Appendix S - ALS Biorepository Protocol.pdf

Appendix S

National ALS Biorepository

Version 1 – Amendment 4

Table of Contents

Introduction

Background

ALS Epidemiology and Genetics

ALS Biomarkers

ALS Biorepositories

Best Practices for Biorepositories

Anticipating Research Uses

Pilot Study Results

Approach

In-home Biorepository Component

Selecting prospective participants

Enrolling participants

Preparing for specimen collection

Collecting specimens

After specimens are collected

Event collection Biorepository Component

Postmortem Biorepository Component

Selecting prospective participants

Enrolling participants

Preparing for specimen collection

Collecting specimens

After specimens are collected

Human Subjects Considerations

Distribution of specimens

Specimen Analysis Conducted by the National ALS Registry

Results of research

References

Tables

Table 1. Factors previously studied for association with ALS, with selected examples citing relevant biomarkers of exposure

Table 2. Frequencies of mutations in five genes associated with familial ALS in a study of 162 families

Table 3. Pathophysiologic processes with hypothesized roles in ALS, with selected examples citing approaches to measurement

Table 4. Proposed desirable minimum clinical dataset for ALS biomarker studies

Table 5. Biorepositories and brain banks with samples from people with ALS

Table 6. UK Biobank specimen collection protocol

Table 7. Relative advantages of different specimen types for biomarker research

Table 8. Proposed minimum specimen collection protocol

Figures

Figure 1. Genetic association studies of sporadic ALS, by year, 2001-2011

Appendices

Appendix A: Request for additional information

Appendix B: In-home Component

Appendix B-1. Communications B-1a. Email/letter to those expressing interest in biorepository

B-1a(1). Email/letter (full collection)

B-1a(2). Email/letter (saliva only)

B-1b. Phone script to schedule appointment, review instructions, and obtain consent

B-1c. Letter/email appointment confirmation and instructions

B-1d. Phone script to reschedule appointment

B-1e. Email/letter blood draw failure

B-1f. Phone script for failed blood draw or unusable blood

B-1g. Email/letter for saliva collection

B-1h. Phone script follow-up saliva collection

B-1i. Email/letter thank you for participating in the biorepository

Appendix B-2. Other participant materials

B-2a. Fact Sheets

B-2a(1). Fact Sheet Full Collection

B-2a(2). Fact Sheet without Hair and Nails

B-2a(3). Fact Sheet Saliva only

B-2b. Instructions for opening the specimen collection kit

B-2c. Instructions for participant urine collection

B-2d. Instructions for saliva collection

Appendix B-3. Consent forms

B-3a. In-home collection (biospecimens)

B-3b. In-home collection (saliva)

Appendix B-4. In-home specimen collection procedures

B-4a. Urine

B-4b. Blood

B-4c. Hair

B-4d. Fingernails

B-4e. Specimen Processing Form

Appendix B-5. Email/letter announcement event collections

Appendix C: Postmortem Component

Appendix C-1. Communications – Postmortem component

C-1a. Email/letter to those interested in biorepository

C-1b. Phone script to begin eligibility screening

C-1c. Phone script to provide eligibility screening update

C-1d. Phone script to inform potential participants that physician is not responding

C-1e. Phone script to notify ineligible participants

C-1f. Phone script to schedule consent appointment

C-1g. Email/letter to confirm appointment

C-1h. Phone script to obtain consent

C-1i. Email/letter to those who have not agreed to participate

C-1j. Phone script to contact those who have not agreed to participate

C-1k. Email/letter thank you for agreeing to participate

C-1l. Email/letter thank you and Donation Plan

C-1m. Phone script to get health status updates

C-1n. Email/letter thank you for family

C-1o. Email/letter thank you for family where donation failed

Appendix C-2. Other participant materials – Postmortem component

C-2a. Fact Sheet

C-2b. HIPAA Authorization form

C-2c. Eligibility Checklist

C-2d. Family Authorization form

Appendix C-3. Consent form – Postmortem component

C-3a. Consent Form Addendum

Appendix C-4. Postmortem specimen collection procedures

C-4a. Brain, spinal cord, and CSF

C-4b. Muscle and bone

C-4c. Postmortem collection of skin specimens

Appendix C-5. Processing of brain, spinal cord, and CSF specimens

Appendix C-6. Neuropathology data collection form

Introduction No single study or database can provide answers to the most pressing questions about ALS, such as:

How many people have ALS? What are the underlying causes? How can understanding these causes lead to prevention and treatment? What biomarkers are useful for predicting disease progression and treatment response? Answering such questions requires the integration of epidemiologic, clinical, and basic research findings to place individual measurements in context, suggest hypotheses, and provide a basis for inference. This biorepository will enhance the Registry’s utility for research on ALS. It will differ from existing biorepositories because it is population-based and nationally comprehensive, rather than defined by geographic area, exposure, or clinical characteristics. The National ALS Biorepository will increase the number of biological samples available for research and will make them available to ALS researchers regardless of institutional affiliation. Persons with ALS have expressed interest in providing biological specimens through the National ALS Registry.

Background

ALS Epidemiology and Genetics

ALS occurs worldwide but because of its rarity, variation and trends in ALS incidence are difficult to measure. The only consistently identified risk factors for ALS are increasing age and male sex. Although tobacco smoking has been proposed as an additional “established risk factor” (Armon 2009), findings among studies are inconsistent and the effect on risk (if any) is small (Alonso 2010). Epidemiologic studies have examined many other potential risk factors for ALS, including occupation, trauma, infections, exposure to metals and other chemicals, and electric shock (Table 1). Potential biomarkers have been proposed for some of these exposures or their pathologic sequelae; however, so far few epidemiologic studies have incorporated biomarkers.

Like other degenerative neurologic diseases, including Alzheimer’s and Parkinson’s diseases, ALS is thought to develop as a result of complex interactions among multiple genetic and environmental risk factors (Ahmed 2011; Siddique 2011). The clinical heterogeneity of ALS suggests that different underlying mechanisms and pathways could be involved.

Familial ALS. Approximately 10% of people diagnosed with ALS have an affected first-degree relative

(Fang 2009; Siddique 2011). Fang et al. conducted a retrospective cohort study of familial ALS by analyzing the Swedish Multi-Generation Register from 1961 to 2009. They found that the risk of ALS was increased about 10-fold in siblings and children of affected persons; risk was further increased if the proband was diagnosed at a younger age. Clustering of ALS within families suggests that genetic factors contribute to susceptibility.

Beginning in the 1990’s, genetic studies of families with multiple affected members implicated mutations in several genes that typically confer risk in an autosomal dominant manner, although autosomal recessive inheritance has been observed in some families (OMIM, 2012). Variations in superoxide dismutase 1 (SOD1) were first associated with familial ALS in 1993; since then, more than

130 SOD1 mutations have been implicated, accounting for approximately 10–20% of multiply affected

ALS families (Millecamps 2010).

A recent French study of 162 distinct families with ALS examined the frequencies of mutations in five previously associated genes; results are summarized in Table 2 (Millecamps 2010). A mutation in at least one of these five genes was found in only 36 (22%) of the 162 families; of the 31 distinct mutations identified, 7 (23%) had not been previously reported. The finding of largely private mutations in multiple genes attests to the heterogeneity of genetic susceptibility to ALS.

Recently, two studies implicated expansion of a GGGGCC hexanucleotide repeat in C9ORF72, a non-coding region of chromosome 9, in families of European ancestry affected by both ALS and frontotemporal dementia (FTD) (DeJesus-Hernandez 2011; Renton 2011). DeJesus-Hernandez et al.

found this repeat expansion in 22.5% of familial ALS seen in three clinics (Mayo Clinic, University of

British Columbia, and University of California—San Francisco). Renton et al. found the same expansion in nearly half of all familial ALS in Finland and in one-third of 268 familial ALS probands of European ancestry.

The ALS Online Database (ALSoD, http://alsod.iop.kcl.ac.uk), an open source data repository for sharing

ALS genotype and phenotype data, was developed by a consortium within the World Federation of

Neurology (Lill 2011). Consortium members submit their own data and extract data from scientific publications; users of the database can also upload their own data. The focus is on familial ALS, although data from non-familial cases are also accepted. As of January 2012, the database included more than

450 records for people with familial ALS; mutations in 17 genes were represented, including the five studied by Millecamps.

Discoveries of additional mutations in families affected by ALS continue to offer clues to underlying disease processes. For example, Wu et al. in 2012 used exome sequencing in families with ALS to discover associated mutations within the profilin 1 (PFN1) gene.

Sporadic ALS. New technologies and information stemming from the Human Genome Project have spurred genetic association studies of “sporadic” ALS in persons without a family history (Figure 1).

These population case-control studies are the focus of the ALSGene database (www.alsgene.org), which as of January 2012 included more than 250 polymorphisms in more than 90 genes, including some previously identified in family studies (Lill 2011).

In genetic association studies of sporadic ALS, observed odds ratios are typically small (between 1.1 and

2.0), even for variants of genes implicated in familial ALS. Such findings are typical for genetic association studies of multifactorial chronic diseases, including atherosclerosis and cancer. No genetic association with sporadic ALS has been found to have effects as large and consistent as the association of APOE genotype with Alzheimer disease (meta-analysis odds ratio [OR] 3.68, 95% CI 3.30–4.11, www.alzgene.org, January 2012) and even strong genetic associations serve only as the starting point for further investigations of pathogenesis (Storandt 2012). Since 2005, genome-wide association studies

(GWAS) have been a powerful tool for identifying new candidate genes in complex diseases. The results for GWAS of ALS have offered only a few leads and replication of the promising association with DPP6 has been inconsistent (Chio 2009).

http://alsod.iop.kcl.ac.uk/ http://www.alsgene.org/ http://www.alzgene.org/

ALS Biomarkers

Advances in molecular biology and neuroimaging technologies are providing an increasingly detailed picture of neuropathology in ALS. Biomarkers of contributory exposures, disturbed physiologic processes, and tissue damage may be useful in discovering underlying physiologic mechanisms. Several proposed mechanisms and categories of potential biomarkers are summarized in Table 3. Many of these have been identified in basic research using neural tissue obtained postmortem.

A vital goal of pathophysiologic research is to identify biomarkers that can be used clinically, to diagnose

ALS earlier and reduce diagnostic delay, predict disease progression, stratify patient populations for clinical trials, and assess response to treatment (Bowser, 2011, Otto 2012). Desirable attributes of clinical biomarkers include their presence in accessible specimens (e.g., blood, urine); detectability early in disease; high sensitivity and specificity; and high predictive value for relevant clinical outcomes. The successful development of such biomarkers requires integrating findings from epidemiologic, clinical, and basic science research.

A recently published article, “Roadmap and standard operating procedures for biobanking and discovery of neurochemical markers in ALS,” followed from a 2010 workshop sponsored by the World Federation of Neurology (Otto 2012). This report systematically describes key considerations in the design and conduct of research on ALS biomarkers and the need for international collaboration and standardization. The authors point out that “biomarker studies are of limited or no use without reliable clinical characterization of patients and propose a “desirable minimum clinical dataset,” which is included as Table 4 of this protocol.

Bowser et al. (2011) recently reviewed candidate protein-based, neurophysiological, and neuroimaging biomarkers in ALS. Although all three types of biomarkers are potentially measurable in clinical settings, only protein-based biomarkers will be available from samples collected for the National ALS

Biorepository. Candidate protein-based biomarkers include various molecules with a role in innate immunity (e.g., interleukins, chemokines), complement factors, and markers of neural tissue damage.

Quantitative methods for measuring candidate protein biomarkers include antibody-based methods

(quantitative ELISA, multiplex antibody capture) and various combinations of protein purification procedures (gel electrophoresis, liquid chromatography) with mass spectrometry.

The discovery and validation of biomarkers is an area of ongoing, active research. As of January 2012, the NIH clinical trials database (http://clinicaltrials.gov/) included at least 18 studies of ALS that included collection of biomarkers, of which four were actively recruiting. For example, enrollment began in

November 2011 for a new longitudinal study of ALS biomarkers (NCT01495390) at Massachusetts

General Hospital.

ALS Biorepositories

Understanding the complex pathobiology of ALS, which appears to involve multiple complex, interdependent processes over time, requires the capacity to detect factors with small effects on overall risk. Thus, there is a strong incentive for ALS clinical researchers to collaborate in establishing http://clinicaltrials.gov/ standardized diagnostic criteria (such as the El Escorial criteria, first published in 1994) and frameworks for sharing clinical data and biological research specimens.

Many ALS clinical registries have been established at academic and other research centers; typically, these registries consist of data and specimens collected from patients at the time of diagnosis and treatment or during the course of clinical trials. The data and specimens may be shared in ad hoc research collaborations. ARISLA, an Italian foundation for research on ALS, maintains a list of European and U.S. biobanks and repositories (with brief descriptions and links) at http://www.alscience.it/?view=52#v52.

Tissue banks containing postmortem specimens of brain, spinal cord, and muscle tissue are also important resources for ALS research. The International Brain Banking Network (IBBN)

(http://www.intbbn.org/) facilitates communication among brain banks and conducts annual workshops in association with the American Association of Neuropathologists. The IBBN website includes a directory of brain banks in the U.S. and other countries (http://www.intbbn.org/registry-of-brain-banks.aspx); most of these banks are at universities and many have an Alzheimer research focus.

Several independent organizations offer specimen retrieval services that will query registries and tissue banks for specimens with specific characteristics and request them for use by outside researchers. For example, the National Disease Research Interchange (http://www.ndriresource.org) is a not-for-profit corporation funded mostly by NIH that locates biological research materials and maintains a specimen bank for the NIH Office of Rare Diseases Research (ORDR, http://biospecimens.ordr.info.nih.gov/).

SpecimenCentral.com (http://specimencentral.com/) is a commercial broker, connecting researchers with biobanks holding the types of biological samples they are seeking.

Prospective collaborations in the US and elsewhere have developed clinical research resources for ALS, including biospecimen collections. The principal examples are summarized briefly below and in Table 5.

New England ALS (NEALS) Consortium. The NEALS Consortium is a network for ALS clinical research that was formed in 1994 and has grown to more than 90 members. NEALS provides infrastructure for multi-center clinical trials and other clinical research studies. The Massachusetts General Hospital (MGH) serves as the Coordinating Center for many of these studies and houses an associated biofluid repository containing a variety of blood, urine, plasma, cerebrospinal fluid (CSF), and extracted DNA samples. Biorepository samples are linked to clinical data and stored under controlled conditions, providing potentially larger sample sizes for research than are available from single studies. The NEALS

Sample Sharing Committee oversees sample sharing policies and reviews data and sample requests. A scalable “virtual biobank” platform has been developed to allow investigators in ALS clinical research centers to share and search for samples while retaining control of their own specimens and data

(Sherman 2011).

National Institute of Neurologic Diseases and Stroke (NINDS), NIH. In 2004, NINDS added ALS to the conditions included in a DNA bank and cell line repository established at the Coriell Institute to support large-scale research on the genetics of neurologic diseases. This effort was a public-private collaboration http://www.alscience.it/?view=52#v52 http://www.intbbn.org/ http://www.intbbn.org/registry-of-brain-banks.aspx http://www.intbbn.org/registry-of-brain-banks.aspx http://www.ndriresource.org/ http://biospecimens.ordr.info.nih.gov/ http://specimencentral.com/ of NINDS with the ALS Association (ALSA), the Muscular Dystrophy Association (MDA), and academic researchers from 62 centers across the United States, including many members of the ALS Research

Group (http://www.alsrg.org) (Gwinn 2007). The repository is a national research resource of biological samples linked to individual phenotypic datasets. For all included subjects, NINDS Clinical Data Elements

(CDE) must be complete; these include demographic, clinical, and medical and family history data

(Gwinn 2007, Appendix S1B). With the exception of smoking (optional), data on potential environmental exposures are not collected. The repository includes specimens from approximately 2000 people with

ALS, including blood, immortalized lymphocytes, and fibroblasts. Some samples from unaffected and affected blood relatives of subjects, spouses, and healthy individuals (including population and convenience controls) are also included. The repository is not actively adding specimens, although researchers may apply to submit their own collections. More information is available at http://ccr.coriell.org/Sections/Collections/NINDS/Motor.aspx?PgId=192&coll=ND.

Medical Research Council (MRC) London Brain Bank for Neurodegenerative Diseases. The MRC

London Brain Bank was established in 1989 in the Department of Neuropathology, Institute of

Psychiatry, King’s College London, UK, to make clinically and pathologically well-characterized brain and spinal cord tissue available to researchers worldwide

(http://www.iop.kcl.ac.uk/departments/?locator=380). The MRC London Brain Bank focuses on neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, and motor neuron disease (including ALS). Currently, specimens are available from 189 persons with motor neuron disease and the bank maintains an ongoing program to “facilitate brain donation through an ethically approved program of informed consent for cohort studies and ad-hoc donations.”

Several epidemiologic studies of ALS have used population-based registries to identify potential participants, who are contacted to request biological specimens (Chio 2009). Very few population-based

ALS registries have been designed to collect specimens prospectively. In the US, the only large population-based ALS registry with an associated biorepository is the National Registry of Veterans with

ALS. This biorepository includes blood (or buccal cell) specimens collected for DNA analysis, as well as a brain bank; because it offers the most relevant model for development of the National ALS

Biorepository, it is described in additional detail here.

National Registry of Veterans with ALS. The registry was established by the Veterans Administration

(VA) after increased incidence of ALS was reported in veterans deployed to the Persian Gulf in 1990–91

(Kasarskis 2004). The objectives of the registry were to identify living US military veterans with ALS, follow their health status, collect data for epidemiologic research, and inform veterans about clinical trials for which they could be eligible (Allen 2008). Veterans with ALS were identified from VA medical databases and via nationwide solicitation; a brief telephone interview screened potential participants for eligibility. During the enrollment period (April 2003–September 2007) more than 2,000 participants aged 23–93 years were enrolled, spanning combat eras from World War II to the 1990–91Gulf War.

Registrants were asked to provide contact information for all providers of healthcare since their onset of

ALS symptoms. Their medical records were retrieved and abstracted by trained personnel, who recorded results of physical and electromyographic examinations, pulmonary function tests, and http://www.alsrg.org/ http://ccr.coriell.org/Sections/Collections/NINDS/Motor.aspx?PgId=192&coll=ND http://www.iop.kcl.ac.uk/departments/?locator=380 laboratory and imaging studies, as well as medical history (including surgery and physical trauma) and family history of ALS. An integral component of the registry is the DNA bank, which is a collaboration of the ALS Registry (Epidemiologic Research and Information Center, Durham VAMC), the VA DNA

Coordinating Center (Palo Alto VAMC), and the Genetic Tissue Core Laboratory at the Massachusetts

Veterans Epidemiology Research and Information Center (Boston VAMC). All registry participants were asked to contribute a specimen, although it was not required. DNA specimens were collected in participants’ homes by trained nurses associated with a nationwide home health agency. Nurses were supplied with blood collection kits, as well as mouthwash for use in collecting buccal cells when no blood specimen could be obtained. Of the more than 2,000 veterans enrolled in the registry, 76% provided written consent for participation in the DNA bank; 8% refused and the remainder were excluded for other reasons. More than 1,000 specimens were obtained, of which 85% were blood and 15% were buccal cells only.

A brain bank component was added to the registry in 2006. The VAB Brain Bank is coordinated at the

Massachusetts Veterans Epidemiology Research and Information Center (MAVERIC) at VA Boston

Healthcare System (VABHS). Tissue is analyzed, processed, and stored at the Southern Arizona Core

Tissue Laboratory (SACTL) at the Southern Arizona VA Healthcare System (SAVAHCS) in Tucson, AZ.

Although enrollment in the registry ended in 2007, regular telephone follow-up of enrolled Veterans has been continued by the VAB. This follow-up includes semi-annual administration of the ALS Functional

Rating Scale. Postmortem collection of brain tissue continues for previously enrolled participants.

Additionally, the VAB continues to enroll Veterans with ALS via referrals from the Neurology Service at

VA Boston, and other VAs. A Scientific Review Committee provides expert review for all requests to use registry data and samples. Multiple studies have already been conducted on a range of topics, including genetics, environmental exposures, disease progression, psychosocial issues, and a brain-computer interface for patients (Allen 2008). The National Registry of Veterans with ALS is also the source of cases for the GENEVA Study, a case-control study of genes, environmental exposures, and gene-environment interactions in ALS (Schmidt 2008; Schmidt 2010). The VAB has begun to release tissue and associated data to investigators.

Best Practices for Biorepositories

International Society for Biological and Environmental Repositories (ISBER). ISBER publishes best practices for the collection, storage, retrieval and distribution of biological materials for research. The second and most recent edition was published in 2012 and is available online at https://c.ymcdn.com/sites/www.isber.org/resource/resmgr/Files/ISBER_Best_Practices_3rd_Edi.pdf

(ISBER 2012). Guidelines in this document address all aspects of biorepository management, including administration and records management, facilities and equipment, safety, quality assurance, specimen tracking and management, and ethical and legal issues, as well as best practices for human biospecimen collection, transport, and storage. A key point notes the value of pilot or feasibility studies for identifying problems in specimen collection and handling before beginning a larger study.

National Cancer Institute (NCI). NCI has invested in extensive biorepository programs to support basic, epidemiologic, and clinical research on cancer. Recognizing the increased value and changing uses of biospecimens in clinical research, NCI commissioned the National Biospecimen Network Blueprint https://c.ymcdn.com/sites/www.isber.org/resource/resmgr/Files/ISBER_Best_Practices_3rd_Edi.pdf

(2003), which recommends ways to enhance the collection of biospecimens and the management of biorepositories. The NCI Clinical Trials Cooperative Group Program, which includes more than 3,000 institutions and more than 14,000 investigators, also maintains the network of Cooperative Group Banks

(http://cgb.cancer.gov/). Banked biospecimens include formalin-fixed, paraffin-embedded tumor and normal tissue, fresh frozen tumor and normal tissue, blood, serum, plasma, urine, genomic DNA, and bone marrow. A directory of participating Cooperative Group Banks is available online at http://cgb.cancer.gov/contacts/index.html.

In 2006, NCI’s Office of Biorepositories and Biospecimen Research (OBBR) established the Biospecimen

Research Network to assess the effects of pre-analytical factors (e.g., specimen types, collection methods, transport, processing, and storage) on the outcomes of genomic and proteomic studies in cancer research. OBBR has issued Best Practices for Biospecimen Resources that address scientific and technical considerations, as well as ethical and legal issues. Last updated in 2011, these Best Practices are available online at http://biospecimens.cancer.gov/bestpractices.

Public Population Project in Genomics (P3G). P3G is an international non-profit organization that promotes collaboration among researchers in population-based genomic research. The P3G Observatory

(http://www.p3gobservatory.org/) is an online repository of information and tools for harmonization among individual biobanks. The Observatory includes a directory of relevant guidelines and best practices at http://www.p3gobservatory.org/repository/sampleCollection.htm. In addition to the ISBER and NCI guidelines already mentioned, these include Best Practice Guidelines for Biological Resource

Centers published by the Organization for Economic Co-operation and Development (OECD) and other national and international research organizations.

Anticipating Research Uses

Anticipating future uses of biorepository samples is challenging, especially because technology continues to expand the realm of possible analyses. Although versatile specimens—such as whole blood, serum, and plasma—are likely to be most useful in the long run, collecting specimens for future use always requires trade-offs: for example, between collecting a larger variety and volume of specimens

vs. cost of collection and storage, or between asking participants to consent to unspecified future analyses vs. their willingness to participate.

Choosing the types and quantities of specimens to be collected for a biorepository depends on multiple considerations. A recently published biobanking “roadmap” for ALS biomarker research compared the advantages and disadvantages of several types of specimens, which are summarized in Table 7 (Otto

2012). In addition to blood and cerebrospinal fluid (CSF), which have been studied most often, the roadmap considered urine, saliva, skin, and muscle tissue. Urine and saliva offer practical advantages relative to blood: collection procedures are less invasive and analysis is simpler (although also more limited) because of their less complex molecular composition. Urine has potential added value as a specimen for measuring levels of drugs and other environmental chemicals and their metabolites. Saliva provides an alternative source of DNA when blood collection is not possible. CSF, skin, and muscle tissues can only be collected in a clinical setting. Nail and hair clippings can provide information about http://cgb.cancer.gov/ http://cgb.cancer.gov/contacts/index.html http://biospecimens.cancer.gov/bestpractices past environmental exposures (such as those in Table 1), mostly limited to near- and intermediate-term exposures (Goullé 2009).

Whole blood remains the preferred specimen type for biobanks intended to serve as long-term research resources. Its advantages include:

DNA yield (quality and quantity);

versatility (e.g., for analysis of RNA and other biomolecules, as well as markers of environmental exposures);

straightforward creation of aliquots for sample sharing;

long-term stability in storage; and potential for creating cell lines.

Whole blood specimens must be collected with the intended analyses in mind to prevent interference by anticoagulants. For example, collecting blood in anticoagulant-containing tubes causes the release of cytokines, resulting in spuriously elevated concentrations. Whole blood specimens collected on filter paper (Guthrie cards) share many of these advantages with conventional blood specimens; however, their small volume limits the numbers and types of possible analyses and they can’t be used to establish cell lines.

Pilot Study Results

Through a contract ATSDR conducted a study to pilot methods for collecting and banking biological specimens from participants in ATSDR’s National ALS Registry. The Pilot Study included two specimen collection components: biological specimens from living participants (in-home) and postmortem specimens. The in-home component enrolled 330 participants, from whom specimens were collected on two occasions, approximately six months apart. The postmortem component enrolled 30 participants, who could also participate in the in-home study. In-home collection included blood, urine, hair, and nails. The postmortem collection included the collection of brain, spinal cord, CSF, muscle, bone, and skin specimens for the creation of cell lines.

Persons participating in the in-home collection were asked to provide consent over the phone. Those participating in the postmortem collection were visited in their homes and asked to provide consent in person. When skin specimens were added to the tissue types collected postmortem, a consent addendum was created and approved by the IRB. Participants who provided consent to participate before the addition of skin specimens were contacted about participating in this additional specimen type and asked to provide consent over the phone. For all parts of the consent process that took place over the phone, the participant returned a signed copy of the consent form to McKing before any study procedures took place. Participants were provided a copy of the signed consent form for their records.

Three hundred and thirty-nine individuals provided consent to take part in the Pilot Study, 202 (59.5%) male and 137 (40.5%) female. Of the 339 biospecimen participants, 221 (64.8%) were recruited and 118

(35.2%) were volunteers. Nine of the 339 participants were unable to schedule an appointment, resulting in 330 participants completing at least one specimen collection. Thirty individuals provided consent to take part in the postmortem part of the study. Of the 30 postmortem participants, 5 (16.7) were recruited and 25 (83.3) were volunteers. All 30 postmortem participants provided consent for the overall donation of brain, spinal cord, bone, muscle, and CSF. In addition, 27 of the 30 the postmortem participants provided consent for skin sample collection. Three participants did not provide consent for skin sample collection (one died before skin sample collection was added to the protocol, one refused, and one died before the signed skin consent form was received).

There were 330 participants who completed first specimen collections. Three hundred and eleven participants provided at least one vial of blood and 19 were unable to provide any blood for the first specimen collection.

There were 272 participants that completed second specimen collections. Two hundred fifty-five provided at least one vial of blood and 17 were unable to provide any blood during the second specimen collection. Fifty–eight of the participants who had a first specimen collection did not have a second specimen collection. The reasons for not completing the second draw included death (36), too ill or unable to contact (9), and no longer interested or scheduling difficulties.

Three hundred and twenty-one participants provided at least one blood specimen and 15 provided a saliva sample. Nine participants were unable to provide any blood; however, eight provided hair samples, nine provided nail samples, nine provided a urine sample, and four provided a saliva sample.

Most (78%) participants were able to provide specimens at both collection appointments.

Eighteen postmortem participants have donated specimens as of November 12, 2015. The length of time in the study for these participants from date of consent to date of death ranged from 1-24 months and the length of time in the study for participants that are still living, ranged from 20-27 months. The age at death for the deceased participants ranged from 43-76 years of age. Two participants are deceased but withdrew from the postmortem part of the study and did not provide postmortem donations. However, these participants did take part in the biospecimen part of the study.

Creating a geographically-diverse biorepository had unique challenges. Recruitment was slower than expected, finding reliable phlebotomists across the country was difficult, and there were unexpected issues related to shipping specimens including higher than average temperatures and mechanical failure. In addition, after the first specimens were collected there was a larger than expected number of individuals that were too sick or deceased and could not participate in the second specimen collection, decreasing the number of paired specimens. However, we were able to recruit the target sample size and process the varied specimen types. In March 2015, the Expert Panel extensively discussed the results of the pilot study and endorsed adding specimen collection as a component of the National ALS

Registry. The Expert panel felt that the specific specimens collected should remain flexible so that there could be changes as research priorities and technology changed. In addition, they felt that because of the logistics and costs specimens should be collected at only one point in time. The discussion on the types of samples that should be collected as well as some changes to the logistics have been incorporated into the current proposal.

Approach

We will collect specimens from ALS patients enrolled in the National ALS Registry. The specimens to be collected will be determined on a yearly basis depending on the scientific demand. Currently, McKing is accessing demand by reviewing the literature and talking with key ALS researchers. In subsequent years, we will evaluate the actual researcher requests. For example, although hair and nails are easy to collect there is still a cost to the laboratory for inventory control and storage. Therefore, we may not collect any additional hair or nail samples until the ones from the pilot study have been used up.

Specimen types that could be included for in-home collection are blood, urine, hair, and nails. The amount of blood collected will not exceed 50 ml. Types of blood specimens could include blood for serum and DNA, blood for plasma, metals free collection of blood, blood for RNA extraction, and blood to create cell lines. For the postmortem collection, we will collect the brain, spinal cord, CSF, bone, muscle, and skin.

Population. Eligible participants for either component must have enrolled in the congressionally mandated National ALS Registry. After an individual has joined the Registry he/she will have the opportunity to ask for additional information about the biorepository and provide needed contact information (Appendix A). Additional specific eligibility requirements are outlined for each component below.

In-home component. Specimens will primarily be collected in participants’ homes by trained phlebotomists. This was the process used by the biorepository pilot study and VA ALS Registry specimen collection. This approach is more convenient for participants and less likely to discourage those whose mobility is severely restricted. Minimizing limitations on when or where specimen collection can take place improves participation.

It is possible that ATSDR will want to collect blood specimens that need immediate processing and special handling. For example, some analytic techniques require plasma samples that have been centrifuged within 15 minutes of collection and then frozen. If this is the case, we will arrange for some participants to come to central locations for these specialized collections.

The phlebotomist will use a commercially available kit to collect the specimens and will be responsible for sending collected specimens to the laboratory according to the shipping protocol. Practical issues preclude the collection of specimen types that require a medical procedure or immediate processing

(e.g., skin or muscle biopsy, lumbar puncture for CSF). Table 8 indicates minimum specimen collection requirements, as well as potential additional specimens that could be self-collected and shipped by the participant or caregiver.

In-home saliva only component. DNA is one of the most used specimen types as more and more genes associated with ALS are identified. In order to increase the number and diversity of DNA specimens available for analysis, we will collect only saliva from an additional group of ALS patients enrolled in the Registry. We will sent a salvia collection kit to the participant’s home with instructions for collection along with a pre-paid shipping label to return it to the lab.

Postmortem component. Research on the pathobiology of ALS has naturally emphasized analysis of brain, spinal cord, and CSF, the tissues most proximal to the disease process. The Boston University

Alzheimer's Disease Center Brain Bank has developed procedures based on ISBER best practices (ISBER

2012) to recover these tissues postmortem. Their procedures will be used for the postmortem component of the biorepository with additions to include collection of muscle and bone tissue specimens.

Sample size. The number of participants will vary from year to year depending on funding. The

National ALS Biorepository would like to collect a full set of biological specimens from 250 – 325 participants per year. Based on the blood failure rate in the pilot study, we anticipate that about 10% of the participants will be unable to give blood and will be offered the opportunity to provide a saliva sample. In addition, we will collect saliva only on 350 participants per year. Those who provide saliva only will be a combination of those who were unable to provide a blood specimen and those selected for saliva only collection. Therefore in a year with 325 participants in the full biological specimen component and 350 saliva only collections, we are expecting 675 participants. We would like to obtain postmortem tissue from 40 participants, i.e., approximately 10 per year.

Training staff. All biorepository staff will be trained on biorepository procedures and data security by the Senior Scientist. All biorepository staff will be required to provide documentation of training in

HIPAA or take a certified course in HIPAA regulations that pertains to research. All biorepository staff will also take approved Human Subjects Protection training.

Outreach. Staff from the National ALS Biorepository will attend ALS related events (e.g., ALS Walks and

ALS Association meetings) to answer questions about the project and handout IRB approved materials such as the factsheet and introductory letter. These activities will increase the biorepository visibility and better establish the connection with the National ALS Registry.

In-home Biorepository Component

Selecting prospective participants

Eligibility. To be eligible to donate specimens, prospective participants must have enrolled in the

National ALS Registry and requested additional information about the biorepository

Identifying prospective participants. To achieve the target sample size of 675 participants per year, we estimate that it will be necessary to contact approximately 325-350 persons enrolled in the National ALS

Registry for the full collection and 350-400 for saliva only collection. After providing consent to take part in the Registry, a potential participant will be asked if he/she would like to more information about the Biorepository (Appendix A). On a monthly basis, ATSDR will provide a list of those in the National

ALS Registry who have expressed an interest in the biorepository. This list will include the date of birth, sex, current address, telephone number, and date of diagnosis for each registrant. Each month individuals will be selected to receive an invitation to participate in the biorepository based on geographic area. If a person with ALS calls and wants to provide specimens for the biorepository, he/she will be provided information on how to join the Registry and indicate interest in the biorepository.

Selection full collection and saliva. The number of participants to be recruited each month will be determined by the number for the entire year. Each month ATSDR will provide McKing with a listing of

Registry participants interested in the biorepository. To better address the congressional mandate to examine genetic and environmental risk factors that may cluster by geographic area, we will select a convenience sample from those who are interested in the biorepository proportional to state population and with at least one person from each state. We will recruit from the harder to fill states, e.g., Wyoming, Rhode Island, first and then distribute the cases throughout the other states. Because recruitment tends to get individuals from the same town to enroll during the same time period, selection in states where we are recruiting multiple participants will be distributed across the states in any given month. Based on our experience with the pilot study, this will give a good distribution of those living in urban and rural areas in addition to good state representation. At the time of selection, potential participants will be assigned to full collection or saliva only and receive the appropriate recruitment materials and consent form.

Demographic composition of biorepository population. A spreadsheet will be developed to track recruitment which includes age, sex, city, and state. We will attempt to assure the participants are diverse as possible within those participating in the National ALS Registry. After each round of participant selection, biorepository staff will examine the distribution of enrolled participants by geographic area, age, and sex, so that subsequent recruitment can target enrollment of the underrepresented groups. We will determine the number of individuals that should be recruited per state based on state population.

Enrolling participants

Recruitment. A sample of persons indicating an interest in the biorepository (full collection or saliva only) will be mailed a packet of information that includes an introductory letter (Appendix B-1a(1) or B-

1a(2)), factsheet (Appendix B-2a(1) or B-2a(2)), and consent form (Appendix B-3a or B-3b).

Approximately one week (5-10 days) after the packet is mailed, McKing biorepository staff will contact the potential participant to determine if he/she has received the packet, answer any questions, and if interested in participating, go over the consent form. The consent form will be signed, by the participant or witness, at the time of the phone call and returned in a self-addressed stamped envelope.

The witness is just attesting that the consent form was explained to the participant and he/she agreed.

Only the participant can provide consent. If a participant decides after reviewing the consent form that he/she does not want to participate, the individual will be removed from future contact lists for the biorepository. We will attempt to reach the person by phone up to 3 times.

Scheduling specimen collection. McKing biorepository staff will answer all questions about the biorepository and maintain all direct contact with participants except for collecting the specimens. Each prospective participant who verbally confirms his/her willingness to participate will be offered a range of dates and times to schedule a visit by a phlebotomist to collect the specimens (Appendix B-1b). We anticipate that most participants will choose to have specimens collected at their place of residence;

however, acceptable alternative locations will be found for those who prefer for specimen collection to occur elsewhere. Biorepository staff will assign a unique identification number (Biorepository ID) to each participant at the time his/her first appointment for specimen collection is scheduled.

Within one week after scheduling an appointment for specimen collection, biorepository staff will send the participant a confirmation letter with the appointment location, date, and time (Appendix B-1c). The letter will include and instructions for how to open the specimen collection kit to remove the urine collection portion (Appendix B-2b). If the participant is only providing a saliva sample, a kit will be mailed within one week of receiving the signed consent form.

No appointment will be scheduled for the saliva only collection.

Preparing for specimen collection

Arranging the home visit. McKing has hired phlebotomy companies to provide phlebotomy services.

They have the capability to provide nationwide specimen collection by hiring experienced local, licensed phlebotomists to collect specimens from participants who live in various regions of the U.S. McKing will provide information packets about the biorepository and collection requirements that will be provided to the contract phlebotomist. The phlebotomist will be provided via a secure method the name of the participant, date, time, address, phone number and location for specimen collection, at least one day prior to the scheduled in-home visit. The specimen collection kit, which includes detailed instructions for collecting the specimens, will be sent to the participant’s home.

The phlebotomist will contact the participant via telephone introduce herself and confirm the appointment for the next day. If there are any changes at this time, the phlebotomist contacts McKing to notify them of any changes. If the participant states on this call that he/she has decided against participating in the biorepository, the appointment will be cancelled and the participant will be removed from the biorepository contact list. Otherwise, the participant will be reminded about the procedures that will take place and given basic instructions related to eating and drinking before the specimen collection.

If the participant does not feel well enough on the day of specimen collection but still wants to participate in the biorepository, biorepository staff will reschedule the appointment (Appendix B-1d).

The participant may call the McKing biorepository coordinator or notify the phlebotomist upon arrival at the participant’s home. Appointments will be rescheduled up to two times.

Specimen collection kits. Kits will be created that contain the necessary instructions and equipment for collecting each type of specimen (blood, urine, hair, nail clippings, saliva), as well as appropriate shipping containers with instructions for transporting specimens to the laboratory for processing.

McKing will distribute and track kits mailed to the participants.

All materials shipped to and received back from the phlebotomist will be tagged with a barcode. The barcode will contain information about the type of sample and the Biorepository ID for each participant, which does not contain personally identifying information. A list of the barcodes and a data collection form will be included with each specimen shipment.

Collecting specimens

Detailed information on specimen collection and processing can be found in Appendix B-4.

Urine. Specimen collection will be a random or “spot” urine, because a 24-hour urine or timed urine is not feasible. Participants will receive instructions on how to open the specimen collection kit and remove the urine collection container (Appendix B-2b) before the visit from the phlebotomist with instructions on how to obtain the specimen (Appendix B-2c). Participants will be encouraged to collect the specimen in the morning prior to the appointment to obtain the first morning void.

Blood. Blood will be collected using a 21-gauge x ¾” multi-sample butterfly needle blood collection set, with 12” tubing…