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Mass spectrometry (MS) is widely used in science and industry. It allows accurate, specific, sensitive, and reproducible detection and quantification of a huge range of analytes. Across MS applications, quantification by MS has grown most dramatically, with >50 million experiments/year in the USA alone. However, quantification performance varies between instruments, compounds, different samples, and within- and across runs, necessitating normalization with analyte-similar internal standards (IS) and use of IS-corrected multipoint external calibration curves for each analyte, a complicated and resource-intensive approach, which is particularly ill-suited for multi-analyte measurements. We have developed an internal calibration method that utilizes the natural isotope distribution of an IS for a given analyte to provide internal multipoint calibration. Multiple isotope distribution calibrators for different targets in the same sample facilitate multiplex quantification, while the emerging random-access automated MS platforms should also greatly benefit from this approach. Finally, isotope distribution calibration allows mathematical correction for suboptimal experimental conditions. This might also enable quantification of hitherto difficult, or impossible to quantify, targets, if the distribution is adjusted in silico to mimic the analyte. The approach works well for high resolution, accurate mass MS for analytes with at least a modest-sized isotopic envelope. As shown herein, the approach can also be applied to lower molecular weight analytes, but the reduction in calibration points does reduce quantification performance.
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Isótopos , Espectrometria de Massas em Tandem , Calibragem , Padrões de ReferênciaRESUMO
OBJECTIVES: Matrix differences among serum samples from non-pregnant and pregnant patients could bias measurements. Standard Reference Material 1949, Frozen Human Prenatal Serum, was developed to provide a quality assurance material for the measurement of hormones and nutritional elements throughout pregnancy. METHODS: Serum from non-pregnant women and women in each trimester were bottled into four levels based on pregnancy status and trimester. Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and applied to the measurement of thyroid hormones, vitamin D metabolites, and vitamin D-binding protein (VDBP). Copper, selenium, and zinc measurements were conducted by inductively coupled plasma dynamic reaction cell MS. Thyroid stimulating hormone (TSH), thyroglobulin (Tg), and thyroglobulin antibody concentrations were analyzed using immunoassays and LC-MS/MS (Tg only). RESULTS: Certified values for thyroxine and triiodothyronine, reference values for vitamin D metabolites, VDBP, selenium, copper, and zinc, and information values for reverse triiodothyronine, TSH, Tg, and Tg antibodies were assigned. Significant differences in serum concentrations were evident for all analytes across the four levels (p≤0.003). TSH measurements were significantly different (p<0.0001) among research-only immunoassays. Tg concentrations were elevated in research-only immunoassays vs. Federal Drug Administration-approved automated immunoassay and LC-MS/MS. Presence of Tg antibodies increased differences between automated immunoassay and LC-MS/MS. CONCLUSIONS: The analyte concentrations' changes consistent with the literature and the demonstration of matrix interferences in immunoassay Tg measurements indicate the functionality of this material by providing a relevant matrix-matched reference material for the different stages of pregnancy.
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Selênio , Oligoelementos , Biomarcadores/sangue , Cromatografia Líquida , Cobre , Feminino , Humanos , Gravidez , Espectrometria de Massas em Tandem , Tireoglobulina/sangue , Glândula Tireoide , Tireotropina , Oligoelementos/sangue , Vitamina D/sangue , Vitaminas , ZincoRESUMO
Insulin-like growth factor-1 (IGF-1) measurement by high-resolution accurate mass-mass spectrometry (HRAM-MS) is replacing IGF-1 immunoassays and allows for identification of single amino acid variants; by contrast, both normal and deleterious sequence variants might be missed by immunoassays or non-HRAM-MS methods. We have developed an intact molecule HRAM-MS method to identify IGF-1 variants, distinguishing them by a center of mass (COM) calculation, followed by various tandem-MS activation techniques (HCD, ETD, ETciD, EThcD, UVPD). We found single amino acid variants in 841 of 146â¯620 patient samples (0.57%). Most were benign (A67T, A70T). We also observed a pathogenic variant (V44M), likely pathogenic variants (A38V, V17M), and a likely benign variant (A67V). For 207 samples from unique patients with residual serum, the MS variant results were confirmed by cell-free DNA sequencing. Our approach allows accurate quantitative reporting of functional IGF-1 in the presence of single amino acid variants. The COM approach potentially enables omission of tandem-MS for known, common variants, while the combination of COM and tandem-MS allows accurate identification in all cases we encountered. This approach should be applicable to qualitative and quantitative analyses of other peptides/proteins in clinical and research settings and might lend itself to the characterization of other protein variations.
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Fator de Crescimento Insulin-Like I , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Aminoácidos , Humanos , Fator de Crescimento Insulin-Like I/genética , Polimorfismo GenéticoRESUMO
BACKGROUND: Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS: We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS: For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS: Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.
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Química Clínica/normas , Análise Mutacional de DNA/normas , Biópsia Líquida/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Adulto , Idoso , Ácidos Nucleicos Livres , Análise Mutacional de DNA/métodos , Receptores ErbB/genética , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Valores de ReferênciaRESUMO
BACKGROUND: Recessive genes cause disease when both copies are affected by mutant loci. Resolving the cis/trans relationship of variations has been an important problem both for researchers, and increasingly, clinicians. Of particular concern are patients who have two heterozygous disease-causing mutations and could be diagnosed as affected (one mutation on each allele) or as phenotypically normal (both mutations on the same allele). Several methods are currently used to phase genes, however due to cost, complexity and/or low sensitivity they are not suitable for clinical purposes. METHODS: Long-range amplification was used to select and enrich the target gene (CYP21A2) followed by modified mate-pair sequencing. Fragments that mapped coincidently to two heterozygous sites were identified and used for statistical analysis. RESULTS: Probabilities for cis/trans relationships between heterozygous positions were calculated along with 99% confidence intervals over the entire length of our 10 kb amplicons. The quality of phasing was closely related to the depth of coverage and the number of erroneous reads. Most of the error was found to have been introduced by recombination in the PCR reaction. CONCLUSIONS: We have developed a simple method utilizing massively parallel sequencing that is capable of resolving two alleles containing multiple heterozygous positions. This method stands out among other phasing tools because it provides quantitative results allowing confident haplotype calls.
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Haplótipos/genética , Análise de Sequência/métodos , Heterozigoto , Reação em Cadeia da Polimerase , Probabilidade , Projetos de Pesquisa , Esteroide 21-Hidroxilase/genéticaRESUMO
BACKGROUND: Maintaining consistency of results over time is a challenge in laboratory medicine. Lot-to-lot reagent changes are a major threat to consistency of results. METHODS: For the period October 2007 through July 2012, we reviewed lot validation data for each new lot of insulin-like growth factor 1 (IGF-1) reagents (Siemens Healthcare Diagnostics) at Mayo Clinic, Rochester, MN, and the University of Virginia, Charlottesville, VA. Analyses of discarded patient samples were used for comparison of lots. For the same period, we determined the distributions of reported patient results for each lot of reagents at the 2 institutions. RESULTS: Lot-to-lot validation studies identified no reagent lot as significantly different from the preceding lot. By contrast, significant lot-to-lot changes were seen in the means and medians of 105 668 reported patient IGF-I results during the period. The frequency of increased results increased nearly 2-fold to a high of 17%, without detectable changes in the underlying patient demographics. Retrospective statistical analysis indicated that lot-to-lot comparison protocols were underpowered and that validation studies for this assay required testing >100 samples to achieve 90% power to detect reagent lots that would significantly alter the distributions of patient results. CONCLUSIONS: The number of test samples required for adequate lot-to-lot validation protocols is high and may be prohibitively large, especially for low-volume or complex assays. Monitoring of the distributions of patient results has the potential to detect lot-to-lot inconsistencies relatively quickly. We recommend that manufacturers implement remote monitoring of patient results from analyzers in multiple institutions to allow rapid identification of between-lot result inconsistency.
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Fator de Crescimento Insulin-Like I/análise , Kit de Reagentes para Diagnóstico/normas , Humanos , Medições Luminescentes , Controle de Qualidade , Valores de Referência , Estudos Retrospectivos , Adulto JovemRESUMO
Introduction: Steroid measurements are important for diagnosis and monitoring of many conditions and treatment regiments; however, due to structural and chemical similarities amongst steroids, these analyses are challenging, even for highly specific techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential mobility spectrometry (DMS) has the potential to improve these analyses by providing an orthogonal and complementary separation technique. Methods: Initially, the potential for DMS to improve signal-to-noise ratio (S/N) and reduce interference was tested by comparing chromatograms acquired with and without DMS when performing measurements of six different steroids. Subsequently, a full clinical validation of cortisol and cortisone in urine was performed with the LC-DMS-MS/MS method. Results and Discussion: DMS significantly reduced interferences observed in the chromatograms and boosted S/N by between 1.6 and 13.8 times. Additionally, DMS improved the agreement between quantifier/qualifier fragment ion results for cortisol and cortisone as indicated by the increase in R2 from approximately 0.81 to 0.98. All validation studies met acceptance criteria and we observed exceptional analytical performance in terms of precision, with % CVs less than 8%. Conclusions: DMS improved the specificity of the steroid measurements by reducing interferences and improving S/N. The validation studies prove that these benefits did not come at the expense of other aspects of analytical performance. This study indicates that DMS has the potential to benefit not just clinical measurements of challenging analytes, but many clinical LC-MS/MS analyses.
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Pyridoxal-5'-phosphate (PLP), the active form of vitamin B6, is required for numerous enzymatic reactions. Vitamin B6 deficiency or exceptionally high levels of PLP have negative implications, making measurements of PLP imperative for diagnoses and monitoring in many clinical scenarios. Traditional assays are enzymatic, ELISA based, or employ HPLC with various detection modalities; all of these are prone to interferences and crossreactivity with other compounds. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used to overcome these issues, but the high polarity of PLP raises chromatographic challenges. Using ion pairing reagents in the mobile phases is a possible solution, but these reagents often have deleterious effects on instrumentation. An alternative strategy is the addition of an ion pairing reagent after extraction, but prior to injection. To prove this, we used 1-octanesulfonic acid (OSA) without changing the LC method or column. With this technique, we observed a 2-4 fold increase in signal-to-noise ratio. Intraday and interday precision of replicate measurements also improved drastically compared to analyses without OSA, while also yielding a dramatic improvement in column life compared to our previous approach and to this point no deleterious effects on instrument hardware commonly associated with traditional ion pairing reagent techniques have been observed.
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Cromatografia Líquida , Fosfato de Piridoxal , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Fosfatos , Fosfato de Piridoxal/análise , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: COVID-19 is a multi-system disorder with high variability in clinical outcomes among patients who are admitted to hospital. Although some cytokines such as interleukin (IL)-6 are believed to be associated with severity, there are no early biomarkers that can reliably predict patients who are more likely to have adverse outcomes. Thus, it is crucial to discover predictive markers of serious complications. METHODS: In this retrospective cohort study, we analysed samples from 455 participants with COVID-19 who had had a positive SARS-CoV-2 RT-PCR result between April 14, 2020, and Dec 1, 2020 and who had visited one of three Mayo Clinic sites in the USA (Minnesota, Arizona, or Florida) in the same period. These participants were assigned to three subgroups depending on disease severity as defined by the WHO ordinal scale of clinical improvement (outpatient, severe, or critical). Our control cohort comprised of 182 anonymised age-matched and sex-matched plasma samples that were available from the Mayo Clinic Biorepository and banked before the COVID-19 pandemic. We did a deep profiling of circulatory cytokines and other proteins, lipids, and metabolites from both cohorts. Most patient samples were collected before, or around the time of, hospital admission, representing ideal samples for predictive biomarker discovery. We used proximity extension assays to quantify cytokines and circulatory proteins and tandem mass spectrometry to measure lipids and metabolites. Biomarker discovery was done by applying an AutoGluon-tabular classifier to a multiomics dataset, producing a stacked ensemble of cutting-edge machine learning algorithms. Global proteomics and glycoproteomics on a subset of patient samples with matched pre-COVID-19 plasma samples was also done. FINDINGS: We quantified 1463 cytokines and circulatory proteins, along with 902 lipids and 1018 metabolites. By developing a machine-learning-based prediction model, a set of 102 biomarkers, which predicted severe and clinical COVID-19 outcomes better than the traditional set of cytokines, were discovered. These predictive biomarkers included several novel cytokines and other proteins, lipids, and metabolites. For example, altered amounts of C-type lectin domain family 6 member A (CLEC6A), ether phosphatidylethanolamine (P-18:1/18:1), and 2-hydroxydecanoate, as reported here, have not previously been associated with severity in COVID-19. Patient samples with matched pre-COVID-19 plasma samples showed similar trends in muti-omics signatures along with differences in glycoproteomics profile. INTERPRETATION: A multiomic molecular signature in the plasma of patients with COVID-19 before being admitted to hospital can be exploited to predict a more severe course of disease. Machine learning approaches can be applied to highly complex and multidimensional profiling data to reveal novel signatures of clinical use. The absence of validation in an independent cohort remains a major limitation of the study. FUNDING: Eric and Wendy Schmidt.
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COVID-19 , Biomarcadores , COVID-19/diagnóstico , Estudos de Coortes , Citocinas , Humanos , Lipidômica/métodos , Lipídeos , Metabolômica/métodos , Pandemias , Prognóstico , Proteômica/métodos , Estudos Retrospectivos , SARS-CoV-2RESUMO
BACKGROUND: The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography-tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold. METHODS: After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS: TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 µg/L, 25(OH)D2], 2.2 nmol/L [0.9 µg/L, 25(OH)D3], and 10 nmol/L [4 µg/L, 25(OH)D2], 5 nmol/L [2 µg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 µg/L). Interassay CVs across the reportable range were 3.7%-15.2%. Spiked recoveries were 94%-119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n=1733) and 25(OH)D3 (n=7614) (R2=0.96 and 0.97), respectively. CONCLUSIONS: Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.
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25-Hidroxivitamina D 2/sangue , Calcifediol/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , 25-Hidroxivitamina D 2/química , Automação , Calcifediol/química , Calibragem , Cromatografia Líquida/instrumentação , Cromatografia Líquida/normas , Humanos , Estrutura Molecular , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/normasRESUMO
BACKGROUND: Molecular testing of thyroid malignancies, in combination with cytologic and histologic examination, is becoming increasingly attractive as a tool for refining traditional morphologic diagnosis. The molecular changes associated with follicular thyroid carcinoma (FTC) are point mutations in RAS oncogenes or the presence of PAX8/PPARG (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement. METHODS: We developed and validated a clinical assay for the detection of PAX8/PPARG rearrangements that uses a 4-color reverse-transcription PCR (RT-PCR) assay and high-resolution fragment analysis. RESULTS: The RT-PCR assay is applicable for detecting the various described fusion transcripts of PAX8/PPARG in formalin-fixed, paraffin-embedded thyroid tissue and in fine-needle aspirate biopsy washes from thyroid nodules. The analytical sensitivity of the assay is 1 abnormal cell in a background of 100-10 000 translocation-negative cells. A comparison of the RT-PCR assay with dual-fusion fluorescence in situ hybridization showed an overall concordance of 95%. With this assay, we obtained a prevalence for the PAX8/PPARG rearrangement in FTC of 62% (13 of 21 cases), compared with a 5% prevalence (3 of 55) for other follicular cell-derived neoplasms. CONCLUSIONS: The introduction of this assay into clinical practice could provide useful information for the diagnosis and possibly for the prognosis and treatment of thyroid cancer in the future.
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Adenocarcinoma Folicular/genética , Regulação Neoplásica da Expressão Gênica , PPAR gama/genética , Fatores de Transcrição Box Pareados/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/patologia , Humanos , Fator de Transcrição PAX8 , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Sensibilidade e Especificidade , Neoplasias da Glândula Tireoide/patologiaRESUMO
CONTEXT: The initial diagnosis of pheochromocytoma relies on plasma fractionated metanephrines levels. Normal levels exclude pheochromocytoma, but positive tests have a low positive predictive value due to the disease's rarity. OBJECTIVES: The objective of the study was to evaluate three approaches to distinguish between true-positive and false-positive tests: 1) increased cutoff for plasma fractionated metanephrines, 2) measurement of serum/plasma chromogranin A (CGA), and 3) urine fractionated metanephrine testing. DESIGN: We studied retrospectively all Mayo Clinic patients with positive plasma fractionated metanephrine tests over a 15-month period and determined their final diagnosis based on histology, imaging, additional biochemical tests, and more than 1 yr follow-up. For a subgroup, urine fractionated metanephrine results were available. All original plasma samples were retested for CGA. RESULTS: Of 140 patients, 40 had a chromaffin tumor confirmed and 100 excluded, indicating a positive predictive value of plasma fractionated metanephrines of 28.6%. Increasing the threshold for a positive test improved specificity to 98% but missed eight cases (20%). Incorporation of urine fractionated metanephrine testing as follow-up test achieved 80% specificity and 91% sensitivity. The corresponding figures for CGA were 71 and 87% for all patients and 89 and 87% when patients taking proton pump inhibitors were excluded. CONCLUSIONS: Unless plasma fractionated metanephrines levels are elevated more than 4-fold above the upper limit of normal, patients with a positive plasma fractionated metanephrines test should be evaluated with urine fractionated metanephrines and serum/plasma CGA assays before being subjected to imaging or invasive diagnostic tests.
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Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/urina , Cromogranina A/sangue , Metanefrina/sangue , Metanefrina/urina , Feocromocitoma/sangue , Feocromocitoma/urina , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Minnesota , Valor Preditivo dos Testes , Curva ROC , Estudos RetrospectivosRESUMO
The American Cancer Society estimates 30,180 new cases of thyroid cancer in the United States in 2006. Of all thyroid cancers, 15-20% are follicular thyroid carcinoma (FTC), making this the second most common thyroid malignancy (after papillary carcinoma). A proportion of FTC has been found to be associated with a chromosomal translocation, t (2, 3)(q13;p25), which fuses the thyroid-specific transcription factor paired box-8 with the peroxisome proliferator-activated receptor-gamma nuclear receptor, a ubiquitously expressed transcription factor. This fusion event causes expression of a paired box-8/peroxisome proliferator-activated receptor-gamma fusion protein (PPFP). PPFP is detected in approximately 30% of FTC. In this report we review data on the role of PPFP in FTC, its mechanism of oncogenesis, and PPFP targeting as a strategy in thyroid cancer treatment.