quantification precision
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Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Quantification of monoclonal immunoglobulin free light chains (FLCs) in serum is used increasingly in clinical practice for the diagnosis, prognostic assessment, and treatment monitoring of monoclonal gammopathies. It is used as an adjunct to standard serum protein electrophoresis and immunofixation. However, methods for FLC quantification need further standardization and validation. METHODS: The Czech Myeloma Group and the Czech Society of Clinical Biochemistry have initiated an interlaboratory study where six laboratories collaborating with the primary myeloma treatment centres measured FLC concentrations in 12 serum samples from patients with monoclonal gammopathies. RESULTS: Repeatability of the measurements in five laboratories was calculated based on differences between the results of duplicate measurements. We found that repeatability depended more on the laboratory than on the device used for measurement. CONCLUSIONS: The study revealed several weak points in the methodology, including the need for a uniform sample dilution procedure. Interlaboratory reproducibility was comparable with values achieved in the NEQAS programme. Because the κ/λ ratio cannot be measured with high precision, κ and λ FLC concentrations should be used where possible. Due to its impact on the clinical management of patients with gammopathy, FLC quantification needs to become a part of the regular quality control cycle in myeloma centres.
- MeSH
- klinické laboratorní techniky metody normy MeSH
- lehké řetězce imunoglobulinů analýza krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mnohočetný myelom krev diagnóza MeSH
- paraproteinemie krev diagnóza MeSH
- referenční standardy MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Point-of-care systems based on microchip capillary electrophoresis require single-use, disposable microchips prefilled with all necessary solutions so an untrained operator only needs to apply the sample and perform the analysis. While microchip fabrication can be (and has been) standardized, some manufacturing differences between microchips are unavoidable. To improve analyte precision without increasing device costs or introducing additional error sources, we recently proposed the use of integrated internal standards (ISTDs): ions added to the BGE in small concentrations which form system peaks in the electropherogram that can be used as a measurement reference. Here, we further expand this initial proof-of-principle test to study a clinically-relevant application of K ion concentrations in human blood; however, using a mock blood solution instead of real samples to avoid interference from other obstacles (e.g. cell lysis). Cs as an integrated ISTD improves repeatability of K ion migration times from 6.97% to 0.89% and the linear calibration correlation coefficient (R2 ) for K quantification from 0.851 to 0.967. Peak area repeatability improves from 11.6-13.3% to 4.75-5.04% at each K concentration above the LOQ. These results further validate the feasibility of using integrated ISTDs to improve imprecision in disposable microchip CE devices by demonstrating their application for physiological samples.
BACKGROUND: Different types of analytical methods, with different characteristics, are applied in metabolomics and lipidomics research and include untargeted, targeted and semi-targeted methods. Ultra High Performance Liquid Chromatography-Mass Spectrometry is one of the most frequently applied measurement instruments in metabolomics because of its ability to detect a large number of water-soluble and lipid metabolites over a wide range of concentrations in short analysis times. Methods applied for the detection and quantification of metabolites differ and can either report a (normalised) peak area or an absolute concentration. AIM OF REVIEW: In this tutorial we aim to (1) define similarities and differences between different analytical approaches applied in metabolomics and (2) define how amounts or absolute concentrations of endogenous metabolites can be determined together with the advantages and limitations of each approach in relation to the accuracy and precision when concentrations are reported. KEY SCIENTIFIC CONCEPTS OF REVIEW: The pre-analysis knowledge of metabolites to be targeted, the requirement for (normalised) peak responses or absolute concentrations to be reported and the number of metabolites to be reported define whether an untargeted, targeted or semi-targeted method is applied. Fully untargeted methods can only provide (normalised) peak responses and fold changes which can be reported even when the structural identity of the metabolite is not known. Targeted methods, where the analytes are known prior to the analysis, can also report fold changes. Semi-targeted methods apply a mix of characteristics of both untargeted and targeted assays. For the reporting of absolute concentrations of metabolites, the analytes are not only predefined but optimized analytical methods should be developed and validated for each analyte so that the accuracy and precision of concentration data collected for biological samples can be reported as fit for purpose and be reviewed by the scientific community.
- MeSH
- hmotnostní spektrometrie * metody MeSH
- lidé MeSH
- metabolomika * metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Hippocampal dysfunction is known to be associated with several neurological and neuropsychiatric disorders such as Alzheimer's disease, epilepsy, schizophrenia and depression; therefore, there has been significant clinical interest in studying hippocampal neurochemistry. However, the hippocampus is a challenging region to study using (1) H MRS, hence the use of MRS for clinical research in this region has been limited. Our goal was therefore to investigate the feasibility of obtaining high-quality hippocampal spectra that allow reliable quantification of a neurochemical profile and to establish inter-session reproducibility of hippocampal MRS, including reproducibility of voxel placement, spectral quality and neurochemical concentrations. Ten healthy volunteers were scanned in two consecutive sessions using a standard clinical 3 T MR scanner. Neurochemical profiles were obtained with a short-echo (T(E) = 28 ms) semi-LASER localization sequence from a relatively small (~4 mL) voxel that covered about 62% of the hippocampal volume as calculated from segmentation of T1 -weighted images. Voxel composition was highly reproducible between sessions, with test-retest coefficients of variation (CVs) of 3.5% and 7.5% for gray and white matter volume fraction, respectively. Excellent signal-to-noise ratio (~54 based on the N-acetylaspartate (NAA) methyl peak in non-apodized spectra) and linewidths (~9 Hz for water) were achieved reproducibly in all subjects. The spectral quality allowed quantification of NAA, total choline, total creatine, myo-inositol and glutamate with high scan-rescan reproducibility (CV ≤ 6%) and quantification precision (Cramér-Rao lower bound, CRLB < 9%). Four other metabolites, including glutathione and glucose, were quantified with scan-rescan CV below 20%. Therefore, the highly optimized, short-echo semi-LASER sequence together with FASTMAP shimming substantially improved the reproducibility and number of quantifiable metabolites relative to prior reports. In addition, the between-session variation in metabolite concentrations, as well as CRLB, was lower than the between-subject variation of the concentrations for most metabolites, indicating that the method has the sensitivity to detect inter-individual differences in the healthy brain.
- MeSH
- algoritmy MeSH
- biopolymery metabolismus MeSH
- dospělí MeSH
- hipokampus anatomie a histologie metabolismus MeSH
- lidé MeSH
- molekulární zobrazování metody MeSH
- protonová magnetická rezonanční spektroskopie metody MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- studie proveditelnosti MeSH
- tkáňová distribuce MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Anthracyclines, e.g. doxorubicin, pirarubicin, are widely used as cytostatic agents in the polymer nanotherapeutics designed for the highly effective antitumor therapy with reduced side effects. However, their precise dosage scheme needs to be optimized, which requires an accurate method for their quantification on the cellular level in vitro during nanocarrier development and in body fluids and tissues during testing in vivo. Various methods detecting the anthracycline content in biological samples have already been designed. Most of them are highly demanding and they differ in exactness and reproducibility. The cellular uptake and localization is predominantly observed and determined by microscopy techniques, the anthracycline content is usually quantified by chromatographic analysis using fluorescence detection. We reviewed and compared published methods concerning the detection of anthracycline nanocarriers.
AbstractA proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein.
- Publikační typ
- časopisecké články MeSH
A proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein.
- MeSH
- biotest metody normy MeSH
- izotopové značení MeSH
- kinetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- peptidy chemická syntéza chemie MeSH
- proteiny analýza chemie MeSH
- proteolýza MeSH
- proteomika metody MeSH
- referenční standardy MeSH
- rozpouštědla MeSH
- rozpustnost MeSH
- sekvence aminokyselin MeSH
- trypsin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
This study compares alternative approaches for analyzing phytocannabinoids in different plant materials. Three chromatographic analytical methods (ultra-high-performance liquid chromatography with tandem mass spectrometric detection and gas chromatography with mass spectrometric and flame ionization detection) were evaluated regarding selectivity, sensitivity, analytical accuracy, and precision. The performance of the methods was compared and all three methods were demonstrated to be appropriate tools for analyzing phytocannabinoids in cannabis. Gas chromatography coupled with mass spectrometric detection showed slightly better accuracy in determining phytocannabinoid acids, which are often difficult to quantify owing to their limited stability. Aspects of sample preparation, such as material homogenization and extraction, were also considered. A single ultrasonic-assisted ethanolic extraction of dried and powdered plant samples of cannabis was shown to be exhaustive for extracting the samples prior to analysis.
- MeSH
- Cannabis chemie MeSH
- kanabinoidy analýza MeSH
- laboratoře organizace a řízení MeSH
- limita detekce MeSH
- plamínková ionizace metody MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7β-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.
- MeSH
- androgeny MeSH
- androstendion * MeSH
- chromatografie kapalinová metody MeSH
- estron MeSH
- lidé MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
