Sample Preparation Protocol Research Articles

Changes in thyroid fine needle aspiration practice during the COVID-19 pandemic.

PurposeTo investigate the diagnostic accuracy of a different sample preparation protocol for fine needle aspiration cytology (FNAC) of thyroid nodules established during the COVID‐19 pandemic.MethodsFrom April 2020, conventional smears during FNAC were ceased according to World Health Organization recommendations due to the increased infection risk for operators, and a new protocol using only liquid‐based cytology (LBC) was adopted. FNACs performed between April and July 2020 (COVID‐19 group) were retrospectively compared with those from December 2019 through March 2020 (Pre‐COVID‐19 group). The distribution of diagnoses based on SIAPEC‐IAP categories and the concordance between cytological and histological results were compared using the chi‐squared test.ResultsCategories based on FNAC for 90 and 82 thyroid nodules in the Pre‐COVID‐19 and COVID‐19 groups showed no significant difference in distribution (P = .081), with the following respective cases (and percentages): TIR1, 7 (8%) and 8 (10%); TIR1C, 0 (0%) and 6 (7%); TIR2, 59 (66%) and55 (67%); TIR3A, 8 (9%) and 5 (6%); TIR3B, 1 (1%) and 2 (3%); TIR4, 5 (6%) and 1 (1%); and TIR5, 10 (12%) and 5 (7%). Among patients with potentially malignant lesions, surgery was performed for 12/16 (75%) nodules in the Pre‐COVID‐19 and 7/8 (88%) nodules in the COVID‐19 groups, with no significant differences between cytological and histological diagnoses (P = .931).ConclusionThe new LBC‐only protocol provided similar diagnostic accuracy in comparison with conventional smears, and can be effectively applied during a viral pandemic improving operator safety.

The use of MALDI­-TOF mass­spectrometry for the identification of fungal pathogens

Here we review modern data on the advantages and disadvantages of identifying mycoses pathogens using the MALDI-TOF mass spectrometry method. The advantages of this method are the high accuracy of the results obtained, the simplicity of the analysis, and the ability to determine the indicators of sensitivity to antifungal drugs. Limitations in using the method are associated with the lack of reference mass spectra of many micromycetes in the databases and standardized protocols for preparing micromycete cultures for MALDI-TOF mass spectrometry. Additional difficulties arise in the identification of pathogens of especially dangerous mycoses due to the limited representation of reference mass spectra in databases. When developing unified protocols for sample preparation, it is also necessary to take into account the growth phase of dimorphic micromycetes.

Verification of water presence in graphene liquid cells

Graphene liquid cells (GLCs) present the thinnest possible sample enclosures for liquid phase electron microscopy. However, the actual presence of liquid within a GLC is not always guaranteed. Of key importance is to reliably test the presence of the liquid, which is most frequently water or saline. Here, the commonly used methods for verifying the presence of water were evaluated. It is shown that depending on the type of sample, applying a single criterion does not always conclusively verify the presence of water. Testing liquid filling for a specific GLC sample preparation protocol should thus be considered critically. The most reliable method is direct observation of the water exciton peak using electron energy loss spectroscopy (EELS). But if this method cannot be carried out, water filling of the GLC can be verified from a combination of higher contrast in the image, the presence of bubbles, and an oxygen signal in the EEL spectrum, which can be accomplished at a high electron dose in spot mode. Nanoparticle movement does not always occur in a GLC.

Microplastic extraction protocols can impact the polymer structure

Although microplastics are ubiquitous in today’s natural environments, our understanding of the materials, quantities, and particle sizes involved remains limited. The recovery of microplastics from different types of environmental matrices requires standardized matrix digestion protocols that allow inter-laboratory comparisons and that have no effect on the polymers themselves. A number of commonly used digestion methods rely on oxidation with concentrated hydrogen peroxide solutions to remove organic matter from the matrix. However, this can alter the nature of polymers through hydrolysis and often does not lead to a complete matrix removal. We have therefore investigated the use of two altered matrix digestion protocols, an acidic (Fenton) protocol and a new alkaline (Basic Piranha) protocol, focusing mainly on the effect on biodegradable polymers (polylactide, polybutylene adipate terephthalate, polybutylene succinate) and polymers with known degradation pathways via hydrolysis (thermoplastic polyurethanes, polyamide). Comparing the initial surface textures, chemical compositions, and particle size distributions with those obtained after digestion revealed that the Fenton protocol left most of the polymers unchanged. The ferrous residue that remains following Fenton digestion had no effect on either the polymer composition or the particle size distribution, but could disturb further analytics (e.g. Raman microscopy due to auto-fluorescence). While increasing the chance of complete matrix removal, the more powerful Basic Piranha protocol is also more likely to affect the polymer properties: Polylactide polymers in particular showed signs of degradation under alkaline digestion (reduced polylactide content, holes in the polymer matrix), indicating the unsuitability of the Basic Piranha protocol in this specific case. Polyamide, however, remained stable during the Basic Piranha treatment, and the surface chemistry, the particle size as well as the molar mass distribution of the investigated thermoplastic polyurethanes were also not affected. Hence, this protocol offers a powerful alternative for microplastic analysis with focus on particle size in more complex environmental matrices (e.g. removal of cellulose in soil), while avoiding ferrous Fenton residue. Unexpectedly, also tire rubber, a frequent target analyte in microplastic monitoring, was found to be susceptible to artefact structures by both oxidation protocols. In summary, controls for the specific combination of polymer and sample preparation protocol are highly recommended to select the most fitting protocol. Here selected suitable combinations are reported.

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline.

The mounting of microcrystals (<10 µm) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise. Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (<10 µm) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise. Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

Development and Optimisation of HILIC-LC-MS Method for Determination of Carbohydrates in Fermentation Samples.

Saccharides are the most common carbon source for Streptococcus thermophilus, which is a widely used bacterium in the production of fermented dairy products. The performance of the strain is influenced by the consumption of different saccharides during fermentation. Therefore, a precise measurement of the concentrations of saccharides in the fermentation media is essential. An 18-min long method with limits of quantitation in the range of 0.159–0.704 mg/L and with 13C labelled internal standards employing hydrophilic interaction chromatography coupled to mass spectrometric detection-(HILIC-LC-MS) allowed for simultaneous quantification of five saccharides: fructose, glucose, galactose, sucrose, and lactose in the fermentation samples. The method included a four-step sample preparation protocol, which could be easily applied to high-throughput analysis. The developed method was validated and applied to the fermentation samples produced by Streptococcus thermophilus.

Serum metabolic fingerprinting of psoriasis and psoriatic arthritis patients using solid-phase microextraction\u2014liquid chromatography\u2014high-resolution mass spectrometry

IntroductionPsoriatic arthritis (PsA), an inflammatory arthritis that develops in individuals with psoriasis, is associated with reduced quality of life. Identifying biomarkers associated with development of PsA as well as with PsA disease activity may help management of psoriatic disease.ObjectivesTo use metabolomic fingerprinting to determine potential candidate markers of disease conversion (psoriasis to PsA) and/or PsA activity.MethodsA novel sample preparation protocol based on solid-phase microextraction (SPME) was used to prepare serum samples obtained from: (1) individuals with psoriasis, some of whom develop psoriatic arthritis (n = 20); (2) individuals with varying PsA activity (mild, moderate, severe; n = 10 each) and (3) healthy controls (n = 10). Metabolomic fingerprinting of the obtained extracts was performed using reversed-phase liquid chromatography coupled to high resolution mass spectrometry.ResultsPsoriasis patients who developed PsA had similar metabolomic profiles to patients with mild PsA and were also indistinguishable from patients with psoriasis who did not develop PsA. Elevated levels of selected long-chain fatty acids (e.g., 3-hydroxytetradecanedioic acid) that are associated with dysregulation of fatty acid metabolism, were observed in patients with severe PsA. In addition, 1,11-undecanedicarboxylic acid—an unusual fatty acid associated with peroxisomal disorders—was also identified as a classifier in PsA patients vs. healthy individuals. Furthermore, a number of different eicosanoids with either pro- or anti-inflammatory properties were detected solely in serum samples of patients with moderate and severe PsA.ConclusionA global metabolomics approach was employed to analyze the serum metabolome of patients with psoriasis, PsA, and healthy controls in order to examine potential differences in the biochemical profiles at a metabolite level. A closer examination of circulating metabolites may potentially provide markers of PsA activity.

Fast Antibiotic Susceptibility Testing via Raman Microspectrometry on Single Bacteria: An MRSA Case Study.

Despite recent advances in molecular diagnostics, ultrafast determination of the antibiotic susceptibility phenotype of pathogenic microorganisms is still a major challenge of in vitro diagnostics (IVD) of infectious diseases. Raman microspectroscopy has been proposed as a means to achieve this goal. Previous studies have shown that susceptibility phenotyping could be done through Raman analysis of microbial cells, either in large clusters or down to the single-cell level in the case of Gram-negative rods. Gram-positive cocci such as Staphylococcus aureus pose several challenges due to their size and their different metabolic and chemical characteristics. Using a tailored automated single-cell Raman spectrometer and a previously proposed sample preparation protocol, we acquired and analyzed 9429 S. aureus single cells belonging to three cefoxitin-resistant strains and two susceptible strains during their incubation in the presence of various concentrations of cefoxitin. We observed an effect on S. aureus spectra that is weaker than what was detected on previous bacteria/drug combinations, with a higher cell-to-cell response variability and an important impact of incubation conditions on the phenotypic resistance of a given strain. Overall, the proposed protocol was able to correlate strains’ phenotype with a specific modification of the spectra using majority votes. We, hence, confirm that our previous results on single-cell Raman antibiotic susceptibility testing can be extended to the S. aureus case and further clarify potential limitations and development requirements of this approach in the move toward industrial applications.

Environmental implications of one-century COPRs evolution in a single industrial site: From leaching impact to sustainable remediation of CrVI polluted groundwater

The Stoppani factory manufactured chromium for more than one century, dumping millions of tons of Chromite Ore Processing Residues (COPRs) over decades. The massive presence of COPRs resulted in an intense CrVI leaching and consequent contamination of percolating groundwater. The site offers a unique opportunity to follow COPRs evolution from the primary roasting process to the aged Cr-bearing mineral phases. Herein, new insights on COPRs mineralogy evolution and their role in CrVI release are provided by a dry sample preparation protocol, coupled with in-depth multi-technique characterization. Besides typical COPRs mineral assemblages, highly soluble Na2CrO4 and the first evidence of crocoite (PbCrO4) in a COPR contaminated site are revealed. Selective extraction experiments confirmed a strong reactivity for Cr-bearing minerals as confirmed by concentrations as high as 375 mg L−1 of leached CrVI. The mineralogical approach was combined with a nanotechnological solution for CrVI wastewater remediation. The application of naked colloidal maghemite (γ-Fe2O3) nanoparticles (SAMNs) on the complex industrial wastewater, led to > 90% CrVI removal, either under acidic or in-situ conditions. The present case study of a highly polluted site, ranging from mineral characterization to wastewater remediation, highlights the use of multidisciplinary approaches to cope with complex environmental issues.

Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development.

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

A preparation sequence for multi‐analysis of µm‐sized extraterrestrial and geological samples

AbstractWith the recent and ongoing sample return missions and/or the developments of nano‐ to microscale 3‐D and 2‐D analytical techniques, it is necessary to develop sample preparation and analysis protocols that allow combination of different nanometer‐ to micrometer‐scale resolution techniques and both maximize scientific outcome and minimize sample loss and contamination. Here, we present novel sample preparation and analytical procedures to extract a maximum of submicrometer structural, mineralogical, chemical, molecular, and isotopic information from micrometric heterogeneous samples. The sample protocol goes from a nondestructive infrared (IR) tomography of ~10 to ~70 µm‐sized single grains, which provides the distribution and qualitative abundances of both mineral and organic phases, followed by its cutting in several slices at selected sites of interest for 2‐D mineralogical analysis (e.g., transmission electron microscopy), molecular organic and mineral analysis (e.g., Raman and/or IR microspectroscopy), and isotopic/chemical analysis (e.g., NanoSIMS). We also discuss here the importance of the focused ion beam microscopy in the protocol, the problems of sample loss and contamination, and at last the possibility of combining successive different analyses in various orders on the same micrometric sample. Special care was notably taken to establish a protocol allowing correlated NanoSIMS/TEM/IR analyses with NanoSIMS performed first. Finally, we emphasize the interest of 3‐D and 2‐D IR analyses in studying the organics–minerals relationship in combination with more classical isotopic and mineralogical grain characterizations.

A massively multi-scale approach to characterizing tissue architecture by synchrotron micro-CT applied to the human placenta.

Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure–function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure–function relationships in both normal and diseased placentas, allowing us to optimize therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in the characterization of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.

High-throughput phenotyping methods for quantifying hair fiber morphology

Quantifying the continuous variation in human scalp hair morphology is of interest to anthropologists, geneticists, dermatologists and forensic scientists, but existing methods for studying hair form are time-consuming and not widely used. Here, we present a high-throughput sample preparation protocol for the imaging of both longitudinal (curvature) and cross-sectional scalp hair morphology. Additionally, we describe and validate a new Python package designed to process longitudinal and cross-sectional hair images, segment them, and provide measurements of interest. Lastly, we apply our methods to an admixed African-European sample (n = 140), demonstrating the benefit of quantifying hair morphology over classification, and providing evidence that the relationship between cross-sectional morphology and curvature may be an artefact of population stratification rather than a causal link.

Protein Signatures of Remodeled Airways in Transplanted Lungs with Bronchiolitis Obliterans Syndrome Obtained Using Laser-Capture Microdissection

Bronchiolitis obliterans syndrome, a common form of chronic lung allograft dysfunction, is the major limitation to long-term survival after lung transplantation. The histologic correlate is progressive, fibrotic occlusion of small airways, obliterative bronchiolitis lesions, which ultimately lead to organ failure. The molecular composition of these lesions is unknown. In this sutdy, the protein composition of the lesions in explanted lungs from four end-stage bronchiolitis obliterans syndrome patients was analyzed using laser-capture microdissection and optimized sample preparation protocols for mass spectrometry. Immunohistochemistry and immunofluorescence were used to determine the spatial distribution of commonly identified proteins on the tissue level, and protein signatures for 14 obliterative bronchiolitis lesions were established. A set of 39 proteins, identified in >75% of lesions, included distinct structural proteins (collagen types IV and VI) and cellular components (actins, vimentin, and tryptase). Each respective lesion exhibited a unique composition of proteins (on average, n = 66 proteins), thereby mirroring the morphologic variation of the lesions. Antibody-based staining confirmed these mass spectrometry-based findings. The 14 analyzed obliterative bronchiolitis lesions showed variations in their protein content, but also common features. This study provides molecular and morphologic insights into the development of chronic rejection after lung transplantation. The protein patterns in the lesions were correlated to pathways of extracellular matrix organization, tissue development, and wound healing processes.

Comparison of different digestion methods for proteomic analysis of isolated cells and FFPE tissue samples

Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients’ treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

Sample preparation strategy for the detection of steroid-like compounds using MALDI mass spectrometry imaging: pulmonary distribution of budesonide as a case study

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard’s reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples.Graphical abstract

Monitoring the carryover of egg proteins in pasta making to support allergen risk management

ABSTRACT Egg proteins are among the major food allergens. Very often, the same pasta-making plants are used for industrial production of egg-based pasta (EBP) and semolina-only pasta (SP), so that residual egg proteins may be present in SP. This calls for defining the amount of semolina pasta that should be discarded when switching production lines. In this study, the egg proteins content was measured in pasta samples taken at various times after switching production lines from EBP to SP Both long and short pasta shapes were sampled before and after a drying step. Protocols meant to circumvent the difficulties associated with detecting egg proteins in a complex matrix after processing were set up for using commercial ELISA kits to monitor the disappearance of egg proteins from the products. The use of both denaturants and disulphide reductants to solubilise egg proteins was found to be mandatory, as verified by ovalbumin detection by ELISA and by using mass spectrometry to assess residual egg white lysozyme. Appropriate sample preparation protocols were used to monitor the progressive disappearance of egg proteins in the products when shifting production lines in an industrial pasta plant, providing a basis for credible, reliable, and consistent self-control procedures. For lines with a production capacity of 2200–2400 kg h−1, the amount of material to be discarded to ensure that products meet the strictest analytical requirements has been found to be around 2000–3000 kg (for long pasta) and 3000–4000 kg (for short pasta).