Introduction of BD Vacutainer Barricor™ tubes in clinical biobanking and application of amino acid and cytokine quality indicators to Barricor plasma.

Objectives: The use of BD Vacutainer Barricor™ tubes (BAR) can reduce turnaround time (TAT) and improve separation of plasma from cellular components using a specific mechanical separator. Concentrations of amino acids (AAs) and cytokines, known to be labile during pre-analytical time delays, were compared in heparin (BAR, BD Heparin standard tube [PST]), EDTA and serum gel tubes (SER) to validate previously identified quality indicators (QIs) in BAR.

Methods: Samples of healthy individuals (n=10) were collected in heparin, EDTA and SER tubes and exposed to varying pre- and post-centrifugation delays at room temperature (RT).

Standardization of the preanalytical phase of DNA extraction from fixed tissue for next-generation sequencing analyses.

Next-generation sequencing (NGS) analyses on DNA derived from archived Formalin-Fixed Paraffin-Embedded (FFPE) clinical material can provide a powerful tool in oncology research and clinical diagnostics. Although several studies have established that NGS can be performed using DNA from FFPE tissue, the accuracy and reproducibility of such analyses, as well as their robustness to the biomolecular quality of the samples used, remains a matter of debate. Excellent reviews have recently been published, providing evidence-based best practices for FFPE DNA extraction.

Quality Assessment of the Preanalytical Workflow in Liquid Biobanking: Taurine as a Serum-Specific Quality Indicator for Preanalytical Process Variations.

The scientific impact of translational biomedical research largely depends on the availability of high-quality biomaterials. However, evidence-based and robust quality indicators (QIs) covering the most relevant preanalytical variations are still lacking. The aim of this study was to identify and validate a QI suitable for assessing time-to-centrifugation (TTC) delays in human liquid biospecimens originating from both healthy and diseased individuals.

Cold Ischemia Score: An mRNA Assay for the Detection of Extended Cold Ischemia in Formalin-Fixed, Paraffin-Embedded Tissue.

Although there are thousands of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown preanalytical variables. We analyzed FFPE tissue biospecimens as part of the National Cancer Institute (NCI) Biospecimen Preanalytical Variables program to identify mRNA markers denoting cold ischemic time. The mRNA was extracted from colon, kidney, and ovary cancer FFPE blocks (40 patients, 10-12 hr fixation time) with 1, 2, 3, and 12 hr cold ischemic times, then analyzed using qRT-PCR for 23 genes selected following a literature search.

Method Validation for Extraction of Nucleic Acids from Peripheral Whole Blood.

Background: This article is the fifth in a series of publications providing formal method validation for biospecimen processing. We report the optimization and validation of methodology to obtain nucleic acids of sufficient quantity and quality from blood.

Methods: DNA was extracted using the Chemagic DNA Blood Kit on an MSM I.

Method optimization for fecal sample collection and fecal DNA extraction.

Background: This is the third in a series of publications presenting formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report here optimization of a stool processing protocol validated for fitness-for-purpose in terms of downstream DNA-based analyses.

Methods: Stool collection was initially optimized in terms of sample input quantity and supernatant volume using canine stool.

Method validation for automated isolation of viable peripheral blood mononuclear cells.

Background: This article is part of a series of publications providing formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks. We report the optimization and validation for fitness-for-purpose of automated and manual protocols for isolating peripheral blood mononuclear cells (PBMCs) from whole blood, and compare the two methods.

Methods: The manual method was optimized for whole blood centrifugation speed, gradient type (Ficoll, Leucosep, CPT), and freezing method (Mr Frosty, Controlled Rate Freezing).

Method validation for preparing urine samples for downstream proteomic and metabolomic applications.

Background: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints.

Methods: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C.

Method validation for preparing serum and plasma samples from human blood for downstream proteomic, metabolomic, and circulating nucleic acid-based applications.

Background: Formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. Serum and plasma processing protocols were validated for fitness-for-purpose in terms of key downstream endpoints, and this article demonstrates methodology for biospecimen processing method validation.

Methods: Serum and plasma preparation from human blood was optimized for centrifugation conditions with respect to microparticle counts.

Automatic buffy coat extraction: methodology, feasibility, optimization and validation study.

Buffy coat isolation from whole blood has typically been a long, manual process. We tested and evaluated the feasibility, efficiency, and reproducibility of extracting buffy coat by an automated process with the Tecan pipetting robot Freedom Evo200.

Short-term stability study of RNA at room temperature.

The quality of RNA preserved in different stabilization matrices was investigated after 2 weeks of storage at room temperature. RNA samples in RNAstable (Biomatrica), GenTegra (IntegenX), and RNAshell (Imagene) were compared to RNA stored at -80°C (the current gold standard for RNA preservation) and with liquid or dried RNA stored at room temperature without additives in this multi-center study. One center prepared all of the RNA samples, and five participating laboratories applied the samples to the matrices and stored them for 2 weeks at room temperature.

Transcriptional profiling after lipid raft disruption in keratinocytes identifies critical mediators of atopic dermatitis pathways.

Lipid rafts are cholesterol-rich cell signaling platforms, and their physiological role can be explored by cholesterol depletion. To characterize transcriptional changes ongoing after lipid raft disruption in epidermal keratinocytes, a cell type that synthesizes its cholesterol in situ, we performed whole-genome expression profiling. Microarray results show that over 3,000 genes are differentially regulated.

Heparin-binding EGF-like growth factor is induced by disruption of lipid rafts and oxidative stress in keratinocytes and participates in the epidermal response to cutaneous wounds.

Epidermal homeostasis and repair of the skin barrier require that epidermal keratinocytes respond to alterations of their environment. We report that cellular stress with methyl-beta-cyclodextrin (MBCD), a molecule that extracts membrane cholesterol and thereby disrupts the structure of lipid rafts, strongly induces the synthesis of heparin-binding EGF-like growth factor (HB-EGF) in keratinocytes through the activation of p38 mitogen-activated protein kinase. Interesting parallels between lipid raft disruption and oxidative stress can be drawn as hydrogen peroxide induces p38 activation and HB-EGF synthesis in keratinocytes.