CHILD Banner

METHODS

CHILD is generating important new data on maternal and infant health in Canada, serves as a national resource for multiple research endeavours, and will continue to be a national and international maternal-child health resource for decades to come. A national, general population- based, longitudinal birth cohort study provides the optimum setting for study of the multiple, interrelated, time-dependent determinants of allergy and asthma.

The study will provide knowledge needed to determine how to reduce the prevalence and impact of these diseases. A made-in-Canada longitudinal birth cohort study is of critical importance not only because of the impact of asthma and allergy in the population and the need to study the link between environment and children's health, but also for its enormous possibilities in many other areas of Canadian research. These areas include childhood obesity, diabetes mellitus, early cardiovascular risk factors. Key methods for each component of the CHILD study are listed below. Specific lead investigators will be responsible for quality assurance and quality control. See CHILD Methods presented in detail following the chart for further details relating to the methods.

Summary of CHILD Methods by Topic

Methods Summary
Clinical assessments Clinical assessment of infants at 1, 3 and 5 years in entire cohort. Birth anthropometric data extracted from birth chart. Anthropometric measures made at 3 month home visit. Anthropometrics, skin testing and clinical exam occur in a clinic setting (1, 3 and 5 years).
Immune Function

Vancouver and Toronto sub-cohorts: innate immune function is assessed using cord blood samples.

Adaptive immune function will be assessed in child blood samples collected at 1 and 5 years using high-throughput, multiplexed analyses.

Nutrition

Pre-and post-natal nutrition in mothers and children collected by questionnaires and FFQs.

Breast-feeding questionnaires and breast milk collected (3 months).

Microbiome Child stool is collected at birth (meconium), 3 months and 1 year for microbiota profiling.
Respiratory Infections Toronto sub-cohort: respiratory symptom diary, biannual time -activity logs, health questionnaires; nasal swabs (3 months, at 1 year and during acute viral lower respiratory infection in first year). Full CHILD cohort: nasal swabs (3 months, 1 year).
Pulmonary Function Testing Toronto sub-cohort: lung function assessed using raised volume rapid thoraco-abdominal compression and multiple breath washout (birth, 3 months, 1 year and 2 years), and spirometry (4 years). Entire CHILD cohort: lung function assessed using spirometry 5 years.
Psychosocial, Stress, SES Standardized detailed pre-and post-natal assessments focused on family's socioeconomic (SES) status, stress present in the home, and the quality of the parent-child relationship.
Environmental exposure assessments

Comprehensive questionnaires (3 months, 1, 3 and 5 years) along with biannual updates (time-activity, smoking and changes in residence).

Home assessment at 3 months with dust sampling (for analysis of allergens, endotoxin, mould, air polution, phthalates) and questionnaires;

Smoke exposure assessed by questionnaires, urinary cotinine measurements.

Genetics, Epigenetics

Maternal blood is collected prenatally.

Paternal blood is collected when available.

Child cord blood is collected at birth and venous blood is collected at 1 and 5 years

 

 

SPECIFIC METHOD DETAILS - WORK-IN-PROGRESS

Study subjects were required to meet the following inclusion and exclusion criteria:

 

Inclusion Criteria:
[1] pregnant women aged 18 years and older (19 in Vancouver);
[2] residence in reasonable proximity to the delivery hospital;
[3] able to read, write, and speak English;
[4] willing to provide informed consent;
[5] willing to consent to cord blood collection for the study;
[6] planning to give birth at a designated recruitment centre participating hospital;
[7] infants born at or after 35 weeks;
[8] able to provide name, address and telephone numbers of two alternate contact individuals.

 

Exclusion Criteria:
[1] children born with major congenital abnormalities or respiratory distress syndrome (RDS);
[2] expectation of moving away from a recruitment area within 1 year;
[3] children of multiple births;
[4] children resulting from in vitro fertilization;
[5] children who will not spend at least 80% of nights in the index home.
[6] children born before 35 weeks gestation

 

Final enrolment is confirmed postpartum. Paternal participation was not mandatory.

CHILD DATA And TASK

Primary Outcome = Doctor Diagnosed Asthma at Age 5 Years:

 

Each child will be assessed by a physician who is an asthma expert using a semi-structured interview with the child and parents, based in part on the International Study of Asthma and Allergy in Children (ISAAC) questionnaire. Individuals will be classified as definite, probable, possible, or no asthma. We have previously validated expert physician diagnosis in two cohorts: at age 7 in the Canadian Asthma Primary Prevention Study against the combination of the primary ISAAC question (Has your child wheezed [a whistling noise in the chest] in the past 12 months?) plus airway hyperresponsiveness and in children 8 years of age when expert physician diagnosis was compared with healthcare data base definition.

 

Objective Evidence to be Used in Assessing the Primary Outcome:


ATOPY

Atopy is being assessed at 1, 3 and 5 years of age using skin testing. Highly standardized ALK allergens include tree, grass and weed pollens, cat, dog, D. pteronyssinus, D. farinae, cockroach, Alternaria, Cladosporium, Aspergillus, and Penicillium. Food allergens including cow's milk, egg white, soy, wheat and peanut allergens are also tested, using trained staff and Standardized Operating Procedures for uniformity across all sites.

 

SPIROMETRY

At 5 years, all children will perform forced expiratory flow-volume loops using American Thoracic Society (ATS) established standard techniques and test criteria. Success with this technique has been over 80% in 5-year-old children.

 

SECONDARY OUTCOMES:

Multiple intermediate outcomes are being assessed as the children develop. These include early manifestations of atopy including atopic dermatitis, food intolerance or allergy, rhinitis, infant wheezing syndromes, and biological markers including development of skin sensitization, serum IgE, and pulmonary function measurements. Some of these outcomes will be available at age 1 year, and others by age 3 years.

 

 

The comprehensive longitudinal assessments within CHILD allow application of more sophisticated statistical modeling whereby critical periods during which genetic, environmental, biological and social risk factors have the greatest impact on the subsequent development of asthma, and the pathways by which these factors interact and mediate the development of asthma are explored. We propose a combination of statistical analyses to better understand the early life determinants of asthma.

 

  1. CHILD investigators will define clusters of asthma phenotypes based on a combination of; repeatedly collected symptoms data (wheezing at 3mo, 6mo, 1 year), objective measures of allergic response (IgE levels, skin prick testing at 1 , 3 , 5 years) and, objective measures of lung function (infant pulmonary function tests (3, 12, 18 months) and preschool spirometry (3, 5 years)). We will use longitudinal latent class analysis whereby patterns in these measures are grouped together for children that exhibit similar patterns. Resulting trajectories will be used to model risk factors distinguishing between different phenotypes.

  2.  

  3. CHILD researchers will then use hierarchal modeling to identify the factors which best distinguish between the proposed trajectories. Multi-level models will allow investigation of time dependent variables (viral infections, environmental exposures, nutrition) and time-independent variables (gender, family history) thereby identifying risk factors and timing of exposures which have the greatest influence on the development of asthma. In a cohort of 3000 at 5 years, with a minimum of two post-natal observations per child, assuming 10% attrition, and adjusting for 10 simultaneous exposures and categorical outcomes, we will have greater than 80% power to detect associations with the outcome.

  4.  

  5. The independent risk factors and the timing of exposures for each asthma phenotype will subsequently be used to propose pathways by which genetic, environmental, biological and social risk factors interact. CHILD investigators will use structural equation modeling to test alternative explanations for how the proposed factors within the hypothesis influence the development of asthma. A series of structural equation models (SEM), ranging from simple independent associations to saturated multidimensional associations, will be compared to determine which pathway best explains different sequences of events that lead to asthma. SEMs are a series of multiple linear equations used to specify causal relationship between observed and latent variables, which are not directly measured, but rather inferred from several measured variables. In addition to the factors derived from the multi-level analysis, we will use factor analysis to identify latent variables which best describe these underlying constructs.

 

In addition to several specific pathways, we will assess the over-arching hypothesis and determine the contribution of genetics, socioeconomic status, immunity, environment and nutrition, each as latent variables, to the development of asthma. Preliminary analysis will identify the measured variables that best explain the variability and error for each of the latent variables.

 

Several statistics (including chi-squared test, non-normal fit index, comparative fit index, root mean square error) will be used to evaluate the differences between two models. The method allows for the comparison of the validity of competing explanations that specify different patterns of relations among variables of interest. With a sample size of 3000, of which some 300 will develop clinically defined asthma by age 5, we will have more than 80% power to investigate approximately 6 latent variables in a pathway at one time, with 4 or more measured variables contributing to each latent variable. These complementary methods utilizing the rich databases of CHILD will provide further insight into timing of exposures and inter-relationships between factors that predispose a child to develop allergy and asthma.

 

For many of the intermediate outcomes, where prevalence rates are higher than the 10% expected for doctor-diagnosed asthma (e.g. allergic sensitization 30-40%, atopic dermatitis 15-20%, early childhood wheezing 40-50%) a sample size of 3000 provides over 80% power for detecting significant interactions and associations. To ensure all statistical analyses are carefully developed and validated based on reasonable hypotheses, CHILD statistics and publications committees will work with lead investigators to review analysis plans, and provide necessary assistance (including examining model assumptions and feasibilities of analyses).

The result of this series of tests will be a list of immune parameters with reliable statistical evidence for association with risk for asthma. Multivariate logistic regression models will then be used to explore the joint effects and possible interactions amongst the various innate immune parameters on risk for asthma or atopy. Host genetics and environmental exposures will then be added to the best multivariate model to assess the extent to which the observed gradient between host vs. environment in risk for asthma and atopy can be explained by the measured innate immune response profile. Finally, the most promising (i.e. most significantly associated with asthma or atopy) innate parameters will be included in the stepwise analysis of the entire data set obtained in CHILD.

 

INNATE IMMUNE EFFECTOR FUNCTION:

In addition to a complete blood count for eosinophil numbers (done on fresh blood), we will 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 1and 5 years of age. Besides B-cell serum immunoglobulin levels (including IgE, IgG4), several T-cell functional assays will be 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 and NK/T 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 will allow 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 will be 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 will be 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 will be 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 will be run in the same experiment.

MATERNAL DIET DURING PREGNANCY

Prenatal and postnatal nutrition are assessed in addition to specific measures related to breast-feeding and breast milk. A Food Frequency Questionnaire (FFQ) developed by the nutritional epidemiologists at the Fred Hutchinson Cancer Research Center in Seattle, WA and based on the questionnaires used in two large NIH funded studies, the Selenium and Vitamin E Cancer Prevention Trial (SELECT) and the VITamins and Lifestyle study (VITAL) was modified to reflect Canadian ethnic food choices. In addition, the database developed by the University of Minnesota Nutrition Data Systems for Research (NDSR) for data entry and nutrient analysis was "Canadianized" for use in CHILD. This questionnaire is a self-administered FFQ asking pregnant mothers to report the frequency of consumption and portion size of approximately 175 line items during their current pregnancy. These questionnaires are completed by the mother at the time of enrolment.

 

BREAST-FEEDING ASSESSMENT:

Breast-feeding status is assessed by questionnaire at 3, 6, 12 and 18 months of age. Items on the questionnaire include duration of exclusive breast-feeding, age of first supplementation, and the age of first introduction of solids.

 

BREAST MILK SAMPLE:

Breast-feeding mothers are asked to collect (by hand express or pump) and refrigerate 10 ml of breast milk in a collection tube within 24 hours of the 3-month home inspection visit, when it is collected and transported in a cooler with a cold pack to the central laboratory facility, centrifuged and the aqueous and lipid phases separated, aliquoted, and stored at -80oC for subsequent analyses. Hand expressed breast milk is collected from 1 day prior to the visit to include at least 2 separate feedings with milk collected before and after feeding. A total of 10 mls of breast is dispensed by study staff into 6 x 1.5 ml aliquots and frozen within 8 hours.

 

CHILDHOOD DIET:

Child diet will be assessed using the Hutchinson FFQ at 3 and 5 years of age. Anthropometric measures: Weight and length/height are recorded at birth (from the delivery chart), 3 months, 1 year, 3 years and 5 years of age.

 

SAMPLE COLLECTION :

Meconium and fecal samples will be collected from infants at 3 months of age and repeated at 1 year of age. Samples consisting of 5–10 g stool (1–2 g for meconium) will be collected aseptically from a freshly soiled diaper using a sterile spatula, divided into aliquots and stored at –80oC in sterile containers. One frozen fecal sample will be thawed and be homogenized in phosphate buffered saline and the homogenate will be divided into two aliquots, one of which will be frozen for later analysis of fecal IgA.

ANTIBIOTIC EXPOSURES MEASUREMENT

Antibiotic and other prescription medication records for infants from provincial databases and data collection instruments used in the cohort. To protect patient confidentiality, all prescription records in provincial databases are anonymized and contain encrypted versions of the health identification number. Linkage between the prescription data, microbiota profiles and survey data from the CHILD study will be achieved through a CHILD identification number, which has been attached to the encrypted health identification number. The child's health identification number will be encrypted by the respective provincial health departments for data linkage purposes. Parent permission for this data linkage and the child's health identification number has been obtained on the consent form. The wording on the consent from to request this access was approved by the Human Research Ethics boards at the site-specific universities. Province-specific processes will be followed to receive approval for database access, to achieve the encryption of the health identification number and to obtain the prescription data sets.

Other environmental exposure measures: Variables will be created to measure the events around birth including mode of delivery (vaginal unassisted, forceps and extraction; elective or emergency elective Caesarean section), premature rupture of membranes, gestational age, birth weight and type of feeding (breast, formula or both). Maternal use of antibiotics and other medications (e.g. probiotic supplements) and diet during pregnancy are determined from questionnaires during pregnancy at 18 and 36 weeks. During the home visit at 3 months of age and clinic visit at one year, a child health questionnaire is administered to inquire about type and duration of feeding (breast, formula, both), and if applicable, the name of the formula and amount used to supplement breast-feeding These questionnaires also query infections, episodes of wheezing, and medication use (over-the-counter, physician-provided samples, prescription) and its indication.

 

 

Respiratory tract infections are being assessed in the full cohort and in more detail in the Toronto sub-cohort.

 

DIRECT VIRAL DETECTION: A nasal swab sample, collected in all children at 3 months and 1 year of age, is separated into 3 aliquots and frozen at -80oC. Subsequently, with separate funding, nucleic acids will be extracted from the aliquots 1 and 2 using the bioMérieux MiniMag extractor (bioMérieux, Marcyl'Etoile, France). The ID-TagTM respiratory viral panel (RVP) (Tm Bioscience [Luminex] Corporation, Toronto, Ontario) bead-based microarray method will be used to detect the presence of common respiratory viruses (influenza A and B, parainfluenza types 1-4, RSV, enterovirus/rhinovirus, coronaviruses, metapneumovirus and adenovirus) in aliquot 1. For samples which are negative by the RVP, aliquot 2 will be tested on the Virochip microarray which tests for all known viruses simultaneously. The third aliquot will be used for virus-specific PCR tests to confirm the results of the RVP or Virochip.

 

In the Toronto sub-cohort, viral infections are being monitored and captured by two methods:

DIARY - A respiratory symptom diary is given to the mother at birth, and information transferred to study records at each follow-up visit. Exposure to other children is assessed through bi-annual time/activity logs that include daycare questions, age when the child first started daycare, and hours/days spent at the daycare. Other infections (urinary tract, gastrointestinal, and otitis media) are obtained by questionnaire every 6 months.

 

ASSESSMENT OF ACUTE VIRAL INFECTIONS - Acute infections in the first year of life are reported by parents to the Toronto CHILD study team via a respiratory infection call hotline. A symptom assessment is administered via standardized questionnaire by a nurse. Lower respiratory infections of sufficient severity as assessed by questionnaire will result in a clinical assessment of the child by a trained nurse and administration of an acute viral swab.

 

The CHILD study is uniquely designed to address many of the unanswered questions regarding lung function in infancy and development of asthma.

 

In the Toronto sub-cohort, infant pulmonary function will be measured using state of the art methods. Raised volume rapid thoraco-abdominal compression technique has been shown to be more sensitive at detecting lower airway obstruction than older methods used in earlier studies. Furthermore, novel methods such as multiple breath washout, which is a more sensitive measure of small airway obstruction are being utilized. Inflammation measured using tidal breathing exhaled nitric oxide is assessed at birth and at all infant lung function assessments.

 

Furthermore in the CHILD Toronto sub-cohort we will be taking viral swabs not only at screening visits at 3 months and 12 months but also at times of acute viral lower respiratory infection in the first year of life. Our study is the first to prospectively study the role of RSV and other viruses in the development of wheezy illness in a general population cohort by looking at immune, lung function and airway inflammatory markers not only post infection but, most importantly, pre-infection. See a video of the Infant Pulmonary Function Lung Assessment at SickKids

 

 

SOCIO_ECONOMIC STATUS

Each method of characterizing SES will be evaluated independently and jointly within the analyses:


SUBJECTIVE SES: Parents are asked to place their household (relative to their community) on a ladder using the MacArthur Scale of Subjective Social Status.


RESOURCE - BASED SES: Parents are asked about family assets (e.g. income, savings, housing status (rent/own), number of bedrooms, and number of cars). Responses will be standardized (z-scored for the sample) and the z-scores summed to create one composite asset score.


PRESTIGE - BASED SES: Years of education and highest educational degree attained for each parent is recorded. Parental job characteristics are also recorded and will be used to index occupational prestige using standardized Canadian coding systems. Psychological stress will be assessed along a number of dimensions, both prenatally (at 18 and 36 weeks gestational age (GA) and annually from the time of the index child's birth:


Chronic stress in multiple life domains will be assessed: the nuclear family, the broader family/social network, in work and/or academic settings, or from health problems in self/family. Acute life events in family, social network, work life, health domains will be assessed Global perceived stress, reflecting the extent to which life is viewed as unmanageable, and unpredictable, will also be assessed. Multiple aspects of maternal mood will be measured, including symptoms of depression, and positive and negative mood states. Factors that can protect against stress, or accentuate its impact, will also be measured. These factors include social support, social capital, marital quality, and social integration.

The following standardized instruments are currently being employed in the CHILD Study: NOTE: Post-natal questionnaire sets are identical to the 36 week prenatal set with the addition of the Parenting Questionnaire:

 

Pre-natal: 18-34 Weeks GA

  1. Profile of Mood States (POMS)
  2. Perceived Stress Scales (PSS)
  3. Centre for Epidemiological Studies - Depression Scale (CES-D)

 

Pre-natal Assessment: 36 (± 4 ) Weeks GA

  1. Chronic Stressor Evaluation
  2. Recent Life Events (RLE)
  3. Profile of Mood States (POMS)
  4. Perceived Stress Scales (PSS)
  5. Centre for Epidemiological Studies - Depression Scale (CES-D)
  6. Dyadic Adjustment Scale (DAS)
  7. Interpersonal Support Evaluation List (ISEL)
  8. Socio Economic Status Index (SES)
  9. MacArthur Subjective Community Standing Scale
  10. MacArthur Subjective Socioeconomic Standing Scale

Post-natal: 1 year and subsequently

  1. Chronic Stressor Evaluation
  2. Recent Life Events (RLE)
  3. Parenting Questionnaires (PQ)
  4. Profile of Mood States (POMS)
  5. Perceived Stress Scales (PSS)
  6. Centre for Epidemiological Studies - Depression Scale (CES-D)
  7. Dyadic Adjustment Scale (DAS)
  8. Interpersonal Support Evaluation List (ISEL)
  9. Socio Economic Status Index (SES)
  10. MacArthur Subjective Community Standing Scale
  11. MacArthur Subjective Socioeconomic Standing Scale
 

Indoor and outdoor environmental exposures are assessed by multiple methods, including questionnaires, home inspections, dust sampling for multiple allergens and pollutants and outdoor exposure modeling.

 

Questionnaires:

A comprehensive baseline (during pregnancy) and comprehensive updates at 3 months, 1 year, 3 years and 5 years of age are administered regarding address, housing structure, function, condition, maintenance history and cleaning habits, presence and use of attached garage, renovations, source and extent of dampness indicators, mould growth, new furnishings, appliance and household cleaner emissions, presence and type of air conditioning and cleaning, conditions of use, etc. Questions related to the child's and families' time activity include time spent in different rooms in the house and indoors vs. outdoors, time in transit, mode of transport, frequency/duration of visits to daycare, indoor pools, other microenvironments, exposure to smoke, etc. Shorter followup questionnaires are administered at 6 and 18 months, 2, 2.5 and 4 years of age and focus mostly on time activity of the child and major changes such as home renovations. A list of the questionnaires can be found following this link.

 

Environmental Tobacco Smoke:

Environmental tobacco smoke exposure is assessed on the comprehensive prenatal and postnatal questionnaires with validation in a subset by cord blood and 3 month urinary cotinine measurements.

 

Home Assessment:

When the infant is approximately 3 months old, RAs trained by Canada Mortgage and Housing Corporation (CMHC) and lead CHILD environment investigators visit the home to measure geographical coordinates (GPS), 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, furnishings, ventilation (including presence of air-conditioning and type of heating/cooling), humidification, microbial and chemical contaminant burdens, and basement conditions. CHILD has published a 158 page Home Assessment Training manual and Interpretive Guide for use in the study. A sample of this document can be found following this link.

 

Dust Sample Collection:

During the home assessment, a dust sample is collected from a 2 (sqmetres) area of flooring using a standardized consumer model vacuum cleaner fitted with a specially designed 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, as well as markers of indoor chemicals (e.g., phthalates) and infiltration of outdoor traffic particles. The dust sample is collected from the child's bedroom (bed and floor) and the living space where the child spends the most time. Total dust weight and fine dust weight is determined and multiple aliquots are prepared and stored at -80ºC. At present, analytical protocols use less than 100 mg of dust and thus a considerable amount is archived for future considerations.

 

Dust sample analysis for allergens:

One aliquot of fine dust (at least 50 mg) is extracted at room temperature for 2 hr using a standard protocol. After analysis for endoxin and B glucan the remainder is used for allergen analysis with standard ELISA protocols for Der p 1, Der f 1, Mite group 2, Fel d 1, Can f 1, Mus m 1, Rat n 1, Bla g 2, Alt a 1, Bet v 1.

 

Outdoor Exposure Modeling:

Family residence history and time-activity information is collected through the questionnaires. Outdoor air exposure modeling will be based upon available monitoring data and city-specific land-use regression (LUR) models focused mainly on NO2. More advanced data fusion/assimilation methodologies using all available data are constantly evolving within the CHILD/AllerGen network and Canadian exposure research community. This includes government monitoring data, LUR output and 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 will include models that take into account outdoor-indoor exchange of pollutants given the amount of time the children spend indoors.

 

Urine Sample Collection:

Urine is collected at the 3 month home visit and at the 1, 3 and 5 year clinics. At the beginning of the 3 month and 1 year visits, a tegaderm liner is placed in the child's diaper followed by the placement of cotton pads to absorb the urine. At the end of the visit (approximately 2 hours) the urine is collected by placing the cotton pads in a large syringe and squeezing out 0.5 ml of urine into 6 pre-labelled cryovials. The last drop is placed on a refractometer and the specific gravity recorded. Samples are kept cold and frozen within 8 hours. At 3 years, urine is collected in a similar manner for those children not yet toilet trained. For those who are toilet trained, a potty, with an appropriate insert, will be provided in the clinic. A volume of 1.5 ml of urine is stored in each of 6 cryovials.

 

Breast Milk Sample Collection:

Breast milk is collected from mothers using a collection jar provided with instructions prior to the 3 month home visit. Hand expressed breast milk is collected from 1 day prior to the visit to include at least 2 separate feedings with milk collected before and after feeding. A total of 10 mls of breast is dispensed by study staff into 6 x 1.5 ml aliquots and frozen within 8 hours.

 

Stool Sample Collection:

Meconium is collected at birth, by a nurse who transfers the meconium from the baby's diaper into a disposable container. Stool is collected by mothers who provide a soiled diaper at the 3 month and 1 year visits. The meconium/stool specimen collected is divided equally into 4 preweighed cryovials and frozen within 8 hours.

Aliquots are shipped weekly to the the Clinical Research and Clinical Trials Laboratory (CRCTL) for long term liquid nitrogen storage.

Gene-environment interactions identified in the previous literature and in the ongoing metaanalyses in consortia such as EVE and GABRIEL, will be examined in the CHILD participants. In addition, we will test polymorphisms in genes that have been robustly associated with asthma in published GWAS i.e. those showing overlap between the large-scale studies. For the gene-environment interactions we will test those polymorphisms in the Toll-like receptor genes previously associated with asthma and related traits.

Genetic and environmental factors will be associated with the outcome of physician-diagnosed asthma at 5 years of age and we expect that ~10% of the CHILD participants. We will also utilize wheeze as an outcome that is expected to have a prevalence of 17-20% at 5 years of age. Additional phenotypes will be other allergic diseases such as atopic dermatitis.

Blood samples are being collected in EDTA tubes and buffy coat white blood cells are stored frozen until DNA extraction. Each clinical phenotype will be investigated using logistic regression with genotypes considered under an additive model. We will test for gene-environment interactions by including a product term in these models, with the null hypothesis that each potential risk factor (genotypes and environmental exposures) is independent. Due to the large number of comparisons we will estimate the false discovery rate i.e. the proportion of false-positive results in the set of rejected hypotheses. Gene-environment interactions will be tested in the entire CHILD population including ethnicity as a covariate and in the Caucasians only to guard against false positive associations.

 

In order to standardize the methods used at different sites, CHILD developed Standard Operating Procedures (SOPs) and then trained people. The following is a list of SOPs developed for the CHILD study.

 

CHILD STANDARD OPERATING PROCEDURES

1. Cord Blood Collection
2. Cord Blood Processing
3. Meconium/Stool Collection
4. Nasal Secretions Collection
5. Saliva Collection
6. Home Visit/Dust Collection
7. Urine Collection
8. Breast Milk Collection
9. Parental Venipuncture
10. Parental Spirometry
11. Parental Allergy Skin Testing
12. Children's Allergy Skin Testing
13. Child's blood processing at 1 Year
14. Dry Vapour Shipper protocol
15. Dry Ice Shipping protocol
16. 1 Year Child Clinical Assessment
17. 3 Year Child Clinical Assessment (includes spirometry and blood pressure measurement)

 

Other reference material developed:


1. CHILD Study Protocol
2. Prenatal maternal FFQ + booklet ("Canadianized" Hutchinson GNA questionnaire and database)
3. Child Birth Chart Extraction Manual
4. Medications and Supplements prompt list (for Allergy skin tests)
5. Home Visit Information for Mother Booklet
6. Home Assessment Interpretive Guide (158pg)
7. Home Volume Worksheet
8. Dust Collection and Boxing Manual
9. Tablet Computer User Reference Manual
10. HealthDiary Sample Recording Manual
11. HealthDiary User Reference Manual
12. HealthDiary Pivot Grid Training Manual
13. Data Entry Guidelines
14. Data Dictionary

 

 

CHILD Logo and Tag