11) Tang,�2015�(12) 3/8 4/9 F2-isoprostane Wiśniewski,�2020�(19) Wiśniewski,�2017�(20) 6/9 6/9 Haptoglobulin�2-1 Kim,�2018�(8) Leclerc,�2015�(9) Ohnishi,�2014�(10) 3/8 5/8 6/8 Haptoglobulin�2-2 Kim,�2018�(8) Leclerc,�2015�(9) Ohnishi,�2014�(10) 3/8 5/8 6/8 NLR Liu,�2020�(13) Al-Mufti,�2019�(14) Wu,�2019�(16) Tao,�2017�(15)� 6/8 6/8 7/8 5/8 P-selectin Wang,�2011�(17) Frijns,�2006�(36) 7/9 5/8 vWF Li,�2017�(11) Tang,�2015�(12) 3/8 4/9 Radiological DCI LPR Kofler,�2020�(26) Torné,�2020�(27) 4/8 2/8 ADAMTS13�=�ADAM�metallopeptidase�with�thrombospondin�type�1�motif�13;� DCI = delayed�cerebral�ischemia;�LPR�=�lactate/pyruvate�ratio;�NLR�=�neutrophil/ lymphocyte�ratio;�vWF�=�von�Willebrand�factor. (Supplementary material, Table S2, page 13-34). A summary of the studied and significant biomarkers and specific trends observed within each biomarker group is given in Table V. Of the significant biomarkers, 56 were investigated by one study and 7 by multiple studies. Meta-analyses were not possible. Based on the biomarker-specific figures, one biomarker, lac- tate/pyruvate ratio (LPR) in blood, was considered validated as two independent studies showed that an LPR higher than 40 was associated with DCI, measured during the first weeks following SAH (26,27). The quality of the two studies on LPR was 4/8 and 2/8 (Table 4). The biomarker-specific figures can be found in the supplemental data (Supplementary material, Figure S59-S77, page 46-48). Among the 56 significant biomarkers investigated by one study, 7 nongenetic blood biomarkers were investigated by a high-quality study: monocyte LFA-1, monocyte Mac-1, mono- cyte PSGL-1, neutrophil LFA-1, neutrophil Mac-1, neutrophil PSGL-1, and osteopontin (22,28). Clinical vs. Radiological DCI Of all biomarkers, 89 were studied for both clinical and radiological DCI (Supplementary material, Table S2, page 13-34). Eight blood biomarkers were significantly associ- ated with both clinical and radiological DCI (d-dimer, platelet activating factor, interleukin-17, lectin complement pathway activity, osteopontin, brain natriuretic peptide, urea/creati- nine ratio and serine proteinase inhibitor family E member 1 SNP rs2227631 + rs1799889 + rs6092 + rs6090 + rs2227684 + rs7242). Forty-five biomarkers were classified as nonsignifi- cant for both clinical and radiological DCI. Of the remaining 36 biomarkers, the classification into significant, inconclusive, or nonsignificant differed between clinical and radiological DCI. Discussion This review has selected seven biomarkers of clinical DCI (haptoglobin polymorphisms 2-1 and 2-2, ADAMTS13, NLR, P-selectin, and vWF in blood and F2-isoprostane in urine) and one biomarker of radiological DCI (LPR in MD) as most prom- ising biomarkers of DCI. Of note, the majority of significant biomarkers (121/141 of clinical DCI and 56/63 of radiological DCI) were only evaluated by one study, and although being potential biomarkers, especially the 12 biomarkers from high-quality studies, are in need of validation. In 2018, a review on biomarkers of DCI yielded a panel of 21 predictive biomarkers (3); however, this review had three substantial limitations, which may have misguided the selected panel of biomarkers: (1) patients with solely angi- ographic vasospasm and increased flow velocities on TCD ultrasound, as indication of DCI, were also included, (2) the results of biomarkers measured in different media (CSF and blood) were combined, and (3) no differentiation between predictive and diagnostic biomarkers was made. In our present review, the most up-to-date definition of DCI was strictly applied during the selection of eligible articles and a biological substance measured in multiple media was con- sidered a separate biomarker for each medium. As a result, our review yielded less studies (209 vs. 270). Although we found more biomarkers (724 vs. 257), our review yielded Tjerkstra et al J Circ Biomark 2023; 12: 23 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu only eight validated biomarkers, as opposed to 21 by Jabbarli et al., of which two (ADAMTS13 and NLR) were in agree- ment (3). The discrepancy between the panels of biomark- ers selected by both reviews emphasizes the importance of a uniform definition of DCI, especially in the absence of solid diagnostic criteria. The eight validated biomarkers of DCI are all related to known pathways of the multifactorial pathophysiology of DCI. In clinical DCI, increased platelet aggregability, thereby potentially leading to the formation of microthrombi, was observed by decreased admission levels of ADAMTS13 and increased levels of vWF on day 4 after SAH. As DCI usu- ally occurs between days 4 and 10 after SAH (4), the early onset of significantly differing levels makes these biomarkers potentially predictive of DCI. Notwithstanding, the quality of the studies was rather low (NOS scores of 3/8 and 4/9), therefore a high-quality study is necessary to further eluci- date matters. Most inflammation and immune system biomarkers seem to be significantly elevated early in the course of SAH. Accordingly, elevated NLR (a validated biomarker) in the first couple of days following SAH was found to be significantly associated with clinical DCI in four independent studies. But, the exact predictive ability, including determination of an optimal cut-off value with accompanying accuracy, specific- ity, and sensitivity, remains to be determined. Whether NLR could also serve as a preventive treatment target is debat- able, as NLR is a highly nonspecific biomarker and nonspecific anti-inflammatory therapies have thus far proven disappoint- ing for the prevention of DCI (29). P-selectin was found to significantly increase during clinical DCI onset in two stud- ies, which suggests that P-selectin may be a potential can- didate for diagnosis or therapeutic treatment, for example, P-selectin antagonism. Thus far, therapies targeting P-selectin have only been performed in experimental settings of isch- emic stroke, showing a reduction in infarct volume and bet- ter clinical outcome (30), and myocardial infarction, showing significantly reduced myocardial damage after percutaneous coronary intervention (31). P-selectin antagonism has not been studied in SAH, yet. Three validated biomarkers of oxidative stress were found for clinical DCI in our review: F2-isoprostane in urine and genetic biomarkers haptoglobin polymorphism 2-1 and 2-2. Genetic markers often require extensive processing time and TABLE V - Number of studied and significant biomarkers and specific trends observed within each biomarker group of radiological DCI. Biomarker group Biomarkers of radiological DCI Specific trends observed among significant biomarkers within biomarker groupStudied Significant Nongenetic biomarkers 100 39 Brain injury  6  2 All�indicated�more�brain�injury�in�patients�with�DCI.�No�specific�trend� in�time. Coagulation�cascade  16  9 All four biomarkers of primary hemostasis indicated increased platelet�activation/aggregation,�three�of�four�secondary�hemostasis� biomarkers showed hypercoagulability, one biomarker showed hyperfibrinolysis�in�patients�with�DCI.�No�specific�trend�in�time.� Cortical�spreading�depressions – – – Endothelial injury  4  2 Both biomarkers indicated increased endothelial injury in DCI. Inflammation�and�immune� system  33 15 12�of�15�inflammation�and�immune�system�markers�indicated� increased�inflammation�and�elevated�immune�response�in�patients� with�DCI.�No�specific�trend�in�time.� Oxidative�stress  4  2 Both�biomarkers�indicated�increased�oxidative�stress�in�patients�with� DCI. Vascular�tone  8  1 Vasoconstriction�between�days�0�and�14�after�SAH�in�DCI.� Volemic�status  2  2 More diuresis and natriuresis were observed in DCI. More DCI in hyponatremia vs. no hyponatremia. Other  27  6 Subcategories�were:�angiogenesis,�cerebral�metabolism,�lipid� metabolism, stress response, and protein metabolism. Genetic biomarkers  63 24 Genomic MicroRNA Methylation� Polymorphism RNA Single�nucleotide� polymorphism  3  28 –  5 –  27  0 19 –  1 –  4 Function�often�unclear;�however,�four�biomarkers�were�involved�in� processes�of�coagulation,�inflammation,�and�vascular�tone. DCI�=�delayed�cerebral�ischemia;�RNA�=�ribonucleic�acid;�SAH�=�subarachnoid�hemorrhage. Biomarkers of DCI after SAH24 © 2023 The Authors. Journal of Circulating Biomarkers - ISSN 1849-4544 - www.aboutscience.eu/jcb are therefore unsuitable for acute diagnosis; however, early determination of genetic profiles, for example, on admission, may aid in stratification of patients at high risk of clinical DCI. As such, the haptoglobin polymorphisms 2-1 and 2-2 may be useful for patient stratification, and could perhaps pro- vide a potential preventive treatment target, for example, the administration of exogenous haptoglobulin (32). For F2-isoprostane, whether the observed increased urine lev- els have preceded the onset of clinical DCI in all patients is unknown; therefore, we cannot distinguish whether these biomarkers are predictive or diagnostic. Further research should be performed to explore the temporal relationship of these markers in regard to clinical DCI development. The remaining validated biomarker, LPR in MD, allows for continuous neuromonitoring providing direct metabolic information, which can be used to detect an ischemic state, that is, radiological DCI. Since DCI currently is a clinical diag- nosis per exclusion, LPR may be a helpful additive in coma- tose patients, as it may detect metabolic distress in an early phase already, before hypodensities are visible on noncon- trast head CT and when ischemic brain injury may still be pre- ventable. However, this invasive technique yields metabolic information only locally, resulting in a detection failure of at least 20% (27). The difficulty of DCI is in the incomplete understanding of its pathophysiology and lack of hard diagnostic criteria. Currently, two definitions are used to identify patients with DCI: clinical and radiological DCI (4). Even though adher- ence to one definition yields a different selection of patients than the other, both are strongly related to poor clinical outcome at follow-up, therefore both seem to be relevant (33-35). Differences in the two definitions are clearly observed in this review, too, as 36 of 89 biomarkers studied for both definitions showed dissimilar results. The clinical definition seems to be the most relevant, given that early detection and correction of altered biomarkers may prevent the onset of clinical deficits. However, not all patients are neurologically assessable, therefore in comatose patients we currently rely on the radiological definition for DCI diagnosis. Considering that radiological appearance of ischemia or infarction is not immediately visible, once radiological DCI is diagnosed the treatment window for ischemic disease has often elapsed. Diagnosing radiological DCI based on biomarkers may lead to earlier detection of radiological DCI, perhaps even within the treatment window. Limitations of this review are the heterogeneity in defini- tions used for DCI in literature, which complicates the inclu- sion of relevant studies, the methodological heterogeneity among included studies, which has resulted in a largely narra- tive review with a suboptimal method used for classification of biomarkers, and a high proportion of poor quality studies. The quality of studies was not implemented in our method used for classification. As a result, even though the results of studies of validated biomarkers have been reproduced by a second group, some of the selected validated markers rely on studies with relatively poor quality (e.g., ADAMTS13, vWF, and LPR). Strengths of this review are the strict use of the most up-to-date definition of DCI and the inclusion of all types of laboratory markers in blood as well as CSF. Conclusion In this review, seven biomarkers of clinical DCI (haptoglo- bin polymorphisms 2-1 and 2-2, ADAMTS13, NLR, P-selectin, and vWF in blood and F2-isoprostane in urine) and one bio- marker of radiological DCI (LPR in MD) were selected as the most promising biomarkers of DCI. Future research should focus on determining the exact predictive or diagnostic abili- ties of these biomarkers, and exploring whether these bio- markers could serve as preventive or therapeutic treatment targets for DCI. Disclosures CRediT author statement: Maud A. Tjerkstra conceptualization, methodology, formal analysis, investigation, writing—original draft Homeyra Labib conceptualization, methodology, formal analysis, investigation, writing—review and editing Bert A. Coert writing— review and editing René Spijker conceptualization, methodol- ogy, writing—review and editing Jonathan M. 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