Microabrasive-based lapping is widely used in the manufacturing of single-crystal substrates such as sapphire, SiC, and GaN. Although many studies have been conducted to improve the lapping process characteristics, most of them focused on process conditions or consumables. In this study, the effect of the lapping platen groove density on the lapping characteristics was studied using a sapphire substrate. Groove density was defined as the ratio of groove width to groove pitch, and the displacement of the lapping head was measured to calculate the oil film thickness. It was confirmed that, for groove densities below 0.30, hydroplaning occurs when the oil film thickness increases. When the oil film thickness is larger than the abrasive particle size, the material removal rate is low because the abrasive does not participate in the lapping process. When the oil film was developed, the experimental results showed a high surface roughness and poor flatness of the substrate, as only large abrasive particles participated in the lapping process. Therefore, to improve the lapping characteristics, it is important to reduce the groove density by reducing the groove pitch, which prevents the development of the oil film. Lapping using microabrasives improves the flatness of the substrate being machined and reduces the thickness of the subsurface damaged layer in the manufacturing of single-crystal substrates such as sapphire, SiC, and GaN, which in turn reduces the post processing time of chemical mechanical polishing (CMP) ,2,3,4. MThe lapping characteristics are represented by the material removal rate (MRR), the substrate surface roughness, and the flatness of the substrate. The MRR is an indicator of lapping efficiency and generally represents the decrease in thickness of the substrate per unit time . The surThe platen grooves are depressions created on the platen surface, as shown in To address this knowledge gap, in this study, the effect of groove density on the state of the contact surface between the substrate and the platen, i.e., the magnitude of the oil film thickness and frictional force, were investigated and the effect of the change in the oil film thickness on lapping characteristics, including MRR, surface roughness, and substrate flatness, was studied.G) was fixed at 2.0 mm and the groove density (Dg) was modified by adjusting the groove pitch (P) and land width (L). Groove density is defined as:Concentric square grooves were selected to study the effect of groove density on the oil film thickness and the lapping process characteristics. To vary the groove density, the groove width  having a resolution of 0.1 μm was used. The average thickness was obtained by measuring the substrate thickness of 21 points, and the MRR was calculated by dividing the processing time by the change in average thickness. The surface roughness of the substrate was measured using a noncontact surface roughness measuring device , and the surface roughness value was calculated by averaging the surface roughness values of 21 points after lapping. The platen surface was analyzed using a scanning electron microscope .To determine the condition of the contact interface between the substrate and the platen during the lapping process, an oil film thickness measuring system was used; its configuration is shown in To confirm the oil film thickness changes, the frictional force between the lapping platen and the substrate was measured using a frictional force measuring system, as shown in The oil film thickness between the platen and the substrate was measured to confirm the contact interface state change with groove density during lapping process. The oil film thickness was highest when the groove density was 0.10 and gradually decreased as the groove density increased. When the groove density was 0.30 or higher, the oil film thickness converged to approximately 1 µm. This indicates that when the groove density was less than 0.30, hydroplaning occurred at the contact interface between the substrate and the platen, resulting in the development of the oil film.To verify the formation of the oil film, the frictional force generated between the substrate and the platen was measured at each groove density value under the same experimental conditions used for oil film thickness determination; the average values of the measured frictional forces are shown in By comparing the changes in oil film thickness and frictional force, when the groove density was less than 0.3, it can be assumed that the contact interface experienced mixed lubrication, in which the pressure load was supported by the abrasive particles and the slurry, and the smaller the groove density, the larger the oil film thickness. When the groove density was 0.3 or higher, boundary lubrication occurred, in which the abrasive particles on the platen surface were in contact with the substrate. In the boundary lubrication region, the load applied to the substrate is supported by the abrasive particles. In addition, some large particles are deeply embedded in the platen or broken when subjected to a force higher than the breaking strength due to the characteristics of the polycrystalline diamond particles . The scaEnergy dispersive analysis of X-ray (EDAX) was performed to confirm what the particles on the surface were. The results are shown in To confirm the effect of the oil film thickness change on the lapping process characteristics, lapping experiments were performed for each groove density. The MRR was calculated by dividing the processing time by the amount of removed material; the results obtained are shown in This can be confirmed through the differential interference contrast images of the substrate surface presented in The change in the film thickness caused by groove density variation also affected the flatness  of the substrate after lapping. As shown in Thakurta  presenteTo determine the effect of groove density on the lapping process characteristics, the change in the oil film thickness at the contact interface between the substrate and the platen was investigated. When the groove density was less than 0.3, a mixed lubrication state was observed; moreover, it was confirmed that the smaller the groove density, the thicker the oil film formed. When the groove density was 0.3 or higher, a boundary lubrication state was observed because the platen, the abrasive particles, and the substrate were in direct contact. In the friction force measurements, even when the groove density was less than 0.3, the frictional force in the wet state was lower than that in the dry state, since hydroplaning phenomenon occurs between the substrate and the platen. At groove densities above 0.3, it was confirmed that the frictional force in the wet state increased and became larger than that in the dry state owing to the lapping force of the abrasive particles.The lapping platen groove density affects the lapping process characteristics by changing the oil film thickness at the contact interface between the substrate and the platen. The MRR gradually decreases as the groove density decreases, and then rapidly decreases at groove densities below 0.3, since the load pressing the substrate is mostly supported by the oil film, in a mixed lubrication state. On the contrary, it was confirmed that the surface roughness increases rapidly at groove densities below 0.3. This is because large abrasive particles participate in the lapping process to produce thick and deep scratches when the oil film thickness is large. In addition, since most of the pressure is supported by the oil film, a runout phenomenon in which the lapping head shakes around the gimbal occurs, thereby deteriorating the flatness of the substrate.Therefore, in order to improve the lapping process characteristics, it is important to increase the groove density by reducing the groove pitch to prevent the development of the oil film. Since the oil film thickness is related to the slurry properties, it is possible to prevent the development of the oil film by adjusting the slurry viscosity. When the groove density is high, it is possible to achieve a boundary lubrication state; however, the contact area of the platen may be reduced, thereby reducing the lifespan of the platen. Therefore, it is important to select an appropriate groove density to achieve the desired lapping characteristics. In further work, it is necessary to study the effects of kinematics factors such as the rotational speed and the pressure load on the oil film thickness, which will enhance the understanding of the hydrodynamic phenomenon occurring at the contact interface between the platen and the substrate.


Loss of function of triggering receptor expressed on myeloid cell 2 (TREM2), a key receptor selectively expressed by microglia in the brain, contributes to the development of Alzheimer’s disease (AD). Whether TREM2 levels are pathologically altered during the preclinical phase, and whether cerebrospinal fluid (CSF) soluble TREM2 protein (sTREM2) has a relationship with major pathological processes including Aβ and tau deposition are still unclear.1–42, T-tau and P-tau), stage 1 , stage 2 (low Aβ1–42 and high T-tau or P-tau), and suspected non-AD pathology (SNAP) , to examine changes of CSF sTREM2 in the preclinical AD. Biomarker cut-off was based on the assumption that one-third of adults with normal cognition have AD pathology.According to the NIA-AA criteria, 659 cognitively normal participants from the Chinese Alzheimer’s Biomarker and LifestylE (CABLE) cohort were divided into four groups, stage 0 , and then increased in the stage 2 (P = 0.008). SNAP individuals also had significantly increased CSF sTREM2 (P < 0.001). Results of multiple linear regressions also showed positive correlations of CSF sTREM2 with Aβ1–42 , T-tau  and P-tau .The level of CSF sTREM2 in the stage 1 decreased compared with the stage 0 (CSF sTREM2 levels are dynamic in preclinical AD. Aβ pathology is associated with a decrease in CSF sTREM2 in the absence of tau deposition and neurodegeneration. However, tau pathology and neurodegeneration are associated with an increase in CSF sTREM2. Recent neuropathologic, epidemiologic and genetic studies have demonstrated that alterations of the innate immune system play a crucial role in the development of Alzheimer’s disease (AD) , 2. TrigPrevious studies of patients with late-onset AD reported that CSF sTREM2 increased in a disease-stage-dependent manner and peaked in the early symptom phase , 11. In 1–42), aggregated tau  and neurodegeneration . We also tested whether CSF sTREM2 levels were associated with the core AD CSF biomarkers Aβ1–42, Aβ1–40, T-tau and P-tau in healthy controls (HC) and preclinical AD groups [Therefore, we used the biomarker-based hypothetical staging model to investigate CSF sTREM2 levels in different groups of cognitive normal divided by AD biomarkers , 14. ThiD groups . We limiD groups , 17.Chinese Alzheimer’s Biomarker and LifestylE (CABLE) study is an ongoing large-scale cohort study initiated in 2017, mainly focusing on Alzheimer’s disease risk factors and biomarkers in the northern Chinese Han population. CABLE is aimed to determine the genetic and environmental modifiers of AD biomarkers and to identify lifestyle factors that may affect the risk of AD in the non-demented northern Chinese Han population, thus providing a basis for disease prevention and early diagnosis. In conjunction with the investigation, oral informed consent for future use of their CSF and blood samples for research purposes was obtained. All patients were later instructed to withdraw their permission if they changed their minds. The design of the study was approved by the Institutional Review Board of Qingdao Municipal Hospital, and the study procedure was conducted in accordance with the Declaration of Helsinki.All enrolled participants in the CABLE were Han Chinese aged between 40 to 90 years old. The exclusion criteria include: (1) central nervous system infection, head trauma, multiple sclerosis, neurodegenerative diseases other than AD , or other major neurological disorders; (2) major psychological disorders; (3) severe systemic diseases  that may affect CSF or blood levels of AD biomarkers including Aβ and tau; (4) family history of genetic diseases. All participants received clinical and neuropsychological assessments, biochemical testing, as well as blood and CSF sample collection. Comprehensive questionnaire, electronic medical record system, and a laboratory inspection management system were used to collect demographic information, AD risk factor profile and medical history. The diagnostic criteria of preclinical AD was defined according to the 2011 National Institute on Aging–Alzheimer’s Association (NIA-AA) workgroup reports, that is, normal cognition but abnormal AD biomarkers . We firsA total of 1000 cognitively normal participants from CABLE had available information on covariates. Sixty-nine participants were excluded who had no information about CM-MMSE, and 215 participants without available CSF sTREM2 data were excluded. Fifty-seven participants without CSF biomarkers and those had data outside the standard deviation (SD) of four times were removed. Finally, 659 participants were included in this cross-sectional analysis. Frequency histogram of age distribution of populations was shown in APOE) genotyping in this study. Two specific loci related to APOE status (rs7412 and rs429358) were selected for genotyping with restriction fragment length polymorphism (RFLP) technology.Collection and storage of fasting lumbar CSF samples and blood samples were performed at Qingdao Municipal Hospital. CSF samples were processed immediately within 2 h after standard lumbar puncture. Each sample was centrifuged at 2000×g for 10 min, and CSF samples were separated and stored in an enzyme-free EP (Eppendorf) tube  at − 80 °C for further use in the subsequent steps of this study. The samples were subjected to a maximum of two freeze-thaw cycles. The DNA in the blood samples extracted using the QIAamp®DNA Blood Mini Kit (250) was separated and stored in an enzyme-free EP tube at − 80 °C until the apolipoprotein E . CSF sTREM2 measurements were done with ELISA kits  and CSF core biomarkers measurements were done with other ELISA kits . All ELISA measurements were performed by experienced technicians in strict accordance with the manufacturer’s instructions. They were blinded to the clinical information. The samples and standards were measured in duplicate, and the means of duplicates were used for the statistical analyses. All the antibodies and plates were from a single lot to exclude variability between batches. Moreover, the within-batch CV was < 5% and the inter-batch CV was < 15%. Furthermore, problems .1–42), aggregated AD-tau (P-tau) and neurodegeneration (T-tau). And each biomarker is binarized based on whether they are normal or abnormal. The observation that approximately one-third of cognitively normal older adults have AD pathology in their brains has been approved by previous amyloid imaging [1–42, > 40.05 pg/ml (upper one-third) for P-tau, and > 187.32 pg/ml (upper one-third) for T-tau, respectively Aggregated tau and neurodegeneration groups were merged to reduce the number of groups to be compared, which resulted in four different biomarker group combinations, including stage 0, stage 1, stage 2, and suspected non-AD pathology (SNAP). Individuals with normal measures of Aβ1–42, P-tau and T-tau are classified as stage 0; individuals with abnormal Aβ1–42 but no abnormal P-tau or T-tau are classified as stage 1; during stage 2, abnormal Aβ1–42, and abnormal P-tau or T-tau are evident. Individuals with evidence of aggregated tau or neurodegeneration but with normal levels of amyloid are classified as having SNAP [continuum category [The scheme comprises 3 biomarkers: aggregated Aβ  and visual inspection of the Q-Q plot  and preclinical AD (stage 1 and stage 2) groups, with a multiple linear regression adjusted for age, gender, educational level, and APOE ε4 carrier status. The analyses were performed in the total sample and then in subgroups stratified by age, gender and APOE ε4 carrier status.CSF sTREM2 didn’t follow a normal distribution as assessed by Kolmogorov-Smirnov test  and IBM SPSS Statistics 23.Statistical significance was defined as APOE ε4 positive percentage of 16%. As for cognitive performance, the study population had CM-MMSE of 27.8 ± 2.1 scores. There were no differences between the four groups in terms of gender, educational level, CM-MMSE score. However, individuals in stage 2 group were older and had lower cognitive composite scores than the HC group.We studied a total of 659 participants  of the CABLE study. The demographical and clinical characteristics are described in Table P < 0.001)     . The resn = 242), stage 1 (n = 148), stage 2 (n = 70), and SNAP (n = 199). The results of one-way ANCOVA showed significant differences in CSF sTREM2 levels between the four groups. The results of the comparison between groups showed that the stage 1 group had the significantly lowest CSF sTREM2 compared to other groups  and the SNAP (P < 0.001) groups had significantly increased CSF sTREM2 concentrations compared to the stage 1 group . Both the stage 2 (P = 0.021) and the SNAP (P < 0.001) groups had higher CSF sTREM2 concentrations compared to the stage 1 group. We also repeated the same analyses in groups divided by participants’ Aβ pathology (Aβ1–42) and tau pathological status (P-tau) or in groups divided by participants’ Aβ pathology (Aβ1–42) and neurodegenerative state (T-tau). The results are shown in Because the results suggest that age is an important influencing factor for CSF sTREM2, we conducted another ANCOVA controlling for age, and obtained the same results as follows. The stage 1 group had significantly lowest CSF sTREM2 compared to other groups (P < 0.001) and the preclinical AD  groups , increased CSF sTREM2 was associated with higher levels of Aβ1–42      and preclinical AD groups (n = 218)   Fig. a and Aβ1r status . There wu/Aβ1–42 . Outlier1–42) is associated with a decrease in CSF sTREM2, while tau pathology or neurodegeneration is associated with elevated CSF sTREM2. This seems to confirm the potential role of microglial inflammatory response in the pathogenesis of AD. Our data provide support for this hypothesis: Aβ deposition occurs independently of the inflammatory process, but the type and extent of the inflammatory response to Aβ deposition in the brain may trigger or affect subsequent neurodegeneration [In the present study, we combined biomarker-based classification with clinical stages to assess changes in the microglial-activity marker CSF sTREM2 within the early phases of AD. The application of this classification system enabled us to explore the associations of microglial inflammatory response with AD pathophysiology . Our study extends previous evidence by showing that CSF sTREM2 levels did change dynamically with pathological processes. The simple Aβ pathology  even in the absence of Aβ pathology, as indicated by the differences between HC and SNAP individuals , 30. Thi1–42, Aβ1–40, T-tau and P-tau on the entire data set and in preclinical AD, which further suggests an early involvement of reactive microgliosis. In HC subjects, the correlation with Aβ1–42 disappeared, while the correlations of sTREM2 with T-tau and P-tau remained. These findings suggest that changes in CSF sTREM2 may indeed be associated with neuronal injury. It is also suggested that the increase in CSF sTREM2 during normal aging may be a protective response to mild neuronal injury. Furthermore, we showed that sTREM2 did not seem to depend on the APOE ε4 genotype, as there was no difference in CSF sTREM2 concentration between APOE ε4 carriers and non-carriers; sTREM2 also did not depend on gender because there was no difference in CSF sTREM2 concentration between males and females.Exploring relations between CSF biomarkers may provide better pathogenic understanding by hinting to simultaneously ongoing processes in the brain. Our results are consistent with previous research studies –12, 32. The underlying mechanism of dynamic changes in CSF sTREM2 remains to be investigated throughout the disease. The mechanism underlying the reduction of CSF sTREM2 in the early stage of Aβ pathology is still unclear. One explanation is that microglia initially form a barrier around the plaque, and sTREM2 released by microglia remains in the plaque until the barrier fails, followed by neural injury , 34. AnoThe strength of the current study is a large sample of well-characterized subjects with biomarker evidence of AD pathology. We used an AD biomarker classification system in line with NIA-AA research diagnostic guidelines for AD. This allowed us to study patients in the preclinical stages of AD. Previous studies mainly focused on AD patients, with a few participants in the preclinical phase, and had no biomarker classification , 44. HowIn conclusion, this study is based on an independent cohort. The results indicate that CSF sTREM2 is dynamic in preclinical AD. Aβ pathology is associated with a decrease in CSF sTREM2 in the absence of tau deposition and neurodegeneration, whereas tau pathology and neurodegeneration is associated with an increase in CSF sTREM2. Future studies should use biomarkers to further categorize the preclinical AD stage and explore the biological mechanisms underlying the dynamic CSF sTREM2 changes.Additional file 1: Figure S1. Histogram of age distribution of CABLE populations. Figure S2. Associations of age and the presence or absence of APOE ε4 for CSF measures of Aβ1–42. Figure S3. The Quantile-Quantile plot of CSF sTREM2. Figure S4. Association of CSF sTREM2 and age. Figure S5. One of the standard curves for CSF sTREM2. Figure S6.CSF sTREM2 in groups defined only by CSF Aβ1–42 and P-tau. Figure S7. CSF sTREM2 in groups defined only by CSF Aβ1–42 and T-tau. Table S1. Association of CSF sTREM2 and CSF core biomarkers. Table S2. Subgroup analyses of whole participants by age, gender, and APOE ε4 carrier status.


Diagnosis of dementia with Lewy bodies (DLB) is challenging, largely due to a lack of diagnostic tools. Cerebrospinal fluid (CSF) biomarkers have been proven useful in Alzheimer’s disease (AD) diagnosis. Here, we aimed to identify novel CSF biomarkers for DLB using a high-throughput proteomic approach.n = 20) and controls (n = 20). Validation was performed using (1) the identical proteomic workflow in an independent cohort (n = 30), (2) proteomic data from patients with related neurodegenerative diseases (n = 149) and (3) orthogonal techniques in an extended cohort consisting of DLB patients and controls (n = 76). Additionally, we utilized random forest analysis to identify the subset of candidate markers that best distinguished DLB from all other groups.We applied liquid chromatography/tandem mass spectrometry with label-free quantification to identify biomarker candidates to individual CSF samples from a well-characterized cohort comprising patients with DLB (p < 0.05). Independent cohort replication confirmed VGF, SCG2, NPTX2, NPTXR, PDYN and PCSK1N as candidate biomarkers for DLB. The downregulation of the candidate biomarkers was somewhat more pronounced in DLB in comparison with related neurodegenerative diseases. Using random forest analysis, we identified a panel of VGF, SCG2 and PDYN to best differentiate between DLB and other clinical groups (accuracy: 0.82 (95%CI: 0.75–0.89)). Moreover, we confirmed the decrease of VGF and NPTX2 in DLB by ELISA and SRM methods. Low CSF levels of all biomarker candidates, except PCSK1N, were associated with more pronounced cognitive decline .In total, we identified 1995 proteins. In the discovery cohort, 69 proteins were differentially expressed in DLB compared to controls (We identified and validated six novel CSF biomarkers for DLB. These biomarkers, particularly when used as a panel, show promise to improve diagnostic accuracy and strengthen the importance of synaptic dysfunction in the pathophysiology of DLB. Dementia with Lewy Bodies (DLB) is a common cause of dementia in the elderly, accounting for up to 20% of dementia cases . Clinica1–42) combined with increased levels of total and phosphorylated tau  protein levels supports the diagnosis of AD  single photon emission computed tomography (DaT-SPECT) were performed by indication. Diagnoses were made by consensus in a multidisciplinary meeting according to standard diagnostic criteria. Probable DLB was diagnosed according to the 2005 clinical consensus criteria [DLB patients and controls enrolled in the current study were selected from the Amsterdam Dementia Cohort and the Erasmus Medical Center. All subjects underwent extensive clinical examination including physical and neurological examination, neuropsychological assessment, electroencephalogram, structural brain imaging and laboratory tests . Additiocriteria . All patcriteria , and preFor the biomarker discovery phase, 20 DLB patients and age- and sex-matched controls were selected from the Amsterdam Dementia Cohort according to the criteria described above. In addition, DLB patients in cohort 1 fulfilled the following additional inclusion criteria: (1) DaT-SPECT scan showing presynaptic dopaminergic deficits and (2) normal AD biomarker levels in the CSF .n = 27) and the Erasmus Medical Center (n = 3) was used for validation using an identical proteomics workflow. The DLB patients in cohort 2 had less stringent inclusion criteria, namely DLB patients were not selected on the basis of normal CSF AD biomarker levels and a DaT-SPECT scan was not required.A second cohort consisted of an independent set of 17 DLB patients and 13 age- and sex-matched controls selected from the Amsterdam Dementia Cohort (http://biofind.loni.usc.edu/) [For the validation of the identified candidate biomarkers in related neurodegenerative diseases, we analyzed proteomic data previously generated in 20 patients with AD and 20 patients with frontotemporal dementia (FTD) as part of a parallel study (PRODIA Memorabel Project). In addition, proteomic data from 109 PD patients were provided by the Fox Investigation for New Discovery of Biomarkers (“BioFIND”) database (sc.edu/) .A subset of the identified candidate biomarkers was validated by orthogonal analytical techniques in CSF samples from DLB patients and controls. Cohort 3B consisted of 48 DLB patients and 28 controls selected from the Amsterdam Dementia Cohort. Cohort 3B was not completely independent from cohort 1 and 2, such that 15 controls and 18 DLB patients were overlapping between cohort 3B and cohort 1 and 3 controls and 6 DLB patients were overlapping between cohort 3B and cohort 2. DLB patients in cohort 3B fulfilled similar inclusion criteria as DLB patients in cohort 2.1–42, total tau and p-tau were measured with commercially ELISA’s . The remaining CSF was centrifuged at 1800 g at 4 °C for 10 min, aliquoted in polypropylene tubes of 0.5 mL and stored at − 80 °C [In line with international biobanking guidelines , CSF was − 80 °C  until fuThe workflow for mass-spectrometry biomarker discovery analysis and validation is summarized in Fig. CSF samples were coded and analyzed in a blinded fashion. The depletion of the top-14 high abundant proteins, i.e. albumin, IgG, antitrypsin, IgA, transferrin, haptoglobulin, fibrinogen, α2-macroglobulin, α1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3 and transthyretin, was performed as previously reported . Deplete6 charges. The top 10 peptide signals (charge-states 2+ and higher) were submitted to MS/MS in the higher-energy collision cell . MS/MS spectra were acquired at resolution 17.500 (at m/z 200) in the Orbitrap using an AGC target value of 2 × 105 charges and an underfill ratio of 0.1%. Dynamic exclusion was applied with a repeat count of 1 and an exclusion time of 30 s.Before NanoLC-MS/MS analysis, separated proteins were in-gel digested as previously described . PeptideMS/MS spectra were searched against the Swissprot human 2018 reference proteome using MaxQuant 1.6.0.16 . Enzyme To identify differentially expressed proteins among the DLB and control groups raw intensities were processed using the label-free quantification (LFQ) algorithm in MaxQuant and MaxLFQ intensities were obtained . Missinguniprot.org) with Mascot.We obtained proteomic data from CSF samples from AD and FTD patients that were generated in previous, published  and yet 373–417 peptide were measured by quantitative competitive ELISA [For Neurosecretory protein VGF (VGF) and Neuronal pentraxin 2 (NPTX2), we performed additional validation experiments using orthogonal techniques  in cohort 3B. For VGF, CSF levels of the VGFve ELISA –29 and bve ELISA .373–417 ELISA was carried out as described [375–420 assay [373–382 , was used for rabbit immunizations. Briefly, plates were coated with the corresponding synthetic peptide in carbonate/bicarbonate buffer (pH 9.6), blocked in PBS-Tween 20 containing normal donkey serum (90 mL/L), aprotinin (20 nmol/L) and EDTA (1 g/L), and incubated with a mixture of primary antibody (in the same medium), and serial dilutions of either standard peptide, or samples. For the standard curve, a range of concentrations (50 nmol/L to 50 fmol/L) of either VGF373–382 or VGF373–417 (GGEE-45) synthetic peptide was used. The latter was identified as a natural peptide in human CSF [372 in the di-basic site: human VGF Arg371-Arg372 immediately preceding the natural peptide VGF373–417 and iplicated in its N-terminal cleavage). The data showed a < 0.5% cross-reactivity for this peptide, hence indicating a high specificity of the assay for the N-terminally cleaved peptide.The human VGFescribed , 28, on 20 assay . A synthuman CSF , hence wFor SRM analysis of VGF in CSF samples, 200 μL of CSF was spiked with TEAB buffer and a quantitative protein epitope signature tag  of VGF as internal standard. Samples were reduced and alkylated with 1 mM TCEP and 1 mM CAA at 95 °C for 10 min. Proteins were digested for 16 h at 37 °C by adding 1.2 μg trypsin/LysC (Promega). Digestion was stopped by addition of 800 μL 1.25% TFA and peptides were transferred to strong cation exchange STAGE-Tips  by centrAll statistical analyses were performed in R v.3.5.1 ‘Feather Spray’. Demographics were compared using Student’s t-test, Wilcoxon signed-rank test or Fisher’s Exact Test. Correlations between identified CSF biomarkers and age, sex and MMSE were assessed with Spearman partial correlation, adjusted for cohort. For the validation of the identified candidate biomarker levels in related neurodegenerative diseases (cohort 3A), all protein levels were first normalized according to the mean and standard deviation values of their corresponding control group. The obtained z-scores were compared using general linear models corrected for age. In addition, we performed random forest analyses  with theTable p < 0.05). Forty-six proteins were downregulated and 23 proteins were upregulated in DLB . A total of 69 unique proteins showed significantly different abundances  showed six proteins with same direction and magnitude of change in both cohorts, i.e. Neurosecretory protein (VGF), Secretogranin-2 (SCG2), Neuronal pentraxin-2 (NPTX2), Neuronal pentraxin receptor (NPTXR), Proenkephalin-B (PDYN) and ProSAAS (PCSK1N)  to validate the results . Next, we explored whether these six candidate biomarkers were associated with age, sex and cognitive impairment. Partial spearman correlation analysis adjusted for cohort revealed that lower CSF levels of all proteins, except PCSK1N, were associated with lower MMSE scores at time of lumbar puncture . CSF NPTXR levels were lower in DLB than in PD (p < 0.05). CSF levels of PCSK1N were lower in DLB compared to both PD and FTD (p < 0.05). CSF levels of PDYN, SCG2 and VGF were lower in DLB compared to all related neurodegenerative diseases studied (p < 0.05). CSF levels of all proteins were comparable between the other neurodegenerative disease, i.e. AD, PD and FTD, except VGF for which levels were lower in AD compared to FTD (p < 0.05). The identified markers still showed considerable overlap between groups, suggesting limited ability for diagnostic purposes as single markers. Therefore, we performed random forest analyses to study whether a combination of biomarkers improved discrimination between DLB and all non-DLB individuals. VGF, SCG2 and PDYN best differentiated between DLB and all non-DLB, with accuracy of 0.82, specificity of 0.83 and sensitivity of 0.69  and NPTX2 were confirmed using these alternative analytical methods .Finally, VGF and NPTX2 were selected for validation in cohort 3 based on the availability of orthogonal analytical methods (ELISA and SRM). As shown in Fig. Using a state-of-the-art rigorous proteomic approach and validation in a completely independent cohort, we identified and positively validated six promising CSF biomarker candidates for DLB, namely VGF, SCG2, NPTX2, NPTXR, PDYN and PCSK1N (proSAAS). All six biomarker candidates were downregulated in DLB and levels were consistently lowest in DLB patients compared to related neurodegenerative diseases studied, i.e. AD, PD and FTD. Additionally, we utilized machine learning to identify the biomarker panel best capable of classifying DLB patients. The combination of VGF, SCG2 and PDYN best differentiated between DLB and related neurodegenerative diseases with acceptable specificity and sensitivity. In a second validation step, we confirmed the decrease of CSF VGF  and NPTX2 (ELISA) using orthogonal analytical techniques. Low CSF levels of all biomarker candidates, except PCSK1N, were associated with more pronounced cognitive decline. We will discuss these validated biomarker candidates below.26–62) or amyotrophic lateral sclerosis (ALS) (VGF398–411). In addition, VGF peptides were also reduced in brain tissue of patients with PD [23–62 were increased in schizophrenia patients  are members of the chromogranin/secretogranin family and play a role in the regulated secretory pathway of peptides, hormones, neurotransmitters and growth factors. VGF topped the list of potential biomarker candidates. Biologically active peptides derived from VGF play an important role in diverse processes, for example, hormone, neurotrophin and neurotransmitter release, energy homeostasis and regulation of gastrointestinal function , 35. Alt with PD . Conversynuclein , 38, supNPTX2 and NPTXR are members of the neuronal pentraxin family . The neuThe sixth biomarker candidate that we identified and validated was PDYN. The large decrease (fold change of > 4) suggest that PDYN is an on/off marker (i.e. subjects either have low PDYN levels or have high PDYN levels). More DLB patients than controls have very low PDYN levels resulting in an average decreased expression in DLB Fig. . PDYN isThe identification of these candidate biomarkers highlight the importance of synaptic dysfunction in DLB. This is in line with previous research indicating that this biological process is a central feature in DLB pathogenesis –52. HoweLoss of synapses and a decrease in synaptic proteins are constant features of neurodegenerative diseases. This is also supported by similar synaptic protein changes in post-mortem brain tissue revealed by proteomic analysis from patients with AD, PD dementia and DLB . The ide1–42, were not detected, since their concentration are below the typical limit of detection of mass spectrometry methods. In addition, these proteins can be highly post-translationally modified, which further compromises mass spectrometry-based identification by default search strategies. Hence, the possibility cannot be excluded that we may have missed some potentially interesting proteins. Furthermore, proteomic data of the PD patients were obtained from a different mass spectrometry platform than proteomic data of the AD, DLB and FTD patients, which could have introduced some noise. However, normalization of the biomarker candidate values relative to the corresponding control group could restrict the methodological differences. Additionally, the random forest classifier was generated and tested in the same cohort which may lead to over-optimistic classification results. Although we have used bootstrapping to reduce such over-fitting, larger cohorts are needed to validated these findings as well as to examine the added diagnostic value of the (combination of) candidate proteomic markers in relation to the established AD biomarkers and α-synuclein. Such a study would be of tremendous value to the field by optimizing biomarker panel for fit-for-use purposes, as well as to evaluate the role of synaptic dysfunction in the pathogenesis of neurodegeneration.Among the strengths of the current study are the use of a rigorous in-depth proteomic approach, replication in an independent cohort, validation of the biomarker candidates in related neurodegenerative diseases, including AD, PD and FTD, validation of a subset of biomarker candidates using orthogonal techniques, and the strict inclusion criteria for patients and controls. For example, an abnormal DAT-SPECT scan and normal AD biomarker levels were obligatory for all DLB patients in the discovery cohort. On the other hand, DLB patients in the validation cohorts were more heterogeneous, i.e. DLB patients were more representative of DLB patients in daily memory clinic practice, as they were not selected based on their CSF AD biomarker values and almost half of the DLB patients had a CSF profile compatible with AD (in line with previous literature ). The usIn conclusion, we identified and positively validated six novel proteins  as promising biomarkers for DLB. Our results might suggest that these candidate biomarkers, particularly when used as a panel, show promise to improve diagnostic accuracy for DLB, which should be explored in future prospective validation studies. Moreover, our validation using orthogonal techniques (i.e. high-throughput immunoassays or SRM) of a subset of the candidate biomarkers revealed by the proteomics approach supports the robustness of our findings. Therefore, future studies should include a replication in independent cohorts including patients with different neurodegenerative diseases , and testing our panel of synaptic biomarkers, in combination with the AD biomarkers, using such higher throughput techniques (e.g. SRM). Identification of these candidate biomarkers also strengthens the importance of synaptic dysfunction in the pathophysiology of DLB, which warrant further research as potential therapeutic target. On the applicative and clinical side, the identification of novel CSF biomarkers for DLB, can be expected to enhance clinical diagnostic accuracy, especially early in the disease course, and might thereby accelerate the development of new disease-modifying and neuroprotective agents.Additional file 1.


During competitive events, the pacing strategy depends upon how an athlete feels at a specific moment and the distance remaining. It may be expressed as the Hazard Score (HS) with momentary HS being shown to provide a measure of the likelihood of changing power output (PO) within an event and summated HS as a marker of how difficult an event is likely to be perceived to be. This study aimed to manipulate time trial (TT) starting strategies to establish whether the summated HS, as opposed to momentary HS, will improve understanding of performance during a simulated cycling competition. Seven subjects (peak PO: 286 ± 49.7 W) performed two practice 10-km cycling TTs followed by three 10-km TTs with imposed PO (±5% of mean PO achieved during second practice TT and a self-paced TT). PO, rating of perceived exertion (RPE), lactate, heart rate (HR), HS, summated HS, session RPE (sRPE) were collected. Finishing time and mean PO for self-paced , fast-start , and slow-start  TT were not different. There was a significant interaction between each secondary outcome variable  for starting strategy and distance. The evolution of HS reflected the imposed starting strategy, with a reduction in PO following a fast-start, an increased PO following a slow-start with similar HS during the last part of all TTs. The summated HS was strongly correlated with the sRPE of the TTs (r = 0.88). The summated HS was higher with a fast start, indicating greater effort, with limited time advantage. Thus, the HS appears to regulate both PO within a TT, but also the overall impression of the difficulty of a TT. Pacing is most simply defined as the distribution of energy expenditure over time intended to accomplish a desired goal without excessive fatigue or negative health effects ,2. A varThe basis of pacing reaches back to a hunter–gatherer society, where hunters had to make effort/reward decisions when pursuing game . This prThe concept of pacing highlights the importance of controlling intensity throughout an exercise bout in order to avoid unacceptably large homeostatic disturbances ,19,20,21Contemporary studies have extended this concept by looking at changes in energy expenditure relative to the details of specific athletic competitions. During shorter events, particularly when the primary retarding factor is air resistance (cycling/skating), it appears best to utilize anaerobic energy quickly to compensate for the short race duration, as velocity at the end of a race can be viewed as wasted kinetic energy . The oppThe process through which athletes spontaneously select their pacing strategy is called teleoanticipation . TeleoanWhile objective physiological measures can be used to measure homeostatic disturbance, exercise intensity can also be appreciated through the rating of perceived exertion (RPE) . RPE hasThis reciprocal relationship between RPE and changes in PO, and the abrupt decrease in PO after the starting segment of track cycling races ,38 led tThe subjects for this study were seven well-trained (7–10 h per week), recreational level, cyclists, age 25–61 years. The subjects were mostly long distance “tourists”, and performed limited competitive cycling, but regularly participated in “tours” of up to 160 km. Within the classification scheme of De Pauw et al.  and DelcFor subject characterization, each subject performed maximal incremental exercise on an electronically braked cycle ergometer . Tests were conducted to provide peak PO, maximal oxygen uptake, ventilatory threshold, maximal heart rate, and maximal RPE. After a warm-up stage of 3-min at 25 W, PO was increased of 25 W/min until pedaling cadence could not be maintained within the range of 70–90 rpm.init). During the self-paced TT, the subject was only instructed to finish the TT as quickly as possible. During the fast-start TT, the PO during the initial 3-km was 5% greater than POinit. During the slow-start TT, the PO during the initial 3-km was 5% less than POinit. This was reinforced by a visual display visible to the rider and verbal feedback from the investigator. The remaining 7-km were finished as rapidly as possible. A small monetary reward ($10), based on improving final TT performance versus the 2nd practice TT, was offered to provide a “competitive incentive” during TTs 3–5. PO was measured continuously by the ergometer, and integrated every 0.5-km. The RPE was measured every 1-km using the Category Ratio (0–10) RPE scale [Following the maximal test each subject performed a total of five 10-km (km) cycling TTs on a Velotron cycle ergometer . Prior to all TTs, there was a self-selected warm-up of 15–30 min, which included 2–3 bursts of 30–60 s at the anticipated starting velocity. The first two TTs were practice 10-km TTs to allow the athletes to become habituated to the 10-km cycling TT [PE scale . Blood lPE scale .p < 0.05 to achieve statistical significance.Descriptive characteristics of subjects were calculated as mean ± standard deviation. Time and average PO of the three experimental TTs  were compared using a one-way Analysis of Variance (ANOVA) with repeated measures. The HS was calculated by multiplying momentary RPE by the remaining fraction of the race . Summatep > 0.05). On average, the self-paced TT was 15.6 s faster than the slow-start TT and 12.6 s faster than the fast-start TT. sRPE, which included the warm-up, the TT, and cool-down, was the greatest for the fast-start TT and least for the slow-start TT.Descriptive data from the maximal tests are presented in The pattern of PO, RPE, blood lactate concentration, and HR within the three TTs is shown in p = 0.034), RPE (p = 0.027), blood lactate concentration (p = 0.043), and HR (p = 0.046).A significant interaction between the starting strategy and the distance covered was shown for PO  and summated HS (p = 0.031). HS during the fast-start TT was significantly greater than the self-paced TT at kilometers 1 and 3. HS during the fast-start TT was significantly greater than the slow-start TT at kilometers 1 and 2. HS during the self-paced TT was significantly greater than the slow-start TT at kilometers 1, 2, and 3 . This corresponds with Cohen et al.  who showIn the present study, while summated HS was different between TTs, the finish time was not significantly different  reflecting that since knowledge of the endpoint was present and distance remaining was not relatively large, the athletes were able to generate an end spurt despite the large summated HS. RPE has been shown to increase when athletes are aware of the distance to the endpoint ,31. ThisOptimal race performance is not always about who has the highest PO, but who can maintain optimal PO in order to perform a successful end spurt. de Koning et al.  comparedIn this particular study, the specific application was to a maximal effort TT. However, an equivalent argument may be made toward training bouts. Very high early PO can lead to increases in RPE  and leadOne limitation to this study included a limited sample size and task habituated tourist type cyclists rather than experienced TT athletes. Although we have shown that task habituation leads to stable performances , more acThe results of this study indicate that despite a reduction in PO following a fast-start TT, the summated HS remains higher with a fast-start strategy. This indicates that summated HS is a powerful predictor to better understand accumulated fatigue on pacing pattern during simulated competition. The sum of all HS from the beginning of the race to the present point have a cumulative effect on the outcome of the event, the physiological state, and the sRPE experienced by the exerciser.


Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques (n = 11) than in the non-culprit plaques (n = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques (p < 0.0001). VEGF-A expression was co-localized with CD34, a marker for angiogenesis (p < 0.001). Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo.Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques ( The annual stroke risk due to carotid atherosclerosis is increased by 10–20% compared to patients without carotid artery stenosis2. A study based on the large population-based Rotterdam cohort study demonstrated that 80% of the cohort over 55 years of age had some degree of carotid artery atherosclerosis3. Risk factors for the development of atherosclerosis include hypertension, dyslipidemia, diabetes mellitus, obesity and smoking. Most of these risk factors are increasingly prevalent amongst the world population as the result of ageing and the earlier emergence of comorbidities4.Carotid artery atherosclerosis is a leading cause for ischemic stroke, accounting for approximately one in four stroke cases5. Currently, there is no imaging modality available to stratify the risk of rupture in vivo. This leads to unnecessary surgical intervention in many patients, which is not desirable as the mortality rate is approximately 3% for this type of operation6. Identifying intraplaque biomarkers might possibly reveal the extent of plaque (in)stability and therefore aid in risk-stratification. Besides the composition and volume of the plaque, pathological plaque properties are crucial in assessing its stability. These properties include inflammation, lipid accumulation, angiogenesis, calcium precipitation, proteolysis, apoptosis, and thrombosis8. Ultimately, a combination of increased endothelial shear stress and increased tensile  stress facilitates rupture of the unstable plaque9. Specific molecular imaging techniques can aid in predicting the risk of stroke by visualizing and quantifying plaque characteristics. Currently, carotid endarterectomy (CEA) is primarily performed on eligible patients with carotid stenotic lesions and signs of transient ischemic attacks or minor ischemic stroke to reduce the risk of future events. Imaging techniques might enable reliable selection for CEA surgery before symptom onset, in for example a high-risk patient population.Formation of an atherosclerotic plaque within the vessel wall can cause partial or total vessel occlusion. Plaque rupture inevitably causes downstream ischemic damage in other organs18F-FDG uptake was associated with inflammation in the symptomatic plaque, a clear cut-off point for the vulnerable plaque could not be established due to the heterogeneity of data previously described10. Another study reported that 18F-FDG uptake in recent transient ischemic attack (TIA) or amaurosis fugax patients did not differ significantly from asymptomatic patients, rendering it a questionable clinical indicator for plaque vulnerability11. 18F-sodium fluoride was investigated as a tracer for plaque calcification in both myocardial infarction and symptomatic carotid stenosis patients. The former observed uptake in almost all culprit plaques, but also in half of their non-culprit plaque group (stable angina pectoris patients)12. The latter did not observe a statistical difference between uptake of culprit and non-culprit carotid plaques, based on time interval between symptom onset and CEA13.Positron emission tomography (PET) and single-photon emitted computed tomography (SPECT) have been investigated in detecting the vulnerable plaque. Although a recent meta-analysis found that 14 demonstrated an accuracy of 83% in symptomatic patients with ultrasound imaging of plaque surface irregularities in combination with the degree of stenosis, whereas plaques from asymptomatic patients have a significantly smoother surface. However, it is unclear whether plaque surface irregularities were true plaque ulcerations or other, incidental, surface defects. Additionally, identification of irregularities depends on scanning resolution and might be missed due to two-dimensional ultrasound imaging.Ultrasound techniques have also been investigated in atherosclerotic plaques. Plaque surface irregularity was also investigated as risk indicator for cerebrovascular events. Kanber et al.15. A potential new target for plaque categorization is intraplaque angiogenesis17. Angiogenesis within the atherosclerotic plaque is considered to arise from initial inflammation of the arterial wall and subsequent influx of macrophages resulting in a relative localized hypoxia, the latter being the most important driver through expression of HIF-1α for vascular endothelial growth factor-A (VEGF-A) expression18,19. Consequently, intraplaque release of VEGF-A is stimulated, prompting the formation of immature, leaky blood vessels prone to rupture causing hemorrhage and thrombus formation20, which results in plaque instability. The Athero-Express study demonstrated plaque neovascularization and plaque hemorrhage to be significantly related to adverse cardiovascular outcome21. Previously, Shah et al.22 demonstrated the use of contrast-enhanced ultrasound imaging to detect neovascularization in carotid atherosclerotic plaques and found a moderate correlation with histopathological staining. Practical implication remains a challenge, however, due to lack of an optimal enhancement quantification method and standardized protocols23. Accurate detection of the angiogenic process, and thus progression towards a vulnerable plaque, might contribute to the identification of high-risk patients.A non-invasive clinical follow-up study was performed in patients who underwent carotid endartectomy, collecting test and imaging results from the hospitals concerned. During this study, a yearly risk assessment was performed based on analysis of the endarterectomized samples and the clinical patients characteristics, demonstrating that plaque characteristics such as macrophage infiltration and lipid core extent alone were not predictive of future cardiovascular events due to other atherosclerotic plaques24. Relatively new in the  cardiovascular field is near-infrared fluorescence (NIRF) imaging. This modality uses fluorophores as imaging compound instead of the generally used radioactive imaging agents. In and ex vivo near-infrared fluorescent (NIRF) imaging can provide real-time information about molecular tissue composition with high sensitivity26. In order to image angiogenesis, previously a VEGF-A targeted antibody (bevacizumab) has been conjugated to a NIR fluorescent dye (IRDye800CW) for successful fluorescent imaging in solid tumors 30. Bevacizumab-800CW showed first of all to be safe, to have a high sensitivity and specificity, and allowed for accurate and reproducible imaging of angiogenesis. Previously, our research group has shown that radiolabeled 89Zr-bevacizumab is an effective and specific method for the detection of VEGF-A in carotid endarterectomy specimens31. The advantage of bevacizumab-800CW over 89Zr-bevacizumab is that the former has no radiation burden and is relatively less expensive than the latter. In this study, the feasibility of bevacizumab-800CW-based optical imaging for the detection of carotid plaque instability was assessed. The aim of this study was to determine whether the optical tracer bevacizumab-800CW is suitable for ex vivo visualization of intraplaque angiogenesis by means of identifying VEGF-A presence, and thereby able to distinguish between culprit and non-culprit atherosclerotic plaques.Conventional current imaging techniques possess a central role in visualizing the atherosclerotic plaque, such as Computed Tomography Angiography (CT-A) and duplex ultrasound (US) as part of the standard of caren = 17), of which three were used for dose-finding analysis. The optimal dose was used in 15 plaques: 11 plaques in the culprit group and three in the non-culprit group, based on the period of time between symptom onset and carotid endarterectomy (CEA), and one plaque was retrieved from an asymptomatic patient and was analyzed separately.Carotid artery plaque specimens from 17 patients were included in the study (2. He had no previous history of cardiovascular problems. Indication for CEA surgery was significant stenosis (≥ 80%) of the carotid artery.Patient characteristics of the culprit and non-culprit groups are shown in Table background = 1961) was similar to non-culprit plaques (MFIbackground = 1883). Specimens were then incubated within 20 min after surgical excision at room temperature with bevacizumab-800CW 1 µg/ml for 1 h. After incubation, the specimens were carefully rinsed, and subsequently imaged within 10 min. All PEARL images of culprit plaques showed clear fluorescent hot spots , whereas non-culprit plaques displayed less distinct, weak hot spots . MFI was calculated based on three to five measuring points per plaque depending on plaque size. After formalin-fixation and paraffin-embedding (FFPE) and micrometer tissue sectioning, flatbed scanning showed again a clear fluorescence pattern in culprit plaques with clear overlap of VEGF-A expression and the fluorescent signal pattern   was found. The H-score of the plaque from the asymptomatic patient was 236, indicating an intense staining for VEGF-A. VEGF-A expression levels were observed to be clearly higher in the culprit plaques than in the non-culprit plaques . No intraplaque micro vessels were observed in the non-culprit plaques.CD34 staining for microvessel density confirmed the presence of microvasculature in all culprit plaques. In addition, in all culprit plaques, including the asymptomatic plaque, VEGF-A staining was strongest in areas of high microvessel density. A strong association with VEGF-A staining was demonstrated . Of the non-culprit plaques, 6 sections were stained with Von Kossa and 3 specimens revealed microcalcifications in both the intimal and medial layers. No significant association was found between microcalcifications and VEGF-A expression in both the culprit and non-culprit group (p > 0.05).Von Kossa staining for (micro)calcifications was performed in 22 sections of the culprit plaques (2 per specimen), and revealed small calcium deposits around the VEGF-A and micro vessel areas in 73% of the sections (n = 15/17). Weak to moderate staining was found in non-culprit plaques. A moderate association was found between VEGF-A expression scores and CD68 staining scores . The average CD68 score in the culprit group was higher than in the non-culprit group (2.83 ± 0.389 vs 1.27 ± 0.577).Macrophage infiltration (CD68 staining) was most abundant in the VEGF-A ROIs in culprit plaques (p = 0.023).Strong expression of smooth muscle actin (SMA) staining was observed in the non-culprit plaques as opposed to mostly weak staining in culprit plaques. The asymptomatic plaque also displayed low SMA expression. There was a strong inverse correlation between H-scores and SMA staining . This complements earlier research in which diminished SMA staining was related to unstable coronary plaques37. Conversely, the frail integrity of 3 µm tissue slides results in disrupted tissue sections due to tearing and breaking of the tissue. This could attribute for a weaker bevacizumab-800CW fluorescent signal, imaged by the flatbed scanner, at the corresponding sites of VEGF-A expression. Another reason could be the submersion method of incubation with bevacizumab-800CW as the tracer might not be able to penetrate the full thickness of the specimens.The fluorescent signals found at sites of clear visual macrocalcification without VEGF-A expression at those specific sites could be explained by autofluorescence of calcified areas, as described previously in human atherosclerotic arteries39. Additionally, symptomatic plaques are normally removed within 2 weeks after symptom onset as standard of patient care in the Netherlands, which could be an explanation for the low number of non-culprit plaques.A limitation of this study is the low number of asymptomatic and non-culprit atherosclerotic plaques, which posed a difficulty in comparing symptomatic to non-symptomatic plaques and assessing statistical significance between culprit and non-culprit plaques. Asymptomatic plaques are normally not resected as standard of care because of the patient risk involved in the procedure. The number-needed-to-treat (NNT) to prevent major CVA or death is 6 for symptomatic patients, whereas asymptomatic patients have an NNT of 20, emphasizing patient risk89Zr-bevacizumab and the use of bevacizumab in CEA specimens31, as the monoclonal antibody bevacizumab was able to target VEGF-A ex vivo in human carotid atherosclerotic plaques in this study too. In addition, this study adds knowledge as we demonstrate the ability of the conjugate bevacizumab-800CW to identify intraplaque angiogenesis by targeting VEGF-A, and to be capable of distinguishing between culprit and non-culprit plaques. Furthermore, we demonstrate that angiogenesis correlates with macrophage infiltration using CD68 immunohistochemistry. An advantage is that a systemic dose of bevacizumab-800CW is already proven to be safe and effective in humans, according to various cancer studies30, which facilitates further in vivo research in the cardiovascular field. In general, the benefit of NIRF-tracers over PET-tracers is that the production process of NIRF-tracers is generally simpler and less expensive, due to the long shelf life and the elimination of radioactive infrastructure in the production process. NIRF-imaging allows for non-invasive imaging techniques, which is favorable in this fragile patient group. Additionally, with NIRF imaging, it is easier to image in real-time, there is no radiation burden, and the imaging time itself is relatively short31, however whole body imaging and deep signal penetration is lacking.This study complements previous research by our group with . We believe that this tracer might allow for improved risk-stratification of carotid atherosclerosis and therefore a reliable selection method for CEA. Benefits would be prevention of disease and mortality caused by acute vascular events. Ultimately, clinical implementation of this tracer to select for CEA should rely on the predictive value of angiogenesis in high-risk plaques.In conclusion, the NIRF tracer bevacizumab-800CW is promising in visualizing VEGF-A expression in human carotid atherosclerotic plaques ex vivo. Our results demonstrate the potential of bevacizumab-800CW to define vulnerable (VEGF-positive) and non-vulnerable (VEGF-negative) plaques. The validation of the latter hypothesis should take place in a clinical study. Future studies should focus on the clinical proof of principle in vivo of the use of bevacizumab-800CW in this particular patient group, to allow for its validation in vivo40. Odds ratios of present plaque characteristics as risk indicators for stroke were demonstrated to be significantly greater for plaques retrieved from patients operated within 30 days after the ischemic event than in plaques retrieved after 30 days after symptom onset. For high microvessel density, these odds ratios were 4.52 and 1.47, respectively32. In this study, we adopted this dichotomy: ‘positive’ culprit plaques were defined as plaques in patients with recent events , and ‘negative’ non-culprit plaques were defined as plaques in patients with no events in the last 4 weeks. For all patients, clinical and demographic data were collected from medical records, using the hospital’s electronic medical record system, including medication use and cardiovascular risk factors that were present before symptom onset. The study was released by the Medical Ethical Committee of the UMCG and is in accordance with the Helsinki Declaration (2013). It was decided that patient informed consent was not required as the study uses waste tissue-material, that is not restricted by the Dutch WMO . The IRB of the UMCG approved this study. For all patients, it was checked if objection statements about the use of waste material were ever recorded by the hospital. Data analysis was performed anonymously.For this ex vivo feasibility study, fresh human carotid specimens were obtained from patients undergoing carotid endarterectomy between September 2017 and April 2018 in the University Medical Center Groningen (UMCG). Previous literature demonstrated that ‘positive’ plaques corresponded to the patients with recent events (i.e. culprit plaques) and ‘negative’ plaques to patients with a remote history of ischemic stroke (i.e. non-culprit plaques)Primary endpoints were the accumulation of ex vivo incubated bevacizumab-800CW in carotid plaques by fluorescence macro- and microscopy, and its correlation with VEGF-A expression, macrophage infiltration (CD68), microvessel density (CD34), microcalcifications (Von Kossa) and structural integrity of the vessel wall , determined by semi-quantitative analysis of (immuno)histochemical stainings. For these parameters, culprit and non-culprit plaques were compared.41. An optimal dose of 1 μg/ml was determined by submersing the specimens in various solutions  for one hour in a dark room to prevent photobleaching. The used concentrations were based on theoretical blood concentrations after intravenous administration of 4.5 mg, 10 mg, and 25 mg bevacizumab-800CW, respectively. The imaging procedures with the optimal dose were as follows. Immediately after excision, the specimens were rinsed with PBS. Subsequently, images of the whole tissue were acquired with the PEARL imager . The specimens were then submersed in a 1 μg/ml bevacizumab-800CW solution for one hour at room temperature. PEARL images were obtained from the whole tissue specimen, after which hot spots  were selected. The MFI was only calculated from the subsequent PEARL images. All images of all specimens were scaled to the same intensity. The spots were formalin-fixed, paraffin-embedded (FFPE) and cut in 3 μm sections.Bevacizumab  conjugation with the fluorescent dye IRDye800CW was performed at the department of Clinical Pharmacy and Pharmacology, as described earlierThe FFPE blocks were analyzed with the Odyssey fluorescent flatbed scanner . In order to cross-correlate a fluorescent signal within the corresponding 3 μm slides with additional histopathological staining, the sections were deparaffinized (10 min xylene) and analyzed with the Odyssey scanner. Afterwards, these 3 μm slides were stained with hematoxylin and eosin (H&E) for cell morphology and to determine what part of the plaque displayed fluorescent signal.29. Leica NIR fluorescence microscopy was carried out to colocalize bevacizumab-800CW and VEGF-A. For image processing and analysis, LAS-AF software  and Fiji/ImageJ  were used as described previously42.In additional consecutive 3 μm slides cell nuclei were visualized with Hoechst staining  in the same manner as we described previously1 κ monoclonal, Dako, clone QBEnd/10). Slides were incubated with anti-CD68  for the staining of macrophages. Smooth muscle actin in the vessel walls was stained with anti-SMA . All slides were incubated for one hour at room temperature. Consecutively, the slides were incubated with horseradish peroxide (HRP) secondary antibodies, and afterwards chromogenic staining with 3,3′-diaminobenzidine (DAB) was performed for color development. Hematoxylin was used to counterstain the slides.To allow for examination of the pathological status (i.e. vulnerability) of the plaques, for each spot slides were double stained for VEGF-A and either Von Kossa, CD34, CD68 or SMA. The latter three immunohistochemical stainings were performed by standardized machine protocols. For the staining of microvessels, slides were incubated with anti-CD34  and graded alcohols. For heat-induced antigen retrieval (15 min), 100 mM Tris/EDTA buffer (pH 9.0) was used. Endogenous peroxidase was blocked for 30 min with 30% hydrogen peroxide, followed by avidin–biotin blocking. Subsequently, the slides were incubated overnight at 4 °C with a polyclonal rabbit anti-human VEGF-A antibody . Slides were incubated with goat anti-rabbit biotin in phosphate-buffered saline with 1% bovine serum albumin [PBS 1%BSA] with 1% AB serum, and consecutively with alkaline-phosphatase conjugated streptavidin in PBS 1% BSA with 1% AB serum, both for 30 min. For color development, alkaline-phosphatase activity was visualized using naphthol AS-MX phosphate and Fast Blue salt (30 min). The slides were washed with demineralized water and cover slipped with Kaiser’s glycerin. PBS was used for washing, and all steps were performed at room temperature unless indicated otherwise.For the Von Kossa staining, the slides were first submersed with 1% silver nitrate solution for 60 min in broad daylight, followed by 3% sodium thiosulfate (5 min). The slides were counterstained with nuclear fast red (3 min), after which dehydration was performed with graded alcohols. The slides were then air-dried (30 min) and cover slipped with mounting medium. Demineralized water was used throughout washing.30, determined by semi-quantitative combined assessment of the percentage of stained cells and the staining intensity . Von Kossa, CD34, CD68 and SMA stainings were categorized semi-quantitatively . The pattern of fluorescent signal was compared to corresponding VEGF-A-stained slides by fitting the VEGF-A region of interest (ROI) on the corresponding fluorescence image and determining the extent of overlap between VEGF-A expression and fluorescent signal.The stained slides were digitized with the NanoZoomer Digital Pathology Scanner . The imaged slides were then analyzed and evaluated with Aperio ePathology  software. To score the degree of VEGF staining, the H-score was calculated in the same manner as described previously by our groupp value ≤ 0.05 was considered to be statistically significant.Data were analyzed with SPSS Statistics 24  and Fiji/ImageJ . Regions of interest (ROIs) were drawn based on the VEGF-A staining . The mean fluorescence intensity of this ROI was measured quantitatively. Categorical data was tested with Chi-Square association tests. A