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Assessment of a multisite standardized biospecimen collection protocol for immune phenotyping in neurodevelopmental disorders – Scientific Reports


Participants

Participants were recruited through the POND Network (pond-network.ca) at 5 sites: Holland Bloorview Kids Rehabilitation Hospital, Toronto; The Hospital for Sick Children, Toronto; Queens University, Kingston; Lawson Health Research Institute, London; and McMaster Children’s Hospital, Hamilton. Primary caregivers and participants provided either written informed consent or assent after a complete description of the study was provided in accordance with the Tri Council Policy Statement, the Declaration of Helsinki, and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH)–Good Clinical Practice (GCP) guidelines and institutional policy. Approval for collection and sample banking were obtained from the research ethics boards of each institution including Holland Bloorview Research Ethics Board, Sick Kids Research Ethics Board, General Research Ethics Board at Queen’s University, Western University’s Health Sciences Ethics Board, and Hamilton Integrated Research Ethics Board.

A sub-cohort of children and young adults with NDDs and TD participants that contributed immune samples between January 2017 and July 2019 were enrolled. Eligible participants were between the ages of 1 and 23 years with a diagnosis including ASD, ADHD, OCD, and ID, in addition to TD controls. Diagnoses were confirmed using standardized assessment tools. ASD was confirmed using the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview-Revised (ADI-R), ADHD was confirmed using the Parent Interview for Child Symptoms (PICS), and OCD was confirmed using the Children’s Yale-Brown Obsessive–Compulsive Scale (CY-BOCS)33,34,35,36. Exclusion criteria for the immune platform were as follows: fever greater than 38 °C in the preceding 3 days, vaccination in the preceding 7 days, oral or intravenous steroids in the preceding 14 days, oral or intravenous antibiotics in the preceding 21 days, chemotherapy, intravenous immunoglobulins of monoclonal antibodies in the preceding 12 months and known primary or secondary immunodeficiency.

Immune metrics

The immune medical history of participants was assessed using the Child Immune History Questionnaire (Supplemental File 1) to collect information about the participants’ immune status. The questions were grouped into three sections for analysis: autoimmune/allergy, childhood infection, and medication. Autoimmune/allergy-related questions ascertained if the participant had environmental, food, or other allergies, asthma, autoimmune cytopenia, or other autoimmune diseases. The childhood infection questions collected information about the participant’s history of infections, including oral thrush, ear infections, skin warts, chickenpox, or other self-described “significant” infections that required antibiotic use. Lastly, the medication section questions determined if the participant had previously used immunosuppressants or oral/IV steroids. Sections were considered incomplete if at least one question was left blank. Participants with incomplete responses were removed using case-wise deletion.

Biospecimen collection

Venous blood was drawn from participants: (1) in a 10 mL heparinized blood tube for the isolation of PBMCs, granulocytes and plasma (367,874, Becton, Dickinson and Company, Mississauga, ON); (2) in two 5 mL silica-coated serum tubes for the isolation of serum (366,434 G, Becton, Dickinson and Company, Mississauga, ON); (3) 4 mL EDTA treated tube for complete blood counts (367,844, Becton, Dickinson and Company, Mississauga, ON). Anonymized samples (1 and 2) were transported from the blood collection site to the nearest processing lab, where biospecimens were cryopreserved. The EDTA samples (3) were transported to 3rd party medical labs for CBC analysis. The date and time of the blood draw were recorded. Biospecimen samples were processed with minimal delay after collection. Samples collected in Hamilton and London areas were immediately transported to the processing lab at McMaster University. Samples collected in Toronto were immediately transported to the processing lab at The Hospital for Sick Children. Samples collected in Kingston were immediately processed in the lab on site.

PBMC and granulocyte isolation

PBMCs and granulocytes were isolated using Ficoll Paque Plus (17-1440-03, GE Healthcare, Mississauga, ON). Ficoll Paque Plus was pipetted into the bottom of the processing tube, below the blood, according to the manufacturer’s protocol with minor alterations. Blood layers were separated by centrifugation at 514 RCF for 25 min with the brake off. PBMCs were collected from the buffy coat interphase and washed with phosphate buffered saline (PBS) (see Supplementary Protocol 1). Following PBMC isolation, RBCs and granulocytes were isolated from the Ficoll Paque separation. Red blood cells were lysed, and pelleted granulocytes were washed in PBS. Concentrations of PBMCs and granulocytes were determined using a hemocytometer. Cells were resuspended in 10% dimethyl sulfoxide (DMSO) (Caledon, 4100-1-05) human AB (hAB) (VWR, CA45001-062) serum freezing media and frozen with 1 mL aliquots of 5–10 × 106 cells/mL per vial. PBMCs and granulocytes were frozen at − 80 °C using a cell freezer for storage37.

Biospecimen banking

Samples were stored locally and shipped to the POND McMaster Biobank (Hamilton, ON). Documentation of the biospecimen collection and aliquots was submitted electronically and in paper format with each shipment. Samples were stored in the POND McMaster Biobank at St. Joseph’s Healthcare in Hamilton, ON. Biobank sample IDs were assigned to individual blood sample aliquots, and an up-to-date inventory of samples is maintained. Upon research proposal approval from platform leads, POND associated labs and collaborators request samples to be sent for research purposes.

Flow cytometry and PBMC viability measures

A two-step sample viability check was used for quality control. Single aliquots of PBMCs were thawed in warm RPMI 1640 medium (SLM-240-B, Sigma-Aldrich, Oakville, ON). In step one, live cells were counted on a hemocytometer using the Trypan Blue exclusion test38. Samples with less than or equal to 5 × 105 cells measured using the Trypan Blue exclusion test were considered to fail quality control. Samples that failed the Trypan Blue exclusion test were not used in subsequent flow cytometry. Samples that passed the trypan blue exclusion test criteria proceeded to step two where they were washed with fluorescence-activated cell sorting (FACS) wash (0.5% (w/v) BSA, 5 mM EDTA (pH 7.4–7.6), for 500 mL 2.5 g BSA, 5 mL of 0.5 M EDTA) prior to staining. 5 × 105 cells were stained with Zombie Red Fixable Viability Kit (423,109, Biolegend, Cedarlane, Burlington, ON) and fluorochrome-conjugated antibodies against CD45 (BV510), each diluted 1:100 in a final volume of 50 µL of FACS wash, for 30 min at room temperature. Cells were then fixed using 1-step Fix/Lyse Solution (1X) for 10 min. PBMCs were washed with FACS wash and filtered through 0.45 µm mesh to ensure single-cell suspension before analysis on an LSR II flow cytometer (Becton, Dickinson and Company, Mississauga, ON). Cells stained Zombie Red positive were considered dead cells, while Zombie Red negative cells were considered viable. Samples with a Zombie Red viability of 70% or less were considered to fail quality control.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 8.4.3. Analyses were conducted to assess differences in responses to the Child Immune Questionnaire, and differences between samples that passed and failed quality control. Fisher’s Exact Tests were used to compare responses to the Child Immune History Questionnaire between TD and NDD, and to compare quality control pass rate by site. The Shapiro–Wilk Goodness-of-fit test was applied to continuous variables to assess normality. If the data were normally distributed, then the parametric t-test or one-way ANOVA was used. If the data was not normally distributed, then the non-parametric Mann–Whitney U test was used. All tests were 2-tailed, and p < 0.05 was considered significant.



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