The standardization of clinical studies using extracellular vesicles (EVs) has mainly focused on the procedures employed for their isolation and characterization; however, preanalytical aspects of sample collection, handling and storage also significantly impact the reproducibility of results. We conducted an online survey based on SPREC (Standard PREanalytical Code) among members of GEIVEX (Grupo Español de Investigación en Vesiculas Extracelulares) to explore how different laboratories handled fluid biospecimens destined for EV analyses. We received 70 surveys from forty-three different laboratories: 44% focused on plasma, 9% on serum and 16% on urine. The survey indicated that variability in preanalytical approaches reaches 94%. Moreover, in some cases, researchers had no access to all relevant preanalytical details of samples, with some sample aspects with potential impact on EV isolation/characterisation not coded within the current version of SPREC. Our study highlights the importance of working with common standard operating procedures (SOP) to control preanalytical conditions. The application of SPREC represents a suitable approach to codify and register preanalytical conditions. Integrating SPREC into the SOPs of laboratories/biobanks will provide a valuable source of information and constitute an advance for EV research by improving reproducibility and credibility.

UNASSIGNED: Plasma concentrations of glucagon, GLP-1 and GIP are reported in numerous clinical trials as outcome measures but preanalytical guidelines are lacking. We addressed the impact of commonly used blood containers in metabolic research on measurements of glucagon, GLP-1 and GIP in humans.
UNASSIGNED: Seventeen overweight individuals were subjected to an overnight fast followed by an intravenous infusion of amino acids to stimulate hormonal secretion. Blood was sampled into five containers: EDTA-coated tubes supplemented with DMSO (control), a neprilysin inhibitor, aprotinin (a kallikrein inhibitor) or a DPP-4 inhibitor, and P800 tubes. Plasma was kept on ice before and after centrifugation and stored at -80 Celsius until batch analysis using validated sandwich ELISAs or radioimmunoassays (RIA).
UNASSIGNED: Measures of fasting plasma glucagon did not depend on sampling containers, whether measured by ELISA or RIA. Amino acid-induced hyperglucagonemia was numerically higher when blood was collected into P800 tubes or tubes with aprotinin. The use of p800 tubes resulted in higher concentrations of GLP-1 by RIA compared to control tubes but not for measurements with sandwich ELISA. Plasma concentrations of GIP measured by ELISA were higher in control tubes and negatively affected by P800 and the addition of aprotinin.
UNASSIGNED: The choice of blood containers impacts on measurements of plasma concentrations of glucagon, GLP-1 and GIP, and based on this study, we recommend using EDTA-coated tubes without protease inhibitors or P800 tubes for measurements of glucagon, GLP-1 and GIP in clinical trials.

The cumulative pool of cell-free DNA (cfDNA) molecules within bodily fluids represents a highly dense and multidimensional information repository. This \"biological mirror\" provides real-time insights into the composition, function, and dynamics of the diverse genomes within the body, enabling significant advancements in personalized molecular medicine. However, effective use of this information necessitates meticulous classification of distinct cfDNA subtypes with exceptional precision. While cfDNA molecules originating from different sources exhibit numerous genetic, epigenetic, and physico-chemical variations, they also share common features that complicate analyses. Considerable progress has been achieved in mapping the landscape of cfDNA features, their clinical correlations, and optimizing extraction procedures, analytical approaches, bioinformatics pipelines, and machine learning algorithms. Nevertheless, preanalytical workflows, despite their profound impact on cfDNA measurements, have not progressed at a corresponding pace. In this perspective article, we emphasize the pivotal role of robust preanalytical procedures in the development and clinical integration of cfDNA assays, highlighting persistent obstacles and emerging challenges.

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Recent studies evaluating the preanalytical factors that impact the outcome of nucleic-acid based methods for the confirmation of SARS-CoV-2 have illuminated the importance of identifying variables that promoted accurate testing, while using scarce resources efficiently. The majority of laboratory errors occur in the preanalytical phase. While there are many resources identifying and describing mechanisms for main laboratory testing on automated platforms, there are fewer comprehensive resources for understanding important preanalytical and environmental factors that affect accurate molecular diagnostic testing of infectious diseases. This review identifies evidence-based factors that have been documented to impact the outcome of nucleic acid-based molecular techniques for the diagnosis of infectious diseases.

There is growing interest in proteomic analyses of tissue biopsies to reveal pathophysiology and identify biomarkers. The current gold standard for collecting tissue biopsies for preserving the proteome and post-translational modifications is flash freezing in liquid nitrogen (LN2). However, in many clinical settings, this is not an option due to unavailability of LN2 nor trained personnel for rapid biospecimen processing. To address this need, we developed a proof-of-concept quick-freeze prototype device to rapidly freeze biospecimens at the point-of-care to preserve the phosphoproteome without the need for LN2. Our objectives were to develop the device, demonstrate the ease of use, confirm the ability to ship through existing cold chain logistics, and evaluate the cooling performance (i.e., cool a tissue sample to <0°C in <60 seconds, below -8°C in <120 seconds, and maintain temperature <0°C for >60 minutes) in the context of preserving the proteome in a tissue biospecimen. To demonstrate feasibility, the performance of the prototype was benchmarked against flash freezing in LN2 using a murine melanoma patient-derived xenograft model subjected to total body irradiation to elicit phosphosignaling in the DNA damage response network. Tumors were harvested and quadrisected, with two parts of the tumor being snap frozen in LN2, and the remaining two parts being rapidly cooled in the prototype quick-freeze biospecimen containers. Phosphoproteins were profiled by liquid chromatography tandem mass spectrometry and quantified by targeted multiple reaction monitoring MS. Overall, the phosphoproteome was equivalent in biospecimens processed using the quick-freeze containers to those using the LN2 gold standard, although the measurements of a subset of phosphopeptides in the device-frozen specimens were more variable than LN2-frozen specimens. The prototype device forms the framework for development of a commercial device that will improve tissue biopsy preservation for measurement of important phosphosignaling molecules.

Given the crucial role of mitochondria as the main cellular energy provider and its contribution towards tumor growth, chemoresistance, and cancer cell plasticity, mitochondrial DNA (mtDNA) could serve as a relevant biomarker. Thus, the profiling of mtDNA mutations and copy number variations is receiving increasing attention for its possible role in the early diagnosis and monitoring therapies of human cancers. This applies particularly to highly aggressive pancreatic cancer, which is often diagnosed late and is associated with poor prognosis. As current diagnostic procedures are based on imaging, tissue histology, and protein biomarkers with rather low specificity, tumor-derived mtDNA mutations detected from whole blood represents a potential significant leap forward towards early cancer diagnosis. However, for future routine use in clinical settings it is essential that preanalytics related to the characterization of mtDNA in whole blood are thoroughly standardized, controlled, and subject to proper quality assurance, yet this is largely lacking. Therefore, in this study we carried out a comprehensive preanalytical workup comparing different mtDNA extraction methods and testing important preanalytical steps, such as the use of different blood collection tubes, different storage temperatures, length of storage time, and yields in plasma vs. whole blood. To identify analytical and preanalytical differences, all variables were tested in both healthy subjects and pancreatic carcinoma patients. Our results demonstrated a significant difference between cancer patients and healthy subjects for some preanalytical workflows, while other workflows failed to yield statistically significant differences. This underscores the importance of controlling and standardizing preanalytical procedures in the development of clinical assays based on the measurement of mtDNA.

Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized diploid cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified.
Tissue sections that fail more frequently show low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces are more likely to show FFPE-specific errors, akin to \"edge effects\" seen in histology, while the inner samples display no quality degradation related to fixation time.
To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.

Sampling of blood in the supine position for diagnosis of pheochromocytoma and paraganglioma (PPGL) results in lower rates of false positives for plasma normetanephrine than seated sampling. It is unclear how inpatient vs outpatient testing and other preanalytical factors impact false positives.
We aimed to identify preanalytical precautions to minimize false-positive results for plasma metanephrines.
Impacts of different blood sampling conditions on plasma metanephrines were evaluated, including outpatient vs inpatient testing, sampling of blood in semi- vs fully recumbent positions, use of cannulae vs direct venipuncture, and differences in outside temperature. A total of 3147 patients at 10 tertiary referral centers were tested for PPGL, including 278 with and 2869 without tumors. Rates of false-positive results were analyzed.
Outpatient rather than inpatient sampling resulted in 44% higher plasma concentrations and a 3.4-fold increase in false-positive results for normetanephrine. Low temperature, a semi-recumbent position, and direct venipuncture also resulted in significantly higher plasma concentrations and rates of false-positive results for plasma normetanephrine than alternative sampling conditions, although with less impact than outpatient sampling. Higher concentrations and rates of false-positive results for plasma normetanephrine with low compared with warm temperatures were only apparent for outpatient sampling. Preanalytical factors were without impact on plasma metanephrines in patients with PPGL.
Although inpatient blood sampling is largely impractical for screening patients with suspected PPGL, other preanalytical precautions (eg, cannulae, warm testing conditions) may be useful. Inpatient sampling may be reserved for follow-up of patients with difficult to distinguish true- from false-positive results.

Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined.
We analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing.
Neither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods.
Fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.