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1.
Medicine (Baltimore) ; 100(37): e27220, 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34664859

RESUMO

ABSTRACT: The visualization of intrahepatic hepatitis B virus (HBV) DNA by in situ hybridization (ISH) has uncovered some interesting aspects of HBV life cycle at the single-cell level. In the current study, we intend to evaluate the reliability and robustness of this assay in the real-world clinical scenario and its relationship with currently available clinical biomarkers in chronic hepatitis B (CHB) patients.In this cross-sectional study, 94 CHB patients and 10 patients with non-HBV related liver diseases were enrolled. Liver biopsies and routine histopathology analysis were performed. Intrahepatic HBV DNA and viral antigens (HBsAg and HBcAg) were detected by ISH and immunohistochemistry (IHC), respectively. The basic biochemical and virological parameters such as alanine transaminase, serum HBV DNA, and serum HBsAg were measured.The HBV DNA-ISH assay showed 55.8% (53/94 cases) positive rate in CHB patients, no false positive was found in non-HBV related hepatitis. The IHC of HBsAg and HBcAg showed a positive rate of 94.7% (89/94 cases) and 19.5% (17/87 cases), respectively. Quantification of HBV DNA-ISH signal showed a significant correlation with serum HBV DNA (rs = 0.6223, P < .0001). In addition, the staining pattern of HBV DNA in situ in the context of collagen deposition informed the histopathological progression of chronic liver disease.The application of this ISH assay in evaluating intrahepatic viral replication in real-world CHB patients showed favorable performance. It can be a complementation to conventional liver histopathology examination and IHC detection of viral antigens. This methodology provides an intuitive assessment of virological and pathological state of CHB patients, and further supports clinical diagnosis and management.


Assuntos
Bioensaio/normas , Vírus da Hepatite B/genética , Hepatite B Crônica/diagnóstico , Adulto , Bioensaio/métodos , Bioensaio/estatística & dados numéricos , China , Estudos Transversais , Feminino , Antígenos do Núcleo do Vírus da Hepatite B/análise , Antígenos de Superfície da Hepatite B/análise , Hepatite B Crônica/fisiopatologia , Humanos , Fígado/patologia , Fígado/fisiopatologia , Masculino , Pessoa de Meia-Idade
2.
Ann Clin Lab Sci ; 51(4): 584-586, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34452901

RESUMO

In this study, we evaluated the analytical interference of glycolic acid on several lactate assays that use lactate oxidase and dehydrogenase. Herein, we tested the effect of different concentrations of glycolic acid (0.01-46mM) on the lactate assay by using central lab and point of care (POCT) analyzers: Radiometer ABL 800, Beckman AU480, Roche Cobas c502, and Abbott i-STAT. Glycolic acid concentrations as low as 0.12mM resulted in a ≥20% positive bias in lactate assay on the ABL 800 and a concentration of approximately 0.23mM resulted in >20% on the Roche Cobas c502 and Abbott i-STAT. A significant lactate gap is found at concentrations >0.06mM between the Radiometer ABL 800 and Roche Cobas c502/Abbott i-STAT. However, at concentrations ≥0.92mM, the lactate gap is very significant among all three platforms. Falsely elevated lactate levels could result in misdiagnosis.


Assuntos
Bioensaio/normas , Erros de Diagnóstico/prevenção & controle , Glicolatos/envenenamento , Ácido Láctico/sangue , Sistemas Automatizados de Assistência Junto ao Leito/normas , Diagnóstico Diferencial , Humanos
3.
Parasit Vectors ; 14(1): 347, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210362

RESUMO

BACKGROUND: Long-lasting insecticidal nets (LLINs) have played an important role in reducing the global malaria burden since 2000. They are a core prevention tool used widely by people at risk of malaria. The Vector Control Prequalification mechanism of the Word Health Organization (WHO-Vector Control PQ) established the testing and evaluation guidelines for LLINs before registration for public use. In the present study, two new brands of deltamethrin-impregnated nets (Yahe® LN and Panda® Net 2.0) were evaluated in an experimental hut against wild pyrethroid-resistant Anopheles gambiae s.l. in M'Bé nearby Bouaké, central Côte d'Ivoire. METHODS: The performance of Yahe® LN and Panda® Net 2.0 was compared with that of PermaNet 2.0, conventionally treated nets (CTN), and untreated net to assess the blood-feeding inhibition, deterrence, induced exophily, and mortality. RESULTS: Cone bioassay results showed that Panda® Net 2.0, PermaNet 2.0 and Yahe® LN (both unwashed and washed 20 times) induced > 95% knockdown or > 80% mortality of the susceptible Anopheles gambiae Kisumu strain. With the pyrethroid-resistant M'Bé strain, mortality rate for all treated nets did not exceed 70%. There was a significant reduction in entry and blood feeding (p < 0.05) and an increase in exophily and mortality rates (p < 0.05) with all treatments compared to untreated nets, except the CTNs. However, the personal protection induced by these treated nets decreased significantly after 20 washes. The performance of Panda® Net 2.0 was equal to PermaNet® 2.0 in terms of inhibiting blood feeding, but better than PermaNet® 2.0 in terms of mortality. CONCLUSION: This study showed that Yahe® LN and Panda® Net 2.0 met the WHO Pesticide Evaluation Scheme (WHOPES) criteria to undergo phase III trial at the community level. Due to an increasing spread and development of pyrethroid resistance in malaria vectors, control of malaria transmission must evolve into an integrated vector management relying on a large variety of efficient control tools.


Assuntos
Anopheles/efeitos dos fármacos , Bioensaio/normas , Resistência a Inseticidas , Mosquiteiros Tratados com Inseticida/normas , Inseticidas/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Nitrilas/farmacologia , Piretrinas/farmacologia , Animais , Anopheles/fisiologia , Bioensaio/métodos , Ensaios Clínicos Fase II como Assunto , Costa do Marfim , Malária/parasitologia , Malária/prevenção & controle , Controle de Mosquitos/métodos , Controle de Mosquitos/normas , Mosquitos Vetores/parasitologia
4.
Sci Rep ; 11(1): 10398, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001964

RESUMO

We report a shipping container that enables a disruptive logistics for cytogenetic biodosimetry for radiation countermeasures through pre-processing cell culture during transportation. The container showed precise temperature control (< 0.01 °C) with uniform sample temperature (< 0.1 °C) to meet the biodosimetry assay requirements. Using an existing insulated shipping box and long shelf life alkaline batteries makes it ideal for national stockpile. Dose curve of cytogenetic biodosimetry assay using the shipping container showed clear dose response and high linear correlation with the control dose curve using a laboratory incubator (Pearson's correlation coefficient: 0.992). The container's ability of pre-processing biological samples during transportation could have a significant impact on radiation countermeasure, as well as potential impacts in other applications such as biobanking, novel molecular or cell-based assays or therapies.


Assuntos
Bancos de Espécimes Biológicos/normas , Liberação Nociva de Radioativos , Manejo de Espécimes/normas , Transportes/normas , Bioensaio/normas , Análise Citogenética/normas , Citogenética/normas , Humanos , Navios/normas
5.
Front Immunol ; 12: 636420, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936049

RESUMO

The expanded availability of adalimumab products continues to widen patient access and reduce costs with substantial benefit to healthcare systems. However, the long-term success of these medicines is highly dependent on maintaining consistency in quality, safety and efficacy while minimizing any risk of divergence during life-cycle management. In recognition of this need and demand from global manufacturers, the World Health Organization (WHO) Expert Committee on Biological standardization established the WHO 1st International standard (IS) for Adalimumab (coded 17/236) in October 2019 with a defined unitage ascribed to each of the individual bioactivities evaluated in the study namely, TNF-α binding, TNF-α neutralization, complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity. For development of the IS, two candidate standards were manufactured as per WHO recommendations. Analysis of extensive datasets generated by testing of a common set of samples including the candidate standards by multiple stakeholders including regulatory agencies using their own qualified assays in a large international collaborative study showed comparable biological activity for the tested candidates for the different activities. Use of a common standard significantly decreased the variability of bioassays and improved agreement in potency estimates. Data from this study clearly supports the utility of the IS as an important tool for assuring analytical assay performance, for bioassay calibration and validation, for identifying and controlling changes in bioactivity during life-cycle management and for global harmonization of adalimumab products. In addition, in a separate multi-center study which included involvement of hospital and clinical diagnostic laboratories, the suitability of the adalimumab IS for therapeutic drug monitoring assays was examined by analysis of data from testing of a common blind coded panel of adalimumab spiked serum samples representative of the clinical scenario along with the IS and in-house standards in diverse immunoassays/platforms. Both commercially available and in-house assays that are routinely used for assessing adalimumab trough levels were included. Excellent agreement in estimates for adalimumab content in the spiked samples was observed regardless of the standard or the method with inter-laboratory variability also similar regardless of the standard employed. This data, for the first time, provides support for the extended applicability of the IS in assays in use for therapeutic drug monitoring based on the mass content of the IS. The adalimumab IS, in fulfilling clinical demand, can help toward standardizing and harmonizing clinical monitoring assays for informed clinical decisions and/or personalized treatment strategies for better patient outcomes. Collectively, a significant role for the adalimumab IS in assuring the quality, safety and efficacy of adalimumab products globally is envisaged.


Assuntos
Adalimumab/uso terapêutico , Bioensaio/normas , Medicamentos Biossimilares/uso terapêutico , Monitoramento de Medicamentos/normas , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab/efeitos adversos , Animais , Especificidade de Anticorpos , Medicamentos Biossimilares/efeitos adversos , Medicamentos Biossimilares/normas , Células CHO , Cricetulus , Células HEK293 , Humanos , Células Jurkat , Controle de Qualidade , Padrões de Referência , Equivalência Terapêutica , Inibidores do Fator de Necrose Tumoral/efeitos adversos , Inibidores do Fator de Necrose Tumoral/normas , Fator de Necrose Tumoral alfa/imunologia , Células U937 , Organização Mundial da Saúde
6.
AAPS J ; 23(3): 64, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33942188

RESUMO

In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.


Assuntos
Bioensaio/normas , Desenvolvimento de Medicamentos/normas , Guias como Assunto , Anticorpos Biespecíficos/análise , Anticorpos Biespecíficos/uso terapêutico , Bioensaio/métodos , Terapia Baseada em Transplante de Células e Tecidos , Cromatografia Líquida/normas , Desenvolvimento de Medicamentos/métodos , Citometria de Fluxo/normas , Terapia Genética , Espectrometria de Massas/normas , Oligonucleotídeos/análise , Oligonucleotídeos/uso terapêutico , Pró-Fármacos/análise , Pró-Fármacos/uso terapêutico , RNA/análise , RNA/uso terapêutico , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/uso terapêutico
7.
J Steroid Biochem Mol Biol ; 212: 105917, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34010687

RESUMO

An intralaboratory study assessing assay variability and bias for determination of serum total 25-hydroxyvitamin D [25(OH)D] was conducted by the Vitamin D Standardization Program (VDSP). Thirteen assays for serum total 25(OH)D were evaluated in a single laboratory including 11 unique immunoassays and one liquid chromatography - tandem mass spectrometry (LC-MS/MS) assay. Fifty single-donor serum samples, including eight samples with high concentrations of 25(OH)D2 (> 30 nmol/L), were assigned target values for 25(OH)D2 and 25(OH)D3 using reference measurement procedures (RMP). Using four replicate measurements for each sample, the mean total percent coefficient of variation (%CV) and mean % bias from the target values were determined for each assay using the 50 single-donor samples and a 42-sample subset, which excluded 8 high 25(OH)D2 concentration samples, and compared with VDSP performance criteria of ≤ 10 % CV and ≤ ±5 % mean bias. All 12 assays achieved the performance criterion for % CV, and 9 of the 12 assays were within ≤ ±5 % mean bias. The Fujirebio Inc. assay exhibited the lowest %CV and highest percentage of individual measurements within ≤ ±5 % mean bias. Ten immunoassays exhibited changes in response due to the high 25(OH)D2 samples with Abbott, Biomérieux, DiaSorin, DIAsource, and IDS-iSYS assays having the largest deviations. The Fujirebio Inc. and Beckman Coulter assays were only minimally affected by the presence of the high 25(OH)D2 samples. Samples with high concentrations of 25(OH)D2 provided a critical performance test for immunoassays indicating that some assays may not have equal response or recovery for 25(OH)D2 and 25(OH)D3.


Assuntos
Bioensaio/normas , Imunoensaio/normas , Vitamina D/análogos & derivados , Vitaminas/sangue , Viés , Cromatografia Líquida , Humanos , Laboratórios , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Vitamina D/sangue
8.
Sci Rep ; 11(1): 10880, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035340

RESUMO

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.


Assuntos
Bioensaio/métodos , Peptídeos/química , Proteínas/análise , Sequência de Aminoácidos , Bioensaio/normas , Humanos , Marcação por Isótopo , Cinética , Peptídeos/síntese química , Conformação Proteica , Proteínas/química , Proteólise , Proteômica/métodos , Padrões de Referência , Solubilidade , Solventes , Tripsina/metabolismo
9.
Bioanalysis ; 13(8): 609-619, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33847160

RESUMO

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.


Assuntos
Anticorpos/análise , Bioensaio/normas , Humanos , Padrões de Referência
10.
Basic Clin Pharmacol Toxicol ; 128(5): 642-648, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33665955

RESUMO

Predictive biomarkers play an important role in our efforts to individualize pharmacotherapy, and within recent years, a number of different types of assays have been introduced. These biomarkers may potentially support the selection and dosage of specific drugs in order to maximize efficacy and minimize adverse reactions in the individual patient. However, in many instances, the scientific and clinical evidence is insufficient to support the prescribing decision. When predictive biomarkers are used to guide pharmacotherapy, it is important to secure that decisions are based on solid clinical evidence. Here, the regulatory authorities, especially the FDA, have been at the forefront in relation to regulate this type of biomarker assay in order to secure patient safety. The approval process for companion diagnostics is an example of this effort, where the scientific validity of the biomarker and assay is in focus. With the approaching implementation of the new IVD Regulation, greater attention will also be paid to analytical and clinical validity of biomarker assays in the EU. For any type of predictive biomarker assay, including pharmacogenetic and tumour profiling tests, the clinical evidence needs to be in place before they are used routinely in the clinic.


Assuntos
Bioensaio/instrumentação , Biomarcadores/análise , Testes Farmacogenômicos/instrumentação , Bioensaio/métodos , Bioensaio/normas , Aprovação de Teste para Diagnóstico , União Europeia , Testes Farmacogenômicos/métodos , Testes Farmacogenômicos/normas , Medicina de Precisão/métodos , Kit de Reagentes para Diagnóstico/normas , Estados Unidos , United States Food and Drug Administration/normas
11.
Artigo em Inglês | MEDLINE | ID: mdl-33461699

RESUMO

This present review described the validation method of in-vitro bioassay for its application in herbal drug research. Seven sequencing steps that can be taken for performing a valid bioassay include: literature survey, sample stability evaluation, Biosystem performance testing, Sample performance evaluation, determination of 50% effective concentration or cytotoxic concentrations, selective index evaluation, and determination of accurate relative potency of sample. Detailed methods and acceptance criteria for each step are described herein. Method calculations of the relative potency of sample using European Pharmacopeia 10.0, 5.3 (2020) were recommended instead of using United States Pharmacopeia 42 (2019). For having reliable data and conclusions, all methods (chemical and bioassay) need to be first validated before any data collection. Absence of any validation method may results in incorrect conclusions and bias.


Assuntos
Bioensaio/normas , Preparações de Plantas
12.
Int J Hematol ; 113(4): 530-536, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33417140

RESUMO

Patients with congenital protein S (PS) deficiency show a hereditary predisposition for thrombosis, and PS deficiency is prevalent among Japanese populations. Diagnosis is based on symptoms of thrombosis and reduced PS activity. Three reagents that use different measurement principles for determining PS activity are available in Japan. This study aimed to confirm the possibility of harmonization of these three reagents to establish a universal standard for PS activity in Japanese populations. Commercial normal plasma and plasma samples obtained from healthy individuals and healthy pregnant women were tested at three facilities using three reagents for measuring PS: STA-Staclot Protein S (STA-PS), HemosIL Protein S (Clotting) (IL-PS), and a total PS assay (SNT-PS). The within-run precision of each reagent was good, as each had a coefficient of variation of ≤ 3.8%. The dilution linearity for each reagent was also good. The correlation coefficient was 0.94 for STA-PS vs. IL-PS, 0.93 for SNT-PS vs. STA-PS, and 0.90 for SNT-PS vs. IL-PS, indicating a good correlation. Although the three reagents available in Japan for measuring PS activity use different measurement methods, each showed good performance, and large differences were not observed between the obtained values. Harmonization among them appears possible.


Assuntos
Bioensaio/métodos , Bioensaio/normas , Proteína S/metabolismo , Kit de Reagentes para Diagnóstico , Coagulação Sanguínea , Humanos , Deficiência de Proteína S/sangue , Deficiência de Proteína S/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Valores de Referência , Reprodutibilidade dos Testes
13.
Clin Biochem ; 87: 39-45, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33188771

RESUMO

BACKGROUND: The commutability of control materials used for external quality assessment (EQA) programs is of great importance. Evaluating the commutability of control materials is crucial to assess their suitability for EQA programs. METHODS: Forty-eight individual patient serum samples, commercial EQA samples, human serum pools (HSPs), commercially available sterile filtered charcoal stripped serum (CS) and swine serum were analyzed using the isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) comparative method and six immunoassays for progesterone. The commutability was assessed according to the EP14-A2 guideline and the difference in bias approach, respectively. RESULTS: According to the EP14-A2 guideline, HSPs and CS were commutable for all the tested immunoassays, while swine serum showed positive matrix effects in some assays. Based on the difference in bias approach, a large number of inconclusive and noncommutable results appeared. CONCLUSIONS: The commutability of the processed materials varied depending on which evaluation approach and criterion was applied. Noncommutability of the EQA materials was observed. And HSPs and CS were possible commutable candidate control materials according to the EP14-A2 guideline.


Assuntos
Bioensaio/métodos , Cromatografia Líquida/métodos , Progesterona/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Bioensaio/normas , Cromatografia Líquida/normas , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Controle de Qualidade , Padrões de Referência , Suínos , Espectrometria de Massas em Tandem/normas
14.
Neuroendocrinology ; 111(5): 490-504, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32392558

RESUMO

BACKGROUND: The NETest is a multigene assay comprising 51 circulating neuroendocrine tumor (NET)-specific transcripts. The quotient of the 51-gene assay is based upon an ensemble of machine learning algorithms. Eight cancer hallmarks or "omes" (apoptome, epigenome, growth factor signalome, metabolome, proliferome, plurome, secretome, SSTRome) represent 29 genes. The NETest is an accurate diagnostic (>90%) test, but its prognostic utility has not been assessed. In this study, we describe the expansion of the NETest omic cluster components and demonstrate that integration amplifies NETest prognostic accuracy. METHODS: Group 1: n = 222; including stable disease (SD, n = 146), progressive disease (PD, n = 76), and controls (n = 139). Group 2: NET Registry NCT02270567; n = 88; prospective samples (SD, n = 54; PD, n = 34) with up to 24 months follow-up. We used PubMed literature review, interactomic analysis, nonparametric testing, Kaplan-Meier survival curves, and χ2 analyses to inform and define the prognostic significance of NET genomic "hallmarks." RESULTS: 2020 analyses: In-depth analyses of 47 -NETest genes identified a further six omes: fibrosome, inflammasome, metastasome, NEDome, neurome, and TFome. Group 1 analysis: Twelve omes, excluding the inflammasome and apoptome, were significantly (p < 0.05, 2.1- to 8.2-fold) elevated compared to controls. In the PD group, seven omes (proliferome, NEDome, epigenome, SSTRome, neurome, metastasome, and fibrosome) were elevated (both expression levels and fold change >2) versus SD. Group 2 analysis: All these seven omes were upregulated. In PD, they were significantly more elevated (p < 0.02) than in SD. The septet omic expression exhibited a 69% prognostic accuracy. The NETest alone was 70.5% accurate. A low NETest (≤40) integrated with epigenome/metastasome levels was an accurate prognostic for PD (90%). A high NETest (>40) including the fibrosome/NEDome predicted PD development within 3 months (100%). Using decision tree analysis to integrate the four omes (epigenome, metastasome, fibrosome, and NEDome) with the NETest score generated an overall prognostic accuracy of 93%. CONCLUSIONS: Examination of NETest omic gene cluster analysis identified five additional clinically relevant cancer hallmarks. Identification of seven omic clusters (septet) provides a molecular pathological signature of disease progression. The integration of the quartet (epigenome, fibrosome, metastasome, NEDome) and the NETest score yielded a 93% accuracy in the prediction of future disease status.


Assuntos
Bioensaio/normas , Biomarcadores Tumorais/genética , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/genética , Transcriptoma/genética , Análise por Conglomerados , Seguimentos , Humanos , Tumores Neuroendócrinos/metabolismo , Valor Preditivo dos Testes , Prognóstico
15.
Ann Clin Transl Neurol ; 7(11): 2262-2271, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33185334

RESUMO

OBJECTIVE: Real-time quaking-induced conversion (RT-QuIC) assays detect prion-seeding activity in a variety of human biospecimens, including cerebrospinal fluid and olfactory mucosa swabs. The assay has shown high diagnostic accuracy in patients with prion disorders. Recently, advances in these tests have led to markedly improved diagnostic sensitivity and reduced assay times. Accordingly, an algorithm has been proposed that entails the use of RT-QuIC analysis of both sample types to diagnose sporadic Creutzfeldt-Jakob disease with nearly 100% accuracy. Here we present a multi-center evaluation (ring trial) of the reproducibility of these improved "second generation" RT-QuIC assays as applied to these diagnostic specimens. METHODS: Cerebrospinal fluid samples were analyzed from subjects with sporadic Creutzfeldt-Jakob (n = 55) or other neurological diseases (n = 45) at multiple clinical centers. Olfactory mucosa brushings collected by multiple otolaryngologists were obtained from nine sporadic Creutzfeldt-Jakob disease cases and 19 controls. These sample sets were initially tested blindly by RT-QuIC by a coordinating laboratory, recoded, and then sent to five additional testing laboratories for blinded ring trial testing. RESULTS: Unblinding of the results by a third party indicated 98-100% concordance between the results obtained by the testing of these cerebrospinal fluid and nasal brushings at the six laboratories. INTERPRETATION: This second-generation RT-QuIC assay is highly transferrable, reproducible, and therefore robust for the diagnosis of sporadic Creutzfeldt-Jakob disease in clinical practice.


Assuntos
Bioensaio/normas , Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Síndrome de Creutzfeldt-Jakob/diagnóstico , Técnicas de Diagnóstico Neurológico/normas , Mucosa Olfatória/metabolismo , Príons/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
16.
Protist ; 171(6): 125773, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33243724

RESUMO

The marine protozoan parasite Neoparamoeba perurans has been established as the causative agent for amoebic gill disease (AGD) in Atlantic salmon (Salmo salar). Freshwater bathing is the only routinely used treatment for AGD in Australia while hydrogen peroxide (H2O2) is used in countries with cooler water temperatures. The identification of new treatments that do not rely on either freshwater or H2O2 bathing is highly sought. However, in vitro based methods for high throughput screening of antiparasitic compounds have not been established for this parasite. To this end the present study evaluated two in vitro bioassays based on metabolic energy production and cellular membrane integrity to distinguish between amoebistatic (crenated or pseudocyst forms with recovery possible) and amoebicidal (death) activity. Amoebae were subject to either freshwater, H2O2 or chloramine-T for 4h treatment and assessed 24h after recovery. Visualization by microscopy and bioassay assessment 24h post-treatment confirmed that H2O2 and freshwater are 95% amoebicidal albeit due to different mechanisms of action. These data are consistent with other studies where amoebae have been observed to recover following exposure to these compounds and provide evidence for the inclusion of a recovery component to differentiate between the mechanism of action of amoebicidal and amoebistatic treatments. Together these bioassays are a critical tool for high throughput screening of novel and more effective treatments against AGD.


Assuntos
Amebíase/parasitologia , Amoeba/fisiologia , Bioensaio/normas , Doenças dos Peixes/parasitologia , Ensaios de Triagem em Larga Escala/métodos , Amoeba/citologia , Animais , Organismos Aquáticos , Pesqueiros , Ensaios de Triagem em Larga Escala/normas , Viabilidade Microbiana
17.
Mod Rheumatol Case Rep ; 4(1): 156-160, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-33086964

RESUMO

Alpha-defensin (αD), an antimicrobial peptide released by neutrophils in response to bacterial pathogens, was proposed as a novel diagnostic biomarker in synovial fluid. Several reports have shown that αD can serve as a reliable biomarker in the diagnosis of periprosthetic joint infection (PJI). We assessed whether αD could also serve to diagnosis of septic arthritis, a similarly difficult to diagnose PJI. To our knowledge, besides PJI, few reports exist assessing the utility of αD for septic arthritis. We have attempted to diagnose several cases of suspected septic arthritis using the Synovasure® αD detection lateral flow device. We report a false-positive case and a false-negative case. The false-negative case we experienced was caused by Staphylococcus capitis, which is coagulase-negative, and possibly represents a low virulence micro-organism infection. The false-positive case was ultimately diagnosed as seronegative rheumatoid arthritis and possessed calcium pyrophosphate depositions. False positives have been suggested to occur in conditions where neutrophils are mobilised. As for PJI, in cases where diagnosis is difficult, αD can be an additional diagnostic indicator. However, making a definitive diagnosis using the αD lateral flow device alone was found to be difficult. The utility of αD in assessing septic arthritis is inconclusive; therefore, larger prospective clinical studies should be considered for a better assessment.


Assuntos
Artrite Infecciosa/diagnóstico , Artrite Infecciosa/metabolismo , Bioensaio/métodos , Biomarcadores , Líquido Sinovial/metabolismo , alfa-Defensinas/biossíntese , Artrite Infecciosa/etiologia , Bioensaio/instrumentação , Bioensaio/normas , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus capitis
19.
Acta Virol ; 64(3): 271-275, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32985203

RESUMO

Transfer factor (TF) is a heterogeneous mix of low-molecular weight molecules obtained from dialyzed leukocyte extract that is capable of transferring cell-mediated immunity. As an immunostimulatory drug TF is used to improve treatment of infectious diseases, allergies, cancer and immune deficiencies. The main benefit of TF preparations as immunotherapeutic agents is the induction of a rapid immune response and the potential of TF as an adjuvant in combination with other drugs might lead to development of novel approaches to combat various diseases in the future. The process of TF preparation is rather simple. However, with respect to fact that TF is composed by several multifunction molecules, it is likely that during the activity measurement based only on one single parameter, other TF biological activities might be overlooked. In addition, separated TF components might display synergetic activity effect. According to recent European Pharmacopoeia there is no general protocol for immuno-stimulatory drugs (including TF) activity measurement available. Nevertheless, for the process of TF preparation, control of input material and for final pharmaceutical product batches it is inevitable to guaranty proper quality control including appropriate in vivo or in vitro test(s) for TF biological activity assay. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic problem, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. The currently used method for testing biological activity of TF is the in vitro MTT cells proliferation assay that is recognized by control authorities in Slovak Republic. Keywords: immune system; transfer factor; dialysable leukocyte extract; diseases; MTT cells proliferation assay.


Assuntos
Bioensaio/normas , Imunidade Celular , Fator de Transferência/normas , Adjuvantes Imunológicos , Animais , Reprodutibilidade dos Testes , Eslováquia
20.
Reprod Biol Endocrinol ; 18(1): 86, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32799874

RESUMO

BACKGROUND: There is a need for a reference material to support the development and ensure the quality of immunoassays for human AMH. A batch of ampoules, coded 16/190, containing lyophilised recombinant AMH was evaluated in a WHO Collaborative Study. The aims of the study were to determine the AMH content in terms of the calibration of each immunoassay method, to predict long-term stability and to assess the suitability of the preparation to calibrate AMH immunoassays. METHODS: Study participants were asked to report the AMH content of specific dilutions of coded ampoules of 16/190 and a comparator preparation containing approximately half the AMH content. In each assay, participants also reported the AMH content of 22 patient samples to assess commutability. A robust all-laboratory geometric mean of the content estimates was determined using the laboratory geometric mean estimates. Commutability was assessed using a difference in bias approach. Stability was predicted by the measurement of thermally accelerated degradation samples. RESULTS: Seven laboratories performed twenty-one immunoassay method-platform combinations, sixteen of which provided data which met the validity criteria, giving a consensus geometric mean estimate of AMH content of 511 ng/ampoule (95% CI, 426-612, n = 16, GCV 42%) and a robust geometric mean of 489 ng/ampoule. By contrast, the GCV% for the all-laboratory geometric mean of the relative content estimates for the comparator sample to 16/190 was 12%. Commutability was assessed using 20 of the 22 representative patient samples. Of the valid assays, 16/190 was within the limits of acceptable commutability for 6 methods, partially commutable for a further 3 methods and non-commutable when measured by 7 methods. The preparation was predicted to be highly stable when stored at - 20 °C. CONCLUSION: The majority of methods met the validity criteria. Content estimates showed a high between-method variability, yet assays exhibited a similar proportionality of response as demonstrated using the comparator sample. 16/190 was commutable in some but not all methods. On the basis of these results, it was agreed by the WHO Expert Committee on Biological Standardization to establish 16/190 as a WHO Reference Reagent for AMH with a content defined by consensus immunoassay of 489 ng/ampoule.


Assuntos
Hormônio Antimülleriano/análise , Bioensaio/normas , Indicadores e Reagentes , Organização Mundial da Saúde , Animais , Hormônio Antimülleriano/sangue , Bioensaio/métodos , Células CHO , Calibragem/normas , Serviços de Laboratório Clínico/normas , Cricetulus , Feminino , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Indicadores e Reagentes/análise , Indicadores e Reagentes/isolamento & purificação , Cooperação Internacional , Internacionalidade , Ensaio de Proficiência Laboratorial/normas , Padrões de Referência
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