Canadian Healthy Infant Longitudinal Development (CHILD) …

Canadian Healthy Infant Longitudinal Development (CHILD) Study

CHILD STUDY PROTOCOL: Version Date: January 5, 2018

Canadian Healthy Infant Longitudinal Development Study (CHILD)

Page 2 CHILD Study Protocol Version Date: January 5, 2018

Protocol Objective: The purpose of the CHILD Study Protocol is to describe specific procedures performed during the conduct of the CHILD research project involving human subjects. Distribution of the Protocol will be to the Investigators and Institutions named in the CHILD study. Permission to distribute to other parties requires written permission from the Principal Investigator(s). Amendments: Any suggested changes to the protocol, questionnaires and standard operating procedures (SOPs) will be submitted in writing to the Director of the Study, Dr. Malcolm R. Sears or to Diana Lefebvre, Study Manager. Content change requests will be reviewed by Dr. Malcolm Sears and his executive team and any resulting amendments will be recorded below. Log of Protocol Versions and Amendments Version Date to Update Description of Update Current Version Date

Original Original January 8, 2011

Jan 8, 2011

Update contact information. Added section on collaborators, updated organizational structure, questionnaires and timelines. Removed Methacholine at 5 years. Replaced with spirometry. Revised mother’s blood collection at 1 year. Updated sample collection and visit task summary.

May 8, 2013

May 8, 2013

Updated contact information, revised 5 year child blood collection volumes, added 3 year spirometry (Toronto), updated list of SOPs.

December 1, 2014

December 1, 2014

Revised wording for planned genetic analyses to include whole genome sequencing and “Next Generation sequencing technologies”.

January 5, 2018

Canadian Healthy Infant Longitudinal Development Study (CHILD)


Study Synopsis This national longitudinal birth cohort study proposed to recruit up to 5000 pregnant women from several study centers across Canada. Children born will be assessed to age five years. The primary purpose of the study is to examine gene-environment factors in relation to children’s health and development, specifically in relation to allergy and asthma. The study was developed in response to the RFA from CIHR in 2005, entitled “Indoor Air Exposures, Genes, and Gene-Environment Interactions in the Etiology of Asthma and Allergy in Early Childhood”.

National Director

Dr. Malcolm R. Sears, McMaster University Firestone Institute for Respiratory Health St. Joseph’s Healthcare, Juravinski Tower T3209 50 Charlton Ave. East Hamilton, Ontario L8N 4A6 [email protected] Tel: 905-522-1155 ex. 33286 Fax: 905-521-6132

Site Investigators Toronto Dr. Padmaja Subbarao

The Hospital for Sick Children Division of Respiratory Medicine 555 University Avenue Toronto, Ontario M5G 1X8 [email protected] Tel: 416-813-6247

Winnipeg Dr. Allan Becker University of Manitoba AE101-840 Sherbrook Street Winnipeg, MB R3A 1S1 [email protected] Tel: 204-787-2455

Edmonton Dr. Piush Mandhane University of Alberta 2034 Research Transition Facility 8308-114 St. Edmonton, AB, T6G 2V2 [email protected] Tel: 780-407-2753

Vancouver Dr. Stuart Turvey Division of Infectious & Immunological Diseases BC Children’s Hospital Child and Family Research Institute Rm. 371 950 West 28th Avenue Vancouver, BC, V5Z 4H4 [email protected] Tel: 604-875-2345

mailto:[email protected]







TABLE OF CONTENTS SECTION 1: Summary and Investigators 5

Executive Summary 5 Scientific Investigators 6 National Coordinating Centre Contacts 8 Senior Coordinator Contacts 9

SECTION 2: CHILD Study Overview 10 Primary Objective, Study Design, Investigators, Partners, Collaborators 10 Primary Outcome, Secondary Outcomes 12

Inclusion and Exclusion Criteria 12 Timeline 13 Principles and Practices 13

Study Assessments 14

SECTION 3: Organization, Committees and Scientific Working Groups 20

Organizational Chart 20 Committees 21 Scientific Working Groups 22

SECTION 4: Procedures 25 Procedures 25 Schedule of Visits 26 Questionnaires 27 Standard Operating Procedures 28 Sample Summary of Aliquots 29 Collection of Samples 30 Data Management 32 Sample Shipping 32



SECTION 1: SUMMARY AND INVESTIGATORS: Study Executive Summary In December 2007, following international peer review, CIHR awarded $6M over 6 years to a consortium of investigators led by Professor Malcolm Sears, McMaster University. This Canadian birth cohort study, launched in 2008, focuses primarily on the environment and gene-environment interactions, underlying the development of allergies and asthma in childhood. CIHR funding was matched by $6M from the Allergy, Genes and Environment (AllerGen) Network of Centres of Excellence. CHILD is a multidisciplinary, longitudinal, population-based birth-cohort study. This study is powered to test multiple hypotheses within the theme that specific environmental exposures together with genetic and immunological determinants, lead to pathophysiological allergic responses, and that clinical outcomes including asthma may be further modified by lung growth, hormonal and metabolic influences and psychosocial environment. The cohort will provide a platform for many current and future studies of childhood allergy and asthma. Initial plans were for 5000 women to be recruited during pregnancy in 4 centres. Recruitment started for the Vanguard Study August 2008, and June 2009 for the Main CHILD Study cohort. Recruitment ended April 2012, with a total of 3629 subjects recruited. Infants will be studied at birth, at a home visit at 3 months, and at clinic visits at ages 1, 3 and 5 years (with questionnaires at 6 months, 1½, 2, 2½ and 4 years). Participants in Toronto will also have two additional visits for infant pulmonary function testing at 3 and 18 months. Indoor and outdoor environmental exposures (questionnaires, home inspections, dust sampling) will be assessed, along with immunological studies, genetic and epigenetic testing of DNA from child and parents, and longitudinal measures of pulmonary function, airway inflammation, nutrition and infections, focusing particularly on the critical window of the first year of life. Environmental assessments will include sampling indoor allergens, endotoxin, beta (1,3)-D-glucans, phthalates, hopanes, and tobacco smoke. Outdoor exposure assessment will involve models employing land use regression techniques and new methodologies supported by Geographic Information Systems. Environmental and biologic material will be assayed both in real time and stored for later analyses using family, cohort and nested case-control designs. The study is powered on the primary outcome of diagnosed asthma at 5 years, but intermediate outcomes of food allergy, allergic eczema, atopy and recurrent wheeze will be assessed throughout. For practical reasons, pulmonary function and airway inflammation in infancy and at 3 years of age will be assessed in a subset of children participating at the Toronto centre, but in all children at age 5 years. Cellular and molecular readouts of acquired and innate immunity will be assessed in a subset of infants at Toronto and Vancouver only and consist of approximately 1000 infants. Psychosocial factors will be assessed in all children, with detailed evaluation of stress and its relation to immune function in a subset of children participating in Vancouver. Family-based analyses will be used to validate previously identified asthma- or allergy-related genes, and support hypothesis generating studies. Inflammatory cell gene expression will be related to gene sequence variation, environmental exposures, epigenetic phenomena and outcome variables. Storage of biological material will allow testing of multiple hypotheses, and promote the formulation and testing of novel hypotheses not yet conceived. The study will not only provide unique Canadian data essential to determining environmental effects on childhood allergy and asthma, but will also enable examination of important parental health research questions.



SCIENTIFIC INVESTIGATORS

Name Affiliation City

Allen, Ryan Simon Fraser University Vancouver

Anand, Sonia McMaster University Hamilton

Becker, Allan University of Manitoba Winnipeg

Befus, Dean University of Alberta Edmonton

Brauer, Michael University of British Columbia Vancouver

Brook, Jeff Environment Canada Toronto

Cyr, Michael McMaster University Hamilton

Chen, Edith Northwestern University Chicago

Daley, Denise James Hogg iCAPTURE Centre Vancouver

Dell, Sharon Hospital for Sick Children Toronto

Denburg, Judah McMaster University Hamilton

Elliott, Susan University of Waterloo Waterloo

Grasemann, Hartmut Hospital for Sick Children Toronto

HayGlass, Kent University of Manitoba Winnipeg

Hegele, Richard University of Toronto Toronto

Holness, Linn St. Michael’s Hospital Toronto

Kobor, Michael University of British Columbia Vancouver

Kollmann, Tobias University of British Columbia Vancouver

Kozyrskyj, Anita University of Alberta Edmonton

Laprise, Catherine Chicoutimi University Hospital Quebec



Larché, Mark McMaster University Hamilton

Lou, Wen-Yi Wendy University of Toronto Toronto

Macri, Joseph McMaster University Hamilton

Mandhane, Piush University of Alberta Edmonton

Miller, Gregory Northwestern University Chicago

Moqbel, Redwan University of Manitoba Winnipeg

Moraes, Theo Hospital for Sick Children Toronto

Paré, Peter University of British Columbia Vancouver

Ramsey, Clare University of Manitoba Winnipeg

Ratjen, Felix Hospital for Sick Children Toronto

Ritchie, Bruce University of Edmonton Edmonton

Sandford Andrew James Hogg iCAPTURE Centre Vancouver

Scott, James University of Toronto Toronto

Scott, Jeremy University of Toronto Toronto

Sears, Malcolm McMaster University Hamilton

Silverman, Frances University of Toronto Toronto

Stanojevic, Sanja Hospital for Sick Children Toronto

Subbarao, Padmaja Hospital for Sick Children Toronto

Tebbutt, Scott James Hogg iCAPTURE Centre Vancouver

Takaro, Tim Simon Fraser University Vancouver

Tang, Patrick University of British Columbia Vancouver

To, Teresa Hospital for Sick Children Toronto

Turvey, Stuart University of British Columbia Vancouver



NATIONAL COORDINATING CENTRE (NCC) CONTACTS – HAMILTON SITE Malcolm R. Sears Tel: 905-522-1155 ext. 33286

Principal Investigator Fax: 905-521-6132 FIRH, St. Joseph’s Healthcare [email protected]

T3209 – Juravinski Tower 50 Charlton Avenue East Hamilton ON L8N 4A6 Diana Lefebvre Tel: 905-522-1155 ext. 32866 Research Manager Fax: 905-521-6132 FIRH, St. Joseph’s Healthcare [email protected] T3218 – Juravinski Tower 50 Charlton Avenue East Hamilton ON L8N 4A6 Sherri Smith CHILD Project Administrative Assistant Fax: 905-521-6132 ex. 33286 FIRH, St. Joseph’s Healthcare [email protected] T3219 – Juravinski Tower 50 Charlton Avenue East Hamilton ON L8N 4A6

Melanie St. John Data Entry Supervisor Tel: 905-522-1155 ext. 35091

FIRH, St. Joseph’s Healthcare Fax: 905-521-6132 T3216 – Juravinski Tower [email protected]

50 Charlton Avenue East Hamilton, ON L8N 4A6







SENIOR COORDINATOR CONTACTS Toronto

Aimée Dubeau Tel: 416-813-7765 Site Coordinator Fax: 416-813-6246 The Hospital for Sick Children [email protected] 7292-555 University Avenue Division of Respiratory Medicine Toronto, ON M5G 1X8

Winnipeg Rishma Chooniedass Site Coordinator John Buhler Research Centre Tel: 204-789-3978

504H – 715 McDermot Avenue Fax: 204-789-3986 Winnipeg MB R3E 3P4 [email protected]

Edmonton

Joyce Chikuma Tel: 780-407-8084 Site Coordinator Fax: 780-407-8286 University of Alberta [email protected] 2034 Research Transition Facility 8308-114 St. Edmonton AB, T6G 2V2

Vancouver

Mary Ann Mauro Site Coordinator Tel: 604-875-2000 Ext. 7882

Child and Family Research Institute [email protected] 950 West 28th Avenue Vancouver, BC, V5Z 4H4







SECTION 2: CHILD STUDY OVERVIEW (source Grant Application, June 28, 2007) PRIMARY OBJECTIVE The primary objective of this national longitudinal birth cohort study is to determine the roles of a range of environmental factors and their interactions with genetic and host factors in the development of allergy and asthma in children. To meet this goal we will be obtaining multiple objective measurements especially focusing on the indoor environment (home inspection, dust collection), child responses (immune function, endocrine, pulmonary function, infections, allergies and clinical assessment) and genetic factors, together with extensive questionnaire data (demographics, family history, diet, activity, housing environment, psychosocial environment and stress) in a prospective longitudinal birth cohort of children recruited in pregnancy in a general population. STUDY DESIGN This study is a general population based prospective cohort study. This longitudinal design will allow prospective evaluation of data regarding multiple indoor and outdoor environmental exposures, serial measurements of immunological responses, lung function and airway inflammation, infections, allergies, socioeconomic factors, stressful environments, genetics and epigenetic phenomena, and clinical outcomes. The primary unit of study is the family based trio – child and parents. Following a new national birth cohort from pregnancy allows tracking of the timing of a wide variety of potentially important environmental exposures through pregnancy, infancy and into childhood. The design allows evaluation of neonatal predictors of allergy and asthma, and examination of outcomes in relation to changing environments. Genetic investigations in this cohort will focus on validating gene-environment interactions from other studies; the prospective and detailed information collected on the environment as well as in-depth phenotype characterization will enhance the strength of such validations and may lead to discovery of new and important gene-environment interactions particularly relating to the indoor environment. The study design provides the following advantages. • This design allows for the measure of exposures (environmental and clinical) before the

outcome of interest occurs, which is likely to reduce measurement bias in comparison with the less costly case-control study design. The study of allergy and asthma is a challenge in that many factors are considered as contributing causal factors, including environmental exposures that are difficult to measure, even prospectively. It provides the opportunity to take repeated measures, of particular value for cellular and physiological measures, which can be intermediate outcomes of great interest. It is also highly relevant to model environmental exposures based on more than one measure, as models will gain precision and validity.

• Linkage with rich Canadian health databases and possibly other cohort studies will eventually allow the study of many other health outcomes.

• Enables inferences to be made on the relationship between the exposure and outcome assuming that the relationship is the same among those in the study as it would be among those who did not participate.



INVESTIGATORS A network of over 40 investigators, the majority already members of the AllerGen Network of Centres of Excellence, has come together under the overall direction of the Principal Investigator. Disciplines represented include pediatric and adult respirology, neonatology, population health, epidemiology, allergy and immunology, infectious diseases, statistics, environmental assessment, geographic information systems, environmental hygiene, occupational medicine, obstetrics, pulmonary physiology and pathology, genetics, genetic epidemiology and epigenetics, nutrition, medical geography, molecular biology, psychology, air quality, toxicology, sociology, neuroimmunology, biostatistics, and bioethics.

PARTNERS This CHILD study has been funded by the Canadian Institute of Health Research (CIHR) and AllerGen Network Centres of Excellence. The Canadian Mortgage and Housing Corporation (CMHC) have strong interests in this proposal because of the potential that findings will provide information relevant to building standards and design. CMHC provided financial and in-kind support for training of personnel to conduct the walk-through inspection of the home, administration of the home environment questionnaire and dust sampling. Environment Canada has been a supporter of AllerGen since before its inception. For the CHILD study Environment Canada will provide relevant information and analyses. For example, Environment Canada currently monitors outdoor levels of many pollutants and has networks of monitoring stations in each of the recruitment sites for this study. Environment Canada has committed to provide these data at no cost to the study and to work in ensuring they are used appropriately and to the fullest extent possible. In future years, Environment Canada, along with Health Canada and other partners, aims to develop new techniques for spatial characterization of pollutant levels and chronic exposure estimation. The results of this work will be tailored to support exposure estimation for the full CHILD cohort. COLLABORATORS This CHILD study has begun the process of collaboration and developing alliances with other children’s health studies that create increased sample size providing more power for health outcomes and associations. The CHILD Study has collaborated with the South Asian birth Cohort. (START) led by Dr. Sonia Anand and Dr. Gupta. This study is enrolling South Asian mothers with their newborns and examining effects of the environment on early life adiposity, growth and cardiovascular measurements. The CHILD Study and START Study will work to align themselves on several outcomes, including adiposity, growth, asthma, allergies, cardiovascular disorders, diabetes, genetics and epigenetic markers and will study the influence of environmental factors, such as mother’s nutrition, on these outcomes.



PRIMARY OUTCOME The primary outcome on which the study is powered is specialist-physician diagnosed asthma at age five years (anticipated to occur in approximately 10% of the population-based cohort). Other more common, important allergy-related outcomes (i.e., atopy (IgE/skin prick responses), atopic eczema, food allergy, transient wheezing, hay fever) will be assessed as secondary outcomes. SECONDARY OUTCOMES

• Preschool wheeze • Eczema • Allergic Rhinitis • Food allergy • Atopy • Preschool lung function: • Lung inflammation markers • Urine • Immunologic outcomes • TLR function • T-cell stimulation and cytokine assays • Innate immune effector cell assays • Obesity • Cardiovascular disorders • Metabolic outcomes

INCLUSION AND EXCLUSION CRITERIA Inclusion Criteria

• Pregnant women aged 18 years or older (19 in Vancouver) • Residence in reasonable proximity to a recruitment centre • Able to read and speak English • Willing to provide informed consent • Planning to give birth at the recruitment centre • Infants born at 35 weeks or more gestation (min 34 weeks and 4 days accepted) • Must be able to provide a:

Valid address and telephone number Names and phone numbers of two alternate contact individuals

Exclusion Criteria

• Children with major congenital abnormalities • Expectation of moving away from a recruitment area within 1 year • Children of multiple births • Children resulting from in-vitro fertilization (artificial insemination and hormonal therapy will be

accepted) • Children who do not spend at least 80% of nights in the index home



TIMELINE FOR CHILD STUDY DATES ACTIVITY Jan 2007-Sept 2007 Grant application process

Dec 2007 Approval of Funding

Jan 2008 – May 2008 Funding to Universities, Start up process

Sept 2008 – Dec 2008 Vanguard Recruitment of up to 200 mothers

Jan 2009 – June 2009 Review of Vanguard, Assess study collection tools, procedures

June 2009 – April 2012 Recruitment of main CHILD study, follow-up

Sept 2008 – Dec 2017 Visits, Follow up to 5 years

PRINCIPLES AND PRACTICES The CHILD Study follows the International Conference on Harmonization (ICH) guidelines of conduct regarding research involving humans and Good Clinical Practice (GCP) as outlined by Health Canada. The CHILD Study sites are responsible to ensure all their staff have GCP training. The full guidelines ICH1 Guidance E6: Good Clinical Practice: Consolidated guideline can be found on Health Canada’s web site www.hc-sc.gc.ca.

• The rights, safety, and well-being of the trial subjects are the most important considerations and should prevail over interests of science and society.

• The study will be conducted in compliance with the protocol that has received prior institutional review board (IRB)/independent ethics committee (IEC) approval/favourable opinion.

• The medical care given to, and medical decisions made on behalf of, subjects should always be the responsibility of a qualified physician.

• Each individual involved in conducting the study should be qualified by education, training, and experience to perform his or her respective task(s).

• Freely given informed consent must be obtained from every subject prior to participation.

• All information should be recorded, handled, and stored in a way that allows its accurate reporting, interpretation and verification.

• The confidentiality of records that could identify subjects should be protected, respecting the privacy and confidentiality rules in accordance with the applicable regulatory requirement(s).

• Systems with procedures that assure the quality of every aspect of the study should be

implemented.

http://www.hc-sc.gc.ca/dhp-mps/prodpharma/applic-demande/guide-ld/ich/efficac/e6-eng.php#fn_1

http://www.hc-sc.gc.ca/



STUDY ASSESSMENTS The CHILD study is a general population-based cohort study. This longitudinal design allows prospective evaluation of data regarding multiple indoor and outdoor environmental exposures, serial measurements of immunological responses, lung function and airway inflammation, infections, allergies, socioeconomic factors, stressful environments, diet and nutrition, genetics and epigenetic phenomena, and clinical outcomes. The primary unit of study is the family based trio – child and parents. Following a new national birth cohort from pregnancy allows tracking of the timing of a wide variety of potentially important environmental exposures through pregnancy, infancy and into childhood. The design will allow evaluation of neonatal predictors of allergy and asthma, and examination of outcomes in relation to changing environments. Genetic investigations in this cohort will focus on validating gene-environment interactions from other studies; the prospective and detailed information collected on the environment as well as in-depth phenotype characterization will enhance the strength of such validations and may lead to discovery of new and important gene-environment interactions particularly relating to the indoor environment.



CLINICAL AND BIOLOGICAL ASSESSMENT OF THE PARENTS Maternal factors and exposures: A general health questionnaire is administered during pregnancy regarding past medical history, family history, pregnancy history and complications, medications, gestational diabetes, smoking, exercise and diet. Information will be obtained regarding family size, duration at present residence, education, and other relevant demographics, the home environment during pregnancy, psychosocial and stress factors. Maternal diet during pregnancy is assessed using a standardized food frequency questionnaire modified for Canadian diets. Paternal health is assessed with a standardized questionnaire. Parental evidence of asthma: Asthma (ever and current) in the mother and father is assessed by completion of a Health Questionnaire based on the ECRHS questionnaire. Each parent will be classified as definite asthma, probable asthma, possible asthma, or no asthma using similar definitions as those used for children at age 5. Parental evidence of atopy: Atopy in the mother and father is assessed by skin testing. Parental lung function: Spirometry is undertaken in fathers at the first or subsequent visit and in mothers when the child is seen at the 1 year visit. CLINICAL ASSESSMENT OF THE CHILD Final enrolment in the study will be confirmed after the birth of the child, based on the inclusion and exclusion criteria. Children with major congenital anomalies and respiratory distress syndrome will be excluded. Children with transient tachypnea of the newborn period (estimated to be between 1 and 2% of newborns) will be included. The mode of delivery, complications of delivery, fetal status at birth, and associated stress will be documented. Cord blood will be collected and processed for both immediate testing and for storage. ENVIRONMENTAL ASSESSMENTS Exposures relating to the indoor and outdoor environments will be assessed using multiple tools, including questionnaires, home inspections, and dust sampling for multiple allergens and pollutants. Questionnaires: A comprehensive baseline (during pregnancy) and annual follow-up questionnaires are administered regarding housing structure, function, condition and maintenance history, renovations, source and extent of dampness indicators, mould growth, new furnishings, appliance emissions, presence and type of air conditioning, conditions of use, etc. Questions related to the child’s and families’ time activity is included in follow-up, gathering information such as time spent in transit, mode of transport, frequency/duration of visits to indoor pools, exposure to wood smoke, etc. Home Assessment: When the infant is approximately three months old, a physical home assessment is carried out to evaluate the structure, function and exposure sources within the home with specific emphasis on the evaluation of the house in terms of cleanliness and cleanability, ventilation including presence of air-conditioning and type of heating/cooling, microbial and



chemical contaminant burdens, and basement condition. Changes in address and geographical coordinates will be tracked along with other changes in family members, housing and appliances. Dust Sample Collection: In conjunction with the 3-month home assessment, dust samples are collected from a 2 m2 area of flooring using a standardized consumer model vacuum cleaner fitted with a dust collection device. The measured area is enlarged as necessary to collect a minimum of 1-2 g fine dust in a standardized manner for use in allergen and endotoxin testing, together with markers of outdoor exposure. The dust sample is collected from the child’s bedroom and most used living area. The total recovered mass of bulk dust samples and collection area is determined. Dust extracts are sieved to provide a <300 μm size fraction, weighed, labeled, aliquoted, frozen at -80oC, and subsequently made available to appropriate laboratories for later analyses. Sieved dust samples will subsequently (using a case-control design) be analyzed for:

• Allergens • Endotoxins and other environmental TLR ligands • Beta (1,3)-D-glucans

Environmental Tobacco Smoke exposure: Environmental tobacco smoke (ETS) exposure is assessed annually with a questionnaire. Outdoor Air Pollution and Infiltration Modeling: Family residence history and time-activity information is collected with baseline questionnaire and periodically throughout the study. Outdoor air exposure modeling is based upon available monitoring data and land-use regression (LUR) techniques (published models currently exist for Toronto and Vancouver, focused mainly on NO2 and are being developed for Edmonton and Winnipeg. More advanced data fusion/assimilation methodologies using all available data are evolving and new progress, potentially funded by Environment and/or Health Canada, and are expected within the next few years. This includes NAPS monitoring data, LUR output and, if necessary, air quality model output and satellite-based observations. In time series fashion, these data will be linked to the geographic information collected on the family and combined with indoor exposure information. These assessments include a range of outdoor-indoor exchange models, which are currently being assessed to estimate total asthma hazard exposure.

GENETICS Study Designs: The primary design for the genetic studies is a family-based design consisting of parents and offspring. This design circumvents the pitfalls of hidden population stratification. DNA is collected from the mother and father at the first pre-natal study visit, and from the child at the time of delivery (cord blood). Nested case-control designs, parent of origin analyses and case-only analyses will also be used. Genotyping: Candidate genes and SNPs have been identified through pilot studies supported by AllerGen NCE across several cohorts. The CHILD study will provide facilities for the collection and storage of biological material. Investigators will apply to a scientific DNA-use committee for use of the stored samples and will be required to outline the hypothesis to be tested, study design, power, and secure sufficient funds to genotype the requested samples. Genotyping will be performed using appropriate platforms including Taqman, Illumina, Sanger sequencing, whole genome sequencing and “Next Generation” sequencing technologies.



Gene Expression: Discovery of factors that control gene expression can be approached on a gene-by-gene, or pathway-by-pathway, hypothesis-testing basis or on a genome wide basis. We will investigate the genetic and epigenetic control of specific gene expression. Stored m-RNA will provide a resource for future studies. Future Genetic Methodologies: The MethylLight method will be used for gene-specific interrogation of DNA methylation190, and Chromatin immunoprecipitation (ChIP)191 to test for gene-specific histone modifications. The technology to do genome wide assays for DNA methylation and histone modification will shortly be available. It is likely that the epigenome of blood leukocytes will be the first to be systematically investigated since it is accessible. Since peripheral blood leukocytes and in particular lymphocytes will be available from infants in the birth cohort and because these immune/inflammatory cells are key effectors of allergy and asthma phenotypes we feel we are uniquely positioned to collect the appropriate samples for a comprehensive epigenetic analysis. We will initially concentrate on DNA methylation and chromatin modification of key candidate genes in maternal and fetal blood cells, but we will store the samples for future genome-wide interrogation of DNA methylation and histone modification. As the understanding of the effects of epigenetics expands, we will be ideally placed to make important contributions to this rapidly developing field and in particular to answer the important questions concerning exactly how the environment acts to influence genes and risk for allergy. IMMUNOLOGY The following assessments are conducted using cord blood, and peripheral blood at 1 and 5 years of age. Because of fiscal and practical constraints, innate immune function tests are performed in a subset of children in Toronto and Vancouver. Hence the bloods from these children (approximately 1000) are activated, plated and frozen for innate immune function testing. Innate Immune Function: a) TLR function: To determine TLR function, we use 5 - 10 ml heparinized blood with the following readouts: TLR expression on relevant cells (e.g. dendritic cells, monocytes/macrophages, and CD34+ hematopoietic progenitors); single cell cytokine profiling in response to TLR stimulation (by multiplex Flow Cytometry) and global blood cytokine profiles (by Cytometric Bead Arrays). This is done on fresh blood at birth (cord blood), and at 1 and 5 years of age (peripheral blood). The stimulated samples are stored frozen, and analyzed in pools at a later time point in one centralized facility. b) Innate effector function: In addition to a CBC for eosinophil numbers, we subsequently measure eosinophil and mast cell granule proteins and cytokines (including LTC4 production), as well as functional IDO levels, on cryopreserved samples taken at birth, and at 1 and 5 years of age. c) Adaptive immune function: Besides B-cell serum immunoglobulin levels (including IgE, IgG4), several T-cell functional assays are combined in one single Flow Cytometry assay with the following readouts: Antigen/Allergen specific T cell proliferation; Antigen/Allergen specific single cell cytokine (Th1/Th2) profiling; Antigen/Allergen specific global cytokine profile; and Regulatory T cell characterization. We expect to confirm and extend findings of previous birth cohorts that focused primarily on T cells (particularly low T cell IFN-gamma production). This antigen/allergen specific assessment allows us to determine when sensitization occurs, which has important public health implications in terms of advising mothers on, e.g., their diet during pregnancy. Furthermore, allergen specific immune function is assessed in the context of the entire immune system and



environmental milieu in each individual, determining whether children who develop allergy/asthma have a generally different immune response, or a selective allergic reaction to specific allergens. This is accomplished by comparing allergen to vaccine antigens (Tetanus Toxoid, Pertussis Toxin; selected allergens will be identified through skin testing). Determination of allergen specific acquired immune function is undertaken at birth, 1 and 5 years of age, using peripheral blood frozen leukocyte samples taken and stored frozen on all children. All samples from one individual are run in the same experiment. BIOLOGICAL EXPOSURE ASSESSMENT Breast Milk: On the day of the home visit, mothers are asked to collect 10 ml of breast milk. Estrogen can be measured using gas chromatography and mass spectrometry (GC/MS) method. Stool Composition: Stool samples are collected from infants at birth, 3 months and 1 year, frozen and subsequently analyzed for species of bacteria making up the microbiota of the intestine. Urine samples are collected at 3 months, and at 1, 3 and 5 years to examine urinary markers of environmental exposures including smoke and plasticizers. Markers for inflammation using Nuclear Magnetic Resonance-based metabolomics methods are currently under validation. Urine samples will be collected at additional visits in the Toronto sub-cohort PFT group at the 3 month and 18 month visit. Blood samples are collected at birth (cord blood), 1 and 5 years of age in children for genetic and epigenetic analysis. Childhood diet: Childhood diet is assessed using a food questionnaire at each follow up. Exposure to other children: This is assessed through time-activity logs that include questions about daycare such as age when the child first started daycare, age when the child left daycare, and the number of hours per week spent in daycare. Post-natal exposures of children to common respiratory pathogens: Detailed assessments of respiratory infections are done in the first year of life in Toronto only. Parents will be encouraged to call the viral hotline whenever signs or symptoms of viral infection are present. The study nurse will assess the severity of the viral symptoms using the Respiratory Illness score card and a nasal swab will be collected within a week of the call if the cold symptoms score is ≥ 5. Nasal Samples are collected at 3 months and 1 year in all children. Toronto will collect additional nasal swabs before age 1 as part of their post-natal respiratory infection assessments as required according to the protocol.

PSYCHOSOCIAL ASSESSMENT

Four sets of psychosocial factors are explored: socioeconomic status (objective and subjective); stress; maternal depression; and social support/social capital Key Measures for assessment of SES • Resource-based SES. Parents are asked about family assets, including income, savings,

housing status (rent/own), number of bedrooms, and number of cars owned. Answers to each



question are standardized (z-scored for the sample) and the z-scores summed to create one composite asset score.

• Education SES. Years of education, and highest educational degree attained for each parent. • Subjective SES. Using the MacArthur Scale of Subjective Social Status parents are asked to

place their household (relative to their community) on a ladder, with higher rungs indicating higher status. Social support/social capital. The Social Provision Scale, a simple, well-validated 24-item scale in several dimensions (attachment, social integration, reassurance of worth, reliable alliance, guidance, opportunity for nurturance), is administered to parents.

Key Measures for assessment of stress

• Chronic stress in the context of the nuclear family, in the broader family/social network, in work and/or academic settings, and arising from health problems in self/family

• Occurrence of acute life events in family, social network, work life, health domains • Global perceived stress – extent to which life is unmanageable, unpredictable • Social support/social capital as a potential mediator in the stress-outcome

relationship Key Measures for assessment of depression

• Center for Epidemiology Studies-Depression scale (CED-D) questionnaire • Dyadic Adjustment Scale, Interpersonal Support Evaluation, Perceived stress scale • Comprehensive Index of Life and Chronic Stress Interview • Life stress UCLA and mood Structured clinical interview (Vancouver only) • Prescription and health care utilization data bases (in Manitoba)

PULMONARY FUNCTION AND ATOPY Consenting study members in the sub-cohort in Toronto undergo early infancy assessment of their pulmonary function. This includes:

• Raised volume RTC with bronchodilator responsiveness using standardized methodology at 3 months, 1 year and 18 months of age in the Collins Baby Box. Children at age 3 and 5 years perform spirometry where possible with bronchodilator response.

• Exhaled nitric oxide use the tidal breathing methodology at the 3 month, 12 months, and 18 month assessments.

• Incentive spirometry at age 3.

• Multiple Breath Washout using tidal breathing methodology at the 3 month, 1 year, 18 month, 3 year and 5 year assessments.

All centres to perform:

• Spirometry - where possible with bronchodilator response at 5 years of age.

• Atopy - skin testing to assess atopy is performed at 1, 3 and 5 years of age.



SECTION 3: ORGANIZATION; COMMITTEES AND SCIENTIFIC WORKING GROUG

CHILD STUDY – ORGANIZATIONAL CHART

Edmonton siteP Mandhane, site leader

Vancouver siteS Turvey, site leader

Strategic Planning and PartnershipCouncil

Scientific Working Groups

- Environment- Genetics- Immunology- Infection- Lung Function- Nutrition- Psychosocial- Statistics- Epidemiology

CHILD cohort

Toronto siteP Subbarao, site leader

Manitoba sitesA Becker, site leader

Scientific Advisory Committee

Biological Samples Committee

Executive CommitteeP Subbarao (co-PI, U Toronto)

Deputy DirectorS Anand (McMaster U)A Becker (co-PI, U Manitoba)D Befus (U Alberta)J Brook (U Toronto; Env Canada)W Lou (U Toronto)P Mandhane (co-PI, U Alberta)G Miller (UBC)A Sandford (UBC)S Turvey (co-PI, UBC)

Data Centre Quality Control

Senior coordinator Research assistants

Laboratory personnel

Ethics and Legal Committee

PublicationsCommittee

HealthDiaryBioinformaticsManagement

BiologicalSamples

Management

Finance Committee

National Coordinating Centre

Nominated PI and DirectorM Sears (McMaster U)

Senior coordinator Research assistants Laboratory personnel



3.1



3.2 COMMITTEES Executive Committee: Chair: Malcolm Sears

Members: PJ Subbarao, Sonia Anand, Allan Becker, Dean Befus, Jeff Brook, Wendy Lou, Piush Mandhane, Gregory Miller, Andrew Sandford, Stuart Turvey NCC: Diana Lefebvre Scientific Advisory Committee: Chair: Paul O’Byrne

Members: Mark Raizenne, Peter Sly, Fernando Martinez, Felix Ratjen, Erika von Mutius

NCC: Malcolm Sears, Diana Lefebvre

Ethics / Legal Committee: Members: Tim Caulfield, Nola Reis, Susan Elliott, Malcolm Sears Biological Samples Committee: Chair: Theo Moraes, Dean Befus Members: Judah Denburg, Tobias Kollmann, Jeff Brook, Richard Hegele, Mark Larche NCC: Malcolm Sears, Diana Lefebvre Sample requests by researchers for analyses pertinent to the CHILD Study will be reviewed by this Committee in conjunction with the Executive Committee. Publication Committee: Chair: Felix Ratjen Members: Allan Becker, Sharon Dell, Susan Elliott, Kent Hayglass, Anita Kozyrskyj, Piush Mandhane, Wendy Lou, Andrew Sandford, Malcolm Sears, PJ Subbarao, Stuart Turvey NCC: Diana Lefebvre Finance Committee: Chair: Malcolm Sears Members: Allan Becker, PJ Subbarao, Piush Mandhane, Stuart Turvey, Diana Lefebvre This Committee of the Director, Site Leaders and Administrative Manager reviews and determines allocation of funding across the components of the study



3.3 SCIENTIFIC WORKING GROUPS To facilitate the many areas of investigations, CHILD has developed various Scientific Working Groups consisting of researchers from related disciplines and specialties to develop the assessments, questionnaires and tools fundamental to meeting the objectives of the CHILD study. These groups work in conjunction with the National Coordinating Centre to develop Standard Operating Procedures for tests, biological sample collection and training for recommendation to, and approval by, the Executive Committee. Genetics Working Group: Chair: Andrew Sandford Members: Denise Daley, Andrew Sandford, Scott Tebbutt, Tobias Kollmann, Michael Brauer, Stuart Turvey, Sonia Anand NCC: Malcolm Sears, Diana Lefebvre The genetics working group advises on sample collection, storage and analysis of genetic material from parents and children from the CHILD Study. Immunology Working Group: Chair: Stuart Turvey, Dean Befus Members: Padmaja Subbarao, Theo Moraes, Kent HayGlass, Tobias Kollmann, Stuart Turvey, Redwan Moqbel, Judah Denburg, Mark Larché, Jean Marshall, John Gordon NCC: Malcolm Sears, Diana Lefebvre The Immunology working group works with the National Coordinating Centre to develop standard operating procedures for collecting, processing and quality control of blood samples. Blood components from cord blood and child’s blood will be processed to provide RNA, DNA, serum, plasma, cord blood mononucleocytes, and peripheral blood mononucleocytes. Immunology investigations will focus on a comprehensive analysis of genetic, epigenetic, gene expression, innate and adaptive immunity and toll-like receptor profiles of children to understand the biological basis of allergic disease. Environmental Working Group: Chair: Jeff Brook Members: Tim Takaro, James Scott, Ryan Allen, Michael Brauer, Anita Kozyrskyj, Francis Silverman, Sharon Dell, Amanda Wheeler, Greg Evans, Tedd Konya. Administrative Support: Agatha Blancas NCC: Malcolm Sears, Diana Lefebvre



The CHILD Study is particularly interested in exposures relating to the indoor and outdoor environments which will be assessed using multiple tools, including questionnaires, home visit assessments, and home dust sampling for multiple allergens and pollutants. The Environmental Working group works with the National Coordinating Centre and Canada Mortgage and Housing Corporation to develop Standard Operating Procedures for assessments and home dust sampling procedures to support the CHILD study objective of understanding environmental factors involved in allergic disease. Questions related to the child’s and families’ activity will be included in follow-up, gathering information such as time spent in transit, mode of transport, frequency/duration of visits to indoor pools, exposure to wood smoke, etc. Dust samples will be processed at the Gage Occupational & Environmental Health Unit, Toronto, Ontario and will subsequently be analyzed for endotoxins, allergens, other environmental TLR ligands and Beta (1,3)-d-glucans. Outdoor air pollution will be assessed based on traffic-related air pollution estimated by modeling. Psychosocial Working Group Chair: Greg Miller Members: Edith Chen, Anita Kozyrskyj NCC: Malcolm Sears, Diana Lefebvre The CHILD Study is interested in exploring psychosocial factors including the mental and physical health of families making the transition to parenthood. The goal is to better understand the thoughts, feelings, and experiences that families have during this period, and how they come to influence the well-being and health status of their child. The psychosocial working group will recommend validated psychosocial questionnaires and conduct interviews to meet the objective of understanding why some family experiences might allow some people to remain healthy while others develop a disease, such as asthma or allergies. Lung Function and Allergy Working Group Chair: Padmaja Subbarao Members: Allan Becker, Sanja Stanojevic, Rishma Chooniedass NCC: Malcolm Sears, Diana Lefebvre Assessments of lung function and allergies are an important measure in the CHILD study to meet the primary objective of understanding allergic disease in children. This working group recommends to the CHILD Executive the times during child development when lung function and allergy skin tests will be performed to assess children’s health status. Procedures used for lung function tests include exhaled nitric oxide to examine lung inflammation, infant lung function tests conducted at 3 months, 1 year and 18 months (in Toronto only), child lung function tests at 5 years of age in all children, allergy skin testing at 1, 3 and 5 years of age.



Infection Working Group: Chair: Padmaja Subbarao Members: Rick Hegele, Patrick Tang, Stuart Turvey NCC: Malcolm Sears, Diana Lefebvre A primary focus of this working group is the detailed examination of respiratory infections particularly in the first year of life. All children will have nasal swabs examined at three months and one year, while a sub-cohort in Toronto will be offered more frequent review at the time of acute symptoms suggesting infection. Nutrition and Endocrine Working Group Chair: Allan Becker Members: Piush Mandhane, Anita Kozyrskyj, Sonia Anand NCC: Malcolm Sears, Diana Lefebvre Information about mother’s diet during pregnancy and while breastfeeding is important in assessing baby’s nutrition, as well as what the child eats. The nutrition working group works in conjunction with the National Coordinating Centre to develop assessment tools and to implement Food Frequency Questionnaires for the collection of nutritional information in mothers and children. Breast milk will be collected for subsequent hormone analyses. Epidemiology and Statistics Working Group: Chair: Wendy Lou Members: Allan Becker, PJ Subbarao, Piush Mandhane, Stuart Turvey NCC: Malcolm Sears, Diana Lefebvre Recruitment data, data collection and management, and subsequent data analysis are complex issues under continuing development and review by this Working Group in liaison with all recruitment sites and investigators.



SECTION 4: PROCEDURES The Birth Cohort Study initially planned to recruit up to 5,000 pregnant mothers from 18 weeks of pregnancy from 4 provinces across Canada. The infants will be followed until they are five years old. The cities for recruitment are Toronto, Winnipeg, Morden, Winkler, Edmonton and Vancouver. The study consists of a Vanguard Cohort (Pilot) of 200 women across Canada. After a 6 month period of assessment and review, the General cohort has enrolled the remainder to add up to a total of 3629 women (Vanguard and General). The General cohort includes sub-cohorts that will carry out more in depth investigations in various areas. These sub-cohorts include a Pulmonary Function Testing (PFT) sub-cohort in Toronto with a viral study in the first year of life, a Psychosocial sub-cohort in Vancouver, a sleep add-on study in Edmonton and various blood and home environment add-on studies in Winnipeg. Province/Cities Ontario

Toronto Manitoba Winnipeg, Morden, Winkler

Alberta Edmonton

British Columbia Vancouver

Universities Toronto Manitoba Alberta UBC, Simon Fraser

Centre location Hospital for Sick Kids.

Manitoba Institute of Child Health

College Plaza BC Children’s

Subject ID numbers

50001 - 50865 40001 - 41107 30001 - 30841 20001 - 20817

Sub Cohorts at centre

Infant pulmonary function assessments, viral study to 1 year

Vitamin D and dental health 2nd home visit Child blood at 3 years

Sleep and neurological assessments

Additional in depth psychosocial assessments

Recruitment OB/GYN locations

Ob/Gyn clinics at hospitals

Various hospital and local OB/GYB, doctors’ offices, ultrasounds

Various hospital and local OB/GYB, doctors’ offices

Hospital based ultrasound clinics, offices

Delivery hospitals

Mt. Sinai, Sunnybrook

St. Boniface, Health Science Centre, Boundary Trails

Grey Nuns, Misericordia, Sturgeon, Royal Alexandra

BC Children’s and Womens, St. Paul’s Hospitals


Page 26 CHILD Study Protocol

Version Date: December 1, 2014

18 WK

36 WK

Birth 3 MTH home

3 MTH TO

clinic

6 MTH

1 YR clinic

1.5 YR

1.5 YR TO

clinic

2 YR

2.5 YR

3 YR clinic

4 YR

5 YR clinic

Q: Mother Profile/Residence √ √ √ √ √ √ √Q: Mother Health √ √ √Q: Mother Nutrition √ √ √ √Q: Mother Vitamins √ √ √ √Q: Mother Medications √ √ √ √ √Q:Mother's Stress √ √ √ √ √ √ √ √Q: Mother Psychosocial (multiple Qs) √ √ √ √ √Q: Mother Life Stress Interview √ √ *Q: Parenting √ √ √ √ √Q: Father Health √ √Q: Socio-economic status (SES) √ √ √ √ √Q: Child Delivery Chart Extraction √Q: Child Health √ √ √ √ √ √ √ √ √ √Q: Child Nutrition and Diet √ √ √ √ √ √ √ √ √ √Q: Child Medications √ √ √ √ √ √ √ √ √ √Q:Child Clinical Assessment √ √ √Q: Home Environment Questionnaire √ √ √ √ √ √ √ √ √ √Q: Home Assessment done by RA √Q:Food packaging and prep √ √ √ √T: Mother Skin Prick Test √T: Mother Spirometry √T: Father Skin Prick Test √T: Father Spirometry √T: Child Skin Prick Test √ √ √T: Child eNO √ √ √ √ √T: Child Pulmonary Function Tests √ √ √ √ √S: Cord Blood √S: Mother Breast Milk √S: Mother Venous Blood √ √**S: Mother/Father Buccal Swab √***S: Father Venous Blood √S: Child Venous Blood √ √**** √S: Child Buccal Swab √***S: Child Nasal Swab √ √S: Child Urine √ √ √ √ √ √S: Child Meconium/Stool √ √ √S: Home Dust Collection √

CHILD Study Schedule from Prenatal Recruitment to Age 5

VANCOUVER ONLY

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stSa

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TORONTO ONLY

Visit

* Completed to October 30/2013 ** 1 yr col lected unti l March 31, 2013 in Toronto and Edmonton. Al l col lected in Vancouver and Winnipeg*** Bucca l Swab col lected i f not able to col lect blood at 1 year **** Chi ld blood col lected only in Winnipeg at age 3




CHILD STUDY QUESTIONNAIRES SUMMARY

Screening and Enrolment 3 Month Home Visit 1,3,5 Year Visits Mother's Consent Home Environment Residential History Mother’s Profile Environmental Assessment RA Home Environment Reason for Ineligibility Residential History New Home Characteristics (3 year only) Enrolment Mother's Nutrition Mother's Health (1 & 5 year only) Physician Profile Mother's Medications to 3 months Mother's Medications (1 year only) Pharmacy Information Mother's Vitamins & Supplements Mother's Stress Supplemental Residential History Child's Health Mother's Psychosocial CSR DAS ISE PREN RLE

Withdrawal Form Child's Medications Parenting Mother Breast Milk Mother's SES 18-Week Prenatal Visit Child Nasal Food Packaging and Prep (3 & 5 Year Only) Mother’s Health Child Urine Child's Nutrition / Food Frequency Prenatal Home Environment Child Stool Child's Health Hutchinson’s Food Frequency Questionnaire Dust Child's Bedroom Sample Child's Medications Mother's Prenatal Medications Dust Most-Used Living Room Sample Global Sleep Assessment (Edmonton Only) Mother's Vitamins & Supplements Control Dust 3 months Brief Infant Sleep (Edmonton Only) Prenatal Mother's Stress Brief Infant Sleep (Edmonton only) Child's Clinical Assessment Socioeconomic Status (SES) Pediatric Sleep (Edmonton only) Child's Skin Allergy Tests Mother’s Venous Blood Child's Spirometry (Toronto 3 Year only)

PFT Visit Assessment (Toronto Only)

36-Week Prenatal Visit 3, 18 Month Visits (Toronto only) Child's Spirometry (5 Year only) Mother's Psychosocial CSR, PRENS, DAS, ISE, RLE

Infant Pulmonary Function Testing (IPFT) Assessment

Child's Venous Blood (1 & 5 yr all, 3 Winnipeg)

Mother's Life Stress Interview (Vancouver Only) Child Urine Child’s Urine Child's Stool (1 year only) Father Visit 0.5, 1.5, 2, 2.5, 4 Year Mail/ Online Child's Nasal (1 year only) Father's Consent Residential History (2 & 4 year only) Child's CBC Results (1 & 5 year only) Father’s Skin Prick Home Environment Child's Skin Test Meds/ Supplements Father's Health Post Natal Mother's Stress Mother's Stress Interview (Vancouver only) Father Skin Test Meds/Supplements Child's Nutrition / Food Frequency Mother's Skin Prick (1 year only) Father's Spirometry Parenting (2 & 4 year only) Mother's Spirometry (1 year only) Father's Venous Blood Child's Health Mother's Venous Blood (1 year only) Global Sleep Assessment (Edmonton only) Child's Medications Mother's Skin Test Meds/ Supplements (1 yr) Food Packaging and Prep (4 year only) Father’s Health (recruitment, 5 yr) Birth Mother's Medications (6 month only) Child Birth Chart Data Mother's SES (4 year only) Cord Blood Mother's Psychosocial CSR, DAS, ISE, RLE Child Meconium Cord Blood Hematology Results Exhaled NO at birth (Toronto only) Child Medications Birth Mother Medications to Birth




Approved Standard Operating Procedures:

Standard Operating Procedures Version Date Sample Handling SOPs Cord Blood Collection SOP 9-Sep-09 Cord Blood Processing SOP 13-Oct-11 Parental Blood Collection and Processing SOP 26-Nov-09 Breast Milk Collection 3 Months SOP 14-Aug-09 Child Blood Processing 1 Year SOP 21-Jan-10 Child Blood Processing 5 years SOP 27-Oct-13 CHILD Home Visit/Dust Collection SOP 18-May-10 Saliva Collection SOP 10-Aug-10 Urine Collection SOP 8-Jul-10 Test Procedure SOPs Child Allergy Skin Testing SOP 15-May-10 3 year Clinical Assessment SOP 13-May-13 5 Year Clinical Assessment (Vanguard cohort) SOP 8-Oct-13 5 Year Clinical Assessment (General cohort) SOP 5-Nov-14 Child Spirometry 5 Years SOP (KoKo system) 4-Mar-14 Child Spirometry 5 Years SOP (NDD system) 2-Oct-14 Meconium/Stool Collection SOP 7-Jul-10 Nasal Swab Collection SOP 7-Jul-09 Parental Allergy Skin Testing SOP 8-Jul-10 Parental Spirometry SOP 26-May-10 Procedural SOPs Query SOP Sample Shipping SOP 1-Feb-13 Participant Withdrawal SOP 16-Oct-12 Participant Site Transfer SOP 1-Feb-13




CHILD Study Sample Collection Summary – (not including add on studies)




BLOOD COLLECTIONS

1. Cord Blood - Birth - processed within 24 hours a. Collecting in vacutainers after delivery of placenta in Winnipeg, Vancouver, Edmonton.

Collection in a syringe before placental delivery in Toronto b. Store - transport at room temperature to designated CHILD blood processing lab c. Aliquot serum, plasma, RNA, DNA, CBMC, CBMCRNA, innate plates d. Test for CBC

2. Parental blood - for mom and dad - processed within 24 hours. Mom’s blood collected prenatally and 1

year up to March 31, 2013. After March 31, 2013, Toronto and Edmonton to collect only those mom’s bloods at 1 year that have not had blood collected prenatally. Vancouver and Winnipeg will continue to collect all mom’s blood at 1 year. Father’s blood to collect any time and as soon as possible during the study.

a. Collecting in vacutainers b. Store - transport at room temperature to designated CHILD blood processing lab c. Aliquot serum, plasma, DNA

3. Child blood - 1 and 5 years - processed within 24 hours

a. Collecting in vacutainers b. Store - transport at room temperature to designated CHILD blood processing lab c. Aliquot serum, plasma, DNA, CBMC, CBMCRNA, innate plates

OTHER NON BLOOD BIOLOGICAL COLLECTIONS

4. Child Meconium – birth - processed within 24 hours a. Collection - spoon collection jar/Edmonton/Winnipeg - diaper Vancouver/Toronto b. Store - fridge/cooler c. Aliquot - at the site into 4 cryovials using stainless steel depyrogenized spatulas

5. Child Stool 3 month and 1 year - same day as visit - within 8 hours

a. Collection – diaper in a plastic bag b. Store - fridge at subject’s home up to 3 days before, transport to centre by cooler c. Aliquot - at the site into 4 cryovials using stainless steel depyrogenized spatulas

6. Child Nasal swab 3 month visit, 1 year - processed same day as visit - within 8 hours

a. Collection UTM media 3 mL b. Store - transport by cooler c. Aliquot - into 6 cryovials, 0.5 mL in each

7. Child Breast milk 3 month visit - processed same day as visit - within 8 hours

a. Collection - at least 10 mL collected in a 30 mL blue lid polypropylene collection jar b. Store - transport by cooler c. Aliquot - into 6 cryovials, 1.5 mL in each.




8. Child Urine 3 month, 1 year, 3 year and 5 year - processed same days as visit - within 8 hours

a. Collection - no collection container b. Aliquot - directly in the home into 6 cryovials using a 60 mL syringe. 1 mL will be aliquoted to

each of 6 cryovials if 6 mLs of urine is obtained. If less than a 6 mL volume is collected, ensure 1 mL is aliquoted into the first cryovial labeled 1-1 and aliquot 0.5 mL in the next five cryovials. If there is still urine left, top up the other cryovials to 1 mL starting with aliquot number 1-2.

c. Store – transport in cooler

9. Child Buccal Swab: Collect at 1 year if there is no cord blood and no 1 year blood a. Using Oragene kit for children, OG-575 assisted kit with sponges b. Collect saliva inside the cheek using the sponges until enough saliva to reach the “fill line”.

There should be about 1.5 mLs total after saliva is mixed with liquid from lid. c. Aliquot 0.75 mLs into each of two cryovials d. Freeze aliquots

10. Parental Buccal Swab Collect at 1 year if blood was not obtained at this or previous visits.

a. Using Oragene kit OG-500 b. Collect saliva by having donor spit into collection tube until saliva reaches the “fill line”. There

should be about 3 mLs total after saliva is mixed with liquid from lid. c. Aliquot 1.5 mLs into each of two cryovials

Freeze aliquots

11. Urine Control - same day as visit - within 8 hours a. Take one water cryovial “field blank” to the home visit, room temperature at home, remove top

when ready to aliquot the urine sample. Leave the “blank” open while aliquoting urine samples. Once finished aliquoting the urine, close the “field blank” cryovial.

b. Store - transport in cooler along with urine samples

12. Dust a. Two thimbles of child’s bed/child’s floor (vacuumed in that order), and two thimbles of the most

used living area b. Store- transport at room temperature c. Shipped to Tedd Konya at the Gage Occupational & Environmental Health Unit (GAGE) d. Aliquot into 7 cryovials with the following amounts, Cryovial 1-1: 50 mg, 1-2 to 1-5: 25 mg, then

splitting the rest evenly into cryovials 1-6 and 1-7.

13. Dust control a. NIST dust – scoop ~200 mg (size of a quarter) using a stainless steel depyrogenized spatula,

into a dust collection bottle b. When starting to vacuum, open NIST container in the home during the first 5 minute of

vacuuming then close it. c. Store - transport at room temperature d. Shipped to Tedd Konya (as above) e. Aliquot into 1 cryovial




Data management and data entry

Data are collected on either online or by paper based questionnaire forms developed by the National Coordinating Centre. The original questionnaires will be scanned and uploaded in the web based electronic data capture system, HealthDiary. The original questionnaire will remain at the site and the scanned copy will be downloaded by the NCC for data entry except for the allergy skin testing form and food frequency questionnaire. As a result of data review process, corrections or clarifications may be required. The data supervisor at the NCC will send to the site a Data Query. Queries need to be resolved within a reasonable time period. After all data have been collected and all queries have been answered, the data will be entered into HealthDiary by the data team at the NCC.

The food frequency questionnaire and the allergy skin testing sheets are to be photocopied at the site and the original sent to NCC through the on line Fed Ex system.

Shipping Biological Samples to Central Storage

Samples will be collected by the sites. These samples will be processed and aliquoted according to the standard operating procedures and aliquoted into 2 mL Corning cryovials. These cryovials will be stored in cryoboxes, each with a capacity to store 81 cryovials, and kept at -80 degrees temporarily at the site until 5 cryoboxes are full.

Dry Shippers will be used for the transport of all frozen biological specimens collected for the CHILD Study from each coordinator and blood laboratory to the Central Storage Facility in Hamilton, Ontario.

Documents

Canadian Healthy Infant Longitudinal Development (CHILD) …