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Multisite collection and preservation of peripheral blood mononuclear cells (PBMCs) for centralized analysis is an indispensable strategy for large cohort immune phenotyping studies. However, the absence of cross-site standardized protocols introduces unnecessary sample variance. Here we describe the protocol implemented by the Province of Ontario Neurodevelopmental Disorders (POND) Network's immune platform for the multisite collection, processing, and cryopreservation of PBMCs. We outline quality control standards and evaluate the performance of our PBMC processing and storage protocol. We also describe the Child Immune History Questionnaire results, an assessment tool evaluating pre-existing immune conditions in children with neurodevelopmental disorders (NDDs). Cell viability was assessed in samples from 178 participants based on strict quality control criteria. Overall, 83.1% of samples passed quality control standards. Samples collected and processed at the same site had higher quality control pass rates than samples that were collected and subsequently shipped to another site for processing. We investigated if freezer time impacted sample viability and found no difference in mean freezer time between samples that passed and failed quality control. The Child Immune History Questionnaire had a response rate of 87.1%. The described protocol produces viable samples that may be used in future immune phenotyping experiments.

Perhaps it is their scale and diversity-they inhabit most Yellowstone region waters and come in sizes ranging from #10 in Montana's Hebgen and Earthquake lakes to the tiny #20-#26 olive and black midges emerging six months of the season on the Madison, Yellowstone, Henry's Fork, and Paradise Valley spring creeks. EMERGER STRATEGIES Because midges emerge all year, you need to be prepared to find fish feeding on them, and have properly designed fly patterns on hand. In late February last winter, I met a couple who had moved to the Madison River Valley to cross-country ski, create software, and fish. Observing a trout's head slowly and completely coming out of the water perpendicular to the surface is a solid clue that the fish is taking single emerging midges.