Biospecimen Qualification in a Clinical Biobank of Urological Diseases.

Development of novel biomarkers for diagnosis of disease and assessment of treatment efficacy utilizes a wide range of biospecimens for discovery research. The fitness of biospecimens for the purpose of biomarker development depends on the clinical characteristics of the donor and on a number of critical and potentially uncontrolled pre-analytical variables. Pre-analytical factors influence the reliability of the biomarkers to be analyzed and can seriously impact analytic outcomes. Sample quality stratification assays and tools can be utilized by biorepositories to minimize bias resulting from samples' inconsistent quality. In this study, we evaluated the quality of biobanked specimens by comparing analytical outcomes at 1, 5, and 10 years after collection. Our results demonstrate that currently available assays and tools can be used by biobank laboratories to support objective biospecimen qualification. We have established a workflow to monitor the quality of different types of biospecimens and, in this study, present the results of a qualification exercise applied to fluid samples and their derivatives in the context of urological diseases.

In Vitro Comparative Study of Platelets Treated with Two Pathogen-Inactivation Methods to Extend Shelf Life to 7 Days.

BACKGROUND AND OBJECTIVES: Since 2015, platelet products have been pathogen-inactivated (PI) at the Luxemburgish Red Cross (LRC) using Riboflavin and UV light (RF-PI). As the LRC should respond to hospital needs at any time, platelet production exceeds the demand, generating a discard rate of 18%. To reduce this, we consider the extension of storage time from 5 to 7 days. This study's objective was to evaluate the in vitro 7-day platelet-storage quality, comparing two PI technologies, RF-PI and amotosalen/UVA light (AM-PI), for platelet pools from whole-blood donations (PPCs) and apheresis platelets collected from single apheresis donation (APCs). MATERIALS AND METHODS: For each product type, 6 double-platelet concentrates were prepared and divided into 2 units; one was treated with RF-PI and the other by AM-PI. In vitro platelet-quality parameters were tested pre- and post-PI, at days 5 and 7. RESULTS: Treatment and storage lesions were observed in PPCs and APCs with both PI methods. We found a higher rate of lactate increase and glucose depletion, suggesting a stronger stimulation of the glycolytic pathway, a higher Annexin V binding, and a loss of swirling in the RF-PI-treated units from day 5. The platelet loss was significantly higher in the AM-PI compared with the RF-PI units. CONCLUSIONS: Results suggest that RF-PI treatment has a higher deleterious impact on in vitro platelet quality compared to AM-PI, but we observed higher loss of platelets with AM-PI due to the post-illumination amotosalen adsorption step. If 7-day storage is needed, it can only be achieved with AM-PI, based on our quality criteria.

Method Optimization of Skin Biopsy-Derived Fibroblast Culture for Reprogramming Into Induced Pluripotent Stem Cells.

Background: Fibroblasts can be isolated from skin biopsies using a chemical dissociation, a physical dissociation, or a combination of both techniques. They can be reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of defined sets of key transcription factors. This study aimed to identify the optimal protocol for skin biopsy dissociation, fibroblast culture, and fibroblast cryopreservation in the scope of reprogramming into iPSCs and in the context of biobank accreditation. Methods: First, four dissociation techniques typically used in the laboratory (explant based, enzymatic, and/or mechanical) and two cryopreservation media containing 10% dimethyl sulfoxide, either commercial or homemade, were evaluated in terms of post-thaw recovery, viability, growth curves, and karyotyping analyses of the fibroblasts. Next, the clones reprogrammed from the fibroblasts isolated with the two optimal dissociation methods and cryopreservation media were further assessed by reprogramming quality before cryopreservation and post-thaw pluripotency comparison. Results: Fibroblasts isolated from skin biopsies using an explant-based or enzymatic dissociation method showed higher viability, higher proliferative potential, and higher genome stability post-thaw compared to the other dissociation techniques. Fibroblasts obtained by the explant-based dissociation technique showed a slightly higher reprogramming quality. The iPSC reprogrammed from explant-based dissociated fibroblasts showed successful recovery of iPSC clones. No difference between the two cryopreservation media was detected for the tested endpoints, with the exception of a higher visual count of colonies at the end of the reprogramming for the explant-based dissociation method. Conclusions: This article presents a formal method optimization for biospecimen processing in the context of accreditation in laboratories and biobanks. We validated skin biopsy-derived fibroblast isolation, culture, and cryopreservation for downstream mRNA reprogramming into iPSCs. The explant-based dissociation technique and homemade medium are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.

Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?

We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF-ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF-ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9-2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF-ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem.

Effect of Different Proteinase K Digest Protocols and Deparaffinization Methods on Yield and Integrity of DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue.

DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer's protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ2 = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100-400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.

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. Alternative fixatives exist, although their implementation in clinical practice is difficult. In this article, we present (i) a review of fixed tissue DNA preanalytics with a special focus on DNA extraction and fixed tissue sample qualification and (ii) results from comparisons between different methods of DNA extraction from tissue samples that have been fixed or stabilized by different methods, in terms of NGS metrics and different DNA quality metrics.

Preanalytical robustness of blood collection tubes with RNA stabilizers.

Background Efficient blood stabilization is essential to obtaining reliable and comparable RNA analysis data in preclinical operations. PAXgene (Qiagen, Becton Dickinson) and Tempus (Applied Biosystems, Life Technologies) blood collection tubes with RNA stabilizers both avoid preanalytical degradation of mRNA by endogenous nucleases and modifications in specific mRNA concentrations by unintentional up- or down-regulation of gene expression. Methods Sixteen different preanalytical conditions were tested in PAXgene and Tempus blood samples from seven donors: different mixing after collection, different fill volumes and different 24-h transport temperature conditions after collection. RNA was extracted by column-based methods. The quality of the extracted RNA was assessed by spectrophotometric quantification, A260/A280 purity ratio, RNA Integrity Number (Agilent Bioanalyzer), miRNA quantative real time polymerase chain reaction (qRT-PCR) on two target miRNAs (RNU-24 and miR-16), mRNA quality index by qRT-PCR on the 3' and 5' region of the GAPDH gene, and the PBMC preanalytical score, based on the relative expression levels of the IL8 and EDEM3 coding genes. Results When PAXgene RNA and Tempus blood collection tubes were used following the manufacturers' instructions, there was no statistically or technically significant difference in the output RNA quality attributes. However, the integrity of the RNA extracted from Tempus collection tubes was more sensitive to fill volumes and effective inversion, than to storage temperature, while the integrity of RNA extracted from PAXgene collection tubes was more sensitive to effective inversion and storage temperature than to fill volumes. Conclusions Blood collection tubes with different RNA stabilizers present different robustness to common preanalytical variations.

IL8 and EDEM3 gene expression ratio indicates peripheral blood mononuclear cell (PBMC) quality.

BACKGROUND: Uncontrolled preanalytical variables can reduce the accuracy and reproducibility of downstream analytical results from peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were isolated from EDTA and citrate-anticoagulated blood samples, obtained from healthy subjects and patients with inflammatory and infectious conditions. PBMC-derived RNA samples were examined for gene expression changes induced by extended blood pre-centrifugation delays at 4 °C and RT. We used Taqman RTqPCR to evaluate the combination of two target genes for their "diagnostic performance" in identifying EDTA and citrate-anticoagulated PBMC samples with extended pre-centrifugation times. RESULTS: We established the PBMC preanalytical score, a gene expression metric to asses the PBMC quality related to the pre-centrifugation delay at room temperature for different anticoagulants. The PBMC preanalytical score measurement can identify: CONCLUSION: The proposed PBMC preanalytical score may enable objective PBMC sample qualification for downstream applications, which may be influenced by blood precentrifugation delays.

IL8 and IL16 levels indicate serum and plasma quality.

BACKGROUND: Longer pre-centrifugation times alter the quality of serum and plasma samples. Markers for such delays in sample processing and hence for the sample quality, have been identified. METHODS: Twenty cytokines in serum, EDTA plasma and citrate plasma samples were screened for changes in concentration induced by extended blood pre-centrifugation delays at room temperature. The two cytokines that showed the largest changes were further validated for their "diagnostic performance" in identifying serum or plasma samples with extended pre-centrifugation times. RESULTS: In this study, using R&D Systems ELISA kits, EDTA plasma samples and serum samples with a pre-centrifugation delay longer than 24 h had an IL16 concentration higher than 313 pg/mL, and an IL8 concentration higher than 125 pg/mL, respectively. EDTA plasma samples with a pre-centrifugation delay longer than 48 h had an IL16 concentration higher than 897 pg/mL, citrate plasma samples had an IL8 concentration higher than 21.5 pg/mL and serum samples had an IL8 concentration higher than 528 pg/mL. CONCLUSIONS: These robust and accurate tools, based on simple and commercially available ELISA assays can greatly facilitate qualification of serum and plasma legacy collections with undocumented pre-analytics.

Integrated multi-omics of the human gut microbiome in a case study of familial type 1 diabetes.

The gastrointestinal microbiome is a complex ecosystem with functions that shape human health. Studying the relationship between taxonomic alterations and functional repercussions linked to disease remains challenging. Here, we present an integrative approach to resolve the taxonomic and functional attributes of gastrointestinal microbiota at the metagenomic, metatranscriptomic and metaproteomic levels. We apply our methods to samples from four families with multiple cases of type 1 diabetes mellitus (T1DM). Analysis of intra- and inter-individual variation demonstrates that family membership has a pronounced effect on the structural and functional composition of the gastrointestinal microbiome. In the context of T1DM, consistent taxonomic differences were absent across families, but certain human exocrine pancreatic proteins were found at lower levels. The associated microbial functional signatures were linked to metabolic traits in distinct taxa. The methodologies and results provide a foundation for future large-scale integrated multi-omic analyses of the gastrointestinal microbiome in the context of host-microbe interactions in human health and disease.

LacaScore: a novel plasma sample quality control tool based on ascorbic acid and lactic acid levels.

INTRODUCTION: Metabolome analysis is complicated by the continuous dynamic changes of metabolites in vivo and ex vivo. One of the main challenges in metabolomics is the robustness and reproducibility of results, partially driven by pre-analytical variations. OBJECTIVES: The objective of this study was to analyse the impact of pre-centrifugation time and temperature, and to determine a quality control marker in plasma samples. METHODS: Plasma metabolites were measured by gas chromatography-mass spectrometry (GC-MS) and analysed with the MetaboliteDetector software. The metabolites, which were the most labile to pre-analytical variations, were further measured by enzymatic assays. A score was calculated for their use as quality control markers. RESULTS: The pre-centrifugation temperature was shown to be critical in the stability of plasma samples and had a significant impact on metabolite concentration profiles. In contrast, pre-centrifugation delay had only a minor impact. Based on the results of this study, whole blood should be kept on wet ice and centrifuged within maximum 3 h as a prerequisite for preparing EDTA plasma samples fit for the purpose of metabolome analysis. CONCLUSIONS: We have established a novel blood sample quality control marker, the LacaScore, based on the ascorbic acid to lactic acid ratio in plasma, which can be used as an indicator of the blood pre-centrifugation conditions, and hence the suitability of the sample for metabolome analyses. This method can be applied in research institutes and biobanks, enabling assessment of the quality of their plasma sample collections.

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. Three DNA extraction methods (PerkinElmer MSM I®, Norgen Biotek All-In-One®, MoBio PowerMag®) and six collection container types were evaluated with human stool in terms of DNA quantity and quality, DNA yield, and its reproducibility by spectrophotometry, spectrofluorometry, and quantitative PCR, DNA purity, SPUD assay, and 16S rRNA gene sequence-based taxonomic signatures. RESULTS: The optimal MSM I protocol involves a 0.2 g stool sample and 1000 µL supernatant. The MSM I extraction was superior in terms of DNA quantity and quality when compared to the other two methods tested. Optimal results were obtained with plain Sarstedt tubes (without stabilizer, requiring immediate freezing and storage at -20°C or -80°C) and Genotek tubes (with stabilizer and RT storage) in terms of DNA yields (total, human, bacterial, and double-stranded) according to spectrophotometry and spectrofluorometry, with low yield variability and good DNA purity. No inhibitors were identified at 25 ng/µL. The protocol was reproducible in terms of DNA yield among different stool aliquots. CONCLUSIONS: We validated a stool collection method suitable for downstream DNA metagenomic analysis. DNA extraction with the MSM I method using Genotek tubes was considered optimal, with simple logistics in terms of collection and shipment and offers the possibility of automation. Laboratories and biobanks should ensure protocol conditions are systematically recorded in the scope of accreditation.