scholarly journals Pursuing Experimental Reproducibility: An Efficient Protocol for the Preparation of Cerebrospinal Fluid Samples for NMR-Based Metabolomics and Analysis of Sample Degradation

Metabolites ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 251
Author(s):  
Benjamin Albrecht ◽  
Elena Voronina ◽  
Carola Schipke ◽  
Oliver Peters ◽  
Maria Kristina Parr ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Sample Preparation ◽  
Ph Control ◽  
Sample Storage ◽  
Sample Collection ◽  
Experimental Conditions ◽  
Sample Handling ◽  
Sample Stability ◽  
Standard Operating ◽  
New Biomarkers

NMR-based metabolomics investigations of human biofluids offer great potential to uncover new biomarkers. In contrast to protocols for sample collection and biobanking, procedures for sample preparation prior to NMR measurements are still heterogeneous, thus compromising the comparability of the resulting data. Herein, we present results of an investigation of the handling of cerebrospinal fluid (CSF) samples for NMR metabolomics research. Origins of commonly observed problems when conducting NMR experiments on this type of sample are addressed, and suitable experimental conditions in terms of sample preparation and pH control are discussed. Sample stability was assessed by monitoring the degradation of CSF samples by NMR, hereby identifying metabolite candidates, which are potentially affected by sample storage. A protocol was devised yielding consistent spectroscopic data as well as achieving overall sample stability for robust analysis. We present easy to adopt standard operating procedures with the aim to establish a shared sample handling strategy that facilitates and promotes inter-laboratory comparison, and the analysis of sample degradation provides new insights into sample stability.

scholarly journals The Impact of Delayed Storage on the Measured Proteome and Metabolome of Human Cerebrospinal Fluid

2011 ◽  
Vol 57 (12) ◽  
pp. 1703-1711 ◽  
Author(s):  
Therese Rosenling ◽  
Marcel P Stoop ◽  
Agnieszka Smolinska ◽  
Bas Muilwijk ◽  
Leon Coulier ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Cystatin C ◽  
Room Temperature ◽  
Close Contact ◽  
Selected Reaction Monitoring ◽  
Sample Collection ◽  
Metabolome Analysis ◽  
Sample Handling ◽  
The Stability ◽  
The Impact

BACKGROUND Because cerebrospinal fluid (CSF) is in close contact with diseased areas in neurological disorders, it is an important source of material in the search for molecular biomarkers. However, sample handling for CSF collected from patients in a clinical setting might not always be adequate for use in proteomics and metabolomics studies. METHODS We left CSF for 0, 30, and 120 min at room temperature immediately after sample collection and centrifugation/removal of cells. At 2 laboratories CSF proteomes were subjected to tryptic digestion and analyzed by use of nano-liquid chromatography (LC) Orbitrap mass spectrometry (MS) and chipLC quadrupole TOF-MS. Metabolome analysis was performed at 3 laboratories by NMR, GC-MS, and LC-MS. Targeted analyses of cystatin C and albumin were performed by LC–tandem MS in the selected reaction monitoring mode. RESULTS We did not find significant changes in the measured proteome and metabolome of CSF stored at room temperature after centrifugation, except for 2 peptides and 1 metabolite, 2,3,4-trihydroxybutanoic (threonic) acid, of 5780 identified peptides and 93 identified metabolites. A sensitive protein stability marker, cystatin C, was not affected. CONCLUSIONS The measured proteome and metabolome of centrifuged human CSF is stable at room temperature for up to 2 hours. We cannot exclude, however, that changes undetectable with our current methodology, such as denaturation or proteolysis, might occur because of sample handling conditions. The stability we observed gives laboratory personnel at the collection site sufficient time to aliquot samples before freezing and storage at −80 °C.

scholarly journals Integrating automation and LC/MS for drug discovery bioanalysis

2002 ◽  
Vol 24 (1) ◽  
pp. 1-7 ◽  
Author(s):  
David T. Rossi
Keyword(s):  
Drug Discovery ◽  
Sample Preparation ◽  
Biological Fluid ◽  
Multiple Reaction Monitoring ◽  
Feasibility Studies ◽  
Integrated Approach ◽  
Sample Collection ◽  
High Concentration ◽  
Sample Handling ◽  
Flight Mass Spectrometry

A novel, integrated approach for automated sample handling in drug discovery bioanalysis is described. The process includes aspects of animal study design, biological sample collection, sample processing and high-throughput APILC/MS operating in under multiple reaction monitoring (MRM). A semi-automated 96-well liquid—liquid extraction technique for biological fluid sample preparation was developed and used in conjunction with the integrated sample-handling approach. One plate of samples could be prepared within 1.5 h compared with 4 h for a manual approach, and the resulting 96-well plate of extracts was directly compatible with the LC/MS. Feasibility studies for the development of the process included sample collection map generation and information management, sample collection formatting, evaluation of alternative dilution schemes for high-concentration samples, choice of biological fluid, and evaluating the capabilities of the two liquid-handling workstations. Numerous comparisons between the new approach and conventional sample-handling approaches gave equivalent drug-quantitation results for several example compounds. This new sampling process has approximately doubled the efficiency (as measured by studies assayed per month) of drug discovery bioanalysis in our laboratory. The approach was also used in conjunction with time-of-flight mass spectrometry instrumentation (LC/TOF/MS) to quantify and characterize the disposition of simultaneously dosed example drug compounds in the rat. Likely strategies for future automated sample preparation workstations are described.

Evaluation of Acoustic Analyses of Voice in Nonoptimized Conditions

2020 ◽  
Vol 63 (12) ◽  
pp. 3991-3999
Author(s):  
Benjamin van der Woerd ◽  
Min Wu ◽  
Vijay Parsa ◽  
Philip C. Doyle ◽  
Kevin Fung
Keyword(s):  
Repeated Measures ◽  
Voice Quality ◽  
Data Sets ◽  
Acoustic Measurements ◽  
Sample Collection ◽  
Experimental Conditions ◽  
Environment Analysis ◽  
Acoustic Measures ◽  
Recording Conditions ◽  
Cepstral Peak Prominence

Objectives This study aimed to evaluate the fidelity and accuracy of a smartphone microphone and recording environment on acoustic measurements of voice. Method A prospective cohort proof-of-concept study. Two sets of prerecorded samples (a) sustained vowels (/a/) and (b) Rainbow Passage sentence were played for recording via the internal iPhone microphone and the Blue Yeti USB microphone in two recording environments: a sound-treated booth and quiet office setting. Recordings were presented using a calibrated mannequin speaker with a fixed signal intensity (69 dBA), at a fixed distance (15 in.). Each set of recordings (iPhone—audio booth, Blue Yeti—audio booth, iPhone—office, and Blue Yeti—office), was time-windowed to ensure the same signal was evaluated for each condition. Acoustic measures of voice including fundamental frequency ( f o ), jitter, shimmer, harmonic-to-noise ratio (HNR), and cepstral peak prominence (CPP), were generated using a widely used analysis program (Praat Version 6.0.50). The data gathered were compared using a repeated measures analysis of variance. Two separate data sets were used. The set of vowel samples included both pathologic ( n = 10) and normal ( n = 10), male ( n = 5) and female ( n = 15) speakers. The set of sentence stimuli ranged in perceived voice quality from normal to severely disordered with an equal number of male ( n = 12) and female ( n = 12) speakers evaluated. Results The vowel analyses indicated that the jitter, shimmer, HNR, and CPP were significantly different based on microphone choice and shimmer, HNR, and CPP were significantly different based on the recording environment. Analysis of sentences revealed a statistically significant impact of recording environment and microphone type on HNR and CPP. While statistically significant, the differences across the experimental conditions for a subset of the acoustic measures (viz., jitter and CPP) have shown differences that fell within their respective normative ranges. Conclusions Both microphone and recording setting resulted in significant differences across several acoustic measurements. However, a subset of the acoustic measures that were statistically significant across the recording conditions showed small overall differences that are unlikely to have clinical significance in interpretation. For these acoustic measures, the present data suggest that, although a sound-treated setting is ideal for voice sample collection, a smartphone microphone can capture acceptable recordings for acoustic signal analysis.

CHAPTER 9: Immunology of TBEV-Infections

2020 ◽  
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Cerebrospinal Fluid ◽  
Nervous System ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Sample Collection ◽  
Tick Borne Encephalitis ◽  
Viral Infectious Disease ◽  
The Central Nervous System

Tick-borne encephalitis (TBE) is a viral infectious disease of the central nervous system caused by the tick-borne encephalitis virus (TBEV). TBE is usually a biphasic disease and in humans the virus can only be detected during the first (unspecific) phase of the disease. Pathogenesis of TBE is not well understood, but both direct viral effects and immune-mediated tissue damage of the central nervous system may contribute to the natural course of TBE. The effect of TBEV on the innate immune system has mainly been studied in vitro and in mouse models. Characterization of human immune responses to TBEV is primarily conducted in peripheral blood and cerebrospinal fluid, due to the inaccessibility of brain tissue for sample collection. Natural killer (NK) cells and T cells are activated during the second (meningo-encephalitic) phase of TBE. The potential involvement of other cell types has not been examined to date. Immune cells from peripheral blood, in particular neutrophils, T cells, B cells and NK cells, infiltrate into the cerebrospinal fluid of TBE patients.

scholarly journals Cerebrospinal Fluid Biomarkers of Alzheimer’s Disease: Current Evidence and Future Perspectives

2021 ◽  
Vol 11 (2) ◽  
pp. 215
Author(s):  
Donovan A. McGrowder ◽  
Fabian Miller ◽  
Kurt Vaz ◽  
Chukwuemeka Nwokocha ◽  
Cameil Wilson-Clarke ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cerebrospinal Fluid ◽  
Late Onset ◽  
Amyloid Β ◽  
Current Evidence ◽  
Csf Biomarkers ◽  
Diagnostic Efficacy ◽  
Neurofilament Light ◽  
New Biomarkers

Alzheimer’s disease is a progressive, clinically heterogeneous, and particularly complex neurodegenerative disease characterized by a decline in cognition. Over the last two decades, there has been significant growth in the investigation of cerebrospinal fluid (CSF) biomarkers for Alzheimer’s disease. This review presents current evidence from many clinical neurochemical studies, with findings that attest to the efficacy of existing core CSF biomarkers such as total tau, phosphorylated tau, and amyloid-β (Aβ42), which diagnose Alzheimer’s disease in the early and dementia stages of the disorder. The heterogeneity of the pathophysiology of the late-onset disease warrants the growth of the Alzheimer’s disease CSF biomarker toolbox; more biomarkers showing other aspects of the disease mechanism are needed. This review focuses on new biomarkers that track Alzheimer’s disease pathology, such as those that assess neuronal injury (VILIP-1 and neurofilament light), neuroinflammation (sTREM2, YKL-40, osteopontin, GFAP, progranulin, and MCP-1), synaptic dysfunction (SNAP-25 and GAP-43), vascular dysregulation (hFABP), as well as CSF α-synuclein levels and TDP-43 pathology. Some of these biomarkers are promising candidates as they are specific and predict future rates of cognitive decline. Findings from the combinations of subclasses of new Alzheimer’s disease biomarkers that improve their diagnostic efficacy in detecting associated pathological changes are also presented.

scholarly journals Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation

1999 ◽  
Vol 338 (3) ◽  
pp. 615-618 ◽  
Author(s):  
Xiaoke YANG ◽  
N. Dennis CHASTEEN
Keyword(s):  
Ph Control ◽  
Alternative Mechanism ◽  
Experimental Conditions ◽  
Iron Storage ◽  
Ferroxidase Activity ◽  
Effects Of Ph ◽  
Ph Stat ◽  
Ph Buffer ◽  
Horse Spleen Ferritin

It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin.

Influence of Storage Conditions, Sample Handling, Sample Preparation, and Repeated Analysis on the Measured Flash Point and Hydrocarbon Composition of Jet A

Fuel ◽  
2021 ◽  
Vol 305 ◽  
pp. 121399
Author(s):  
Brent A. Modereger ◽  
Sarah E. Nowling ◽  
Wan Tang Jeff Zhang ◽  
Mackenzie L. Jones ◽  
Nathan Chapman ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Hydrocarbon Composition ◽  
Storage Conditions ◽  
Flash Point ◽  
Sample Handling ◽  
Repeated Analysis

Chapter 9: Immunology of TBEV-Infection

2021 ◽  
Author(s):  
Sara Gredmark-Russ ◽  
Renata Varnaite
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Cerebrospinal Fluid ◽  
Nervous System ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Sample Collection ◽  
Tick Borne Encephalitis ◽  
Viral Infectious Disease ◽  
The Central Nervous System

Tick-borne encephalitis (TBE) is a viral infectious disease of the central nervous system caused by the tick-borne encephalitis virus (TBEV). TBE is usually a biphasic disease and in humans the virus can only be detected during the first (unspecific) phase of the disease. Pathogenesis of TBE is not well understood, but both direct viral effects and immune-mediated tissue damage of the central nervous system may contribute to the natural course of TBE. The effect of TBEV on the innate immune system has mainly been studied in vitro and in mouse models. Characterization of human immune responses to TBEV is primarily conducted in peripheral blood and cerebrospinal fluid, due to the inaccessibility of brain tissue for sample collection. Natural killer (NK) cells and T cells are activated during the second (meningo-encephalitic) phase of TBE. The potential involvement of other cell types has not been examined to date. Immune cells from peripheral blood, in particular neutrophils, T cells, B cells and NK cells, infiltrate into the cerebrospinal fluid of TBE patients.

scholarly journals Searching for New Biomarkers of Renal Diseases through Proteomics

2012 ◽  
Vol 58 (2) ◽  
pp. 353-365 ◽  
Author(s):  
Ana Konvalinka ◽  
James W Scholey ◽  
Eleftherios P Diamandis
Keyword(s):  
Mass Spectrometry ◽  
Prognostic Markers ◽  
Renal Diseases ◽  
Sample Collection ◽  
Technological Advances ◽  
Regulatory Bodies ◽  
New Biomarkers ◽  
Study Designs ◽  
Treatment Responsiveness ◽  
Proteomic Methods

Abstract BACKGROUND Technological advances have resulted in a renaissance of proteomic studies directed at finding markers of disease progression, diagnosis, or responsiveness to therapy. Renal diseases are ideally suited for such research, given that urine is an easily accessible biofluid and its protein content is derived mainly from the kidney. Current renal prognostic markers have limited value, and renal biopsy remains the sole method for establishing a diagnosis. Mass spectrometry instruments, which can detect thousands of proteins at nanomolar (or even femtomolar) concentrations, may be expected to allow the discovery of improved markers of progression, diagnosis, or treatment responsiveness. CONTENT In this review we describe the strengths and limitations of proteomic methods and the drawbacks of existing biomarkers, and provide an overview of opportunities in the field. We also highlight several proteomic studies of biomarkers of renal diseases selected from the plethora of studies performed. SUMMARY It is clear that the field of proteomics has not yet fulfilled its promise. However, ongoing efforts to standardize sample collection and preparation, improve study designs, perform multicenter validations, and create joint industry–regulatory bodies offer promise for the recognition of novel molecules that could change clinical nephrology forever.

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