A total of 17 cases were identified as probably caused by specific etiological factors (F06.2), of which ten cases fulfilled the criteria for a probable autoimmune psychosis linked to the following autoantibodies: amphiphysin, CASPR2, CV2, LGl1, NMDA, zic4, and titin. Two cases presented with anti-thyroid tissue autoantibodies. In four cases, further probable causal factors were identified: COVID-19, a frontal intracranial tumor, multiple sclerosis (n = 2), and neurosyphilis. Twenty-one cases remained with "no reliable diagnostic classification". Age at onset of psychotic symptoms differed between patients diagnosed with F2 and F06.2 (p = 0.014), with the latter group being older (median: 44 vs. 28 years). Various CSF parameters were analyzed in an exploratory analysis, identifying pleocytosis and oligoclonal bands (OCBs) as discriminators (F06.2 vs. F2) with a high specificity of > 96% each. No group differences were found for gender, characteristics of psychotic symptoms, substance dependency, or family history. This study emphasizes the great importance of a detailed diagnostic workup in diagnosing psychotic disorders, including CSF analysis, to detect possible underlying pathologies and improve treatment decisions.In current international classification systems , the diagnostic criteria for psychotic disorders (e.g. schizophrenia and schizoaffective disorder) are based on symptomatic descriptions since no unambiguous biomarkers are known to date. However, when underlying causes of psychotic symptoms, like inflammation, ischemia, or tumor affecting the neural tissue can be identified, a different classification is used  or psychosis caused by medical factors (DSM5)). While CSF analysis still is considered optional in current diagnostic guidelines for psychotic disorders, CSF biomarkers could help to identify known physiological conditions. In this retrospective, partly descriptive analysis of 144 patients with psychotic symptoms and available CSF data, we analyzed CSF examinations' significance to differentiate patients with specific etiological factors (F06.2) from patients with schizophrenia, schizotypal, delusional, and other non-mood psychotic disorders (F2). In 40.3% of all patients, at least one CSF parameter was out of the reference range. Abnormal CSF-findings were found significantly more often in patients diagnosed with F06.2 (88.2%) as compared to patients diagnosed with F2 (23.8%,  In current disease classification systems  or Diagnostic and Statistical Manual of Mental Disorders 5(DSM-5)), psychiatric disorders are primarily classified using symptom descriptions3. However, in cases in which a specific morphologically comprehensible pathophysiological process is assumed to cause psychotic symptoms , a causal attribution is made4.The terminology for these cases has changed in recent years. Currently, the DSM-5 defines them as "psychotic symptoms caused by a medical factor"; the ICD-10 as “psychotic disorder with delusions due to known physiological condition (F06.2)” (or organic psychosis). Exclusion diagnostics are maintained to distinguish between psychotic symptoms as part of disorders classified as F2 "Psychotic disorder with delusions due to known physiological condition" (ICD-10/DSM-5) and psychotic symptoms as part of F06.2. Exclusion diagnostics of alcohol-/drug-associated, systemic, or other brain organic causes are based on a physical examination, neuropsychological testing, routine blood testing, urine drug screening, breath/blood alcohol test, electroencephalography (EEG), and brain structural imaging (MRI) at initial manifestation5. Cerebrospinal fluid (CSF) analysis is considered optional. However, according to the recently updated guidelines of the German Psychiatric Association, it should be offered to patients, particularly when clinical signs indicate that psychotic symptoms might be caused by a pathological process that is associated with specific known biomarkers6. Psychosis was already identified as a possible immunological disease in 1930 by Lehmann-Facius amongst others, leading to the autoimmune hypothesis of schizophrenia9. In a recent consensus statement, the concept of autoimmune psychosis was updated, including the increasingly important group of autoantibody-associated autoimmune encephalitis12.Despite extensive research, the pathophysiology of schizophrenia and schizoaffective disorders has not been clarified yet5. Before, patients were mainly tested for antineuronal autoantibodies against intracellular antigens in the context of paraneoplastic processes. Lately, several other antineuronal antibodies against cell surface antigens  have been associated with psychotic symptoms.After discovering the anti N-methyl-D-aspartate receptor encephalitis in 2007, autoimmune psychosis rapidly became an important field of study13. Thus, the identification of specific underlying pathophysiologies is crucial since this may allow for more specific treatment and therefore better outcomes. Currently, immunomodulatory treatments in autoimmune psychosis10 or multiple sclerosis14, search and removal of extracranial tumors16, vitamin substitution17, or antibacterial treatment e.g. in syphilis19 are considered complementary to therapies for psychotic symptoms.Specific etiological processes are estimated to be present in up to 25% of patients with psychotic symptomsPsychotic symptoms as part of F2 "Schizophrenia, schizotypal, delusional, and other non-mood psychotic disorders" according to ICD-10 (abbr.: F2)Psychotic symptoms as part of F1 "Mental and behavioral disorders due to psychoactive substance use"/F3 "Mood [affective] disorders", F4 "Anxiety, dissociative, stress-related, somatoform and other nonpsychotic mental disorders", F5 "Behavioral syndromes associated with physiological disturbances and physical factors", and F6 "Disorders of adult personality and behavior" in ICD-10 (abbr.: F1/F3-F6)Psychotic symptoms as part of F06.2 "Psychotic disorder with delusions due to known physiological condition" in ICD-10 (abbr.: F06.2)"No reliable classification"In this publication, we will use a descriptive approach in our nomenclature:20; therefore, our secondary aim was to confirm F06.2 patients being older at disease onset than F2 patients. We aimed to identify the CSF parameters with the highest discriminatory power for F06.2 vs. F2 in a further exploratory approach. We also analyzed demographics, psychopathological symptoms, comorbidities, and family history of the cohort.In this retrospective analysis, we aimed to assess the significance of CSF examinations to differentiate psychotic disorder with delusions due to known physiological condition (label used: F06.2) from schizophrenia, schizotypal, delusional, and other non-mood psychotic disorders (label used: F2) in patients with psychotic symptoms. We hypothesized that the rate of abnormal CSF findings would be higher in patients diagnosed with F06.2 than in patients diagnosed with F2. Later age of onset is suggested to be a red flag for F06.2This study is a retrospective monocentric  analysis of a cohort of 144 psychotic inpatients with CSF analysis, between 01/2016 and 06/2020 (42 months). For the patients recruiting scheme, see Fig. All patients admitted to any of the four acute admission wards or two wards specialized in the psychotherapy of psychotic disorders (n = 4041) were screened for CSF examination lab data. From this cohort of all CSF analyses (n = 401), patients with a discharge diagnosis of dementia  were excluded. The remaining patients were screened for any psychotic symptom  and delusion ) in their history or admission report ; this yielded n = 144 for further in-depth analysis.23. In contrast, for hallucinogens  and stimulants , psychotic symptoms usually persist for up to one month and rarely up to three months or longer despite ongoing abstinence25. The necessary abstinence periods were chosen depending on the substance used (up to six months) and a diagnosis of F20 was assigned when psychotic symptoms persisted for an additional four weeks as required by the ICD-10.Patients were assessed during their hospitalization, including a neurological examination. Psychiatric symptoms were assessed and documented by the attending physician as well as a resident physician or clinical psychologist following ICD-10 diagnostic guidelines. Additional information from previous hospital admissions was acquired, particularly regarding substance abuse/dependency, previous neurological diseases, and family history. We evaluated the plausibility for drug-induced psychosis with regard to the specific substance. For alcohol, cannabis, and cocaine, psychotic symptoms usually subside within one to four weeks after substance withdrawal26. For antibody screening, the commercially available IIFT kit, including hippocampus and cerebellum (FA 111 m-###-3) was used; for antibody differentiation, we utilized the IIFT Biochip Mosaic (FA 112d-###-6) and the anti-neuronal blot (DL 1111–1601-7 G) .Patients' samples (blood and CSF as paired samples) were routinely analyzed in the Institute for Clinical Chemistry and Pathobiochemistry; neurodegenerative markers and oligoclonal bands (OCBs) were analyzed in the Clinical and Chemical Laboratory "CSF laboratory", Center for Neurology, University Hospital Tübingen. OCBs were classified as types I-IV, according to the 2005 consensus statement27. Age-dependent upper normal CSF values, as published by Yilmaz were used , cutoffs are specified in Fig. The NF-LIGHT ASSAY ELISA was performed as previously publishedAs an operationalized group assignment, a dichotomous categorization into normal and abnormal CSF was done  patients with psychotic symptoms and a presumed causal origin. (4) Cases fulfilling criteria for possible autoimmune psychosis (according to Pollak) as well as cases that were not sufficiently explained with the examination results available were assigned to the "no reliable classification" category.A second group allocation was based on all available clinical information, including ICD-10 diagnostic criteria, MRI and EEG findings, clinical examination, laboratory results , and patients' histories. In some cases, the discharge diagnosis was adapted according to examination results to allocate the patients into four groups . According to German law and the Institutional Review Board, there was no necessity to retroactively obtain informed consent due to the importance of this study subject and the retrospective study design.p-values29.SPSS 26  was used. The Shapiro–Wilk Test tested Gaussian distribution due to 3 < n < 3000. For the demographic data analysis, all variables were tested using the Mann–Whitney-U-test , and the chi-square test analyzed gender as nominal values using a two-sided exact p-value. For group comparisons of the four disease classifications , the Kruskal–Wallis-test was used with Dunn-Bonferroni-testing as posthoc analysis. Bonferroni correction was applied to correct for multiple testing for the two main hypotheses  presented in Table All further data analyses were exploratory and no further correction for multiple testing was applied. Bivariate correlations were analyzed using Pearson's coefficient or Spearman's rank correlation coefficient, respectively.p < 0.00001 (Table s (144) = 0.72, p = 0.388.We were able to confirm our primary hypothesis of a significantly higher frequency of abnormal CSF findings in F06.2 compared with F2 with p = 0.11) or gender. There was a group difference regarding the presumed underlying pathology when comparing F06.2 with F2 for age at examination  higher age at onset in F06.2 than F2 patients (44 vs. 28 years) (Table 4 higher All psychotic symptoms (according to ICD-10 diagnostic criteria for schizophrenia) did not differ between the groups in our exploratory analysis (see Table p = 0.09) when comparing "no reliable classification" (median: 1 ± IQR: 2) [range: 0–3]) vs. F2 (1 ± 1 [0–3]) as well as hallucination modalities  for "no reliable classification" (1 ± 1 [0–2]) vs. F2 (0 ± 1 [0–3]) as well as  F06.2 (depression strength (1 ± 2 [0–2]); hallucination modalities (0 ± 2 [0–2])) vs. F2.In addition, there were no group differences between the F2 and the "no reliable classification" group neither for psychotic symptoms nor any comorbidity or positive family history. Severity of depressive symptoms  was not different at a group level (probable autoimmune psychosis (according to Pollak10) (n = 10) and b) psychosis of probable other etiology (n = 8). Since we did not find well-operationalized criteria for non-autoimmune symptomatic psychosis in the literature, we tried to apply analogous criteria and counted six cases, which presumably have an identifiable underlying pathology (non-bold cases in Table 10 stated that the significance of thyroid antibodies for autoimmune psychosis is still unknown. One case (#16—SREAT) had highly elevated TPO-antibodies and TRAK-antibodies in serum; the other case (#77) is mentioned above in the positive CSF findings.This group is composed of two subgroups: a) 10) as probable autoimmune psychosis and are listed in Table probable autoimmune psychosis cases (57.1%): anti-CASPR2 associated psychosis with ovarian teratoma , anti-CASPR2 associated psychosis without confirmed tumor (#116), serum anti-zinc finger protein 4 (zic4) and anti-titin (#139), and anti-amphiphysin- (#98). Three cases were without serum or CSF antibodies, but CSF pleocytosis as the probable autoimmune psychosis defining parameter according to Pollak10.A total of ten cases (6.9% of the cohort with psychotic symptoms of n = 144) were classified , but no differences for total protein, albumin quotient (CSF/serum), as well as IgG quotient (CSF/serum). OCB prevalence in the F06.2 group was higher for types II and III than in F2 as tested by chi-square testing X2  = 23.974, p < 0.0001. There were no group differences in CSF neurodegenerative markers .The exploratory comparison of different CSF parameters with regard to their discriminatory power between F06.2 and F2 revealed pleocytosis and OCB to be the most relevant parameters  = 27.66, p < 0.0001, and there was a significant higher NfL level as tested by Mann–Whitney-U-test (p = 0.014) in the "no reliable classification" compared to F2.A total of 21 cases remained unresolved despite intensive diagnostics and were therefore categorized as "no reliable classification". The psychotic symptoms were found not to be sufficiently explained by the abnormal examination results, including 5 for possible autoimmune encephalitis as well as Pollak's10 for probable autoimmune psychosis. In contrast, our cohort's specificity for pleocytosis (F06.2 vs. F2) was relatively high with 96.4% and only three cases (3.6%) being false-positive. CSF protein was elevated in 18.1% of cases, markedly less than a similar cohort published by Endres7 or half of the prevalence (40%) in n = 188 first episode schizophreniform syndromes30.There was a high prevalence of abnormal CSF findings  in the study cohort. According to our primary hypothesis we observed a higher prevalence in F06.2 (88.2%) compared to F2 (23.8%). Pleocytosis seems not to be very sensitive (sensitivity of 47.1% to distinguish between F06.2 and F2) for the identified autoimmune psychosis cases despite being one of Graus' criteria26, seem to be relatively common in patients with psychotic symptoms30 . In our "no reliable classification" group  to unravel further anti-neuronal targets in autoimmune psychosis.OCBs with specific (type II) or partially specific (type III) intrathecal IgG production, according to Freedman33), 4% (n = 5034) to up to 7% (n = 4135). In contrast, in all four operationalized groups combined, about 11% of cases had OCB type II or III; this is almost double the occurrence rate noted in healthy controls. Endres et al.30 found 10% of OCB type II and III in n = 188 first episode schizophreniform syndromes. Healthy siblings of MS patients had 18–20% positive OCBs, probably vastly overestimating OCBs in healthy subjects, as there are shared genetic and environmental influences36; but not all OCB-positive cases should routinely be considered an autoimmune psychosis. OCB-negative MS cases depend on genetic variants being major determinants of CSF antibody levels37. We presume genetic predisposition plays a role concerning antibody levels and OCBs in psychosis.To our knowledge, the occurrence of OCBs in healthy control subjects has not yet been systematically studied; findings vary from 0%  to discriminate F06.2 from F2  had NfL levels above the upper reference of two standard deviations. Therefore, even in healthy controls, increased NfL levels can occur, explaining some of the "no reliable classification" cases with increased NfL levels.NfL is a rather unspecific biomarker for axonal degeneration, showing increased CSF concentrations in neurodegenerative diseases41. Therefore, the high number of ANA-positive cases in our "no reliable classification" group is not surprising. ANA have been discussed to play a role in affective disorders, but studies showed mixed results (reviewed in 42); this also applies for schizophrenia (11% in an exemplary study43 for n = 28) with an extensive study of n = 368 patients and n = 283 controls showing no differences in ANA occurrence, but a prevalence of 11% of ANA positive cases in healthy controls44.The occurrence of antinuclear antibodies (ANA) in healthy individuals seems to be stable throughout different age groups and ranges from 31.7% (1:40 serum dilution), 13.3% at 1:80, 5.0% at 1:160, to 3.3% at 1:320 in a published putative normal population46, leading to categorization in the F06.2 category. The patient presented with acute onset of psychotic symptoms combined with respiratory symptoms (coughing and dyspnea), hyposmia, CSF pleocytosis, and elevated total protein. There was a close coincidence of psychotic exacerbation and PCR detection of the SARS-CoV2 virus in a throat swab with simultaneous improvement of psychotic and respiratory symptoms through the course of the disease. Parra et al.47 report further evidence of a causal relationship of COVID-19 and psychosis beyond acting as a stress factor.Case #11 was initially categorized in the "no reliable classification" group. However, the association of COVID-19 and psychotic symptoms was just recently described in larger cohortsprobable autoimmune psychosis cases (n = 10) with regard to psychiatric symptoms and clinical features, only the CASPR2 case15 had the "red flag symptom" hyperkinesia, as described by Herken and Prüss 48. No catatonia was found in the subgroup of probable autoimmune psychosis. Two anti-thyroid autoantibody cases  were not classified as probable autoimmune psychosis. Pollak et al.10 rate thyroid antibodies for autoimmune psychosis to be still of unknown significance. When disregarding the anti-thyroid antibodies, the two cases did not meet criteria for a probable autoimmune psychosis according to Pollak.When analyzing our 49. Endres30 reports up to 65% of MRI imaging abnormalities, including e.g. white matter lesions, which are not necessarily related to psychosis. Despite our smaller sample sizes and even without systematic analysis of brain imaging in this cohort, we identified six cases (4.2%) with structural brain abnormalities  which more than doubles the previously reported prevalence related to psychosis.In a cohort of > 300 MRI and CT brain images in first episode psychosis, a prevalence of approximately 2% of structural brain abnormalities related to psychosis was reported50. With two patients with psychotic symptoms possibly relating to MS, one case (= 50%) had a previous history of MS (> 13 years). In contrast, the second case (= 50%) presented with psychotic symptoms as the initial MS manifestation. Case #98 , most patients did not have a history of MS or psychiatric disease prior to manifestation of psychotic symptoms15). We furthermore identified a small number of cases with other serum antibodies only  used ; thus, there could be some diagnostic uncertainty there as well.This study stands out for its large overall sample size, extensive file research and antibody analysis, as well as the explorative data on CSF levels of neurofilament light chain and non-autoimmune cases with psychotic symptoms in patients with "psychotic disorders with delusions due to known physiological condition (F062.)", including COVID-19. The lack of computerized automated recognition of symptoms from medical files and structured assessment of psychopathology constitutes one of its major limitations. Because of its retrospective nature and open and uncontrolled design, not all patients received the same diagnostic regime. This also precluded analysis of positive findings in healthy adults, similar to a recently published cohort52. In our cohort, 40.3% of cases had abnormal CSF findings (n = 58) with a significantly higher prevalence in the psychotic disorders with delusions due to known physiological condition (labeled: F06.2) (83.3%) than schizophrenia, schizotypal, delusional, and other non-mood psychotic disorders (labeled: F2) (23.8%) group. Of the ten cases with an autoimmune psychosis, nine were identified by CSF examinations. Later onset was found in patients diagnosed with F06.2 compared to F2 (median age difference between patient-groups: 16 years), emphasizing the importance of CSF analysis in patients with advanced age at disease onset. We were able to identify pleocytosis and OCBs to be good discriminators between F06.2 and F2 with high specificity (> 96%). As mounting evidence points to specific underlying pathophysiological processes in psychotic disorders, serum and CSF antibodies should be routinely used for diagnostics.Preventing damage to patients in our care is of utmost priority in medicine. A balance must be struck between economic costs and associated risks for patients to administer medical procedures effectively. The extraction of CSF using atraumatic lumbar punctures has a sensible benefit/risk ratio and should be offered to patients with psychotic symptomsSupplementary Information


The prevalence of idiopathic intracranial hypertension (IIH), a complex disorder, is increasing globally in association with obesity. The IIH syndrome occurs as the result of elevated intracranial pressure, which can cause permanent visual impairment and loss if not adequately managed. CSF diversion via ventriculoperitoneal and lumboperitoneal shunts is a well-established strategy to protect vision in medically refractory cases. Success of CSF diversion is compromised by high rates of complication; including over-drainage, obstruction, and infection. This review outlines currently used techniques and technologies in the management of IIH. Neurosurgical CSF diversion is a vital component of the multidisciplinary management of IIH. Idiopathic intracranial hypertension (IIH) is a rare disorder affecting approximately 0.9–3/100,000 of adults ,3. This Symptomatically, IIH is characterised as a headache syndrome that has significant crossover with migraine disorders, often existing in conjunction . It typiThe underlying aetiology of IIH remains obscure; however, interplay between BMI, raised cranial venous pressure and/or outflow obstruction, and resistance to CSF resorption is the most popular hypothesis . The comIIH continues to present unique challenges to clinicians involved in its management. It is a truly multi-disciplinary condition requiring coordinated input from neurology, ophthalmology, optometry and neurosurgery. For the majority of patients, non-surgical treatments that include medication and weight management suffice in controlling the disease; however, up to a quarter of IIH patients require surgical or interventional radiology treatment in response to visual deterioration .While headache associated morbidity is significant, the threat to vision presents the most alarming consequence of inadequately managed IIH . FortunaCSF diversion constitutes the mainstay of neurosurgical management of IIH. The purpose of this article is to review the practice of neurosurgical CSF diversion in CSF and to outline a ‘what-to-do-when’ algorithm for suspected shunt failure.We performed a comprehensive literature search on the electronic database MEDLINE (PubMed) to identify original research articles and reviews on the neurosurgical management of IIH. A further literature search for published systematic reviews and meta-analyses were performed using MEDLINE (PubMed) and the Cochrane database. Databases were searched using relevant combinations of appropriate keywords, including “idiopathic intracranial hypertension”, “pseudotumor cerebri”, “benign intracranial hypertension”, “CSF diversion”, “ventriculoperitoneal”, “lumboperitoneal”, and “shunt”. Additional studies were found by reviewing the reference lists of the identified publications. A narrative review was produced based on these searches.Neurosurgeons have a very defined role in the management of IIH, and are consulted when there is evidence of papilloedema or visual deficits that are progressive ,21. In tHistorically, a variety of cranial vault expansion and decompression techniques have been applied to the management of IIH. In particular, subtemporal decompression (STD), which is performed via either unilateral or bilateral temporal craniectomy, found some broad application ,25. The The practice of lumboperitoneal shunting was historically the preferred CSF diversion technique as the ventricles in IIH patients are typically small with attendant technical challenges ,31. AcceThe catheter can be sited within the lumbar subarachnoid cistern via a lumbar puncture or under direct vision via an open incision. This catheter is anchored and connected in line with a valve and distal tubing tunnelled to the abdomen. In very rare circumstances, the pleural cavity has also been described as an alternative CSF resorption site .LP shunts have demonstrated efficacy in improving visual symptoms in 55–100% of cases reported and headache symptoms are likewise responsive, up to 76–92% cases report improvement, at least initially ,33,34,35LP shunts are prone to complications, more so than VP shunts. Up to 40% of patients will require shunt revision, with many requiring multiple revisions; median revision per patient rate = 4.3 in a recent systematic review compared with 1.83 for VP shunts . Over-drInsertion of VPS is the most common CSF diversion procedure performed for IIH nowadays and is proven effective ,41,42,43Under general anaesthetic, a ventricular catheter (thin silicon catheter) is advanced into the ventricle, most commonly the right lateral ventricle. Use of antibiotic (rifampicin + clindamycin) impregnated Bactiseal® catheters to reduce the risk of shunt infection is supported by level 1 evidence . This caThe ventricles in IIH patients are not enlarged and typically may even be compressed  41,45].,45.41,45Serious complications associated with CSF diversion techniques are rare, although ventricular shunt placement is associated with more serious complications, including intracranial and intraventricular haemorrhage, infection, and CSF leak . FurtherLP shunt placement is associated with a less serious operative risk as it avoids traversing neural tissue and, thus, the cranial risks of haemorrhage and seizures, and the potential restrictions on driving are negated. Over-drainage and development of secondary Chiari type I malformation are the most commonly reported complications . Injury The vast majority of complications related to VP and LP shunts are mechanical failures and infections. Shunt revision rates overall are up to 25% within the first year and up to 85% within the patient’s lifetime ,55,56. RShunts may paradoxically cause complications due to over-drainage. Development of low-pressure symptom impacts on both VP and LP shunts though LP shunts are more severely affected due to the hydrostatic pressure effect of gravity on the fluid column within the neuroaxis in an upright position . CSF leaThe approach to management of over-draining shunts depends very much on the constituents of the implanted system. Conservative strategies include increasing the opening pressure of programmable valves where inserted, the addition of anti-siphon devices to shunt systems, or the use of gravitational valves. Disconnection and ‘tying-off’ of shunts can be performed, though this requires a further surgery to reconnect the shunt if needed at a later stage.The consensus guidelines on the management of IIH patients produced by the UK IIH special interest group produced a helpful algorithm for management of shunted IIH patients with headache exacerbation and suspected shunt malfunction  12]. Pa. Pa12]. Confidently excluding shunt malfunction or obstruction can be challenging in IIH patients, and surgical exploration to confirm patency or perform a shunt revision may be required, particularly if vision is threatened. As the neurosurgeon must be prepared to revise any or all components of the shunt, the patient is consented and prepped/draped in anticipation of a full shunt revision. Any hardware that may be required  is brought to the theatre in case of need. In the majority of circumstances, only one component requires revision, most frequently the proximal catheter, followed by the valve, and rarely the distal catheter. Attempts should be made to explant failed components; however, this may prove difficult. Ventricular catheters commonly have choroid plexus adherent to them, which can bleed if avulsed. Tunnelled distal tubing that has been in situ for a number of years can be very difficult to extract, is occasionally disconnected, and left in place with new tubing connected to the functioning shunt. Malfunctioning valves should be replaced or correctly functioning valves exchanged for alternative models, e.g., with a higher opening pressure or a valve with a programmable facility in the context of over-drainage. The addition of an in-line anti-siphon device may also be considered.At their inception, the flow rate through CSF diversion systems was regulated simply by the resistance provided by the length of tubing, a slit valve at the very distal end, and by the pressure on the internal compartment the shunt drains to . Since tNon-programmable differential pressure (DP) valves can be considered as the standard CSF shunt valve. DP valves will open and permit flow of CSF once a threshold pressure (opening pressure) has been attained. This opening pressure in a DP valve is not an absolute pressure but rather the difference between inlet and outlet pressures. It holds then that transient increases in intracranial pressure or drops in outlet pressure (due to change in body position for example) can result in valve opening and drainage at lower relative intracranial pressures due to a ‘siphoning’ effect . The ratDP valves may come as fixed-pressure units with defined opening pressures, manufacturers typically providing a range of low, medium, and high opening pressure models to cater for the spectrum of hydrocephalic conditions. In addition, there are programmable models, which have an in-built adjustable component, which importantly can be altered post-implantation to fine-tune the CSF pressure/shunt flow dynamic, according to symptom control ,71,72. TIn the closed (non-shunted) CSF compartment, CSF circulates from the site of production to reabsorption. An immediate drop in ICP is seen with change from recumbent to upright posture due to orthostatic changes in venous drainage. This drop is however attenuated, it is believed, by subsequent collapse of large draining neck veins . The intThe BASICS trial evaluated the efficacy of antibiotic impregnated catheters against standard and silver impregnated catheters establishing superiority of antibiotic catheters in reducing infection rates . The triIdiopathic intracranial hypertension is a difficult condition to manage that can cause serious morbidity in otherwise healthy young adults if not adequately managed. Comprehensive guidance on the diagnosis and management of IIH has been produced, which emphasises the importance of a multidisciplinary approach . CSF divWhile CSF diversion techniques are not reliable at controlling headache morbidity in the long-term, they are seen to improve symptoms in the short-term in the majority of patients. This can then serve as an adjunct to permit increased activity, positive change in mental state and result in sustainable weight loss. Over-drainage and the symptoms of low intracranial pressure can be problematic and difficult to manage. The evidence overall supports the use of VPS over LPS though a large recent retrospective analysis of 1082 shunts  did however demonstrate no difference in revision rates . This hiInsertion of a conduit into the CSF system that bypasses normal circulation will fundamentally alter CSF pressure dynamics. Some patients can be acutely sensitive to such changes though individual responses and tolerance of symptoms are not uniform or predictable. These challenges have driven development of valve technology in an attempt to recreate and maintain a physiological CSF pressure dynamic in shunted patients. There is comprehensive literature reporting on in-vitro performance of these technologies; however, evaluation in patients is difficult, not least because of the wide array of devices available . The pasSelection of specific valves and shunt technology should be done on a patient-by-patient basis. No one configuration has been demonstrated superior. Given the plethora of options available, management by a neurosurgeon with an interest in CSF disorders is advisable. These decisions should ideally be made in conjunction with the patient, explaining the features, advantages, and drawbacks of each option. It hardly bears mention that increasing complexity does not necessarily equate to superiority. Implantation of programmable components for example appears to offer only advantages. The programmable element can be immensely useful in certain circumstances, for example in managing symptoms of over-drainage. The programmable element can have the unwanted effect of increasing the number of consultations with anxious patients however, which has attendant impact on personnel and resources.Implanted pressure monitoring devices can also be problematic. Evidence to support their use in IIH is limited . In the This review focused on CSF diversion strategies to manage the impact of IIH and deliberately avoids discussion of alternate surgical and interventional strategies, such as optic nerve sheath fenestration (ONSF) and intracranial sinus stenting. Both are viable and efficacious approaches with their own indications, advantages, and disadvantages . There iCSF diversion constitutes the standard of practice for neurosurgical management of papilloedema and visual loss in IIH. It is safe and effective with both techniques; LPS and VPS showing equivalent and satisfactory efficacy. VPS has superseded LPS due to lower complication rates, in particular mechanical failure and over-drainage. Stereotactic (image guided) placement of ventricular catheters is strongly recommended in IIH. Increasing complexity of shunt valves available affords the surgeon many options to tailor shunt function to the patient’s needs. This requires a working knowledge of these devices, and as such, patients with IIH should be managed by a surgeon with an interest in CSF disorders.


Though many previous studies have indicated immunological alterations in psychotic disorders, the role and prevalence of neuroinflammation is still unknown. Studies previously investigating immune related biomarkers in the cerebrospinal fluid (CSF) of these patients are mainly small studies on few markers, and many have not compared patients to healthy controls.We will conduct a large case-control study including at least 100 patients with recent onset psychotic disorders and 100 sex- and age matched healthy controls. The cases will include patients diagnosed with a psychotic disorder according to ICD-10 (F20/F22-29) within a year prior to inclusion. We will collect both CSF, blood and fecal samples, to gain insight into possible immunological alterations. The psychopathology of all participants will thoroughly be evaluated using the SCAN interview, and multiple rating scales covering different symptom groups. All participants will partake in a detailed neurological examination, including the Neurological Evaluation Scale assessing neurological soft signs. Additionally, we will assess cognitive functioning, evaluate quality of life and level of functioning, and collect data on a broad array of possible confounders. Our primary outcomes will include CSF leucocytes, CSF/serum albumin ratio, CSF total protein, IgG index, CSF levels of IL-6 and IL-8, and presence of antineuronal autoantibodies in CSF and blood. For our secondary outcomes, exploratory analyses will be performed on a broader panel of neuroimmunological markers. All participants will be invited for a follow-up visit to assess longitudinal changes. The current study is part of a larger CSF biobank build-up for severe mental disorders (PSYCH-FLAME).This study will represent the largest investigation of CSF in patients with psychotic disorders compared to healthy controls to date. We expect the study to contribute with new, important knowledge on pathophysiological mechanisms, and to help pave the way for future investigations of individualized treatment options.The study is approved by The Regional Committee on Health Research Ethics  and The Danish Data Protection Agency . Psychotic disorders are a heterogeneous group of mental disorders with massive impact on both the affected individuals, relatives and the society . A substMounting evidence now suggests involvement of inflammatory pathways in at least a subset of patients with psychotic disorders. Higher levels of pro-inflammatory cytokines in the blood of acutely ill patients with psychosis have been found , peripheSome studies have additionally found indications of neuroinflammation in the cerebrospinal fluid (CSF) of patients with psychotic disorders; a recent meta-analysis  showed iHowever, the evidence on immunological alterations in CSF, and prevalence of immune components such as antineuronal antibodies in the CSF of patients with psychotic disorders, is still limited, and as CSF is the bodily fluid that can tell us the most about neuroinflammation, this knowledge is important to move further in the immunopsychiatric field. Most studies investigating immunological findings in the CSF of psychotic patients have assessed only single or few biomarkers of neuroinflammation, and while some cytokines such as IL-6 have been investigated in multiple studies, even when all available data hereon is combined in a meta-analysis, the total number of cases and healthy controls still do not exceed 250 . The larRegarding NMDA-receptor antibodies, which is currently the most well-studied antineuronal autoantibody in psychiatric disorders, a meta-analysis found these autoantibodies in the peripheral blood of 8% of patients with schizophrenia . HoweverIt seems plausible that inflammation is present and immunological pathways are altered in at least a subgroup of patients with psychotic disorders. Identification of this potential subgroup is highly relevant, as it can improve our understanding of the etiological processes involved in psychosis and give rise to a wide range of new treatment options in psychiatry; amongst other the possibility of personalized medicine, and treatment with immunomodulating drugs. To gain more knowledge in this field, and move closer to clinically relevant conclusions, large, systematic, comprehensive studies of both CSF and blood of patients with psychotic disorders and matched healthy controls, with thorough assessment of psychopathology, level of functioning and cognition, analyses of a wide array of neuroimmunological biomarkers, and at least one follow-up visit in order to assess longitudinal changes and correlation with clinical measurements, are needed.We aim to investigate the role of neuroinflammation in patients with psychotic disorders, in order to obtain increased knowledge on the immunological pathways hypothesized to play a role in the development of psychosis in at least a subgroup of patients. With thorough evaluation of clinical measures such as psychopathology and level of functioning, and longitudinal measurements of immunological alterations, we additionally aim to assess the clinical relevance of these neuroimmunological findings.This will be the hitherto most extensive investigation of immune related changes in CSF and blood from people with psychotic disorders compared to healthy controls, and the first study to assess these changes longitudinally.As part of the larger PSYCH-FLAME study, we will investigate neuroimmunological differences in a new cohort of at least 100 adults with recent onset of non-affective psychotic disorders , and 100 sex and age matched healthy controls. All clinical data and biological samples from the participants will be saved as part the establishment of a new biobank with focus on research in biological and precision psychiatry at the Mental Health Centre Copenhagen, Denmark. The study was approved by The Regional Committee on Health Research Ethics in the Capital Region of Denmark (De Videnskabsetiske Komiteer) , September 7, 2016, and February 1, 2019 after funding was obtained. The study was additionally approved by the Danish Data Protection Agency  on August 31, 2016 and updated July 16, 2018.A prospective case-control study of at least 100 patients with recent onset of a non-affective psychotic disorder  and 100 healthy controls matched on sex and age.Inclusion criteria for cases:Patients with a first-time diagnosis within the past year of a non-affective psychotic disorder Age between 18 and 50 yearsObtainment of written informed consentFor exclusion criteria, see Inclusion criteria for healthy controls:Age between 18 and 50 yearsObtainment of written informed consentFor exclusion criteria, see For an overview of the study procedure, see www.forsøgsperson.dk).The patients will be recruited via psychiatric departments and outpatient clinics in the Capital Region of Denmark  , coverinAll participants will be rated on multiple psychopathology rating scales as listed in the following: Positive and Negative Symptom Scale (PANSS) , Scale fDuring the visit, all participants will be asked to complete a questionnaire assessing depressive symptoms, the Major Depression Inventory (MDI) , and a qCognitive testing of all participants will be done using the Brief Assessment of Cognition in Schizophrenia (BACS) , MontreaAll participants will undergo a thorough neurological examination including scoring on the Neurological Evaluation Scale (NES)  for deteDuring the visit, all participants will be asked to complete a Danish questionnaire on diet and exercise . AdditioTime points for collection of all biological samples will be noted. To minimize diurnal variation in the biomarkers, we aim to collect all blood samples between 9.30 and 11 am, and all CSF samples between 10 and 12 am. For both blood and CSF, a sterile culture swab will be used to perform a swab sample of the skin above the point of needle entry prior to sampling. The swab sample will be stored at -80 degrees Celsius.The procedures are described below. For further details, see text section 4 in Venous blood samples from all participants will be collected prior to the lumbar puncture. A maximum of 44 mL of blood will be sampled.Lumbar puncture will be carried out according to current consensus guidelines for lumbar puncture . It willAs part of the construction of the larger biobank, each participant is asked to provide a fecal sample, either during the visit for inclusion, or afterwards from home. Fecal samples are collected in two ways, both in a Sarstedt Faeces Container and with the OMNIgene Gut Sample Collection Kit. If done at home, the fecal samples will be sent via mail, and stored at -80 degrees Celsius when received at the research unit. Samples collected during the visit will be frozen immediately. The Bristol Stool Scale score, time and date of sampling, as well as time and date of freezing is noted.All included patients will be offered a follow-up visit, which will take place at least one year after the first visit. Controls that matched patients who agreed to a follow-up visit, will also be offered a second visit. Follow-up visits will consist of the same procedures as those mentioned above.Eight mL of the collected blood and 2 mL of the collected CSF are initially analyzed. Initial blood analyses include white blood cell (WBC) count, differential WBC count, hemoglobin, platelet count, HbA1c, glucose, hs-CRP, albumin, and IgG. Initial CSF analyses include WBC count, differential WBC count, erythrocyte count, protein, glucose, albumin, and IgG. Analyses of cells in CSF and blood are done using Sysmex XN9000, with distribution of cells in the CSF analyzed using CellaVision DM 96. HbA1c is measured using TOSOH G8, albumin and glucose using an ABL800 FLEX radiometer and the rest of the initial CSF and blood analyses are performed on Cobas 8000 module c502 (Roche). We aim to perform all initial CSF analyses and freeze all the remaining CSF no later than 1 hour after sampling. All laboratory personnel will be blinded to all clinical information.A total of 36 mL of blood and 14 mL of CSF are processed for storage for later analyses, both as droplets on PKU cards, and as small aliquots frozen at -80°C. For details on handling and storage, see text section 6 in The initial analyses of the biobank samples stored in small aliquots will be conducted when the first 100 patients with psychotic disorders and 100 healthy controls have been included.Cytokines and chemokines will be analyzed using the Mesoscale V-PLEX Neuroinflammation Panel 1 Human Kit, by Statens Serum Institut, according to the manufacturer’s instructions.Antineuronal autoantibodies will be analyzed at Odense University Hospital, with the Euroimmun Mosaic 1 kit  combined with the Euroimmun Gad65 Elisa kit, both used according to the manufacturer’s instructions.All analyses will be performed in a blinded fashion, with no laboratory staff having access to clinical information on participants. Analyses will be performed according to a statistically designed experimental schedule based on a randomized block design or similar, to minimize the risk of confounding by time of analysis, batch, and other factors.The four co-primary outcomes for our initial screening analyses of CSF and blood will be CSF leukocytes, blood/CSF albumin ratio, CSF total protein and IgG index. For cytokine and chemokine CSF levels, IL-6 and IL-8 are the two co-primary outcomes. For antineuronal autoantibodies the two co-primary outcomes will be presence of any one of seven antineuronal autoantibodies  in 1) CSF and 2) in either blood or CSF.As, secondary outcomes, more exploratory analyses will be performed on broader panels of cytokines, specific antineuronal autoantibodies, and other neurological/inflammatory markers. Possible associations between all biomarker outcomes and several clinical measurements will additionally be analyzed.In our primary publication we will furthermore include reports of adverse events to the lumbar puncture procedure.In order to assess the risk of adverse events associated with lumbar puncture, participants are contacted by telephone the day after the intervention. Adverse event that could be due to the procedures are noted, with specific focus on possible post lumbar headache and infections.Data in the biobank will eventually be linked to the nationwide Danish registers to obtain information on, amongst others, prior infections treated in the primary care sector, comorbidities, treatment outcome (e.g. psychiatric readmissions and change in medication), and later psychiatric diagnoses.t-tests using standardized mean differences (SMDs) from our prior meta-analysis [Power and sample size calculations based on two-sample analysis  of CSF st-tests when outcome is continuous, and χ2 test when categorical (e.g. sex).Possible differences in demographic data will be examined using two-sample Possible differences in continuous variables (such as level of IL-6 or IgG index) between patients and healthy controls will be analyzed using linear models with group, age, and sex as covariates. Outcomes measured either as concentrations (e.g. IL-6) or ratios  will be log-transformed prior to analyses. Associations between levels of biomarkers and clinical measures such as psychopathology , cognitive measures, medication status, duration of illness, and level of functioning will also be investigated. Differences between patients and healthy controls of dichotomous variables  will be analyzed using Pearson chi-square tests. Two-sided tests with p<0.05 will be considered significant. If a continuous outcome variable has measurements censored below the lower limit of quantification a censored Gaussian model will be used instead of linear modelsAll statistical analyses will be performed in R . For theThe first publications will follow the inclusion of the first 100 participants with recent debut of non-affective psychosis and 100 sex and age-matched controls. This will initially result in three publications; one comprising all initial screening analyses, one investigating all cytokines and chemokines and one on antineuronal autoantibodies . With our thorough SCAN-interview and multiple rating scales, we can adequately evaluate the severity of any possible symptoms. Recruiting healthy volunteers from the website forsøgsperson.dk allows us to include individuals from the same broad community as the cases.When including a vulnerable group of patients, a risk of selection bias is difficult to avoid. The most severely ill patients might be less likely to agree to participate, particularly since the intervention is somewhat comprehensive. To overcome this to the greatest possible extent, we have tried to keep our intervention as brief as possible, whilst including all relevant variables, and have managed to limit the duration of the intervention to a single day. When recruiting patients, we will strongly emphasize the possibility of multiple breaks, and that the patients may leave at any time, should they feel the need to stop the intervention. Additionally, we arrange and pay for transport via taxi to and from the inclusion, and for those who are admitted to hospital wards, we offer to perform the intervention at their ward. Another possibility of selection bias stems from our strict inclusion and exclusion criteria. However, the nature of the study, investigating multiple biomarkers that broadly represent many aspects of the immune system, establishes the need for stringent exclusion of other factors with impact hereon.Some confounders such as smoking, alcohol use, and eating habits, rely on self-reported data, raising the issue of possible recall bias, and limits the validity of some of these data.The potential side effects of this study are limited to adverse events following the procedures during the sampling of blood and CSF, since medical treatment is not included in the trial. All participants will be given information on how to contact the doctors responsible for PSYCH-FLAME in case any adverse events occur, and to thoroughly assess these possible adverse events, participants will furthermore be contacted via telephone by one of our doctors on the day after inclusion.The risk of infection associated with the procedure of a common blood test is very low and the risk of infection after a lumbar puncture is considered extremely small, with some authors estimating an incidence of less than 0.01% . The bloSome of the clinical examinations and biological samples will result in unexpected findings regarding the health status of patients or healthy controls. If a consent to be contacted regarding such findings has been given, the participant will be contacted and referred to further relevant investigations . If the findings are found to be severe or life threatening, the participant will be contacted regardless of consent.The study will be conducted in accordance with the principles of the Declaration of Helsinki , and other applicable laws and regulations. Participation in the study is preceded by elaborate information on the entire course of the study, potential individual benefits and personal risks. It is emphasized that participation is absolutely voluntary. Patients are given sufficient time to read all provided information, and no procedures will take place before written consent is obtained. This consent can be revoked at any time. Before the lumbar puncture is performed, all participants are investigated for contraindications. All individuals will be anonymized when analyzing the data. The identity of participants will only be used for merging of the data and retrieving the biological samples. Data will only be published in a manner where no individuals can be identified.This study and the PSYCH-FLAME biobank construction will represent the largest, most comprehensive study to date on immunological alterations and differences in levels of neurological biomarkers in the CSF between patients with psychotic disorders and healthy controls. Additionally, it will be the first time that biomarkers in the CSF of patients with psychosis and matching healthy controls are assessed longitudinally. This will provide unique insight into possible correlations between clinical status and neuroinflammation. A substantial part of patients with psychotic disorders experience low treatment response of current antipsychotic treatment, and psychotic disorders have huge impact on the patients’ functioning and quality of life, causing suffering on both themselves and relatives, while bearing great costs to society . The ideThe study is ongoing. Participants are currently being recruited, and inclusion for this first part is expected until August 1, 2021, after which the first analysis described in this protocol will initiate, and inclusion will continue hereafter in order to build a larger biobank.The study is approved by The Regional Committee on Health Research Ethics  and The Danish Data Protection Agency .Before any collection of data, participants will receive thorough information about the study , both orally and in writing. All participants will be given sufficient time to consider participation before consent is given. The patient can at any time retract their consent and will then no longer be part of the study unless the material has already been analyzed. This will have no influence on their further treatment in the psychiatric department/clinic.All participants will be identified using their unique Civil Registration Number which is assigned to all Danish citizens at birth or at the time of immigration. When informed consent has been given, participants will be registered by their Civil Registration Number in an identification key kept separately from all other data, and data will be pseudoanonymized by a study ID-number, as according to the approval from the Danish Data Protection Agency. This will make it possible to later expand the project with data from the many different Danish registers.S1 Table(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


Conclusions: AI allows a fully automated volumetric quantification of pleural plaques showing volumetric progression of PP over a five-year period. The reproducible PP volume evaluation may enable further investigations for the comprehension of the unclear relationships between pleural plaques and both respiratory function and occurrence of thoracic malignancy.Objective: This study aimed to develop and validate an automated artificial intelligence (AI)-driven quantification of pleural plaques in a population of retired workers previously occupationally exposed to asbestos. Methods: CT scans of former workers previously occupationally exposed to asbestos who participated in the multicenter APEXS (Asbestos PostExposure Survey) study were collected retrospectively between 2010 and 2017 during the second and the third rounds of the survey. A hundred and forty-one participants with pleural plaques identified by expert radiologists at the 2nd and the 3rd CT screenings were included. Maximum Intensity Projection (MIP) with 5 mm thickness was used to reduce the number of CT slices for manual delineation. A Deep Learning AI algorithm using 2D-convolutional neural networks was trained with 8280 images from 138 CT scans of 69 participants for the semantic labeling of Pleural Plaques (PP). In all, 2160 CT images from 36 CT scans of 18 participants were used for AI testing versus ground-truth labels (GT). The clinical validity of the method was evaluated longitudinally in 54 participants with pleural plaques. Results: The concordance correlation coefficient (CCC) between AI-driven and GT was almost perfect (>0.98) for the volume extent of both PP and calcified PP. The 2D pixel similarity overlap of AI versus GT was good (DICE = 0.63) for PP, whether they were calcified or not, and very good (DICE = 0.82) for calcified PP. A longitudinal comparison of the volumetric extent of PP showed a significant increase in PP volumes ( The association between occupational asbestos exposure and the increase in benign and malignant respiratory diseases has been demonstrated through several studies in the last decades . Among tIn the last few years, Deep learning (DL) has been considered as the most reliable technique for medical image segmentation . HoweverThis is a retrospective cohort study involving retired workers exposed to asbestos during their working life and without already compensated asbestos-related disease. The hospital ethics committee approved the study (CPPRB Paris-Cochin n°1946 (2002), CPP Ile De France III n°1946/11/02-02 (2010)). All participants received information about the study and gave their written informed consent.Asbestos-Related Diseases COhort (ARDCO) is a French surveillance program where retired workers exposed to asbestos during their working life were included between October 2003 and December 2005 in four regions. They benefited from a free medical check-up, including chest CT scan and pulmonary function tests repeated every 5 years. Chest CT scans were sent to the regional coordinating centers and constituted the Asbestos Post EXposure Survey (APEXS) study.The study analyses the second and third round of screening (collected in 2010 and 2017 respectively) since CT scans of the first round were of insufficient quality . Finally, participants with pleural plaques identified by expert radiologists at the 2nd and the 3rd CT screenings were included .We extracted data from the APEXS survey, managed by Institut Interuniversitaire de Médecine du Travail de Paris-Ile de France, Créteil, France. Data contain patients’ CT scans and characteristics  and Time since first exposure (years); Related conditions on CT scans: Lung nodule/Asbestosis/Lung cancer).To establish the diagnosis of pleural plaques, a consensus of three expert radiologists was used as previously reported . PleuralBriefly, trained thoracic radiologists blinded to the participants’ characteristics have manually delineated pleural plaques on standard kernel reconstructed CT images in a random fashion. The manual segmentation was considered as the ground truth (GT). A pre-processing using thin maximum intensity projection (MIP) was used to limit the number of images for the manual segmentation. A five-millimeter slice thickness for MIP was used. Therefore, the number of chest CT slices decreased from 300 to 60 approximately.A 2D-convolutional neural network (CNN) was trained utilizing the Training cohort. We used one single input channel . We used the U-Net architecture presented in y (LBCE)  and DiceE) [LDL)  which isE) [LDL) .L was dThe following parameters were employed: input resolution = 512 × 512, batch size = 1, optimizer = Adam , learnin2D-similarity and 3D-concordance assessments between AI-driven and GT test labels were performed. Calcified pleural plaques were identified by using a threshold of 100 Hounsfield units (HU) on the masks of PP, and their similarity and volume concordance was also assessed. A longitudinal comparison of PP volume progression was evaluated. Correlation with the visual extent score of PP was assessed.Longitudinal paired-comparison analyses of PP volumes were performed. AI-driven PP volume quantification was performed on both native CT slices (slice thickness 1–1.25 mm), and MIP processed CT images. In addition, correlation with PP visual extent score was evaluated.AI-driven measurements were performed twice in the test cohort, while two observers blinded to any other data and the other observer labels, manually segmented a random subset of 20 CT exams. For intra-observer reproducibility, the subset of 20 CTs was re-segmented manually by the first observer a month later to avoid recall bias.Data were expressed as medians with a 95% confidence interval (CI). The similarity was assessed by calculating the balanced accuracy, Sorensen–DICE similarity coefficient (DICE), precision, and recall . Concordn = 8280 CT slices from 138 CT examinations of 69 participants) and independent Test cohort (n = 2160 CT slices from 36 CT examinations of 18 participants) without cross-validation. There were 13 CT models from four major manufacturers .Finally, to independently evaluate the clinical relevance and validity of the AI-driven PP quantification, a clinical validation cohort was constituted  were shuffled randomly before being segmented by the 2D-CNN. The 2D pixel similarity overlap of AI versus GT was good (DICE = 0.63). The similarity overlap for calcified pleural plaques was found very good (DICE = 0.82)  , Table 2n = 36 CTs), the 3D concordance between AI-driven volume calculations and GT labels was almost perfect for both PP and calcified PP (CCC > 0.98 95%CI: 0.96–099)  . At the .96–099) .p < 0.001). The correlation coefficient was similar to that of the GT label . Similarly, in the clinical validation cohort, there was a significant correlation between the AI-driven PP volume and the visual PP extent .There was a significant moderate correlation between the AI-driven PP volumetric quantification and the PP visual extent score in the test cohort  (p < 0.001). (p = 0.003)  , Table 3 0.001). . There w= 0.003) .Moreover, there was no significant difference between AI-driven PP volume quantification using native CT images and thin MIP pre-processed CT images .As expected, the quantitative measurements of AI-driven segmentation had an almost perfect reproducibility when repeated twice in all 36 CT scans  of the Test cohort (DICE > 0.99) . The simThis study shows that AI-driven quantitative measurement of pleural plaques is a reliable, fully automatic technique that can provide relevant information about pleural plaques progression in a population of retired workers previously occupationally exposed to asbestos. The method showed good similarity and very good concordance with manual segmentation of pleural plaques. Volumetric measurements of pleural plaques demonstrated a significant increase between two rounds of CT screening with an average delay of five years. In addition, the PP volume quantification showed a significant correlation with expert visual CT score of PP extent.This study has some limitations. First, this was a retrospective first attempt to evaluate the novel method. Large prospective studies may bring more insights into pleural plaques volume quantification and asbestos exposure. Second, detection of pleural plaques was out of scope since the diagnosis of pleural plaque presence is easily made by an expert radiologist in few seconds. However, using the same dataset, AI algorithms could be trained for detection tasks. Further evaluation using alternative strategies such as multiple CNNs with major voting , multiplTo the best of our knowledge, our study is the first that aimed to develop and validate AI-driven quantification of pleural plaques in a population of retired workers previously occupationally exposed to asbestos. Indeed, Sousa et al.  have repManual segmentation of pleural plaques could be a tedious and time-consuming task, which may have prevented the radiologists’ community from tackling the issue of automatic quantification using DL. In this study, to facilitate manual segmentation, CT scans were pre-processed using maximum intensity projection. Therefore, the number of chest CT slices decreased from 300 to 60 approximatively. The rationale of using a thin MIP over a maximum of five slices  is that pleural plaques show attenuation values higher than the surrounding structures , which facilitate the detection and delineation of PP. On the contrary, the ribs, which are often in contact with PP, show a higher attenuation value. However, a MIP along five-millimeter of thickness would prevent the overlap of PP and costal structures. Moreover, with a thin MIP, partial volume artifacts at the pleura thoracic wall interfaces were limited, especially in non-vertical areas such as the diaphragm and the apices. In addition, we demonstrated that AI-driven pleural plaques volume derived from either native CT images or MIP pre-processed CT images was not different. In the last few years, we have evaluated semi-automated segmentation of pleural plaques  and showThe use of DL in medical imaging in the last few years, allowed a giant leap of image segmentation with less and less of manual editing . Indeed,In our study, an independent Testing cohort of 2160 CT slices was used, including the whole chest without preselecting a few slices with pleural plaques. Thus, this provided an extensive overview of the performance of the technique. We reported a DICE coefficient of 0.63 for PP segmentation, which represents a good overlap index, knowing that pleural plaques may have less than one millimeter of thickness and a very limited extent . Indeed,This is the first time that longitudinal evaluation of pleural plaques has been reported. We showed a progression of pleural plaques volume through the five-year delays between the two rounds of CT screening. This was expected since pleural plaques were demonstrated to be associated with asbestos exposure latency . NeverthRelationships between the prevalence of pleural plaques and benign or malignant respiratory diseases were extensively evaluated in the scientific literature . The preThe AI-driven technique has several advantages in comparison to visual or semi-automated evaluations of pleural plaques extent. A few seconds are needed to accomplish the whole segmentation of pleural plaques with an almost perfect reproducibility. In contrast, the visual/manual scores are time-consuming with moderate reproducibility. This detailed quantification of pleural and calcified pleural plaques may be more relevant in evaluating the relationships with benign and malignant respiratory diseases.Although four manufacturers were represented in this study, additional evaluation with other manufacturers and CT models would be desirable to increase the generalizability of the AI model.We showed for the first time that an automated AI-driven quantification of pleural plaques is feasible, which may help researchers to decipher the unclear relationships between pleural plaques and both respiratory function and chest malignancy.


GDAP1) gene have been associated with demyelinating and axonal forms of Charcot-Marie-Tooth (CMT) disease, the most frequent hereditary peripheral neuropathy in humans. Previous studies reported the prevalent GDAP1 expression in neural tissues and cells, from animal models. Here, we described the first GDAP1 functional study on human induced-pluripotent stem cells (hiPSCs)-derived motor neurons, obtained from normal subjects and from a CMT2H patient, carrying the GDAP1 homozygous c.581C>G (p.Ser194*) mutation. At mRNA level, we observed that, in normal subjects, GDAP1 is mainly expressed in motor neurons, while it is drastically reduced in the patient’s cells containing a premature termination codon (PTC), probably degraded by the nonsense-mediated mRNA decay (NMD) system. Morphological and functional investigations revealed in the CMT patient’s motor neurons a decrease of cell viability associated to lipid dysfunction and oxidative stress development. Mitochondrion is a key organelle in oxidative stress generation, but it is also mainly involved in energetic metabolism. Thus, in the CMT patient’s motor neurons, mitochondrial cristae defects were observed, even if no deficit in ATP production emerged. This cellular model of hiPSCs-derived motor neurons underlines the role of mitochondrion and oxidative stress in CMT disease and paves the way for new treatment evaluation. Mutations in the ganglioside-induced differentiation associated protein 1 ( PMP22 gene, responsible of the so-called CMT1A, remains the main genetic cause of this pathology.Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of sensory-motor disorders belonging to the larger class of genetic neuropathies. With an estimated prevalence of 1:2500, it is considered as the most frequent inherited pathology of the peripheral nervous system. It indifferently affects both sexes, of any geographical origin and age, and it is clinically defined by muscular weakness and atrophy, foot deformities such as pes cavus, and sometimes sensory loss and balance issues [GDAP1 (ganglioside-induced differentiation protein 1) gene have been reported for the first time in 2001 [GDAP1, located on chromosome 8 of the human genome, encodes a 358 aa protein, expressed on the outer mitochondrial membrane of neurons and, at lower levels, of myelinating Schwann cells [GDAP1 CMT-inducing mutations have been largely described in their clinical aspects and associated phenotypes, fewer studies have deeply investigated the molecular mechanisms altered in motor neurons and responsible for the neural degeneration.Mutations in  in 2001 ,4, and a in 2001 ,6. GDAP1nn cells ,8,9. Eve2+ homeostasis, as shown in SH-SY5Y and transfected HeLa cells [Three main model organisms have been developed to elucidate GDAP1 role in cellular physiology, through its up- or down- regulation, in Drosophila melanogaster ,11, its La cells ,24. MoreLa cells ,26. HoweNELF, MFN2 [HSPB1 [PMP22 duplication [GDAP1 are reported in three studies [Human induced-pluripotent stem cells (hiPSCs), created for the first time in 2006 , have beLF, MFN2 ,30, HSPB2 [HSPB1 , but alslication . HiPSCs  studies ,29,33, a studies .GDAP1, underlining the role of mitochondria and oxidative stress in this human GDAP1-defective CMT disease.Here, we report the first functional study on human hiPSCs-derived motor neurons from a CMT2H patient, carrying the homozygous c.581C>G (p.Ser194*) mutation in GDAP1 gene genetic analyses detected the c.581C>G (p.Ser194*) mutation, homozygous in the patient and his affected brother, and heterozygous in the other family members. No other mutation in CMT- and peripheral neuropathies-associated genes was detected by targeted Next Generation Sequencing (NGS) , as well as the informed consent of all participants. The study was conducted in accordance with the Declaration of Helsinki. The family of the CMT-patient presented two cases . The proGS) , with 10% Fetal Bovine Serum (FBS) . After two weeks, once fibroblasts (FBs) have migrated from the skin fragment and grew in the culture dish, they were isolated using trypsin. In the first three days, fibroblasts were cultivated combining the Chang Medium® D (25%) with the RPMI 1640 medium (75%) , supplemented with 10% FBS. Then, Chang medium D was completely replaced by RPMI medium and FBS.Skin biopsies were obtained from patient, Ctrl-1, and Ctrl-2, and incubated in CHANG Medium2 density. The second day, 600,000 fibroblasts, from patient and controls, were reprogramed with three plasmids , through the Nucleofector II device . Reprogramed cells were cultured in DMEM+GlutaMAX medium , supplemented with 10% FBS, 1% MEM non-essential amino acids (Thermo Fisher SCIENTIFIC), 1% sodium pyruvate (Thermo Fisher SCIENTIFIC), and, at day 1, with 0.1% gentamycin (Thermo Fisher SCIENTIFIC). Culture medium was replaced every day. After 14–21 days, colonies were selected, using a needle, and transferred in new gelatin/MEF coated dishes. HiPSCs colonies grew in a KO-DMEM medium , with 20% KnockOut Serum Replacement , 1% MEM non-essential amino acids, 1% Glutamine , 0.1% β-mercaptoethanol , and 0.1% gentamycin. It was replaced every day, extemporaneously supplemented with 20 ng/mL Fibroblast Growth Factor (FGF2) . After hiPSCs amplification, all quality controls were performed  protocol. First day, CF-1 Mouse Embryonic Fibroblasts (MEF), Mitomycin-C treated , were seeded on gelatin coating , at 25,000 cells/cmerformed .2, using the same coating and the same culture medium.Differentiation protocol was applied as previously described by Faye et al. . After n®, Venlo, The Netherlands). After verifying RNA integrity with the Bioanalyzer 2100 system (Agilent Technologies), it was converted in cDNA with the QuantiTect® Reverse Transcription kit (QIAGEN®). For the quantitative PCR , primers were designed between the fifth and the sixth exon of GDAP1, and between the fifth and the sixth exon of TBP (TATA-Box Binding Protein), chosen as reference gene. Reactions were prepared with the Rotor-Gene SYBR-Green PCR Kit (400) (©QIAGEN) and performed on the Corbett Rotor-Gene 6000 Machine (© QIAGEN). All qPCR reactions were performed four times.Total RNA was extracted from fibroblasts, hiPSCs, NPs and MNs of Ctrl-1 and patient, using the miRNeasy Mini kit   for 10 min and permeabilized with 0.1% Triton X-100  for one hour. They were incubated overnight at 4 °C with the primary antibody, prepared in 3% BSA, and then the next day with the secondary antibody for one hour at room temperature. PFA, Triton X-100 and BSA were diluted in Dulbecco’s phosphate-buffered saline 1X (DPBS) . Nuclei were stained with 2 µg/mL 4′,6′-diamidino-2-phénylindole dihydrochloride (DAPI) . Images captures were performed using a fluorescence microscope  or a confocal microscope , while their processing and analysis were made with NIS Element BR software, Zen Black and Zen Bleu software, and Image J software. All antibodies’ dilutions and references are reported in ® Elite ABC kit  was used for the avidin-biotin/peroxidase detection. The DAB+ chromogen, i.e., the peroxidase substrate solution, was added to induce the formation of the brown precipitate, visualized with a light microscope.The chromogenic 3,3′-Diaminobenzidine (DAB) staining was used as complementary ICC method to immunofluorescence, by reason of its high sensitivity. For the DAB staining, MNs were fixed, permeabilized, and incubated with the GDAP1 primary antibody overnight. Next day, the VECTASTAINAll manipulations for the electron microscopy were performed in Neurology and Anatomic Pathology departments at University Hospital of Limoges. Cells were fixed in 2.5% glutaraldehyde, then incubated 30 min, at RT, in 2% OsO4 . After washing them with distilled water, they were dehydrated 10 min in a series of ethanol dilutions  and three times in 100% ethanol. At the end, they were embedded overnight in Epon 812. Thin blocks were selected and stained with uranyl acetate and lead citrate and examined using a Jeol 1011 electron microscope.® Luminescent Cell Viability Assay kit , and the luminescent signal was recorded with the Fluoroskan Ascent®FL  following manufacturer instructions. DAPI staining was used to normalize luminescence’s values to the number of cells. Reactions were performed in triplicate, and experiments were repeated three times.ATP was dosed using CellTiter-GloSuccinate dehydrogenase activity was measured using the Cell Proliferation Kit I , following manufacturer conditions. Absorbance of formazan crystals, at 595 nm, was recorded with the Multiskan™ FC Microplate Photometer (Thermo Fisher SCIENTIFIC), and normalized to the number of cells, measured with the DAPI staining. Reactions were performed in triplicate, and experiments were repeated three times.2O2 solution, prepared in culture medium. After the treatment, 5 µM MitoSOX™ Red mitochondrial superoxide marker  was added to the whole of the plate and incubated for 10 min at 37 °C. Fluorescent signal was detected using the Leica DM IRB microscope and normalized to the number of cells, measured with the DAPI staining. Reactions were performed in triplicate, and experiments were repeated three times.Fibroblasts and MNs were analyzed in basal conditions as well as in stressed conditions. Stressed wells were treated two hours with 1 mM Hp < 0.05 was considered significant.All statistical analyses were performed using the GraphPad Prism 5 software . Data were expressed as mean ± SEM (Standard Error of the Mean). They were compared using the nonparametric Mann–Whitney U test; Fibroblasts of Ctrl-1, Ctrl-2, and the CMT2H patient were reprogramed in hiPSCs. After validating hiPSCs of the three subjects for all quality controls , our difGDAP1 mRNA, from fibroblasts to hiPSCs, neural progenitors, and motor neurons, of patient and Ctrl-1 subject. In Ctrl-1, we showed that GDAP1 is differently expressed in the four cell types , and slightly more expressed in hiPSCs . In contrast, GDAP1 mRNA levels significantly increased in neural cell types, associated with the progression of neural differentiation. In particular, GDAP1 expression was around 33-fold higher in NPs , and 56-fold higher in MNs , compared to fibroblasts’ levels. In patient’s cells, the same expression trend was observed in the four cell types, thus GDAP1 mRNA was nearly absent in patient’s fibroblasts (0.01 ± 0.003), and significantly higher in hiPSCs , NPs , and MNs  .In order to investigate GDAP1 functions and GDAP1-associated mechanisms, we evaluated the expression of To complete the expression study, we evaluated GDAP1 protein expression on MNs, the cellular type known to express GDAP1, in comparison with fibroblasts. The experiment was performed for the two control subjects and the CMT patient .For the immunofluorescence analysis, MNs were stained with GDAP1 antibody (red), and Tuj-1 antibody (green), specific of neural cells. As shown in In fibroblasts, GDAP1 staining (red) was associated to Prolyl 4-hydroxylase subunit-β antibody (P4HB) (green). As expected, according to the RNA measurement, a weak GDAP1 signal was observed, both in controls’ and patient’s fibroblasts B.These results were supported by the DAB staining showing the higher expression of GDAP1 protein in MNs of Ctrl-1 compared to patient .p < 0.01). At d4, proliferating cells were slightly lower than 60% in Ctr-1 (57.7 ± 1.37%) and Ctrl-2 (59.38 ± 3.69%), and considerably reduced to 40% in patient’s cells   B.p < 0.001) or Ctrl-2   .HiPSCs-derived motor neurons of controls and patient were analyzed by electron microscopy. Surprisingly, in the cytoplasm of multiple patient’s MNs, we observed the emergence of several round structures, of various sizes, suspected to be lipid droplets (LDs) F. These Given the mitochondrial localization of GDAP1 protein, we investigated mitochondrial morphology and structure. Looking at MNs’ ultrastructure, any difference in mitochondrial size and shape was remarked between controls and patient. Moreover, MNs of both subjects presented elongated and fragmented mitochondria. However, focusing on mitochondrial cristae, we observed that their organization was altered in mitochondria of patient’s MNs. In particular, cristae’s regular distribution and thickness were perturbed, preventing to discriminate their structure in the internal mitochondrial compartment. Swollen cristae were also observed F. This dp < 0.05)  complexes and the ATP production. Given the limited availability of hiPSCs-derived MNs, we performed the MTT test to evaluate the activity of the succinate dehydrogenase (complex II). In both fibroblasts and motor neurons, succinate dehydrogenase activity seemed to be slightly increased in patient’s cells, compared to Ctrl-1 and Ctrl-2, and it reached significant difference in fibroblasts  . In cont < 0.05) .p < 0.05). This significant difference was even observed in fibroblasts . The same trend emerged in the comparison with Ctrl-2 cells, but significant difference was not reached.Mitochondria are also the main producers of reactive oxygen species (ROS), such as superoxide anion, inducing oxidative stress. A perturbation of mitochondrial cristae could promote redox imbalance. As expected, and shown in GDAP1 gene have already been reported to be responsible for demyelinating, axonal, and intermediate forms of Charcot-Marie-Tooth disease, and associated to heterogeneous phenotypic manifestations [GDAP1 role in cellular functions and processes has not been clearly elucidated, although several relevant studies have been performed [GDAP1 mutations surely represent the most accessible and easier tool to mimic GDAP1-induced pathophysiology. Murine and human GDAP1 proteins share 94% of amino acid homology supporting the relevance of this rodent model in expression and localization studies [GDAP1-ortholog gene (CG4623) [GDAP1 [GDAP1  [GDAP1, notably neurons and Schwann cells. These cell types cannot be obtained from humans, and used in vitro as cellular models. Given the inability to culture human neural cells, the only model available in these conditions was represented by human fibroblasts [GDAP1 [GDAP1, to investigate GDAP1 functions and GDAP1-associated mechanisms in CMT disease.More than 80 mutations in stations . Howevererformed ,16,22,38 studies ,9, struc studies , functio studies ,13. Furt(CG4623) ,11, or y) [GDAP1 ,40. The ) [GDAP1 ,9,15, or5, HT22) ,8,22,24,5, HT22) ,21. Inderoblasts ,18,22, wGDAP1 expression. In animal models, such as mice and rats, GDAP1 has been shown to be largely expressed in neurons. In particular, the higher expression was detected in cerebellum, cerebral cortex, hippocampus, olfactory bulb, spinal nerve, but also in sciatic nerve, and motor and sensory neurons [GDAP1 expression in Schwann cells was controversial, whereas, in non-neural tissues, it was poorly explored [GDAP1 mRNA expression in four human cell types of the same control subject (Ctrl-1): fibroblasts, hiPSCs, NPs, and MNs. Our study revealed, on Ctrl-1 cells, that GDAP1 is weakly expressed in fibroblasts and hiPSCs, while its expression was significantly higher in NPs, and, above all, in MNs. It is interesting to note that GDAP1 mRNA in fibroblasts represented only 3% of NPs-GDAP1 mRNA, and 1.8% of MNs-GDAP1 mRNA. This is in agreement with Noack et al. work, who showed that control human fibroblasts expressed only 2.6% of GDAP1 mRNA compared to embryonic stem cells-derived motor neurons [GDAP1 mRNA was only 10–20% of mRNA estimated in each cell type of Ctrl-1, up to 6- and 8-fold smaller than those assessed in Ctrl-1 NPs and Ctrl-1 MNs. Our data seem to suggest that GDAP1 mRNA is degraded in patient’s cells. As mutated GDAP1 mRNA contains a premature termination codon (PTC), the nonsense-mediated mRNA decay (NMD) system could be activated and induce its degradation, preventing the synthesis of a truncated, and maybe non-functional, protein [GDAP1 neural expression, we chose MNs and NPs, as cellular models to investigate its functions, and evaluate its role in Charcot-Marie-Tooth disease development.We first questioned about  neurons ,8,9. GDAexplored ,8,9. Her neurons . In cont protein . In any GDAP1 expression detected in fibroblasts does not exclude a GDAP1 role in this cell type, and the possibility of conducting functional studies on them [However, the weak  on them ,17,18. IGDAP1-associated pathophysiology. First, we investigated mitochondrial morphology. Electron microscopy revealed, only in patient’s MNs, a general disorganization of mitochondrial cristae, which could affect the inner mitochondrial space and subsequent metabolism. The disruption of cristae structure has already been associated to other pathological conditions induced by mutations or lack of proteins involved in mitochondrial dynamics, such as optic atrophy 1 (OPA1) protein [GDAP1 [GDAP1 mutations [On the other hand, morphological and oxidative stress analyses, allowed highlighting, exclusively in MNs, two main mechanisms which could play a key role in CMT disease progression: the deregulation of mitochondria morphology and dynamics, and the redox imbalance. These aspects support the relevance of our MNs cellular model in the study of  protein , or mito protein . Cristaen [GDAP1 , but alsutations .GDAP1 seems to be expressed, even if at a low level. Moreover, we have demonstrated that patient’s neural cells present a lower proliferation rate, compared to controls. Both these aspects could potentially be related to GDAP1 involvement in mitochondrial dynamics. Indeed, it is now well known that the proper preservation of mitochondrial dynamics is a fundamental condition for cell cycle progression, and fragmentation of mitochondrial network is required during the mitosis phase [GDAP1 knockout, altering the fission machinery, impairs OSKM (Oct4/Klf4/Sox2/cMyc) reprogramming, and cell cycle progression, in murine iPSCs [Interestingly, some additional findings of our cell culture need to be pointed out. We observed in patient’s hiPSCs carrying the p.Ser194* mutation a higher spontaneous differentiation and a reduced maintenance of stemness compared to controls’ hiPSCs, where is phase . Thus, tne iPSCs . Based oGDAP1 knockout mice [GDAP1 p.Ser194* mutation. In fact, GDAP1 has been suggested to have an antioxidant role in cellular homeostasis [GDAP1 is weakly expressed and LDs lacking. The overproduction of superoxide anion, in GDAP1-mutated fibroblasts, has also been reported in a recent work [GDAP1-mutated fibroblasts presented also a reduced expression of Sirtuin 1 (SIRT1) enzyme, which activates the PPARgamma coactivator-1alpha (PGC1 α) [In the cytoplasm of patient’s MNs, round electron-dense structures were observed, suspected to be lipid droplets. LDs could be considered as an accumulation of energetic substrate, such as triglycerides, linked to a defect of mitochondrial beta oxidation, or a hallmark of cellular stress, previously observed in nutrient imbalance, inflammation and oxidative stress . Moreoveout mice , in accoout mice . This pheostasis ,22. Consent work . Moreove(PGC1 α) . PGC1 α (PGC1 α) ,52,53,54GDAP1 impairment in Charcot-Marie-Tooth pathophysiology through mitochondrial dysfunction and oxidative stress development was underlined in an original human model of motor neuron from patient’s fibroblasts, carrying the homozygous codon-stop c.581C>G (p.Ser194*) mutation. The results underlined that GDAP1 is mostly expressed in neural cell types such as MNs and PNs, but also, at lower levels, in fibroblasts and hiPS cells. In patient’s cells, 80–90% of GDAP1 mRNA would be degraded by the NMD system, leading to the considerable reduction of GDAP1 protein. Taken together, these results demonstrated that hiPS cells can be a powerful tool to recreate any suitable cellular model from patients carrying mutations and are essential for understanding the pathophysiological role of the altered protein, but also necessary to develop new therapeutic strategies.In conclusion, the role of


Anaemia has been reported to be associated with cognitive decline and Alzheimer’s disease (AD), but the associations between anaemia and cerebrospinal fluid (CSF) AD biomarkers are still unknown. This study aimed to investigate the associations between anaemia and CSF AD biomarkers.APOE ε4 alleles, comorbidities  and glomerular filtration rate.Participants were included from the Chinese Alzheimer’s Biomarker and LifestylE (CABLE) study. The associations of anaemia and its severity with CSF AD biomarkers including β-amyloid 1–42 (Aβ42), total tau (t-tau) and phosphorylated tau (p-tau) were analysed by multiple linear regression models. Adjusted for age, gender, educational levels, p = 0.035). Besides, participants with more severe anaemia had lower CSF Aβ42 levels (p = 0.045). No significant association of anaemia with CSF t-tau and p-tau levels was found.A total of 646 cognitively normal older adults, consisting of 117 anaemia patients and 529 non-anaemia individuals, were included in this study. Anaemia patients had lower levels of CSF Aβ42 than individuals without anaemia (Cross-sectionally, anaemia was associated with lower CSF Aβ42 levels. These findings consolidated the causal close relationship between anaemia and AD.The online version contains supplementary material available at 10.1186/s12883-021-02487-z. As a very common disease in elders, anaemia has a prevalence of 17% in individuals aged 65 or older . In addiThe formation of extracellular amyloid plaques and intracellular neurofibrillary tangles is the typical pathological characteristic of AD, which can be reflected by alterations in cerebrospinal fluid (CSF) biomarkers including reduction of β-amyloid 1–42 (Aβ42), and increased total tau (t-tau) . These cIn this study, we aimed to investigate the associations between anaemia and CSF biomarkers of Alzheimer’s disease pathology in a sample of 646 cognitively intact individuals from the Chinese Alzheimer’s Biomarker and LifestylE (CABLE) study.Our study only included cognitively normal individuals from the Chinese Alzheimer’s Biomarker and LifestyLE (CABLE) study. Since 2017, CABLE is a large-scale cohort study mainly focusing on Alzheimer’s risk factors and biomarkers in Chinese Han population . CABLE aAll participants were Han Chinese aged between 50 to 90 years. The exclusion criteria were (1) central nervous system infection, head trauma, epilepsy, multiple sclerosis, or other major neurological disorders; (2) major psychological disorders ; (3) severe systemic diseases  that may affect CSF or blood levels of AD biomarkers including Aβ and tau; (4) family history of genetic disease. All participants underwent clinical and neuropsychological assessments, and blood and CSF sample collection for the next day. Participants were required to fast for at least 8 h prior. Demographic information, AD risk factor profile, and medical history were also collected by a comprehensive questionnaire and an electronic medical record system. Participants in the study were outpatients and inpatients. We excluded participants with serious medical conditions under the guidance of a professional and experienced physician.Haemoglobin was measured from blood samples using an automated haematology analyser in the laboratory at Qingdao Municipal Hospital. Anaemia was defined as haemoglobin concentrations < 13 g/dl for men and < 12 g/dl for women. The severity of anaemia was classified as mild (11–12.9 g/dl for men and 11–11.9 g/dl for women), moderate 8–10.9 g/dl), or severe (< 8 g/dl) according to World Health Organization criteria [.9 g/dl, The collection and management of CSF samples conform with the international consensus on standardization of CSF research . CSF wasAPOE ε4 alleles. Participants were classified as APOE ε4 noncarriers (participants with no copies of the APOE ε4 gene), and APOE ε4 carriers . DNA was extracted from the blood samples using QIAamp DNA Blood Mini Kit (250). Restriction fragment length polymorphism (RFLP) technology was used to genotype rs7421 and rs429358 to define APOE ε4 alleles. Hypertension, diabetes mellitus and dyslipidemia were determined using self-report, medication use, and verification of hospital records by an adjudicator according to prescribed algorithms. History of stroke and coronary heart disease were based on self-report, clinic data, and medication use. Glomerular filtration rate was estimated using the Cockcroft-Gault equation. Cognitive function was assessed using the adopted China-Modifed Mini-Mental State Examination (CM-MMSE).Fundamental covariates included age, gender, education years, APOE ε4 alleles, history of coronary heart disease, history of stroke, history of hypertension,history of diabetes mellitus, and history of dyslipidaemia, etc) between groups respectively. Multiple linear regression models were used to investigate the associations of anaemia with CSF AD biomarkers. Age, gender, educational levels, APOE ε4 alleles comorbidities , and glomerular filtration rate were included as covariates. P < 0.05 was considered statistically significant. All statistical analyses were performed with R software (version 3.6.1).Wilcoxon tests and chi-squared tests were used to examine continuous variables  and categorical variables . There were less APOE ε4 carriers in anaemia group (6.0%) than non-anaemia group (18.1%). Besides, participants without anaemia had higher CM-MMSE scores than anaemia patients (p < 0.01). Lower glomerular filtration rate and higher incidence of stroke were also observed in those with anaemia compared with those without anaemia. There was no significant difference in incidence of coronary heart disease, hypertension, dyslipidemia between those with anaemia and those without anaemia (p > 0.05).A total of 646 cognitively normal elders were included in this study, consisting of 171 anaemia patients and 529 participants without anaemia. The characteristics of included participants were summarized in Table APOE ε4 alleles, comorbidities , and glomerular filtration rate .Firstly, we investigated the associations between anaemia and CSF biomarkers. Anaemia patients had lower levels of CSF Aβ42 (145.03 ± 58.07 pg/ml) than participants without anaemia (157.39 ± 70.32 pg/ml) after adjusting for age, gender, educational levels, APOE ε4 alleles, comorbidities , and glomerular filtration rate, patients with more severe anaemia had lower CSF Aβ42 levels .The associations between the severity of anaemia and CSF biomarkers were also investigated. Among 117 anaemia patients in our study, there were 74 participants with mild anaemia, 41 participants with moderate anaemia and 2 participants with severe anaemia, according to the WHO criteria. These two participants with severe anaemia were not included in the analysis due to the small sample size. After adjusting for age, gender, educational levels, Increasing attention has been paid to environmental risk factors of AD in order to find out effective ways to prevent AD. Many chronic diseases including anaemia were found to be associated with AD risk and may aggravate AD pathology. In this study, we investigated the associations between anaemia and CSF AD biomarkers. Anaemia patients had lower CSF Aβ42 levels than participants without anaemia. Besides, the severity of anaemia was also associated with the levels of CSF Aβ42. A decreasing trend of CSF Aβ42 was found in patients with more severe anaemia. Anaemia and AD are common diseases in the elderly. Our study focuses on cognitively normal individuals, which help us explore that the association of AD and anaemia arises at a pre-clinical stage. These findings supported the close relationships between anaemia and AD risk.Previous studies showed that low haemoglobin and anaemia were associated with cognitive impairment , 14. AnaSeveral possible mechanisms underlying how anaemia was involved in AD have been proposed. Long-term reductions in haemoglobin may lead to craniocerebral hypoxic injury. Increasing evidence suggests that hypoxia facilitates the pathogenesis of AD through accelerating the accumulation of Aβ, increasing the hyperphosphorylation of tau . The maiThere were some limitations of this study. Our analysis was cross-sectional, therefore limiting causal inferences and raising the possibility that brain changes may precede the development of anaemia. Furthermore, due to the limitation of sample size and the insufficient data, we were unable to analyse the associations of haemoglobin concentration and the anaemia type with CSF AD biomarkers. Additionally, there was no information on the causes of anaemia  and a neurodegeneration biomarker such as CSF neurofilament light chain or MRI measures of atrophy in this study.Our study showed further evidence for the associations of anaemia with CSF AD biomarkers in cognitively normal elders. The treatment of anaemia would be useful in the prevention of AD. Further studies are still necessary to investigate the longitudinal associations between anaemia and the changes in AD biomarkers.These results indicated that anaemia might have a detrimental effect on AD core pathology. The treatment for anaemia may be useful in the prevention of AD.Additional file 1: Table S1. Associations between anaemia and AD biomarkers. Table S2. Associations between the severity of anaemia and AD biomarkers. Table S3. Association of anemia with CSF Aβ42. Table S4. Association of anemia with CSF Aβ40. Table S5. Association of anemia with CSF t-tau. Table S6. Association of anemia with CSF p-tau. Table S7. Association of anemia with CSF t-tau/Aβ42 ratio. Table S8. Association of anemia with CSF p-tau/Aβ42 ratio. Table S9. Association of anemia with CSF Aβ42/Aβ40 ratio. Table S10. Associations between the severity of anaemia and CSF Aβ42. Table S11. Associations between the severity of anaemia and CSF Aβ40. Table S12. Associations between the severity of anaemia and CSF t-tau. Table S13. Associations between the severity of anaemia and CSF p-tau. Table S14. Associations between the severity of anaemia and t-tau/Aβ42 ratio. Table S15. Associations between the severity of anaemia and p-tau/Aβ42 ratio. Table S16. Associations between the severity of anaemia and Aβ40/Aβ42 ratio.