Biospecimen Research Database (BRD) Recent Papers

ctDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV+ oropharyngeal cancer.

This paper compared the stability of a low-range DNA ladder in urine stored at room temperature for up to 24 h when not stabilized and when stabilized with EDTA (10 mM, or 40 mM) and/or Tris (10 mM, or 30 mM), and in urine containing 100 mM EDTA stored for up to 7 days at room temperature. Additionally, the authors evaluated the effect of urine cell-free DNA (cfDNA) fragment size on the detection of circulating tumor DNA (ctDNA) and copy number variations (CNV) using next generation sequencing (NGS). The study also compared the detection of Human Papillomavirus (HPV) DNA in matched plasma and urine specimens using a stem-loop 42 bp ddPCR assay and a conventional 77 bp ddPCR assay and investigated the ability of the stem-loop assay to detect HPV DNA prior to clinical recurrence.

Effect of Delayed Centrifugation on the Levels of NMR-Measured Lipoproteins and Metabolites in Plasma and Serum Samples.

This paper compared the metabolite profiles and lipoprotein subfractions of serum and plasma from healthy patients and obese patients obtained after a centrifugation delay of up to 8 h at room temperature. Additionally, serum and plasma profiles from the same individual were compared, and the authors explored whether the ratio of lactate to glucose could be used to identify specimens obtained after a centrifugation delay.

Influence of hemolysis, lipemia and bilirubin on biobank sample quality- origin and interference in the use for extracellular vesicle (EV) and MiRNA analyses.

This paper compared patient characteristics (injury severity score, hemoglobin levels, triglyceride levels and survival), treatment (blood transfusions, propofol use, intensive care unit time), miR-16-5p levels and extracellular vesicle size and concentration between serum specimens with spectrophotometrically detected hemolysis, lipemia, or bilirubin and unmatched serum without. Serum was collected from polytraumatized patients in the emergency room (ER) and over the course of 10 days of hospitalization.

Frozen section histopathology and preanalytical factors affecting nucleic acid integrity in biobanked fresh-frozen human cancer tissues.

This paper investigated the effects of cold ischemia and frozen storage duration on DNA and RNA integrity by comparing unmatched colon adenocarcinoma (COAD), hepatocellular carcinoma (HCC), and renal cell carcinoma (RCC) specimens obtained after cold ischemia times ranging from <30 min to >120 min or frozen storage for 1-10 years. The authors also investigated potential correlations between DNA/RNA integrity and histopathologic parameters (tumor cell percentage, presence of normal tissue, extent of inflammatory cell infiltration, extent of stromal fibrosis, percentage necrosis or percentage extracellular mucin pool).

Quality Control of RNA Extracted from PAXgene� Blood RNA Tubes after Long-Term Cryopreservation.

This paper compared the yield, purity and integrity of RNA extracted from 217 unmatched PAXgene blood specimens that were stored frozen (-80°C) for 7, 9 or 11 years before extraction. The authors also investigated if storage duration affects the incidence of clot formation using 517 PAXgene blood specimens that were stored for 1 month to 11 years and compared the yield and integrity of RNA extracted from clotted and non-clotted specimens.

Impact of pre-analytical variables - temperature, agitation, storage duration, and blood-to-anticoagulant ratio - on complete blood count test reliability.

This paper compared complete blood count (CBC) parameters in case-matched blood specimens from optimally and suboptimally (50%) filled tubes that were transported with or without agitation and then stored at 4°C or room temperature for up to 24 h.

Serum and Plasma miRNA Expression Levels in Sudden Sensorineural Hearing Loss.

This paper compared levels of eight microRNAs (miRNA, miR) in case-matched serum and plasma collected from men and women. The eight miRNAs were selected due to their implicated role in sudden sensorineural hearing loss.

Short- and medium-term pre-analytical stability of human serum insulin-like growth factor-1.

This paper compared insulin-like growth factor-1 (IGF-1) levels in serum that was stored at −20°C, 4°C, 20–25°C and 30°C for up to 672 h and in serum obtained after an 8 or 24 h centrifugation delay followed by storage at either 4°C or 20–25°C for up to 672 h.

Systematic comparison of quantity and quality of RNA recovered with commercial FFPE tissue extraction kits.

This paper compared the yield and integrity of RNA obtained from formalin-fixed, paraffin-embedded (FFPE) tonsil, appendix and lymph node specimens following extraction with seven different kits: RNeasy FFPE, ReliaPrep FFPE Total RNA Miniprep System, Norgen FFPE RNA Purification, GenElute FFPE RNA Purification, PureLink FFPE RNA Isolation, AllPrep DNA/RNA FFPE, and High Pure FFPET RNA Isolation Kits.

Evaluation of urinary cfDNA workflows for the molecular profiling of malignant disease.

This paper compared the stability of cell-free DNA (cfDNA) in unpreserved urine and urine preserved with PAXgene or Streck urine preservative during 4 days of room temperature storage. The authors then compared the detection of mutations in breast cancer, prostate cancer and colorectal cancer in patient plasma with that of case-matched urine that was stored for 0 or 3 days unpreserved or preserved with PAXgene or Streck preservatives. Mutations were detected using different platforms for each cancer type investigated.

Optimization and comparison of genomic DNA extraction from whole blood collected in PAXgene blood RNA tube using automated platforms.

This paper used PAXgene-preserved blood to investigate the potential effects of blood thaw duration, freeze-thaw cycling, storage at 4°C, centrifugation speed and duration, washing the cell pellet, and DNA extraction method on the yield, integrity and size of genomic DNA. The effects of DNA extraction method on DNA amplificability were also investigated. The morphology of PAXgene-preserved blood cells was compared to cells from EDTA blood.

Comparison of enzymatic and bisulfite conversion of circulating cell-free tumor DNA for DNA methylation analyses.

This paper compared DNA recovery, fragment size and methylation results between enzymatic and bisulfite-converted cfDNA from pooled plasma and cell line DNA; investigated the effects of using just the conversion module rather than the full kit; and explored the effects of using different ratios and manufacturers of magnetic beads in the clean-up step. DNA recovery, fragment size and methylation results were also compared between enzymatic and bisulfite-converted cfDNA from the plasma of five patients diagnosed with colorectal cancer and known BCAT1 methylation.

Impact of Centrifugation Time Reduction in GLP Systems.

This study compared levels of 30 clinical chemistry and 9 immunological analytes as well as hemolysis, icterus and lipemia in lithium heparin plasma obtained from blood collected in Vacutainer and Vacuette tubes that were centrifuged using the tube type-specific recommended protocol (1300 g for 10 min and 2200 g for 10 min, respectively) and those centrifuged at 2700 g for 5 or 7 min.

Selected miRNAs in Urinary Extracellular Vesicles Show Promise for Early and Specific Diagnostics of Diabetic Kidney Disease.

This paper compared the uEV morphology, protein expression, and RNA size among normoalbuminuric, microalbuminuric, and macroalbuminuric urine. Additionally, the paper aimed to identify microRNA (miRNA, miR) biomarkers of diabetes in urinary extracellular vesicle (uEVs) that met the following criteria: (i) stable for a given case when controlled for specimen type and time of collection (24 h versus overnight collections); (ii) comparable between two cohorts (24 h specimens frozen without centrifugation versus overnight specimens centrifuged prior to freezing); and (iii) comparable between RNA samples that were and were not treated with DNase. Identified miRNAs were validated in multiple urine specimen cohorts, including those collected from males and females, and patients with type I and type II diabetes. Potential correlations with clinical measurements and the sensitivity for predicting eGFR decline were also investigated.

Comparative Evaluation of Centrifugation Speed and Its Impact on Diagnostic Accuracy.

This study compared hemolysis, lipemia, icterus, and the levels of 34 clinical chemistry and 3 coagulation analytes in plasma (lithium heparin and sodium citrate tubes) and serum (without gel or clot activator) obtained by fast (4000 g for 5 min) and standard (3200 g for 10 min) centrifugation.

Cell free RNA detection of pancreatic cancer in pre diagnostic high risk and symptomatic patients.

This paper investigated the relative contributions of extrinsic factors (collection tube type, processing delays and centrifugation protocol) on the cell-free RNA (cfRNA) expression profiles of plasma, developed a cfRNA normalization strategy that accounted for preanalytical handling and investigated whether the normalized data could be used to identify a pancreatic ductaladenocarcinoma (PDAC) signature.

Urinary microbiome in non-muscle invasive bladder cancer: impact of sample types and sex differences.

This paper compared the microbiome of case-matched specimens from the bladder mucosa and from urine collected midstream and via a catheter; Patients with (35) and without (15) bladder cancer were included in the study. Potential effects of patient sex, collection method/specimen type and bladder cancer on the microbiome were investigated.

Comparative DNA extraction methods to use for LAMP assay as molecular diagnosis of human schistosomes from urine samples.

This paper compared the detection of cell-free repeat DNA of two parasites (S. mansoni and S. haematobium) by loop-mediated isothermal amplification (LAMP) using DNA extracted from urine by four different methods.

Analysis of urine cell-free DNA copy number and fragment size from healthy individuals.

This paper compared cell-free (cfDNA) size profiles in case-matched plasma and urine from healthy volunteers following DNA extraction or direct amplification without extraction. Additionally, the size profiles and copy numbers of nuclear (Glyceraldehyde-3-phosphate dehydrogenase, GAPDH) and mitochondrial (ND1) cfDNA were compared among urine specimens that were frozen immediately with and without EDTA and stored with and without EDTA at room temperature and 4°C for 0 h, 1.5 h, 3 h and 5 h before freezing. Potential correlations between the copy number of amplicons of different sizes and clinical parameters, including blood cell counts, urinalysis results, and levels of hepatic/renal function biomarkers, were investigated.

The effect of inadequate drying of blood spots on newborn screening analyte concentrations.

This paper compared levels of amino acids, thyroid-stimulating hormone, immunoreactive trypsinogen and decanoyl-carnitine in air-dried blood spots (DBS) and matched blood spots that were inadequately dried due to storage in a glassine envelope between the pages of a book or in a plastic bag.

Refined Procedure to Purify and Sequence Circulating Cell-Free DNA in Prostate Cancer.

This paper described optimization of DNA extraction (addition of proteinase K step, elution solution and number of elutions), sequencing library construction (input amount and number of amplification steps) and sequencing depth for analysis of cell-free DNA (cfDNA) from the plasma of prostate cancer patients. cfDNA yield and fragment size were compared among plasma specimens obtained from patients at radical prostatectomy (RP), and from patients that were disease-free for >6 years post-RP and with metastatic castration-resistant prostate cancer. Additionally, the relationships between cfDNA concentrations and patient age, blood PSA, tumor grade or stage, progression on treatment (Abiraterone or Docetaxel) and patient survival were investigated.

Analysis of genome-wide 5-hydroxymethylation of blood samples stored in different anticoagulants: opportunities for the expansion of clinical resources for epigenetic research.

This paper compared cfDNA yields and 5-hydroxymethylcytosine Selective Chemical Labelling (5hmC-Seal) sequencing metrics results and profiles in case-matched EDTA and heparin plasma from patients diagnosed with colorectal cancer within the past 1 y (cases) and individuals that were free of cancer in the 15 years following blood collection (controls). The effect of patient age, sex and diagnosis on the 5hmC-Seal profile was also investigated.

Stability of microRNAs in serum and plasma reveal promise as a circulating biomarker.

This paper compared levels of microRNAs (miRNAs) in (i) plasma and serum that were stored post-separation at room temperature or on wet ice for up to 24 h and in (ii) plasma from blood that was stored at room temperature up to 6 h prior to plasma separation. Effects of plasma and serum storage were investigated by real-time PCR analysis of 5 miRNAs and two housekeeping RNAs in specimens from five volunteers and by next-generation sequencing (NGS) using the plasma of two volunteers. Effects of storing blood for up to 6 h prior to plasma separation on miRNA levels were investigated by NGS of plasma from a single volunteer.

Impact of ambient temperature exposure on miRNA stability in human plasma.

This paper compared levels of six miRNAs by digital PCR in archival plasma that had not been thawed with plasma samples that were thawed and stored at room temperature for 24, 48 or 72 h before refreezing. Plasma specimens from nine pregnant women were stored at -80°C for 12 years prior to the study. Effects of elution order, extraction batch and digital PCR (dPCR) plate on levels of a spike-in extraction control were also investigated.

Evaluation of pre-analytical factors impacting urine test strip and chemistry results.

This paper compared results of semi-quantitative urinalysis (10 analytes test strip) and urine clinical chemistry (13 analytes) analysis between matched leftover urine specimens that were stored in plain and preservative tubes at room temperature and 2–8°C for up to 96 h. Additionally, effects of fill volume (25%, 50%, 75% and 100%) and centrifuging preservative tubes on semi-quantitative urinalysis results were also investigated.

Faecal amino acids stability: investigating optimal sampling conditions for analysis.

This paper investigated the stability of 20 amino acids in fecal specimens stored at 4°C and 20°C for up to 7 days, at -20°C for up to 90 days, in an OMNImetGUT sampling device at room temperature for up to 7 days, and over the course of three freeze-thaw cycles. Inter- and intra-day variability in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, as well as variability attributable to the sampling location, were also investigated.

Effects of certain pre-analytical factors on the performance of plasma phospho-tau217.

This paper compared phosphorylated -tau217 (p-Tau217) levels in plasma from amyloid beta 42 (Aβ42)/amyloid beta 40 (Aβ40) positive and negative patients that were thawed at room temperature versus on wet ice, centrifuged versus not centrifuged before analysis, and in plasma that was subjected to 1, 2 or 3 freeze-thaw cycles. Additionally, the authors compared the strength of the correlation between plasma p-tau217 levels and cerebrospinal fluid (CSF) Aβ42/Aβ40 and p-tau217 levels among plasma specimens that were thawed at room temperature or on ice and those that were centrifuged or not centrifuged before analysis.

Surgical Tumor Resection Deregulates Hallmarks of Cancer in Resected Tissue and the Surrounding Microenvironment.

This paper compared RNA expression profiles of tumor and normal adjacent biopsies that were collected at the beginning (fully vascularized), during (partially devascularized) and following (devascularized) surgical resection for breast or head and neck cancer.

EDTA tubes are suitable for insulin and C-peptide measurement in resource-limited settings and can be stored at room temperature for up to 24 hours.

This paper compared insulin and C-peptide levels in serum and plasma from blood stored for up to 24 h at room temperature or in a cooler box (-5-15°C) before or after centrifugation.

The influence of hemolysis in patient samples on biochemical tests analyzed using Roche Cobas� 8000 Analyzer.

This paper compared levels of 23 clinical chemistry analytes in 678 pairs of case-matched hemolyzed and non-hemolyzed blood specimens that were retrospectively collected.

A comparative study of four cell-free DNA assays for detecting circulating tumor DNA in early breast cancer.

This paper compared the detection of circulating tumor DNA (ctDNA) in plasma using four different next-generation sequencing (NGS) assays and investigated whether patient age or tumor characteristics influenced detection.

Rapid one-step protocol for exploring immune cells in cerebrospinal fluid.

This paper compared immune cell populations in stained blood and cerebrospinal fluid (CSF) specimens that were analyzed by flow cytometry immediately and after storage for up to 3 days at room temperature. Effects of storing CSF for up to 4 h at room temperature prior to staining with a one-step protocol on immune cell populations and apoptotic markers were also investigated.

Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization.

This paper compared different methods to extract cell-free DNA (cfDNA) from urine and investigated sources of variability during cfDNA extraction from the urine and plasma of healthy volunteers.

Preanalytical stability of 17 analytes in whole blood during transportation.

This paper investigated the stability of 17 analytes in whole blood transported and stored at room temperature for up to 10 h before serum separation.

Challenges in circulating miRNA analysis in adrenocortical tumors.

This paper compared the quantified levels of four endogenous microRNAs (miRNA, miR) and one spike-in miRNA using real-time PCR and ddPCR in matched K₂EDTA and K₃EDTA plasma of healthy patients and those with endocrine disorders.

Storage Time and DNA Quality Determine BRCA1/2 Sequencing Success in Prostate Cancer: A Multicentre Analysis with Therapeutic Implications.

This paper compared the BRCA1/2 sequencing success rate, concentration and fragmentation of DNA from formalin-fixed, paraffin-embedded (FFPE) prostate cancer biopsy and resection specimens that had been stored for <1 year, 1-2 years, or > 2 years. The effect of biopsy site on NGS success rate was also investigated.

RNA degradation patterns in cardiac tissues kept at different time intervals and temperatures before RNA sequencing.

This paper compared RNA integrity and RNA sequencing (RNAseq) metrics among nine case-matched cardiac tissue specimens stored for up to 28 days at 4°C or room temperature before RNA extraction.

Optimization of bile preparation for liquid biopsy in cholangiocarcinoma focusing on circulating tumor DNA and protein stability.

This paper examined how the duration of room temperature storage (1-7 h) affects protein concentration, copies of mutant and wildtype KRAS cell-free DNA (cfDNA), and E- and N-Cadherin activity in case-matched bile specimens from six patients with extrahepatic cholangiocarcinoma.

Comparison of three blood collection tubes for 16 biochemical analytes and stability assessment of selected analytes: VACUETTE(�) CAT serum Sep clot activator tube, VACUETTE(�) LH lithium heparin Sep tube, and VACUETTE(�) CAT serum fast separator tube.

This paper compared the levels of 16 analytes in case-matched serum/plasma that were collected in VACUETTE CAT Serum Fast Separator Tubes (SFT), CAT Serum Sep Clot Activator Tubes (SST) and lithium heparin Sep (LiHep) tubes. Effects of storing serum/plasma in the original tube for up to 5 days at 4°C on levels of glucose, potassium (K), lactate dehydrogenase (LDH), aspartate transaminase (AST), intact parathyroid hormone (iPTH), and cardiac troponin I (cTnI) were also investigated. Blood was collected from both healthy volunteers and cardiology patients.

Evaluation of automatic cell free DNA extraction metrics using different blood collection tubes.

The paper compared cell-free DNA (cfDNA) levels and fragment size in plasma from blood collected in EDTA, Streck, Norgen and PAXgene tubes and stored for 0, 48 and 168 h before centrifugation. The effect of isolating plasma using single versus two-step centrifugation was also investigated. Blood was collected from healthy individuals, and cfDNA was quantified using fluorometry, single-locus real-time PCR and multi-locus real-time PCR quantification; fragment size was analyzed by real-time PCR and capillary electrophoresis.

Cell-Free DNA Blood Collection Tubes Crosslinking Cellular DNA Impeding Nanopore Long-Read Sequencing.

This paper compared DNA fragment length and Nanopore long read sequencing metrics from buffy coats collected in EDTA and Streck tubes and OCT-preserved tumor (unspecified type) specimens. In a follow-up study, the authors compared the same metrics using DNA from buffy coats collected in Streck tubes, EDTA tubes, and EDTA tubes with formaldehyde. The effect of adding a decrosslinking step to the extraction was also investigated.

Effect of pH on stability and solid phase extraction of urinary free metadrenaline measurement by liquid chromatography tandem mass spectrometry.

This paper compared the stability of free metadrenalines in acidified and unacidified spot urine specimens during storage at room temperature, 4°C and -20°C for 28 days using liquid chromatography tandem mass spectrometry. The authors also investigate the effect of extraction method on metadrenaline loss to plate flowthrough.

Single-Cell RNA Sequencing on Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Identified Multi-Ciliary Cells in Breast Cancer.

This paper compared single-cell RNA sequencing (scRNAseq) profiles of case-matched formalin-fixed, paraffin-embedded (FFPE) and fresh specimens (fixed during workflow) obtained from one invasive ductal carcinoma (IDC) and one invasive lobular carcinoma (ILC). scRNAseq findings were compared with those from immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), electron microscopy and massively parallel sequencing from the same specimens.

Methodological influences on circulating cell-free-mitochondrial and nuclear DNA concentrations in response to chronic stress.

This paper compared cell-free mitochondrial DNA (cf-mtDNA) and nuclear DNA (cf-nDNA) levels that were quantified in (i) plasma directly versus after DNA isolation, and (ii) directly in plasma obtained by a single centrifugation at 600 x g and plasma obtained by centrifugation at 600 x g then 2,500 x g or 2,500 x g then 16,000 x g. The authors also investigated potential correlations between psychological stress scores and levels of cf-mtDNA and cf-nDNA.

Value of urine cytology versus bladder washing in bladder cancer.

This paper compared the detection of bladder cancer by cytological evaluation of matched voided urine and bladder wash specimens and investigated whether tumor grade influences detection rate.

DNA extraction from long-term stored urine.

This paper assessed the concentration of cell-free DNA (cfDNA) in freeze-dried urine supernatants using two different methods and evaluated if cfDNA concentration was correlated with proteinuria (assessed prior to storage) or the duration of storage at room temperature (6-28 years). cfDNA was isolated from 24-h urine specimens obtained from patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis with pauci-immune necrotizing crescentic glomerulonephritis.

Pre-analytical pitfalls: How blood collection tubes influence exercise-induced cell-free DNA concentrations.

This paper compared cell-free DNA (cfDNA) levels in serum, lithium heparin plasma and EDTA plasma collected before and 5- and 30-min post-exercise. The eleven participants completed a treadmill exercise protocol the first day of the study and then completed two others on the same day a week later.

Detection of fusion events by RNA sequencing in FFPE versus freshly frozen colorectal cancer tissue samples.

This paper compared the detection of fusions between case-matched formalin-fixed, paraffin-embedded (FFPE) and frozen RNAlater-preserved colorectal cancer specimens by next-generation RNA sequencing (NGS). The presence of a single fusion was verified by PCR followed by Sanger sequencing.

A cost-effective and scalable approach for DNA extraction from FFPE tissues.

This paper compared the yield and purity of DNA, methylation profiles, and exome sequencing results of DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue using the Maxwell RSC FFPE Plus DNA Purification Kit (Maxwell Kit) or a manual or automated version of the in-house manual method (HiTE) with either bead- or column-based purification. The effects of increasing the cross-link reversal incubation from 1 h to 16-24 h on DNA yield and purity were also examined. Additionally, the authors compared quantification of DNA with Qubit versus Nanodrop and investigated the potential influences of FFPE block storage duration and extraction method. The study used archival FFPE blocks of sarcoma (89 specimens) and used FFPE blocks of lung cancer specimens for methylation profiling (3 specimens) and exome sequencing (2 specimens).

miRNA Library Preparation Optimisation for Low-Concentration and Low-Volume Paediatric Plasma Samples.

This paper sought to optimize next-generation microRNA (miRNA) sequencing library preparation for pediatric plasma specimens by comparing library concentrations following extraction from 100 or 200 µL plasma using two different RNA extraction kits (miRNeasy Serum/Plasma Kit and the MagMAX miRVana Total Isolation Kit). Further, optimization of miRNA concentration, adapter and primer dilution and the number of PCR cycles was conducted using miRNA extracted with the miRNeasy Serum/Plasma Kit from 100 µL plasma. The effect of patient diagnosis, age and gender on the concentration of the miRNA library was also investigated using the optimized protocol.