Bottom-up/cross-linking mass spectrometry via simplified sample processing on anion-exchange solid-phase extraction spin column

Capturing proteins on anion-exchange discs facilitates concentration of diluted samples and removal of contaminants, allowing more efficient sample pretreatment for bottom-up/cross-linking mass spectrometry than in-solution and in-gel.

Significant Plasma Cell Loss (up to 90%) Despite Preserved Overall Cell Viability - Time Dependent Changes Observed in Multiparametric Flow Cytometry Analysis of Bone Marrow Samples in Multiple Myeloma

Multiparametric flow cytometry (MPFC) is a mainstream laboratory method used in the diagnosis of multiple myeloma. Minimal residual disease (MRD) assessment by EuroFlow next-generation flow cytometry allows assessment down to an assay sensitivity of 1x10 -5. Delayed sample processing remains a common challenge due to logistical limitations. Specialized tests performed in central pathology laboratories are frequently located a considerable distance from healthcare providers. Our study aims to evaluate the impact of delayed sample processing on plasma cell yield and bone marrow sample stability. There is little published data available. Plasma cell yield and bone marrow sample stability were investigated in patients with multiple myeloma who underwent bone marrow biopsy. Participants were included based on ³10% plasma cell burden by morphological quantification on the bone marrow aspirate smear. Bone marrow aspirates were collected in EDTA (with three samples also collected in lithium heparin) and stored at four degrees Celsius. Samples were analyzed by MPFC within four hours of collection, at 24 and at 48 hours after collection. CD138 and CD38 co-expression were used to identify plasma cells, and absence of 7-AAD to determine cell viability. Mean fluorescence intensity (MFI) of CD138 and CD38 was recorded. Statistical analyses were performed using two-tailed Wilcoxon signed-rank tests and repeated measures ANOVA with significance assigned at p<0.05. Bone marrow aspirate samples of nine participants were evaluated. Significant reduction in plasma cell yield was observed over time (p<0.001) while sample integrity remained unchanged (p>0.05). The most marked reduction in plasma cell detection was seen between initial processing and 24 hours (median absolute reduction 9%, range 0 to 23% and median relative reduction 37%, range -8 to 90%, p<0.01). Further significant reduction of plasma cells occurred after an additional 24 hours (p=0.025). At 48 hours, the median absolute reduction in plasma cell yield from initial testing was 12% (range 1 to 24%) and median relative reduction was 40% (range 18 to 90%). Sample integrity remained constant. The median viability at collection, 24 hours and 48 hours was 91%, 93% and 95% respectively. The most significant specimen deterioration observed was 13% viability reduction to 75% overall by 48 hours. Three of the participants had additional samples collected in lithium heparin anticoagulant media that were analyzed in parallel with their EDTA samples. Plasma cell yield remained similar across the two different anticoagulants with overall cell viability remaining high in lithium heparin (³90%). A trend of time-dependent reduction of CD138 MFI was observed with lithium heparin but not with EDTA. This study demonstrates the significance of time to processing as a pre-analytical variable in MPFC in multiple myeloma. The greatest loss of plasma cells occurs within the first 24 hours after collection but continues to fall significantly out to 48 hours. Reductions of up to 90% were observed in our small cohort and represent a potential 1 log reduction in yield. This decrease in plasma cell yield raises questions of reliability and validity of flow cytometry, whereby the sensitivity depth may be compromised if the sample cannot be processed on the same day of collection. It is a technical limitation of flow cytometry in comparison to polymerase chain reaction methods where sensitivity is unaffected by delays in processing. The overall viability of cells within the samples remained stable over time, despite the decline in plasma cells. A reduction in CD138 MFI is observed in lithium heparin storage medium that may impact on standardized gating techniques. Further validation studies are warranted to explore these phenomena. MRD monitoring in multiple myeloma is rapidly becoming an accepted standard of care in the evaluation of treatment response and represents an independent prognostic maker of progression free survival that can be used to guide further therapy. Our findings indicate the potential of false negative MRD results with delays in sample processing. This questions the current consensus guidelines that recommend samples can be processed up to 2 days after collection. These guidelines may need to be revised in the near future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

Searching Nanoplastics: From Sampling to Sample Processing

Nanoplastics (NPs) are considered emerging pollutants, namely unregulated contaminants whose toxic effect on humans and the environment has been demonstrated or suspected. They are the result of the physical fragmentation of the plastics that over time reach smaller dimensions (<100 nm). The issues related to the characterization and quantification of NPs in the environmental matrices are mainly related to the infinitesimal size, to the fact that they are found in bulk, and to the different physico-chemical forms in which the same polymer can evolve over time by degradation. To deal with the study of a new class of pollutants it is necessary to assess the entire analytical method, carefully considering every single step (sampling, cleanup, qualitative, and quantitative analysis) starting from the validation method in the laboratory. This paper reviews the analytical method steps, focusing on the first ones, which the current literature often underestimates: laboratory tests, sampling, and sample processing; in fact, most errors and the quality of the analyses often depend on them. In addition, all newly introduced sample processing methods were examined.

Explain the law: When the evidence is not enough

The article responds to the current variability of research into linguistic laws and the explanation of these laws. We show basic features to approach linguistic laws in the field of quantitative linguistics and research on linguistic laws outside the field of language and text. Language laws are usually explained in terms of the language system—especially as economizing—or of the information structure of the text (Piantadosi 2014). One of the hallmarks of the transmission of linguistic laws outside the realm of language and text is that they provide other kinds of explanations (Torre et al. 2019). We want to show that the problem of linguistics in the explanation of linguistic laws lies primarily in its inability to clarify the internal structure of language material, and the influence of the theory or method used for sample processing on the result of law analysis—which was formulated by Peter Grzybek (2006). We would like to show that this is the reason why linguistics avoids explanations of linguistic laws.

Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. Methods Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. Conclusions We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Graphical Abstract