Background: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. Methods: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 10⁶ cells/mL to 1.67 × 10⁶ cells/mL. Results: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 10⁶ cells/mL. Conclusion: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Introduction The Environmental Determinants of Islet Autoimmunity (ENDIA) Study is an ongoing Australian prospective cohort study investigating how modifiable prenatal and early-life exposures drive the development of islet autoimmunity and type 1 diabetes (T1D) in children. In this profile, we describe the cohort’s parental demographics, maternal and neonatal outcomes and human leukocyte antigen (HLA) genotypes. Research design and methods Inclusion criteria were an unborn child, or infant aged less than 6 months, with a first-degree relative (FDR) with T1D. The primary outcome was persistent islet autoimmunity, with children followed until a T1D diagnosis or 10 years of age. Demographic data were collected at enrollment. Lifestyle, clinical and anthropometric data were collected at each visit during pregnancy and clinical pregnancy and birth data were verified against medical case notes. Data were compared between mothers with and without T1D. HLA genotyping was performed on the ENDIA child and all available FDRs. Results The final cohort comprised 1473 infants born to 1214 gestational mothers across 1453 pregnancies, with 80% enrolled during pregnancy. The distribution of familial T1D probands was 62% maternal, 28% paternal and 11% sibling. The frequency of high-risk HLA genotypes was highest in T1D probands, followed by ENDIA infants, and lowest among unaffected family members. Mothers with T1D had higher rates of pregnancy complications and perinatal intervention, and larger babies of shorter gestation. Parent demographics were comparable to the Australian population for age, parity and obesity. A greater percentage of ENDIA parents were Australian born, lived in a major city and had higher socioeconomic advantage and education. Conclusions This comprehensive profile provides the context for understanding ENDIA’s scope, methodology, unique strengths and limitations. Now fully recruited, ENDIA will provide unique insights into the roles of early-life factors in the development of islet autoimmunity and T1D in the Australian environment. Trial registration number ACTRN12613000794707.