A 38-year-old Chinese Han man presented with fever, headache and difficulty in language expression. The initial cerebrospinal fluid (CSF) analysis revealed lymphocytic-predominant pleocytosis with a normal glucose level, and magnetic resonance imaging (MRI) showed extensive cortical edema in left cerebral hemisphere. He received the antiviral treatment. However, one week later, he developed psychomotor agitation and seizures. Lumbar puncture was performed again and further testing for autoantibodies was conducted in both the CSF and serum. His CSF was positive for anti-myelin oligodendrocyte glycoprotein (MOG), anti-N-methyl-D-aspartate receptor (NMDAR) and anti-metabotropic glutamate receptor 5 (mGluR5) antibodies. He was diagnosed with overlapping syndrome of MOG antibody-related cerebral cortical encephalitis and anti-NMDAR, anti-mGluR5 autoimmune encephalitis. He received intravenous methylprednisolone and immunoglobulin, followed by oral prednisone and mycophenolate mofetil. His psychomotor agitation and seizures were relieved, and he gradually recovered his language expression ability. We reported for the first time a case that was positive for coexistent MOG, NMDAR, mGluR5 antibodies, which was initially misdiagnosed as infectious meningoencephalitis. This case widens the clinical spectrum of the overlapping syndrome recently reported. Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is an inflammatory demyelinating disease of the central nervous system (CNS), and typically presents as optic neuritis, transverse myelitis, brainstem demyelination and acute disseminated encephalomyelitis . Recentl9/L). Routine biochemical tests, antinuclear antibody and thyroid function testing were normal. Human immunodeficiency virus and rapid plasma reagin were negative. Workup for malignancies  was negative. Cerebrospinal fluid (CSF) analysis indicated an elevated opening pressure (380 mm H2O), pleocytosis , an elevated protein level (1.417 g/L) and a normal glucose concentration. CSF Gram staining, ink staining as well as bacterial and fungal cultures were negative. Testing for infectious encephalitis was unremarkable . He was empirically treated for presumptive viral meningoencephalitis with intravenous acyclovir, and mannitol was used for the reduction of intracranial pressure.A 38-year-old right-handed Chinese Han man presented with complaints of difficulty in language expression with no past medical history and no family history of neurological disease. He reported a 12-day history of intermittent fever and headache after catching a cold. On admission, a neurological examination revealed unremarkable except for motor aphasia and neck stiffness. His mini mental score examination (MMSE) was 28/30 . Brain magnetic resonance imaging (MRI) showed extensive cortical edema in left cerebral hemisphere (6/L) and a high protein level (1.132 g/L). Meanwhile, CSF and serum testings for MOG antibody and autoimmune encephalitis panel  were conducted on a cell based assay (CBA). MOG antibodies were positive in the serum (1:32) and CSF (1:10)  was given and antiviral treatment with intravenous acyclovir continued. Lumbar puncture was performed again, and the repeated CSF test still indicated leukocytosis  for the publication of any potentially identifiable images or data included in this article.All authors have contributed significantly to the manuscript and approved it for publication. JF and BR conceived the case report idea. JF, LP, YY and YX collected and analyzed the case materials. The manuscript was written by JF, and reviewed by ZL and BR. All authors contributed to the article and approved the submitted version.This study was supported by the Doctoral Research Initiation Fund of Affiliated Hospital of Southwest Medical University (No. 21024).We appreciate the technical assistance provided by the KingMed Diagnostic laboratory in Antibody test and Cell-Based Assay.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Neurological manifestations of autoimmune connective tissue diseases (CTD) are poorly understood and difficult to diagnose. We here aimed to address this shortcoming by studying immune cell compositions in CTD patients with and without neurological manifestation.Using flow cytometry, we retrospectively investigated paired cerebrospinal fluid (CSF) and blood samples of 28 CTD patients without neurological manifestation, 38 CTD patients with neurological manifestation (N-CTD), 38 non-inflammatory controls, and 38 multiple sclerosis (MS) patients, a paradigmatic primary neuroinflammatory disease.+ T cells and the CD4+/CD8+ ratio were elevated in the blood of N-CTD compared to CTD. Several B cell-associated parameters partially overlapped in the CSF in MS and N-CTD. We built a machine learning model that distinguished N-CTD from MS with high discriminatory power using either blood or CSF.We detected an expansion of plasma cells in the blood of both N-CTD and CTD compared to non-inflammatory controls and MS. Blood plasma cells alone distinguished the clinically similar entities N-CTD and MS with high discriminatory performance (AUC: 0.81). Classical blood monocytes indicated higher disease activity in systemic lupus erythematosus (SLE) patients. Surprisingly, immune cells in the CSF did not differ significantly between N-CTD and CTD, while CD4We here find that blood flow cytometry alone surprisingly suffices to distinguish CTD with neurological manifestations from clinically similar entities, suggesting that a rapid blood test could support clinicians in the differential diagnosis of N-CTD.The online version contains supplementary material available at 10.1186/s12974-023-02733-w. Systemic lupus erythematosus (SLE), granulomatosis with polyangiitis (GPA), and primary Sjögren's syndrome (SS) are three of the most common autoimmune connective tissue diseases and vasculitides, which we will refer to as connective tissue diseases (CTDs) for simplicity. Although their exact triggers are still under debate, both genetic and microbiota/environmental factors are likely to contribute .Neurological manifestations can occur in CTD patients and generally indicate tissue inflammation and an unfavorable prognosis , 3. NeurDifferentiating CTD without neurological manifestation from CTD patients with neurological manifestations (hereafter referred to as N-CTDs) is often challenging but important to guide treatment decisions because patients with N-CTD have increased mortality  and loweWhile the systemic pathogenesis of CTDs has been extensively studied , neuroloFlow cytometry analysis of CSF leukocyte composition is a powerful tool for diagnosing and understanding brain immunity –26. CSF We screened patients who were admitted to the University Hospital Münster between 2011 and 2020 and received a diagnostic lumbar puncture, including flow cytometry, for the ICD-10 diagnoses M32 (systemic lupus erythematosus), M35.0 (Sjögren syndrome), and M31.3 (granulomatosis with polyangiitis). Flow cytometry of the CSF and blood is routinely performed in our center. CTD patients were selected according to the ACR/EULAR criteria. SLE patients fulfilled the 2019 ACR/EULAR criteria for SLE , SS patig for 15 min, the supernatant was removed, and CSF cells were stained with different antibodies as described previously [®, Siemens). A Reiber scheme was created and evaluated for the presence of a blood–CSF barrier disruption (BCBD). Oligoclonal bands (OCBs) were detected via isoelectric focusing followed by silver nitrate staining. In detail, a mixture of 5 µl serum and 100 µl cell-free CSF was diluted to a concentration of 25 mg/l IgG using IgG Sample Diluent (Serva). 20 µl of the diluted sample was then added to the wells of a FocusGel 6–11 24S (Serva) on a Multiphor II cooling plate (Pharmacia Biotech). The mixture was then electrophoresed. Next, the gel was transferred to a processor and gel stainer , fixed, and covered. The staining process is automated, and upon completion, the gel was dried at room temperature and sealed with Mylar conservation sheets .LPs were performed under sterile conditions, and CSF was promptly analyzed. CSF was centrifuged at 300eviously –25. BrieR 4.1.1. The Mann–Whitney U test was used when comparing two groups and the Kruskal–Wallis test with the Dunn test as a post hoc test when comparing multiple groups. The Benjamini–Hochberg method was used for multiple testing corrections. The significance level was set at 0.05. We used propensity scores estimated by logistic regression and nearest neighbor matching with the MatchIt R package [datawizard R package. Briefly, a linear model was fitted for each variable with the therapy status as a predictor. The residuals were computed and used for further analysis. The box plots, the variable importance plot, and the ROC curves were generated with the ggplot2 R package. The correlation plot was produced with the R package corrplot based on the Spearman coefficient. The data were clustered hierarchically with complete linkage and the Euclidean distance measure. The heatmaps were created with the R package pheatmap. In a first step, Yeo-Johnson transformation was performed, followed by centering and scaling with the recipes R package. Next, the mean of each parameter for each cohort was calculated and visualized in a heatmap. Rows were clustered hierarchically with complete linkage and the Euclidean distance measure. We performed receiver operating characteristic (ROC) analysis with the R package pROC [tidymodels R package. First, the data were split into training (75%) and test (25%) data stratified according to the outcome. The data were then preprocessed by removing sparse variables and performing a Yeo-Johnson transformation, centering, and scaling. Lasso regression was performed with the glmnet package [R package vip.The computational analysis was carried out with  package  to identage pROC . A ROC aage pROC . The mac package . We traiWhether N-CTDs display immune cell alterations distinct from CTDs without neurological symptoms and other neuroinflammatory diseases remains unknown. We therefore retrospectively analyzed available flow cytometry data of paired blood and CSF samples and routine CSF parameters of 28 CTD and 38 N-CTD patients, who had been admitted to our center during the past 10 years and underwent a lumbar puncture as part of the diagnostic workup , while classical monocytes were decreased in N-CTD (81.8% vs 87.4%)  and N-CTD (0.22%) compared to Ctrl (0.07%) and MS (0.05%) patients  and severe (n = 25) severity based on their ACR/EULAR scores as described in the literature [p = 0.03). All other blood and CSF parameters did not differ significantly between severe and mild SLE patients. The group size of the other CTDs was insufficient to subset based on severity. Collectively, this provides evidence that the decrease in classical monocytes in N-CTD is not disease specific but rather reflects disease severity. In contrast, the increase in CD4+ T cells and the CD4+/CD8+ ratio in the blood likely represent “neurologic-specific” surrogates of CTD.Disease severity in SLE shows high inter-individual variability. SLE patients with an ACR/EULAR score of ≥ 20 have higher disease activity, less remission, and more often require immunosuppressive therapy . We aimeterature . In our We next investigated whether analysis of the CSF, which ensheaths the brain, could shed more light on neurology-specific mechanisms in CTDs. However, no single CSF parameter differed significantly between N-CTD and CTD Fig. A, B. In +/CD8+ ratio were reduced, while granulocytes, monocytes, and double-positive T cells were elevated in N-CTD compared to MS patients  and N-CTD (9/38) patients were in remission at the time of CSF collection. We wondered whether this difference introduced a relevant bias in our results. Therefore, we subdivided CTD and N-CTD patients into patients in remission and relapse  was the best parameter to distinguish N-CTD from CTD patients . In contrast, when distinguishing N-CTD from CTD, the multivariable lasso regression performed similarly to the single variables  with the AUC of the ROC analysis as a performance measure. When differentiating between N-CTD and MS, the lasso regression approach showed the best performance. Additionally, lasso regression inherently removes unimportant features, thus making the model easier to use and interpret. In blood, the resulting model had a high AUC in both the training 0.84) and test (0.89) datasets , but also notably distinct . We were able to construct a machine learning model that differentiated N-CTD from MS with high accuracy using either blood or CSF. Rapid flow cytometric analysis of the blood could potentially assist clinicians in differentiating N-CTD from clinically similar diseases.The pathogenesis of CTD and its neurological manifestations is poorly understood and the differentiation from other neuroinflammatory diseases remains challenging. Capitalizing on existing multidimensional flow cytometry data, we identified an expansion of plasma cells in the blood in both N-CTD and CTD compared to non-inflammatory controls and MS. The differential diagnosis between N-CTD and MS can be challenging . ImportaMany of our observations were consistent with previous observations. We found a BCBD in CTD and N-CTD patients, as previously described in neuropsychiatric SLE . AlteredAutoreactive B cells, plasma cells, and the resulting antibodies are known to be one of the main drivers of most CTDs , 41. BloOur study is limited by its retrospective design; associated confounding factors, especially treatment; a biased flow cytometry panel; a selection bias, as all patients were enrolled in a tertiary referral center and received a lumbar puncture; and a relatively small sample size. However, we adjusted for treatment with a regression model. We also found no evidence that inhomogeneities in disease status and progression biased our main results. Further prospective multicenter studies with larger cohorts are needed to validate our findings.Collectively, we here provide a comprehensive CSF and blood analysis of the immune cell composition of patients with connective tissue diseases with and without neurological manifestations. Our analysis adds to the understanding of discrepancies and similarities between the immune profiles of N-CTD and CTD in CSF and blood. Moreover, we provide evidence that flow cytometric analysis of peripheral blood can support the clinically challenging but important differentiation between N-CTD and MS. Mechanistic understanding of diverse immune compartments can thus supplement the armamentarium in neuroimmunological diseases that are difficult to diagnose.Additional file 1: Figure S1. Gating scheme. Figure S2. Comparison of blood and CSF parameters between CTD and N-CTD in relapse and remission. Figure S3. Correlation analysis between disease duration and blood and CSF parameters in CTD and N-CTD. Figure S4. Multivariable models differentiating N-CTD from CTD.Additional file 2: Table S1. Patient characteristics table.Additional file 3: Table S2. ROC AUC values corresponding to Fig.


Preclinical studies provided a strong rationale for a pathophysiological link between cell-free hemoglobin in the cerebrospinal fluid (CSF-Hb) and secondary brain injury after subarachnoid hemorrhage (SAH-SBI). In a single-center prospective observational clinical study, external ventricular drain (EVD) based CSF-Hb proved to be a promising biomarker to monitor for SAH-SBI. The primary objective of the HeMoVal study is to prospectively validate the association between EVD based CSF-Hb and SAH-SBI during the first 14 days post-SAH. Secondary objectives include the assessment of the discrimination ability of EVD based CSF-Hb for SAH-SBI and the definition of a clinically relevant range of EVD based CSF-Hb toxicity. In addition, lumbar drain (LD) based CSF-Hb will be assessed for its association with and discrimination ability for SAH-SBI.HeMoVal is a prospective international multicenter observational cohort study. Adult patients admitted with aneurysmal subarachnoid hemorrhage (aSAH) are eligible. While all patients with aSAH are included, we target a sample size of 250 patients with EVD within the first 14 day after aSAH. Epidemiologic and disease-specific baseline measures are assessed at the time of study inclusion. In patients with EVD or LD, each day during the first 14 days post-SAH, 2 ml of CSF will be sampled in the morning, followed by assessment of the patients for SAH-SBI, co-interventions, and complications in the afternoon. After 3 months, a clinical follow-up will be performed. For statistical analysis, the cohort will be stratified into an EVD, LD and full cohort. The primary analysis will quantify the strength of association between EVD based CSF-Hb and SAH-SBI in the EVD cohort based on a generalized additive model. Secondary analyses include the strength of association between LD based CSF-Hb and SAH-SBI in the LD cohort based on a generalized additive model, as well as the discrimination ability of CSF-Hb for SAH-SBI based on receiver operating characteristic (ROC) analyses.We hypothesize that this study will validate the value of CSF-Hb as a biomarker to monitor for SAH-SBI. In addition, the results of this study will provide the potential base to define an intervention threshold for future studies targeting CSF-Hb toxicity after aSAH.NCT04998370. Date of registration: August 10, 2021.ClinicalTrials.gov Identifier  After an aneurysmal subarachnoid hemorrhage (aSAH) up to two-thirds of patients develop aSAH-related secondary brain injury (SAH-SBI), which negatively influences clinical outcome . SAH-SBIThe pathophysiology of SAH-SBI is believed to be multifactorial involving macro- and microvascular dysfunction, neuroinflammation, neuronal apoptosis, and pathological electrical activity of the brain . After tIn a single center observational study, we found strong evidence for a positive association between daily measured EVD based CSF-Hb and the occurrence of SAH-SBI . EVD basThe strong underlying pathophysiological rationale, the preliminary clinical evidence for a positive association and high discrimination ability, and its easy measurability, position CSF-Hb as an attractive biomarker to monitor for SAH-SBI. The primary objective of this prospective international multicenter cohort study is to validate the positive association between EVD based CSF-Hb and SAH-SBI during the first 14 days post-SAH. Secondary objectives include the assessment of the discrimination ability of EVD based CSF-Hb for SAH-SBI and the definition of a clinically relevant range of EVD based CSF-Hb toxicity. Also, LD based CSF-Hb will be assessed for its association with and discrimination ability for SAH-SBI.The HeMoVal study is designed as a prospective international multicenter observational study. The study population consists of patients admitted to a tertiary care center due to an aneurysmal subarachnoid hemorrhage.inclusion criteria are eligible for the study:Participants fulfilling all of the following a. Age ≥ 18 years.b. Hospital admission due to an aneurysmal subarachnoid hemorrhage exclusion criteria will lead to exclusion of the participant:c.Non-aneurysmal subarachnoid hemorrhage .d.Participation in another study with CSF sampling or an interventional medical product within the 30 days preceding and during the present study.e.Previous enrollment into the current study.The presence of any of the following Patients with SAH admitted to one of the study centers will be screened for study inclusion as soon as possible after hospital admission  by a member of the clinical study team at the respective center. The screening will be based on the initial CT imaging of the head to confirm SAH and vascular imaging  for detection of a ruptured aneurysm. In patients fulfilling the inclusion and not meeting any of the exclusion criteria, informed consent will be obtained as required per local regulations.a patient who was incapable of giving informed consent at the time of inclusion does not agree to participate in the study once she/he is capable of giving consent. Any samples collected until the participant’s study withdrawal will be destroyed. Any data collected until the participant’s study withdrawal will be destroyed or, if destruction is not possible as in the study database, de-identified by removal of the patient ID. De-identified data will not be included in the analysisa patient who was incapable of giving informed consent at the time of inclusion and who remains permanently unable to judge and his/her legal representative does not agree to study participation Any samples collected until the participant’s study withdrawal will be destroyed. Any data collected until the participant’s study withdrawal will be destroyed or, if destruction is not possible as in the study database, de-identified by removal of the patient ID. De-identified data will not be included in the analysisa patient withdraws his/her study participation. Any samples and data collected until study withdrawal will remain coded for the analysis. It is not possible to anonymize the data and samples upon withdrawal as this would involve disproportionate effort.A study participant who discontinues study participation prematurely for any reason is defined as dropout. This may happen when:The primary objective of this study is to evaluate the strength of association between EVD based CSF-Hb (measured in the morning) and SAH-SBI (assessed in the afternoon of the same day) during the first 14 days post-SAH.i.The temporal profile (day 1 to day 14 post-SAH) of EVD based CSF-Hb in the EVD cohort.ii.The temporal profile (day 1 to day 14 post-SAH) of LD based CSF-Hb in the LD cohort.iii.Difference  between EVD and LD based CSF-Hb from day 1 to day 14 post-SAH.iv.The strength of association between EVD based CSF-Hb (measured in the morning of day 1 to 14 post-SAH) and aneurysm location (assessed at study inclusion), hematoma volume (assessed at study inclusion), presence of intraventricular hemorrhage (assessed at study inclusion) and day post-SAH in the EVD cohort.v.The strength of association between LD based CSF-Hb (measured in the morning of day 1 to 14 post-SAH) and aneurysm location (assessed at study inclusion), hematoma volume (assessed at study inclusion), presence of intraventricular hemorrhage (assessed at study inclusion) and day post-SAH in the LD cohort.Temporal profiles and dependence of EVD and lumbar drain (LD) based CSF-Hb:i.The temporal profiles (day 1 to day 14 post-SAH) of SAH-SBI, aVSP, DCI, and DIND in the EVD cohort.ii.The temporal profiles (day 1 to day 14 post-SAH) of EVD based CSF-Hb stratified by the presence or absence of SAH-SBI, aVSP, DCI, or DIND in the EVD cohort.iii.The strength of association between EVD based CSF-Hb (measured in the morning) and aVSP, DCI, and DIND (assessed in the afternoon of the same day) during the first 14 days post-SAH in the EVD cohort.iv.The discrimination ability of EVD based CSF-Hb (measured in the morning) for SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of the same day) during the first 14 days post-SAH in the EVD cohort.v.The discrimination ability of EVD based CSF-Hb (measured in the morning) for the first occurence of SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of the same day) during the first 14 days post-SAH in the EVD cohort.vi.The discrimination ability of EVD based CSF-Hb (measured in the morning of day X) for the first occurence of SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of day X + 1) during the first 14 days post-SAH in the EVD cohort.vii.The discrimination ability of EVD based CSF-Hb (measured in the morning) for SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of the same day) during day 4 and day 14 post-SAH in the EVD cohort.EVD based CSF-Hb and SAH-SBI, aVSP, DCI, and DIND in the EVD cohort:i.The temporal profiles (day 1 to day 14 post-SAH) of SAH-SBI, aVSP, DCI, and DIND in the LD cohort.ii.The temporal profiles (day 1 to day 14 post-SAH) of LD based CSF-Hb stratified by the presence or absence of SAH-SBI, aVSP, DCI, or DIND in the LD cohort.iii.The strength of association between LD based CSF-Hb (measured in the morning) and SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of the same day) during the first 14 days post-SAH in the LD cohort.iv.The discrimination ability of LD based CSF-Hb (measured in the morning) for SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of the same day) during the first 14 days post-SAH in the LD cohort.v.The discrimination ability of LD based CSF-Hb (measured in the morning) for the first occurence of SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of the same day) during the first 14 days post-SAH in the LD cohort.vi.The discrimination ability of LD based CSF-Hb (measured in the morning of day X) for the first occurence of SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of day X + 1) during the first 14 days post-SAH in the LD cohort.vii.The discrimination ability of LD based CSF-Hb (measured in the morning) for SAH-SBI, aVSP, DCI, and DIND (assessed in the afternoon of the same day) during day 4 and day 14 post-SAH in the LD cohort.LD based CSF-Hb and SAH-SBI, aVSP, DCI, and DIND in the LD cohort:i.The strength of association between EVD based CSF-Hb during the first 14 days post-SAH and chronic hydrocephalus at 12 weeks follow-up in the EVD cohort.ii.The strength of association between EVD based CSF-Hb during the first 14 days post-SAH and functional outcome  at 12 weeks follow-up in the EVD cohort.EVD based CSF-Hb and 3-months follow-up status:i.The strength of association between LD based CSF-Hb during the first 14 days post-SAH and chronic hydrocephalus at 12 weeks follow-up in the LD cohort.ii.The strength of association between LD based CSF-Hb during the first 14 days post-SAH and functional outcome  at 12 weeks follow-up in the LD cohort.LD based CSF-Hb and 3-months follow-up status:The secondary objectives are to investigate:Here we provide a summary of the endpoints assessed in this study. A detailed list with characterization of the variables can be found online (10.5281/zenodo.5556317).CSF-Hb constitutes the biomarker assessed in this study. CSF-Hb levels (oxyhemoglobin) will be quantified in the CSF samples obtained from patients after aSAH using spectral deconvolution, as described previously , 13.The primary endpoint of this study is the occurrence of SAH-SBI during the monitoring period. SAH-SBI is a categorical variable that will be derived as a composite from the categorical secondary endpoints aVSP, DCI, and DIND: If any one or more of the three secondary endpoints  is present on the corresponding day, SAH-SBI is considered to be present. If neither information on aVSP (no imaging performed), nor DCI (no imaging performed) or DIND  could be obtained, SAH-SBI is considered to be non-assessable. In any other instance, SAH-SBI is considered not to be present.Secondary endpoints, which will be assessed during the first 14 days post-SAH on a daily basis, are: angiographic vasospasms (aVSP), delayed cerebral ischemia (DCI), delayed ischemic neurologic deficits (DIND), elements of triple H therapy, spasmolysis, surgical decompression.no imaging performed. Categorical variable .The definition of aVSP comprises a narrowing of cerebral arteries based on a DSA, CTA or MRA. In the absence of an appropriate imaging procedure on the respective day, this will be noted as no imaging performed. Categorical variable .DCI is defined as new perfusion deficit or new infarction in native and/or contrast-enhanced CT/MRI. In the absence of an appropriate imaging procedure on the respective day, this will be noted as non-assessable). Categorical variable .DIND is defined as a new focal neurological deficit or a decrease in GCS of at least 2 points for at least 2 hours. In case the patient cannot be clinically assessed , this will be noted as Secondary endpoints, which will be assessed at the 12 weeks (+/− 2 weeks) follow-up: Chronic hydrocephalus, Glasgow Outcome Scale Extended (GOSE), modified Rankin Scale (mRS).Safety endpoints, which will be assessed during the first 14 days post-SAH on a daily basis in the afternoon, always at approximately the same time: CSF infection, revision due to surgical site infection at the EVD skin entrance.Measures assessed at the time of study inclusion : Patient demographics , medical history , clinical assessment , pupil status, cranial nerve deficit, focal neurological deficit, headache, neck stiffness), clinical SAH scores at admission , Hunt & Hess grade), Radiological features , radiological SAH scores at admission  grade), aneurysm therapy.baseline assessment, post-SAH monitoring, 3-months follow-up, and measurements. Table A detailed definition and description of each variable, including levels (for factors) and range (for continuous variables), and assessment procedure, can be found online (10.5281/zenodo.5556317). It is ensured that all members of the clinical study team will be specifically trained in the study-specific assessments. The assessments in this study are divided into Gathering of demographic  and baseline  patient data. Performed directly after study inclusion by a trained member of the clinical study team.CSF sampling and clinical assessments will be performed on a daily basis between day 1 and day 14 post-SAH, always at approximately the same time of the day  by a trained member of the clinical study team.CSF sampling will be performed via EVD or LD if present. The decision regarding the insertion of an EVD or LD is made on a clinical basis by the treating physician independently of this study. If an EVD or LD is present, 2 ml of CSF will be sampled daily, centrifuged at 1500 G for 15 minutes, and subsequently stored at − 80 °C.no imaging performed. DCI is defined as a new perfusion deficit or a new infarction in native and/or contrast-enhanced CT/MRI. In the absence of an appropriate imaging procedure on the respective day, this will be noted as no imaging performed. DIND is defined as a new focal neurological deficit or a decrease in GCS of at least 2 points for at least 2 hours. In case the patient cannot be clinically assessed , this will be noted as non-assessable). Other secondary endpoints assessed include elements of triple H therapy, spasmolysis, and surgical decompression. CSF infection and revision due to surgical site infection at the EVD skin entrance comprise safety endpoints.The clinical assessment of patients for the primary and secondary endpoints will be blinded to CSF sampling, since the macroscopic aspect of CSF after centrifugation may provide an indication of the CSF-Hb level. The definition of aVSP comprises a narrowing of cerebral arteries based on a DSA, CTA or MRA. In the absence of an appropriate imaging procedure on the respective day, this will be noted as The assessment will be performed at the 12 weeks (+/− 2 weeks) follow-up visit. This will be done on site if possible. If this is not possible, a consultation by telephone will be conducted. The presence of chronic hydrocephalus is defined as the dependence on permanent CSF diversion which was not present before aneurysm rupture. The functional status scores will be assessed with the Glasgow Outcome Scale Extended (GOSE) and the modified Rankin Scale (mRS).CSF-Hb measurements and hematoma volumetry will be performed after completion of the monitoring period of the last included patient in this study.CSF-Hb measurements: CSF-Hb measurements will be performed centralized in the Preclinical Research in Internal Medicine (PRIME) Laboratory, Schlieren, Switzerland. Absorption spectra in the visual range between 350 and 650 nm will be measured and quantification of the oxyhemoglobin in CSF will be performed using spectral deconvolution, as described previously , 13. CSFHematoma volume: Assessed on the initial CT scan of each patient. The CT scan will be uploaded by the local investigators in coded form (identifying information replaced by patient ID) to a HIPAA compliant platform , from where automated centralized quantification of hematoma volume is performed. The quantification of the hematoma volume is performed by a person blinded to the patient’s CSF sampling/CSF-Hb values and their clinical assessment/endpoints.requires inpatient treatment not envisaged in the protocol or extends a current hospital stay,results in permanent or significant incapacity or disability, oris life-threatening or results in death.A serious event is defined as any adverse event where it cannot be excluded, that the event is attributable to the sampling of biological material or the collection of health-related personal data, and which:If a serious event occurs, the research project will be interrupted. The responsible Ethics Committee(s) and Competent Authority will be notified according to the locally relevant regulations, if required.For quality assurance representatives of the responsible Ethics Committee may visit the research sites. Direct access to the source data and all project related files and documents will be granted on such occasions. Central and on-site monitoring  will be part of the quality control activities implemented for this study. During the study, the central data monitor will check the eCRFs on a weekly basis and lock complete visits. All involved parties will keep participant data strictly confidential.https://www.project-redcap.org/). The EDC system was activated for the study after successfully passing a formal test procedure. All data entered in the CRFs are stored on a Linux server in a dedicated mySQL database. Responsibility for hosting the EDC system and the database lies with CTU Berne. The initial CT scan of each patient will be uploaded and stored for the duration of the study in coded form (identifying information replaced by patient ID) on the HIPAA compliant platform Augsafe. The data from the CSF-Hb analysis are recorded in the EDC system of the Preclinical Research in Internal Medicine (PRIME) Laboratory, Schlieren, Switzerland. For the statistical analysis, the data will be merged to an export of the eCRF, based on the patient ID. Source data will be available at each study site in the respective patient files, either electronically or on paper. The source data will include the original documents relating to the study, as well as the medical treatment and medical history of the patients. In the Investigator Site File and Trial Master File the informed consent forms and other relevant study-specific documents will be recorded. Any patient files and source data will be archived for the longest possible period of time according to the feasibility of the investigational site.The CRFs in this trial are implemented electronically using a dedicated electronic data capturing (EDC) system  regulates permission for each user to use the system and database as he/she requires. Authorized to enter data into the eCRF are the local trial team staff according to the database access list. The local project leader is responsible for proper training and instruction of the trial personnel filling data into the eCRF. Study-related data of the patient will be collected in a coded manner. The names of the patients will not be disclosed. A code  will be attributed to each patient registered. All data entered into the CRFs are transferred to the database using Transport Layer Security (TLS) encryption. Each data point has attributes attached to it identifying the user who entered it with the exact time and date. Retrospective alterations of data in the database are recorded in an audit table. Time, table, data field and altered value, and the person are recorded (audit trail). A multi-level back-up system is implemented. Back-ups of the whole system including the database are run internally several times per day and on external harddrives once a day. The back-up tapes are stored in a secure place in a different building.Biological material in this project will be coded using a barcode system that will be linked to the patient ID and sampling attributes. Biological material is appropriately stored in a restricted area only accessible to authorized personnel. CSF samples from local sites will be shipped to Research Biobanking Service Center (RBSC), University Hospital Zurich, Zurich, Switzerland.At final analyses, data files will be extracted from the database into the statistical software R to be analyzed. After database lock, the status of the database is recorded in special archive tables. The sponsor will keep the Trial Master File, the extracted data, the meta data and final reports for at least 10 years. Each site will archive its study documents for at least 10 years. Leftover CSF samples will be stored for a maximum time period of 10 years. At the end of this period they will be destroyed according to local law and regulations.The primary objective of the study focuses on patients included with EVD (EVD cohort) while secondary objectives also consider patients with LD (LD cohort) and patients irrespective of any drainage system (full cohort). The sample size estimation is based on an anticipated effect size of 0.1, a power of 90% and accounts for between-site variability using a generalized linear mixed model. This calculation results in a minimum of 6 participating study sites and 30 patients per site with an EVD. Considering an anticipated drop-out rate of 15% and heterogenous recruitment rates at the sites, we plan to include 8 sites and a maximal study population of 250 patients with EVD. Based on our clinical experience, we assume that only every second subject receives an EVD. Thus, we assume to include a maximum of 500 subjects. The recruitment will be stopped as soon as 250 patients with EVD are included.p-value will be used to test the null hypothesis of no effect of the biomarker at a 5% significance level. In the primary analysis, no adjustment of the significance level for multiple testing is needed. Detailed description on summary statistics, secondary and safety endpoint analyses can be found online in the statistical analysis plan (10.5281/zenodo.5556317). For secondary analyses, no level of statistical significance will be defined. Instead, the results of secondary analyses will be interpreted based on the level of evidence  and conThe HeMoVal study is conducted in accordance with the Declaration of Helsinki and national and international standards of ICH-GCP E6(R2). Potential participants or their legal representatives receive detailed written and oral information on the study procedures and all participants or their legal representatives provide written informed consent. The study protocol will be approved by the local ethics committees of all participating study sites. To date, the study protocol has been approved by the ethics committee of the Canton of Zurich, Switzerland (KEK 2021–01023) for Zurich (CH), Aarau (CH), and St. Gallen (CH). The primary and secondary results of this study will be published in peer-reviewed journals. Authors of these publications are persons who planned and/or conducted the study and/or did parts of the statistical analysis.ClinicalTrials.gov Identifier NCT04998370. Date of registration: August 10, 2021.Recruitment for the HeMoVal study is currently starting at multiple centers across Switzerland, Germany and the Netherlands. At the University Hospital of Zurich (Switzerland) the recruitment started on August 15, 2021, and at the Kantonsspital Aarau (Switzerland) on August 25, 2021. At the time of submission of this protocol, 21 patients have been enrolled in the HeMoVal study.Preclinical studies provide a strong rationale for a causal pathophysiological link between CSF-Hb and SAH-SBI –14. AlsoThere is considerable heterogeneity in the use of the terms aVSP, DCI and DIND in both literature and clinical practice . The comThe HeMoVal study will enroll all patients who are diagnosed with aSAH at admission, thus ensuring that start of CSF sampling from a newly introduced EVD or LD is possible at each day of the 14-day monitoring period. For the statistical analysis, the cohort will be stratified into an EVD cohort (at least one CSF sample obtained via an EVD), LD cohort (at least one CSF sample obtained via an LD), and a full cohort . Distinguishing between EVD and LD cohorts anticipates potential differences in the temporal profiles of EVD and LD based CSF-Hb, and hence their association to and discrimination ability for SAH-SBI. Despite the anatomical communication between the intraventricular and lumbar CSF spaces, substantial differences in the protein levels of samples obtained from the two compartments were described in patients with multiple sclerosis . The fulThe strength of association between CSF-Hb and SAH-SBI, aVSP, DCI, and DIND will be quantified using a GAM. GAMs blend properties of generalized linear models with additive models . The dat. Further, we don’t expect an increased risk of symptomatic CSF overdrainage due to the sampling regimen in the HeMoVal study, since the collected 2 ml of CSF per day represent only a fraction of the regular hourly drainage volume needed for the treatment of posthemorrhagic hydrocephalus [The collection of CSF samples for the HeMoVal study is a manipulation at the closed EVD/LD drainage system, which may raise concerns about an increased risk for CSF infections. Reported infection rates of EVD/LD systems in clinical routine range from 0 to 32%. However, rates around 10% are usually described –23. Sinccephalus  and becaIn this study, a timed imaging protocol was deliberately avoided since it would have had to be performed at high frequency due to the variability of the temporal occurrence of aVSP and DCI and would therefore have been associated with an unacceptable additional burden for the patients . Moreover, it would not have been a compelling substitute for clinically indicated imaging in the setting of neurologic deterioration. Thus, in the HeMoVal study, we follow the real clinical situation with imaging according to clinical indication, which increases the variability of the results, but also their generalizability.The HeMoVal study has the potential to strengthen the evidence for a positive association between CSF-Hb levels and the occurrence of SAH-SBI. In the future, CSF-Hb may serve as a biomarker to monitor for SAH-SBI. CSF processing and photospectrometry based measurements constitute easy to conduct procedures, which could be readily implemented as point-of-care test. The HeMoVal study is intended to provide generalizable data on the discrimination ability of CSF-Hb for SAH-SBI and thus to provide the basis to define an intervention threshold. Final choice on a clinical intervention threshold will additionally depend on the individual burden and the economic costs associated with false-positive and false-negative results.Given the hypothesized causal pathophysiological link, CSF-Hb also represents a potential drug target. Targeting CSF-Hb through intrathecal application of the Hb-scavenger haptoglobin offers both preventive and therapeutic potential , 13. By


Psychotic disorders are currently diagnosed by examining the patient’s mental state and medical history. Identifying reliable diagnostic, monitoring, predictive, or prognostic biomarkers would be useful in clinical settings and help to understand the pathophysiology of schizophrenia. Here, we performed an untargeted metabolomics analysis using ultra-high pressure liquid chromatography coupled with time-of-flight mass spectroscopy on cerebrospinal fluid (CSF) and serum samples of 25 patients at their first-episode psychosis (FEP) manifestation (baseline) and after 18 months (follow-up). CSF and serum samples of 21 healthy control (HC) subjects were also analyzed. By comparing FEP and HC groups at baseline, we found eight CSF and 32 serum psychosis-associated metabolites with non-redundant identifications. Most remarkable was the finding of increased CSF serotonin (5-HT) levels. Most metabolites identified at baseline did not differ between groups at 18-month follow-up with significant improvement of positive symptoms and cognitive functions. Comparing FEP patients at baseline and 18-month follow-up, we identified 20 CSF metabolites and 90 serum metabolites that changed at follow-up. We further utilized Ingenuity Pathway Analysis (IPA) and identified candidate signaling pathways involved in psychosis pathogenesis and progression. In an extended cohort, we validated that CSF 5-HT levels were higher in FEP patients than in HC at baseline by reversed-phase high-pressure liquid chromatography. To conclude, these findings provide insights into the pathophysiology of psychosis and identify potential psychosis-associated biomarkers. Schizophrenia is a detrimental illness with a lifetime prevalence of 0.7% and is associated with increased mortality and a shortened lifespan. The disorder is characterized by positive  and negative symptoms  as well as a loss of cognitive functions . Schizophrenia symptoms usually first appear in adolescence or early adulthood, and as early detection and care of subjects with psychosis significantly improve clinical outcomes, it is essential to identify afflicted persons at an early stage . The diaFor long, the pathophysiology of schizophrenia has been attributed to brain dopaminergic dysfunctions . HoweverMetabolomics is the comprehensive analysis of small molecule metabolites in biological samples and has emerged as an evolving technology with a promising approach for identifying potential biomarkers for diagnosis, monitoring, prediction, or prognosis. In line with this, we recently identified an index of baseline biomarkers with high positive predictive values for acamprosate treatment-response in alcohol-dependent patients . MetabolThis study utilized ultra-high pressure liquid chromatography coupled with time-of-flight mass spectroscopy (UPLC-ToF/MS) to perform global metabolomic analyses on CSF and serum samples collected from patients presenting their first-episode psychosis (FEP) and at a follow-up assessment 18 months after. CSF and serum samples collected from healthy controls (HC) at corresponding time points were also analyzed. The objective was to identify clinically relevant biomarkers associated with psychosis, thereby providing important insights into the pathophysiology of schizophrenia. Furthermore, we aimed to identify putative catalogs of metabolic biomarkers that may aid in diagnosing schizophrenia spectrum disorders or be used for objectively monitoring disease progression. Using Ingenuity Pathway Analysis (IPA), we also identified candidate signaling pathways that may contribute to the pathophysiology of schizophrenia spectrum disorders. Finally, by identifying changes in CSF metabolites, we validated the identified metabolites as markers for diagnosis and outcome predictability.The Stockholm Regional Ethics Committee (Dnr 2010/879-31/1) approved the protocol of this study. All participants had given written informed consent before any procedures according to the Declaration of Helsinki. FEP patients and HC were recruited from four psychiatric clinics under the Stockholm City Council for the Karolinska Schizophrenia Project (KaSP). At recruitment, all FEP patients met the DSM-IV criteria for psychotic disorders while HC were deemed healthy by medical history and examination with no history and no first-degree relatives of psychotic illness. Participants with a history or current use of illegal substances  were excluded but were allowed to use nicotine products  with peripheral venous blood was sampled using standard venipuncture techniques at the same time as the CSF collection. Details of sample collection and processing procedures are described in Supplementary methods.Untargeted metabolomic analysis of the CSF and serum samples was performed by UPLC- TOF/MS  at the Mayo Clinic Metabolomics Core . UPLC-TOp < 0.05.Pathway analysis was performed on metabolites significantly different between groups at baseline and between time points by the Ingenuity Pathway Analysis program . Metabolite identifiers from various metabolite databases were submitted to IPA to maximize metabolite recognition (Supplementary methods). Significantly enriched pathways/processes were considered at Benjamini-Hochberg adjusted To follow up with our major findings, we measured the levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) by reversed-phase HPLC and tryptophan by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in baseline CSF samples of an extended cohort  was used to perform these analyses.Subject demographics and basic clinical characteristics were compared between groups at baseline by binary logistic regression for continuous variables and Fisher’s exact test for categorical variables. Psychiatric scales and cognitive test results of FEP patients were compared between baseline and 18-month follow-up by paired p < 0.15  with a fold change that exceeded a certain empirical cutoff  in GraphPad Prism 8.One-way ANCOVA used to compare levels of serotonin and related metabolites measured by HPLC and LC-MS/MS between FEP patients and HC adjusting for age, antidepressant use and nicotine use. Statistical significance was set at Benjamini-Hochberg-adjusted p > 0.05). However, FEP patients were older than HC in the metabolomics and extended cohort (p < 0.05). FEP patients showed significant alleviations in symptoms and cognitive impairments at the 18-month follow-up compared to baseline, including reductions in PANSS positive, general and total scores, GAF symptom and function scores, and CGI scores, as well as elevations in BACS-SC, CPT-IP, and BVMT-R scores  and serum 5-HT levels  and GAF functioning subscale scores , while the change in CSF 5-HT levels were negatively correlated with PANSS negative , general , and total scores  as well as MSCEIT-ME score . Some significantly enriched biological functions overlap between CSF and serum psychosis-associated metabolites, such as “lipid metabolism”, “neurological disease”, and “cell death and survival” , 5-HT in 15 samples (8 FEP and 7 HC), and 5-HIAA in 67 samples (47 FEP and 20 HC). Consistent with metabolomics analysis, we found that baseline CSF 5-HT levels were higher in the FEP patients than the HC after adjusting for age, anti-depressant treatment, and nicotine use  biosynthesis, 5-HT receptor signaling, 5-HT degradation, and tryptophan degradation. An elevation in brain 5-HT was confirmed in our second cohort, where HPLC detection showed higher CSF levels in FEP patients at baseline. Notably, 5-HT levels were normalized to a level similar to the HC at 18-month follow-up, indicating that 5-HT changes are related to FEP onset. Since the FEP patients showed significant improvements in cognitive and psychological domains, these results implicate that 5-HT may be a potential biomarker for diagnosis and treatment progress monitoring. On the other hand, in the FEP patients, we did not observe altered CSF 5-HIAA, a 5-HT metabolite that is generally considered an appropriate predictive marker of schizophrenia –13. In a2A receptor, as well as by the superior antipsychotic action of clozapine which is attributed to its relatively potent antagonistic effect on 5-HT2A receptors [. 5-HT has indirectly shown to be implicated in negative symptoms by studies in treatment-resistant schizophrenia where antipsychotics were found to alleviate negative symptoms when combined with serotonin reuptake inhibitors [Although numerous studies have focused on 5-HT’s role in the origin and progress of schizophrenia, most clinical studies analyzing the neurotransmitter (or 5-HIAA) in CSF or blood from schizophrenia patients were performed decades ago, and contradictory results were reported , 15. Theeceptors , 19. 5-Hhibitors . Our preA large body of clinical and experimental studies implicate dopamine, glutamate, GABA, and kynurenic acid in schizophrenia. In the present study, though, we see no striking changes in these neurotransmitters/modulators, or their related metabolites, neither at baseline nor at the follow-up. However, metabolites related to dopamine signaling were identified in the pathway analysis. The dopamine hypothesis of schizophrenia has become a long-standing theory for psychosis ever since dopamine receptor antagonists were recognized to improve positive symptoms .Considering the significance of dopamine signaling in psychosis –24, it iWe found that 13-eicosenoic acid was significantly increased in serum of FEP patients at follow-up, other lipids such as trans-3-aminocyclopentane-1-carboxylic acid, 10-hydroxy-2E,8E-decadiene-4,6-diynoic acid, taurochenodeoxycholic acid, and S-farnesyl thioacetic acid were also identified as disease- or follow-up associated metabolites. In line with previous studies, our data suggest an altered lipid metabolism in schizophrenia –32. A reN-docosahexaenoyl GABA, an unsaturated fatty acid that belongs to the N-acyl-GABA family and has been shown to increase cellular calcium levels [We further identified significant alterations in several pyridine alkaloids in the CSF and sera of FEP patients, such as acanthicifoline, guvacoline, and theobromine. Interestingly, consumption of guvacoline, a betel alkaloid, has been identified to mitigate positive symptoms in schizophrenia patients . Guvacolm levels , was obsm levels . Drugs am levels . Our patm levels .Several limitations need to be noted. Firstly, our FEP patients were, on average, somewhat older than our HC. Here, we endeavored to avoid false-positive findings due to potential age effects by removing age-related metabolites that were observed to be changed across time in HC. Additionally, we found no significant independent effect of age on our psychosis-associated CSF and serum metabolites using a multivariate model (Supplementary Tables To conclude, we identified several metabolites, such as 5-HT, 13-eicosenoic acid, and citric acid, as clinical meaningful potential psychosis metabolomic biomarkers in serum and CSF using untargeted metabolomics that may provide insights into FEP pathogenesis and help to assess prognosis. The most prominent findings include changes related to serotonergic neurotransmission found in the CSF. The psychosis and follow-up associated metabolites require replication and further clinical investigations to establish their validity as predictive biomarkers. Although beyond the scope of this study, future studies may take advantage of our results by establishing correlations between central and peripheral levels of significant metabolites. Such an approach may enable a rational biomarker analysis valuable for the diagnosis of psychotic disorders.Supplemental MethodsSupplemental Tables S1-23Supplemental Tables S24Supplemental Figures


Here, we have considered different metastable nanoporous and amorphous iridium oxides of different chemical stoichiometries. Using first-principles electronic structure calculations, we investigate the (electro)chemical stability, intercalation properties, and electronic structure of these iridium oxides. Using an empirical regression model between the Ir-O bond characteristics and the measured OER overpotentials, we demonstrate how activated Ir-O bonds (and the presence of more electrophilic oxygens) in these less understood polymorphs of iridium oxides can explain their superior OER performance observed in experiments.To date, the search for active, selective, and stable electrocatalysts for the oxygen evolution reaction (OER) has not ceased and a detailed atomic-level design of the OER catalyst remains an outstanding  problem. Considerable studies on different surfaces and polymorphs of iridium oxides (with varying stoichiometries and dopants) have emerged over the years, showing much higher OER activity than the conventionally reported rutile-type IrO Here the authors use density-functional theory calculations to examine structure-property relations of nanoporous and amorphous iridium oxides and reconcile the superior oxygen evolution reaction catalytic performance reported in previous experiments. Here, the anodic reaction—commonly known as the oxygen evolution reaction (OER), is an important half-cell reaction where H2O is catalytically split to evolve O2. However, due to the intrinsic sluggish kinetics of the OER, this leads to an overall poor catalytic performance in general.To achieve sustainable energy production, solar-driven (electro)conversion of CO4. A promising way to tune and engineer the structure–property relations of these oxide catalysts is to control their stoichiometry and polymorphic phase at the atomic-level9.Thus, to improve the long-term efficacy of this anodic reaction, the search for active, selective, and stable OER electrocatalysts has been on the rise, and amongst them, oxides (and oxyhydroxides) of iridium and ruthenium are known for their outstanding stability and reactivity in acidic environments10. It was also reported that the intercalation of alkaline earth metal cations in the nanopores of these channel-type microstructures adds stability to the overall catalyst structure13. Though these accounts demonstrate improved OER performance over conventional crystalline rutile-structured IrO2, a fundamental atomic-scale understanding of these nanopore-containing amorphous iridium oxides is very much lacking. Thus, it greatly hinders the establishment of a design rule for further performance improvement.Willinger et al. experimentally found that channel-like microstructures (resembling the hollandite- or romanechite-type motifs) contributed to a better OER efficiency and catalytic stability of the amorphous iridium oxide catalyst14  has attempted to investigate the role of polymorphism in both IrO2 and IrO3 (and their surfaces) for OER activity. Using a machine-learning-based surrogate model, they were able to rationalize that under OER technical catalysis conditions, the α-phase of IrO3 was, in fact, predicted to have higher thermodynamic stability and high OER activity, as compared to that of rutile-type IrO2. However, nanoporous  and amorphous iridium oxides—which have been proposed in various experiments13—have not been included nor examined in this study.In an effort to fill this lack, a very recent high-throughput computational study2 molecule proceeds via the adsorbate evolving mechanism 16, or by means of the lattice oxygen mechanism 17. O isotope-labeling experiments have revealed and suggested that the LOM is more likely on the surface of IrO2 where the Mars van Krevelen-type process has also been proposed for non-oxide compounds (e.g. in sulfides and chlorides)17. Specifically, for the LOM, it has been proposed in many reports that the presence of electrophilic oxygen on the surface of iridium oxide will play an important role in the adsorption of the nucleophilic H2O molecule via the so-called flexible charge state of Ir cations in the different phases of iridium oxide21.Moreover, from the OER reaction mechanistic point-of-view, there is still an ongoing debate as to whether the direct formation of O2 desorption and the adsorption of H2O on iridium oxides15. In the same vein, the use of the metal–sulfur bond strength as a descriptor for Mars van Krevelen-type dehydrosulfurization has also been deliberated in a previous report17. These suggested atomistic features  are still poorly understood, especially for experimentally observed nanoporous and amorphous iridium oxides.In addition, the chemical characteristics of the Ir–O bond has been suggested as a key descriptor for both O14, we expand and include experimentally motivated IrO2 polymorphs  as well as amorphous structures of iridium oxides of varying chemical stoichiometry. Here, using first-principles density-functional theory (DFT) calculations, we show that the nonequivalent connectivity in the amorphous iridium oxide structures greatly improves the flexibility of the Ir charge states, and hence promoting the presence of electrophilic oxygens in them, when compared to their crystalline counterparts. We also demonstrate that a Pauling-like relation between the Ir–O bond length versus bond strength for the Ir–O bonds exist in amorphous iridium oxides, corroborating with the proposal of flexible charges states and activated bonds for more efficient OER catalysis. Via an empirical regression model between the Ir–O bond characteristics and the measured OER overpotentials, we propose that these less understood metastable nanoporous and amorphous iridium oxides may indeed afford a lower OER overpotential, reconciling their superior OER catalytic performance in recent experiments.In this work, to bolster the work of Flores et al. 14, we have adopted the low-energy polymorphs of IrO2 and IrO3—namely, the rutile phase of IrO2 —in particular, the hollandite (Ho-IrO2), romanechite (Ro-IrO2), and todorokite (To-IrO2) phases , the K ion intercalated IrO2 romanechite structure (2K + Ro-IrO2), and the K ion intercalated IrO2 todorokite structure (4K + To-IrO2), respectively. To further extend our theoretical investigation on non-stoichiometric iridium oxides, fictitious crystal structures of IrO1.5 (or Ir2O3) have been included in this work—namely the corundum phase  for all crystalline phases of IrO2, IrO1.5, and IrO3 polymorphs and listed their values in Table a, b, and c lattice constants for rutile R-IrO2 are 4.51, 4.51, and 3.16 Å, respectively)22. It is worth noting that there has been a previous attempt to experimentally expound on the crystallography of the hollandite-type iridates23. For the hollandite-type iridates, our DFT calculations consistently favor the lower symmetry monoclinic C2/m phase , regardless of ion intercalation. We also find that the volumetric changes to the intercalated nanoporous IrO2 defer according to channel size. For instance, the volume of 2K + Ro-IrO2 shrinks by about 3% while that of 4K + To-IrO2 expends by about 5%.Using the optB86b x for OER, recent experimental reports of an amorphous IrOx phase could well be responsible for the high OER activity observed24. To generate approximate atomistic models of amorphous IrOx, we perform aiMD calculations (following the melt-and-quench method25) for various chemical stoichiometries  while ensuring numerical convergence with supercell size.Besides the commonly reported crystalline phases of active IrOg(r) of Ir–O (in black), O–O (in red), and Ir–Ir (in blue) bond pairs  peaks  are shown in Fig. 12. When considering the much larger supercell of 216 atoms  peaks are somewhat unchanged, and thus lending support that our smaller 96-atom supercell may be appropriate for modeling a-IrO2. Likewise, for both a-IrO1.5  peak for a-IrO3 near 1.5 Å is attributed to small oxygen clusters in the model.We calculate and plot the calculated partial radial distribution function, tary Eq.  for a-Ira-IrO2, a-IrO1.5, and a-IrO3 are found to be 10.7, 12.2, and 8.9 g/cm3, respectively. When compared to the experimentally reported mass density of crystalline IrO2 (i.e. 11.7 g/cm3),28 our calculated value for a-IrO2 appears somewhat smaller and this is inline with that found for other materials29. This difference may be attributed to the lower average coordination number of cations in the amorphous phase when compared to the crystalline phase30. Moreover, a weak correlation between our MD-determined mass densities and the oxygen content is also suggested from our calculations31.In addition, the calculated mass densities for Hf, via Suppplementary Eq. (2) and are tabulated in Table 15, rutile IrO2 is found to be the thermodynamic ground state structure for iridium dioxide, while both R-IrO3 (3 (P6322) are the representative ground state structures for iridium trioxide (differing only by 1 meV/f.u.). For the hypothetical IrO1.5, the corundum phase, C-IrO1.5 is taken as the lowest energy structure.To address the thermodynamic stability for the various iridium oxide structures we considered in this work, we calculate the formation enthalpy, Δ2 are all found to be metastable with respect to rutile IrO2 and additional stability is gained upon K intercalation. To analyze the intercalation energetics in these nanoporous iridium oxide structures, we have also calculated their intercalation energy, ΔHint  are well discussed in literature.The nanoporous Ho-, Ro-, and To-IrOtary Eq. ) and areFvib  and plo34. To do this, we construct the DFT-derived Pourbaix phase diagram by considering the reaction energy, Δμ and various relevant ionic species  may form when assuming the kinetic hinderance of R-IrO2 formation, and has been realized in recent electrochemical experiments12. It has been argued that the residual presence of K+ ions in the nanoporous framework of amorphous iridium oxide  might improve the overall catalytic performance and stability10.Within the considered range of pH and the electrode potentials in Fig. 3 coincides well with previous studies14. Given that many non-equilibrium structures may be obtained by controlled synthesis conditions and a careful choice of reactants/precursors24, we are hopeful that more metastable iridium oxides  is correlated to the enhanced activity of iridium oxides for OER. However, a clear theoretical consensus is yet to be reached. Moreover, an apparent relationship between the flexible charge state of Ir and its structural motifs (e.g. in nanoporous and amorphous iridium oxides) is still lacking.For the adsorption of the nucleophilic water molecule on the iridium oxide surface, the presence of an electrophilic oxygen atom may play a pivotal role as the susceptible adsorption site to bind the water molecule on the iridium oxide surfacetary Eq. ). In pas2, the Bader charge of the Ir atom is calculated as +2.06e, while that of crystalline IrO1.5 and IrO3 are determined to be between +1.52–+1.66 and +2.33–+2.42e, respectively. In the case of nanoporous IrO2 polymorphs, the Bader charges of Ir are calculated to be within the range of +1.81–+1.93e, which is somewhat less than that in R-IrO2 (+2.06e). Upon intercalation of K, the Bader charges of Ir (+1.52–+1.85e) are slightly reduced when compared to their pristine counterparts.To uncover the influence and relationship of the atomic charges of Ir and O atoms and the associated structural motifs, in Fig. d states are calculated and plotted according to their Wyckoff positions. Noting the down-shift in the Ir 5d states to lower energy and the decreased Bader charges, it is indicative that electron transfer has occurred from the intercalated K atom to the nanoporous IrO2 host structure12.This reduction in the Bader charges of Ir can be correlated to the changes in the projected density-of-states (pDOS) for Ir in both the pristine and K-intercalated iridium oxides. In Supplementary Fig. e for R-IrO2, and the corresponding values for O atoms in crystalline IrO1.5, and IrO3 are between −1.14 to −1.02 and −0.85 to −0.74e, respectively. Here, we find that the oxygen atoms in the K-intercalated nanoporous iridium oxides become more nucleophilic  when compared to that of the pristine counterparts. For instance, the calculated Bader charges of O atoms in 1K + Ho-IrO2 are between −1.05 to −1.00e, as compared to that in pristine Ho-IrO2 (−0.99 to −0.94e). Referring to Fig. 2 and 4K + To-IrO2. It now appears that K intercalation in these nanoporous iridium oxides adds extra thermodynamic stability but may not have a positive effect in generating more electrophilic oxygens needed for better OER performance.Now, turning to the atomic charges of O atoms, we find a negative Bader charge value of −1.04a-IrO2, a-IrO1.5, and a-IrO3). From Fig. a-IrO2 are calculated to be between +1.62–+2.61 and −1.29 to −0.62e, respectively (in contrast to +2.06 and −1.04e for R-IrO2). Similarly, the spread  of the calculated Bader charges for a-IrO1.5 , and +1.75–+2.54 (for Ir) and −1.17 to +0.12e (for O), respectively.Along the same vein of discussion, we notice a more flexible range of charge states for both the Ir and O atoms in the amorphous iridium oxides  is determined as a key descriptor for enhanced OER performance in amorphous iridium oxides19.It is worth noting that the very small positive values in the Bader charges of O atoms for 15. To tie the electronic structure argument back to its chemical bonding characteristics, we calculate and report the averaged projected crystal orbital Hamilton population  between2 structures, a down-shift to lower energies in the pCOHP plot is observed, corroborating well with the pDOS in Supplementary Fig. 41 in iridium oxides, we calculate the integrated pCOHP (IpCOHP) by integrating the pCOHP of specific bonding pairs of interest.From Fig. 2 is reduced compared to that of R-IrO2, indicating a corresponding reduction in bond strength for the Ir–O bonds. Specifically, K-intercalation further reduces the −IpCOHP values, alluding that the intercalation of K+ ions in hollandite-type IrO2 is also experimentally shown to reduce the OER overpotential42. In the case of amorphous a-IrO2, a-IrO1.5, and a-IrO3 , respectively. This then further lends support to the presence of flexible charge states of Ir  in the amorphous iridium oxides, and explaining the enhancement in OER performance.More importantly, from Fig. 2(110), Ho-IrO2(100), a-IrO2(001), and a-IrO1.5(001)—and their calculated −IpCOHP values as a function of Ir–O bond length are shown in Fig. G for the AEM and LOM mechanistic steps36 via the computational hydrogen electrode (CHE) approach44 while considering the effect of surface coverages under reaction conditions46. The Gibbs energy diagrams for O*-covered Ho-IrO2(100) and O*-covered a-IrO1.5(001) are presented in Fig. 2(110) and O*-covered a-IrO2(001) can be found in Supplementary Fig. Extending this argument from the bulk phases of iridium oxides to their surfaces, we have chosen and constructed four different surface models—namely, R-IrO2 (including the nanoporous IrO2 systems), the calculated value of ηLOM (~2 V) is almost two times larger than that of ηAEM (~1 V), as shown for O*-covered Ho-IrO2(100) in Fig. 2(110) in Supplementary Fig. 36, and is in line with recent experiments47 where the contribution of the LOM mechanism to the OER on polycrystalline IrO2 is deemed negligible. In contrast, the calculated overpotential values for the amorphous (non)stoichiometric IrO2 phases differ less  are 0.59 and 0.44 V, respectively; see Fig. ηAEM and ηLOM for OER on the amorphous IrOx surfaces may stem from their ability to display a more flexible range of Ir–O bond lengths (and hence bond strengths) as captured by the Pauling’s empirical relation in Fig. Generally, for the crystalline phases of IrO12 for R-IrO2 (0.40 V) and 1K + Ho-IrO2 (0.34 V) and correlate that to our DFT-calculated −IpCOHP for R-IrO2 (3.54) and 1K + Ho-IrO2 (3.21). From literature51, we have further collected the measured OER overpotentials for various oxides—namely, RuO2, SrIrO3, Sr2IrO4, Sr4IrO6, CuFeO3, SrFeO3, and CaFeO3—and performed −IpCOHP calculations using their DFT-optimized lattice parameters . Details of these values are tabulated in Table S3 of the Supplementary Information.To provide a perspective of how activated Ir–O bonds (i.e. stretched Ir–O bonds) may be correlated to the overall OER performance, we put forth a simple linear regression model to estimate the OER overpotential of these amorphous and nanoporous iridium oxides. From literature, we obtain the experimentally measured overpotential2, 1K + Ho-IrO2, SrIrO3, Sr2IrO4, and Sr4IrO6, a least-squared linear regression is plotted in Fig. 2 and the iridates  scatter close to the linear regression line while the ferrites  deviate from this linear behavior, highlighting our simple regression line may capture the needed chemistry to estimate the OER potentials of iridium oxides studied in this work. In this line of argument, RuO2 exhibits a larger overpotential due to stronger Ru–O bonds while the iridates may show comparable or even better OER performance as compared to the reference R-IrO2.By using the experimentally measured overpotential values and our DFT-calculated −IpCOHP values of bulk R-IrO2 are predicted to exhibit a slightly lowered overpotential of 0.37, 0.37, and 0.36 V, respectively, in comparison to experimentally measured overpotential 0.4 V for R-IrO212. Upon K-intercalation, a further decrease in overpotential values to 0.32 and 0.29 V is seen for 2K + Ro-IrO2 and 4K + To-IrO2, respectively. For crystalline IrO1.5, the estimated overpotential values are comparatively lower at 0.26 V while that for crystalline IrO3 is the largest at 0.52 V.Using this empirical linear regression model (based on the Ir-based oxides), we can now empirically estimate the OER overpotential of the crystalline, nanoporous, and amorphous iridium oxides from their corresponding calculated −IpCOHP values. In particular, pristine nanoporous Ho-, Ro-, and To-IrO2. However, given the presence of flexible atomic charges and activated/stretched Ir–O bonds in these amorphous iridium oxides, a much lower limit of ~0.20 V may be attained . This is qualitatively inline with our calculated overpotential trends when comparing the crystalline versus amorphous iridium oxide phases , a schematic perspective plot Fig. b may be 2 and IrO3. This report marks the first time amorphous oxides of iridium are discussed. Under an electrochemical environment, we predict that the intercalation of K in nanoporous IrO2 may be stable if the formation of rutile-type IrO2 is kinetically hindered. When considering the atomic Bader charges of these IrOx polymorphs, it becomes apparent that nonequivalent connectivity in the amorphous structures strongly enhance the flexibility of the charge states of Ir, and hence promoting the presence of electrophilic oxygens in them, as compared to their crystalline counterparts. Using the COHP-bonding analysis, we obtain a Pauling-like relation between the Ir–O bond length versus bond strength for the Ir–O bonds in amorphous iridium oxides while a fairly linear trend is found for the crystalline analogs. This also corroborates with the proposal of flexible charges states and activated bonds for more efficient OER catalysis. Lastly, using an empirical regression model between the Ir–O bond characteristics and the measured OER overpotentials, we provide a perspective as to how these less understood metastable nanoporous and amorphous iridium oxides can offer a breakthrough in OER performance by a notable lowering of the anode overpotential. Here, we offer a fundamental atomistic picture to explain and reconcile the superior OER performance of sub-stoichiometric amorphous iridium oxides (where some channel-like microstructures have been observed) in recent OER experiments.In conclusion, we have expanded and considered various nanoporous  and amorphous iridium oxides of different chemical stoichiometries. They are considered metastable with reference to the commonly reported ground state structures of IrO53 as implemented in the Vienna Ab initio Simulation Package (VASP)55. The 6s and 5d states of Ir, 2s and 2p states of O, and 3s, 3p, and 4s states of K are explicitly considered as the valence states within the PAW approach. The optB86b exchange-correlation (xc) functional is used, treating the DFT xc energy using a self-consistent van der Waals-corrected semi-local generalized gradient approximation56. The optB86b xc functional has been shown to adequately describe various physicochemical properties of iridium oxides57. All DFT calculations have been tested for convergence of kinetic energy cutoff and k-points, where total energies and forces do not change more than 20 meV and 0.02 eV Å−1, respectively. A planewave kinetic energy cutoff of 500 eV and a Γ-centered k-point grid spacing of 0.15 Å−1 are used.The DFT calculations are performed employing the projector augmented wave (PAW) methodaiMD) calculations are conducted at constant pressure (i.e. using the NPT ensemble) following the method of Parrinello and Rahman59. The Langevin thermostat is used to control the temperature modulation with a friction coefficient of 10 ps−1. To model the amorphous phases of iridium oxide with different stoichiometry, the 96-atom and 216-atom supercells of the cubic IrO2 pyrite phase, the 80-atom cell IrO1.5 bixbite phase, the 240-atom IrO1.5 corundum phase, and the 96-atom and 192-atom supercells of the 3 are adopted as initial atomic configurations for the respective stoichiometries. For these large supercell structures, the Brillouin zone is folded to the Γ-point and the kinetic cutoff energy is lowered to 300 eV. The initial structures are randomized at 3000 K for 10 ps (mimicking the melting process) and then quenched from 3000 to 100 K in 5 ps, with a cooling rate of 580K/ps. We further equilibrate the quenched structures at 300 K for another 10 ps in order to get more representative geometries at the temperature. For all aiMD calculations, a time step of 1 fs is used for the integration of the equations of motion.Ab initio molecular dynamics  thermodynamic stability, and chemical bonding and orbital population via the LOBSTER code63 are detailed in the Supplementary Information (SI).The methods used to analyze the structural descriptorsSupplementary Information


Genetic diagnostic methods for evaluation of chimerism after HSCT, such as STR-PCR and XY-FISH, have limited sensitivity. When donor chimerism is in the micro range (< 1%), deviations in the accuracy of assessment are the most significant disadvantage of these common methods. We developed a highly sensitive method that applies SNPs based on NGS in order to explore the value of donor cell microchimerism in microtransplantation (MST). This improved SNP-NGS approach has higher sensitivity (0.01–0.05%) and only requires a small amount of DNA (8–200 ng). We retrospectively analyzed the clinical data of 48 patients with AML who received HLA-mismatched stem cell MST at our center to assess the impact of microchimerism on clinical prognosis. Patients whose duration of microchimerism was > 10.5 months (median) had a relapse rate of 26.1%, and had better 5-year LFS and OS (73.4% and 82.6%). In contrast, patients whose duration of microchimerism was < 10.5 months had a higher relapse rate (69.6%), and their 5-year LFS and OS were 30.4% and 43.5%. In conclusion, duration of donor chimerism is highly valuable for assessment of survival and prognosis in patients with AML who have received HLA-mismatched stem cell MST, especially the intermediate-risk group.The online version contains supplementary material available at 10.1007/s12185-022-03415-8. Allogenic hematopoietic stem cell transplantation  is the predominant treatment used to cure malignant and non-malignant hematological disorders. Analysis of chimerism following allogeneic HSCT has been a routine method for the assessment of engraftment and early detection of graft failure. It is helpful in guiding risk-adapted immunotherapy for prophylaxis and interventional treatment of graft-versus-host disease (GVHD) or imminent relapse , 2. HLA-Polymerase chain reaction for short tandem repeats (STR-PCR), revealed by conventional agarose-acrylamide gel electrophoresis, is the most commonly used method for analysis of chimerism due to the high informativity and discrimination between donor and recipient. This method has a moderate sensitivity (between 1 and 5%) , 9. ChimThe main aim of the present investigation was to develop a custom bioinformatic pipeline for chimerism and microchimerism quantification based on SNP-NGS. We retrieved SNP loci with a frequency of 45–55% in the East Asian population from GenBank and performed linkage disequilibrium analysis to screen 48 SNP loci from 22 different autosomes. It is expected that the resolution of these sites to distinguish donors and recipients is 99.99%. We designed a primer pool for SNPs to achieve the probability of discriminating between donors and recipients. Meanwhile we compared the efficacy of SNP-NGS with that of STR-PCR and XY-FISH for chimerism quantification. In addition, we explored the application of this method in prognosis assessment of MST. To further explore the value of donor cell microchimerism in MST, our center retrospectively analyzed the clinical data of young AML patients who used MST consolidation therapy after the first complete remission and used SNP-NGS to monitor the donor microchimerism post MST and assess the impact of microchimerism on the clinical prognosis of patients.Studies were approved by the Human Ethics Committee of the First Affiliated Hospital of Soochow University in accordance with the Declaration of Helsinki protocol. Written informed consent was obtained from all participants. A total of 48 eligible AML patients, 12–59 years of age, were enrolled in this study. Patient clinical characteristics are shown in Table Samples of peripheral blood from two healthy volunteer donors (V1–V2) were collected for sensitivity testing of chimerism detection. All peripheral blood samples were separated into mononuclear cells by Ficoll density gradient centrifugation. Mononuclear cell samples from V1  and V2  were paired as “donor” and “recipient,” respectively. Seven artificial chimeric mononuclear cell mixtures, as donor/recipient chimerism, were mixed by counting at seven percentages  for each artificial mixture (aCh).http://www.ncbi.nlm.nih.gov/SNP/). The SNP loci of the patients are shown in Fig. S1. 48 primer pairs (Table S1) were designed in the multiplex reaction of amplification donor DNA. A total of 4–6 base sequences were used as label regions for primer design in Primer Pool, such as AGTC, CCAA, GTCT, TAGG, ACAG, TGCT, CTTC, or GAGA. By introducing label regions, it is possible to remove contaminant interference. The multiplex PCR mixture contained 8–200 ng genomic DNA (quantified by Qubit® dsDNA HS Assay Kit), 8 μl PrimerMix (100 nmol/l), and 15 μl high-fidelity PCR Master Mix  in a total volume of 30 μl. The reaction conditions were as follows: 95 ℃ for 3 min (holding stage), 18 cycles of 95 ℃ for 15 s, and 60 ℃ for 4 min (cycling stage). The purification operation was performed using nucleic acid purification kit magnetic beads (Beckman AMPure XP Beads) as target DNA product is bound to magnetic beads. A second round of PCR purification was performed. The SNP-PCR mixture contained the DNA product bound to the beads from the previous step, 1 μl forward universal sequencing primer , 1 μl barcode sequencing primer (R_index: CAAGC AGAAG ACGGC ATACG AGAT) (10 μmol/l), and 15 μl high-fidelity PCR Master Mix  in a total volume of 30 μl. The reaction conditions were as follows: 95 ℃ for 1 min (holding stage), 6 cycles of 95 ℃ for 15 s, and 60 ℃ for 15 s and 72 ℃ for 15 s (cycling stage). Subsequently, the purification operation was also performed using nucleic acid purification kit magnetic beads (Beckman AMPure XP Beads). Finally, the target PCR product was eluted from the beads and recovered for high-throughput sequencing.Forty eight single-nucleotide, biallelic, polymorphisms were selected from the Single Nucleotide Polymorphism database (dbSNPs)  . For the8/kg) and 1.32 × 106/kg , respectively.Donors were subcutaneously injected with 10 μg/kg G-CSF for 5 days to mobilize peripheral hematopoietic stem cells. Apheresis of donor peripheral mononuclear cells was performed using a COBE SPECTRA cell separator  and aliquoted into three parts. One of three fresh donor cells was used for the first course, and the remaining parts were cryopreserved in liquid nitrogen for the second and third courses. The median numbers of mononuclear and CD34 + cells infused per course were 2.39 × 108/kg -mobilized donor consolidation treatment of peripheral blood stem cells (G-PBSCs) on day 7. No preventive treatment of GVHD was given after G-PBSC infusion. The interval between the cycles was 2–2.5 months. G-CSF was given (300 μg/d) when the neutrophil count (ANC) was < 0.5 × 109/L. When the level of hemoglobin was < 70 g/L and platelet count was < 20 × 109/L, red blood cell and platelet transfusion were given, respectively. Antibacterial drugs, antifungal drugs, and antiviral drugs were administered according to institutional guidelines.Remission induction chemotherapy consisted of maintenance 24 h intravenous drip of Ara-C 100 mg/m2 daily for 7 days or idarubicin 12 mg/m2 daily for 3 days. If the patient did not achieve complete remission (CR) after the first course of treatment, a second cycle of the same induction therapy was recommended. Patients who achieved complete remission after one or two cycles of induction therapy were enrolled in this study. These 48 patients who achieved CR1 were assigned to receive three further courses of consolidation chemotherapy consisting of the following: Ara-C 2.0 g/mP < 0.05 indicates that the difference is statistically significant.SPSS version 19.0  software was used for all statistical analyses. Survival data were analyzed by means of log-rank test, and survival curves were generated using the Kaplan–Meier method. Survival curves were generated using the Kaplan–Meier method. The probabilities of OS and LFS were estimated by means of the log-rank test. A total of 7 artificial chimerism (aCh1-7) were prepared in a fixed detection range (0.01–10.0%). Sequencing results were fully consistent with the known genotype of the donors and recipients. Subsequent to Illumina sequencing and using the custom tool, quantitative data for all SNPs of each artificial chimerism were obtained. The detection results were visually inspected to confirm the presence of donor chimerism or microchimerism.The SNP-NGS analysis of artificial chimerism or microchimerism showed that the limit of detection for a minor cell population is nearly 0.01%. Figure Multiple STR-PCR was performed using artificial chimerism aCh1-3) ranging from 1.0 to 10.0%  or the number of CD34+ cells  (data not shown). Among 227 samples from 48 patients, 219 samples had microchimerism of less than 1%.All patients were consecutively monitored for donor chimerism or microchimerism using SNP-NGS technology. The donor chimerism or microchimerism were detected at + 14 d, + 28 d, + 2 m, + 3 m, + 6 m, + 9 m, + 12 m, + 18 m, + 24 m, + 30 m, + 36 m after GPBSC infusion. Forty-six patients received long-term chimerism monitoring after completing all consolidation treatments. The remaining two patients only tested for donor chimerism two months after the treatment, one in the low-risk group and another was in the intermediate-risk group. The donor cell chimerism or microchimerism dynamics of all patients are shown in Fig. χ2 = 8.712, P = 0.003) was found in patients whose persistence of chimerism or microchimerism > 10.5 months. Thirteen patients received long-term monitoring of donor chimerism or microchimerism in intermediate risk group. The median of nonrelapsed and relapsed patients was 15 months (9–30 months) and 6 months (2–12 months), respectively (P = 0.009). The 13 cases were divided into two subgroups by a median time of 10.5 months. The relapse rate of the subgroup < 10.5 months was 85.7% (6/7), and lower relapse rates   were seen in the other subgroup. These data suggested that patients with a long persistence of donor microchimerism have a lower relapse rate.The persistence of chimerism or microchimerism varied from 2 to 30 months. The median was 10.5 months. The median duration of donor chimerism or microchimerism in patients with relapsed disease was 6 months (2–18 months), and the median in patients without relapsed was 12 months (2–30 months). In patients whose persistent time of donor chimerism or microchimerism was < 10.5 months, the relapse rate was 69.6% (16/23). A lower relapse rate  , and the median OS was 53.5 months (4–84 months). The 5-year LFS and OS rates were 51.6% and 62.5%, respectively . No patient was lost during the follow-up period. The incidence of relapse in low-risk patients was 35.7% (10/28), of which 3 patients reached CR2 after reinduction therapy, successfully completing myeloablative allo-HSCT to achieve long-term survival. The incidence of relapse in intermediate-risk patients was 57.1% (8/14), of which only one patient achieved long-term survival after treatment after myeloablative allo-HSCT. Five patients in the high-risk group  experienced relapse, and eventually, all died of the disease. The 5-year LFS rates were 63.3%, 42.9%, and 16.7% in the low-risk, intermediate-risk, and high-risk groups, respectively. The 5-year OS rates were 78.6%, 50.0%, and 16.7% in the low-risk, intermediate-risk, and high-risk groups, respectively . Pairwise comparisons between low-risk, intermediate-risk, and high-risk groups are statistically significant.For further analysis, the 46 patients who received long-term monitoring were divided into 2 groups based on the median of persistent time of donor chimerism or microchimerism (10.5 months). Among them, patients with persistence of chimerism or microchimerism > 10.5 months were group 1, and those with persistence of chimerism or microchimerism < 10.5 months were group 2. The 5-year LFS and OS of group 1 were 73.4% and 82.6%, respectively, and the 5-year LFS and OS of group 2 were 30.4% and 43.5%, respectively . These data highlight the importance of the novel detection method with high sensitivity and accuracy. The detection data confirmed that the persistence of donor microchimerism in patients who did not relapse after MST was significantly longer than that of relapsed patients. Moreover, subgroup analysis showed that the persistence of donor microchimerism had a more significant impact on the survival of AML patients in the intermediate-risk group. Patients at intermediate risk with longer persistent time of donor microchimerism had better LFS and OS. In previous reports from our center and the Affiliated Hospital of the Academy of Military Medical Sciences, the hematopoietic recovery time was shorter in the MST group than in the MSD (HLA-matched sibling donor) group. Other advantages of MST are the lower infection rate and lower incidence of GVHD , 16. TheOur results show that microtransplantation is safe and effective in treating young patients with low- and intermediate-risk AML, and SNP-NGS can accurately detect the microchimerism status of donor cells. Remarkably, the duration of donor microchimerism is of great value for the survival and prognosis assessment of AML patients, especially in the intermediate-risk group. Patients at intermediate risk with longer donor microchimerism have better LFS and OS. Therefore, a well-established universal method is warranted to examine donor-cell microchimerism. Our research mainly focuses on the establishment of methodologies that overcome the limitations of current technologies in sensitivity and accuracy. Improving the accuracy of chimerism detection can help provide important auxiliary means for recurrence monitoring and survival assessment of patients after transplantation.Supplementary file1 (DOCX 62 kb)Supplementary file2 (PDF 85 kb)Supplementary file3 (TIF 2722 kb)Below is the link to the electronic supplementary material.