Background: Donor age affects allograft quality and the prognosis of recipients after liver transplantation (LT). Clinicians have assessed the quality of grafts from older donors based on their appearance and texture, with no reliable quantitative evidence. Our study aimed to assess the quantitative impact of donor age on post-transplant outcomes and its safety threshold for LT, based on the published literature.Methods: Relevant studies were retrieved from the Embase, PubMed, and ISI Web of Science databases. Pooled dichotomous relative risks (RRs) were calculated using metan. Continuous RRs were calculated using a two-stage random-effects model.Results: Eleven studies including 30,691 LT cases were included for further analysis. For categorical comparison, the RR of death within the first post-transplant year was significantly higher among patients who received grafts from older donors. Similarly, the RR of graft failure (GF) was increased within the 3 years after transplantation. For continuous comparison, advanced donor age affected transplant outcomes in a linear manner (P > 0.05). A 10-year increment in donor age was associated with RRs 1.10, 1.12, 1.15, 1.10, and 1.08 for 90-day, 180-day, 1-year, 3-year, and 5-year patient mortality and 1.08, 1.06, 1.10, 1.11, and 1.12, for 90-day, 180-day, 1-year, 2-year, and 3-year GF, respectively . A spline model showed that transplants using grafts from donors <43 years old were not associated with age-related risks (P > 0.05). The risk of GF was increased in subgroups with fewer LT cases, longer cold ischemic time, fewer male donors, and recipients with viral hepatitis (P < 0.05).Conclusion: Donor age might affect post-LT outcomes in a dose-dependent manner. The safety threshold for donor age in terms of GF should be lowered to 43 years as an early warning for the guarantee of satisfactory outcomes. Clinicians should weigh the benefits against the risks carefully for patients receiving grafts from older donors. Further studies are warranted to investigate the mechanisms responsible for the relationship between donor age and graft quality. Marginally suitable grafts are widely utilized by clinicians to overcome organ shortages , and morAllografts from older donors might lead to a poor prognosis for the recipients after LT . In addiDespite being a crucial continuous covariate in the evaluation of allograft quality, the dose-response relationship between donor age and post-transplant prognosis has not been systematically evaluated. The safety cutoff for donor age has not been determined, and the decision to use or refuse grafts from older donors has mainly been decided based on the clinician's experience, with a lack of systematic evaluation . A meta-The study was performed strictly according to the criteria of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . The SupWe searched for articles assessing the impact of donor age on post-LT prognosis. Included articles were required to meet the following criteria: (1) LT cases categorized into three or more groups by donor age; (2) patient mortality and graft failure (GF) reported or could be evaluated by calculation; and (3) follow-up duration > 90-days.Two authors (JQ and WW) extracted the information from all the included studies according to a unified standardized reporting form. Potential inter-author discrepancies were checked and resolved by a third experienced author (LZ). The following information was collected: (1) general information ; (2) reasons for LT and causes of death after LT; (3) donor/recipient factors ; (4) surgery ; and (5) outcomes  stay, patient death, and GF).http://getdata-graph-digitizer.com/index.php). Hazard ratios (HRs) and variance were retrieved from the provided data or were calculated based on the data provided. Accurate HRs were acquired from Kaplan–Meier curves using Engauge Digitizer (version 4.1)  , 31. ForThe quality of the methods in each study was examined by WW and ZL based on the Newcastle-Ottawa Scale (NOS) for non-randomized cohort studies (NOS checklist) . We condWe assessed the risk of donor age on patient mortality, organ failure, and post-transplant complications using HRs and relative risks (RRs). Patients were divided into three or four groups, according to donor age, as indicated in the studies. Donors in the top tertile or highest two quartiles were defined as older, while donors in the lowest tertile/quartile were defined as younger. For categorical comparison, we evaluated the RR based on donor age (older/middle vs. younger). Pooled standardized mean differences were used to evaluate quantitative differences across groups classified by donor age (older/middle vs. younger), and the relationship between donor age and the risk of post-transplant outcomes was shown using a dose-response model. We evaluated the standardized mean differences (SMDs) of body mass index (BMI) based on donor age (older vs. younger) using the metan.We also conducted subgroup analyses to assess the effects of potential confounders on the impacts of donor age on patient/graft survival, classified according to sex distribution (recipient/donor), recipient age/etiology, recipient MELD score, sample size, CIT, WIT, type of study (multicenter vs. single center), year of LT, and origin country.The effects of intermediate confounders  on the association between donor age and post-transplant outcomes were examined by meta-regression .Sensitivity analysis of categorical/continuous risks of donor age on patient mortality, GF, primary graft non-function (PNF), and re-transplantation was performed to evaluate the influence of each study on the overall pooled results.Egger's test was used to evaluate the effects of potential publication bias on the categorical/continuous impact of donor age on patient mortality, GF, PNF, and re-transplantation .U-tests. Distributions between different groups were compared by χ2 test. All analyses were performed using SPSS software .Descriptive data with a normal distribution were presented as mean ± standard deviation and compared by one-way ANOVA. Non-normally distributed data were presented as median (inter-quartile range) and compared by Mann–Whitney https://www.medcalc.org/calc/oddsratio.php) if the data were unavailable. We examined statistical heterogeneity using χ2 Q and I2 tests, with I2 values of 25, 50, and 75% defined as low, moderate, and high heterogeneity, respectively  were calculated using an online calculator  . FurtherP > 0.05). The trends in donor-age-related RRs of patient death across different time points are shown in P < 0.05). In terms of donor-age-related risk of GF, the RR increased in the first 3 years after LT  (P > 0.05). Donor age had no significant effect on the RRs of PNF and re-transplantation.Regarding the time-dependent prognosis in relation to donor age, the risks of mortality in patients receiving a graft from an older donor were significantly increased after 180 days  and 1 year . However, the effect of using an older liver was reduced after prolonged follow-up, and there was no significantly increased risk of death associated with an older donor in long-term survivors  , 2, but P < 0.05) (P > 0.05) (P > 0.05)  . However > 0.05) , and no  > 0.05) . The pooP for non-linearity > 0.05) (P > 0.05) (The pooled continuous risks of donor age on patient outcomes are presented in  > 0.05) . The con > 0.05) . The con > 0.05) . There w > 0.05) .The trend in risk of patient mortality related to donor age at different time-points is shown in P < 0.05) (P < 0.05). The risk of donor-age-related GF was higher in single-center studies  (P < 0.05) (P < 0.05) (P = 0.80) (P > 0.05)  , and the < 0.01) , in stud < 0.05) , and in  < 0.05) . Notably = 0.80) , as vali = 0.80) . No sing > 0.05) .P < 0.05). Studies with a larger sample size had a lower risk of GF  (P < 0.05) (P < 0.05)  . In cont < 0.05) . No cova < 0.05) .Sensitivity analysis was performed to visualize the impact of each study on the overall risks. The trend of pooled categorical risk was consistent. No study had any apparent influence on the overall results . RegardiP > 0.05)  . However > 0.05) .P < 0.05); (2) older donors had a significant effect on post-transplant prognosis, showing a linear trend, with increments in patient death and GF of about 5% and 12% per 10 year increase in donor age (both P < 0.05); (3) the use of older grafts had no significant effect on the incidence of PNF or re-transplantation, as short-term post-transplant complications (both P > 0.05), but use of female donors, prolonged CIT, and primary viral hepatitis might amplify the effects of graft age on GF ; and (4) compared with the previous empirical definition of 60 years  using grafts from older donors significantly increased the pooled risks of patient mortality and GF by about 114 and 66%, respectively .Elder donors may suffer more frequently from diabetes and obesity, which might affect the relationship between donor age and post-transplant outcomes. The pooled BMI and prevalence of diabetes were higher in the older donors compared to youngers (both We suspect obesity and diabetes in elder donors might be responsible for worse outcomes in patients received older organs. Further studies for exact estimation on association between aging organ and post-transplant prognosis by adjustment of donors' metabolic derangement are needed to fully confirm our speculation.The current study had some limitations. First, some studies were performed in single center with only 70 cases , and theIn conclusion, donor age might have adverse dose-response effects on patient death and GF following LT. Factors including primary viral hepatitis in recipients and extended CIT might aggravate the risk of GF associated with increased donor age. The safety for threshold donor age can be lowered to 43 years, as early warning for guarantee of satisfactory post-transplant outcomes. Further studies are needed to clarify the mechanisms underlying this association.The original contributions generated for the study are included in the article/This study was performed in accordance with the Declaration of Helsinki and approved by the ethical board of First Affiliated Hospital, School of Medicine, Zhejiang University.ZL, WW, and SZ conceived and designed the study. WW, JQ, SQ, JX, and ZL extracted information, analyzed the data, and wrote the manuscript. WW, LZho, LZhu, LG, and SZ reviewed the manuscript. All authors approved the final manuscript for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AA6111 aluminum automotive body-sheet alloy has been formulated from 100% Taint Tabor scrap aluminum. Direct chill casting with and without high shear melt conditioning (HSMC) was used to produce the AA6111 alloy billets. Both homogenized and non-homogenized billets were extruded into sheets. The optical micrographs of the melt conditioned direct chill (MC-DC) samples showed refined equiaxed grains in comparison to direct chill (DC) cast and direct chill grain refined (DC-GR) samples. Optical metallography showed extensive peripheral coarse grain (PCG) for the DC, DC-GR and MC-DC planks extruded from the homogenized standard AA6111 billets while planks extruded from modified AA6111 billets  showed thin PCG band. The co-addition of recrystallization inhibitors Mn, Zr, and Cr with elimination of the billet homogenization step had a favorable impact on the microstructure of the AA6111 alloy following the extrusion process where a fibrous grain structure was retained across the whole section of the planks. The mechanical properties of as-cast planks extruded from non-homogenized billets were similar to those extruded from homogenized billets. Eliminating the homogenization heat treatment step prior to extrusion has important ramifications in terms of processing cost reduction. The metal extraction for primary aluminum production consumes about 3.5% of global electricity and emits 1% of the total CO2 emission. The challenge for aluminum production is to cut 50% of CO2 emission by 2050. Re-melting of end-of-life aluminum scrap consumes 5% of the energy required to produce primary aluminum and can saves up to 95% of CO2 emission [There is a growing demand to recycle aluminum end-of-life scrap to save natural resources, reduce production energy, and cut COIn the automotive industry, AA6111 is an automotive body-sheet aluminum alloy that used for structural applications . It was In this study, AA6111 was formulated from 100% Taint Tabor scrap with no adding of primary aluminum. In the UK, Taint Tabor (TT) scrap consists of two or more aluminum alloys and it is free of wire or any non-metallic contamination . FurtherBecause of the disadvantage of using grain refinement in casting some of aluminum alloys, other techniques have been tested such as electromagnetic mixing, ultrasonic and the melt conditioning . At BrunChemical segregation, hot tearing, porosity and non-uniform microstructure are the main defects in direct chill casting . In thisThe aim of this study was to assess the microstructure and tensile properties of AA6111 automotive body-sheet formulated from TT scrap. The end-of-life (TT) scrap was processed into billets in direct chill casting followed by extrusion process, cold rolling, heat treatment, and paint bake simulation.For direct chill casting with and without high shear melt conditioning (HSMC), three batches of TT scrap  were received as RSIs from Norton aluminum . To match the target AA6111 composition, the first batch of TT (A052) was melted and formulated by adjusting Cu and Mg level only. The other two batches of TT (F255 and F256) were melted and alloyed with Cu, Mg, Mn, Cr, and Zr. The aim of adding recrystallization inhibitors Mn, Cr, and Zr to TT-6111 was to minimize the development of a coarse outer band of recrystallized grains during extrusion. The billets casting of TT-6111 alloys were carried out in a conventional direct chill caster assembled with a tilting furnace and launder to pour the melt in the open-hot-top of the system. The casting speed of 152 mm in diameter billets was 110 mm/min and the water flow rate was 116 L/min. The melt temperature in the launder was about 690 °C. ‘Optifine’  (Al–3Ti–1B) grain refiner rod was used for the grain refined direct chill (DC-GR) casting. It was added to the melt in the launder before pouring into the sump. For casting with high shear melt conditioning  solution heat treatment at 560 °C for 10 min; (b) cold water quenching and (c) immediate pre-age at 85 °C for 5 hr to develop the T4P temper.To measure the tensile properties after forming and paint bake treatment and/or after aging to peak properties, the extruded 4.8 mm thick 118 mm wide planks of TT-6111 (2) and TT-6111 (3) for all direct chill casting were selected from the center position of extrusion cast. The TT-6111 (1) planks were not considered suitable for further processing due to excessive peripheral coarse grain. The TT-6111 (2) and TT-6111 (3) planks were annealed for 2 h at 360 °C followed by cold rolling down to 3.0 mm. One half of each 3.0 mm planks were annealed at 360 °C for 2 h followed by cold rolling down to 1.7 mm thickness. One half of each 1.7 mm plank was then cold rolled down to 1.0 mm. Sheet cut from the rolled planks at each gauge was used for the machined tensile specimens. Simulation of industrial heat treatment of AA6111/T4P temper strip was carried at each gauge by the following steps: , TT-6111 (2) and TT-6111 (3), showed the significant effect of high shear melt conditioning in grain refining with homogenous grain size distribution across the billets. The cast of modified TT-6111 (3) provided the best extruded plank microstructure and the peripheral coarse grain was eliminated. Processing the recycled aluminum alloys with intensive melt shearing in direct chill casting results in high grain refinement and a homogenized microstructure. Films or discrete oxide particles form at melt surface and entrained within the melt during melt processing . Due to The optical micrographs of TT-6111 (1), TT-6111 (2) and TT-6111 (3) planks extruded from homogenized or non-homogenized billets are shown in The peripheral coarse grain (PCG) is due to the formation of recrystallized grains layer in the upper and lower surface of the extruded part . PCG is A fibrous structure enhances surface quality, forming characteristics, and corrosion resistance in addition to improving strength, toughness, and fatigue properties. Hence, the elimination of grain recrystallization across the whole section of extruded planks is important ,24. DuriThe optical micrographs of annealed, cold rolled with intermediate heat treatment planks extruded from non-homogenized cast billets of TT-6111 (3) are shown in The mechanical properties of as-cast planks extruded from non-homogenized MC-DC cast billets of TT-6111 (3) in the transverse section were measured. With thermo-mechanical treatment  of planks extruded from non-homogenized MC-DC cast billets of TT-6111 (3), the results of tensile test in the T4P and the T8 temper for the three gauges  are shown in Quainoo et al.  investigThe formability and final component quality of the alloy can be improved by optimizing the pre-aging and aging treatment conditions to lower the yield strength for stamping and then reducing of the natural aging kinetics of the stored product ,30. In oFor the first time, the AA6111 aluminum automotive body-sheet alloy has been alloyed, casted, and processed to sheet successfully from 100% end-of-life recycled aluminum scrap.In comparison with the conventional DC and DC-GR castings of the TT-6111 alloy, the metallography of MC-DC cast showed refined equiaxed grains with no casting defects.Microstructural evaluation for planks extruded from non-homogenized of modified TT-6111 (3) showed eliminated peripheral coarse grain. The co-addition of recrystallization inhibitors  with elimination of the homogenization step has a favorable effect on the recrystallization of AA6111 alloy during the extrusion process where a fibrous grain structure was retained across the whole section of the planks.The tensile properties of as-cast planks from non-homogenized billets were similar to those from homogenized billets. Eliminating the homogenization heat treatment step prior to extrusion has important ramifications in terms of processing cost reduction.The yield strength and tensile strength increased with a decrease in ductility of T8 temper after paint bake simulation in comparison with the T4P temper of TT-6111 alloy.


ASPM) gene cause autosomal recessive primary microcephaly (MCPH) in humans. ASPM is a positive regulator of Wnt/β-Catenin signalling and controls symmetric to asymmetric cell division. This process balances neural progenitor proliferation with differentiation during embryogenesis, the malfunction of which could interfere with normal brain development. ASPM mutations may play a role also in other neurodevelopmental disorders, nevertheless, we lack the details of how or to what extent. We therefore assessed neurodevelopmental disease and circuit endophenotypes in mice with a truncating Aspm1–7 mutation. Aspm1–7 mice exhibited impaired short- and long-term object recognition memory and markedly enhanced place learning in the IntelliCage®. This behaviour pattern is reminiscent of a cognitive phenotype seen in mouse models and patients with a rare form of autism spectrum disorder (ASD) as well as in mouse models of altered Wnt signalling. These alterations were accompanied by ventriculomegaly, corpus callosum dysgenesis and decreased parvalbumin (PV)+ interneuron numbers in the hippocampal Cornu Ammonis (CA) region and thalamic reticular nucleus (TRN). PV+ cell number correlated to object recognition (CA and TRN) and place learning (TRN). This opens the possibility that, as well as causing MCPH, mutant ASPM potentially contributes to other neurodevelopmental disorders such as ASD through altered parvalbuminergic interneuron development affecting cognitive behaviour. These findings provide important information for understanding the genetic overlap and improved treatment of neurodevelopmental disorders associated with ASPM.Neurodevelopmental disorders are heterogeneous and identifying shared genetic aetiologies and converging signalling pathways affected could improve disease diagnosis and treatment. Truncating mutations of the abnormal spindle-like microcephaly associated ( Abnormal spindle-like microcephaly associated (ASPM) truncating mutations are the most common cause of the NDD autosomal recessive primary microcephaly (MCPH) in humans2. This is a rare, genetically heterogeneous, disease where patients exhibit a smaller, albeit structurally normal, brain closely linked with intellectual disability. Limited association study data imply an ASPM contribution also to the pathogenesis of other NDDs including schizophrenia3, communication disorder4 and ASD6. Nevertheless, this has not been reinforced by additional patient data, validated empirically nor is it clear through what mechanism ASPM could contribute to features of these NDDs.The overlap in symptoms between neurodevelopmental disorders (NDDs) suggests that there is at least partial overlap in genetic aetiology and affected signalling networks during brain development. Therefore, the challenge is to understand better the consequences of altered gene expression, shared genetic aetiology and convergent signalling pathways to account for clinical variation and ultimately to improve disease diagnosis, treatment and preventionASPM gene is located on chromosome 1q31, composed of 28 exons and encodes 3477 amino acids2. In mice, it is expressed in the ventricular zone of the neocortex during embryonic neurogenesis where it localises to the mitotic spindle poles and midbody7. There, it controls symmetric to asymmetric cell division, which is important for balancing neural progenitor proliferation with differentiation8. ASPM is also essential for correct neuronal migration during corticogenesis9 and was positively selected during primate evolution where it contributed to the genetic basis of brain size10. Recent evidence indicates that ASPM is a positive regulator of the canonical Wnt/β-catenin signal transduction pathway and overexpression of β-catenin can rescue defective neurogenesis induced by ASPM reduction in mice9. Aspm is also expressed in the adult brain12. While there is correlational data potentially implicating ASPM in adult neurogenesis11, an exact function remains unclear.The Aspm1–7, was generated by gene-trap vector insertion into the intron between exon 7 and 8 producing a MCPH patient-like protein truncation13. The majority of human microcephaly patients with ASPM mutations have protein truncations in or before the region encoded by exon 2614. The truncated protein consists of only the microtubule binding domain and lacks the C-terminal amino acids, the calponin homology domains and the calmodulin-binding isoleucine-glutamine (IQ) repeats. This allele, believed to be loss-of-function, causes mild microcephaly in mice where brain size was ~90% that of controls. Currently, there is only limited information detailing the cognitive alterations consequent to Aspm disruption. A recent report undertook neuropsychologic assessment of MCPH patients with ASPM mutations and revealed mild-to-moderate intellectual disabilities15. They nevertheless observed 50% or more reductions in the surface area of cortical regions, the relative magnitude of which was not observed previously in the Aspm1–7 mice. The aim of this study was thus to assess the causal relationship between this allele, a concomitant mild form of microcephaly and changes in mouse cognitive ability as well as neuronal cell populations. By applying a disease-relevant endophenotype strategy, i.e., focusing on objectively quantifiable components of gene-to-behaviour-to-neural circuit pathways16, we wanted also to ascertain whether Aspm alterations have relevance for other NDDs. We therefore applied a comprehensive analysis that addressed multiple behaviour modalities including aspects of emotionality, social affinity/memory, working, recognition and spatial memory. Given the established Aspm influence on embryonic neurogenesis, we endeavoured to understand the effect of this mutation on adult neurogenesis. As well as quantifying this process, we implemented running wheel exercise, a known inducer of adult hippocampal neurogenesis, to assess potential amelioration of genotypic behavioural effects with this environmental enrichment17.Previously, a mutant mouse line, Aspm1–7 gene trap mutant mouse line was produced as described in detail previously13. It was generated from ES cells  by blastocyst injection and chimeras that had germ line transmission were crossed to C57BL/6JOlaHsd mice for more than 10 backcross generations. For the AA0137 ES cell line, PCR and sequencing of genomic DNA  were used to show that the insertion site for the vector pGT01xr was in the intron between exon 7 and 8, 1386 nt downstream from the intron start site. In this initial validation of the line, RT-PCR and immunohistochemistry were used to establish the expression of the truncated ASPM protein tagged with lacZ. RT-PCR was performed on embryonic day (E) 13.5 whole embryo DNA to show that endogenous Aspm mRNA was lost in the mutant mice. The endogenous WT transcript was present only in the WTs and only the gene-trapped transcript was present in the homozygotes. Furthermore, to show that ASPM protein was tagged with β-geo, immunostaining with β-galactosidase  together with ASPM on E10.5 dorsal telencephalon sections was performed. In homozygous mutant mice, β-gal and ASPM were colocalised at metaphase spindle poles whereas β-gal immunoreactivity was absent in the WT. This demonstrated that ASPM was tagged with β-geo. All experiments involved the use of male homozygous mutant mice and their littermate control wild types. The sample size used for the behavioural analyses was based on that used for similar published analyses. Mice were assessed from the age of 23 weeks according to the sequence and ages shown in Fig. n = 11, Aspm1–7n = 10, WT RW n = 10, Aspm1–7 RW n = 10) were assessed in a series of behavioural assays. Both mice with access to running wheels and sedentary controls from each group were tested in parallel. Only sedentary mice were tested in Intellicage® due to setup limitations in giving equal wheel access and activity quantification to all mice within this environment. We therefore focused our cell population quantifications on sedentary mice only. These analyses remain to be replicated in an independent cohort and no method of randomization was used for either behaviour or histological analyses. As mouse behaviour was analysed in a counter balanced design, the observer was not blind to the genotypes. More detailed methods information is contained in the Supplementary Material.All tests performed were approved for the ethical treatment of animals by the responsible authority of the Regierung von Oberbayern (Government of Upper Bavaria). The Aspm1–7 RW, n = 10) and wildtype control  mice was analysed as described previously using low profile wireless running wheels (RWs) 18. Mice were singly housed throughout the running period from age 23 to 37 weeks and individual running activity recorded for analysis.The home-cage voluntary running wheel behaviour of mutant  and illumination at 150 lux in the corners and 200 lux in the middle.The Open Field (OF) analysis was as described before using the ActiMot system 20. The Y maze was composed of opaque light grey PVC and had three identical arms (30 × 5 × 15 cm) placed at 120° from each other. The illumination in the centre of the maze was 100 lux.Spontaneous alternations, alternate arm returns and entries were assessed using the Y-Maze as reported previously21. Measures included time spent in social investigation  and a social recognition index .The Social Discrimination procedure involved using ovariectomised 129Sv stimulus females as shown previously20. The recognition index is the percentage time spent investigating the unfamiliar novel object/(time spent investigating familiar + unfamiliar objects).The object recognition procedure was as described previously20). It is an automated behavioural analysis apparatus consisting of a relatively large plastic homecage (55 × 37.5 × 20.5 cm3) where the four cage corners are operant conditioning chambers (15 × 15 × 21 cm3).The IntelliCage®  assessed place and reversal learning and working memory (“Patrolling”)  as detailed previously22 was used. The materials used were polyclonal anti-DCX antibody  with a biotinylated goat anti-rabbit IgG  and a primary monoclonal mouse anti-PV antibody  with a biotinylated rabbit anti-mouse IgG . An ABC complex was prepared according to manufacturers instructions . Negative controls, with omission of the primary antibodies, revealed no positive staining.For immunostaining of doublecortin (DCX)+ and parvalbumin (PV)+ cells, an Avidin-Biotin Complex ABC method like that employed previously23. The observer was blind to the experimental groups during analysis. When tissue from an animal was damaged during processing, it was excluded from the analysis. A subset of younger (27 weeks old) sedentary WT and Aspm1–7 mice (n = 3–4) were sacrificed and doublecortin (DCX)+ cell number  was analysed at a timepoint that coincided with the start of the behaviour testing sequence. DCX+ cells were estimated within the dorsal hippocampal dentate gyrus. The subgranular zone of the hippocampal dentate gyrus was selected for analysis because it is one of two main neurogenic niches in the adult mouse brain and has the potential to play a role in spatial memory24. PV+ cell numbers were estimated within brain regions known to be involved in cognitive behaviour and/or dysfunctional in NDDs. These included the hippocampal dentate gyrus, Cornu Ammonis (CA) 1, 2/3 regions and the anterior cingulate cortex (ACC). We also quantified the PV+ cell number in the thalamic reticular nucleus (TRN) because of the dense PV+ cell population here and our previous evidence that it may play a role in NDD endophenotypes23. Volumetric analysis of selected ROIs was performed using the Cavalieri estimator probe as described previously22. The following brain ROIs were analysed: corpus callosum, lateral ventricles, CA1, 2, 3 and DG.DCX+ and PV+ cell numbers were estimated in specific regions of interest (ROIs) with design-based stereology using the Stereo Investigator software system  on every sixth serial 40 µm coronal section with the Optical Fractionator probe as described previouslyt-test ; Grubb’s test  and Pearson’s correlation test using GraphPad Prism version 7.03 for Windows . For all tests, a P value < 0.05 was considered significant and data are presented as means ± SEM. A correction for multiple testing was not performed.Data was analysed using Shapiro-Wilk test for normal distribution and then analysed with two-way ANOVA  and repeated measures ANOVA (with post hoc Sidak’s for Intellicage data); unpaired Student’s Aspm disruption, we gave mutant mice and littermate controls access to running wheels within their homecage. The Aspm1–7 mice showed normal voluntary wheel running activity (Student’s t-test: t(18) = 0.56, n.s., Supplementary Table In order to assess the voluntary exercise behaviour consequent to F = 7.14, p = 0.01, genotype effect velocity: F = 6.56, p = 0.02, Fig. F = 4.24, p = 0.047, post hoc Tukey’s test, WT RW vs. Aspm1–7 RW, p = 0.01). The mutant mice also showed decreased arm entries in the Y maze  = 5.18, p = 0.03, Fig. t-test corner visits: t(14) = 2.55, p = 0.02, nosepokes: t(14) = 2.97, p = 0.01, % visits with nosepokes: t(14) = 2.25, p = 0.04, Fig. t-test nosepokes during hour after lights off: t(12) = 3.67, p = 0.003, Fig. In the open field  = 0.002, p = 0.96, Fig. F = 0.001, p = 0.97, Fig. F = 0.36, p = 0.55, Supplementary Table F = 0.79, p = 0.38, Fig. F = 0.84, p = 0.37, Fig. F = 0.006, p = 0.94; social affinity: two-way ANOVA exercise effect: F = 0.61, p = 0.44).To understand the cognitive ability of the mutant mice, we applied a series of learning and memory assays. In the Y-maze, a test of simple working memory, the truncating effect: F,37 = 0.0F = 5.94, p = 0.02, genotype effect long-term memory: F = 4.57, p = 0.04, Fig. F = 0.14, p = 0.71, exercise effect long-term memory: F = 1.30, p = 0.26). This truncating Aspm mutation therefore causes specific recognition deficits.While neither of these forms of memory were markedly affected by the mutation, from the age of 37 weeks, the mutant mice showed impaired short- and long-term object recognition memory. Here the recognition memory index decreased after both 3 and 24 h  = 12.52, p = 0.004, day effect: F = 17.88, p < 0.0001, interaction effect: F = 2.25, p = 0.10, Fig. Aspm1–7 mice made slightly more corner visits  = 5.03, p = 0.005, post hoc Sidak’s test on day 3 t(52) = 2.86, p = 0.02). The mutant mice made also more nosepokes, an effect that was significant during day 2 and 3 of the place learning  = 3.65, p = 0.02, post hoc Sidak’s test for day 2: t(52) = 3.08, p = 0.01, day 3: t(52) = 3.14, p = 0.01, Fig. Using the IntelliCage® apparatus, place and reversal learning ability were assessed in these mice. During the IntelliCage® place learning trial  = 2.53, p = 0.14, Fig. Aspm mutation led to increased corner activity as indexed by an increased number of corner visits by the mutant mice  = 6.21, p = 0.03, Fig. Aspm1–7 mutation also led to a significantly increased number of nosepokes during day 3 and 4 of the reversal learning phase  = 2.99, p = 0.04, post hoc Sidak’s test day 3 wt vs. hom t(52) = 2.83, p = 0.03, day 4 wt vs. hom t(52) = 3.58, p = 0.003, Fig. There were no genotype effects on behavioural flexibility in the reversal learning phase  = 7.48, p = 0.0005, post hoc Sidak’s test day 2 wt vs. hom: t(52) = 3.02, p = 0.02, day 3 wt vs. hom: t(52) = 4.26, p = 0.0003, day 4 wt vs. hom: t(52) = 5.93, p < 0.0001, Fig. Aspm mutation was associated with a clear improvement (decreased % error rate) between day 1 and day 2 of patrolling (post hoc Sidak’s day 1 vs. day 2 in homs t(39) = 4.27, p = 0.0007, day 3 vs. day 4 t(39) = 2.80, p = 0.047). There was no such improvement in the WT mice. With respect to activity during this testing phase, the truncating Aspm1–7 mutation led to an increased number of corner visits during day 1 of patrolling without significant differences during the other three days  = 13.41, p < 0.0001, post hoc Sidak’s test day 1 wt vs. homs t(52) = 3.53, p = 0.004, Fig. Aspm1–7 mutant mice executed significantly more nosepokes on the first day of patrolling; a pattern of activity not visible during the other three days of this task  = 24.11, p < 0.0001, post hoc Sidak’s test, day 1 wt vs. hom t(52) = 5.78, p < 0.0001, Fig. Aspm mutation leads to clearly enhanced place learning as an index of spatial memory and patrolling ability as an index of working memory.From day 2 to day 4 of patrolling, the mutant mice exhibited a progressively lower % error rate compared to controls  balance. To determine whether alterations in PV+ GABAergic interneurons formed the substrate for the enhanced place learning in these Aspm1–7 mice, we quantified the number of PV+ cells in the hippocampal CA1 and CA2/3 regions. The Aspm truncating mutation led to a clear significant decrease in the number of PV+ cells in the CA1 region (Unpaired t-test: t(13) = 3.92, p = 0.002, Fig. t test: t(13) = 2.34, p = 0.04, Fig. Aspm mutation, we quantified the number of PV+ cells in the ACC, the hippocampal dentate gyrus and the TRN of the thalamus. While there was no clear genotype-related difference in either the ACC (Unpaired t-test: t(10) = 1.65, p = 0.13, Supplementary Table t-test: t(10) = 0.004, p = 0.10, Supplementary Table t-test: t(14) = 3.52, p = 0.003, Fig. Aspm thus alters the number of PV+ cells in specific brain regions.The GABAergic inhibitory interneurons are a key cell population crucial for modulating local circuit activity in the hippocampus and cortex. Of these inhibitory interneurons, the subset of PV+ GABAergic neurons is known to play a role in cognitionAspm is expressed in the proliferative zones of the developing mouse brain where it is necessary for maintaining the spindle pole of mitotic neuroepithelial cells in symmetric proliferation7. Furthermore, there was an upregulation of Aspm in the adult rat hippocampus with increased proliferation suggesting a role for ASPM in adult hippocampal neurogenesis11. We thus quantified the number of DCX+ cells in the dorsal dentate gyrus as a surrogate marker of adult neurogenesis. The dorsal dentate gyrus preferentially mediates cognitive function including pattern separation and spatial learning and memory27. There was a clear decrease in the number of DCX+ cells (t(5) = 8.13, p = 0.0005, Fig. It is known that Aspm1–7 mutant mice exhibit a mild microcephaly analogous to human patients with a truncating ASPM mutation and a thinner neocortex13. Subcortical structures were not assessed in this study, thus we here focused our volumetric analysis firstly on the hippocampus. We established that there were no volumetric differences between the genotypes in any of the hippocampal subfields  = 3.46, p = 0.004, Fig. Aspm1–7 mutant mice indicative of corpus callosum dysgenesis (t(11) = 3.33, p = 0.007, Fig. Given this reduction in adult hippocampal neurogenesis, we assessed whether there were volumetric differences that would be related to such an impairment. It was shown previously that young adult Aspm1–7 mice may be related to and/or a consequence of the altered PV+ cell number, ventriculomegaly and corpus callosum dysgenesis, we performed a Pearson’s correlation analysis. From the highlights of this analysis of all subjects focusing on the parameters that showed alterations in the Aspm1–7 mice . Spatial reference and working memory are also regarded as predominantly hippocampus-based, for both spatial disambiguation and using environmental cues to generate a cognitive map of a location33. During the place learning and patrolling trials in IntelliCage®, the Aspm1–7 mice performed better, exhibiting decreased error rates. Thus, it is likely that the truncating Aspm allele alters neurocircuits recruited for the execution of these cognitive tasks and this is supported by our neuropathological analysis results.34. Furthermore, this phenotypic pattern is present in ASD-related genetic models such as Taok2 KO mice35 and to a degree in the Shank1 KO mice36, neuroligin-3 R451C mutant mice38, Scrib1 KO mice39 and Lrfn2 KO mice40  bolsters this assertion. A feature of certain NDDs including ASD and sensory processing disorders in humans include hypo- or hyper-reactivity to sensory stimuli44. While this locomotor change may represent an altered anxiety response to novelty, it could also signify that this truncating Aspm mutation blunts responses to sensory stimuli including novel situations. For reasons not currently appreciated and requiring further study, the experience of chronic exercise heightens this effect with Aspm mutation. The complex cognitive phenotype also phenocopies alterations in mice with conditional knockout of β-catenin (CTNNB1) in GABAergic PV+ interneurons45. CTNNB1 is a key regulator of the canonical Wnt signalling pathway pivotal to neurodevelopment. Aspm, in turn, is a positive regulator of this canonical Wnt/β-catenin pathway9. Thus, ASPM loss-of-function could lead to such a mixed cognitive phenotype via alterations in this pathway and cell population.While dissociable effects on different memory modalities may seem counterintuitive, such a combination has been described in humans previously, for example, in rare high functioning autistic patients exhibiting superior cognitive and learning ability and performance in map-related spatial tasks46. They are generated in the embryonic medial ganglionic eminence47 where Aspm is highly expressed at E14.5 and E16.548. The evidence described here infers that disrupted ASPM may alter cognitive function by regulating PV+ cell development leading to an altered NDD-related endophenotype. It appears then that the TRN and hippocampal CA are ROIs for understanding IntelliCage® place learning as well as, by extrapolation, enhanced spatial memory in NDDs. The dense shell of PV+ cells of the TRN encapsulates thalamo-cortical neurons, relaying sensory information between thalamus and somatosensory cortex. It suppresses distracting sensory inputs during attentional processing mediating normal cognition50. It is possible that aberrant TRN PV+ cell number leads to thalamic excitation/inhibition imbalance modifying attentional capacity and affecting cognitive ability. Notably, correlational analysis revealed potential opposing roles of TRN PV+ cells in object recognition vs. place learning where a deficit is detrimental to the former yet advantageous for the latter. Further experiments are needed to address the fundamental mechanisms involved and to assess putative thalamocortical and hippocampal excitation/inhibition imbalances in detail. The possible contribution of CA1 PV+ interneurons to impaired object recognition is consistent with a previous observation51. How PV+ cell numbers decrease requires further study. Nevertheless, altered Wnt signalling is a likely explanation given that a similar complex cognitive phenotype occurs in mice with CTNNB1 KO in PV+ cells and the established role of ASPM in regulating this pathway. It may contribute to loss of PV+ cells by interfering with cell cycle regulation, proliferation, differentiation, migration and/or maturation52.Fast-spiking PV+ GABAergic interneurons inhibit and synchronise principal cells during network oscillationsAspm1–7 mice may also relate to the observed ventriculomegaly and corpus callosum dysgenesis that resemble alterations seen in human microcephaly patients with ASPM mutations and in Aspm−/− mice30. As hypothesised by others, the Aspm mutation likely impairs ciliary function and precipitates lateral ventricle enlargement consequent to hydrocephalus53. The inverse correlation between enlarged ventricles and PV+ cell numbers as well as with IntelliCage® place learning and working memory suggests a connection. It is postulated that ASPM participates in cilia-mediated neuronal migration53. The N-terminal domain of Aspm is part of the family of ASH  domains, present in proteins associated with cilia and flagella53. In addition, primary ciliary signalling is necessary for interneuronal connectivity54 and a function of ependymal cilia is to transmit the directional information imperative for cellular migration55. Thus, the region-specific reduction in PV+ cell number may relate to impaired migration during development concomitant to compromised ciliary function. The Aspm1–7 mutation leads also to decreases in adult hippocampal neurogenesis (DCX+ cell number) in young adult mice. While it warrants further enquiry, given the role of ASPM in cell division and Wnt signalling, it could also be that Aspm1–7 spurs a reduction in adult neurogenesis through altered proliferative activity57. Reduced adult hippocampal neurogenesis consequent to disrupted Wnt signalling in rats impairs object recognition58. In addition, adult neurogenesis ablation can enhance hippocampal-dependent working memory under certain conditions59. Thus, whether the adult neurogenesis deficit in Aspm1–7 mice contributes to the object recognition and working memory phenotype needs to be confirmed. Additional analysis of Aspm1–7 mutation-induced developmental effects during embryogenesis would also be necessary to determine the origins of the brain tissue alterations observed here.Loss of PV+ interneurons in Aspm1–7 mice has unearthed a complex cognitive phenotype including mild recognition memory impairment and clearly enhanced spatial memory. This outcome is partially consistent with findings in human MCPH patients. While the memory modalities affected may not overlap, human MCPH patients with ASPM mutations also showed mixed cognitive symptoms with mildly impaired cognition (including working memory) and intact mnesic abilities15. Although not reported previously in MCPH patients , the enhanced spatial memory described here is also a proposed endophenotype of specific rare ASD-subtypes, a disorder often comorbid with microcephaly in general61. ASD is highly complex and heterogeneous, related to variation in multiple genes62. ASPM was not identified as a susceptibility gene for either ASD or schizophrenia in recent genome-wide association studies from European and North American cohorts (that largely excluded Asian and African cohorts)64. Nevertheless, it may be that ASPM mutations, when present in certain rare cases (e.g. see ref. 6), contribute to this specific ASD endophenotype and not the full gamut of typical ASD features such as social avoidance and repetitive behaviour. There are now moves to elucidate convergent signalling pathways affected in NDDs to understand genetic overlap, improve disease diagnosis and facilitate treatment strategies65. In that respect, it is remarkable that the Aspm1–7-induced enhanced spatial memory is therefore also reminiscent of that found in mice with genetic modifications in the Wnt signalling pathway66. As Aspm is a positive regulator of the canonical Wnt/β-catenin pathway it is possible that dysfunctional Wnt signalling could underlie some of the phenotypes described here. We therefore propose the hypothesis that a potential Aspm1–7-induced excitation/inhibition imbalance that may result from altered PV+ interneuron number and/or additional modifications in key brain regions consequent to altered Wnt signalling, underlies the cognitive endophenotype. While confirmation is needed, our findings are consistent with a convergence of NDD-related risk genes onto the Wnt neurodevelopmental pathway and the PV+ interneuron population69. This information therefore has important implications for understanding the genetic overlap and improved treatment of NDDs associated with ASPM. Furthermore, we propose spatial memory alterations as a measurable correlate of TRN circuit activity that may be a viable clinical biomarker to imply disruption of this circuit.In sum, this analysis of Supplementary information


The recent development of IQ-CSF, the second generation of real-time quaking-induced conversion (RT-QuIC) using cerebrospinal fluid (CSF), for the diagnosis of Creutzfeldt-Jakob Disease (CJD) represents a major diagnostic advance in the field. Highly accurate results have been reported with encouraging reproducibility among different centers. However, availability is still insufficient, and only a few research centers have access to the method in developing countries. In Brazil, we have had 603 suspected cases of CJD since 2005, when surveillance started. Of these, 404 were undiagnosed. This lack of diagnosis is due, among other factors, to the lack of a reference center for the diagnosis of these diseases in Brazil, resulting in some of these samples being sent abroad for analysis. The aim of this research study is to report the pilot use of IQ-CSF in a small cohort of Brazilian patients with possible or probable CJD, implementing a reference center in the country. We stored CSF samples from patients with possible, probable or genetic CJD (one case) during the time frame of December 2016 through June 2018. All CSF samples were processed according to standardized protocols without access to the clinical data. Eight patients presented to our team with rapidly progressive dementia and typical neurological signs of CJD. We used CSF samples from seven patients with other neurological conditions as negative controls. Five out of seven suspected cases had positive tests; two cases showed inconclusive results. Among controls, there was one false-positive (a CSF sample from a 5-year-old child with leukemia under treatment). The occurrence of a false positive in one of the negative control samples raises the possibility of the presence of interfering components in the CSF sample from patients with non-neurodegenerative pathologies. Our pilot results illustrate the feasibility of having CJD CSF samples tested in Brazilian centers and highlight the importance of interinstitutional collaboration to pursue a higher diagnostic accuracy in CJD in Brazil and Latin America. C), into an abnormally folded isoform (PrPsc) , encompass a group of rare neurodegenerative conditions secondary to abnormal conversion of a constitutively expressed cellular glycoprotein, the prion protein (PrP (PrPsc) . Sporadi (PrPsc) . Since i (PrPsc) .Sc are still the standard criteria for establishing a definitive diagnosis for CJD  and cerebrospinal fluid (CSF) analysis have increased diagnostic accuracy . The preiagnosis . OverallSc in CSF and other tissue samples  assay was developed in 2010, and since then, its experimental conditions have been improved and tested on a large number of samples in several laboratories worldwide . It ultr samples . The rec samples . In huma samples .RT-QuIC started to be clinically used in 2015 and became a criterion of the Centers for Disease Control and Prevention (CDC) to diagnose CJD as probable . Its higSurveillance of TSE cases has been compulsory in Brazil since 2005 , and 603Patients with suspected CJD were admitted for investigation under a protocol for rapidly progressive dementia (RPD) as previously reported . FollowiThree cases had access to molecular analysis of the PRNP gene for polymorphisms in the 129 codon. Samples were analyzed using denaturing high-performance liquid chromatography. Technical details about this procedure as well as amplification reactions and DNA extraction have been previously described .We analyzed eight CSF samples from seven patients with possible, probable or genetic CJD referred to the Department of Neurology at University of São Paulo from December 2016 to June 2018. One patient (named ABT) had her CSF tested twice at different times. We used CSF samples from seven patients with other neurological conditions as negative controls. CSF samples (10 mL) were collected by lumbar puncture (LP) following a standard procedure. Two milliliters of the CSF sample were then centrifuged at 1000 × g for 10 min and stored in polypropylene tubes at −80°C until blind analysis by researcher TCRGV at UFRJ.The RT-QuIC assay was performed using the improved QuIC-CSF (IQ-CSF) conditions as published . BrieflyUnseeded reaction (not shown) and random CSF samples from patients with other neurological conditions  were selIn the referred time frame, seven patients presented to our team with rapidly progressive dementia and typical neurological signs of CJD. The patient named ABT had her CSF tested twice at different times (ABT1 and ABT2 samples), therefore rendering a total of eight CSF samples. ABT patient results will be better reported in Case 1 description.We randomly used CSF samples from seven patients with other neurological conditions as negative controls. Five out of eight suspected samples had positive RT-QuIC results in our hands . ClinicaSamples from patients LRC and DAS were used as positive controls, given that they were previously analyzed using RT-QuIC in a reference center outside the country . Our RT-QuIC analyses were also positive, corroborating this result . PatientAmong the negative controls, there was one false positive, sample CT2 . The falIn the following, two cases are presented in which the results obtained with RT-QuIC show interesting aspects related to RT-QuIC sensitivity and disease progression.ABT was a 72-year-old woman with 7 years of formal education. She had an atypical presentation characterized by a 4-month history of rapidly progressive cognitive impairment associated with visual hallucinations and gait disturbances with repeated falls. She had no relevant past medical history or family history of any neurological conditions. On neurological examination, she scored 17/30 on the Mini-Mental State Examination (MMSE), and physical tests revealed only a prominent axial syndrome as she could not sit or stand up without bilateral support. There were no pyramidal signs, parkinsonism or visuospatial impairment. A comprehensive investigation with metabolic, inflammatory, paraneoplastic, and infectious tests was unremarkable. Brain magnetic resonance imaging (MRI) revealed symmetric diffusion weighted image (DWI) hyperintensities in the basal ganglia , a findiJJN was a 65-year-old male who presented to our clinic with an 8-month history of behavioral changes. He had 4 years of education and a medical history of hypertension and gouty arthritis. There was no family history of any neurological conditions. His wife described the first symptoms as prominent changes in his food preferences with an unusual demand for rice and chicken. Two months later, he developed visual hallucinations, mostly described as the presence of spiders in the ceiling. In the first evaluation, he was independent of instrumental activities of daily living. Neurological examination revealed an MMSE of 20/30 with an unremarkable physical examination. A laboratory work-up including metabolic, inflammatory and serology studies was negative. At this point, we were surprised by the finding of brain MRI diffusion weighted imaging revealing marked bilateral hypersignal in the frontotemporoparieto-occipital cortex, basal ganglia, thalamus and – less markedly – in the hippocampus, raising the suspicion of sporadic Creutzfeldt-Jakob Disease (sCJD) . The patThe present study reports the results of pilot second-generation RT-QuIC testing in a small patient cohort referred for rapidly progressive neurological syndromes of suspected prion nature. Our center previously reported 61 cases of rapidly progressive dementia among 1648 patients in a 3-year interval. Immune-mediated encephalopathies represented the majority of the samples (46%), followed by CJD (11.5%) . All casAs we described here, IQ-CSF RT-QuIC proved to be an extremely important tool in the diagnosis of ABT and JJN cases (Cases 1 and 2). Both patients presented with rapidly progressive neurological syndromes and could not initially be classified with possible or probable CJD, according to the most recent criteria . Both ABIn the case of ABT patient, the clinical presentation of rapid cognitive impairment with visual hallucinations and gait impairment was considered atypical. In this case, the brain MRI was an important diagnostic clue, with DWI hyperintensities in the basal ganglia leading to a differential diagnosis with metabolic encephalopathies, hypoxic-ischemic lesions or toxic lesions  . A carefsc following disease progression. Although disease duration does not seem to be related to RT-QuIC results  included (1) a presentation with mild behavioral changes; (2) the initial sparing of motor systems, with evidence of motor findings in the neurological examination only almost 12 months after first symptoms; (3) an unremarkable EEG with 7–8 months of evolution; and (4) the presence of a bilateral T2 hypersignal in the hippocampus. The RT-QuIC was inconclusive, but together with MRI, it could suggest early diagnosis of probable CJD. Perhaps with disease progression, JJN’s CSF sample would obtain a positive RT-QuIC result, as observed for ABT patient.in vitro by The occurrence of one false-positive case in our RT-QuIC test weakens our diagnostic accuracy and underscores the need for improvements in the protocol. Despite having an extremely high specificity, IQ-CSF RT-QuIC false-positive results have been reported in the literature. Regarding our false-positive case, we did not find any similar cases reporting the use of intrathecal medications as a possible reason for a positive RT-QuIC result. A positive case in a 5-year-old child without any neurological symptoms would never be expected; an 18-year-old woman was the youngest person diagnosed with probable sCJD using RT-QuIC . TherefoThe present study is limited by its pilot nature, with a modest sample size. Improvements in the establishment of the protocol are necessary, requiring a greater number of analyses. The use of nasal brushes to collect patient samples for RT-QuIC analysis is also valid to improve protocol sensitivity and specificity. However, this is the first study to our knowledge to report specific biomarker-based feasible results performed in a developing country for prion diagnosis, in addition to pointing out new possible interferences in the protocol, and the need to understand how the different current diagnostic approaches can reveal disease progression.The cases reported here illustrate the importance of using RT-QuIC for patients with neurological syndromes, enabling the diagnosis of probable CJD, while no other method was sufficient to support this diagnosis, even with atypical clinical presentation. In addition, the identification of a false positive in a sample from a leukemic pediatric patient undergoing intrathecal treatment with chemotherapy suggests new possible interferences in the method. This will require future investigation of the effect of these chemotherapeutic agents for inclusion or not as a limitation for carrying out the assay.CSF samples are often evaluated only once, due to the invasiveness of CSF collection and the absence of curative treatment for TSEs, in addition to rapid disease progression. One of the cases we report points out the importance of carrying out studies that evaluate the progression of the disease and that RT-QuIC is a useful approach for such studies. In this case, the use of nasal brushes might be prioritized over CSF analysis, since this provides greater sensitivity and enables more frequent sample collection, given the less invasive nature of the procedure.Finally, our study illustrates the feasibility of having CJD CSF samples tested in Brazilian centers, and highlights the importance of inter-institutional collaboration in order to pursue a greater diagnostic accuracy for CJD in developing countries. It also demonstrates that RT-QuIC can be clinically available for testing patients from Brazil and other Latin American countries, and points to the need and feasibility of establishing a national reference center for the diagnosis of these rare but devastating diseases.All datasets generated for this study are included in the article/supplementary material.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.BB: study design, patient data collection, and manuscript writing. BB, BC, RA, MB, HG, and SB: patient data collection and interpretation. JS and RN: study design, data interpretation, and manuscript critical revision. ML and VM: genetic analysis. JLS and TV: RT-QuIC facility implementation. TV: study design, RT-QuIC evaluations, RT-QuIC data analysis, and manuscript writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Do patients discharged from the emergency department after a single troponin test with negative results have similar outcomes to patients undergoing multiple troponin tests?This cohort study found no significant difference in 30-day acute myocardial infarction or cardiac mortality between patients receiving a single troponin test with negative results and those who underwent serial troponin tests .This study suggests that physician discretion to order serial or single troponin tests in managing emergency department patients suspected to have acute coronary syndrome appears safe and is a reasonable strategy to possibly improve efficiency without an adverse association with patient outcomes. Chest pain is among the most common reasons for emergency department (ED) presentations. However, most patients are at low risk for acute coronary syndrome (ACS), with low cardiac adverse outcomes rates. Biomarker testing with troponin levels is key in the initial assessment for ACS. Although serial troponin testing can improve the diagnosis of ACS in clinical practice, some patients deemed to be low risk are discharged after a single negative troponin test result.To report the clinical outcomes of patients discharged after a single negative troponin test result compared with patients discharged after serial troponin measurements.This is a retrospective cohort study of ED encounters from May 5, 2016, to December 1, 2017, across 15 community EDs within an integrated health care system in southern California. The study cohort includes 27 918 adult ED encounters in which patients were evaluated for suspected ACS with a HEART  score and an initial conventional troponin-I measurement below the level of detection (<0.02 ng/mL). Statistical analysis was performed from December 1, 2019, to December 1, 2020.Single troponin test vs multiple troponin tests.The primary outcome was acute myocardial infarction or cardiac mortality; secondary outcomes included coronary artery bypass graft, percutaneous coronary intervention, invasive coronary angiography, and unstable angina within 30 days of discharge. A multivariable logistic regression model was performed to evaluate the association between testing strategies and clinical outcomes.A total of 27 918 patient encounters  were included in the study. Of patients with an initial troponin measurement below the level of detection, 14 459 (51.8%) were discharged after a single troponin measurement, and 13 459 (48.2%) underwent serial troponin tests. After adjustment for cardiac risk factors and comorbidities, there was no statistically significant difference in the primary outcome of acute myocardial infarction or cardiac mortality within 30 days between the 2 groups . Patients discharged after a single troponin test had lower rates of coronary artery bypass graft  and invasive coronary angiography .This study suggests that patients are routinely discharged from the ED after a single negative troponin test result, and when compared with serial troponin testing, a single troponin test appears safe based on current physician decision-making, with no difference in rates of 30-day cardiac mortality and acute myocardial infarction, which are low in both groups. This cohort study reports the clinical outcomes of patients discharged from the emergency department after a single negative troponin test result compared with those discharged after serial troponin measurements. Prompt diagnosis is critical to initiate appropriate evidence-based therapies, while accurate and efficient exclusion of AMI is essential to avoid iatrogenic harms and for the efficient use of health care resources.Chest pain and symptoms concerning for acute myocardial infarction (AMI) are among the most common reasons for emergency department (ED) presentation, accounting for more than 10 million ED visits annually.4 Recent studies measuring high-sensitivity troponin (hsTn) showed that, if the initial troponin level is very low, 1 troponin test with negative results may be sufficient to safely discharge a patient from the ED.7 These studies were performed using hsTn, but the use of hsTn is still low in the US .8 In practice, some clinicians have adopted this strategy of a single troponin test using conventional troponin assays. The safety of this practice is not well studied, to our knowledge.Current guidelines and several diagnostic algorithms recommend the repeated use of troponin tests and the detection of absolute change in the troponin level to safely rule out AMI in the ED.9 Incorporating the HEART score into clinical care pathways has been shown to reduce cardiac testing and length of stay and increase early discharges, while maintaining high sensitivity for detecting AMI.11 A single-center prospective cohort study that compared the performance of 3 troponin assays  using the modified HEART score found that all 3 assays were equally effective in risk stratifying patients with chest pain for safe discharge, yielding a 100% sensitivity for 30-day mortality.12Several decision rules are available to help risk stratify patients presenting to the ED with symptoms concerning for AMI. The HEART  score is a validated risk stratification tool that incorporates clinical history with electrocardiography and troponin testing to help clinicians identify high-risk patients for further observation and low-risk patients who may be discharged and safely avoid further diagnostic testing.Many patients who present to the ED have an initial troponin level that is in the undetectable range. The value of keeping low-risk patients with an initial undetectable troponin level in the ED for serial troponin measurements may be of marginal value. The goal of this study is to evaluate the safety of discharging patients with suspected AMI after a single negative conventional troponin test result , incorporating the HEART score, compared with a strategy of serial troponin measurements for patients with suspected AMI.STROBE) reporting guideline.13We conducted a retrospective cohort study of prospectively collected data from 15 community EDs between May 5, 2016, and December 1, 2017, within Kaiser Permanente Southern California, an integrated health system with more than 4 million health plan members. Kaiser Permanente Southern California hospitals provide care to more than 1 million patients per year presenting to the ED. Of these ED visits, approximately 80% are by health plan members. Health plan members receive comprehensive services, including ambulatory, inpatient, pharmacy, imaging, and laboratory services. All care episodes are captured in a single integrated electronic health record among health plan facilities and through claims data for care outside of the integrated system. This study was approved by the Kaiser Permanente Southern California Institutional Review Board, which waived the need for informed consent given the retrospective nature of this observational study that involved no direct patient contact to evaluate the impact of a quality improvement initiative to optimize care for patients evaluated in the ED. Our study followed the Strengthening the Reporting of Observational Studies in Epidemiology  with a HEART score and an initial conventional troponin I level below the limit of quantification (<0.02 ng/mL). The patients included were with Kaiser Permanente Southern California health plan membership of at least 12 months prior to their ED visit . Patients who were nonmembers or did not have continuous insurance coverage for 1 year prior to their ED visit were excluded because we do not have accurate comorbidity data or follow-up information for their outcomes. Patients with a do-not-resuscitate status or hospice status were also excluded.14All sites used the same conventional troponin I assay . The limit of quantitation is 0.02 ng/mL. The 99th percentile is 0.04 mg/mL. The coefficient of variation at a concentration of 0.04 ng/mL is 10%. The coefficient for variation at a concentration of 0.02 ng/mL is 20%.15 Clinical patient variables were obtained by querying the structured electronic health records. The International Classification of Diseases, Ninth Revision (ICD-9) and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) codes used to define the clinical variables  can be found in the eAppendix in the Baseline demographic information, including age, sex, and race/ethnicity data, were obtained from administrative records. Our administrative data were previously shown to be accurate in describing race/ethnicity.17 as well as the US,19 and recommended by the American Heart Association.9 Scores are categorized as low risk (0-3), moderate risk (4-6), and high risk (7-10) to aid clinical decision-making. Details about the implementation of the HEART score in May 2016 at the participating study sites has been previously reported.21 At the time of the implementation of the HEART score, data on physician education were provided to encourage repeated troponin testing at least 3 hours after symptom onset or after the peak of symptom severity. Physician education was provided via a prerecorded continuing education module recommending physicians order repeated troponin tests for patients with symptoms for less than 3 hours. Physicians were encouraged to use timing of symptoms, initial troponin value, and initial HEART score to assess the benefit associated with repeated troponin testing and further clinical management. The data for the timing of the onset of chest pain are not available for this study.The HEART score was calculated using clinical and demographic data. HEART is a 0- to 10-point scoring system initially developed in the Netherlands and validated in Europe,The primary outcome was 30-day cardiac mortality or myocardial infarction after ED discharge. Secondary outcomes were unstable angina, invasive coronary angiography, percutaneous transluminal coronary angioplasty, or coronary artery bypass graft.ICD-9 or ICD-10 code for myocardial infarction was the principal diagnosis listed at a subsequent inpatient or ED encounter either within the integrated health system or from claims for services provided at an out-of-network facility. Mortality was assessed using a death registry that included data obtained from the California death index, the federal Social Security death index, and membership files. Cardiac or noncardiac cause of death was analyzed by a physician (M.W.) reviewing the stated cause of death in ICD-9 and ICD-10 codes and California state records.Myocardial infarction was considered to have occurred if an t tests  or Wilcoxon rank sum tests when appropriate. Categorical variables were compared using the χ2 test. Multivariable logistic regression models were conducted to investigate the association between a strategy of serial troponin measurements and cardiovascular outcomes. Separate analyses were conducted for each cardiac outcome  and cardiac mortality. Analyses were performed using SAS, version 9.3 (SAS Institute Inc). Reported P values are 2-sided, and the level of statistical significance was P < .05.Statistical analysis was performed from December 1, 2019, to December 1, 2020. Baseline characteristics are summarized using descriptive statistics. A comparison between the group undergoing a single troponin test and the group undergoing serial troponin tests was made by conducting A total of 27 918 patient encounters  were included in the study . BetweenThe mean (SD) age of patients who underwent a single troponin test was 57.1 (16.1) years, and 8617 (59.6%) were women . PatientOf 27 918 patients with an initial negative troponin test result, 14 459 (51.8%) were discharged after a single troponin test, 10 611 (38.0%) had 2 troponin tests performed, and 2848 (10.2%) had 3 or more serial troponin tests performed . Among tFollow-up information was available for 25 702 (92.1%) of patients. In analysis of secondary outcomes, patients discharged after a single negative troponin test result had lower adjusted rates of coronary artery bypass graft  and invasive coronary angiogram . However, there was no statistically significant difference between the groups in the rates of percutaneous coronary intervention  and unstable angina  .In this large retrospective cohort study analyzing the safety of discharging patients after a single nondetectable conventional troponin level, we found that use of a single troponin measurement, when deemed appropriate by the clinician caring for the patient, was safe and not associated with differences in 30-day AMI or cardiac mortality. We also observed that patients discharged after a single troponin test had a lower rate of coronary artery bypass graft and a lower rate of invasive coronary angiography within 30 days.4 Serial testing has been recommended by most to safely rule out AMI. Elevated troponin levels can be detected as early as 2 to 4 hours after the onset of symptoms and can be delayed for up to 8 to 12 hours. Several early studies have demonstrated that the slow increase and late peak of troponin levels make the conventional troponin assay vulnerable to missing early evolving AMI.24 However, most patients presenting to the ED with chest pain are at low risk for ACS and are below a 1% risk threshold of 30-day adverse events.18 Therefore, there may not be a benefit associated with repeated troponin testing for low-risk patients and those with prolonged symptoms, despite guideline recommendations. In our cohort, 51.8% of patients with an initial troponin evaluation did not undergo repeated troponin testing. In addition, among patients who did undergo repeated troponin testing, very few (66 [0.5%]) had a positive test result (troponin level >0.5 ng/mL). Our results also demonstrate that, in clinical practice, the strategy of administering a single troponin test is safe when the test is performed along with a clinician’s clinical assessment and risk stratification by the HEART score.The American Heart Association and European Society of Cardiology guidelines recommend serial cardiac troponin tests to identify and exclude myocardial infarction as well as to assess the acuity of a cardiac event in an individual with suspected ACS.25 A previous multicenter study, in which 64% of troponin measurements were single assesssments, found that 79% of elevated troponin levels were associated with diagnoses other than AMI.26 When the troponin test was first introduced for clinical use, it was thought to be specific for AMI because the population in which it was studied had a significantly higher prevalence of coronary artery disease. However, its current use in the general population and with hsTn has made it less specific for AMI when results are positive. In a prospective cohort study of patients presenting to the ED in the UK and US, only 4.2% had an AMI.27 Our study demonstrates that physicans are able to appropriately identify patients at higher risk based on other clinical factors beyond the results of a second troponin test.Many experts agree that there is broad overuse of troponin tests for clinical presentations even when the suspicion for AMI is low.28 Chest pain evaluation accounts for a substantial health care cost, representing approximately 7 million yearly ED visits, with an estimated cost of $5 billion.29 Strategies allowing rapid and accurate discharge of low-risk patients after a single troponin test can potentially reduce cost and allow resources to be allocated for other patients in need.Furthermore, the ability to effectively and promptly rule out AMI using results of a single troponin test can reduce the need to keep patients in the ED for serial troponin testing. Overcrowding of the ED and increased hospital length of stay are a major health care concern. Crowding of the ED is associated with patient outcomes and the quality of care provided.30 An analysis by Kline et al31 showed that a 2% rate of missed diagnoses of ACS might be considered acceptable considering the risk of harm from further testing. Many physicians are currently using the strategy of a single troponin test, and it appears to be as accurate and safe as serial troponin tests as a diagnostic approach for selected groups of patients. This strategy can prove useful in situations of high health care demand owing to increasing costs, ED crowding, or a limited resource setting, such as the coronavirus disease 2019 pandemic.There was a statistically insignificant difference in cardiac mortality and myocardial infarction between the group of patients who underwent a single troponin test and the group of patients who underwent serial troponin tests, with both groups having very low 30-day cardiovascular outcomes. A survey of ED physicians found the tolerable rate of missed major adverse cardiac events to be less than 1%.32 Compared with the conventional troponin assay, the hsTn assay has higher sensitivity with a more pronounced difference in performance for patients presenting with recent-onset chest pain,22 as well as for those with low to moderate risk.33 The imprecision of the conventional troponin assay is indicated by its high coefficient of variation of −20% at a level of 0.02 ng/mL.34 Given the lower performance of the conventional troponin assay, supplementation with risk-stratification tools, such as the HEART and Emergency Department Assessment of Chest Pain scores, may improve the accuracy of the use of a single troponin test to rule out ACS.14 This approach can improve the patient experience by decreasing the length of stay in the ED and help to relieve ED overcrowding while maintaining safety.This study was performed with the conventional troponin assay. Early studies with hsTn (hsTn level <5 ng/L) show high accuracy for a rapid rule out of patients in the ED.This study has several strengths. It is a large population-based study that reflects the practice pattern in multiple community-based EDs across southern California. The study population is diverse, and follow-up information is available for all patients. Cohort members have comprehensive health insurance, thereby minimizing selection bias.Some limitations also need to be considered in the interpretation of the findings. There are no data on the timing of symptom onset because it is highly associated with the clinician’s decision-making. Given the observational study design, causality cannot be assumed. Residual confounding may be present despite adjustments of multiple variables. The data set used in this analysis lacks the detail available in trials and registries; particularly, we cannot assess the timing of symptom onset, which may be the key variable in determining which patients are most likely to experience a benefit associated with serial troponin testing.The findings of this cohort study suggest that, when compared with serial troponin testing, physician discretion to safely discharge a patient after an undetectable troponin level using a conventional troponin test, together with the HEART score, is a safe and reliable strategy for patients with suspected ACS . Especially as results of hsTn assays become available, clinical recommendations should consider the physician’s discretion to use a single troponin test, which may help to improve resource allocation.


We assessed the concordance of cerebrospinal fluid (CSF) amyloid beta (Aβ) and tau measured on the fully automated Lumipulse platform with pre‐symptomatic Alzheimer's disease (AD) pathology on amyloid positron emission tomography (PET).18F‐florbetapir amyloid PET status (n = 63).In 72 individuals from the Insight 46 study, CSF Aβ40, Aβ42, total tau (t‐tau), and phosphorylated tau at site 181 (p‐tau181) were measured using Lumipulse, INNOTEST, and Meso Scale Discovery (MSD) assays and inter‐platform Pearson correlations derived. Lumipulse Aβ42 measures were adjusted to incorporate standardization to certified reference materials. Logistic regressions and receiver operating characteristics analysis generated CSF cut‐points optimizing concordance with P < .0001); those of t‐tau and t‐tau/Aβ42 correlated moderately . The best concordance with amyloid PET (100% sensitivity and 94% specificity) was afforded by cut‐points of 0.075 for Lumipulse Aβ42/Aβ40, 0.087 for MSD Aβ42/Aβ40 and 17.3 for Lumipulse Aβ42/p‐tau181.Measurements of CSF Aβ, p‐tau181, and their ratios correlated well across platforms (r 0.84 to 0.94, The Lumipulse platform provides comparable sensitivity and specificity to established CSF immunoassays in identifying pre‐symptomatic AD pathology. Guidelines for their use in the clinical setting include the UK National Institute of Clinical Assessment Guideline 2018,A decrease in CSF concentration of soluble Aβ1–42 (Aβ42) peptide is one of the earliest changes in preclinical AD,CSF Aβ42 has a high concordance of 89% to 92% with amyloid PET;Multiple analytical platforms are used for measuring core CSF AD biomarkers, for example, INNOTEST (Fujirebio) provides clinically validated enzyme‐linked immunosorbent assays (ELISA) for Aβ42, total tau (t‐tau), and phosphorylated tau at site 181 (p‐tau181); the Meso Scale Discovery (MSD) Aβ triplex electrochemiluminescence assay simultaneously measures Aβ38, Aβ40, and Aβ42. However, despite efforts to standardize biomarker measurements among multiple platforms and laboratories,Recent studies directly comparing measurements by Lumipulse with INNOTEST ELISAs have shown good concordance between the two platforms but reduced intra‐ and inter‐assay variability on the Lumipulse.Other studies assessed the ability of Lumipulse assays to differentiate clinical AD from non‐AD neurological conditions. No significant difference in diagnostic accuracy has been shown between Lumipulse and INNOTEST assays;A few studies have assessed the concordance of CSF Aβ and tau with amyloid PET.When assessing CSF by Lumipulse in a mixed memory clinic cohort, Alcolea et al. also found a higher concordance with amyloid PET when using the CSF Aβ42/Aβ40 ratio (86%) than when using individual markers (76% to 84%).As yet, no single study has directly compared all four CSF Aβ and tau markers and ratios measured by the Lumipulse platform with established immunoassays and compared platforms according to concordance with amyloid PET in a preclinical setting. In the present study we extend the comparison of individual Lumipulse CSF Aβ40, Aβ42, t‐tau, and p‐tau181 markers to also include ratios, with direct comparison to the INNOTEST and MSD platforms. We supplement existing knowledge about the possible contribution of Aβ40 interference to differences in measurements of Aβ42 by evaluating all three platforms, and assess concordance of individual markers and ratios with amyloid PET imaging in a preclinical cohort.22.1Participants were from the second time point of Insight 46, the neuroscience substudy of the National Survey of Health and Development , for which the study design has previously been described,Systematic review: The authors searched PubMed using the terms “Lumipulse AND (Alzheimer's OR amyloid).” Recent research has compared Lumipulse measurements of cerebrospinal fluid (CSF) amyloid beta (Aβ)42 and t‐tau with established manual immunoassays and assessed concordance of Lumipulse Aβ42, Aβ40, total tau (t‐tau), and phosphorylated tau at site 181 (p‐tau181) with amyloid positron emission tomography (PET). However, measurements of Aβ40 and p‐tau181 by Lumipulse and established immunoassays had not yet been compared, and concordance of Aβ42/Aβ40 and Aβ42/p‐tau181 ratios with amyloid PET had not been assessed in a preclinical cohort.18F‐florbetapir PET was afforded by Lumipulse Aβ42/Aβ40 and Aβ42/p‐tau181 ratios, and Meso Scale Discovery Aβ42/Aβ40 ratio.Interpretation: We compared Aβ42 measurements by three CSF platforms, and Aβ40, p‐tau181, and t‐tau across two platforms, in the same individuals from a British birth cohort aged 72 to 74. The highest concordance with Future directions: Lumipulse quantification of Alzheimer's disease CSF biomarkers may allow for stratification of preclinical cohorts by amyloid status. As certified reference materials for these biomarkers are developed, cross‐platform cut‐point standardization may become achievable.2.2Exclusion criteria for lumbar puncture (LP) were clinical/neuroimaging safety concerns for raised intracranial pressure, known/suspected thrombocytopenia or coagulopathy, use of antiplatelet or anticoagulant medications (apart from aspirin 75 mg daily), congenital spinal malformation, lumbar fixation surgery, active skin inflammation overlying the proposed LP site, or lignocaine allergy. Participants were not instructed to fast, and LP was timed between 08:30 and 10:30 hrs. After local anesthesia with lignocaine, a 22G atraumatic spinal needle was used to collect up to 20 mL of CSF, without active withdrawal, into 2 × 10 mL polypropylene screw top containers (Sarstedt 62.610.018), which were transported on ice within 30 minutes to the laboratory. CSF was centrifuged at 1750 g for 10 minutes at 4°C and the supernatant placed in 0.5 mL aliquots into polypropylene screw top cryovials, to be stored at –80°C within 60 minutes of LP.2.318F‐florbetapir (Amyvid) amyloid PET and magnetic resonance imaging (MRI) data were simultaneously acquired on a single Biograph mMR 3T PET/MRI scanner for all participants . The standardized uptake value ratio (SUVR) between a pre‐defined composite neocortical region of interest and an eroded white matter reference region was calculated, and a SUVR cut‐point of 0.61 was used to define amyloid PET status, as derived by mixture modeling generated at the first study time point.Dynamic Eighty‐five percent of participants had their amyloid PET scan on the day before LP, but in the remaining 15%, either due to lack of tracer availability or participant choice, LP‐scan delay ranged between –13 and +110 days.2.4For each of the four analytes of interest, CSF measurements were undertaken using the Lumipulse platform and at least one other established immunoassay platform that uses manual steps in the measurement protocol Table .For measuring Aβ peptides, three assay platforms were used: INNOTEST β‐amyloid 1‐42 (Fujirebio) for Aβ42, Lumipulse G600II automated assay (Fujirebio) for Aβ42 and Aβ40, and MSD Multi‐spot Aβ 6E10 Triplex assay  for Aβ42 and Aβ40. A single 500 µL aliquot of neat CSF was used to perform the INNOTEST and Lumipulse assays in parallel. INNOTEST required 25 µL/replicate for measuring Aβ42 alone. Lumipulse required 100 µL of dead volume, 50 µL/replicate for measuring Aβ42, and 40 µL/replicate for measuring Aβ40. A different aliquot of CSF from the same individuals was used to perform the MSD assay, with 15 µL/replicate of neat CSF (diluted 1:2 in assay diluent) used to measure all three peptides—Aβ38, Aβ40, and Aβ42—together.T‐tau and p‐tau181 were measured in parallel on the INNOTEST and Lumipulse platforms, using the same aliquot of CSF. The INNOTEST hTau Ag assay required 25 µL/replicate and the INNOTEST phospho‐tau (181P) assay 75 µL/replicate. The Lumipulse assays required 100 µL of dead volume, 75 µL/replicate for measuring t‐tau, and 40 µL/replicate for measuring p‐tau181.Samples were assayed after a single thaw to room temperature. On each platform, a single batch of reagents was used for all samples. Measurements by INNOTEST and MSD assays were performed in duplicate, and sample measurements accepted if coefficients of variation across duplicates were < 30%. Given that the Lumipulse platform required a larger total volume of CSF due to dead volume, measurements by Lumipulse were made once per sample.Two run validation controls (provided with each assay kit) and two control CSF samples  with low and high values of the analyte(s) of interest were used. Intra‐run variation for the run validation controls and inter‐run variation using the control CSF samples are shown in Table S1 in supporting information. Measurements were performed according to the manufacturers’ instructions.2.5After the measurements of Aβ peptides were performed in this study, certified reference materials (CRM) were produced for Aβ1‐42 and data have been published on the commutability of different assay platforms in measuring against these CRM.2.6Investigation of Aβ40 interference with Aβ42 measurements is detailed in the supporting information.2.7All analyses used Stata v14.2 . As individual biomarkers have a positively skewed distribution, log‐transformation was undertaken before assessing Pearson correlations between individual biomarker values across platforms. Such transformation was not required before assessing correlations between ratios. All individuals with available CSF data were included in correlation analyses.Spearman correlation was used to assess the impact of spiking increasing concentrations of Aβ40 on measurements of Aβ42. Significant Aβ40 concentration‐dependent interference was shown if the correlation coefficient (rho) between measured Aβ42 and spiked Aβ40 concentration was significantly less than zero.t tests for age at LP, and χ2 tests for sex  and apolipoprotein E (APOE) ε4 carrier status . Differences between groups in Mini‐Mental State Examination (MMSE) and measured biomarker values were assessed using Wilcoxon rank‐sum tests.In the group with full CSF and amyloid PET data, differences in demographic characteristics between amyloid PET positive and negative groups were assessed using Logistic regression models with amyloid PET status as the outcome and CSF biomarkers or their ratios as predictors were used to perform receiver operating characteristics (ROC) analysis. The area under the ROC curve (AUC) was compared across biomarkers and platforms using De Long tests. Optimal CSF cut‐points for classifying amyloid PET positive versus negative individuals were ascertained using the Youden index.33.1APOE ε4 alleles. Comparing those with full data with those excluded due to incomplete data, there were no significant differences in age, sex, APOE ε4 carrier status, MMSE, or any of the measured CSF biomarkers (Table S2 in supporting information).Of 72 participants with CSF samples, 63 had full CSF and amyloid PET data. Of these, 71.4% were male and 22.4% carried one or two P = .031) and had a higher percentage of APOE ε4 carriers . All three platforms measured significantly lower (by 41% to 47%) CSF Aβ42 in PET–positive individuals (median concentration in pg/mL [interquartile range (IQR)]: Lumipulse PET–positive 711  vs PET–negative 1330 , P < .0001; MSD: 471  vs 801 , P = .0001; INNOTEST 669  vs 1252 , P < .0001). Similarly, the CSF Aβ42/Aβ40 ratio was significantly lower (by ≈50%) in PET–positive individuals  vs 0.113 ).Of the individuals included in the analyses against amyloid PET, 13 20.6%) were PET–positive. Table 0.6% wereP = .069) but the INNOTEST assay did detect significantly higher CSF t‐tau in the PET–positive group . Both platforms detected significantly higher p‐tau181 in the PET–positive group .Differences between amyloid PET groups in CSF t‐tau measured by Lumipulse did not achieve statistical significance . PET–positive individuals also had significantly lower Aβ42/p‐tau181 ratios .The Aβ42/t‐tau ratio was significantly lower in PET–positive individuals . Measurements on the Lumipulse and INNOTEST platforms of p‐tau181 were better correlated than those of t‐tau  but correlations were stronger for Lumipulse versus INNOTEST Aβ42/p‐tau181  and Lumipulse versus MSD Aβ42/Aβ40 .CSF Aβ42 measurements were highly correlated across the three platforms .Spiked Aβ40 did not significantly interfere with Aβ42 measurements by the Lumipulse platform . However, significant negative correlations between measured Aβ42 and spiked Aβ40 were observed for both the MSD and INNOTEST platforms  but t‐tau performed better when measured by Innotest . Figure S2 in supporting information shows scatter plots of the raw data, demonstrating the superiority of the ratios compared to individual biomarkers.The performance of the three platforms in classifying amyloid PET–negative/positive status is shown in Table APOE ε4 carriers. Despite this, incorporating age, sex, and APOE ε4 carrier status as covariates into predictive models did not significantly change the percentage of discordantly classified individuals or type of discordance .Concordance of CSF biomarker ratios with amyloid PET SUVR as a continuous variable is shown in Figure 4In this study we build on previous validations of Lumipulse measurements of CSF Aβ and tau biomarkers against two other established CSF assay platforms. We report good correlations of measurements of individual biomarkers of CSF Aβ40, Aβ42, t‐tau, and p‐tau181 between platforms, in agreement with other studies.Measurements of p‐tau181 correlated better than t‐tau between Lumipulse and INNOTEST, and this was also reflected in the Aβ42/p‐tau181 and Aβ42/t‐tau ratios. Our findings for t‐tau contrast with high correlations (r > 0.9) reported in other studies between Lumipulse and INNOTEST measurementsWe found differences in absolute biomarker values between platforms, as reported by others.APOE ε4 carriage than in the participants of our cohort.We report 100% sensitivity across all three platforms for CSF Aβ42 in predicting cortical amyloid load, but the specificity of the Lumipulse measurements (74%) was lower than that of the INNOTEST (86%) or MSD (80%). In contrast to other studies, which show CSF t‐tau and p‐tau181 to be good individual predictors of amyloid PET,Advantages of the Lumipulse platform over conventional assays include reduction in labor‐intensive steps and manual error; reduced total analysis time due to testing all four biomarkers on the same CSF sample; and improved accuracy for analyte detection, due to the measurement by photon‐counting of directly emitted light rather than wavelength‐based colorimetric absorbance. Furthermore, the Lumipulse platform can process small numbers of CSF samples, without needing to collect enough samples to use in batched assays (as is required for the INNOTEST or MSD). However, a disadvantage of the Lumipulse is its requirement for a large dead volume of 100 µL, relative to sample volumes per replicate for the four biomarkers of 40 to 75 µL, while the INNOTEST and MSD assays use similar volumes per replicate (25 to 75 µL) but have no dead volume requirement.APOE ε4 carrier status, the differences did not reach statistical significance, likely due to this being an interim dataset of samples collected by this point of the ongoing study. This cohort consists mostly of cognitively healthy individuals of the same age. It is possible that some individuals classified as “CSF–positive” (through the use of the ratio cut‐points) but “PET–negative” do actually have subthreshold cerebral amyloid deposition, as CSF changes may precede PET changes.This study has some limitations that might be assessed in future research. MSD analysis took place on a separate day using a separate sample aliquot to that used for INNOTEST and Lumipulse analysis. Lumipulse measurements were performed in singleton due to CSF volume requirements, so precision of the Lumipulse assays was not assessed. We did not compare measurements of all four biomarkers on all platforms, and we focused on comparing Lumipulse measurement with other immunoassays but not with other methods of measurement like mass spectrometry. Although the AUC obtained for prediction of amyloid PET status were higher for CSF Aβ42 and its ratios than that obtained by the model using age, sex, and In summary, this study supports the use of the fully automated Lumipulse platform, particularly for measuring CSF Aβ42/Aβ40 and Aβ42/p‐tau181, to identify cerebral amyloid deposition with excellent sensitivity and high specificity, without Aβ40 interference, even in cognitively normal individuals.Avid Radiopharmaceuticals, a wholly owned subsidiary of Eli Lilly, kindly provided the 18F‐florbetapir tracer (Amyvid) free of cost but had no role in the design, conduct, analysis, or reporting of Insight 46 study findings. We are particularly indebted to the support of the late Chris Clark of Avid Radiopharmaceuticals who championed this study from its outset. Fujirebio provided and set up the Lumipulse platform free of cost but had no role in the design, conduct, analysis, or reporting of this study. The National Survey of Health and Development is funded by the Medical Research Council . TDP was supported by a Wellcome Trust Clinical Research Fellowship (200109/Z/15/Z). NCF is supported by UK Dementia Research Institute at University College London, Medical Research Council, National Institute for Health Research (Senior Investigator award), and Engineering and Physical Sciences Research Council. HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018‐02532), the European Research Council (#681712), Swedish State Support for Clinical Research (#ALFGBG‐720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809‐2016862), and the UK Dementia Research Institute at UCL. HZ has served at scientific advisory boards for Denali, Roche Diagnostics, Wave, Samumed, and CogRx; has given lectures in symposia sponsored by Fujirebio, Alzecure, and Biogen; and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program . JMS is supported by Engineering and Physical Sciences Research Council (EP/J020990/1), British Heart Foundation (PG/17/90/33415), and EU's Horizon 2020 research and innovation programme (666992). MH and JMS are supported by the University College London Hospitals Biomedical Research Centre. NCF and JMS are supported by the National Institute for Health Research Queen Square Dementia Biomedical Research Unit and the Leonard Wolfson Experimental Neurology Centre.Supporting InformationClick here for additional data file.