Translational research in pediatrics II: blood collection, processing, shipping, and storage.

Translational research often involves tissue sampling and analysis. Blood is by far the most common tissue collected. Due to the many difficulties encountered with blood procurement from children, it is imperative to maximize the quality and stability of the collected samples to optimize research results. Collected blood can remain whole or be fractionated into serum, plasma, or cell concentrates such as red blood cells, leukocytes, or platelets. Serum and plasma can be used for analyte studies, including proteins, lipids, and small molecules, and as a source of cell-free nucleic acids. Cell concentrates are used in functional studies, flow cytometry, culture experiments, or as a source for cellular nucleic acids. Before initiating studies on blood, a thorough evaluation of practices that may influence analyte and/or cellular integrity is required. Thus, it is imperative that child health researchers working with human blood are aware of how experimental results can be altered by blood sampling methods, times to processing, container tubes, presence or absence of additives, shipping and storage variables, and freeze-thaw cycles. The authors of this review, in an effort to encourage and optimize translational research using blood from pediatric patients, outline best practices for blood collection, processing, shipment, and storage.

Expression of extracellular matrix components is disrupted in the immature and adult estrogen receptor ß-null mouse ovary.

Within the ovary, Estrogen Receptor ß (ERß) is localized to the granulosa cells of growing follicles. 17ß-estradiol (E2) acting via ERß augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERß-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERß in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERß disrupts gene expression as early as post-natal day (PND) 13, and in particular, we identify a number of genes of the extracellular matrix (ECM) that are significantly higher in ERß-null follicles than in wildtype (WT) follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERß-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERß-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23-29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERß-null follicles at PND 13 into adulthood, and is elevated specifically in the ERß-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERß-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a) that ERß regulates granulosa cell gene expression ovary prior to puberty, and b) that ERß regulates expression of ECM components in the mouse ovary.