Assessment of a multisite standardized biospecimen collection protocol for immune phenotyping in neurodevelopmental disorders.

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.

Test-retest reliability and validity of body composition methods in adults.

Cost-effective and efficient body composition measurement devices that are reliable and valid are necessary for identifying health risk as well as for understanding the effectiveness of lifestyle interventions. The objective of this study was to evaluate the test-retest reliability and validity of three body composition measurement devices. Forty-nine adults (mean age (SD) = 31.5 (10.7) y; BMI = 23.5 (3.0) kg/m2 ) completed a reference air displacement plethysmography (ADP) measure, and duplicate measures using skinfold callipers (Lange), ultrasound (BodyMetrix A-mode) and a 3-dimensional photonic scanner (3DPS; Fit3D ProScanner). Skinfold thickness was measured at seven sites using callipers and ultrasound; percent body fat (%BF) was then estimated using population-specific algorithms. The 3DPS was used to measure body circumferences, and then %BF was estimated using its beta-software. While skinfold callipers showed poor absolute reliability (mean differences (Δ) [95% CI] = 0.54% [0.22, 0.87], standard error of measurement (SEM) = 0.63%), ultrasound and the 3DPS showed excellent absolute (Δ = 0.17% [-0.25, 0.58], SEM = 0.78%; and Δ = -0.01% [-0.43, 0.40], SEM = 0.67%, respectively) and relative reliability (ICC2,1  = 0.988 [0.979, 0.993]; and ICC2,1  = 0.983 [0.968, 0.991], respectively). Compared to ADP (n = 43), skinfold callipers underestimated %BF (Δ = -4.53 [-7.72, -1.34]; p = 0.003), while ultrasound (Δ = -0.32 [-3.51, 2.87]; p = 0.99) and the 3DPS (Δ = 1.06 [-2.12. 4.26]; p = 0.77) were not significantly different. Bland-Altman plots showed a minimal bias of ultrasound [95% limit of agreement (LOA) = -7.87, 7.23] and the 3DPS [95% LOA = -6.66, 8.79]. In conclusion, estimating %BF from subcutaneous fat measurements using ultrasound and body circumferences using a 3DPS may be reliable and valid methods that require minimal technician expertise.

The contribution of muscarinic-receptor-mediated responses to epineurial vascular diameter at the sciatic nerve.

This study used an anaesthetized rat model to directly observe changes in diameter of the vessels supplying the sciatic nerve in response to acetylcholine (10-4 M), a muscarinic receptor agonist, and atropine (10-5 M), a muscarinic receptor antagonist. Topical application of acetylcholine resulted in increases in vessel diameter (baseline: 22.0 ± 2.5 µm, acetylcholine: 28.8 ± 3.3 µm), while topical application of atropine resulted in a decrease in diameter (baseline: 26.6 ± 3.2 µm, atropine: 15.5 ± 3.6 µm) of the epineurial vessels. Mean arterial pressure was not affected by either acetylcholine (baseline: 103.8 ± 1.8 mm Hg, acetylcholine: 102.8 ± 3.2 mm Hg) or atropine (baseline: 104.0 ± 1.9 mm Hg, atropine: 105.2 ± 2.2 mm Hg). These data suggest that muscarinic-receptor-mediated responses can affect the diameter of the epineurial vessels at the sciatic nerve. In addition, muscarinic-receptor-mediated responses appear to contribute to baseline diameter of epineurial vessels at the sciatic nerve.