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Circulating cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress and disease which predicts mortality and is associated with various disease states. To evaluate the contribution of cf-mtDNA to health and disease states, standardized high-throughput procedures are needed to quantify cf-mtDNA in relevant biofluids. Here, we describe MitoQuicLy: Mitochondrial DNA Quantification in cell-free samples by Lysis. We demonstrate high agreement between MitoQuicLy and the commonly used column-based method, although MitoQuicLy is faster, cheaper, and requires a smaller input sample volume. Using 10 µL of input volume with MitoQuicLy, we quantify cf-mtDNA levels from three commonly used plasma tube types, two serum tube types, and saliva. We detect, as expected, significant inter-individual differences in cf-mtDNA across different biofluids. However, cf-mtDNA levels between concurrently collected plasma, serum, and saliva from the same individual differ on average by up to two orders of magnitude and are poorly correlated with one another, pointing to different cf-mtDNA biology or regulation between commonly used biofluids in clinical and research settings. Moreover, in a small sample of healthy women and men (n = 34), we show that blood and saliva cf-mtDNAs correlate with clinical biomarkers differently depending on the sample used. The biological divergences revealed between biofluids, together with the lysis-based, cost-effective, and scalable MitoQuicLy protocol for biofluid cf-mtDNA quantification, provide a foundation to examine the biological origin and significance of cf-mtDNA to human health.
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Ácidos Nucleicos Libres de Células , Masculino , Humanos , Femenino , Saliva , ADN Mitocondrial/genética , Mitocondrias/genética , BiomarcadoresRESUMEN
Targeting and quantifying intact proteins from biological samples is still a very challenging research area. Several crucial steps exist in the analytical workflow, including development of a reliable sample preparation method. Here, we developed and applied for the first time a non-immunoaffinity sample preparation method based on a generally widely available micro-elution solid phase extraction (µSPE) strategy for the extraction of multiple lower molecular weight intact proteins (<30 kDa) from various biological matrices. Omission of a time-consuming drying and reconstitution step after extraction resulted in a more simple and rapid sample preparation procedure. A model set of eleven intact proteins (molecular weights: 5.5-29 kDa; isoelectric points: 4.5-11.3) were analyzed in multiple biological fluids using reversed-phase liquid chromatography with a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode. Various sample pre-treatment reagents, sorbent types, and washing and elution solvents were experimentally tested and optimized to obtain the µSPE clean-up condition for a broad mixture of intact proteins having variable physicochemical properties. 1% trifluoroacetic acid and 0.2% Triton 100-X were selected as suitable sample pre-treatment reagents for releasing protein-protein interactions in human serum/plasma and human urine, respectively. Hydrophilic lipophilic balanced µSPE sorbent was selected as a high performing stationary phase. Addition of 1% trifluoroacetic acid to all washing and elution solutions showed the most beneficial effect for the extraction recovery of the proteins. Under the optimized conditions, reproducible extraction recoveries >65% for all targeted proteins (up to 30 kDa) in human urine and >50% for most of the proteins in serum/plasma were achieved. The selected conditions were applied also for the analysis of clinical serum and urine samples to demonstrate the feasibility of the developed method to target intact proteins directly by more affordable µSPE sample preparation and triple quadrupole mass spectrometry, which could be beneficial in many application fields.
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Proteínas , Extracción en Fase Sólida , Humanos , Peso Molecular , Ácido Trifluoroacético , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Extracción en Fase Sólida/métodos , Cromatografía Líquida de Alta PresiónRESUMEN
Circulating cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress and disease which predicts mortality and is associated with various disease states. To evaluate the contribution of cf-mtDNA to health and disease states, standardized high-throughput procedures are needed to quantify cf-mtDNA in relevant biofluids. Here, we describe MitoQuicLy: Mito chondrial DNA Qu antification in c ell-free samples by Ly sis. We demonstrate high agreement between MitoQuicLy and the commonly used column-based method, although MitoQuicLy is faster, cheaper, and requires a smaller input sample volume. Using 10 µL of input volume with MitoQuicLy, we quantify cf-mtDNA levels from three commonly used plasma tube types, two serum tube types, and saliva. We detect, as expected, significant inter-individual differences in cf-mtDNA across different biofluids. However, cf-mtDNA levels between concurrently collected plasma, serum, and saliva from the same individual differ on average by up to two orders of magnitude and are poorly correlated with one another, pointing to different cf-mtDNA biology or regulation between commonly used biofluids in clinical and research settings. Moreover, in a small sample of healthy women and men (n=34), we show that blood and saliva cf-mtDNAs correlate with clinical biomarkers differently depending on the sample used. The biological divergences revealed between biofluids, together with the lysis-based, cost-effective, and scalable MitoQuicLy protocol for biofluid cf-mtDNA quantification, provide a foundation to examine the biological origin and significance of cf-mtDNA to human health.
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Utilizing biofluids to identify cancer biomarkers has received considerable attention in the past decade. In this regard, serum and urine are convenient biofluids to noninvasively recapitulate information usually indicated by traditional tissue biopsies. In particular, we are interested in exploring the extracellular vesicle (ECV)-containing compartment of these fluids as a targeted source for cancer biomarker discovery. ECVs are membrane-enclosed particles, comprising of various fractions including exosomes, microvesicles, and apoptotic bodies. In both physiological and pathological states such as cancer, ECVs carry a rich load of molecular and protein cargoes, which aid in mediating intercellular communication between cells from various tissue types. Here we successfully enriched ECVs using a simple, low-cost, optimized method that we have developed; it is generalizable for the analysis of ECVs from multiple sample types. Such procedures are necessary as ECVs are nanoparticles that contain a treasure trove of large numbers of biomarkers each present at very low levels. Sample processing procedures can enrich for these vesicles and allow for the enhanced detection of proteins in downstream applications such as comprehensive proteomics methods using data-independent acquisition (DIA) and LC-MS/MS.
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Vesículas Extracelulares , Neoplasias , Biomarcadores de Tumor/metabolismo , Cromatografía Liquida , Digestión , Vesículas Extracelulares/metabolismo , Humanos , Biopsia Líquida , Neoplasias/diagnóstico , Neoplasias/metabolismo , Proteómica/métodos , Espectrometría de Masas en TándemRESUMEN
Introduction: Approximately 40% of patients with uveal melanoma (UM) will develop metastatic disease. Tumors measuring at least 12mm in basal diameter with a class 2 signature, as defined by a widely used gene expression-profiling test, are associated with significantly higher risk of metastasis, with a median time to recurrence of 32 months. No therapy has been shown to reduce this risk. Materials and Methods: This was a single-arm, multicenter study in patients with high-risk UM who received definitive treatment of primary disease and had no evidence of metastasis. Patients were consecutively enrolled to receive 12 four-week cycles of adjuvant crizotinib at a starting dose of 250mg twice daily and were subsequently monitored for 36 months. The primary outcome of this study was to assess recurrence-free survival (RFS) of patients with high-risk UM who received adjuvant crizotinib. Results: 34 patients enrolled and received at least one dose of crizotinib. Two patients were unevaluable due to early withdrawal and loss to follow-up, leaving 32 patients evaluable for efficacy. Eight patients (25%) did not complete the planned 48-week course of treatment due to disease recurrence (n=5) or toxicity (n=3). All patients experienced at least one adverse event (AE), with 11/34 (32%) experiencing a Common Terminology Criteria for Adverse Events (CTCAE) grade 3 or 4 AE. After a median duration of follow up of 47.1 months, 21 patients developed distant recurrent disease. The median RFS was 34.9 months (95% CI (Confidence Interval), 23-55 months), with a 32-month recurrence rate of 50% (95% CI, 33-67%). Analysis of protein contents from peripheral blood extracellular vesicles in a subset of patient samples from baseline, on-treatment, and off-treatment, revealed a change in protein content associated with crizotinib exposure, however without a clear association with disease outcome. Conclusions: The use of adjuvant crizotinib in patients with high-risk UM did not result in improved RFS when compared to historical controls. Analysis of blood extracellular vesicles revealed changes in protein content associated with treatment, raising the possibility of future use as a biomarker. Further investigation of adjuvant treatment options are necessary for this challenging disease.
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BACKGROUND: The AACC Academy revised the reproductive testing section of the Laboratory Medicine Practice Guidelines: Evidence-Based Practice for Point-of-Care Testing (POCT) published in 2007. METHODS: A panel of Academy members with expertise in POCT and laboratory medicine was formed to develop guidance for the use of POCT in reproductive health, specifically ovulation, pregnancy, premature rupture of membranes (PROM), and high-risk deliveries. The committee was supplemented with clinicians having Emergency Medicine and Obstetrics/Gynecology training. RESULTS: Key recommendations include the following. First, urine luteinizing hormone (LH) tests are accurate and reliable predictors of ovulation. Studies have shown that the use of ovulation predicting kits may improve the likelihood of conception among healthy fertile women seeking pregnancy. Urinary LH point-of-care testing demonstrates a comparable performance among other ovulation monitoring methods for timing intrauterine insemination and confirming sufficient ovulation induction before oocyte retrieval during in vitro fertilization. Second, pregnancy POCT should be considered in clinical situations where rapid diagnosis of pregnancy is needed for treatment decisions, and laboratory analysis cannot meet the required turnaround time. Third, PROM testing using commercial kits alone is not recommended without clinical signs of rupture of membranes, such as leakage of amniotic fluid from the cervical opening. Finally, fetal scalp lactate is used more than fetal scalp pH for fetal acidosis due to higher success rate and low volume of sample required. CONCLUSIONS: This revision of the AACC Academy POCT guidelines provides recommendations for best practice use of POCT in fertility and reproduction.
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Fertilidad , Reproducción , Femenino , Humanos , Pruebas en el Punto de Atención , EmbarazoRESUMEN
Background: Human immunodeficiency virus (HIV) testing is the first step in the HIV prevention cascade. The Centers for Disease Control and Prevention HIV laboratory diagnostic testing algorithm was developed before preexposure prophylaxis (PrEP) and immediate antiretroviral therapy (iART) became standards of care. PrEP and iART have been shown to delay antibody development and affect the performance of screening HIV assays. Quantitative results from fourth-generation HIV testing may be helpful to disambiguate HIV testing. Methods: We retrospectively reviewed 38â 850 results obtained at an urban, academic medical center. We assessed signal-to-cutoff (s/co) distribution among positive and negative tests, in patients engaged and not engaged in an HIV prevention program, and evaluated changes in patients with multiple results. Classification and regression tree (CART) analysis was used to determine a threshold cutoff, and logistic regression was used to identify predictors of true positive tests. Results: Ninety-seven percent of patients with a negative HIV test had a result that was ≤0.2â s/co. For patients tested more than once, we found differences in s/co values did not exceed 0.2â s/co for 99.2% of results. CART identified an s/co value, 38.78, that in logistic regression on a unique validation cohort remained associated with the likelihood of a true-positive HIV result (odds ratio, 2.49). Conclusions: Machine-learning methods may be used to improve HIV screening by automating and improving interpretations, incorporating them into robust algorithms, and improving disease prediction. Further investigation is warranted to confirm if s/co values combined with a patient's risk profile will allow for better clinical decision making for individuals on PrEP or eligible for iART.
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Monitoring tacrolimus trough concentrations is important for optimal immunosuppression in solid organ transplant recipients. Available assays generally correlate well with each other but little attention is given to patients in whom tacrolimus metabolite concentrations might be elevated, which could lead to artificially increased tacrolimus concentrations assessed by cross-reacting immunoassays. We addressed this hypothesis by investigating the correlation between four different assays (two immunoassays and two mass-spectrometry assays) in both a population with normal and a population with high dose requirements. Routine blood samples were collected in 37 control (CO) and 72 high dose patients (HD). Tacrolimus was measured with a CMIA, an ECLIA and two LCMS assays. Results were investigated using Deming regression analysis, Pearson correlation coefficients, Bland-Altman plots and by calculating bias. The CMIA demonstrated a positive bias of 23-26% compared with both LCMS assays. The correlation between CMIA and LCMS assays was good for the CO (r = 0.96) but less so for the HD group (r = 0.91). The ECLIA showed a positive bias of 11-13% compared with both LCMS assays. The correlation between ECLIA and LCMS assays was also good for the CO (r = 0.95) but again less for the HD group (r = 0.93). The correlation for both LCMS assays was excellent for either group (r > 0.99) with no bias. CMIA, ECLIA and LCMS assays for tacrolimus therefore correlate well for trough concentrations from solid organ transplant recipients. However, inter-assay differences exist, which seem more pronounced in patients who need a high dose of tacrolimus to reach a trough concentration in the therapeutic range.
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Inmunosupresores , Tacrolimus , Bioensayo , Monitoreo de Drogas/métodos , Humanos , Inmunoensayo/métodos , Espectrometría de MasasRESUMEN
Uveal melanoma is a rare melanoma subtype different from cutaneous melanoma, with high incidence of liver metastasis and poor prognosis. Cancer cell-derived extracellular vesicles have been shown to induce proinflammatory and prometastatic signaling in the tumor microenvironment and at distant sites. The characterization of uveal melanoma exosome cargo and its role in metastatic spread is essential to identify targets and intervene in the early stages of metastatic development. Our study characterizes the proteomic content of uveal melanoma exosomes and identified the presence of markers with metastatic properties. We demonstrated that uveal melanoma exosomes induce activation of cell signaling pathways and the release of cytokines and growth factors from hepatocytes. These exosome-stimulated liver cells could in turn induce migration of uveal melanoma cells, confirming that the exosomes have a functional role in the cross-talk between these two cell types. We found that the proinflammatory cytokine macrophage migration inhibitory factor (MIF) was a major player in these mechanisms and its blockade inhibited cell migration in coculture with exosome-stimulated hepatocytes and prevented the development of metastases in vivo. Targeting MIF in the early stages of metastasis may represent a novel adjuvant drug therapy to prevent metastatic spread in uveal melanoma. IMPLICATIONS: This study provides the first in vivo evidence that MIF inhibition may serve as a novel adjuvant drug therapy to prevent metastasis in uveal melanoma.
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Exosomas , Factores Inhibidores de la Migración de Macrófagos , Melanoma , Neoplasias Cutáneas , Exosomas/metabolismo , Humanos , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Melanoma/patología , Proteómica , Neoplasias Cutáneas/metabolismo , Microambiente Tumoral , Neoplasias de la ÚveaRESUMEN
CONTEXT.: The process for identifying patients with monoclonal gammopathies is complex. Initial detection of a monoclonal immunoglobulin protein (M protein) in the serum or urine often requires compilation of analytical data from several areas of the laboratory. The detection of M proteins depends on adequacy of the sample provided, available clinical information, and the laboratory tests used. OBJECTIVE.: To develop an evidence-based guideline for the initial laboratory detection of M proteins. DESIGN.: To develop evidence-based recommendations, the College of American Pathologists convened a panel of experts in the diagnosis and treatment of monoclonal gammopathies and the laboratory procedures used for the initial detection of M proteins. The panel conducted a systematic literature review to address key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation approach, recommendations were created based on the available evidence, strength of that evidence, and key judgements as defined in the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework. RESULTS.: Nine guideline statements were established to optimize sample selection and testing for the initial detection and quantitative measurement of M proteins used to diagnose monoclonal gammopathies. CONCLUSIONS.: This guideline was constructed to harmonize and strengthen the initial detection of an M protein in patients displaying symptoms or laboratory features of a monoclonal gammopathy. It endorses more comprehensive initial testing when there is suspicion of amyloid light chain amyloidosis or neuropathies, such as POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes) syndrome, associated with an M protein.
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Paraproteinemias , Humanos , Laboratorios , Paraproteinemias/diagnóstico , Revisiones Sistemáticas como AsuntoRESUMEN
OBJECTIVES: Laboratory testing and the measurement of appropriate biomarkers play a critical role in managing patients with coronavirus disease 2019 (COVID-19), allowing for disease diagnosis, monitoring progression, prognostication, prediction of treatment response, and risk stratification. We sought to characterize these effects on a more detailed, mechanistic level. METHODS: We reviewed the literature and identified a multitude of reports that describe the unique effects of this virus and its devastating consequences to multiple organ systems in COVID-19 patients. RESULTS: There are specific alterations in biomarkers related to coagulation, depopulation of T-cell subtypes, the cytokine storm and inflammation, and kidney and cardiac dysfunction. CONCLUSIONS: Laboratory measurement of specific parameters and the use of appropriate prognostic, predictive, and monitoring biomarkers afford clinicians the ability to make informed medical decisions and guide therapy for patients afflicted with this dreaded disease.
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Biomarcadores/análisis , COVID-19/complicaciones , Trastornos de la Coagulación Sanguínea/diagnóstico , Trastornos de la Coagulación Sanguínea/virología , COVID-19/inmunología , COVID-19/patología , Síndrome de Liberación de Citoquinas/diagnóstico , Síndrome de Liberación de Citoquinas/virología , Cardiopatías/diagnóstico , Cardiopatías/virología , Humanos , Inmunidad Celular/inmunología , Inflamación/diagnóstico , Inflamación/virología , Enfermedades Renales/diagnóstico , Enfermedades Renales/virologíaRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma, and it constitutes biologically heterogeneous entities. Standard first-line therapies cure ~60% of patients, the rest being either refractory or experiencing relapse. Currently, there are no robust predictive biomarkers of therapeutic response. Heterogeneity of DLBCL is partly explained by the cell of origin (COO), ie, germinal centre B cell or activated B cell, with the latter exhibiting worse prognosis. While gene expression profiling (GEP) is the gold standard for determining COO, surrogate immunohistochemical algorithms are used clinically, but show significant discordance with GEP. Recently, additional genetic subgroups with different prognoses have been reported. However, the tools/expertise required for analysis prohibit widespread deployment. Liquid biopsy-based assays show promise in providing clinically actionable information, are noninvasive and facilitate serial sampling to assess mechanisms of therapy resistance. Circulating, cell-free DNA analysis has shown enhanced sensitivity for detecting molecular alterations, but this modality cannot determine alterations of the tumor proteome or on signalling pathways. Exosomes are endosomally derived vesicles, are found in high abundance in body fluids and are readily isolated using a variety of methods. Tumour-derived exosomes can yield data regarding genetic, transcriptional, and proteomic changes useful for diagnosis, prognosis, and therapy of DLBCL. At present, standardized techniques for isolating exosomes are lacking and discriminating between exosomes from neoplastic and normal B cells is challenging. Refinements in isolation procedures are required to realize their full potential as precision medicine tools to provide comprehensive information on disease subtypes, identify prognostic factors, allow real-time monitoring of therapy response and delineate novel drug targets.
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Exosomas , Linfoma de Células B Grandes Difuso , Biomarcadores , Biomarcadores de Tumor , Exosomas/genética , Humanos , Biopsia Líquida , Linfoma de Células B Grandes Difuso/diagnóstico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Recurrencia Local de Neoplasia , Pronóstico , ProteómicaRESUMEN
BACKGROUND: Antibody diagnostics play an important role in disease detection and can potentially aid in monitoring of the immune responses to see if an individual has developed immunity. Developing high throughput diagnostics which does not involve handling of infectious material becomes imperative in the case of pandemics such as the recent outbreak of SARS-CoV2. METHODS: A protein microarray technology was used to detect the plurality of antibody response to four novel antigens namely S1 glycoprotein, Receptor binding domain (RBD), S2 glycoprotein and Nucleoprotein of the novel coronavirus named SARS-CoV2 using serum samples. A DBS card was additionally used to compare its performance with a venipuncture-based serum separator tube (SST) draw. RESULTS: The three main subclasses of antibodies IgM, IgA and IgG were analyzed to see the variations in immune responses in the affected population and compared to their microbial RT-PCR based NP swab results. The clinical sensitivity and specificity were determined to be 99.67% and 99.77%. In the matrix comparison study, which would enable patients to test without risk of transmitting the virus, DBS (Dried Blood Spot) matched with higher than 98% accuracy to a venipuncture-based SST collection. CONCLUSION: Multiplex testing enables higher sensitivity and specificity which is essential while establishing exposure on a population scale. This flexible platform along with a discrete collection methodology would be crucial and broadly useful to scale up testing in current and future pandemics. Minimum sample volume that can be collected using DBS cards can be processed in this multiplex pillar plate format enabling the capacity to provide the reliability of high throughput analyzers while having the ease of collection similar to rapid tests.
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Anticuerpos Antivirales/sangre , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/diagnóstico , Isotipos de Inmunoglobulinas/sangre , Tamizaje Masivo/métodos , Neumonía Viral/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Betacoronavirus , COVID-19 , Prueba de COVID-19 , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , SARS-CoV-2 , Estados Unidos/epidemiología , Adulto JovenRESUMEN
Current serological immunoassays have inherent limitations for certain infectious diseases such as Lyme disease, a bacterial infection caused by Borrelia burgdorferi in North America. Here we report a novel method of manufacturing high-density multiplexed protein microarrays with the capacity to detect low levels of antibodies accurately from small blood volumes in a fully automated system. A panel of multiple serological markers for Lyme disease are measured using a protein microarray system, Lyme Immunochip, in a single step but interpreted adhering to the standard two-tiered testing algorithm (enzyme immunoassay followed by Western blot). Furthermore, an enhanced IgM assay was supplemented to improve the test's detection sensitivity for early Lyme disease. With a training cohort (n = 40) and a blinded validation cohort (n = 90) acquired from CDC, the Lyme Immunochip identified a higher proportion of Lyme disease patients than the two-tiered testing (82.4% vs 70.6% in the training set, 66.7% vs 60.0% in the validation set, respectively). Additionally, the Immunochip improved sensitivity to 100% while having a lower specificity of 95.2% using a set of investigational antigens which are being further evaluated with a large cohort of blinded samples from the CDC and Columbia University. This universal microarray platform provides an unprecedented opportunity to resolve a broad range of issues with diagnostic tests, including multiplexing, workflow simplicity, and reduced turnaround time and cost.
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Proteínas Bacterianas/inmunología , Biomarcadores/sangre , Borrelia burgdorferi/inmunología , Borrelia burgdorferi/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Enfermedad de Lyme/diagnóstico , Análisis por Matrices de Proteínas/métodos , Algoritmos , Estudios de Casos y Controles , Estudios de Cohortes , Humanos , Enfermedad de Lyme/sangre , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiología , Pruebas SerológicasRESUMEN
Clinical laboratory testing routinely provides actionable results, which help direct patient care in the inpatient and outpatient settings. Since December 2019, a novel coronavirus (SARS-CoV-2) has been causing disease (COVID-19 [coronavirus disease 2019]) in patients, beginning in China and now extending worldwide. In this context of a novel viral pandemic, clinical laboratories have developed multiple novel assays for SARS-CoV-2 diagnosis and for managing patients afflicted with this illness. These include molecular and serologic-based tests, some with point-of-care testing capabilities. Herein, we present an overview of the types of testing available for managing patients with COVID-19, as well as for screening of potential plasma donors who have recovered from COVID-19.
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Betacoronavirus/genética , Infecciones por Coronavirus/diagnóstico , Inmunoensayo/métodos , Técnicas de Diagnóstico Molecular/métodos , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Pruebas Serológicas/métodos , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , COVID-19 , Infecciones por Coronavirus/sangre , Humanos , Inmunoensayo/normas , Técnicas de Diagnóstico Molecular/normas , Pandemias , Neumonía Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , SARS-CoV-2 , Sensibilidad y Especificidad , Pruebas Serológicas/normasRESUMEN
In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancer-related intact proteins was evaluated. Seven growth factors (5.5-26.5 kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.
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Fluoroacetatos/química , Péptidos y Proteínas de Señalización Intercelular/análisis , Proteínas de Neoplasias/análisis , Cromatografía Liquida , Humanos , Espectrometría de Masas , Proteínas Recombinantes/análisisRESUMEN
Exosomes are small (30-200nm) membrane-bound vesicles released by all cells. They have been found in many clinical sample types, particularly those that are obtained in minimally-invasive fashion, including serum/plasma, urine, cerebrospinal and other fluids. They serve as an ideal source of biomarkers as their contents reflect the cell of origin and disease status of patients. Exosomes can serve as a "liquid biopsy," a non-invasive means to assess disease/health status in real-time. They can provide insights into disease mechanisms as they carry and transfer important signaling molecules, which may induce changes in the recipient cells. This is particularly relevant for metastatic cancers, as exosomes can prime the pre-metastatic niche. Many different approaches can be used to characterize the effects of the transfer of exosome content into the recipient cells, including global, untargeted approaches and protein-specific, targeted approaches. We describe herein our studies on the use of antibody arrays to probe for protein expression changes in hepatocytes that result from treating these cells with exosomes derived from uveal melanoma cells.
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Exosomas , Vesículas Extracelulares , Anticuerpos , Biomarcadores , Humanos , Biopsia LíquidaRESUMEN
PURPOSE: This guideline provides recommendations for available tissue-based prostate cancer biomarkers geared toward patient selection for active surveillance, identification of clinically significant disease, choice of postprostatectomy adjuvant versus salvage radiotherapy, and to address emerging questions such as the relative value of tissue biomarkers compared with magnetic resonance imaging. METHODS: An ASCO multidisciplinary Expert Panel, with representatives from the European Association of Urology, American Urological Association, and the College of American Pathologists, conducted a systematic literature review of localized prostate cancer biomarker studies between January 2013 and January 2019. Numerous tissue-based molecular biomarkers were evaluated for their prognostic capabilities and potential for improving management decisions. Here, the Panel makes recommendations regarding the clinical use and indications of these biomarkers. RESULTS: Of 555 studies identified, 77 were selected for inclusion plus 32 additional references selected by the Expert Panel. Few biomarkers had rigorous testing involving multiple cohorts and only 5 of these tests are commercially available currently: Oncotype Dx Prostate, Prolaris, Decipher, Decipher PORTOS, and ProMark. With various degrees of value and validation, multiple biomarkers have been shown to refine risk stratification and can be considered for select men to improve management decisions. There is a paucity of prospective studies assessing short- and long-term outcomes of patients when these markers are integrated into clinical decision making. RECOMMENDATIONS: Tissue-based molecular biomarkers (evaluating the sample with the highest volume of the highest Gleason pattern) may improve risk stratification when added to standard clinical parameters, but the Expert Panel endorses their use only in situations in which the assay results, when considered as a whole with routine clinical factors, are likely to affect a clinical decision. These assays are not recommended for routine use as they have not been prospectively tested or shown to improve long-term outcomes-for example, quality of life, need for treatment, or survival. Additional information is available at www.asco.org/genitourinary-cancer-guidelines.
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Biomarcadores de Tumor/análisis , Guías de Práctica Clínica como Asunto , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/terapia , Biomarcadores de Tumor/genética , Humanos , Masculino , Estadificación de Neoplasias/métodos , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Patología Molecular/métodos , Pronóstico , Prostatectomía/métodos , Neoplasias de la Próstata/genéticaRESUMEN
CONTEXT.: The performance of laboratory testing has recently come under increased scrutiny as part of important and ongoing debates on regulation and reimbursement. To address this critical issue, this study compares the performance of assay methods, using either commercial kits or assays designed and implemented by single laboratories ("home brews"), including next-generation sequencing methods, on proficiency testing provided by the College of American Pathologists Molecular Oncology Committee. OBJECTIVE.: To compare the performance of different assay methods on College of American Pathologists proficiency testing for variant analysis of 3 common oncology analytes: BRAF, EGFR, and KRAS. DESIGN.: There were 6897 total responses across 35 different proficiency testing samples interrogating 13 different variants as well as wild-type sequences for BRAF, EGFR, and KRAS. Performance was analyzed by test method, kit manufacturer, variants tested, and preanalytic and postanalytic practices. RESULTS.: Of 26 reported commercial kits, 23 achieved greater than 95% accuracy. Laboratory-developed tests with no kit specified demonstrated 96.8% or greater accuracy across all 3 analytes (1123 [96.8%] acceptable of 1160 total responses for BRAF; 848 [97.5%] acceptable of 870 total responses for EGFR; 942 [97.0%] acceptable of 971 total responses for KRAS). Next-generation sequencing platforms (summed across all analytes and 2 platforms) demonstrated 99.4% accuracy for these analytes (165 [99.4%] acceptable of 166 total next-generation sequencing responses). Slight differences in performance were noted among select commercial assays, dependent upon the particular design and specificity of the assay. Wide differences were noted in the lower limits of neoplastic cellularity laboratories accepted for testing. CONCLUSIONS.: These data demonstrate the high degree of accuracy and comparable performance across all laboratories, regardless of methodology. However, care must be taken in understanding the diagnostic specificity and reported analytic sensitivity of individual methods.