Picoliter Single-Cell Reactor for Proteome Profiling by In Situ Cell Lysis, Protein Immobilization, Digestion, and Droplet Transfer.

Global profiling of single-cell proteomes can reveal cellular heterogeneity, thus benefiting precision medicine. However, current mass spectrometry (MS)-based single-cell proteomic sample processing still faces technical challenges associated with processing efficiency and protein recovery. Herein, we present an innovative sample processing platform based on a picoliter single-cell reactor (picoSCR) for single-cell proteome profiling, which involves in situ protein immobilization and sample transfer. PicoSCR helped minimize surface adsorptive losses by downscaling the processing volume to 400 pL with a contact area of less than 0.4 mm2. Besides, picoSCR reached highly efficient cell lysis and digestion within 30 min, benefiting from optimal reagent and high reactant concentrations. Using the picoSCR-nanoLC-MS system, over 1400 proteins were identified from an individual HeLa cell using data-dependent acquisition mode. Proteins with copy number below 1000 were identified, demonstrating this system with a detection limit of 1.7 zmol. Furthermore, we profiled the proteome of circulating tumor cells (CTCs). Data are available via ProteomeXchange with the identifier PXD051468. Proteins associated with epithelial-mesenchymal transition and neutrophil extracellular traps formation (which are both related to tumor metastasis) were observed in all CTCs. The cellular heterogeneity was revealed by differences in signaling pathways within individual cells. These results highlighted the potential of the picoSCR platform to help discover new biomarkers and explore differences in biological processes between cells.

Evaluation of a novel lysis-based sample processing method to optimize Vibrio vulnificus detecting by loop-mediated isothermal amplification assay.

BACKGROUND: Vibrio vulnificus exists as one of the most serious foodborne pathogens for humans, and rapid and sensitive detection methods are needed to control its infections. As an emerging method, The Loop-Mediated Isothermal Amplification (LAMP) assay has been applied to the early detection of various foodborne pathogens due to its high efficiency, but sample preprocessing still prolongs the complete detection. To optimize the detection process, our study established a novel sample preprocessing method that was more efficient compared to common methods. RESULT: Using V. vulnificus as the detecting pathogen, the water-lysis-based detecting LAMP method shortened the preprocessing time to ≤ 1 min with 100% LAMP specificity; the detection limits of the LAMP assay were decreased to 1.20 × 102 CFU/mL and 1.47 × 103 CFU/g in pure culture and in oyster, respectively. Furthermore, the 100% LAMP specificity and high sensitivity of the water-lysis method were also obtained on detecting V. parahaemolyticus, V. alginolyticus, and P. mirabilis, revealing its excellent LAMP adaption with improvement in sensitivity and efficiency. CONCLUSION: Our study provided a novel LAMP preprocessing method that was more efficient compared to common methods and possessed the practical potential for LAMP application in the future.

Challenges to the effectiveness of next-generation sequencing in formalin-fixed paraffin-embedded tumor samples for non-small cell lung cancer.

INTRODUCTION: Next-generation sequencing (NGS) of Formalin-Fixed and Paraffin-Embedded (FFPE) specimens is routine in precision oncology practice. However, results are not always conclusive, and it is important to identify which factors may influence FFPE tumor sequencing success. MATERIALS AND METHODS: Here, we evaluated the influence of pre-analytical factors on 705 samples of non-small cell lung cancer specimens that underwent NGS testing. Factors such as tumor site, tumor cell percentage, fragment size, primary tumor or metastasis, presence of necrosis, DNA purity, DNA concentration, sample origin and year of testing. RESULTS: The overall NGS success rate was 84.9 % (n = 599). Bone site specimens had a very low success rate (42.1 %), differing from lung samples (79.8 %) (P < 0.05). Samples with tumor percentages <5 % (success rate of 44.4 %) represented 14.1 % of failed sequencings. Moreover, samples with tumor percentages >10 %-20 % (82 %) did not differ from those with >30 % (88.9 %) on sequencing outcomes (P = 0.086). Specimens that provided DNA concentrations >2.0 ng/uL, 1.0-2.0 ng/uL, 0.5-1.0 ng/uL and <0.5 ng/uL had success rates of 92 %, 77.1 %, 61.3 % and 20.4 %, respectively. Small fragments (≤0.2 cm2) had a success rate of 74.7 % and were more prevalent in the unsuccessful group (P < 0.05). CONCLUSIONS: Our results suggest that tumor percentage, fragment size, decalcified bone specimens, and DNA concentration are potential modifiers of NGS success rates. Interestingly, specimens with tumor percentages between 10 % and 20 % have the same sequencing outcome than specimens with >30 %. These results can strengthen the understanding of factors that lead to NGS success variability.

Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine.

Rapid advances are being made in cancer drug therapy. Since molecularly targeted therapy has been introduced, personalized medicine is being practiced, pathological tissue from malignant tumors obtained during routine practice is frequently used for genomic testing. Whereas cytological specimens fixed mainly in alcohol are considered to be more advantageous in terms of preservation of the nucleic acid quality and quantity. This article is aimed to share the information for the proper handling of cytological specimens in practice for genomic medicine based on the findings established in "Guidelines for Handling of Cytological Specimens in Cancer Genomic Medicine (in Japanese)" published by the Japanese Society of Clinical Cytology in 2021. The three-part practical guidelines are based on empirical data analyses; Part 1 describes general remarks on the use of cytological specimens in cancer genomic medicine, then Part 2 describes proper handling of cytological specimens, and Part 3 describes the empirical data related to handling of cytological specimens. The guidelines indicated proper handling of specimens in each fixation, preparation, and evaluation.

Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples.

Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.

Circular RNAs in peripheral blood mononuclear cells are more stable than linear RNAs upon sample processing delay.

Circular RNAs (circRNAs) are a novel class of RNAs with closed loop structure. Blood circRNAs are widely acknowledged to be more stable than linear mRNAs, which show promising prospect to be liquid biopsy biomarkers for clinical applications. However, accumulating studies have demonstrated that sample processing delays have profound effects on blood transcriptome expression profiles, wherein knowledge remains elusive about the impacts of prolonged sample processing on blood expression profiles of circRNAs. We collected whole blood samples from three donors and isolated peripheral blood mononuclear cells (PBMCs) at six different incubation time points. We measured total RNA expression profiles using RNA sequencing (RNA-seq) and investigated the differentially expressed circRNAs, mRNAs and lncRNAs upon blood processing delay. Meanwhile, we explored the underlying inducement of aberrant expression of circRNAs against their corresponding mRNA transcripts. Finally, we utilized rMATS-turbo and CIRI-AS, respectively, to screen out differential alternative splicing (AS) events in linear mRNAs and circRNAs. Sample incubation at 4°C lasting to 48 hours (h) led to minimal effects to circRNAs' expression. However, it induced extensive alterations for mRNAs and lncRNAs when the incubation time was beyond 12 h. Additionally, only 2 h processing delays may result in profound impacts on AS events of linear mRNAs, while less impact on the equivalence of circRNAs. Our results suggested that PBMC circRNAs are stable upon sample processing delay, which are more suitable to be liquid biopsy biomarkers.

Variable blood processing procedures contribute to plasma proteomic variability.

BACKGROUND: Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC-MS/MS proteomic analysis of plasma. METHODS: Initially two batches of patient plasma samples (120 and 204 samples, respectively) were analyzed using LC-MS/MS shotgun proteomics. Follow-up experiments were designed and carried out on healthy donor blood in order to examine the effects of different centrifugation conditions, length of delay until first centrifugation, storage temperature and anticoagulant type on results from shotgun proteomics. RESULTS: Variable levels of intracellular proteins were observed in subsets of patient plasma samples from the initial batches analyzed. This observation correlated strongly with the site of collection, implicating variability in blood processing procedures. Results from the healthy donor blood analysis did not demonstrate a significant impact of centrifugation conditions to plasma proteome variation. The time delay until first centrifugation had a major impact on variability, while storage temperature and anticoagulant showed less pronounced but still significant effects. The intracellular proteins associated with study site effect in patient plasma samples were significantly altered by delayed processing also. CONCLUSIONS: Variable blood processing procedures contribute significantly to plasma proteomic variation and may give rise to increased intracellular proteins in plasma. Accounting for these effects can be important both at study design and data analysis stages. This understanding will be valuable to incorporate in the planning of protein-based biomarker discovery efforts in the future.

Automatic medium exchange for micro-volume cell samples based on dielectrophoresis.

Cell medium exchange is a crucial step for life science and medicine. However, conventional cell medium exchange methods, including centrifuging and filtering, show limited ability for micro-volume cell samples such as circulating tumor cell (CTC) and circulating fetal cell (CFC). In this paper, we proposed an automatic medium exchange method for micro-volume cell samples based on dielectrophoresis (DEP) in microfluidic chip. Fresh medium and cell suspension were introduced into the microfluidic channel as the laminar flow. Plane stair-shaped interdigital electrodes were employed to drive the cells from the cell suspension to fresh media directly by DEP force. Additionally, we characterized and optimized the cell medium exchange according to both the theory and experiments. In the end, we achieved a 96.9% harvest rate of medium exchange for 0.3 µL samples containing micro-volume cells. For implementing an automatic continuous cell medium exchange, the proposed method can be integrated into the automatic cell processing system conveniently. Furthermore, the proposed method is a great candidate in micro-volume cell analysis and processing, cell electroporation, single cell sequencing, and other scenarios.

The Japanese Society of Pathology Practical Guidelines on the handling of pathological tissue samples for cancer genomic medicine.

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in "Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)" published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be ≥30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.

New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis.

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

Occurrence and distribution of microplastic particles and the concentration of Di 2-ethyl hexyl phthalate (DEHP) in microplastics and wastewater in the wastewater treatment plant.

Wastewater treatment plant (WWTP) is one of the significant sources of Microplastics (MPs) release to the environment. Di 2-ethyl hexyl phthalate (DEHP) is used as an additive for more flexibility of plastics. In this study, we determined the number, size, shape, and color distribution of MPs as well as the concentration of DEHP in MPs and wastewater during the wastewater treatment process in WWTP. Samples were collected from 4 stations of different treatment stages of WWTP. The microplastic particles and the concentration of DEHP were detected via the fluorescence and polarized light microscopy and GC/MS instrument, respectively. The number of MPs decreased from 9.2 (station 1) to 0.84 MP/L (the final treated effluent) during the wastewater treatment process. Also, the size of MPs at the last station was smaller than the other stations. The mean concentrations of DEHP in MPs in stations 1, 2, 3 and 4 had the respective values of 83.3, 61.05, 30.62 and 17.49 µg/g, while the mean concentrations of DEHP in wastewater in stations 1, 2, 3 and 4 were 30.08, 25.07, 9.56, and 8.13 µg/L, respectively. This study shows that despite the removal of high amounts of MPs and DEHP in the final effluent of WWTP, due to the high volume of this effluent (2.828 × 108 L/d), significant amount of MPs (2.419 × 107 MP/day) and DEHP enter the aquatic environment daily, which may threaten the health of the fish and aquatic organisms and ultimately on the health of the local population.

HEnRY: a DZIF LIMS tool for the collection and documentation of biomaterials in multicentre studies.

BACKGROUND: Well-characterized biomaterials of high quality have great potential for acceleration and quality improvement in translational biomedical research. To improve accessibility of local sample collections, efforts have been made to create central biomaterial banks and catalogues. Available technical solutions for creating professional local sample catalogues and connecting them to central systems are cost intensive and/or technically complex to implement. Therefore, the Translational Thematic Unit HIV of the German Center for Infection Research (DZIF) developed a Laboratory Information and Management System (LIMS) called HIV Engaged Research Technology (HEnRY) for implementation into the Translational Platform HIV (TP-HIV) at the DZIF and other research networks. RESULTS: HEnRY is developed at the University Hospital of Cologne. It is an advanced LIMS to manage processing and storage of samples and aliquots of different sample types. Features include: monitoring of stored samples and associated information data selection via query tools or Structured Query Language (SQL) preparation of summary documents, including scannable search lists centralized management of the practical laboratory part of multicentre studies (e.g. import of drawing schemes and sample processing steps), preparation of aliquot shipments, including associated documents to be added to shipments unique and secure identification of aliquots through use of customizable Quick Response (QR) code labels directly from HEnRY support of aliquot data transmission to central registries. In summary, HEnRY offers all features necessary for a LIMS software. In addition, the structure of HEnRY provides sufficient flexibility to allow the implementation in other research areas. CONCLUSION: HEnRY is a free biobanking tool published under the MIT license. While it was developed to support HIV research in Germany, the feature set and language options, allow much broader applications and make this a powerful free research tool.

On-Chip Sample Preparation Using a ChipFilter Coupled to NanoLC-MS/MS for Bottom-Up Proteomics.

Sample preparation is a crucial step in bottom-up proteomics. Analytical performances of bottom-up proteomics can be improved by the miniaturization of sample preparation. Many microfluidic devices have been designed in the field of proteomics, but many of them are not capable of handling complex samples and do not integrate the processing and digestion steps. We propose a ChipFilter Proteolysis (CFP) microfluidic device as a proteomics reactor for the miniaturization of protein sample processing and digestion steps, whose design is closely related to the experimental setup of filter-aided sample processing, even if no denaturing surfactant is required. The microchip has two reaction chambers of 0.6 µL volume separated by a protein filtration membrane in regenerated cellulose (10kD cutoff) that will concentrate or retain large polypeptides and will release small molecules. Cell lysis, protein concentration, and rapid chemical or enzymatic treatment can be performed in the ChipFilter. Complex proteomic samples like yeast protein extract or whole human cells proteome have been successfully analyzed with our microchip. Compared with the membrane-based commercial ultracentrifugation cartridge, our microfluidic device offered a better proteome coverage with 10 times less starting material and 8 times faster protocol duration.

Validation of a simplified procedure for convenient and rapid quantification of reduced and oxidized glutathione in human plasma by liquid chromatography tandem mass spectrometry analysis.

Endogenous glutathione (GSH) and glutathione disulfide (GSSG) status is highly sensitive to oxidative conditions and have broad application as a surrogate indicator of redox status in vivo. Established methods for GSH and GSSG quantification in whole blood display limited utility in human plasma, where GSH and GSSG levels are ~3-4 orders of magnitude below those observed in whole blood. This study presents simplified sample processing and analytical LC-MS/MS approaches exhibiting the sensitivity and accuracy required to measure GSH and GSSG concentrations in human plasma samples, which after 5-fold dilution to suppress matrix interferences range from 200 to 500 nm (GSH) and 5-30 nm (GSSG). The utility of the methods reported herein is demonstrated by assay performance and validation parameters which indicate good sensitivity [lower limits of quantitation of 4.99 nm (GSH) and 3.65 nm (GSSG), and high assay precision (intra-assay CVs 3.6 and 1.9%, and inter-assay CVs of 7.0 and 2.8% for GSH and GSSG, respectively). These methods also exhibited exceptional recovery of analyte-spiked plasma samples (98.0 ± 7.64% for GSH and 98.5 ± 12.7% for GSSG). Good sample stability at -80°C was evident for GSH for up to 55 weeks and GSSG for up to 46 weeks, with average CVs <15 and <10%, respectively.

A comparison of methods for DNA preparation prior to microarray analysis.

Microarrays are a valuable tool for analysis of both bacterial and eukaryotic nucleic acids. As many of these applications use non-specific amplification to increase sample concentration prior to analysis, the methods used to fragment and label large amplicons are important to achieve the desired analytical selectivity and specificity. Here, we used eight sequenced ESKAPE pathogens to determine the effect of two methods of whole genome amplicon fragmentation and three methods of subsequent labeling on microarray performance; nick translation was also assessed. End labeling of both initial DNase I-treated and sonication-fragmented amplicons failed to provide detectable material for a significant number of sequence-confirmed genes. However, processing of amplicons by nick translation, or by sequential fragmentation and labeling by Universal Labeling System or Klenow fragment/random primer provided good sensitivity and selectivity, with marginally better results obtained by Klenow fragment labeling. Nick-translation provided 91-100% sensitivity and 100% specificity in the tested strains, requiring half as many manipulations and less than 4h to process samples for hybridization; full sample processing from whole genome amplification to final data analysis could be performed in less than 10h. The method of template denaturation before amplification did affect detection sensitivity/selectivity of nick-labeled amplicons, however.

A sample processing method for immunoassay of whole blood tacrolimus.

Current sample processing (SP) methods for tacrolimus (FK506) immunoassays are mainly based on extraction of drug by organic solvent and divalent metal ions. Although these methods are effective for drug extraction and interference elimination, they suffer from drawbacks including inconvenience for operation, difficulties for automation and potential measurement bias. To overcome these limitations, this study describes a new SP reagent for blood cell lysis and protein denaturation. A TRFIA (time-resolved fluorescence immunoassay) was developed by using this SP reagent for whole blood FK506 quantification. Results show that blood samples could be turned into homogeneous solution after being treated by this SP reagent, and so could be directly applied to immunoassays without centrifugation. The analytical sensitivity of the FK506-TRFIA was 0.57 ng/mL, the within-run and between-run coefficient of variations (CVs) were both less than 10%. The FK506 values of 126 samples obtained by FK506-TRFIA correlated excellently with that obtained by ABBOTT FK506-CMIA (R2 = 0.982). Comparison studies also show that the FK506-TRFIA was highly resistant to endogenous interferences. These results suggest that the present SP method is a more promising chose for FK506 immunoassay, and in the meantime, its simplicity makes the whole-process immunoassay automation more feasible by obviating the necessary for centrifugation.

Optimization of fecal sample processing for microbiome study - The journey from bathroom to bench.

Although great interest has been displayed by researchers in the contribution of gut microbiota to human health, there is still no standard protocol with consensus to guarantee the sample quality of metagenomic analysis. Here we reviewed existing methodology studies and present suggestions for optimizing research pipeline from fecal sample collection to DNA extraction. First, we discuss strategies of clinical metadata collection as common confounders for microbiome research. Second, we propose general principles for freshly collected fecal sample and its storage and share a DIY stool collection kit protocol based on the manual procedure of Human Microbiome Project (HMP). Third, we provide a useful information of collection kit with DNA stabilization buffers and compare their pros and cons for multi-omic study. Fourth, we offer technical strategies as well as information of novel tools for sample aliquoting before long-term storage. Fifth, we discuss the substantial impact of different DNA extraction protocols on technical variations of metagenomic analysis. And lastly, we point out the limitation of current methods and the unmet needs for better quality control of metagenomic analysis. We hope the information provided here will help investigators in this exciting field to advance their studies while avoiding experimental artifacts.

Establishing Statistical Equivalence of Data from Different Sampling Approaches for Assessment of Bacterial Phenotypic Antimicrobial Resistance.

To assess phenotypic bacterial antimicrobial resistance (AMR) in different strata (e.g., host populations, environmental areas, manure, or sewage effluents) for epidemiological purposes, isolates of target bacteria can be obtained from a stratum using various sample types. Also, different sample processing methods can be applied. The MIC of each target antimicrobial drug for each isolate is measured. Statistical equivalence testing of the MIC data for the isolates allows evaluation of whether different sample types or sample processing methods yield equivalent estimates of the bacterial antimicrobial susceptibility in the stratum. We demonstrate this approach on the antimicrobial susceptibility estimates for (i) nontyphoidal Salmonella spp. from ground or trimmed meat versus cecal content samples of cattle in processing plants in 2013-2014 and (ii) nontyphoidal Salmonella spp. from urine, fecal, and blood human samples in 2015 (U.S. National Antimicrobial Resistance Monitoring System data). We found that the sample types for cattle yielded nonequivalent susceptibility estimates for several antimicrobial drug classes and thus may gauge distinct subpopulations of salmonellae. The quinolone and fluoroquinolone susceptibility estimates for nontyphoidal salmonellae from human blood are nonequivalent to those from urine or feces, conjecturally due to the fluoroquinolone (ciprofloxacin) use to treat infections caused by nontyphoidal salmonellae. We also demonstrate statistical equivalence testing for comparing sample processing methods for fecal samples (culturing one versus multiple aliquots per sample) to assess AMR in fecal Escherichia coli These methods yield equivalent results, except for tetracyclines. Importantly, statistical equivalence testing provides the MIC difference at which the data from two sample types or sample processing methods differ statistically. Data users (e.g., microbiologists and epidemiologists) may then interpret practical relevance of the difference.IMPORTANCE Bacterial antimicrobial resistance (AMR) needs to be assessed in different populations or strata for the purposes of surveillance and determination of the efficacy of interventions to halt AMR dissemination. To assess phenotypic antimicrobial susceptibility, isolates of target bacteria can be obtained from a stratum using different sample types or employing different sample processing methods in the laboratory. The MIC of each target antimicrobial drug for each of the isolates is measured, yielding the MIC distribution across the isolates from each sample type or sample processing method. We describe statistical equivalence testing for the MIC data for evaluating whether two sample types or sample processing methods yield equivalent estimates of the bacterial phenotypic antimicrobial susceptibility in the stratum. This includes estimating the MIC difference at which the data from the two approaches differ statistically. Data users (e.g., microbiologists, epidemiologists, and public health professionals) can then interpret whether that present difference is practically relevant.

Use of a quality control approach to assess measurement uncertainty in the comparison of sample processing techniques in the analysis of pesticide residues in fruits and vegetables.

In routine monitoring of foods, reduction of analyzed test portion size generally leads to higher sample throughput, less labor, and lower costs of monitoring, but to meet analytical needs, the test portions still need to accurately represent the original bulk samples. With the intent to determine minimal fit-for-purpose sample size, analyses were conducted for up to 93 incurred and added pesticide residues in 10 common fruits and vegetables processed using different sample comminution equipment. The commodities studied consisted of apple, banana, broccoli, celery, grape, green bean, peach, potato, orange, and squash. A Blixer® was used to chop the bulk samples at room temperature, and test portions of 15, 10, 5, 2, and 1 g were taken for analysis (n = 4 each). Additionally, 40 g subsamples (after freezing) were further comminuted using a cryomill device with liquid nitrogen, and test portions of 5, 2, and 1 g were analyzed (n = 4 each). Both low-pressure gas chromatography-tandem mass spectrometry (LPGC-MS/MS) and ultrahigh-performance liquid chromatography (UHPLC)-MS/MS were used for analysis. An empirical approach was followed to isolate and estimate the measurement uncertainty contribution of each step in the overall method by adding quality control spikes prior to each step. Addition of an internal standard during extraction normalized the sample preparation step to 0% error contribution, and coefficients of variation (CVs) were 6-7% for the analytical steps (LC and GC) and 6-9% for the sample processing techniques. In practice, overall CVs averaged 9-11% among the different analytes, commodities, batches, test portion weights, and analytical and sample processing methods. On average, CVs increased up to 4% and bias 8-12% when using 1-2 g test portions vs. 10-15 g. Graphical abstract Efficient quality control approach to include sample processing.

An Overview of Pre-Analytical Factors Impacting Metabolomics Analyses of Blood Samples.

Discrepant sample processing remains a significant challenge within blood metabolomics research, introducing non-biological variation into the measured metabolome and biasing downstream results. Inconsistency during the pre-analytical phase can influence experimental processes, producing metabolome measurements that are non-representative of in vivo composition. To minimize variation, there is a need to create and adhere to standardized pre-analytical protocols for blood samples intended for use in metabolomics analyses. This will allow for reliable and reproducible findings within blood metabolomics research. In this review article, we provide an overview of the existing literature pertaining to pre-analytical factors that influence blood metabolite measurements. Pre-analytical factors including blood tube selection, pre- and post-processing time and temperature conditions, centrifugation conditions, freeze-thaw cycles, and long-term storage conditions are specifically discussed, with recommendations provided for best practices at each stage.