A method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm as well as marker substances and the use thereof as a screening test

Abstract

A method for the in vitro diagnosis of the risk of delayed cerebral ischemia (DCI) following subarachnoid hemorrhage from a ruptured aneurysm (aSAH) which includes the following steps: collection of a blood sample from the patient; quantitative determination of 8-iso-prostaglandin F2α (F2-IsoP) concentration in plasma; comparison of the concentration thereof in plasma with the reference value; collection of at least one further blood sample; quantitative determination of the concentration of endothelial nitric oxide synthase (eNOS) and / or nicotinamide adenine dinucleotide phosphate (NADPH) in plasma; comparison of the concentration thereof in plasma with the reference value; analysis of the obtained results and establishing diagnosis. A marker substance for the in vitro diagnosis of the risk of occurrence of DCI selected from 8-iso-prostaglandin F2α (F2-IsoP), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) and combinations thereof and the use thereof as a screening test for the in vitro diagnosis of DCI following aSAH.

Description

A method for in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm as well as marker substances and the use thereof as a screening testTechnical FieldThis disclosure concerns in general the field of medical diagnostics in terms of delayed cerebral ischemia ( DCI ) using immunoenzyme assays . More precisely, it concerns a method for the in vi tro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm and marker substances for the in vi tro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage as well as the use of combinations of the substances as a screening test .Background ArtThe pathophysiology of delayed cerebral ischemia ( DCI ) remains unknown . The most common cause of the occurrence of said condition is hemorrhage , including subarachnoid hemorrhage , for example occurring as a result of rupture of a cerebral aneurysm .Cerebral aneurysm ( also known as intracranial aneurysm, cerebrovascular aneurysm) is a disease of cerebral arteries . It forms when one of the layers constituting an artery is locally weakened . Blood flowing under pressure forces the weakened fragment of the artery toward the outside and forms bulging . The bulging may increase in si ze or rupture over time .Aneurysms typically form in the sites of arterial bi furcation into distal branches or in the takeof f sites of speci fic arteries . Most aneurysms ( approx . 88 % ) mani fest through subarachnoid hemorrhage ( SAH) , 8 % results in symptoms of brain tumor and 4 % are detected incidentally, e . g . during non-invasive cerebral imaging performed due to other causes .Delayed cerebral ischemia ( DCI ) is a complication of subarachnoid hemorrhage from a ruptured aneurysm ( aSAH, aneurysmal subarachnoid hemorrhage ) caused by aneurysm rupture and it remains the principal cause of mortality and disability in patients with a history of subarachnoid hemorrhage from a ruptured aneurysm .A review paper by U . Fdrstermann and T . Mtinzel "Endothelial nitric oxide synthase in vascular disease : from marvel to menace"published in Circulation 2006, 113(13) , pp . 1708-1714 reports the role of oxidative stress, free radicals and tetrahydrobiopterin (BH4) , a nitric oxide synthesis cofactor, in the normal function of vascular endothelium. Absence of nitric oxide results in vasoconstriction. The effect has been reported in animal models. The document does not disclose how oxidative stress and free radical concentrations may be used for the development of a diagnostic method of delayed cerebral ischemia (DCI) .A paper by K. Wisniewski, M. Poppda, B. Price et al. "Glucose- 6-phosphate dehydrogenase and 8-1 so-prostaglandin F2a as potential predictors of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage" published in J. Neurosurg. 2023, 139(3) , pp . 698-707 underscores the role of 8-iso-prostaglandin F2a (F2- IsoP) and glucose- 6-phosphate dehydrogenase (G6PD) as well as the severity of the clinical status based on the Hunt-Hess scale and the amount of blood following subarachnoid hemorrhage from a ruptured aneurysm (aSAH) based on the modified Fisher scale as markers for the occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm. The report integrates objective laboratory markers with subjective clinical markers. The markers are statistically significant only when analyzed together. Their separate prognostic value is low. The paper does not disclose the role of endothelial nitric oxide synthase (eNOS) or nicotinamide adenine dinucleotide phosphate (NADPH) in the diagnosis of delayed cerebral ischemia.S. S. Ramesh, A. Prasanthi, D. I. Bhat et al. in the paper: "Correlation between plasma total nitric oxide levels and cerebral vasospasm and clinical outcome in patients with aneurysmal subarachnoid hemorrhage in Indian population" published in Journal of Neurosciences in Rural Practice 2014, 5(S1) , pp . S22-S27 disclosed a positive correlation of a low nitric oxide concentration with occurrence of delayed cerebral ischemia. In addition, a positive correlation between low NO concentrations and poorer therapeutic outcomes was found. The paper does not specify any details that could result in the development of a diagnostic method based on the disclosures it contains.In the publication of U.S. Patent Application US 2008 / 0227776 Al (VANDERBILT UNIV.) of 2008-09-18, J. A. Oates, L. J. Roberts, N. A. Porter et al. suggested the use of measuringthe content of F2-isoprostanes (F2-IsoP) in cerebrospinal fluid as a biomarker of lipid peroxidation caused by subarachnoid hemorrhage. The authors of the paper measured the concentration of F2-isoprostanes (F2-IsoP) in cerebrospinal fluid in patients with subarachnoid hemorrhage. The concentration of F2-isoprostanes (F2- IsoP) was significantly increased in subarachnoid hemorrhage, and the rate of the increase was a function of the severity of subarachnoid bleeding (on the Fisher scale) and whether the hemorrhage resulted in a vegetative state. The highest concentration was reported on day seven, that is, during delayed vasoconstriction. The paper did not mention the role of nicotinamide adenine dinucleotide phosphate (NADPH) or endothelial nitric oxide synthase (eNOS) and there were no practical suggestions that could be used for the development of a practical and simple test based on ELISA. In addition, the authors did not test the content of F2-isoprostanes (F2-IsoP) in the patient's blood .A review by S. Batista, J. E. Bocanegra-Becerra, B. Claassen et al. "Biomarkers in aneurysmal subarachnoid hemorrhage: A short review" published in World Neurosurgery : X 2023, 19, 100205 suggested potential markers of delayed cerebral ischemia. No substances of the present disclosure are listed as potential markers. In addition, the paper does not mention the aspect of potential synergy effects between 8-1 so-prostaglandin F2c< (F2- IsoP) , endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) .Summary of InventionTechnical ProblemAs shown above, the pathophysiology of delayed cerebral ischemia (DCI) remains unknown. Because no suitable diagnostic tools are available, delayed diagnosis and cessation of treatment are more likely. The currently used criteria are subjective and the proposed laboratory tests are of low effectiveness.The problem to be solved was to provide an alternative quantitative method for in vitro diagnosis which would be more objective, more rapidly executed and more convenient for retesting, when needed, in a laboratory without contact with the patient. Designing such a diagnostic method first of requiressolving the problem of finding relationships that could be used to design a biochemical marker and, as a consequence , also a screening test to enable the detection of starting delayed cerebral ischemia before neurological symptoms occur . Availability of such a test would enable therapeutic therapy to be initiated sooner and monitoring of the course of delayed cerebral ischemia, and in the future , it would help in introducing of a speci fic treatment to directly prevent delayed cerebral ischemia .So far, no in vi tro screening tests are available that could be used in clinical practice for the diagnosis of delayed cerebral ischemia following subarachnoid hemorrhage with a comparable ef fect as the diagnostic criteria based on the evaluation of the patient ' s status using the Hunt-Hess scale (W . E . Hunt and R . M . Hess "Surgical risk as related to time of intervention in the repair of intracranial aneurysms" , J. Neurosurg. 1968 , 28 ( 1 ) , pp . 14-20 ) and the modi fied Fisher scale ( J . A. Frontera, J . Claassen, J . M . Schmidt et al . "Prediction of symptomatic vasospasm after subarachnoid hemorrhage : the modi fied Fisher scale" , Neurosurgery 2006, 59 ( 1 ) , pp . 21-27 ) , which is due to the lack of marker substances so far that would provide credible and reliable indications in terms of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage . Therefore , the current diagnostic measures and methods are insuf ficient .The technical problem is therefore also to provide non- invasive biomarkers that would enable credible and reliable evaluation of the patient ' s health status in terms of the risk of occurrence of delayed cerebral i schemia following subarachnoid hemorrhage and would provide patients with a diagnostic test in terms of delayed cerebral ischemia following subarachnoid hemorrhage based on the aforementioned marker substance of the present disclosure .Solution to ProblemIt was surprisingly found that there was a relationship between 8-1 so-prostaglandin F2a( F2- IsoP ) as a biomarker of oxidative stress , endothelial nitric oxide synthase ( eNOS ) and nicotinamide adenine dinucleotide phosphate (NADPH) and the occurrence of delayed cerebral i schemia caused by subarachnoid hemorrhage . The relationship is not a simple sum of the threeeffects, but it shows synergy of action. The mechanism of action is still not completely known.The problem presented above is solved by providing the method for the in vitro diagnosis having features of Claim 1 and by providing the marker substances having features of Claim 8 as well as providing the use having features of Claim 10 of the marker substances defined in Claims 8 to 9.The preferred variants of the method of the invention are defined in dependent Claims 2 to 7, the preferred variants of the marker substances of the invention are defined in Claim 9, while the preferred variants of the use of the invention are defined in Claims 11 to 12.The subject matter of the invention is the method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm which includes the following steps:- collection of a first blood sample;- quantitative determination of the concentration of 8-iso- prostaglandin F2a(F2-IsoP) in the plasma of the first blood sample and comparison of the determined concentration value of 8-1 so-prostaglandin F2a (F2-IsoP) in the plasma of the first blood sample with a predefined reference value for the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) , wherein if the determined value is higher than the predefined reference value for the concentration of 8-1 so-prostaglandin F2a(F2- IsoP) , the individual is in the group of patients with an increased risk of the development of delayed cerebral ischemia;- collection of at least one further blood sample;- quantitative determination of a) the concentration of endothelial nitric oxide synthase (eNOS) in the plasma of the at least one further blood sample and comparison of the determined concentration value of endothelial nitric oxide synthase (eNOS) in the plasma of the at least one further blood sample with a predefined reference value for the concentration of endothelial nitric oxide synthase (eNOS) , wherein if the determined value is higher than the predefined reference value for the concentration of endothelial nitric oxide synthase (eNOS) ,the individual is in the group of patients with an increased risk of the development of delayed cerebral ischemia ; and / or b ) the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) in the plasma of the at least one further blood sample and comparison of the determined concentration value of nicotinamide adenine dinucleotide phosphate (NADPH) in the plasma of the at least one further blood sample with a predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , wherein i f the determined value is lower than the predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , the individual is in the group of patients with an increased risk of the development of delayed cerebral ischemia ; and- analyzing the obtained results and establishing diagnosis .Wherein in the method for the in vi tro diagnosis of the risk of occurrence of delayed cerebral ischemia of the invention, the collection of the first blood sample may be preferably performed after at least 8 hours of fasting on day two after the occurrence of hemorrhage .Wherein further preferably in the method for the in vi tro diagnosis of the risk of occurrence of delayed cerebral ischemia of the invention, the collection o f the at least one further blood sample may be preferably performed after at least 8 hours of fasting on day six after the occurrence of hemorrhage .Preferably, the method for the in vi tro diagnosi s of the risk of occurrence of delayed cerebral ischemia of any of the preferred embodiments of the invention may further include the following steps each time after the collection of the blood s amp les :- centri fugation of the blood sample to remove solid particles and to obtain plasma ;- trans ferring the plasma into a cryovial and freezing in liquid nitrogen;- trans ferring the plasma from liquid nitrogen to a low- temperature freezer and storing the plasma until further analysis at a temperature in the range of between -100 ° Cand -30 ° C, preferably at a temperature in the range of between -84 ° C and -76 ° C, further preferably at a temperature of -80 ° C ;- bringing the plasma to room temperature and diluting the plasma .Preferably, the method for the in vi tro diagnosi s of the risk of occurrence of delayed cerebral ischemia of any of the preferred embodiments of the invention may further include a step of the evaluation of the patient ' s clinical status according to the criteria of the Hunt-Hess scale .The method for the in vi tro diagnosis of the risk of occurrence of delayed cerebral ischemia of any of the preferred embodiments of the invention may further include a step of the evaluation of the quantity of intracranial blood using computed tomography according to the criteria of the modi fied Fisher scale .In addition, the non-invasive biomarker of the invention is a marker substance for the in vi tro diagnosis of the risk of occurrence of delayed cerebral ischemia, selected from a group including 8-1 so-prostaglandin F2a ( F2- IsoP ) , endothelial nitric oxide synthase ( eNOS ) , nicotinamide adenine dinucleotide phosphate (NADPH) or combinations thereof .Preferably, the marker substance of any of the preferred embodiments of the invention may be a combination of 8 -i so- prostaglandin F2a( F2- I soP ) with endothelial nitric oxide synthase ( eNOS ) and / or nicotinamide adenine dinucleotide phosphate (NADPH) .The subj ect matter of the invention is furthermore the use of the combination of 8-1 so-prostaglandin F2a( F2- I soP ) with endothelial nitric oxide synthase ( eNOS ) and / or nicotinamide adenine dinucleotide phosphate (NADPH) as a screening test for the in vi tro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm .The use of the combination of any of the preferred embodiments of the invention may include the use thereof in combination with clinical tests , preferably with the evaluation of the patient ' s clinical status according to the criteria of the Hunt-Hess scale and / or the evaluation of the quantity of intracranial blood using computed tomography according to the criteria of the modi fied Fisher scale .It is obvious for one skilled in the art that all features preferred according to any of the preferred embodiments of the invention are optional features and they may be present or not in respective preferred embodiments of the invention and they may be combined as needed irrespective of one another, unless it violates the laws of physics and when it is technically feasible .Advantageous Ef fects of InventionSubarachnoid hemorrhage from a ruptured aneurysm ( aSAH) is a severe disease that involves signi ficant morbidity and mortality . Even though initial bleeding is frequently lethal to patients , the ones who survive are still at risk of li fethreatening complications . Delayed cerebral ischemia ( DCI ) af fects almost one-third of patients with subarachnoid hemorrhage from a ruptured aneurysm . It is characteri zed by occurrence of a new neurological deficit or a decrease in the patient ' s consciousness level by at least 2 points on the Glasgow Coma Scale ( GCS ) with no other causes that could account for the worsening of the patient ' s status . Delayed cerebral ischemia is a signi ficant clinical risk, because untreated cases may result in permanent neurological disorders and, in certain cases , even the patient ' s death . However, the exact pathomechanism of delayed cerebral ischemia remains unknown .A number of theories have been proposed to explain the occurrence of delayed cerebral ischemia, related to various aspects , such as microcirculation disorders , microthrombosis , inflammation, blood-brain barrier disorders , electrolyte imbalance and cerebral autoregulation disorders . According to one of the convincing proposals (presented for example in the paper of G . Pradilla, T . Garzon-Muvdi , J . J . Ruzevick et al . "Systemic L- citrulline prevents cerebral vasospasm in haptoglobin 2 -2 transgenic mice after subarachnoid hemorrhage" published in Neurosurgery 2012 , 70, pp . 747-757 ) , extravasated blood results in the development of local inflammation and increased oxidative stress . The increased production of reactive oxygen species , related to oxidative stress , controls redox signaling . The signaling, in turn, regulates vascular tone through various mechanisms known as "redox switches" . Disruptions of the control lead to endothelial dys function and vasoconstriction . Cellular balance is maintained owing to the dynamic cooperation of processesthat involve both the production and removal of reactive oxygen species. The key antioxidizing systems, such as the glutathione system, catalase (CAT) , superoxide dismutase (SOD) and thioredoxin (Trx) , are the integral elements of the balance. The normal function of the systems depends on the availability of nicotinamide adenine dinucleotide phosphate (NADPH) (cf. for example the paper of E. Pigeolet, P. Corbisier, A. Houbion et al. "Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals" published in Mechanisms of Ageing and Development 1990, 51 (3) , pp . 283-297) . Nicotinamide adenine dinucleotide phosphate (NADPH) is also necessary for the normal function of endothelial nitric oxide synthase (eNOS) . Endothelial nitric oxide synthase (eNOS) is an enzyme that produces nitric oxide which plays a significant role in vasodilation. The dysregulation of the aforementioned redox switches, in turn, disturbs the normal function of endothelial nitric oxide synthase (eNOS) , which may result in the insufficient production of nitric oxide and, in consequence, in vasoconstriction. The levels of reactive oxygen species have also been correlated with 8-1 so-prostaglandin F2c< (F2-IsoP) and, therefore, it has been postulated that they are oxidative stress factors (for example in the paper of T. Ahola, V. Fellman, I. Kjellmer et al. "Plasma 8-isoprostane is increased in preterm infants who develop bronchopulmonary dysplasia or periventricular leukomalacia" published in Pediatr . Res. 2004, 56, pp . 88-93) .The basis of the invention is the presumption that the disruption of the elimination of reactive oxygen species is an important factor that contributes to the development of delayed cerebral ischemia, and this may be attributed to imperfect antioxidant mechanisms. Considering the reports that suggest that isoprostanes may serve a role of oxidative stress markers and the antioxidant mechanisms are based on the availability of nicotinamide adenine dinucleotide phosphate (NADPH) and they have a direct effect on the concentration of endothelial nitric oxide synthase (eNOS) , the present inventors, through experiments and diligent clinical studies, found a relationship between changing concentrations of nicotinamide adenine dinucleotide phosphate (NADPH) , endothelial nitric oxide synthase (eNOS) and 8-iso- prostaglandin F2a(F2-IsoP) in patients following subarachnoidhemorrhage from a ruptured aneurysm and the risk of occurrence of delayed cerebral ischemia. Based on this, they developed marker substances of the invention that enable the accuracy of the diagnosis of delayed cerebral ischemia to be increased.Brief Description of DrawingsFig. 1. A method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm of the first preferred embodiment of the invention, in which the concentrations of 8-1 so-prostaglandin F2a(F2-IsoP) and endothelial nitric oxide synthase (eNOS) are determined.Fig. 2. A method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm of the further preferred embodiment of the invention, in which the concentrations of 8-1 so-prostaglandin F2a(F2-IsoP) and nicotinamide adenine dinucleotide phosphate (NADPH) are determined .Fig. 3. A method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm of the still further preferred embodiment of the invention, in which the concentrations of 8-1 so-prostaglandin F2a(F2- IsoP) , endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) are determined .Fig. 4. A scheme that presents registration of patients and followup. Detection of the free form of 8-1 so-prostaglandin F2a(F2-IsoP) , endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) .Fig. 5. A linear chart that shows the concentration of the evaluated marker substances in 3 time points (on days two, four and six after the occurrence of hemorrhage) between a group with delayed cerebral ischemia and a group not showing delayed cerebral ischemia: Fig. 5A - 8-iso- prostaglandin F2a (F2-IsoP) ; Fig. 5B - endothelial nitric oxide synthase (eNOS) ; Fig. 50 - nicotinamide adenine dinucleotide phosphate (NADPH) .Fig. 6. A multiple logistic regression model to predict the occurrence of delayed cerebral ischemia: Fig. 6A - receiver operating characteristics (ROC) curves based on stepwise backward logistic regression for the predilection of delayed cerebral ischemia (AUG = 0.990; confidence interval (95%) = 0.974-1.000; p < 0.01) ; Fig. 6B - precise model .Fig. 7. A multiple logistic regression model (only for clinical variables) to predict the occurrence of delayed cerebral ischemia: Fig. 7A - receiver operating characteristics (ROC) curves based on stepwise backward logistic regression (including clinical variables only) for the predilection of delayed cerebral ischemia (AUG = 0.931; confidence interval (95%) = 0.874-0.989; p < 0.01) ; Fig. 7B- precise model.Fig. 8. A multiple logistic regression model (only for laboratory variables) to predict the occurrence of delayed cerebral ischemia: Fig. 8A - receiver operating characteristics (ROC) curves based on stepwise backward logistic regression (including laboratory variables only) for the predilection of delayed cerebral ischemia (AUG = 0.950; confidence interval (95%) = 0.902-0.998; p < 0.01) ; Fig. 8B- precise model.Fig. 9. Relationships for concentration: Fig. 9A - 8-iso- prostaglandin F2a(F2-IsoP) (day two after the occurrence of hemorrhage) ; Fig. 9C - endothelial nitric oxide synthase (eNOS) and Fig. 9E - nicotinamide adenine dinucleotide phosphate (NADPH) (day six after the occurrence of hemorrhage) between a group with delayed cerebral ischemia, without delayed cerebral ischemia and a control group. The relationships for 8-1 so-prostaglandin F2a (F2-ISOP) and endothelial nitric oxide synthase (eNOS) were evaluated using a Kruskal-Wallis test, while those for nicotinamide adenine dinucleotide phosphate (NADPH) were evaluated using an ANOVA test. Because all p values were < 0.001, the results were further validated using post-hoc tests (Dunn and Tukey tests, respectively) ; relevant p values are listed below. The concentrations were further validated as predictors of delayed cerebralischemia using receiver operating characteristics (ROC) curves and by selecting optimal cut-off points using a Youden J index method; Fig. 9B - AUG = 0.822; confidence interval (95%) = 0.718-0.927; p < 0.01; the cut-off point was evaluated using the Youden J index method; Fig. 9D - AUG = 0.826; confidence interval (95%) = 0.726-0.926; p < 0.01 and Fig. 9F - AUG = 0.789; confidence interval (95%) = 0.679-0, 899; p < 0.01.The figures are only an illustration of the invention and they do not restrict its scope.Description of EmbodimentsThe present inventors unexpectedly found that the increase in the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) on day two after the occurrence of subarachnoid hemorrhage and of endothelial nitric oxide synthase (eNOS) on day six after the occurrence of subarachnoid hemorrhage as well as the decrease in the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) on day six after the occurrence of subarachnoid hemorrhage were correlated with the occurrence of delayed cerebral ischemia. The study shows for the first time three marker substances found in plasma that could improve the accuracy of the diagnosis of delayed cerebral ischemia.The present inventors have also found that a large amount of subarachnoid blood (evaluated according to the modified Fisher scale) , smoking and the patient's poor clinical status on admission (evaluated according to the Hunt-Hess scale) in the study cohort are risk factors for delayed cerebral ischemia, which is consistent with predictions based on information known in the art (cf. e.g. : paper by H. Lee, J. J. Perry, S. W. English et al. "Clinical prediction of delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage" published in J. Neurosurg . 2018, pp . 1-8) .Figure 1 shows the first embodiment of the method of the invention. In the method of the first embodiment, a first blood sample A was collected 100 first from a ruptured aneurysm from a patient following subarachnoid hemorrhage. In the plasma of the first blood sample A, the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) was tested 110 quantitatively and the concentration value of 8-1 so-prostaglandin F2a(F2-IsoP) determined in the plasma of the first blood sample A was compared 120 with the predefinedreference value for the concentration of 8-f so-prostaglandin F2a(F2-IsoP) , wherein if the determined value was higher than the predefined reference value for the concentration of 8-iso- prostaglandin F2a(F2-IsoP) , it indicated that the individual was in the group of patients with an increased risk of the development of delayed cerebral ischemia.Subsequently, a further blood sample B was collected 200 from the same patient. In the plasma of the further blood sample B, the concentration of endothelial nitric oxide synthase (eNOS) was tested 210 quantitatively and the concentration value of endothelial nitric oxide synthase (eNOS) determined in the plasma of the further blood sample B was compared 220 with the predefined reference value for the concentration of endothelial nitric oxide synthase (eNOS) , wherein if the determined value was higher than the predefined reference value for the concentration of endothelial nitric oxide synthase (eNOS) , it indicated that the individual was in the group of patients with an increased risk of the development of delayed cerebral ischemia.Finally the obtained results were analyzed 400 and the final diagnosis in terms of the risk of occurrence of delayed cerebral ischemia was established 500.Figure 2 shows the second embodiment of the method of the invention. In the method of the second embodiment, a first blood sample A was collected 100 first from a ruptured aneurysm from a patient following subarachnoid hemorrhage, similarly as in example one. In the plasma of the first blood sample A, the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) was tested 110 quantitatively and the concentration value of 8-f so-prostaglandin F2a(F2-IsoP) determined in the plasma of the first blood sample A was compared 120 with the predefined reference value for the concentration of 8-f so-prostaglandin F2a(F2-IsoP) , wherein if the determined value was higher than the predefined reference value for the concentration of 8-f so-prostaglandin F2a(F2-IsoP) , it indicated that the individual was in the group of patients with an increased risk of the development of delayed cerebral ischemia.Subsequently, a further blood sample C was collected 300. In the plasma of the further blood sample C, the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) was tested 310 quantitatively and the concentration value of nicotinamide adeninedinucleotide phosphate (NADPH) determined in the plasma of the further blood sample C was compared 330 with the predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , wherein i f the determined value was lower than the predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , it indicated that the individual was in the group of patients with an increased risk of the development of delayed cerebral ischemia .Finally the obtained results were analyzed 400 and the final diagnosis in terms of the risk of occurrence of delayed cerebral ischemia was established 500 .Figure 3 shows the third embodiment of the method of the invention . In the method of the third embodiment , a first blood sample A was col lected 100 first from a ruptured aneurysm from a patient following subarachnoid hemorrhage , s imilarly as in examples one and two . In the plasma of the first blood sample A, the concentration of 8 -1 so-prostaglandin F2a( F2- I soP ) was tested 110 quantitatively and the concentration value of 8 -i so- prostaglandin F2a( F2- I soP ) determined in the plasma of the first blood sample A was compared 120 with the predefined reference value for the concentration of 8-1 so-prostaglandin F2a( F2- I soP ) , wherein i f the determined value was higher than the predefined reference value for the concentration of 8 -1 so-prostaglandin F2a( F2- I soP ) , it indicated that the individual was in the group o f patients with an increased risk of the development of delayed cerebral ischemia .Subsequently, a further blood sample B was collected 200 , similarly as in example one . In the plasma of the further blood sample B, the concentration of endothelial nitric oxide synthase ( eNOS ) was tested 210 quantitatively and the concentration value of endothelial nitric oxide synthase ( eNOS ) determined in the plasma of the further blood sample B was compared 220 with the predefined reference value for the concentration of endothelial nitric oxide synthase ( eNOS ) , wherein i f the determined value was higher than the predefined reference value for the concentration of endothelial nitric oxide synthase ( eNOS ) , it indicated that the individual was in the group of patients with an increased risk of the development of delayed cerebral ischemia .Subsequently, a still further blood sample 0 was collected 300 , similarly as in example two . In the plasma of the stillfurther blood sample C, the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) was tested 310 quantitatively and the concentration value of nicotinamide adenine dinucleotide phosphate (NADPH) determined in the plasma of the still further blood sample C was compared 330 with the predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , wherein i f the determined value was lower than the predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , it indicated that the individual was in the group of patients with an increased risk of the development of delayed cerebral ischemia .Finally the obtained results were analyzed 400 and the final diagnosis in terms of the risk of occurrence of delayed cerebral ischemia was established 500 .Exploratory assumptions and theoretical modelIt was found that in the time intervals tested, the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) was decreased in patients with delayed cerebral ischemia ( DCI ) compared to patients without delayed cerebral ischemia . However, it is di f ficult to explain this situation and it is not obvious for one skilled in the art . One of the most frequent enzyme mutations in humans is the mutation of glucose- 6-phosphate dehydrogenase ( G6PD) which af fects approx . 500 million people worldwide . Glucose- 6-phosphate dehydrogenase ( G6PD) plays a maj or role in the antioxidant system through the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) , a substrate in antioxidant reactions . About 200 mutations of glucose- 6- phosphate dehydrogenase ( G6PD) have been reported so far, and in most cases they result in the decreased activity of the enzyme , remaining however asymptomatic for the whole life . Studies in patients following subarachnoid hemorrhage from a ruptured aneurysm diagnosed with delayed cerebral ischemia showed a decreased concentration of glucose- 6-phosphate dehydrogenase ( G6PD) . This may suggest , without being bound by any theory, that certain patients with delayed cerebral ischemia may also have mutations of glucose- 6-phosphate dehydrogenase ( G6PD) . Unfortunately, no data can be found in the literature whether the deficiency of glucose- 6-phosphate dehydrogenase (G6PD) results from consumption, presumably from the increased demand fornicotinamide adenine dinucleotide phosphate (NADPH) following subarachnoid hemorrhage from a ruptured aneurysm or else it results from a genetic defect . Therefore , it is di f ficult to determine whether the deficiency of nicotinamide adenine dinucleotide phosphate (NADPH) results from problems with its production or from excessive demand .Irrespective of the causes of the deficiency of nicotinamide adenine dinucleotide phosphate (NADPH) , without being bound by any theory, it may be presumed that its deficiency may lead to various disorders considered potential factors that contribute to delayed cerebral ischemia . As mentioned earlier, a low concentration of nicotinamide adenine dinucleotide phosphate (NADPH) disturbs the normal function of antioxidant systems . The glutathione system, catalase ( CAT ) , superoxide dismutase ( SOD) and thioredoxin ( Trx ) require nicotinamide adenine dinucleotide phosphate (NADPH) at certain stages of their action for the neutrali zation process of reactive oxygen species .It is known that neurons require much adenosine triphosphate (ATP ) and, therefore , anaerobic glycolysis leads to low neuron activity and ischemic inj ury . The lack of blood during ischemia is the cause of metabolic crisis . This leads to decreased energy and subsequently to the increased concentration of reactive oxygen species . Neurons are not able to maintain a high glycolysis rate . Glucose is trans ferred to the pentose phosphate pathway which is necessary for the production of nicotinamide adenine dinucleotide phosphate (NADPH) and maintenance of antioxidant concentrations . Nicotinamide adenine dinucleotide phosphate (NADPH) , a metabolic product of the pentose phosphate pathway, is a coenzyme and a classic molecule involved in a number of anabolic reactions in cells . The key role of nicotinamide adenine dinucleotide phosphate (NADPH) is to trans fer hydrogen donors and electrons in the biosynthesis of amino acids , lipids and nucleotides . Furthermore , nicotinamide adenine dinucleotide phosphate (NADPH) ensures the required redox potential in antioxidant systems involved in the response to oxidative stress . Nicotinamide adenine dinucleotide phosphate (NADPH) is also involved in the production of adenosine triphosphate (ATP ) and, along with nicotinamide adenine dinucleotide (NAD+) oxidi zed in the respiratory chain, it maintains homeostasis of cellular energy .In the study proposed by Mei Li and Zhi-Peng Zhou (M . Li , Z . -P . Zhou, M . Sun et al . "Reduced nicotinamide adenine dinucleotide phosphate , a pentose phosphate pathway product , might be a novel drug candidate for ischemic stroke" , Stroke 2016, 47 ( 1 ) , pp . 187-195 ) the administration of exogenous nicotinamide adenine dinucleotide phosphate (NADPH) signi ficantly protected neurons against inj ury caused by ischemia / reperfusion and signi ficantly decreased the extent of infarction following stroke , while also signi ficantly decreasing mortality in rodent models . It was also found that the administration of exogenous nicotinamide adenine dinucleotide phosphate (NADPH) increased the concentration of the reduced glutathione form and adenosine triphosphate (ATP ) , which suggests that nicotinamide adenine dinucleotide phosphate (NADPH) may ef fectively cross the blood-brain barrier and cell membranes to provide biological functions .In this study, it was found in the study cohort that the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) was decreased in patients with delayed cerebral ischemia, and the di f ferences in the concentration between the control group and persons without delayed cerebral ischemia were nonsigni ficant . This suggests that redox potential in patients with delayed cerebral ischemia is lower, which causes several other complications . I t may be presumed, without being bound by any theory, that on one hand subarachnoid hemorrhage from a ruptured aneurysm leads to oxidative stress and the occurrence of "redox switches" and on the other hand, due to the decreased number of proton and electron donors in redox reactions , which results in a low concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , it leads to decreased antioxidant capacity . It is not known at the current stage of research what genetic disorders lead to the condition .Adequate redox s ignaling is probably modi fied by reactive oxygen species and it af fects endothelial function through "redox switches" . One of such "redox switches" most likely af fects the phosphorylation of endothelial nitric oxide synthase ( eNOS ) . The direct oxidation of tetrahydrobiopterin (BH4 ) leads to its decreased concentration . Tetrahydrobiopterin (BH4 ) is a cofactor of endothelial nitric oxide synthase ( eNOS ) . Tetrahydrobiopterin (BH4 ) deficiency impairs the conversion of L-arginine ( L-Arg)guanidine nitrogen to nitric oxide , leading to a decreased concentration of nitric oxide and endothelial dysfunction .In addition, without being bound by any theory, it is known that oxidative stress ( in particular peroxides ) inhibits cyclooxygenase ( COX ) , a key enzyme involved in the synthesis of prostaglandins . This condition facilitates the formation of isoprostane . I soprostanes are compounds similar to prostaglandins and formed during the non-enzymatic free radical peroxidation of polyunsaturated fatty acids , especially arachidonic acid . They are considered oxidative stress markers , because their concentration is directly correlated with the concentration of free oxygen species .I soprostanes initiate the stimulation of endothelial and smooth muscle cell proli feration, mitogenesis and increased platelet aggregation .Therefore , not only vasoconstriction, but also excessive platelet aggregation is involved in microthrombosis that leads to microcirculation disorders , loss of cerebral autoregulation and inflammation, which may be found in the complex pathophysiology of delayed cerebral ischemia .The deficiency of nicotinamide adenine dinucleotide phosphate (NADPH) may also directly af fect the function of endothelial nitric oxide synthase ( eNOS ) . As a substrate of endothelial nitric oxide synthase ( eNOS ) , nicotinamide adenine dinucleotide phosphate (NADPH) is necessary for its normal function . In the present invention, elevated concentrations of endothelial nitric oxide synthase ( eNOS ) were observed, probably resulting from the upregulation mechanism, because it was found that the total plasma nitric oxide concentration was signi ficantly decreased in patients with subarachnoid hemorrhage from a ruptured aneurysm . The mechanism suggests that enzyme production increases with a lack of the substrate to increase overall functionality . However, the increase in the concentration of endothelial nitric oxide synthase ( eNOS ) may also result from the excess of reactive oxygen species as confirmed by S . S . Ramesh, A. Prasanthi , D . I . Bhat et al . in the paper : "Correlation between plasma total nitric oxide levels and cerebral vasospasm and clinical outcome in patients with aneurysmal subarachnoid hemorrhage in Indian population" , J. Neurosci . Rural Pract . 2014 , 5 ( S 1 ) , pp . S22-27 . Ithas been found so far that endothelial nitric oxide synthase ( eNOS ) shows a more complex expression pattern after subarachnoid hemorrhage from a ruptured aneurysm, with decreased expression in the endothelium and increased expression in the parenchyma .Even though nitric oxide is most frequently associated with a vasodilatory action, it is noted that a di f ferent mechanism of action of nitric oxide is known . I t has been found in inflammatory neurodegenerative diseases that inflammation activates microglial cells and astrocytes which in turn activate phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase . This leads to an increased concentration of inducible nitric oxide synthase ( iNOS ) , one of the isoforms of nitric oxide synthase (NOS ) . The activation of the factors leads to the apoptosis of nerve cells as a result of peroxynitrite ( 0N00~) production . In addition, the nitric oxide produced by inducible nitric oxide synthase ( iNOS ) increases cell apoptosis by suppressing cytochrome oxidase , which in turn may lead to increased excitotoxicity . Due to the presence of local inflammation following subarachnoid hemorrhage from a ruptured aneurysm, it is not certain whether nitric oxide may have protective action . On one hand, it may improve microcirculation disorders through vasodilation and on the other hand it may increase cytotoxicity .Results of completed testsAs mentioned above it was found in this study in the study cohort that the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) was decreased in patients with delayed cerebral ischemia, and the di f ferences in the concentration between the control group and persons without delayed cerebral ischemia were non-signi f leant .In the study underlying the present invention, di f ferences in the plasma concentration of endothelial nitric oxide synthase ( eNOS ) were found in the study group in which delayed cerebral ischemia occurred compared to the study group without delayed cerebral ischemia and a control group consisting of healthy volunteers .In addition, the concentration of 8 -1 so-prostaglandin F2c< ( F2- I soP ) on day two after the occurrence of hemorrhage in the study underlying the present invention was a statistically signi ficant predictor of delayed cerebral ischemia . A further lackof free radical scavengers showing normal function facilitates the continuation of oxidative inj uries and, as shown above , leads to microthrombosis , myocyte proli feration, platelet aggregation and increased inflammation . Vasoconstriction is most likely an additional ef fect which occurs mainly in patients with delayed cerebral ischemia, but it is not a prerequisite .Considering the current stage of knowledge , it is desirable to have a laboratory biomarker that could help in establishing a correct diagnosis . The use of at least two marker substances simultaneously showing high sensitivity, a low rate of false positive results and a high area under the curve (AUG ) may be a promising diagnostic strategy for delayed cerebral ischemia . A double or triple test for delayed cerebral ischemia signi ficantly increases the likelihood of the detection of delayed cerebral ischemia and will probably have a high potential in terms of clinical ef ficacy .ExamplesMaterials and methodsEthical statementThe study protocol was developed in line with the Declaration of Helsinki and Good Clinical Practice . I t was subsequently approved by the local Ethics Committee (number RNN / 40 / 20 / KE ) . Signed informed consent was obtained from each patient enrolled in the study .PatientsA prospective study was performed . Inclusion and exclusion criteria were defined, as shown in Fig . 4 . Information collected from patients with confirmed subarachnoid hemorrhage from a ruptured aneurysm within the ongoing prospective clinical , biochemical and radiology database was used in the current study .Clinical evaluationRoutine radiology examinations ( chest radiography, resting electrocardiogram) and laboratory tests were performed in every enrolled patient . The intracranial blood value was evaluated using computed tomography ( CT ) and the modi fied Fisher grading scale (mFisher ) was used . An aneurysm was confirmed using computed tomography angiography ( CTA) or digital subtraction angiography(DSA) . The Hunt-Hess scale was used for the evaluation of the patients' clinical status. Therapeutic decisions (microsurgery or endovascular intervention) were made by a multidisciplinary team (neurosurgeon, neuroradiologist and anesthetist) .The suspicion of delayed cerebral ischemia was taken into consideration each time the patient's status was worsening until evidence to the contrary was obtained. Delayed cerebral ischemia was identified when new symptoms of confusion or a decreased consciousness level occurred (at least by 1 point according to the Glasgow Coma Scale) , with or without focal neurological deficits (speech or motor function with duration of h 1 h) , without other causes of the worsening of the neurological status. Subsequently, transcranial Doppler ultrasound (TCD) and digital subtraction angiography (DSA) were performed to detect radiological symptoms of cerebrovascular spasm. A follow-up neurological examination was performed on discharge and in a routine outpatient clinic (1 and 12 months after hospitalization) by a neurosurgeon blinded with respect to the study results. Both the Glasgow Outcome Scale (GOS) and the modified Rankin scale (mRS) were used.Specimen collectionBlood samples (5-10 mL) were collected from patients after at least 8 hours of fasting on day two, four and six after the occurrence of hemorrhage into commercially available tubes with ethylenediaminetetraacetic acid (EDTA) . After collection, the samples were divided into two portions and centrifuged (7000 x g) for 10 minutes to remove any solid particles. After centrifugation, plasma was harvested into cryovials and was frozen in liquid nitrogen. Subsequently, the samples were transferred from liquid nitrogen to a low-temperature freezer and stored at a temperature of -80 °C until further analysis. Single blood samples were collected from volunteers (control group) and subjected to similar processing .The plasma concentration of the free form of 8-iso- prostaglandin F2a(F2-IsoP) was determined using the STAT-8- Isoprostane ELISA Kit from Cayman Chemical , the concentration of human endothelial nitric oxide synthase (eNOS) was determined using the Human NOS3 / eNOS (nitric Oxide Synthase 3rEndothelial) ELISA Kit from Elabscience®, and the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) was determined using theNADP assay Kit from Biorbyt, respectively. Analyses were performed using manufacturers' protocols. Before analysis, the samples were thawed at room temperature and 1:9 dilutions were prepared. All samples were measured in two replicates. A Synergy 2 multimodal reader from BioTek InstrumentsrInc. with its appropriate software was used. Initial analytical tests were performed using 3 plasma samples from 3 different time points and the concentration of 8- i so-prostaglandin F2a(F2-IsoP) , endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) was evaluated. The obtained results were similar and were within 96% to 98%.Statistical analysisStatistical analyses were performed using Stati stica 13.3PI software (StatSoft , INC., Tulsa, OK, USA) . Statistical significance was defined as p < 0.05. The Shapiro-Wilk test was used to evaluate normal distribution. Continuous variables were expressed as means ± standard deviation (SD) or median with interquartile range (IQR: 25-75 percentile) . Relationships between categorical variables were analyzed using the following tests based on the size of the smallest subgroup: (i) exact Fisher test (nmin < 5) , (ii) x2with Yates correction (5 < nmin < 15) and (iii) X2( nin k 15) . Intergroup differences were evaluated using Student's t test for normal distribution and a nonparametric Mann- Whitney U test for the other cases. The data were visualized using receiver operating characteristics (ROC) curves. Optimal cut-off points were determined using the Youden J index. Variables from monofactorial analysis correlated with delayed cerebral ischemia were further analyzed in a multiple regression model. Multivariable analysis used stepwise logistic regression. Values with p < 0.05 were assumed as statistically significant in all analyses .Sample size analysisThe analysis of sample power and size was performed using G*Power software. The study group (60 patients) enables the identification of the effect size (d) of 0.8 with a power of 85% at a = 0.05 using a two-sided parametric Student's t test. The study group was increased by 8% when there were missing data during sample processing or observation. As a result, the final requiredsample size in the cohort was 65 patients (39 without DCI and 26 with DCI) . In addition, the study included 10 control samples selected with respect to age and gender that fulfilled the inclusion criteria.ResultsClinical status of the patientsThe study enrolled 65 (75.58%) patients out of 86 patients whose eligibility criteria were verified (see Fig. 4) .The study group included 34 (52%) women. Aneurysms were most frequently located in the anterior part of the circle of Willis in 58 (89.2%) of the subjects. Delayed cerebral ischemia was identified in 26 patients; delayed cerebral ischemia occurred on average on day six after the occurrence of subarachnoid hemorrhage. The diagnosis of delayed cerebral ischemia was established in a multidisciplinary team, including neurosurgeons and a radiologist.Biomarker concentrationsThe median and mean concentrations of 8-1 so-pros taglandin F2a (F2-ISOP) , endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) in the study group (after hemorrhage occurred on days two, four, six and overall) and in the control group are shown in Table 1. Patients following subarachnoid hemorrhage showed significantly different concentrations of the analyzed compounds compared to the healthy volunteers on all days. In addition, it was not surprising that the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) was the highest on day two after the occurrence of hemorrhage. It may be accounted for, without being bound by any theory, by a high oxidative stress level in the form of extravasated blood in the subarachnoid space. A decrease was reported on day four after the occurrence of hemorrhage compared to the value determined on day two, followed by a gradual increase. A similar tendency was found for endothelial nitric oxide synthase (eNOS) , while the concentration of nicotinamide adenine dinucleotide phosphate (NAPDH) showed an increase on day four after the occurrence of hemorrhage compared to the value on day two and a slight decrease on day six after the occurrence of hemorrhage.* - Mann-Whitney U test; ** - Student’s t testDi f ferences in the concentration of the biomarkers on days two , four and six after the occurrence of hemorrhage between the group with DCI and the group without DCI are shown in Fig . 5A-C . As for endothelial nitric oxide synthase ( eNOS ) in patients with DCI , the concentration was elevated on all days compared to the group without DCI . As for nicotinamide adenine dinucleotide phosphate (NADPH ) in patients with DCI , the concentration on all days decreased, and the di f ference was particularly obvious on day six after the occurrence of hemorrhage .Subsequently, intergroup comparison between the group with DCI and the group without DCI was performed . The results are shown in Table 2 .Key: * - Student’s t test, ** - Mann-Whitney U test; Relationships between categorical variables were analyzed using the following tests based on the size of the smallest subgroup: exact Fisher test (nmm < 5), x2with Yates correction (5 < nmin < 15) and x2(nmin>15).Subsequently, monofactorial analysis was performed, followed by multifactorial analysis (stepwise backward logistic regression) and the relationship between clinical, radiological and laboratory signs and concentrations of 8-1 so-prostaglandin F2a(F2-IsoP) , endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) were evaluated with respect to delayed cerebral ischemia to find the best predictor of the onset of delayed cerebral ischemia. The multifactorial analysis included only the variables that were significant in monofactorial analysis. The results are shown in Fig. 6. It was found that the most important predictors of delayed cerebral ischemia were the concentration of 8-1 so-prostaglandin F2a (F2-IsoP) on day two after the occurrence of hemorrhage, the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) on day six after the occurrence of hemorrhage, score on the modified Fisher scale (mFisher) and smoking. The receiver operating characteristics curve for the prediction of the occurrence of delayed cerebral ischemia based on the multidimensional model had an area under the curve (AUG) = 0.990; confidence interval (95%) = 0.974-1.000; p value < 0.01 (Fig. 6) .The results of the proposed multidimensional model consist of 4 variables. Two of them: score on the modified Fisher scale (mFisher) and smoking are clinical variables. Therefore, stepwise backward logistic regression was performed to find the best clinical markers of delayed cerebral ischemia in the study cohort. The results showed that smoking, score on the modified Fisher scale (mFisher) and score on the Hunt-Hess scale were the most important clinical prognostic factors, wherein the area under the curve (AUG) = 0.931, confidence interval (95%) = 0.874-0, 989; p < 0.01 (Fig. 7) . The results are consistent with the art in this respect (cf. e.g. paper of H. Lee, J. J. Perry, S. W. English et al."Clinical prediction of delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage" published in J. Neurosurg . 2018, pp . 1-8 ) .Subsequently, the model was developed only for laboratory variables. The most important predictors were the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) on day two after the occurrence of hemorrhage, the concentration of endothelial nitric oxide synthase (eNOS) on day six after the occurrence of hemorrhage and the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) on day six after the occurrence of hemorrhage, wherein the area under the curve (AUG) = 0.950; confidence interval (95%) = 0.902-0.998; p < 0.01 (Fig. 8) , and thus the present inventors surprisingly obtained even better results compared to clinical biomarkers .Both regression models, the model for clinical predictors and the model for laboratory predictors, have the area under the curve (AUG) > 0.9, which means that both models can be used separately and independently predict delayed cerebral ischemia. Laboratory biomarkers improve the prediction accuracy of the occurrence of delayed cerebral ischemia and may be additionally preferably used with clinical variables to improve the diagnosis of delayed cerebral ischemia. Therefore, the combination of the presented laboratory variables may improve the accuracy of prediction in terms of the occurrence of delayed cerebral ischemia.Subsequently, the relationship between the concentration of 8-1 so-prostaglandin F2a (F2-IsoP) , endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) was evaluated in terms of the occurrence and control of treatment of delayed cerebral ischemia. It was decided to perform simultaneous analysis on day two after the occurrence of hemorrhage in terms of the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) and on day six after the occurrence of hemorrhage in terms of the concentration of endothelial nitric oxide synthase (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) , because the days were significant in the regression models developed according to the present disclosure. The present inventors showed a statistically significant difference in the concentration of 8- 1 so-prostaglandin F2a(F2-IsoP) on day two after the occurrence of hemorrhage, the concentration of endothelial nitric oxide synthase(eNOS) on day six after the occurrence of hemorrhage and the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) on day six after the occurrence of hemorrhage. The laboratory parameters were related to the DCI group compared to the patients without DCI and the control group (Fig. 9) .Summary of the study and conclusionsDelayed cerebral ischemia occurs in response to the abnormal action of antioxidant mechanisms in an environment of high oxidative stress which is observed following subarachnoid hemorrhage from a ruptured aneurysm, and the values of the following parameters are: with respect to the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) on day two after the occurrence of hemorrhage: sensitivity = 80.77%, specificity = 71.79%, positive predictive value (PPV) = 65.63% and negative predictive value (NPV) = 84.85%; with respect to the concentration of endothelial nitric oxide synthase (eNOS) on day six after the occurrence of hemorrhage: sensitivity = 92.31%, specificity = 66.67%, positive predictive value (PPV) = 64.86% and negative predictive value (NPV) = 92.86%; and with respect to the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) on day six after the occurrence of hemorrhage: sensitivity = 92.31%, specificity = 64.10%, positive predictive value (PPV) = 63.16% and negative predictive value (NPV) = 92.59%.The marker substances may be used as additional biomarkers separately, but they may also be preferably used together, wherein their predictive values increase to the following level: sensitivity = 80.77%, specificity = 89.74, positive predictive value (PPV) = 84% andnegative predictive value (NPV) = 87.50%, and, therefore, the accuracy of the diagnosis of delayed cerebral ischemia can be further improved.Owing to the laboratory markers, it was possible to leverage more objective values than clinical parameters, such as the modified Fisher scale (mFisher) or the Hunt-Hess scale, but owing to the additional preferred use of clinical markers, the statistical model parameters could also be improved to the following level: sensitivity = 92.31%, specificity = 97.44%, positive predictive value (PPV) = 96% and negative predictive value (NPV) = 95%, and therefore, it is possible to reduce hospitalization cost which also has a positive effect on therapeutic decisions. It is noted that as early as on day two after the occurrence of hemorrhage, the high concentration of 8-1 so-prostaglandin F2a(F2-IsoP) indicated high likelihood of the occurrence of delayed cerebral ischemia, and the predictions were subsequently enhanced by the evaluation of the concentration of endothelial nitric oxide synthase (eNOS) and / or, separately, the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) on day six after the occurrence of hemorrhage. The positive predictive values were even better when additional clinical biomarkers were preferably additionally included.Based on the obtained results, the present inventors found that the evaluation of the concentration of 8-1 so-prostaglandin F2a (F2-ISOP) , endothelial nitric oxide synthase (eNOS) , nicotinamide adenine dinucleotide phosphate (NADPH) and combinations thereof had a predictive value for delayed cerebral ischemia in patients following subarachnoid hemorrhage from a ruptured aneurysm and improved the accuracy of the diagnosis of delayed cerebral ischemia. It seems that the evaluation of the indicators in respective patients following subarachnoid hemorrhage from a ruptured aneurysm, before the development of delayed cerebral ischemia, may lead to establishing a correct diagnosis and could in the future help in the understanding of the pathophysiology and refining of the therapeutic strategies ofdelayed cerebral ischemia targeting the key spots in metabolic pathways .Industrial ApplicabilityThis disclosure is in general applicable to the field of medical diagnostics in terms of delayed cerebral ischemia (DCI) .Citation List1. FORSTERMANN U, MUNZEL T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006-04- 04, Vol. 113, No. 13, pages 1708-1714, <doi: 10.1161 / CIRCULATIONAHA.105.602532, PMID: 16585403>.2. WISNIEWSKI K, POPE]DA M, PRICE B, BIENKOWSKI M, FAHLSTROM A, DRUMMOND K, ADAMIDES AA. Glucose- 6-phosphate dehydrogenase and 8-1 so-prostaglandin F2c< as potential predictors of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage. J. Neurosurg. 2023-01-13, Vol. 139, No. 3, pages 698-707, <doi: 10.3171 / 2022 .12. JNS222332, PMID: 36640097>.3. RAMESH SS, PRASANTHI A, BHAT DI, DEVI BI, CRISTOPHER R, PHILIP M. Correlation between plasma total nitric oxide levels and cerebral vasospasm and clinical outcome in patients with aneurysmal subarachnoid hemorrhage in Indian population. J. Neurosci. Rural Pract . 2014 Nov, Vol. 5, No. SI (Suppl. 1) , pages S22-S27, <doi: 10.4103 / 0976-3147.145196, PMID: 25540533, PMCID: PMC4271376>.4. OATES, JA, ROBERTS LJ, PORTER NA, BOUTAUD 0. Inhibitors of hemeprotein-catalyzed lipid peroxidation. U. S. Pat. Appl . Publ. 2008 / 0227776 Al (VANDERBILT UNIV.) 2008-09-18, paragraphs

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[0074] .5. BATISTA S, BOCANEGRA-BECERRA JE, CLAASSEN B, RUBIAO F, RABELONN, FIGUEIREDO EG, OBERMAN DZ . Biomarkers in aneurysmal subarachnoid hemorrhage: A short review. World Neurosurg. X. 2023-04-22, Vol. 19, article 100205, <doi: 10.1016 / j .wnsx.2023.100205, PMID: 37206060, PMCID:PMC10189293> .6. HUNT WE, HESS RM. Surgical risk as related to time of intervention in the repair of intracranial aneurysms. J. Neurosurg. 1968 Jan, Vol. 28, No. 1, pages 14-20, < doi: 10.3171 / jns.1968.28.1.0014, PMID: 5635959>.FRONTERA JA, CLAASSEN J, SCHMIDT JM, WARTENBERG KE, TEMES R, CONNOLLY ES Jr, MACDONALD RL, MAYER SA. Prediction of symptomatic vasospasm after subarachnoid hemorrhage: the modified fisher scale. Neurosurgery. 2006 Jul, Vol. 59, No. 1, pages 21-27, <doi: 10.1227 / 01.neu.0000243277.86222.6c, PMID: 16823296>. PIGEOLET E, CORBISIER P, HOUBION A, LAMBERT D, MICHIELS C, RAES M, ZACHARY MD, REMACLE J. Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals. Meeh. Ageing Dev. 1990-02- 15, Vol. 51, No. 3, pages 283-297, <doi: 10.1016 / 0047- 6374 (90) 90078-t, PMID: 2308398>. AHOLA T, FELLMAN V, KJELLMER I, RAIVIO KO, LAPATTO R. Plasma 8-isoprostane is increased in preterm infants who develop bronchopulmonary dysplasia or periventricular leukomalacia . Pediatr. Res. 2004 Jul, Vol. 56, No. 1, pages 88-93, <doi: 10.1203 / 01. PDR.0000130478.05324.9D, Epub: 2004-05-05, PMID: 15128912>. LEE H, PERRY JJ, ENGLISH SW, ALKHERAYF F, JOSEPH J, NOBILE S, ZHOU LL, LESIUK H, MOULTON R, AGBI C, SINCLAIR J, DOWLATSHAHI D. Clinical prediction of delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage. J. Neurosurg. 2018-06-01, pages 1-8, <doi: 10.3171 / 2018.1. JNS172715, Epub ahead of print, PMID: 29882700>. LI M, ZHOU ZP, SUN M, CAO L, CHEN J, QIN YY, GU JH, HAN F,SHENG R, WU JC, DING Y, QIN ZH. Reduced Nicotinamide Adenine Dinucleotide Phosphate, a Pentose Phosphate Pathway Product, Might Be a Novel Drug Candidate for Ischemic Stroke. Stroke. 2016 Jan, Vol. 47, No. 1, pages 187-195, <doi:10.1161 / STROKEAHA.115.009687, Epub 2015-11-12, PMID: 26564104>. PRADILLA G, GARZON-MUVDI T, RUZEVICK JJ, BENDER M, EDWARDS L, MOMIN EN, THOMPSON RC, TAMARGO RJ. Systemic L-citrulline prevents cerebral vasospasm in haptoglobin 2-2 transgenic mice after subarachnoid hemorrhage. Neurosurgery. 2012 Mar, Vol. 70. No. 3, pages 747-756, <doi: 10.1227 / NEU.0b013e3182363c2f . PMID: 21915076>.

Claims

Claims1. A method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm which includes the following steps:- collection of a first blood sample (A) (100) ;- quantitative determination of the concentration of 8-iso- prostaglandin F2a(F2-IsoP) in the plasma of the first blood sample (A) (110) and comparison of the determined concentration value of 8-1 so-prostaglandin F2a(F2-IsoP) in the plasma of the first blood sample (A) with a predefined reference value for the concentration of 8-iso- prostaglandin F2a (F2-IsoP) , wherein if the determined value is higher than the predefined reference value for the concentration of 8-1 so-prostaglandin F2a(F2-IsoP) , the individual is in the group of patients with an increased risk of the development of delayed cerebral ischemia (120) ;- collection of at least one further blood sample (B, C) (200, 300) ;- quantitative determination of a) the concentration of endothelial nitric oxide synthase(eNOS) in the plasma of at least one further blood sample (B) (210) and comparison of the determined concentration value of endothelial nitric oxide synthase (eNOS) in the plasma of at least one further blood sample (B) with a predefined reference value for the concentration of endothelial nitric oxide synthase (eNOS) , wherein if the determined value is higher than the predefined reference value for the concentration of endothelial nitric oxide synthase (eNOS) , the individual is in the group of patients with an increased risk of the development of delayed cerebral ischemia (220) ; and / or b) the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) in the plasma of at least one further blood sample (0) (310) and comparison of the determined concentration value of nicotinamide adenine dinucleotide phosphate (NADPH) in the plasma of atleast one further blood sample (C) with a predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , wherein if the determined value is lower than the predefined reference value for the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) , the individual is in the group of patients with an increased risk of the development of delayed cerebral ischemia (330) ; and- analyzing the obtained results (400) and establishing diagnosis ( 500 ) .

2. The method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia of Claim 1, wherein:- the collection of the first blood sample (A) (100) is performed after at least 8 hours of fasting on day two after the occurrence of hemorrhage.

3. The method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia of Claim 1 or 2, wherein:- the collection of a least one further blood sample (B, C) (200, 300) is performed after at least 8 hours of fasting on day six after hemorrhage.

4. The method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia of any of Claims 1 to 3, which further includes the following steps :- each time after the collection of blood samples (A, B, C) (100, 200, 300) :- centrifugation of the blood sample (A, B, C) to remove solid particles and to obtain plasma;- transferring the plasma into a cryovial and freezing in liquid nitrogen;- transferring the plasma from liquid nitrogen to a low- temperature freezer and storing the plasma until further analysis at a temperature in the range of between -100 °C and -30 °C;- bringing the plasma to room temperature and diluting the plasma.

5. The method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia of Claim 4, wherein the plasma is stored until further analysis at a temperature in the range of from -84 °C to -76 °C.

6. The method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia of any of the preceding Claims, which further includes:- evaluation of the patient's clinical status according to the Hunt-Hess scale criteria.

7. The method for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia of any of the preceding Claims, which further includes:- evaluation of the quantity of intracranial blood using computed tomography according to the criteria of the modified Fisher scale.

8. A marker substance for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia, selected from a group including: 8-1 so-prostaglandin F2a(F2-IsoP) , endothelial nitric oxide synthase (eNOS) , nicotinamide adenine dinucleotide phosphate (NADPH) or combinations thereof.

9. The marker substance of Claim 8, wherein the marker substance is a combination of 8-1 so-prostaglandin F2a(F2-IsoP) with endothelial nitric oxide synthase (eNOS) and / or nicotinamide adenine dinucleotide phosphate (NADPH) .

10. A use of a combination of 8-1 so-prostaglandin F2a(F2-IsoP) with endothelial nitric oxide synthase (eNOS) and / or nicotinamide adenine dinucleotide phosphate (NADPH) as a screening test for the in vitro diagnosis of the risk of occurrence of delayed cerebral ischemia following subarachnoid hemorrhage from a ruptured aneurysm.

11. The use of Claim 10, wherein said combination is used in conjunction with at least one clinical test.

12. The use of Claim 11, wherein said combination is used in conjunction with the evaluation of the patient's clinicalstatus according to the criteria of the Hunt-Hess scale and / or the evaluation of the quantity of intracranial blood using computed tomography according to the criteria of the modi fied Fisher scale .