NEwTON is a single center observational prospective case-finding study based at the Alzheimer Center Amsterdam that aims to include 40 participants at risk for CTE (exposed participants) and 40 healthy unexposed control subjects. Exposed participants and unexposed controls are allocated to various study procedures. All exposed participants receive a comprehensive baseline screening and are invited for two follow-up measurements (after one and two years). If the included participant comes from the memory clinic and has received standard diagnostic screening less than six months before inclusion in the NEwTON study, only additional measurements are done, because part of the study procedures overlap with the standard diagnostic screening. The procedure for control subjects is limited to one baseline assessment, including a neuropsychological test battery, blood draw and MRI-scan of the brain. (figure 1a) The rationale behind a single baseline assessment for healthy controls is to diminish participation burden for this population. Besides, the control group is included in the study to identify, validate and compare potential neuro-imaging/fluid biomarkers, which are mainly measured at baseline.
The inclusion criteria for exposed participants are derived from the criteria for TES by Katz et al. :
1) History of repetitive/multiple impacts to the head. Sources of exposure could be:
- Involvement in high exposure contact sports (i.e. combat sports, rugby, soccer) for a minimum period of six years on significant level.
- History of any other significant exposure to repetitive hits on the head (i.e. abuse, head banging behavior, military service)
- Any other activity resulting in multiple TBI (fall, traffic accident)
2) At least one of the following core clinical features of TES must be present and be different from pre-morbid functioning. Symptoms may be self-reported, reported by informant, reported by clinician’s report or objectified by previous standardized clinical testing.
- Cognitive symptoms: memory, executive functioning
- Neurobehavioral dysregulation: emotionally explosive, physically/verbally violent. ‘having a short fuse’
3) Clinical features must be present for a minimum of 12 months.
4) Age above 30 years old
Important to note is that participants with a previous diagnosis of any other neurological or psychiatric disease are also able to participate.
Inclusion criteria for unexposed controls are as follows:
- Age above 30 years old
- No history of participation in organized contact/collision sports*
- No history of military service (professional, with blast exposure)
- No history of clinically significant TBI or concussion
- No history of any neurological, psychiatric or neurodegenerative disease
- No reported complaints of cognition, behavior and depressive mood
* this is indicated as no history of organized participation in any of the following sports: soccer (under age 14 allowed if no significant heading the ball), rugby, boxing, kickboxing, Muay Thai, Mixed Martial Arts, American football, ice hockey, lacrosse, wrestling
Exclusion criteria for participation include an insufficient knowledge of the Dutch language or mentally incompetency to give informed consent. Furthermore, exposed participants are excluded with a Mini-Mental State Examination (MMSE) score of ≤ 18 or when they have reported a clinically significant concussion or traumatic brain injury within one year before inclusion. Control subjects are excluded when there is a contra-indication for MRI according to the hospital protocol. An overview of the participant criteria is given in figure 2.
Several sources of recruitment are used to find eligible participants for NEwTON. Part of the participants are recruited from the memory clinic of the Alzheimer Center Amsterdam, an expertise center in dementia. As part of the standard diagnostic work-up in the Alzheimer Center Amsterdam, patients receive standardized diagnostic tests. Patients are asked to sign a consent form indicating that their clinical information can be used for scientific purposes in the future and that they may be approached for further research participation. If patients fulfill criteria and they are interested in participating, they are invited to undergo additional measurements for the NEwTON study at a later stage.
Other potentially eligible participants are recruited outside the Alzheimer Center’s memory clinic. The NEwTON study has already established collaborations with different national sports federations, such as the Dutch Olympic Committee*Dutch Sports Federation (NOC*NSF), the national soccer union (“Koninklijke Nederlandse Voetbalbond”), the national rugby federation (“Rugby Nederland”), the national combat sports authority (“Nederlandse Vechtsport Autoriteit) and the Dutch Boxing Federation (“Nederlandse Boksbond”). NEwTON is able to promote its study by utilizing their media platforms to reach an eligible audience. Unexposed controls are reached by the media platforms of the Alzheimer Center Amsterdam. Potential candidates are able to contact the researchers via e-mail. When eligible, they are invited to participate to the NEwTON study and undergo all baseline measurements.
For this study, it is complicated to determine a study sample size that achieves adequate statistical power. This is mainly because NEwTON has an exploratory research design, with various outcome measures that have not been well-studied yet in previous research. To illustrate, previous results on fluid and neuro-imaging biomarkers are preliminary and have not been tested in a similar study population. Additionally, there is no available prospective data on clinical progression of CTE. The sample size of 40 exposed participants and 40 unexposed controls is considered sufficient for a pilot study and this number is expected to be expanded in the future. This is in line with the decision of the current two year follow-up period, which also may be extended in the future to make it possible to measure the clinical course over a longer period of time
An overview of all study procedures is displayed in figure 1b.
All exposed participants receive a structured medical interview by a trained physician, to collect data about demographics, neurological symptoms, previous medical history, medication, current or previous substance use, lifestyle history and family history of any neurological or psychiatric diseases. In addition, a comprehensive sports history assessment is performed to identify previous participation in (contact) sports and to collect information about age, era and duration of participation, position played, the highest level of competition and the use of head protection equipment. If the participant consents, his or her informant is also interviewed. Vital signs are administered and neurological examination is performed. Motor symptoms are measured according to the motor score of the unified Parkinson’s disease rating scale (UPDRS), a standardized rating scale to score extrapyramidal signs. These examinations are repeated at follow-up screening.
The MMSE and Montreal Cognitive Assessment (MOCA) are both administered to screen global cognitive functioning. Although there is some overlap in the content of these tests, having the results of both tests in the data set will be of high value for future national and international data exchange. The Frontal Assessment Battery (FAB) and a picture naming test are also included in the protocol, which are screening tests to assess frontal lobe dysfunction and language problems respectively. Additionally, all participants undergo a comprehensive neuropsychological test battery, that includes tests for different cognitive domains: Trail Making Test Part A & B (processing speed, sequencing, mental flexibility and visual-motor skills), Stroop Color Word Test and Digit Span (attention and inhibition), Letter Digit Substitution Test (visual scanning, mental flexibility, sustained attention, psychomotor and processing speed), Verbal Fluency Test (lexical memory, executive functions), Animal Fluency Test (semantic memory), Visual Association Test and Rey Auditory Verbal Learning Test (visual and verbal memory), Rey Complex Figure Test and Recognition trial and recall (visuospatial constructional ability and visual memory), Visual Object and Space Perception Battery (visuospatial constructional ability), Dutch Reading Test (premorbid intelligence level), and Ekman 60 faces test (social cognition). This test battery is applied again to exposed participants after one and two years.
Subjective cognitive impairment and mental health
Multiple questionnaires and rating scales are included for exposed participants in NEwTON regarding subjective cognitive impairment and mental health:
- The Cognitive Change Index – 20 item (CCI-20): this questionnaire has two versions (self-report and informant report), both with 20 questions that reflects subjective cognitive change compared to five years ago. Each item is scored from 1 (no change) to 5 (very severe decline).
- Geriatric Depression Scale (GDS) – 15-item: a questionnaire to test depressive symptoms in the elderly. Each question is answered by yes or no and corresponds to a positive or negative indication of depression. Higher scores reflect more severe depressive symptoms
- The Montgomery Asberg Depression Rating Scale (MADRS): this 10-item rating scale is completed by the physician after the clinical interview with the participant and reflects clinical judgement on different depressive symptoms. Every item is rated from 0 to 6, with higher rates indicating more severe symptoms.
- The Neuropsychiatric Inventory Questionnaire (NPI-Q): this questionnaire is completed by the informant and administered by a trained researcher via a face-to-face interview or telephone call. Twelve different neuropsychiatric symptoms are measured (delusions, hallucinations, agitation/aggression, dysphoria/depression, anxiety, euphoria/elation, apathy/indifference, disinhibition, irritability/lability, aberrant motor behavior, night‐time behavior and appetite/eating). The researcher tests whether the symptom is present and if so, measures the frequency (range 1-4), severity (range 1-3) and emotional distress that the symptom causes (range 1-5).
- The Frontal Behavioral Inventory (FBI): this 24-item inventory assesses behavioral changes and was developed to test symptoms of the behavioral variant of frontal temporal dementia, but is administered in this study because current research lacks validated tools to assess CTE-related behavioral changes. The informant rates every item from 0 (not present) to 3 (severe).
Traumatic brain injury (TBI)
At baseline, a comprehensive assessment of TBI history is applied to exposed participants by means of a translated version of the OHIO State University TBI Identification Method (OSU TBI-ID). The aim of this method is to reveal the life-time history of TBI and comprises three parts. The first part contains questions to identify and recall TBI in history. The second part aims to classify each TBI (cause, age, loss of consciousness or amnesia) and part three identifies any periods in life with repetitive head impacts (for example in contact sports). For each period, the age and cause of the head impacts are listed, as well as the typical effect and the most severe effect of the impacts during this period. In addition, concussion related symptoms are measured by items of the Rivermead Post-Concussion Symptoms Questionnaire.
MRI scans are acquired on a GE 3-Tesla scanner (Discovery MR750) for both exposed participants and unexposed control subjects, with approximately 40 minutes acquisition time. High resolution (1x1x1 mm3) T1-weighted imaging and Fluid Attenuated Inversion Recovery (FLAIR) are used for anatomical imaging. Diffusion weighted images (DWI) are acquired to determine white matter integrity, with a resolution of 2x2x2 mm3 and 2 different b-values (5 b0 and 30 volumes with b1000 s/mm2). Resting-state functional MRI (fMRI) is used to determine brain network connectivity, measured by the degree of synchronized blood-oxygenation-level dependent (BOLD) signal across brain regions. The T2*-weighted echo-planar images are acquired with 3.3 mm3 resolution. With quantitative susceptibility mapping (QSM), a 3D gradient echo sequence is used to determine spatial distribution of magnetic susceptibility. This is sensitive to presence of iron, calcium and myelin in the brain. QSM images are acquired using a 3D multiple echo gradient echo sequence at resolution 0.5x0.5x1.6 mm3. Reference scans with reversed phase-encode direction are acquired for fMRI and DWI, to allow correction of susceptibility induced distortion. For fMRI, DWI and QSM, high-order shimming is performed before each scan.
Prior to the analyses, FLAIR imaging is used to detect white matter hyperintensities and cortical lacerations, which will be corrected for in further evaluations. FreeSurfer is used to measure structural brain features from T1 images, including grey and white matter volume, hippocampal volume and cortical thickness. Additionally, the presence and width of cavum septum pellucidum is measured. For DTI data, FMRIB Software Library (FSL) tools are used for pre-processing, with correction for susceptibility-induced distortion and motion. Four metrics are estimated to identify white matter damage. (1) fractional anisotropy, the most common parameter to detect differences in white matter (2) mean diffusivity, the diffusivity averaged over all directions (3) axial diffusivity, which is sensitive to diffusivity along the axon and (4) radial diffusivity, to determine diffusivity parallel to the axons. For group comparisons, the DTI ToolKit (DTI-TK) is used, which is a spatial normalization and atlas construction toolkit, that takes into account the main diffusion direction. The fMRI images are pre-processed and analyzed using the FSL toolbox. QSM data is visually analyzed for cerebral microbleeds and siderosis and maps are calculated with the help of the online toolbox: Sepia (SuscEptibility mapping PIpeline tool for phAse image), based on the pipeline that has been developed for previous QSM projects using similar input data.  For these analyses, pre-defined region-of-interests, white matter tracts and functional brain networks will be examined, informed by the current literature. In addition, exploratory voxel-wise analyses will be performed to examine more fine-grained regional change.
Body fluid biomarkers
Participants are asked to give separate consent to the researchers for blood collection, CSF collection and storage of these body fluids in the NEwTON biobank for future analysis. However, this is not mandatory for participation in the study.
Venous blood is drawn by a trained physician or researcher at baseline (both exposed participants and controls) and at two years follow-up (exposed participants only). The plasma and serum samples (24 ml in total) are centrifuged (1800g, 10 min) at room temperature within two hours after collection, aliquoted into small vials (0.5 ml) and stored at -80 °C at the NEwTON biobank located at the clinical chemistry laboratory of the Amsterdam University Medical Centers (UMC).  In addition, buffy coat is isolated after centrifugation and stored separately to purify DNA in the future.
All exposed participants are invited to undergo a lumbar puncture (LP), which is performed by a neurologist or trained physician after subject’s consent. Contra-indications for LP are determined according to local hospital guidelines and participants are informed about the procedures and potential complications.  After collection, a small amount of CSF is used for routine analysis on white blood cells, erythrocytes, proteins and glucose. Amyloid beta (aβ) 1-42, total tau and p-tau-181 are determined by using Elecsys® assays. A separate portion of CSF is centrifuged (1200g, 10 min, room temperature) within two hours and aliquoted into small vials and stored at -80 °C in the NEwTON biobank. 
Brain donation program
The NEwTON study has established a national CTE brain donation program in collaboration with the Netherlands Brain Bank (NBB). Former contact sport athletes are able to register as brain donor at the NBB thus consent for post-mortem brain autopsy. All participants of NEwTON are informed about this donation program and the possibility to register. Tissue treatment, sample storage and pathological evaluation of NEwTON brain donors is performed according to standardized protocol of the NBB, including macroscopic evaluation and immunohistochemical staining of multiple brain regions: hematoxylin and eosin (H&E stain), aβ stain, Gallyas, silver-staining, several tau staining (AT8, RD3, RD4), alpha-synuclein and TAR DNA-binding protein-43 (TDP-43). Neuropathological diagnosis will be established according to international guidelines of Brain Net Europe II (BNE) consortium and NIA-AA criteria for AD neuropathological change. [31, 32] Brain tissue is stored frozen or formalin fixed and embedded in paraffin at the NBB for future research purposes.
Multidisciplinary consensus meeting
Every exposed participant is discussed in a multidisciplinary consensus meeting that is attended by at least one neurologist and one neuropsychologist. During this meeting, a consensus diagnosis is made for each participant regarding the TES criteria by Katz et al. (2021).  The panelists also determine the probability of CTE according the TES flow-chart of this study and the disease stage (subjective cognitive decline, mild cognitive impairment or dementia) for each exposed participant.
All statistical analyses of the data will be performed with IBM SPSS Statistics or R Studio. Within exposed participants, explorative analyses are performed to assess associations between cumulative head injury exposure, and cognitive/mental health outcome measures, body fluid biomarkers and MRI data. Correlation coefficients and linear regression analyses are utilized to test these associations, including adjustment for potential confounders. Furthermore, the progression of cognitive and mental health outcome measures in participants over time is assessed using linear mixed models. For group comparisons, neuropsychological test results, blood biomarkers and MRI data are compared at baseline between groups (exposed participants and unexposed controls) by independent T-tests, analyses of variance (ANOVA) or Mann-Whitney U test, where appropriate.
Ethical consideration and data sharing
All research conducted by NEwTON is in accordance with the World Medical Association (WMA) Declaration of Helsinki, Ethical Principles for Medical Research Involving Human Subjects 2013 and has been reviewed by the Medical Ethics Committee from the Amsterdam UMC. Before inclusion, all participants sign written informed consent, including separate permission to store body fluids in the NEwTON biobank for future analysis. Furthermore, explicit consent is obtained from all participants regarding sharing data and/or biomaterials with institutions from abroad, including countries outside the European Union. To illustrate, preliminary collaborations are made with the Boston University CTE Center and the “Diagnostics, Imaging, and Genetics Network for the Objective Study and Evaluation of Chronic Traumatic Encephalopathy” (DIAGNOSE CTE) Project for data sharing in later phases of the project. Handling and sharing of data and/or biomaterials will be in agreement with the General Data Protection Regulation (GDPR) or at the best possible level of confidence, when different regulations apply, such as in the United States of America. All data and/or biomaterial will be shared under a transfer/sharing agreement.