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1.
Biochem Med (Zagreb) ; 29(3): 030708, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31624461

RESUMO

Introduction: The aim of this study was to compare ionized calcium (iCa) concentrations in arterial heparinized blood and venous serum and to investigate time-dependent variation of iCa in serum samples centrifuged and analysed at different times. Materials and methods: Ionized calcium was measured (N = 25) in arterial blood within 20 min after puncture, and in serum within 10 min after centrifugation conducted 30 min after sampling. Effect of time between sampling and centrifugation was examined in three tubes (N = 30) centrifuged 15, 30 and 60 min after sampling, and analysed within 10 min. Effect of time between centrifugation and analysis was investigated in three tubes (N = 31) centrifuged 30 min after sampling and analysed: 0-10, 30-40 and 90-100 min after centrifugation. Ionized calcium was measured on the Siemens RapidLab 348EX analysers. Statistical significance was tested using Wilcoxon test and ANOVA analysis. Clinical significance was judged against reference change values (RCV). Results: No statistically significant difference was found between iCa in arterial blood and serum (P = 0.274). A statistically significant decrease was found: in tubes centrifuged 60 and 15 min after sampling versus 30 min (P = 0.005, P = 0.003); and in tubes analysed 30-40 and 90-100 min after centrifugation versus 0-10 min (P = 0.021, P = 0.027). Clinically significant changes were observed: 60 versus 30 min (centrifugation) and 90-100 versus 0-10 and 30-40 min (analysis). Conclusions: Timely analysed arterial blood and serum samples can be used interchangeably. To avoid clinically significant variations, serum tubes should be centrifuged within 30 min after sampling, and analysis should be performed within 30 min after centrifugation.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Cálcio/sangue , Heparina/sangue , Humanos , Concentração de Íons de Hidrogênio , Manejo de Espécimes/métodos
2.
Lancet ; 394(10202): 967-978, 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31526740

RESUMO

Children bear a substantial burden of suffering when it comes to tuberculosis. Ironically, they are often left out of the scientific and public health advances that have led to important improvements in tuberculosis diagnosis, treatment, and prevention over the past decade. This Series paper describes some of the challenges and controversies in paediatric tuberculosis, including the epidemiology and treatment of tuberculosis in children. Two areas in which substantial challenges and controversies exist (ie, diagnosis and prevention) are explored in more detail. This Series paper also offers possible solutions for including children in all efforts to end tuberculosis, with a focus on ensuring that the proper financial and human resources are in place to best serve children exposed to, infected with, and sick from all forms of tuberculosis.


Assuntos
Tuberculose/terapia , Criança , Busca de Comunicante/métodos , Humanos , Programas de Rastreamento/métodos , Programas de Rastreamento/organização & administração , Manejo de Espécimes/métodos , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Vacinas contra a Tuberculose , Vacinação
4.
Medicine (Baltimore) ; 98(35): e16970, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464941

RESUMO

This study aimed to determine the feasibility of vaginal/cervical nurse-assisted self-sampling (NASS) and the agreement between human papilloma virus (HPV) tests on self-samples versus clinician-taken (CT) specimens.Women participated voluntarily for cervical cancer screening at St. Aklesia Memorial Hospital. Eighty-three women provided a total of 166 coupled self-taken and CT specimens collected. Specimens were stored at room temperature for a maximum of 10 months and analyzed using validated the RIATOL qPCR HPV genotyping test, a quantitative polymerase chain reaction (qPCR) high-throughput HPV E6, E7 assay. The average age of the participating women was 32 years. Seventy-three women (87.9%) felt that NASS was easy to use. An overall HPV, high-risk (HR) HPV, and low-risk HPV prevalence was 22.7% (15/66), 18.2% (12/66), and 6.1% (4/66), respectively. The overall HR HPV prevalence was 17.2% (NASS) and 15.5% (CT). The most prevalent HPV type was HPV51; HPV 16 was only detected in 1 woman (CT+NASS) and HPV18 only in 1 woman (CT). The overall measurement agreement between self-taken and CT samples was moderate with a kappa value of 0.576 (P < .001). Lifetime partnered with >2 men were associated with HR HPV positivity (P < .001). There was a strong statistical association between HR HPV positivity and visual inspection with acetic acid- positive (P < .001). The NASS for HPV testing could be seen as an alternative option and might be acceptable to Ethiopian women. The overall HR HPV prevalence was comparable with Sub-Saharan countries in the general population.


Assuntos
Detecção Precoce de Câncer/métodos , Infecções por Papillomavirus/diagnóstico , Manejo de Espécimes/métodos , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal/métodos , Adulto , Idoso , Etiópia/epidemiologia , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
5.
BMC Public Health ; 19(1): 1026, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366402

RESUMO

BACKGROUND: In the context of WHO's "task shifting" project and growing global consensus on primary HPV-based cervical cancer screening, self-sampling is a promising new tool to expand screening access, uptake and coverage for women worldwide. We aimed to explore perceptions and acceptability of HPV self-sampling-based cervical cancer screening among community members and health professionals in rural northwest Ethiopia and to identify preferences and socio-cultural barriers regarding self-sampling in order to design a suitable high-coverage screening intervention for a rural African setting. METHODS: Four community-based focus group discussions (FGD) were conducted in the rural district of Dabat, Northwest Ethiopia, each comprising 8 to 14 female participants, counting a total of 41 participants. The groups were homogenously composed in terms of their socio-economic status in the community. They included health centre attendees, community members, nurses and health development army leaders (HDAL). Two qualitative data collection experts conducted the interviews in the local language, using a FGD guide with several thematic areas. All participants granted written informed consent prior to the conduct of the interviews. As a concrete example of an existing self-sampling approach for cervical cancer screening we used the Evalyn® Brush. RESULTS: Emerging themes included (i) misconceptions and low awareness about cervical cancer among community residents and primary health care providers in rural northwest Ethiopia, (ii) stigmatization and social exclusion of affected women, (iii) delay in seeking of health care due to poor access and availability of services, and lacking of a concept of early cancer prevention, (iv) need of spousal permission, (v) fear of financial burden and (vi) fear of social marginalization. The self-sampling device was regarded to be acceptable and was judged to be easy to use for most women. The existing Ethiopian health care structure could facilitate a community approach. CONCLUSION: Home-based self-sampling for cervical cancer screening is a socially acceptable and feasible "task shifting" method that will increase cervical cancer screening access and coverage in the Ethiopian study community. Education, awareness creation, community mobilization and family inclusion are identified as key activities to promote, implement and facilitate "task shifting" approaches like self-sampling.


Assuntos
Detecção Precoce de Câncer/métodos , Genitália/virologia , Infecções por Papillomavirus/diagnóstico , Autoexame , Neoplasias do Colo do Útero/diagnóstico , Adulto , Etiópia , Feminino , Grupos Focais , Acesso aos Serviços de Saúde , Humanos , Preferência do Paciente , Pesquisa Qualitativa , População Rural , Manejo de Espécimes/métodos , Adulto Jovem
6.
Parasit Vectors ; 12(1): 353, 2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311591

RESUMO

BACKGROUND: Faecal egg counts (FEC) and the FEC reduction test (FECRT) for assessing gastrointestinal nematode (GIN) infection and efficacy of anthelmintics are rarely carried out on ruminant farms because of the cost of individual analyses. The use of pooled faecal samples is a promising method to reduce time and costs, but few studies are available for cattle, especially on the evaluation of different pool sizes and FECRT application. METHODS: A study was conducted to assess FEC strategies based on pooled faecal samples using different pool sizes and to evaluate the pen-side use of a portable FEC-kit for the assessment of FEC on cattle farms. A total of 19 farms representing 29 groups of cattle were investigated in Italy and France. On each farm, individual faecal samples from heifers were collected before (D0) and two weeks after (D14) anthelmintic treatment with ivermectin or benzimidazoles. FEC were determined individually and as pooled samples using the Mini-FLOTAC technique. Four different pool sizes were used: 5 individual samples, 10 individual samples, global and global on-farm. Correlations and agreements between individual and pooled results were estimated with Spearman's correlation coefficient and Lin's concordance correlation coefficients, respectively. RESULTS: High correlation and agreement coefficients were found between the mean of individual FEC and the mean of FEC of the different pool sizes when considering all FEC obtained at D0 and D14. However, these parameters were lower for FECR calculation due to a poorer estimate of FEC at D14 from the faecal pools. When using FEC from pooled samples only at D0, higher correlation and agreement coefficients were found between FECR data, the better results being obtained with pools of 5 samples. Interestingly, FEC obtained on pooled samples by the portable FEC-kit on-farm showed high correlation and agreement with FEC obtained on individual samples in the laboratory. This field approach has to be validated on a larger scale to assess its feasibility and reliability. CONCLUSIONS: The present study highlights that the pooling strategy and the use of portable FEC-kits on-farm are rapid and cost-effective procedures for the assessment of GIN egg excretion and can be used cautiously for FECR calculation following the administration of anthelmintics in cattle.


Assuntos
Bovinos/parasitologia , Fezes/parasitologia , Infecções por Nematoides/veterinária , Contagem de Ovos de Parasitas/métodos , Animais , Anti-Helmínticos/uso terapêutico , Doenças dos Bovinos/tratamento farmacológico , Doenças dos Bovinos/parasitologia , Feminino , França , Itália , Infecções por Nematoides/diagnóstico , Contagem de Ovos de Parasitas/instrumentação , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Manejo de Espécimes/métodos
7.
J Med Microbiol ; 68(9): 1324-1329, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31355739

RESUMO

Purpose. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK).Methodology. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK.Results. There was no difference in the performance of both primer sets for all HSV-1 dilutions (P=0.83) using a CIM or between a CIM and a swab (P=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (P=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (P=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (P<0.001).Conclusions. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.


Assuntos
Córnea/virologia , Herpes Simples/diagnóstico , Herpesvirus Humano 1/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Primers do DNA/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
8.
Parasit Vectors ; 12(1): 336, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287026

RESUMO

BACKGROUND: Clonorchiasis is caused by eating of raw or undercooked freshwater fish containing the larvae of Clonorchis sinensis; the Kato-Katz method is widely applied in diagnosis. The improvement of repeated Kato-Katz smears from multiple stool samples has been well illuminated in many helminths other than C. sinensis. METHODS: A cross-sectional investigation was implemented to capture the epidemiology and risk factors of clonorchiasis among middle school students in Qiyang county, China. Students with complete data of six Kato-Katz thick smears from two stool samples were included in this analysis. Data on the habits of eating raw freshwater fish were also collected and compared. RESULTS: Altogether, 397 students had complete information of six smears, out of which 394 reported the information on eating habits. According to the 'gold' standard by six smears, 77 students (19.4%) were detected with C. sinensis. However, only 45 (11.3%) were detected using a single smear, with an underestimation of 41.6% compared to the 'gold' standard. However, the geometric mean of eggs per gram of feces in detected cases was 126.4 in a single smear, overestimated by 105.2% compared to 61.6 by the 'gold' standard. The linear relationship between prevalence and infection intensity of detected cases based on different smears was significantly negative. The habits of eating raw freshwater fish in the false negative cases was similar to those in the detected cases, but these two groups had significantly higher levels for habits of eating raw freshwater fish than negative individuals. CONCLUSIONS: In low endemicity situations, underestimation of C. sinensis infection could not be avoided based on a limited number of Kato-Katz smears. Thus, repeated smears from at least two stool samples should be considered when an individual eats raw freshwater fish, drug efficacy is evaluated or elimination of C. sinensis is verified. Additionally, when logistics are insufficient for multiple samples to be taken for diagnosis for survey and surveillance in the areas or populations of low endemicity, prevalence accuracy needs to be corrected.


Assuntos
Clonorquíase/diagnóstico , Clonorchis sinensis/isolamento & purificação , Produtos Pesqueiros/parasitologia , Manejo de Espécimes/métodos , Animais , China/epidemiologia , Clonorquíase/epidemiologia , Clonorquíase/parasitologia , Estudos Transversais , Fezes/parasitologia , Comportamento Alimentar , Feminino , Peixes , Água Doce , Humanos , Masculino , Contagem de Ovos de Parasitas , Prevalência , Inquéritos e Questionários
9.
Am J Vet Res ; 80(8): 787-791, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339768

RESUMO

OBJECTIVE: To evaluate safety of stylet-in and stylet-out techniques for collection of CSF from the cisterna magna and to assess whether there were differences between techniques with regard to contamination of samples, sample quality, and efficiency of collection. ANIMALS: 10 adult purpose-bred research Beagles. PROCEDURES: A prospective crossover study was conducted. Preanesthetic physical and neurologic examinations and hematologic analyses were performed. Dogs were anesthetized, and collection of CSF samples from the cisterna magna by use of a stylet-in or stylet-out technique was performed. Two weeks later, samples were collected with the other sample collection technique. Samples of CSF were processed within 1 hour after collection. RESULTS: Cellular debris was detected in higher numbers in stylet-in samples, although this did not affect sample quality. The stylet-out technique was performed more rapidly. No adverse effects were detected for either technique. CONCLUSIONS AND CLINICAL RELEVANCE: Both techniques could be safely performed in healthy anesthetized dogs. The stylet-out technique was performed more rapidly and yielded a sample with less cellular debris. Both techniques can be used in clinical practice to yield CSF samples with good diagnostic quality.


Assuntos
Líquido Cefalorraquidiano , Cisterna Magna , Cães/líquido cefalorraquidiano , Manejo de Espécimes/veterinária , Punção Espinal/veterinária , Animais , Cisterna Magna/cirurgia , Estudos Cross-Over , Feminino , Masculino , Agulhas , Estudos Prospectivos , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Punção Espinal/instrumentação , Punção Espinal/métodos , Punção Espinal/normas
10.
J Chromatogr A ; 1603: 83-91, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31288928

RESUMO

In the acidic environment of the stomach, nitrosatable pesticide residues may react with nitrite to form potentially carcinogenic pesticide-associated N-nitroso compounds (PANOCs). The objective of this study was to develop a method for the analysis of 10 nitrosatable pesticides and breakdown products in human serum and urine. Three sample preparation methods were evaluated for extraction of target analytes from the biomatrices. Deproteinization by methanol for 300-µL aliquots of serum with a final extract volume of 225 µL resulted in excessive ion enhancement of some analytes and suppression of others. Three types of solid-phase extraction cartridges were tested for optimal analyte retention from 200-µL aliquots of serum with a final extract volume of 400 µL; this approach resulted in significant analyte loss for some compounds. The Quick, Easy, Cheap, Effective, Rugged, and Safe approach resulted in a suitable method for extraction of the analytes from each biomatrix. Biofluid samples (500 µL) were spiked to 100 µg L-1 with analytical standards and extracted using 500 µL of acetonitrile (ACN) with 4% acetic acid (AcOH) for serum and 0.1% AcOH in ACN for urine. For extraction, 200 mg magnesium sulfate (MgSO4) and 50 mg sodium acetate were added for serum and 200 mg MgSO4 and 50 mg sodium chloride were added for urine. Final extract volumes for both biomatrices using the QuEChERS method was 400 µL after dilution. Samples were analyzed via ultra-high pressure liquid chromatography/high-resolution accurate mass orbital ion trap mass spectrometry. Mean recoveries for target analytes in serum and urine ranged between 74 and 120% (%RSD < 12) and 96 to 116% (%RSD ≤ 10), respectively. These methods may be used in large-scale biomonitoring studies to analyze PANNs and their parent compounds in human serum and urine.


Assuntos
Cromatografia Líquida/métodos , Compostos Nitrosos/análise , Resíduos de Praguicidas/análise , Soro/metabolismo , Extração em Fase Sólida/métodos , Manejo de Espécimes/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Soro/química , Urinálise/métodos
11.
Malar J ; 18(1): 192, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185976

RESUMO

BACKGROUND: Mutational analysis of the Plasmodium falciparum kelch 13 (k13) gene is routinely performed to track the emergence and spread of artemisinin resistance. Surveillance of resistance markers has been impeded by the difficulty of extracting sufficient DNA from low parasite density infections common in low-transmission settings, such as Southeast Asia. This problem can be overcome by collecting large volumes of venous blood. Efficient methods for extracting and amplifying k13 from dried blood spots (DBS) would facilitate resistance surveillance. METHODS: Methods for k13 amplification from standard Whatman 3MM DBS (stored for 14 days at 28 °C with 80% relative humidity) were optimized by systematically testing different extraction conditions. Conditions that improved parasite DNA recovery as assessed by quantitative polymerase chain reaction (PCR) of 18S rDNA were then tested for their impact on k13 PCR amplification. RESULTS: The optimized protocol for amplification of k13 from DBS is markedly more sensitive than standard methods using commercial kits. Using this method, k13 was successfully amplified from laboratory-created DBS samples with parasite densities as low as 500 parasites/mL. Importantly, the method recovers both DNA and RNA, making it compatible with RNA-based ultrasensitive techniques currently in use. CONCLUSIONS: The optimized DBS protocol should facilitate drug resistance surveillance, especially in low-transmission settings where clinical malaria infections with high parasite densities are rare.


Assuntos
Artemisininas/farmacologia , Sangue/parasitologia , DNA de Protozoário/isolamento & purificação , Resistência a Medicamentos , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antimaláricos/farmacologia , Ásia Sudeste , DNA de Protozoário/química , DNA de Protozoário/genética , Dessecação/métodos , Humanos , Plasmodium falciparum/efeitos dos fármacos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Manejo de Espécimes/métodos
12.
Adv Exp Med Biol ; 1073: 23-56, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236838

RESUMO

The preanalytical phase of saliva proteomics/peptidomics, which includes sample collection, handling, and storage, represents a major challenge for any researcher that envisions sensitive and high-throughput analyses, coupled to well-controlled study design. The methodology used to collect saliva determines the contribution of each salivary gland to saliva composition with impact on data retrieved from proteomics/peptidomics. The awareness of the importance of this step in the analysis of saliva has prompted the proposal of several collection strategies. Moreover, numerous commercial devices are available in an attempt to routine the procedures. However, whatever the chosen method, procedures should be kept simple, standardized to get better reproducibility and repeatability on saliva proteomics analysis. Sample preservation is also a key step in saliva proteomics/peptidomics, and the implemented lab procedures should avoid posttranslational modifications such as proteolysis, as well as protein precipitation.In this chapter, we provide recommendations for saliva sampling and preservation, envisaging to standardize procedures that facilitate the use of saliva in clinical applications and the translation of proteomics data to diagnosis and/or definition of therapeutic targets.


Assuntos
Proteômica , Saliva/química , Manejo de Espécimes/métodos , Humanos , Processamento de Proteína Pós-Traducional , Reprodutibilidade dos Testes
13.
Adv Exp Med Biol ; 1073: 77-123, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236840

RESUMO

Since the birth of proteomics science in the 1990, the number of applications and of sample preparation methods has grown exponentially, making a huge contribution to the knowledge in life science disciplines. Continuous improvements in the sample treatment strategies unlock and reveal the fine details of disease mechanisms, drug potency, and toxicity as well as enable new disciplines to be investigated such as forensic science.This chapter will cover the most recent developments in sample preparation strategies for tissue proteomics in three areas, namely, cancer, toxicology, and forensics, thus also demonstrating breath of application within the domain of health and well-being, pharmaceuticals, and secure societies.In particular, in the area of cancer (human tumor biomarkers), the most efficient and multi-informative proteomic strategies will be covered in relation to the subsequent application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA), due to their ability to provide molecular localization of tumor biomarkers albeit with different spatial resolution.With respect to toxicology, methodologies applied in toxicoproteomics will be illustrated with examples from its use in two important areas: the study of drug-induced liver injury (DILI) and studies of effects of chemical and environmental insults on skin, i.e., the effects of irritants, sensitizers, and ionizing radiation. Within this chapter, mainly tissue proteomics sample preparation methods for LC-MS/MS analysis will be discussed as (i) the use of LC-MS/MS is majorly represented in the research efforts of the bioanalytical community in this area and (ii) LC-MS/MS still is the gold standard for quantification studies.Finally, the use of proteomics will also be discussed in forensic science with respect to the information that can be recovered from blood and fingerprint evidence which are commonly encountered at the scene of the crime. The application of proteomic strategies for the analysis of blood and fingerprints is novel and proteomic preparation methods will be reported in relation to the subsequent use of mass spectrometry without any hyphenation. While generally yielding more information, hyphenated methods are often more laborious and time-consuming; since forensic investigations need quick turnaround, without compromising validity of the information, the prospect to develop methods for the application of quick forensic mass spectrometry techniques such as MALDI-MS (in imaging or profiling mode) is of great interest.


Assuntos
Medicina Legal , Oncologia , Proteômica , Manejo de Espécimes/métodos , Toxicologia , Cromatografia Líquida , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
14.
Adv Exp Med Biol ; 1073: 125-135, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236841

RESUMO

Urine is a biological fluid that can be collected noninvasively in relatively large quantities which can be used for the search of biomarkers of disease, both diseases of the urological tract and systemic diseases. One of the most important aspects in proteomic studies is sample treatment before further analysis. Methods of preparation of a urine sample depend on the techniques that will be used later for separation and identification of the proteins. Also, urine preparation should be as simple as possible to increase reproducibility. Normal urine has a much diluted protein concentration with a high-salt content, which interferes with proteomic analysis. Thus, an initial step in the handling of urine sample should be to concentrate and eliminate salts. As range of protein concentrations in urine spans several orders of magnitude, effective proteomic analyses require either removal of most abundant protein or enrichment of the less abundant ones. In this chapter, we discuss the aspects related to the collection and treatment of urine for proteomic studies.


Assuntos
Proteômica , Manejo de Espécimes/métodos , Urinálise/métodos , Biomarcadores/urina , Humanos , Proteínas/análise , Reprodutibilidade dos Testes
15.
Adv Exp Med Biol ; 1073: 161-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236843

RESUMO

Because of strong impact of omics in many fields, and the complexity of the samples when focusing on areas such as genomics, (metallo)proteomics, metabolomics, among others, it is easy to rationalize the great importance that sample preparation has for achieving reliable results, mainly considering plant science. Then, this chapter points out applications of the sample preparation focusing on such areas, and a diversity of strategies, techniques, and procedures is highlighted and commented.


Assuntos
Genômica , Metabolômica , Plantas , Proteômica , Manejo de Espécimes/métodos
16.
Adv Exp Med Biol ; 1073: 187-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236844

RESUMO

Meta-omic techniques have progressed rapidly in the past decade and are frequently used in microbial ecology to study microorganisms in their natural ecosystems independent from culture restrictions. Metaproteomics, in combination with metagenomics, enables quantitative assessment of expressed proteins and pathways from individual members of the consortium. Together, metaproteomics and metagenomics can provide a detailed understanding of which organisms occupy specific metabolic niches, how they interact, and how they utilize nutrients, and these insights can be obtained directly from environmental samples. Here, we outline key aspects of sample preparation, database generation, and other methodological considerations that are required for successful quantitative metaproteomic analyses and we describe case studies on the integration with metagenomics for enhanced functional output.


Assuntos
Metagenômica , Consórcios Microbianos , Proteômica , Manejo de Espécimes/métodos , Proteínas
17.
Acta Cytol ; 63(5): 424-430, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31234167

RESUMO

BACKGROUND: Squash cytology is of significant importance in intraoperative consultation of central nervous system (CNS) pathology. There are several studies on squash cytology of CNS lesions, and only a few of them deal with spinal lesions alone. AIMS: (1) To evaluate intraoperative squash cytology of spinal lesions. (2) To correlate cytological diagnosis with histopathological diagnosis and assess the diagnostic accuracy. (3) To study Ki67 expression on squash smears and determine whether it can assist in grading spinal tumours on cytology. MATERIALS AND METHODS: A prospective study was conducted on 68 patients with clinico-radiologically diagnosed lesions of the spine. Intraoperative squash smears were stained with haematoxylin-eosin (H&E) stain, Papanicolaou (Pap) stain, and May-Grünwald-Giemsa (MGG) stain. Subsequently, histological diagnosis was made. Ki67 immunostaining was performed on squash smears and histology sections. RESULTS: The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of squash cytology in spinal lesions were 84.6, 100, 100, 23.1, and 80.88%, respectively. On immunocytochemistry, the mean Ki67 labelling indices for grade I, II, and III tumours were 0, 0.33 and 9%, respectively. CONCLUSION: Squash smear cytology is a rapid intraoperative technique for diagnosing spinal lesions, with high specificity and high positive predictive value. It is more effective in diagnosing neoplasms than non-neoplastic lesions. Ki67 immunostaining can be done on cytology smears to effectively differentiate between WHO grade I and grade II spinal tumours.


Assuntos
Citodiagnóstico/métodos , Imuno-Histoquímica , Cuidados Intraoperatórios/métodos , Antígeno Ki-67/análise , Manejo de Espécimes/métodos , Neoplasias da Coluna Vertebral/química , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Reprodutibilidade dos Testes , Neoplasias da Coluna Vertebral/diagnóstico por imagem , Neoplasias da Coluna Vertebral/patologia , Neoplasias da Coluna Vertebral/cirurgia , Adulto Jovem
18.
Forensic Sci Int ; 301: 331-340, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31202146

RESUMO

The structural identification and the monitoring of the relative concentrations of a wide range of major (3) and minor secondary (16) metabolites used as marker substances for profiling of cannabis resin using GC-FID at the Swedish National Forensic Centre (NFC) has facilitated the mapping of their chemical and physical behaviors over a period of 48months whilst stored under different conditions (exposure to light, exposure to air, temperature). In all cases the behavior of this group of sesquiterpenes, sesquiterpenoids, cannabinoids and waxes could be directly related to their chemical lability/functionality. In particular, the identification of homologue triads for both Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) together with a group of seemingly chemically inert substances (for example, cannabicyclol(CBL) and the waxes (n-alkanes)) has created new tools for the establishment of common origins between samples of cannabis resins aged under different conditions. Since sampling of the resin blocks in NFC's method for profiling of cannabis resin is made below the surface, the effects of light incursion were found to be negligible. The effects of exposure to air (and indirectly temperature) were found to be more significant, not unexpectedly as many of the observed transformations were based on oxidation or rearrangement processes.


Assuntos
Canabinoides/química , Cannabis/química , Resinas Vegetais/química , Manejo de Espécimes/métodos , Cromatografia Gasosa , Escuridão , Luz , Temperatura Ambiente
19.
J Mass Spectrom ; 54(8): 716-727, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31254303

RESUMO

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.


Assuntos
Proteínas/análise , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise Serial de Tecidos/métodos , Formaldeído/química , Humanos , Inclusão em Parafina , Proteólise , Fixação de Tecidos , Tripsina/química
20.
Anal Chim Acta ; 1076: 82-90, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203967

RESUMO

We present a method to preserve and process urine proteins for proteomic analysis in a filter aided sample preparation (FASP) format. The method combines concentration of urine proteins in ultrafiltration devices, their thermal stabilization, allowing long term storage of the samples, and filter aided sample preparation. Proteomes of four different urines were preserved during 48 h and 6 months using (i) the classic freeze preservation at -20 °C, (ii) snap-heated freeze-free preservation at laboratory temperature (20 °C) and (iii) snap-heated preservation at -60 °C. The three storage methods can effetely preserve the urine proteome for at least 6 months without significant alterations. Abundances of more than 500 proteins and specially 24 selected -cleared or -approved protein assayed in serum or plasma were found similar within the three preservation methods assessed. The new method here proposed dramatically simplifies the conditions for preserving the urine proteome for biobanks in terms of space and storage, including lowering the risks of sample degradation caused by misfunction of the freezer. Furthermore, the shipping of large number of samples can be made without the need of freezing. The application of the FASP format to isolate and preserve the proteins facilitates long-term storage and processing of proteome of urine samples.


Assuntos
Proteoma/química , Manejo de Espécimes/métodos , Urina/química , Lesão Renal Aguda/urina , Adulto , Biomarcadores/análise , Análise por Conglomerados , Calefação , Humanos , Biópsia Líquida , Masculino , Estudo de Prova de Conceito , Proteoma/análise , Proteoma/isolamento & purificação , Adulto Jovem
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