The institutional review board at the Boston University (BU) Medical Campus approved
all of our research activities (IRB number 31614). Because all participants are deceased,
consent does not need to be obtained. Figure 1 shows an overall flowchart of the study methodology. The presented methodology resulted
from several iterations of updates based on investigator feedback.
Fig. 1. Overall flowchart of the study methodology
Study recruitment
The U.S. Department of Veterans Affairs (VA)-BU-Concussion Legacy Foundation (CLF)
Brain Donation Registry and Brain Bank is a collaborative effort of the CTE Research
Program within the BU Alzheimer’s Disease Center (ADC), the VA Boston Healthcare System,
and CLF, a non-profit organization dedicated to brain trauma research and education.
Subject recruitment is ongoing and will occur throughout the 4-year study period.
Figure 2 shows recruitment mechanisms in place since UNITE recruitment began in January 2014.
For the majority of the brain donors, the subjects’ next of kin contact the brain
bank and agree to donate near the time of death. While living, some study subjects
agree, through the Brain Donation Registry, to donate their brain and spinal cord
after death. Potential subjects can register at any time, provided they meet specific
inclusion and exclusion criteria (detailed below). The registry currently has nearly
600 potential living subjects. We anticipate that several hundred more will join the
registry over the 4-year study period. On the basis of the rate of brain donation
in recent previous work, we anticipate that 300 subject specimens will be donated
over the 4-year study period, with 20Â % acquired through the registry.
Fig. 2. Recruitment mechanisms in place at the U.S. Department of Veterans Affairs–Boston
University–Concussion Legacy Foundation Brain Donation Registry and Brain Bank since
Understanding Neurologic Injury and Traumatic Encephalopathy project recruitment began.
Next-of-kin recruitment: A potential donor’s legal next of kin contacts the brain
bank near the time of death to ask about participation. Active recruitment: A member
of the brain bank staff contacts a potential donor’s next of kin near the time of
death to ask about participation. Brain Donation Registry: A potential donor contacts
the brain bank and pledges to donate upon death. Medical examiner: A medical examiner
contacts the brain bank upon suspicion of a diagnosis of chronic traumatic encephalopathy
or if an individual’s family member expresses to the medical examiner interest in
brain donation. Consultations: A neuropathologist contacts the brain bank to release
tissue for further evaluation
Eligibility criteria
Regardless of recruitment mechanism, potential subjects are evaluated using the same
inclusion and exclusion criteria (Table 1). The inclusion criteria are based solely on RHI exposure history, regardless of
whether symptoms are present. The criteria allow for breadth of RHI exposure (e.g.,
athletics, military service, abuse) while requiring sufficient intensity such that
there is a reasonable chance for the development of CTE (based on our experience,
acknowledging that the relationship between RHI and CTE is still under investigation).
The inclusion criteria are broader for women, who historically have been investigated
less thoroughly than men, and for individuals with amyotrophic lateral sclerosis (ALS),
a particular research focus at our center. The exclusion criteria prevent inclusion
of brain and spinal cord specimens of poor quality.
Table 1. Inclusion and exclusion criteria for the UNITE study
Consent
Consent for donation of brains and spinal cords is acquired from the decedent’s legal
next of kin or legally authorized representative (LAR). The next of kin or LAR also
may consent for the donation of cerebrospinal fluid (CSF), blood, and/or eye tissue
for use in related studies.
Brain acquisition
A member of the research team coordinates the extraction and shipment of the specimen.
A properly trained individual (pathologist, medical examiner, autopsy technician,
diener) extracts the tissue locally. If immediate shipment is possible, the specimen
is placed on wet ice and shipped using a courier service to minimize the postmortem
interval. If immediate shipment is not possible, the specimen is placed in 10Â % formalin
for fixation for a minimum of 2Â weeks before shipment.
Pathological processing and evaluation
The detailed methodology used for pathological processing and evaluation has been
published previously 28], 29]. The McKee Laboratory evaluates brains obtained from several brain banks, including
-Concussion Legacy Foundation, the Boston University-Alzheimer’s Disease Center, the
Framingham Heart Study, the New England Centenarian Study, the National Registry of
Veterans with Amyotrophic Lateral Sclerosis, and the Veterans Administration National
Posttraumatic Stress Disorder Brain Banks. Regardless of individuals’ brain bank membership,
all brains are processed identically and assigned a random identification number that
does not identify the brain bank to which they belong. Briefly, for fresh tissue,
quality control measures are followed, including RNA integrity number (Agilent Technologies,
Santa Clara, CA, USA) and pH. The brain is hemisected, then one half is sectioned
and frozen and the other half is fixed for 3Â weeks. The fixed tissue is dissected
and processed into tissue sections, including paraffin-embedded tissue sections and
large, fixed coronal slabs. Tissue blocks and stains are detailed in Table 2. If screening regions are positive for ?-amyloid, ?-synuclein, or phosphorylated
transactive response DNA binding protein 43Â kDa (pTDP-43), additional regions are
stained to allow for complete staging of these pathologies. Positive and negative
controls are stained simultaneously to identify improperly stained material.
Table 2. Brain tissue blocks and stains used for neuropathology
The pathological evaluation and diagnosis occur without any knowledge of the subject’s
RHI or clinical history and are confirmed by two neuropathologists (ACM, TDS). Semiquantitative
measures of phosphorylated tau burden (by AT8 immunostaining), ?-amyloid deposition
(by 4G8 immunostaining for Thal phase and Bielschowsky silver stain for Consortium
to Establish a Registry for Alzheimer’s Disease score), ?-synuclein–positive Lewy
body and neurite burden, pTDP-43 burden, vascular disease, and neuronal loss are recorded
for prespecified regions. The validated criteria for diagnosis (2], 27], A.C. McKee) and stages 2] of CTE are summarized in Tables 3 and 4. Well-established pathological criteria are used for diagnosis of all comorbid diseases,
including ALS 30], 31], AD 32]–37], Parkinson’s disease and Lewy body disease 38]–40], FTLD (including progressive supranuclear palsy, corticobasal degeneration, and Pick’s
disease) 41]–45], and multiple system atrophy 43]. A neuropathology report is generated that includes a description of the macroscopic
and microscopic findings and a list of pathological diagnoses, including CTE stage.
Table 3. Pathological criteria used for CTE diagnosis
Table 4. CTE pathological stages
Retrospective clinical evaluation
The goal of the retrospective clinical evaluation is to obtain each subject’s demographic
information; RHI exposure; substance use; and medical, social, and family histories,
with a particular focus on possible neurodegenerative conditions, including symptom
breadth, severity, and progression. The retrospective clinical evaluation comprises
a combination of online surveys and telephone calls between researchers and the family
members and close friends of the subject. Data are collected through an unstructured
interview with either a behavioral neurologist or a neuropsychologist and with modified
(for completion retrospectively, following death, by informants) versions of standardized,
validated scales (Table 5). Preference was given to scales already in use in other relevant BU studies, including
studies of CTE and AD 46]–49], and to scales that are included in the National Institute of Neurological Disorders
and Stroke (NINDS) Common Data Elements. Researchers conducting these evaluations
are completely blinded to the pathological examinations and findings.
Table 5. Administered clinical scales
There are five parts to the clinical evaluation: two online surveys (termed Online
Surveys A and B), two telephone interviews (termed Clinical Interviews A and B), and
a medical record review. Informants may complete the online surveys individually or
as a group (i.e., several members of the decedent’s family responding together). For
the clinical interviews, informants participate as a group. A behavioral neurologist
or neuropsychologist (termed the lead clinician) conducts Clinical Interview A, and
a research assistant conducts Clinical Interview B. To assess informant reliability,
informants answer questions pertaining to the nature and duration of their relationship
with the subject and the frequency with which they were in contact with the subject.
A description of each part of the clinical evaluation (presented in the order in which
the data are collected) is provided in the subsections below.
Online Survey A
Online Survey A queries the subject’s demographic information, educational attainment,
occupational history, living situation before death, athletic history (type of sports
played, level, position, age of first exposure, and duration), and military history
(branch, location of service, and duration of combat exposure). The survey uses a
nested question structure with skip logic to ensure that questions are appropriately
tailored to each subject.
Clinical Interview A
During Clinical Interview A, the clinician (a behavioral neurologist or a neuropsychologist)
obtains a detailed medical history, including traumatic brain injuries (TBIs), and
recreates a timeline of cognitive, behavioral, and mood symptomatology. Specifically,
the clinician asks semistructured questions about cause of death, medical history
(including vascular risk factors), neurological history (including risk factors for
cognitive and motor impairment), and psychiatric history. The clinician then asks
semistructured questions about mild to severe TBIs using the Ohio State University
TBI Identification Method Short Form 50] and two questionnaires, adapted from published studies, that address military-related
head injuries and concussions that result in even the mildest symptoms 47], 48]. Finally, using unstructured questions, the clinician obtains a precise chronology
of deficits in cognition (memory, executive function, attention/concentration, language,
visuospatial function), behavior and/or mood (depression, apathy, mania, anxiety,
irritability/anger, abusiveness, social inappropriateness, psychosis), and daily function
(including instrumental activities of daily living). Motor functioning, sleep, headaches,
substance use, and family history are queried in the same manner. Once the interview
is completed, the clinician answers several summary questions about predominant symptoms
(cognitive, mood, behavior, motor), symptom onset, and disease progression.
Online Survey B
Online Survey B is used to collect data about the subject’s cognition, mood, and behavior
(including impulsivity and apathy) through the administration of the following validated
scales: Geriatric Depression Scale Short Form 51], Cognitive Difficulties Scale (CDS) 52], Behavior Rating Inventory of Executive Function–Adult Version 53], Barratt Impulsivity Scale version 11 (BIS-11) 54], and Apathy Evaluation Scale (AES) 55]. For subjects diagnosed during life with ALS, the ALS Functional Rating Scale 56] is also administered.
Clinical Interview B
In Clinical Interview B, the researcher asks informants semistructured questions to
quantify information obtained qualitatively in Clinical Interview A. Some questions
from Online Survey B are repeated for quality control. Specifically, family history
is obtained using modified questions from the National Alzheimer’s Coordinating Center
Uniform Data Set 49]. Cognitive functioning, including memory, language, attention, executive function,
and visuospatial function, is assessed using selected questions from the informant
section of the CDS 52]. Daily function, cognitive fluctuations, and aggression are assessed using the Functional
Assessment Questionnaire 57], the Clinician Assessment of Fluctuations 58], and the Brown-Goodwin Aggression Scale 59], respectively. Impulsivity and apathy are assessed using selected questions from
the BIS-11 54] and the AES 55], respectively. The presence of major depressive disorder, bipolar disorder, anxiety
disorders (including panic disorder, obsessive compulsive disorder, agoraphobia, social
anxiety disorder, specific phobias, generalized anxiety disorder, and posttraumatic
stress disorder), psychosis, substance use, and somatoform disorders is assessed using
modified questions from the Structured Clinical Interview for DSM-IV 60]. Sleep and headache are briefly assessed using modified questions from the Mayo Sleep
Questionnaire 61] and the Cleveland Clinic Headache Intake Questionnaire, respectively 62]. Motor function is assessed using questions developed internally to query symptoms
of parkinsonism.
Medical record review
For all incoming cases, we request permission from the subject’s legal next of kin
or LAR to review medical records as well as the names and locations of health care
providers. A research assistant contacts the health care providers to request the
records, including original brain imaging (rather than just a report). The research
assistant initially reviews all records and extracts salient information, including
psychiatric, neurological, and neuropsychological evaluations; brain imaging; medical
history; and medications. A behavioral neurologist and a neuropsychologist then review
the extracted information, including original images if available. Information gathered
during the medical record review is combined with the data gathered in the previous
steps to complete the clinical evaluation.
Clinicopathological consensus conference
Clinical consensus methodology is based on recommendations of Bertens et al. 63]. At a twice-monthly clinicopathological consensus conference (CPC), a panel of doctoral-level
clinicians reaches a clinical consensus diagnosis using clinical research criteria
38], 61], 64]–69], including those recently proposed for CTE 5] (Fig. 3). The clinical panel is composed of neuropsychologists, neurologists, psychiatrists,
and neurosurgeons who specialize in neurodegenerative disease and/or TBI. At least
three and upward of six panel members are present for each CPC.
Fig. 3. Adapted clinical diagnostic criteria for traumatic encephalopathy syndrome
a
. Abbreviations: CSF cerebrospinal fluid, CTE chronic traumatic encephalopathy, PET
positron emission tomography, p-tau phosphorylated tau, RHI repetitive head injuries,
TBI traumatic brain injury, TES traumatic encephalopathy syndrome.
a
Criteria adapted from Montenigro et al. 5]
For each case, a lead clinician reads a standardized clinical summary based on the
information collected during the retrospective clinical evaluation. This summary outlines
the disease course. It also includes age at death and cause of death; a subjective
assessment of informant reliability; prior athletic, military, and TBI history; past
medical, educational, and occupational history; living situation before death; substance
use history; and family history. Last, it includes salient features in medical records,
including neuropsychological testing, neuroimaging (including a reading from a behavioral
neurologist if original images are available), CSF biomarkers, diagnoses made during
life, and medications prescribed. At the conclusion of the clinical presentation and
before any formal discussion, each clinical consensus member votes independently,
without discussion, on whether criteria for traumatic encephalopathy syndrome (TES)
5] are met. The diagnosis of TES, which incorporates CTE, is made on the basis of criteria
outlined by Montenigro et al. 5], with modification because neuropsychological testing may not have been conducted
during the subject’s life. Although several groups have proposed CTE clinical criteria
7], 8], use of the Montenigro et al. criteria provides several advantages. To be included
in the core criteria, signs and symptoms needed to be frequent (70Â %) among cases
diagnosed neuropathologically with CTE using the criteria proposed by McKee et al.
2] and free of comorbid neurodegenerative disease. The criteria also are operationalized
for research, explicitly defining the minimum required exposure level, supportive
features, subtype designations, potential biomarkers, and relative likelihood of CTE
(probable, possible, or unlikely). Of note, TES is a broad umbrella term meant to
describe the clinical presentation of CTE as well as other possible long-term consequences
of RHI, including other neurodegenerative diseases. A TES diagnosis neither necessitates
a possible or probable CTE diagnosis nor excludes another clinical neurodegenerative
diagnosis 5].
If the criteria for TES are met, the clinician indicates the subtype designations
and the relative likelihood of underlying CTE (probable, possible, or unlikely) based
upon additional supportive features 5]. The clinicians also record a primary clinical diagnosis and, if appropriate, contributing
clinical diagnoses. Figure 4 shows the diagnostic form that each clinician completes. After each clinician, blinded
to the other clinicians’ diagnoses, completes and submits the diagnostic form, the
group discusses the case. The discussion includes questioning of the lead clinician
about specific details with the goal of reaching a consensus diagnosis using a format
identical to that used for the previous independent voting. To reach consensus, a
majority of the clinicians present must agree on the diagnosis. Once a consensus diagnosis
is reached, panel members again complete a written diagnostic form as a means to record
dissent from the consensus.
Fig. 4. Clinical consensus diagnostic form completed by each clinician. ALS amyotrophic lateral sclerosis, CNS central nervous system, CTE chronic traumatic encephalopathy, FTD frontotemporal degeneration, TBI traumatic brain injury, TES traumatic encephalopathy syndrome
After the clinicians reach a consensus, the neuropathologist who evaluated the case
presents the pathological findings. The presentation includes the brain weight, gross
and microscopic images, and an overall summary. The presentation focuses on 1) regional
patterns of cerebral and white matter atrophy, 2) evidence of septal abnormalities,
including cavum septum pellucidum or fenestrations, 3) pallor of the substantia nigra
and locus coeruleus, 4) extent and anatomic distribution of neuronal loss and gliosis,
5) immunohistochemistry (p-tau, ?-amyloid, pTDP-43 and ?-synuclein) and 6) vascular
pathology. The summary includes pathological diagnoses with staging when appropriate.
All neuropathological diagnoses and associated reports are completed before the consensus
conference and without knowledge of the subject’s antemortem exposure history or clinical
presentation, and they are not changed on the basis of clinicians’ diagnoses or discussions.
After the clinical and pathological presentations, the clinicians and pathologists
discuss clinicopathological correlation. For each case, the physicians and neuropsychologists
identify key summary features of the case that help inform future research directions.
For cases with discrepancies between the clinical and pathological findings, the cause
of the differences and how the discrepancies contribute to diagnostic uncertainty
are discussed.
Last, the lead clinician and pathologist present both the clinical and pathological
diagnoses to the informants by telephone. The informants are also provided with a
written report that summarizes the diagnoses.
Datan analysis
The datan analysis will involve three major phases. Phase 1 will evaluate the reliability
of the consensus raters and the validity of the clinical research criteria for CTE
and the consensus process 63], 70]. For our primary analyses, we will consider a dichotomous clinical diagnosis (i.e.,
possible or probable CTE vs. no CTE). First, we will calculate the pre- and postconsensus
interrater reliability (Cohen’s ?) between clinical consensus members 63], 70], 71], along with the standard errors and 95 % confidence intervals. Next, we will calculate
the pre- and postconsensus sensitivity, specificity, and accuracy of the clinical
diagnosis using the presence of CTE pathology as a gold standard. In secondary analyses,
we will assess the reliability and validity of clinical subtypes and an alternative
operationalization of the likelihood of CTE (i.e., probable CTE vs. possible or no
CTE).
Phase 2 of the analysis involves an evaluation of sources of diagnostic disagreement
between clinical consensus members and sources of diagnostic error between the consensus
diagnosis and the pathological diagnosis. For each case with diagnostic disagreement
(either pre- or postconsensus), we will review the case and identify the issues leading
to the disagreement. We will review how these major issues differ pre- and postconsensus,
paying particular attention to those that remain or that occur postconsensus. Similarly,
for each case with a consensus diagnostic error, we will review the case and identify
the major issue leading to the error. We will calculate the frequency of each type
of diagnostic error (e.g., frequency of clinical consensus diagnosis of CTE with pathological
AD). Finally, we will review differences in diagnostic errors between false positives
and false negatives.
Phase 3 involves the analysis of individual diagnostic features (examples include
but are not limited to contact sport position, years and level of play, number and
severity of individual TBIs, memory impairment, depression, impulsivity, and motor
impairment). We will calculate the sensitivity and specificity of each feature using
pathological CTE as a gold standard. Among cases of pathological CTE, we will calculate
the frequency of each feature overall, as the presenting symptom and/or sign, as an
early symptom and/or sign, and as a late symptom and/or sign. Next, we will create
statistical models of pathological CTE risk, using these diagnostic features as predictors.
We will use conventional logistic regression as well as two machine learning algorithms:
random forests and elastic net penalized logistic regression. The machine learning
algorithms account for the often erroneous assumptions of classical logistic regression
that predictors are not correlated with each other and that their effects are additive
72]. Diagnostic features with the best sensitivity and specificity, and that best predict
CTE pathology, will be critical to include in future iterations of clinical diagnostic
criteria for CTE.