Method for evaluating postoperative delirium possibility of surgical patient

Abstract

Described herein are methods comprising using measurements of perioperative plasma concentrations of Tau-PT217 and / or Tau-PT181 to predict which subjects are likely to develop postoperative delirium, and thereby determine which patients are most likely to benefit from intervention to reduce the risk of postoperative delirium or reduce the severity or consequence of postoperative delirium.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 306,126, filed on February 3, 2022. The entire contents of the foregoing are incorporated herein by reference.

[0003] Federally sponsored research or development

[0004] This invention was made with U.S. Government support under Grant Nos. HD098754, AG062509, and AG070761 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

[0005] Described herein are methods that include using measurements of perioperative plasma concentrations of Tau-PT217 and / or Tau-PT181 to predict which subjects are likely to develop postoperative delirium, and thereby determine which patients are most likely to benefit from intervention to reduce the risk of postoperative delirium or lessen the severity or consequences of postoperative delirium. Background Art

[0006] Postoperative delirium (POD) is one of the most common postoperative complications in elderly patients. 1 , and nosocomial complications 2 , prolonged hospital stay 3 , increased odds of institutional discharge 4,5 , increased incidence 5-8 and increased mortality 9,10 POD can manifest as a hyperactive delirium, where the subject is restless, agitated, hallucinating, and delusional, or a hypoactive delirium, characterized by decreased movement, poor speech, and unresponsiveness. POD usually peaks one to three days after surgery. 47 Because POD can (and often does) occur in subjects without obvious pre-existing delirium or dementia, it is difficult to identify which subjects are likely to experience POD. Postoperative delirium costs the U.S. $32.9 billion in health care costs each year 11 . Summary of the invention

[0007] As shown herein, preoperative plasma concentrations of Tau-PT217 and Tau-PT181 were associated with postoperative delirium, with Tau-PT217 being a stronger indicator of postoperative delirium than Tau-PT181. Thus, measurement of perioperative plasma concentrations of Tau-PT217 and / or Tau-PT181 may be used to predict which subjects are likely to develop postoperative delirium, thereby identifying which patients are most likely to benefit from interventions to reduce the risk or mitigate the severity or consequences of postoperative delirium.

[0008] Provided herein is a method for determining the risk of postoperative delirium (POD) in a subject, preferably, wherein the subject is a human over 60 years of age or 65 years of age. The method comprises optionally obtaining a sample comprising plasma from the subject; determining the level of a Tau isoform phosphorylated at threonine 217 (Tau-PT217) and / or a Tau isoform phosphorylated at threonine 181 (Tau-PT181) in the sample; comparing the level of Tau-PT217 and / or Tau-PT181 in the sample with a reference level of Tau-PT217 and / or Tau-PT181; and determining a subject whose level of Tau-PT217 and / or Tau-PT181 is higher than the reference level as being at risk of postoperative delirium (POD).

[0009] In some embodiments, the subject has no clinical signs of Alzheimer's disease (AD), AD-related dementia (ADRD), and delirium.

[0010] In some embodiments, the subject has a preoperative Mini-Mental State Examination (MMSE) score of greater than 24 points.

[0011] In some embodiments, determining the level of Tau-PT217 and / or Tau-PT181 in a sample comprises using an ultrasensitive detection method. In some embodiments, the ultrasensitive detection method comprises nanoneedle-based assay, single-molecule arrays (SIMOA); Molecular On-bead Signal Amplification for Individual Counting (MOSAIC); Meso Scale Discovery (MSD); Single-Molecule Counting (SMC); Nucleic acid linked immune-sandwich assay (NULISA); LUMINEX; SOMA scan detection (SOMAscan Assays); Mass spectrometry (e.g., MALDI-MS) and / or mass spectrometry flow cytometry (e.g., CyTOF).

[0012] Also provided herein is a method for reducing the risk of postoperative delirium (POD) in a subject. The method includes optionally obtaining a sample comprising plasma from a subject; determining the level of a Tau isoform (Tau-PT217) phosphorylated at threonine 217 and / or a Tau isoform (Tau-PT181) phosphorylated at threonine 181 in the sample; comparing the level of Tau-PT217 and / or Tau-PT181 in the sample with a reference level of Tau-PT217 and / or Tau-PT181; and determining a subject whose level of Tau-PT217 and / or Tau-PT181 is higher than the reference level as being at risk of postoperative delirium (POD); and intervening to reduce the risk of POD in the subject.

[0013] In some embodiments, the subject does not have clinical signs of Alzheimer's disease (AD), AD-related dementia (ADRD), and delirium.

[0014] In some embodiments, the subject has a preoperative Mini-Mental State Examination (MMSE) score of greater than 24 points.

[0015] In some embodiments, determining the level of Tau-PT217 and / or Tau-PT181 in a sample comprises using an ultrasensitive detection method. In some embodiments, the ultrasensitive detection method comprises nanoneedle-based detection, single molecule array (SIMOA); molecular on-bead signal amplification for single counting (MOSAIC); mesoscale discovery (MSD); single molecule counting (SMC); nucleic acid linked immunosandwich detection (NULISA); LUMINEX; SOMA scanning detection; mass spectrometry (e.g., MALDI-MS) and / or mass cytometry (e.g., CyTOF).

[0016] In some embodiments, intervention includes avoiding the use of antihistamines, anticholinergics, tricyclic antidepressants, benzodiazepines, benzodiazepines, muscle relaxants, meperidine, gabapentinoids, and scopolamine; and avoidance of perioperative polypharmacy.

[0017] In some embodiments, the intervention comprises administration of a prophylactic low-dose antipsychotic medication, optionally olanzapine, quetiapine, or risperidone.

[0018] In some embodiments, the intervention comprises sedation with an alpha-2 agonist (optionally clonidine or dexmedotomidine).

[0019] In some embodiments, the intervention comprises administering ketamine during induction of anesthesia; using regional and neuraxial anaesthesia; xenon anesthesia; and / or administering dexmedetomidine.

[0020] In some embodiments, the intervention includes preoperative and postoperative behavioral interventions and additional nursing care measures, optionally following perioperative care based on comprehensive geriatric assessment (CGA), the ABCDE bundle, or the Hospital Elder Life Program (HELP) or modified HELP.

[0021] In some embodiments, behavioral interventions and additional care measures include intentionally guiding the patient to understand the location, time, and reason for hospitalization; encouraging at least daily walking activities; staying hydrated (avoiding prolonged (>6 hours) fasting from fluids); avoiding sleep deprivation; and reminding the patient to use glasses and hearing aids when appropriate.

[0022] In some embodiments, the intervention comprises a pharmacological intervention, optionally selected from pre- and postoperative pain management (optionally using low opioid-sparing analgesia); administration of NSAIDs and paracetamol / acetaminophen; administration of melatonin receptor agonists such as melatonin and ramelteon; and administration of synthetic corticosteroids such as dexamethasone.

[0023] The present invention also describes a method for assessing a patient's risk of postoperative delirium, comprising (a) drawing a blood sample from the patient before surgery; and (b) measuring the preoperative amount of Tau-PT217 and / or Tau-PT181 in plasma, wherein the higher the amount of Tau-PT217 or TauPT181 or both in plasma, the more likely the patient is to have postoperative delirium; and (c) in patients with possible postoperative delirium, administering appropriate postoperative treatment to the patient. Identified high-risk patients will have specific postoperative delirium interventions, including the HELP project, ABCDE bundled treatment, etc.

[0024] In some embodiments, if the blood concentration of Tau-PT217 or Tau-PT181 is high (Tau-PT217 > 75th percentile arbitrary units [au] or 2.5 pg / ml 15 or Tau-PT181>75th percentile au or 8pg / ml 16 ), the patient may have postoperative delirium. In some embodiments, the measuring step is performed using a nanoneedle array as described herein.

[0025] The present invention also provides a method for determining whether a patient suffers from postoperative delirium after surgery, comprising: (a) drawing a blood sample from the patient after surgery; and (b) measuring the amount of Tau-PT217 and / or Tau-PT181 in plasma, wherein a higher amount of Tau-PT217 or TauPT181 or both in plasma indicates that the patient has delirium, in which case the patient should be treated with appropriate intervention.

[0026] In some embodiments, the appropriate intervention or post-operative treatment is selected from HELP, the ABCDE bundle, or a pharmacological intervention or treatment.

[0027] Also provided herein are any and all compositions, articles of manufacture, methods, and uses disclosed and / or described herein.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. The entire contents of all publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference. In the event of a conflict, the present specification (including definitions) shall prevail.

[0029] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1. Flowchart. Flowchart shows that 491 participants were screened for the study and 220 were initially recruited. 81 participants were excluded, resulting in 139 participants for the final data analysis.

[0031] Figure 2A-B .Preoperative plasma concentrations of Tau-PT217 and Tau-PT181 differed between participants with postoperative delirium and participants without postoperative delirium. Preoperative plasma concentrations of Tau-PT217 (A) and Tau-PT181 (B) were higher in participants with postoperative delirium (N=18) than in participants without postoperative delirium (N=121). Student's t-test was used to analyze the data presented in Figures 2A and 2B. P values ​​refer to the difference in preoperative plasma concentrations of Tau-PT217 or Tau-PT181 between participants with postoperative delirium and participants without postoperative delirium. Error bars represent standard deviations. Tau-PT217, Tau phosphorylation at threonine 217; Tau-PT181, Tau phosphorylation at threonine 181; au, arbitrary units.

[0032] Figure 3 . Sensitivity, specificity and AUC (area under the curve) ROC (receiver operating characteristic) curves of preoperative plasma concentrations of Tau-PT217 and Tau-PT181 in predicting postoperative delirium. Tau-PT217 reported a higher discriminatory ability than Tau-PT181. This can be observed from the AUC or area under the ROC curve.

[0033] Figure 4A-B . Peptide array validation of the specificity of Tau-PT217 and Tau-PT181 antibodies. A. Scheme of Tau (2N4R) with phosphorylation sites tested in this study. pT: phosphorylated threonine, pS: phosphorylated serine. B. Synthetic peptides (SEQ ID NOs. 1-12, respectively) with different phosphorylation sites tested against the anti-Tau-PT217 antibody or anti-Tau-PT181 antibody used in this study. 100, 50, 25 and 0 ng of peptides were spotted on nitrogen membrane and measured with 0.5 mg / ml anti-Tau-PT217 antibody or anti-Tau-PT181 antibody. Titration signals were observed at specific residues for both antibodies. No cross-reaction signals with other residues were observed on the dot blots.

[0034] Fig. 5A-FComparison of the nanoneedle technique with the ELISA method in human SY5Y cell lysates. AD. Human SH-SY5Y cells were treated with 3% anesthetic sevoflurane for 0, 3, 6, 9, and 12 hours to induce the pathogenic effects of Tau-PT217 and Tau-PT181. Tau-PT217 (A) and Tau-PT181 (B) were measured with ELISA kits using 50 μL sample volume. Tau-PT217 (C) and Tau-PT181 (D) were further diluted 10-fold in cell extraction buffer and measured with the nanoneedle technique using 10 μL sample volume. The dilution was performed to bring the nanoneedle measurements into a similar detection range (relative concentration in arbitrary units) as the plasma samples in the study. E, F. Correlation of the results of ELISA kit and nanoneedle technique measurements of Tau-PT217 (E) and Tau-PT181 (F). The Pearson correlation coefficients between the nanoneedle and ELISA measurements were 0.88 and 0.83 for Tau-PT217 and Tau-PT181, respectively. These data indicate that the nanoneedle and ELISA methods correlate well. Specific implementation plan

[0035] Population study shows strong bidirectional associations between Alzheimer's disease (AD), AD-related dementia (ADRD), and delirium 12 Specifically, patients with underlying ADRD were 2.5 to 4.7 times more likely to develop delirium, and patients with delirium faced a 12.5-fold increased incidence of newly diagnosed ADRD. 4,13-15 However, the potential pathogenic basis for this association remains unclear, and POD may occur in subjects without clinical evidence of AD, ADRD, or delirium.

[0036] Previous studies have shown that higher preoperative cerebrospinal fluid (CSF) Tau / β-amyloid ratios are associated with a higher incidence and severity of postoperative delirium. 16 These results suggest a potential association of the neuropathogenesis of AD with postoperative delirium. However, in our previous study, we only measured Tau, not phosphorylated Tau, and in CSF rather than plasma. Considering the challenges of obtaining CSF in elderly surgical patients and the fact that plasma biomarkers are both effective and low-cost in conducting clinical studies of delirium, we now investigate emerging blood-based biomarkers of AD and their association with delirium.

[0037] Tauopathies are one of the hallmarks of AD neuropathogenesis 17,18 Tau phosphorylation at threonine 217 (Tau-PT217) and 181 (Tau-PT181) are newly identified plasma biomarkers for AD 19-27Specifically, recent clinical studies have shown that plasma Tau-PT181 can distinguish AD from other neurodegenerative diseases 23 Plasma Tau-PT217 levels are elevated in the early preclinical state of AD patients when insoluble Tau aggregates are not yet detectable by Tau positron emission tomography 26 .

[0038] Interleukin-6 28 , C-reactive protein 29 、neurofilament light chain 30 , chitinase-3-like protein 1 31 and metabolites 32 Plasma concentrations of tau have been shown to be associated with postoperative delirium. In addition, recent studies have shown that the change between preoperative plasma tau concentrations and plasma tau concentrations on postoperative day 1 is greater in patients who develop postoperative delirium and is associated with delirium severity. Plasma tau concentrations also predict recovery from postoperative delirium. 33 However, the relationship between preoperative plasma concentrations of Tau-PT217 or Tau-PT181 and postoperative delirium has not been established.

[0039] This prospective observational group study was conducted to determine the association between Tau-PT217 or Tau-PT181 and the presence or severity of postoperative delirium in patients undergoing surgery under general or spinal anesthesia. It was hypothesized that elevated preoperative plasma concentrations of Tau-PT217 and Tau-PT181 were associated with increased presence and severity of postoperative delirium in patients. The results presented herein revealed that patients with higher preoperative plasma concentrations of Tau-PT217 or Tau-PT181 were more likely to experience delirium and had higher severity of postoperative delirium. These data suggest that Tau phosphorylation, as part of the neuropathogenesis of AD, at least partially contributes to the occurrence of postoperative delirium, and that Tau-based plasma proteins can serve as risk biomarkers for postoperative delirium in patients.

[0040] Increasing evidence suggests that plasma Tau-PT217 and Tau-PT181 are newly identified plasma biomarkers for AD 19-27,38 Previous studies have shown that plasma Tau-PT181 concentrations can distinguish between amyloid β-positive MCI and AD patients (highest levels), Aβ-positive cognitively intact elderly and MCI patients (intermediate levels), and Aβ-negative young people and cognitively intact elderly (lowest levels). 23Furthermore, plasma Tau-PT181 concentrations differentiated AD dementia from frontotemporal dementia, vascular dementia, progressive supranuclear palsy, corticobasal syndrome, Parkinson's disease, or multiple system atrophy. 23 Additional studies have shown that changes in plasma levels of Tau-PT217 correlate with changes in CSF levels of Tau-PT217 24 Associated with the development of AD 25,27 .

[0041] In this study, we found that both Tau-PT217 and Tau-PT181 were associated with the presence and severity of postoperative delirium (Table 2). Our findings suggest that patients with underlying AD neuropathogenesis are more likely to develop delirium. In addition, better preoperative brain health assessment of patients is important.

[0042] In the adjusted model, compared with Tau-PT181 (AUC 0.885; Figure 3 ), plasma Tau-PT217 (AUC 0.969) had an increased ability to distinguish patients with delirium from those without delirium. These results are consistent with previous findings that plasma Tau-PT217 was significantly more accurate than plasma Tau-PT181 in distinguishing neuropathologically defined AD from non-AD, clinical AD dementia from other neurodegenerative diseases, or PSEN1 mutation carriers from non-PSEN1 mutation carriers. 25,27 .

[0043] Previous studies have shown that increases in plasma concentrations of Tau-PT217 occur earlier than changes in positron emission tomography signals of Tau in the cortex 26 . Therefore, the results of this study specifically suggest that preclinical AD neuropathogenesis (such as elevated plasma Tau-PT217) is associated with the development of postoperative delirium. Future studies should evaluate whether the development of postoperative delirium can be a clinical manifestation of preclinical AD. This will become increasingly important as effective treatments for AD become available, especially if they need to be initiated in the preclinical stage to be maximally effective.

[0044] Plasma Tau-PT217 and Tau-PT181 are associated with the development of AD and long-term cognitive impairment 19-27. However, it is not known whether plasma Tau-PT217 and Tau-PT181 can also serve as predictors of postoperative delirium. Therefore, this study focused on evaluating whether preoperative plasma Tau-PT217 and Tau-PT181 are associated with postoperative delirium. In subsequent studies, we will use the established system to determine whether preoperative and postoperative plasma Tau-PT217 and Tau-PT181 are associated with the occurrence of other types of perioperative neurocognitive disorders, including delayed neurocognitive recovery and postoperative neurocognitive impairment.

[0045] The mean Memory Delirium Rating Scale (MDAS) score of the participants in this study was 6.6 (Table 1). Breitbart et al. pointed out that an MDAS score ≥13 indicates the presence of delirium. 36 However, the participants in the study by Breitbart et al. included psychiatry consult patients. 36 On the other hand, Marcantonio et al. showed that the optimal MDAS cutoff for postoperative delirium in participants undergoing hip fracture repair surgery was 5 points. 35 Therefore, the mean MDAS score of 6.6 in this study is reasonable, although on the very mild side.

[0046] Strengths of this study include the application of novel nanoneedle technology to detect phosphorylated Tau in blood samples ( Figure 2A-B , 4A-B and 5A-F). The critical dimension of the nanoneedle sensor is less than 100 nm, i.e., 50-500 times smaller than bead-based detection platforms. Each nanoneedle is a single-molecule biosensor with a capture antibody function, allowing accurate quantification of the analyte by digitally counting the number of nanoneedles with a positive signal. Compared with existing methods, nanoneedles require less sample volume (2-5 μl), have higher sensitivity, and have a lower cost per detection due to its scalable manufacturing process.

[0047] Current data indicate that patients who develop postoperative delirium have higher preoperative plasma concentrations of Tau-PT217 or Tau-PT181 than those who do not develop postoperative delirium. Preoperative plasma concentrations of Tau-PT217 or Tau-PT181 predict the presence and severity of postoperative delirium, with Tau-PT217 being most strongly associated with these results. Therefore, the present invention provides a method for predicting whether a subject is likely to develop postoperative delirium using the preoperative plasma level of one or both of Tau-PT217 and / or Tau-PT181 in the subject. The method may also include recommending or providing intervention to reduce the risk of postoperative delirium or mitigate the severity or consequences of postoperative delirium.

[0048] Methods for determining risk of developing postoperative delirium (POD)

[0049] The methods described herein use the preoperative plasma levels of one or both of Tau-PT217 and / or Tau-PT181 in a subject to predict whether a subject is likely to develop postoperative delirium (POD), e.g., a higher risk of developing POD than a reference subject. The following is an exemplary sequence of a 441-amino acid tau protein (NCBI reference sequence: NP_005901.2, also known as microtubule-associated protein tau isoform 2 (MAPT2)):

[0050]

[0051] The present method can be used for human subjects, such as elderly human subjects aged 60 years or more or 65 years or more, who are planned to undergo surgery, such as surgery requiring general anesthesia or regional anesthesia. In some embodiments, the subject plans to undergo surgery within the next 24-48 hours, 3 days, 4 days, 7 days, one month or longer. Subjects who are "planned" to undergo surgery include subjects who need to undergo non-emergency surgery, emergency surgery, or subjects who are unable to decide on their own surgical plans, including subjects whose medical service providers recommend surgery. In some embodiments, the subject's preoperative cognitive function is normal, for example, the Mini-Mental State Examination (MMSE) score is more than 24 points, or the MMSE score is normal for the individual.

[0052] The method may include obtaining a sample from a subject, evaluating the presence and / or level of Tau-PT217 and / or Tau-PT181 in the sample, and comparing the presence and / or level to one or more references, such as a control reference, which represents normal levels of Tau-PT217 and / or Tau-PT181, such as levels in unaffected subjects; and / or a disease reference, which represents levels of Tau-PT217 and / or Tau-PT181 associated with the risk of developing POD, such as levels in subjects with a risk of developing POD that is higher than the general population or higher than a group of subjects matched by age, sex or other characteristics. Suitable reference values ​​may include at least 1.75 times or 2 times higher than the levels in a control group of subjects who do not develop POD.

[0053] As used herein, the term "sample", when referring to the material to be tested for the presence or absence of a biomarker using the methods of the present invention, includes whole blood, plasma, and serum, among others.

[0054] The method may include isolating and / or purifying Tau-PT217 and / or Tau-PT181 from a sample. Various methods for identifying and / or isolating and / or purifying Tau-PT217 and / or Tau-PT181 from a sample are known in the art. An "isolated" or "purified" biomarker may be substantially free of cells, cellular material, or other contaminants from the source of the biomarker; in some embodiments, a whole blood sample is centrifuged, for example, at 500 g for 10 minutes, to remove cells and cell debris, and the plasma supernatant is collected.

[0055] The presence and / or level of Tau-PT217 and / or Tau-PT181 protein can be assessed using highly sensitive or ultrasensitive protein detection methods known in the art (i.e., detection methods with a limit of detection below 1 picomolar (0.1 femtomolar in 100 μl)). Examples include mesoscale discovery (MSD); single molecule array (SIMOA); droplet digital ELISA (ddELISA) 43 , Molecular On-Bead Signal Amplification for Single Counting (MOSAIC) 44 , single molecule counting (SMC); LUMINEX; SOMA scanning detection; mass spectrometry (e.g., MALDI-MS) and mass spectrometry flow cytometry (e.g., CyTOF) (see, e.g., Cohen and Walt, Chem. Rev. 2019, 119, 293-321), or nanoneedles (see, e.g., WO2022047085, WO2020176793, WO2019051181). The method may include the use of revealing labels such as fluorescent, chemiluminescent, radioactive, and enzymatic or dye molecules that directly or indirectly provide signals. As used herein, the term "label" refers to the coupling (i.e., physical connection) of a detectable substance such as a radioactive agent or fluorophore (e.g., phycoerythrin (PE) or indocyanine (Cy5)) to an antibody or probe, as well as indirect labeling by a probe or antibody (e.g., horseradish peroxidase, HRP) that is reactive with a detectable substance.

[0056] In some embodiments, an enzyme-linked immunosorbent assay (ELISA)-based method can be used, in which a surface such as a bead, a slide, or a well of a microplate is coated with a reagent that binds to Tau-PT217 and / or Tau-PT181, such as an antibody (i.e., a capture antibody). A sample containing or suspected of containing the biomarker is then applied to the surface. After a sufficient time for the antibody-antigen complex to form, the surface is washed to remove the unbound portion, and then a second molecule that can be used to detect the antibody-antigen complex is added, such as a detection antibody that binds to the capture antibody or tau protein, such as a detectable labeled antibody. Similarly, after a sufficient incubation period, the surface is washed to remove any excess unbound molecules, and the presence of the labeled molecule is determined using methods known in the art. In some embodiments, an ELISA method is used, such as SIMOA, MOSAIC, nanoneedle or other ultrasensitive methods. References 53 SIMOA detection of Tau-PT217 or Tau-PT181 is described in. Detectably labeled antibodies may include a tag, such as a label (detectable portion) or a purification portion, such as FLAG, hexahistidine (6-HIS) or hemagglutinin (HA). Examples of detectable substances used as labels include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin, and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0057] Methods for preparing reagents, such as antibodies or antigen-binding fragments thereof, that bind to Tau-PT217 or Tau-PT181 are well known in the art, and antibodies are commercially available or publicly available. For example, anti-Tau-PT217 antibodies can be purchased from Affinity Biosciences, Biorbyt, Bioss Inc., Eli Lilly, GeneTex, Novus Biologicals, Thermo Fisher Scientific, and United States Biological. Anti-Tau-PT181 antibodies can be purchased from Anogen, Antigenix America Inc., BioLegend, Cell Signaling Technology, Creative Biolabs, Creative Diagnostics, dianova GmbH, MyBioSource.com, Thermo Fisher Scientific, and United States Biological. In some embodiments, these antibodies or antigen-binding fragments thereof (such as Fv, Fab, Fab', F(ab')2) can be used in the detection described herein.

[0058] Classical immunoassays monitor signals such as absorbance resulting from the increasing amount of chromogen as the analyte concentration increases. Nanoneedle technology detects the spectral shift of individual nanoneedles resulting from the deposition of additional mass on each nanoneedle to form an antibody-antigen sandwich complex.

[0059] In some embodiments, the capture antibody of Tau-PT217 is ThermoFisher Cat.NO.44-744, or IBA493 (Eli Lilly) or an antigen-binding fragment thereof (such as Fab2), and / or the capture antibody of Tau-PT181 is AT270 (ThermoFisher Cat No.MN1050) or ADx252 (ADx NeuroSciences) or an antigen-binding fragment thereof (such as Fab2). In some embodiments, the detection antibody is an anti-tau antibody, such as LRL or 4G10E2 (Eli Lilly and Company), Dx204 (ADx NeuroSciences) or Tau12 (Sigma Aldrich).

[0060] In some embodiments, mass spectrometry, particularly matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), surface-enhanced laser desorption / ionization mass spectrometry (SELDI-MS) or LC-MS, is useful for detecting Tau-PT217 and / or Tau-PT181 (see, e.g., Barthélemeny et al., Alzheimers Res Ther. 2020 Mar 17; 12(1):26 (quantitative mass spectrometry for detection of Tau-PT217 and / or Tau-PT181); Gobom et al., Mol Neurodegener. 2022 Dec 12; 17(1):81 (LC-MS); U.S. Pat. Nos. 5,118,937; 5,045,694; 5,719,060; 6,225,047).

[0061] In some embodiments, the presence and / or level of Tau-PT217 and / or Tau-PT181 is comparable to the presence and / or level of Tau-PT217 and / or Tau-PT181 in a disease reference, and the subject can be identified as being at risk of developing POD. In some embodiments, the subject has no overt signs or symptoms of dementia or delirium, but the presence and / or level of Tau-PT217 and / or Tau-PT181 is comparable to the presence and / or level of Tau-PT217 and / or Tau-PT181 in a disease reference, and the subject has an increased risk of developing POD. In some embodiments, once a person is determined to have an increased risk of developing POD, treatment can be administered, such as treatments known in the art or as described herein.

[0062] Suitable reference values ​​can be determined using methods known in the art, such as using standard clinical trial methods and statistical analysis. Reference values ​​can have any relevant form. In some cases, the reference includes a predetermined value of a meaningful level of Tau-PT217 and / or Tau-PT181, for example, a control reference level representing a normal level of Tau-PT217 and / or Tau-PT181, for example, a level in a subject who is not at risk of developing POD (e.g., a control level determined from a subject group that does not develop POD), and / or a disease reference representing the level of Tau-PT217 and / or Tau-PT181 associated with a subject group that develops POD.

[0063] The predetermined level can be a single cut-off (threshold) value, such as the median or mean value, or define the upper quartile or lower quartile, tertile or other levels determined as the boundary of the segment of the clinical trial population that is statistically different from other segmentations (segment). It can be a cut-off (or threshold) value range, such as a confidence interval. It can be established according to a comparison group, for example, the association with the risk of onset of a defined group or the presence of a disease is higher or lower than the risk of onset of another defined group or the presence of a disease multiple (for example, about 2 times, 4 times, 8 times, 16 times or more). It can be a range, for example, by grouping the subject population (such as control subjects) on average (or unequally), such as a low-risk group, a medium-risk group and a high-risk group, or divided into quartiles, the lowest quartile is the subject with the lowest risk, and the highest quartile is the subject with the highest risk, or divided into n quartiles (i.e., n regularly spaced intervals), the lowest n quartile is the subject with the lowest risk, and the highest n quartile is the subject with the highest risk.

[0064] In some embodiments, the predetermined level is the level or occurrence in the same subject, eg, at a different time point, such as an earlier time point.

[0065] A subject associated with a predetermined value is often referred to as a reference subject. For example, in some embodiments, a control reference subject will not develop POD.

[0066] A disease reference subject is a subject who has (or has an increased risk of developing) a POD. An increased risk is defined as a risk that is higher than that of subjects in the general population.

[0067] Therefore, in some cases, the level of Tau-PT217 and / or Tau-PT181 of the subject is lower than or equal to the reference level of Tau-PT217 and / or Tau-PT181, indicating that the risk of POD is low or normal. In other cases, the level of Tau-PT217 and / or Tau-PT181 of the subject is higher than or equal to the reference level of Tau-PT217 and / or Tau-PT181, indicating that the risk of POD is increased. In some embodiments, the level of the subject is lower than the reference level by an amount sufficient to distinguish the subject from the control subject, and is optionally statistically significantly lower than the control subject. In some cases, where the level of Tau-PT217 and / or Tau-PT181 of the subject is equal to the reference level of Tau-PT217 and / or Tau-PT181, the "equal" refers to approximately equal (e.g., no statistical difference).

[0068] In some embodiments, the higher the level of Tau-PT217 and / or Tau-PT181 in a subject, the higher the risk of developing more severe POD.

[0069] The predetermined value may depend on the specific subject population selected (e.g., human subjects). For example, an apparently healthy population will have a different "normal" range of Tau-PT217 and / or Tau-PT181 levels compared to a subject population that has, may have, or is at a higher risk of developing the conditions described herein. Therefore, the selected predetermined value may take into account the category to which the subject (e.g., human subject) belongs (e.g., sex, age, health, risk, presence or absence of other diseases). A person of ordinary skill in the art can select appropriate ranges and categories by simply performing routine experiments.

[0070] In characterizing likelihood or risk, a number of predetermined values ​​may be established.

[0071] Intervention

[0072] In some embodiments, once a subject is determined to be at risk for developing POD based on the methods described herein, interventions can be applied. For example, prior to surgery, antihistamines, anticholinergics, tricyclic antidepressants, benzodiazepines, The use of ketamine in the anesthesia induction period may include the use of ketamine infusions, such as 0.5 mg / kg intravenous bolus during anesthesia induction, and the avoidance of perioperative polypharmacy. This may include the administration of preventive medications, including low-dose antipsychotics such as olanzapine, quetiapine, or risperidone. In cases where the subject is sedated, alpha-2 agonists such as clonidine or dexmedetomidine may be used. During surgery, ketamine may also be used during anesthesia infusions such as 0.5 mg / kg intravenously during anesthesia induction. 48 ; regional and neuroaxial anesthesia; xenon anesthesia; and administration of dexmedetomidine to reduce the risk of POD 49 Preoperative and postoperative behavioral interventions and additional care measures, including adherence to comprehensive geriatric assessment (CGA)-based perioperative care, intentional orientation of patients to understand the place, time, and reason for hospitalization; encouragement of at least daily walking activities; staying hydrated (avoiding prolonged (>6 hours) fasting from fluids); avoiding sleep deprivation; reminding patients to use glasses and hearing aids when appropriate; and encouraging family involvement, may be used to reduce the risk of POD in subjects identified as at risk for POD using the methods described herein. 49 For example, you can use the ABCDE bundle 50 or Hospitalized Elderly Living Program (HELP) or Modified HELP 51. See also, e.g., Inouye et al., N Engl J Med. 1999 Mar 4; 340(9): 669-76; Vidán et al., J Am Geriatr Soc. 2009 Nov; 57(11): 2029-36. Pharmacological interventions may include preoperative and postoperative pain management (preferably with low opioid analgesia, e.g., with NSAIDs and paracetamol / acetaminophen); administration of melatonin receptor agonists such as melatonin and ramelteon; and administration of synthetic corticosteroids such as dexamethasone 52 .

[0073] Example

[0074] The present invention is further illustrated in the following examples, which however do not limit the scope of the invention described in the claims.

[0075] Example 1. Preoperative plasma Tau-PT217 and Tau-PT181 are associated with postoperative delirium

[0076] method

[0077] The following materials and methods were used in this example.

[0078] Study Enrollment. This prospective observational study was conducted at Massachusetts General Hospital in Boston, Massachusetts. The Brigham Institutional Review Board at Massachusetts General Hospital approved the study protocol. Patients were included if they were scheduled to undergo elective knee replacement, hip replacement, or laminectomy under general or spinal anesthesia at the study hospital and were 65 years of age or older and proficient in English.

[0079] Subjects were excluded if they had any of the following: (1) a history of neurological and psychiatric disorders, including AD, other forms of dementia, stroke, or psychosis; (2) severe visual or hearing impairment; (3) current smokers; or (4) taking antibiotics within a week of the day of surgery, as disturbances in the gut microbiota may affect brain function. Eligible patients were contacted by the clinical research coordinator during the preoperative clinic. Written informed consent was obtained at enrollment before the start of the study procedures. No major changes in surgical or anesthetic methods have occurred since the start of the study. The manuscript was written in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) standards.

[0080] Anesthesia, surgery, and plasma sample collection. All participants received standardized perioperative care, including standard postoperative pain management (eg, patient-controlled analgesia [PCA] with hydromorphone). The depth of sedation was determined by the attending physician but was not recorded in this study.

[0081] Sample preparation. Five ml of blood was collected from the participants at the time of insertion of the intravenous catheter, before anesthesia and surgery. The blood samples were centrifuged at 500 g for 10 minutes, and the plasma supernatant was collected. The plasma was collected in EDTA tubes and immediately placed on ice. The plasma was then stored in a freezer at -80 °C until the samples were thawed at the time of measurement. All measurements in this study were performed using the same method with a double-blind design.

[0082] Nanoneedle technology: More than 20,000 nanoneedles are integrated on a silicon substrate for the detection of one analyte. Each nanoneedle is a single-molecule biosensor functionalized by an antibody. Since phosphorylated Tau in blood samples is present in low abundance, the binding events of phosphorylated Tau to the nanoneedle sensor array follow Poisson statistics, i.e., one molecule or no molecule is captured on each nanoneedle. This allows accurate quantification of the analyte by digitally counting the number of nanoneedles with positive signals. In this study, we developed sandwich-type Tau-PT217 and Tau-PT181 assays to measure ultra-low concentration protein levels in patient blood samples. The nanoneedle chip was provided by NanoMosaic (Woburn, MA, USA). The nanoneedles were fabricated in an array format with a pitch of 1.8 μm. The diameter of each nanoneedle is less than 100 nm. The nanoneedle surface was modified with 0.5% 3-aminopropyltrimethoxysilane (APTMS) and activated with 2% glutaraldehyde. This allows the antibody to covalently bind to the nanoneedle surface 41 We used capture antibody (anti-Tau46 antibody, Cat# T9450, 1:1000; Sigma-Aldrich, St Louis, MO) at a concentration of 5 μg / ml and incubated it with the chip overnight. We then washed the chip in PBS and incubated it with blocking solution for 1 hour. 5 μL of plasma sample was thawed and diluted 2× with dilution buffer provided by NanoMosaic Inc. The diluted sample was incubated on the chip for 2 hours. After washing, 0.5 μg / ml of biotinylated detection antibody (ThermoFisher 44-744 for Tau-PT217 and ThermoFisher MN1050 for Tau-PT181) was incubated on the chip. Then, 0.5 μg / ml of streptavidin-HRP conjugate was incubated for 30 minutes, and 3,3',5,5'-tetramethylbenzidine (TMB) provided by NanoMosaic was coated on the chip for 15 minutes. In the presence of the sandwich complex, the enzymatic reaction produces an insoluble precipitate on the nanoneedles. The precipitate changes the local refractive index of the nanoneedles and induces a color shift in the intrinsic properties of the nanoneedles. 42 . Tessie was used by NanoMosaic (Woburn, MA, USA) TMNanoneedle detector, using a CMOS color camera in dark field configuration to image the nanoneedles before and after detection. Software provided by NanoMosaic (Woburn, MA, USA) analyzes the color of all nanoneedles and reports the percentage of the number of nanoneedles with color shift, which is expressed as Figure 2A-B The relative concentrations were used as arbitrary units (au) in . We used Tau-PT217 phosphorylation-specific antibody (ThermoFisher 44-744) and Tau-PT181 phosphorylation-specific antibody (ThermoFisher MN1050) as detection antibodies for Tau-PT217 and Tau-PT181. The specificity of the Tau-PT217 and Tau-PT181 phosphorylation-specific antibodies was verified using the peptide array described in the next section.

[0083] Peptide synthesis, purification and analysis: Peptides were synthesized using standard Fmoc solid phase chemistry on an automated robotic peptide synthesizer (Intavis, Model Multipep) (Figure 4B). Phosphorylated amino acids were synthesized using Fmoc-Phospho-Ser(OBzl)-OH and Fmoc-Phospho-Thr(OBzl)-OH. Pre-loaded Wang resin was used for the corresponding C-terminal amino acid. Subsequent amino acids were coupled using an optimized (to generate peptides containing more than 90% of the expected full-length peptide) cycle consisting of removal of Fmoc (deprotection) with 25% piperidine in NMP followed by coupling with Fmoc-AA activated with HBTU / HOBt / DIEA. The resin was washed multiple times with DMF after each deprotection or coupling to remove excess reagents. After peptide assembly and removal of the final Fmoc group, the peptide resin was washed three times with dimethylformamide, dichloromethane, and methanol, respectively, and air dried. The peptide was cleaved from the solid support and deprotected using an odor-free cocktail (TFA / triisopropylsilane / water / DODT; 94 / 2.5 / 2.5 / 1.0 v / v) at room temperature for 2.5 hours. The peptide was precipitated using cold methyl tert-butyl ether (MTBE). The precipitate was washed twice in MTBE, dissolved in a solvent (0.1% trifluoroacetic acid in 30% acetonitrile / 70% water), and then freeze-dried. Purification was performed using preparative reverse phase HPLC. The peptide was characterized by microbore HPLC and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS).

[0084] Validation of Antibody Specificity Using Peptide Arrays: We used peptide arrays to validate the specificity of the antibodies used in this study. Figure 4A-B As shown, 14 amino acid phosphorylated peptides with known phosphorylation sites were synthesized. The sequences of these peptides are as follows; pT indicates phosphorylated tyrosine residues and pS indicates phosphorylated serine:

[0085]

[0086]

[0087] We applied the Bio-Dot system (Bio-Rad, #1706545) to test Tau-PT217 (ThermoFisher44-744) and Tau-PT181 (ThermoFisherMN1050) antibodies against 12 different phosphorylated peptides. First, we pre-wet the nitrocellulose membrane with 100 μl TBS (Tris-buffered saline, Bio-Rad, #1706435) per well and then placed it in the device. Then, we rehydrated the membrane to ensure uniform binding of the designed peptides. The flow valve was adjusted to allow the vacuum chamber to be open to air, and 100 ng, 50 ng, 25 ng and 0 ng of peptides were loaded into the wells respectively. After the peptide solution passed through the membrane thoroughly, we washed the membrane twice with 200 μl TTBS (Tween 20, Tris-buffered saline). Then we added 300 μl of blocking solution to each well. Gravity filtration was allowed to occur until the blocking solution was completely drained from each well, and then we repeated the washing step twice with TTBS solution. 100 μl of anti-Tau-PT217 or anti-Tau-PT181 primary antibody solution (0.5 μg / mL, ThermoFisher #44-744 or ThermoFisher #MN1050) was added to each sample well. After allowing gravity filtration to occur until the antibody solution completely flowed out of the sample well, we then applied vacuum to remove any excess liquid. After washing 3 times, vacuum was used until the wash solution was emptied from the sample well. 100 μl of secondary antibody (rabbit anti-mouse IgG (H+L) secondary antibody, HRP, #61-6520, 1:5000 dilution; Thermo-fisher) was added, drained and washed 3 times. Chemiluminescent solution (Pierce TM ECL Western Blotting Substrate, 32106, Thermo-fisher) and developed with Bio-Rad ChemiDoc TM MP imaging system. As shown in Figure 4B, titration signals of both antibodies to their specific residues were observed. No cross-reaction signals with other residues were observed on the dot blot, supporting the conclusion that the measurement was specific for Tau-PT217 and Tau-PT181.

[0088] Validation of the nanoneedle method with ELISA: The measurements of the nanoneedle technology were compared with the gold standard ELISA in cell line samples. Human SH-SY5Y cells were treated with 3% of the anesthetic sevoflurane for 0, 3, 6, 9, and 12 hours to induce the pathogenic effects of Tau-PT217 and Tau-PT181. Phosphorylated-Tau (Thr181) ELISA kit (Cat#58537, CellSignaling Technology, Danvers, MA) and phosphorylated-Tau (Thr217) ELISA kit (Cat#58672, Cell Signaling Technology) were used to measure the levels of Tau-PT181 and Tau-PT217 in SH-SY5Y cells after treatment. 50 μL of cell lysate samples were loaded into each well on the ELISA plate. The cell lysate was measured 6 times after 0, 3, 6, 9, and 12 hours, respectively. The absorbance was measured at 450 nm using a spectrophotometer (model). The nanoneedle technology was used to measure Tau-PT217 and Tau-PT181 in the same batch of SY5Y cell samples using the method described in the previous section. Compared with the ELISA measurement, the cell samples were further diluted 10× in the cell extraction buffer before loading into the nanoneedle chip. A sample volume of 10 μL was loaded into each well on the nanoneedle chip. This was to ensure that the nanoneedle signal from the current experiment was in the same range as the signal measured in human plasma in this study, so as to validate the nanoneedle technology in the same concentration range. As shown in Figures 5A-D, both ELISA and nanoneedle measurements indicated time-dependent changes in the amount of human Tau-PT217 and Tau-PT181 in SY5Y cells. Specifically, sevoflurane treatment increased the amount of human Tau-PT217 and Tau-PT181 for up to 9 hours after treatment. The decrease in the amount of human Tau-PT217 and Tau-PT181 at 12 hours may be due to cell death after 12 hours of sevoflurane treatment, so fewer cells were collected for measurement. For Tau-PT217 and Tau-PT181, the Pearson correlation coefficients between the nanoneedle and ELISA measurements were 0.88 and 0.83, respectively (Figure 5E-F). These data indicate that the nanoneedle and ELISA methods correlate well. In addition, the nanoneedle technique was able to quantify protein levels with 50× less protein than the ELISA method.

[0089] Reveal - Measurement of Tau-PT217 and Tau-PT181 in plasma by nanoneedles. The main revelation of interest is the measurement of Tau-PT217 and Tau-PT181 from preoperative blood samples. The measurement of phosphorylated Tau was performed in a state of blinded postoperative delirium to avoid bias. Since Tau-PT217 and Tau-PT181 exist in the blood at ultra-low abundance levels that cannot be detected by traditional Western blotting or enzyme-linked immunosorbent assay (ELISA), we developed an in-house phosphorylated Tau detection method using nanoneedle technology to measure preoperative plasma concentrations of Tau-PT217 and Tau-PT181. Since there are currently no recombinant protein standards for Tau-PT217 or Tau-PT181, protein levels are reported in relative units unique to nanoneedle technology (i.e., relative concentrations in arbitrary units). Therefore, the term plasma concentration used in this article refers to the relative concentration of Tau-PT217 or Tau-PT181 in the plasma of participants, rather than the absolute concentration. During the measurement of Tau-PT217 or Tau-PT181, all samples from participants were randomly assigned to different nanoneedle batches. Tau-PT217 and Tau-PT181 were tested in triplicate, with 5ul per well per batch. The average intra-assay coefficients of variation (CV) for plasma Tau-PT217 and Tau-PT181 in the postoperative delirium group were 39.9% and 21.0%, respectively. However, the differences between the plasma Tau-PT217 and Tau-PT181 values ​​obtained by the three measurements were not significant.

[0090] All measurements were above the detection limit of the nanoneedle assay. Dot blot measurements of phosphorylation-specific antibodies against a synthetic peptide array phosphorylated at different residues of the full-length Tau protein confirmed the specificity of the assay ( Figure 4A-B The accuracy of the nanoneedle method was validated by comparing the measurement of Tau-PT217 and Tau-PT181 in cell culture samples using the gold standard ELISA method ( Fig. 5A-F ).

[0091] Results. Participants were interviewed by trained clinical research coordinators on postoperative day 1 and / or day 2. The Confusion Assessment Scale (CAM) is a diagnostic algorithm used to determine the presence or absence of delirium with high reliability. 34,35 . 100 of 139 participants performed CAM on both days, 3 of 18 participants with postoperative delirium and 36 of 121 participants without postoperative delirium performed CAM on one day. Delirium was assessed once daily between 8:00 AM and 12:00 PM using the CAM. The primary outcome was the presence of postoperative delirium defined by CAM presentation on postoperative day 1 or postoperative day 2.

[0092] The secondary outcome was the severity of postoperative delirium, as assessed by the Memory Delirium Rating Scale (MDAS). 35,36 The MDAS score quantifies delirium-related symptoms based on 10 features. Each feature is scored from 0 (best) to 3 (worst symptom), with a maximum score of 30. All patients were evaluated for the MDAS score regardless of whether they met the CAM criteria for delirium that day. In this study, the diagnosis of the presence of delirium was based on the results of the CAM. The MDAS score was used to assess the severity of delirium, independent of the results obtained from the CAM. The postoperative Mini-Mental State Examination (MMSE) was performed as part of the CAM. 34,37 , also used for MDAS calculation on postoperative day 1 and / or day 2 37 .

[0093] Statistical power calculation. We based our estimate on our previous study 16 , calculated and implemented during the study design phase, assuming a two-sided hypothesis test with 80% power and 5% type I error, a sample size of 130 participants would be sufficient to identify a Pearson correlation >0.20 between plasma Tau concentrations and MDAS delirium severity scores.

[0094] Statistical Analysis. Descriptive statistics were performed using methods appropriate to the variables under study. Means and standard deviations were used for normally distributed continuous scale variables. Medians and 25th and 75th percentiles were used for skewed or ordinal data. Frequency counts and percentages or proportions were used for categorical variables. Differences in baseline characteristics between those with and without delirium were assessed using t-tests, Mann-Whitney U tests (for nonnormal continuous data), chi-square tests, or Fisher's exact tests (for cases of small cell counts), as appropriate. Because each plasma sample was measured in triplicate, generalized estimating equations were used to model the variability between measurements. For this model, subject was the clustering variable. Delirium outcomes were specified as binomial variables with logarithmic links. Independent covariance structures were assumed. Robust (i.e., sandwich) estimation was used for estimation. Results are presented as odds ratios (ORs) for each unit change in the biomarker and their associated 95% confidence intervals (CIs). The Youden Index was used to assess the biomarker cutoff that best predicted the presence of delirium. Results are presented as the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity of the Tau-PT217 and Tau-PT181 cutoff values. AUC is calculated by estimating the numerical value of the area under the ROC curve using the trapezoidal rule to assess the probability that the model scores a randomly drawn positive sample higher than a randomly drawn negative sample. The association between biomarkers and delirium severity was estimated using a similar method to delirium, but with a Gaussian distribution and consistency connection. Results are presented as the beta coefficient (β) per unit change of Tau-PT217 or Tau-PT181 value and its associated 95% CI. Models were established to adjust the correlation between biomarkers and age, education, and preoperative MMSE results (both primary and secondary outcomes). The variables used for adjustment were based on previous studies that were considered to be clinically relevant. The assumptions of each model were considered by checking the residuals and calibration curves to ensure that the model fits the data adequately. All analyses were performed using R4.0.5 statistical software (Vienna, Austria). All analyses used two-tailed hypothesis testing when appropriate, with statistical significance interpreted as p < 0.05.

[0095] result

[0096] 491 patients were screened, of whom 220 participants were enrolled between November 2016 and February 2020. 81 participants were excluded because they were ineligible after enrollment (N=3), no longer expressed interest in participating (N=22), plasma samples were not collected (N=51, surgery was cancelled or rescheduled to a conflicting date, complications with plasma collection), or the participant was discharged on the day of surgery or felt unwell and could not complete the test and postoperative delirium was not assessed on either day (N=5). Therefore, 139 participants were included in the final data analysis ( Figure 1 ). Participants experienced no significant complications in the immediate postoperative period.

[0097] Table 1 shows the baseline demographic and clinical characteristics of the 139 participants. There were no significant differences in age, sex, race, type of surgery, type of anesthesia, or preoperative Mini-Mental State Examination (MMSE) scores between participants with postoperative delirium (N = 18) and those without postoperative delirium (N = 121). Participants with postoperative delirium had fewer years of education and lower postoperative MMSE scores than those without postoperative delirium.

[0098] Each plasma sample was randomly assigned to a different batch of nanoneedles to measure Tau-PT217 and Tau-PT181. Measurements were performed in triplicate using the nanoneedle technique. Prior to measurement, the specificity of the antibodies used in the assay was tested using a peptide array ( Figure 4A-B ), and the accuracy of the nanoneedle technique was verified by comparing it with the gold standard ELISA method ( Fig. 5A-F In the present study, there were no significant differences in plasma Tau-PT217 values ​​(F = 0.530, P = 0.589, one-way analysis of variance [ANOVA]) and Tau-PT181 values ​​(F = 0.022, P = 0.978, one-way ANOVA) among three different measurements.

[0099] Participants who developed postoperative delirium had higher preoperative plasma concentrations of Tau-PT217 (11.77 + 4.97 arbitrary units [au] vs. 1.97 + 1.08 au, P < 0.001) and Tau-PT181 (2.25 + 0.61 au vs. 1.06 + 0.63 au, P < 0.001) compared with those who did not develop postoperative delirium ( Table 1 and Figure 2A-B ).

[0100] Preoperative plasma concentrations of Tau-PT217 and Tau-PT181 were associated with postoperative delirium.

[0101] Table 2 shows the results of the unadjusted analysis and multivariate model. After adjustment for age, education, and preoperative MMSE, preoperative plasma concentrations of Tau-PT217 (OR 2.05, 95% CI: 1.61-2.62, P < 0.001) and Tau-PT181 (OR 4.12, 95% CI: 2.55-6.67, P < 0.001) were associated with postoperative delirium in separate models. Preoperative plasma concentrations of Tau-PT217 and Tau-PT181 had high sensitivity (0.956 and 0.882) and variable specificity (0.864 and 0.400) in predicting postoperative delirium ( Figure 3 ). The AUCs of Tau-PT217 and Tau-PT181 were 0.969 and 0.885, respectively ( Figure 3 ).

[0102] Preoperative plasma concentrations of Tau-PT217 and Tau-PT181 were associated with the severity of postoperative delirium.

[0103] In the crude model, increased preoperative plasma concentrations of Tau-PT217 or Tau-PT181 were associated with increased postoperative delirium severity. This association remained after adjustment for age, education, and preoperative MMSE (for Tau-PT217, β coefficient: 0.14, 95% CI: 0.09-0.19, P < 0.001; for Tau-PT181, 0.41, 95% CI: 0.12-0.70, P = 0.006; Table 2).

[0104] Table 1. Demographic characteristics of participants and plasma concentrations of phosphorylated Tau

[0105]

[0106]

[0107] Tau-PT217, Tau phosphorylation at threonine 217; Tau-PT181, Tau phosphorylation at threonine 181; CI, confidence interval.

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[0162] Other Implementations

[0163] It should be understood that although the invention has been described in conjunction with its detailed description, the above description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the claims.

Claims

1. A method for determining the risk of postoperative delirium (POD) in a subject, preferably wherein the subject is a human over 60 years old, the method comprising: optionally obtaining a sample comprising plasma from said subject; determining the level of a Tau isoform phosphorylated at threonine 217 (Tau-PT217) and / or the level of a Tau isoform phosphorylated at threonine 181 (Tau-PT181) in the sample; comparing the level of Tau-PT217 and / or Tau-PT181 in the sample to a reference level of Tau-PT217 and / or Tau-PT181; and Subjects whose levels of Tau-PT217 and / or Tau-PT181 are above a reference level are identified as being at risk for developing postoperative delirium (POD).

2. The method of claim 1, wherein the subject has no clinical signs of Alzheimer's disease (AD), AD-related dementia (ADRD), and delirium.

3. The method of claim 1, wherein the subject has a preoperative Mini-Mental State Examination (MMSE) score of more than 24.

4. The method of claims 1-3, wherein determining the level of Tau-PT217 and / or Tau-PT181 in the sample comprises using an ultrasensitive detection method.

5. The method of claim 4, wherein the ultrasensitive detection method comprises nanoneedle-based detection, single molecule array (SIMOA); molecular on-bead signal amplification for single counting (MOSAIC); mesoscale discovery (MSD); single molecule counting (SMC); nucleic acid-linked immunosandwich assay (NULISA); LUMINEX; SOMA scanning detection; mass spectrometry (e.g., MALDI-MS) and / or mass cytometry (e.g., CyTOF).

6. A method for reducing the risk of postoperative delirium (POD) in a subject, the method comprising: optionally obtaining a sample comprising plasma from said subject; determining the level of a Tau isoform phosphorylated at threonine 217 (Tau-PT217) and / or a Tau isoform phosphorylated at threonine 181 (Tau-PT181) in the sample; comparing the level of Tau-PT217 and / or Tau-PT181 in the sample to a reference level of Tau-PT217 and / or Tau-PT181; and Subjects whose levels of Tau-PT217 and / or Tau-PT181 are above the reference level are identified as being at risk for developing POD; and The subject is given an intervention to reduce the risk of developing POD.

7. The method of claim 6, wherein the subject has no clinical signs of Alzheimer's disease (AD), AD-related dementia (ADRD) and delirium.

8. The method of claim 6, wherein the subject has a preoperative Mini-Mental State Examination (MMSE) score of more than 24.

9. The method of claim 6, wherein determining the level of Tau-PT217 and / or Tau-PT181 in the sample comprises using an ultrasensitive detection method.

10. The method of claim 9, wherein the ultrasensitive detection method comprises nanoneedle-based detection, single molecule array (SIMOA); molecular on-bead signal amplification for single counting (MOSAIC); mesoscale discovery (MSD); single molecule counting (SMC); nucleic acid-linked immunosandwich assay (NULISA); LUMINEX; SOMA scanning detection; mass spectrometry (e.g., MALDI-MS) and / or mass cytometry (e.g., CyTOF).

11. The method of claims 6-10, wherein the intervention comprises avoiding the use of antihistamines, anticholinergics, tricyclic antidepressants, benzodiazepines, antidepressants, muscle relaxants, pethidine, gabapentinoids, and scopolamine; and avoidance of perioperative polypharmacy.

12. The method of claims 6-10, wherein the intervention comprises administration of a prophylactic low-dose antipsychotic medication, which may optionally include olanzapine, quetiapine, or risperidone.

13. The method of claims 6-10, wherein the intervention comprises sedation with an alpha-2 agonist, optionally clonidine or dexmedetomidine.

14. The method of claims 6-10, wherein the intervention comprises administering ketamine during induction of anesthesia; using regional anesthesia and neuraxial anesthesia; xenon anesthesia; and / or administering dexmedetomidine.

15. The method of claims 6-10, wherein the intervention comprises pre- and postoperative behavioral interventions and additional nursing care measures, optionally following perioperative care based on Comprehensive Geriatric Assessment (CGA), the ABCDE bundle, or the Hospitalized Elderly Living Program (HELP) or modified HELP.

16. The method of claim 15, wherein the behavioral intervention and additional care measures include intentionally guiding the patient to understand the location, time, and reason for hospitalization; encouraging at least daily walking activities; staying hydrated (avoiding prolonged (>6 hours) fasting from liquids); avoiding sleep deprivation; and reminding the patient to use glasses and hearing aids when appropriate.

17. The method of claims 6-10, wherein the intervention comprises a pharmacological intervention, optionally selected from pre- and post-operative pain management (optionally using low opioid analgesia); administration of NSAIDs and paracetamol / acetaminophen; administration of melatonin receptor agonists such as melatonin and ramelteon; and administration of synthetic corticosteroids such as dexamethasone.

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