Os autores reconhecem o apoio financeiro de uma National Science Foundation (NSF) conceder CEE-0540832 intitulado &quot;Tecnologias Mid-Infrared da Saúde e do Meio Ambiente (Mirthé)&quot;

1. Preparação de Instrumentos <ol><li> Ligue a fonte de alimentação externa para a amônia plataforma sensor óptico, controlador de laser de diodo, um costume construído unidade eletrônica de controle (CEU), o amostrador ar, bomba de ar, e laptop. </li><li> Verifique se o escape e ventiladores do sensor de amônia estão operando. NOTA: Um está localizado na parte de trás do sensor, o segundo é encontrado dentro do sensor, que é facilmente acessível. </li><li> Assegurar o módulo de detecção acústica e a temperatura da válvula de agulha são a 38,0 ° C, verificando o visor digital localizada no lado da caixa de sensor de amónia. Aguarde cerca de 35 minutos a partir do momento do sensor é fornecido energia para que a temperatura se estabilize. </li><li> Definir tubulação de entrada e temperatura bocal a 55 ° C, clicando no nome de um ícone &quot;ChangeT&quot; encontrado na área de trabalho do amostrador respiração. A temperatura pode ser alterado clicando na seta para cima ou para baixo seguida, clicando em &quot;UPDcomeu Temp &quot;. Clique em &quot;Exit&quot; para retornar à área de trabalho. Permitir que o sistema de, pelo menos, 5 minutos para a estabilização da temperatura. Mantendo a temperatura do dispositivo de amostragem e tubo de 55 ° C minimiza a perda da superfície de amoníaco. </li><li> Abra o software no laptop que controla o sensor de amônia. O programa pode ser acessado dentro de uma pasta chamada &quot;NH3 Programa Sensor respiração&quot; no ambiente de trabalho. Dentro dessa pasta, o usuário deve selecionar o ícone &quot;software LabVIEW principal&quot;. Esta pasta contém várias aplicações, mas o usuário deve selecionar &quot;Mainsequence.vi&quot; para acessar a interface desejada. Selecione &quot;run&quot; no canto superior esquerdo da tela. NOTA: Isto começa a linha de bloqueio sequência de calibração. O laser de monitor QEPAS opera sobre uma corrente ideal, o qual é seleccionado, durante um procedimento de bloqueio de linha automatizada. Este processo demora cerca de 25 min. </li><li> Criar uma nova sessão no amostrador de tal forma que eAsessão assunto ch tem seu próprio arquivo que está salvo em um flash drive conectado. NOTA: Isto é realizado através da abertura do programa &quot;Respiração Sampler&quot; encontrado na área de trabalho do amostrador. Há espaço para identificar a sessão de forma adequada. Todos os dados gerados durante a sessão serão salvos na unidade flash sob este identificador. A data do experimento é normalmente usado como parte do nome do arquivo. A entrada e temperatura bocal deve ser ajustado antes de entrar no programa respiração amostragem. </li><li> Insira um novo bocal descartável para o tubo de entrada. Usar luvas descartáveis ​​para evitar a contaminação bocal com amônia a partir de dedos. </li></ol> 2. Coleta de Amostras Respiração NOTA: O Conselho de Revisão Institucional relevante (Conselho de Ética) deve aprovar qualquer estudo que envolva seres humanos. Há muitos fatores que podem influenciar drasticamente respiração amônia. Esses fatores podem alterar as medições de amônia respiração por diretamente umffecting níveis de amônia sistêmicas ou afectando a jornada de metabólitos de respiração dos pulmões para a instrumentação. <ol><li> Certifique-se de assuntos chegar ao laboratório em jejum, depois de ter consumido nenhum alimento por cerca de 12 horas após a chegada e que eles abstiveram-se de exercício de manhã antes do teste. </li><li> Certifique-se de nenhuma substância foi introduzida na boca durante, pelo menos, 1 hora antes da recolha de dados. Certifique-se de que os sujeitos escovar os dentes maior do que 1 hora antes do teste. </li><li> Assente o assunto na frente do sensor de amônia. Instrua o assunto para segurar o tubo de entrada e certifique-se que não toque no bocal para evitar a contaminação de amônia. </li><li> Clique em &quot;Iniciar&quot; na interface amostrador. Já o tema expirar no bocal durante o tempo que eles podem, ou até que o operador considera que a amostra seja suficiente. NOTA: Esta é uma única expiração completa, com duração mínima de 10 segundos. Boca pressão é medida em tempo real como um substitutopara a taxa de fluxo. Um medidor de pressão com código de cores ajuda a produzir assunto e manter a pressão de expiração desejado de 10 cm de água, o que representa uma taxa de fluxo de expiração de 50 ml / seg. Este fluxo foi selecionado, uma vez que tem sido adotado pela ATS / ERS para o protocolo para determinar Fe NO. Esta taxa de fluxo de expiração é realizável por crianças e adultos. Similarmente três exalações reprodutíveis devem ser obtido que diferem menos do que 10%. </li><li> Clique em &quot;Stop&quot; na interface do amostrador quando a amostra de ar é concluído. </li></ol> 3. Medição amostra de ar <ol><li> Uma vez que uma amostra de respiração foi analisado, o técnico de laboratório operando o monitor, tem a capacidade de analisar qualquer segmento de perfil que respiração. A parte da respiração que é de interesse é o segmento de fase III. Esta caracteriza-se por um &quot;patamar&quot; na concentração de dióxido de carbono e é encontrada no meio de fase tardia da breath. </li><li> Selecione a parte da fase III da amostra, arrastando as linhas verticais na interface amostrador começar quando planaltos de dióxido de carbono e parar logo antes da queda de pressão do ar. Veja a Figura 1 para o esclarecimento. </li><li> Salve os dados para o flash drive, clicando em &quot;Loja&quot; na interface de tela de toque amostrador. </li><li> Uma vez que os dados de respiração foram armazenados, o usuário pode selecionar &quot;Iniciar&quot; para iniciar uma nova amostra de ar. </li></ol> 4. Ilustrando os efeitos da Boca Rinse e pH na respiração de amônia <ol><li> Amostra 3 respirações para estabelecer o nível de amônia linha de base. Certifique-se de que a respiração são tomadas, pelo menos, 5 min afastados uns dos outros. </li><li> Lavar bem a boca com uma alíquota de 30 ml de água por 60 segundos. </li><li> Coletar uma amostra de ar dentro de 60 segundos da lavagem (Seção 2). Recolher amostras de respiração ao longo da hora seguinte, para observar a mudança de amoníaco ao longo do tempo. NOTA: Samples pode ser tomado tão freqüentemente quanto cada minuto, mas intervalos mais longos são normalmente utilizados. </li><li> Enxaguar a boca meticulosamente com uma alíquota de 30 ml de solução básica (bicarbonato de sódio em água) durante 60 segundos e repita 4.2.1. </li><li> Enxaguar a boca meticulosamente com uma alíquota de 30 ml de solução ácida por 60 segundos e repita 4.2.1. </li></ol> 5. Ilustrando os efeitos da entrada e Transporte tubulação Temperaturas em Respiração Amônia <ol><li> Exemplos de três ciclos ao longo de 15 min com a temperatura de entrada ajustada para abaixo da temperatura do corpo, cerca de 30 ° C. </li><li> Aumente as temperaturas de entrada e tubulação de transporte de 55 ° C, respectivamente, usando um ícone no desktop na interface da tela de toque do amostrador respiração. Permitir que o sistema de, pelo menos, 5 minutos para atingir o estado estacionário. </li><li> Amostra 3 respirações, 5 min à parte na entrada aquecida. </li></ol>

<ol>
	<li>Adeva, M. M., Souto, G., Blanco, N., Donapetry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium+metabolism+in+humans.">Ammonium metabolism in humans.</a> Metabolism: clinical and experimental. 61 (11), 1495-1511 (2012).</li><li>Auron, A., Brophy, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperammonemia+in+review:+pathophysiology,+diagnosis,+and+treatment.">Hyperammonemia in review: pathophysiology, diagnosis, and treatment.</a> Pediatric nephrology. 27 (2), 207-222 (2012).</li><li>Blanco Vela, C. I., Bosques Padilla, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+ammonia+concentrations+in+cirrhosis+patients-still+confusing+after+all+these+years.">Determination of ammonia concentrations in cirrhosis patients-still confusing after all these years.</a> Annals of hepatology. 10 Suppl 2, (2011).</li><li>Mpabanzi, L., Ol de Damink, S. W. M., van de Poll, M. C. G., Soeters, P. B., Jalan, R., Dejong, C. H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+pee+or+not+to+pee:+ammonia+hypothesis+of+hepatic+encephalopathy+revisited.">To pee or not to pee: ammonia hypothesis of hepatic encephalopathy revisited.</a> European journal of gastroenterology &amp; hepatology. 23 (6), 449-454 (2011).</li><li>Goggs, R., Serrano, S., Szladovits, B., Keir, I., Ong, R., Hughes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+investigation+of+a+point-of-care+blood+ammonia+analyzer.">Clinical investigation of a point-of-care blood ammonia analyzer.</a> Veterinary clinical pathology / American Society for Veterinary Clinical Pathology. 37 (2), 198-206 (2008).</li><li>Huizenga, J. R., Tangerman, A., Gips, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+ammonia+in+biological+fluids.">Determination of ammonia in biological fluids.</a> Annals of clinical biochemistry. 31 (Pt 6), 529-543 (1994).</li><li>Lewicki, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+ammonia+detection+in+exhaled+human+breath+with+a+quantum+cascade+laser+based+sensor.">Real time ammonia detection in exhaled human breath with a quantum cascade laser based sensor.</a> 2009 Conference on Lasers and ElectroOptics and 2009 Conference on Quantum electronics and Laser Science Conference. 1, (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Thoracic+Society.+European+Respiratory+Society.+Recommendations+for+Standardized+Procedures+for+the+Online+and+Offline+Measurement+of+Exhaled+Lower+Respiratory+Nitric+Oxide+and+Nasal+Nitric+Oxide.">American Thoracic Society. European Respiratory Society. Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide.</a> American journal of respiratory and critical care medicine. 171 (8), 912-930 (2005).</li><li>Solga, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+breath+ammonia+determination.">Factors influencing breath ammonia determination.</a> Journal of breath research. 7 (3), (2013).</li><li>Schmidt, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonia+in+breath+and+emitted+from+skin.">Ammonia in breath and emitted from skin.</a> Journal of breath research. 7 (1), (2013).</li><li>Span&#283;l, P., Dryahina, K., Smith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+study+of+the+influence+of+inhaled+compounds+on+their+concentrations+in+exhaled+breath.">A quantitative study of the influence of inhaled compounds on their concentrations in exhaled breath.</a> Journal of breath research. 7 (1), (2013).</li><li>Boots, A. W., van Berkel, J. J. B. N., Dallinga, J. W., Smolinska, A., Wouters, E. F., van Schooten, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatile+use+of+exhaled+volatile+organic+compounds+in+human+health+and+disease.">The versatile use of exhaled volatile organic compounds in human health and disease.</a> Journal of breath research. 6 (2), (2012).</li><li>Hibbard, T., Killard, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breath+ammonia+levels+in+a+normal+human+population+study+as+determined+by+photoacoustic+laser+spectroscopy.">Breath ammonia levels in a normal human population study as determined by photoacoustic laser spectroscopy.</a> Journal of breath research. 5 (3), (2011).</li><li>Wang, T., Pysanenko, A., Dryahina, K., Span&#283;l, P., Smith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+breath,+exhaled+via+the+mouth+and+nose,+and+the+air+in+the+oral+cavity.">Analysis of breath, exhaled via the mouth and nose, and the air in the oral cavity.</a> Journal of breath research. 2 (3), (2008).</li><li>Amann, A., Smith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Volatile Biomarkers. 1st Edition. , (2013).</li></ol>

Dr. Risby serves as a consultant for Loccioni Humancare the manufacturer of the breath sampler.

Os benefícios de um procedimento não-invasivo capaz de detectar metabólitos traço em tempo real são óbvias. No entanto, o campo da pesquisa respiração tem se esforçado para cumprir esse potencial. Medição da respiração é um processo dinâmico vulneráveis ​​a muitos fatores de confusão. Nossa abordagem tem pontos fortes importantes: especificamente, a sensibilidade ea velocidade do Arroz QEPAS monitor de amônia baseado acoplado ao amostrador fôlego nos permitiram avaliar e identificar os fatores de ...

A amônia é um subproduto onipresente do metabolismo de proteínas 1. Medição de amônia, portanto, pode ajudar os médicos a avaliar várias doenças e estados de bem-estar 2. No entanto, a amônia é difícil medir com precisão, através do sangue ou de respiração, porque é muito reativo. Embora comumente usado, ensaios de sangue têm inúmeras desvantagens, incluindo preocupações básicas sobre precisão 3. Mas o grande problema com os ensaios de sangue é a realidade que eles só já recolhidas episodicamente. Isto é importante porque a fisiologia de amoníaco, bem como a glicose no sangue e muitos outros processos metabólicos, são fluidos e sempre em mudança 4. Em contraste, os ensaios de respiração são totalmente não-invasiva e rápida, permitindo assim facilmente medidas repetidas. Assim, a medição de amônia respiração é atraente porque pode dirigir uma necessidade não atendida sério de uma forma única. Coleta de respiração, no entanto, apresenta preocupações específicas. Considerando flebotomia inerentemente leva o jeopardy de erro de várias maneiras imprevisíveis (por exemplo, tempo de torniquete, a contaminação suor, hemólise de glóbulos, atraso na medição de laboratório, etc 5), os pesquisadores de medição respiração devem lidar com um grupo diferente de novos desafios: variabilidade em respirar, a contaminação com mucosa bucal ou amoníaco bacteriana, a influência do ar e o aparelho de humidade e temperatura ambiente, etc 6. Na verdade, não é prudente subestimar a tarefa em conectar equipamentos experimentais para os seres humanos através de procedimentos experimentais para descobrir biologia desconhecida. Em parte devido a esses obstáculos, a respiração de amônia ainda não atingiu o seu potencial. Aqui, apresentamos o nosso protocolo de medição de amônia respiração por resultados rápidos e precisos. Nosso protocolo tem força em três áreas: o monitor, o amostrador interface, e atenção para as influências humanas. O monitor foi construído por colegas da Universidade Rice, como descrito anteriormente 7. A base do measurement é um quartzo reforçada espectroscopia fotoacústica (QEPAS), técnica que emprega um piezoelétrico afinação quartzo garfo como um transdutor acústico. Efeito fotoacústico ocorre quando as ondas acústicas são produzidos pela absorção da radiação de laser modulado pelo tipo de gás de rastreio alvo. O gás traço é detectada através de uma célula acústica que é acusticamente ressonante com a freqüência modulada. Comprimento de onda de absorção de amônia foi selecionado que é livre de interferências espectrais de espécies interferentes na respiração. Para efeitos de medição de ar exalado humano, as principais funções do monitor incluem uma ampla faixa de medição (de ~ 50 partes por bilhão, ppb, pelo menos, 5.000 ppb) e velocidade (1 medições seg). A velocidade do monitor permite a resolução de tempo ao longo do ciclo de respiração. O monitor está ligado a um amostrador respiração especialmente concebidos. O amostrador consiste de um sensor de pressão e capnógrafo. Ele exibe e arquivos em tempo realmedições de pressão na boca e dióxido de carbono, bem como as concentrações de amoníaco determinados pelo sensor. Este amostrador, por conseguinte, permite que o técnico para avaliar a qualidade de o esforço de respiração quando a respiração é recolhido. Isso nos permite ultrapassar as recomendações para analisar respiração óxido nítrico (NO Fe), proposto pela Força-Tarefa da American Thoracic Society / European Respiratory Society (ATS / ERS) 8. Para todos amostragem respiração, um one-way válvula descartável in-line foi usado na porta boca do amostrador respiração. Por causa da velocidade do monitor e os controles de qualidade fornecido pelo sampler, fomos capazes de avaliar cuidadosamente as influências humanas 9. A maioria dos indivíduos, por exemplo, inicialmente hiperventilar quando instruído a respirar. Outras influências importantes, como o pH oral e enxaguatórios bucais, as temperaturas do sampler, do monitor e toda a tubulação associada, e modo de respiração, foram então estudadas, e são a base for os experimentos ilustrativos abaixo. Finalmente, e talvez mais importante, deve-se enfatizar que vários grupos altamente experientes estão medindo respiração amônia usando completamente diferentes sensores e procedimentos de medição. Estes podem ter vantagens importantes e validade. A comparação completa está além do escopo do presente trabalho 10,11,12.

<table border="0" cellpadding="0" cellspacing="0" width="666">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Rice Ammonia Monitor System</td>
			<td style="width:171px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Not available for commercial purchase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Loccioni Breath Sampler</td>
			<td style="width:171px;">Loccioni Humancare</td>
			<td style="width:119px;">N/A</td>
			<td>Single breath version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Disposable Mouth Piece</td>
			<td style="width:171px;">WestPrime Healthcare</td>
			<td style="width:119px;">G011-200</td>
			<td>Manufacturer is AlcoQuant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Laptop</td>
			<td style="width:171px;">Lenovo</td>
			<td style="width:119px;">N/A</td>
			<td>Old model no longer sold by manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Acid Rinse</td>
			<td style="width:171px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Household acidic drink (coffee, soft drink, citrus juices, etc)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Base Rinse</td>
			<td style="width:171px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Water mixed with a nonexact amount of sodium bicarbonate (Arm &#38; Hammer Baking Soda)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Neutral Rinse</td>
			<td style="width:171px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>Water</td>
		</tr>
	</tbody>
</table>

fast and accurate exhaled breath ammonia measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Assuntos pode ser esperado para produzir uma ampla gama de níveis de amónia respiração basais. Indivíduos saudáveis ​​podem começar o dia com uma medição de amônia de 100-1.000 ppb respiração. Lavar a boca com qualquer fluido imediatamente altera a quantidade de amônia detectável respiração. Fluidos neutros e ácidos normalmente reduzir a quantidade de amônia observáveis ​​por mais da metade. Estes níveis então retornar à linha de base, como os efeitos da lavagem desaparece. Os efeitos da á...

Watch this Scientific Journal Video about Fast and Accurate Exhaled Breath Ammonia Measurement at JoVE.com

Fast and Accurate Exhaled Breath Ammonia Measurement

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

Medição Amônia ar exalado Rápido e Preciso

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

fast-and-accurate-exhaled-breath-ammonia-measurement

Research

JoVE Journal

Medicine

13.1K visualizações.  St. Luke's University Hospital. Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

Vídeo: Fast and Accurate Exhaled Breath Ammonia Measurement

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Medição precisa e simples do pró-inflamatórias IL-1β citocinas usando um ensaio Estimulação Sangue Total

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Medicina

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

Avaliação precisas e simples de anastomoses vasculares em Placenta monocoriônicas usando Dye Colorido

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

Navegação Magnetic remoto para Exato, o posicionamento do cateter em tempo real e Ablação em Eletrofisiologia Cardíaca Procedimentos

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal1,2. This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [13C]-octanoic acid breath test for humans3 has been modified for use in mice4-6 and rats7 and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression8. In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes9. A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach10.
This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.

Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test

Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.

Estudo do esvaziamento gástrico em ratos diabéticos não obesos utilizando a [<sup&gt; 13</sup&gt; C]-octanóico teste de respiração Ácido

Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Lesão Explorer: Um guiada por vídeo, protocolo padronizado para Volumetrics MRI derivados precisos e confiáveis ​​na doença de Alzheimer e idosos normais

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.

The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

Voluntário Técnica de apneia para a Redução Coração Dose em Radioterapia mama esquerda

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Criando anatomicamente precisos e reprodutíveis intracraniana Xenoenxertos de tumores cerebrais humanos

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

Imagens de ultra-som 3D: rápida e econômica de morfometria do tecido músculo-esquelético

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Fast Inspection of Quality of Indigo Naturalis by Multiple Light Scattering

Quality control of Chinese herbal medicine is a crucial component of Chinese herbal medicine research and development. Faced with the challenges of modernization and internationalization of Chinese herbal medicine, it is urgent to establish thorough and effective procedures for quality identification of Chinese herbal medicine, and there is an urgent need for new analytical and testing techniques that are efficient, accurate, and environmentally friendly. 
Multiple light scattering is a cutting-edge and analytical method that can accurately and rapidly assess the quality of Chinese herbal medicine without altering the nature or state of the sample or using organic reagents. Indigo Naturalis is considered a good remedy for pediatric hyperthermia, psoriasis, leukemia, and ulcerative colitis. In this study, the process of addition of Indigo Naturalis powder in water was recorded precisely using a multiple light scattering instrument. 
The qualitative and quantitative measurements of the instrument can be used to accurately capture the overall trajectory and sinking behavior of Indigo Naturalis powder into water and to establish a rapid evaluation method for the quality of Indigo Naturalis with the transmission and backscattering spectrograms of the sample as qualitative indicators and stability index as a quantitative indicator. The analytical technique based on multiple light scattering provides a fast, accurate, green, and environmentally friendly method for the quality evaluation of Indigo Naturalis and supports the development and transformation of high-quality Indigo Naturalis.

Here, we present a quality evaluation method based on a multiple light scattering technique to evaluate the quality of Indigo Naturalis.

Inspeção rápida da qualidade do índigo naturalis por espalhamento múltiplo de luz

Quality control of Chinese herbal medicine is a crucial component of Chinese herbal medicine research and development. Faced with the challenges of ...

Focused Assessment with Sonography for Trauma (FAST) Exam: Image Acquisition

Over the past twenty years, the Focused Assessment with Sonography for Trauma (FAST) exam has transformed the care of patients presenting with a combination of trauma (blunt or penetrating) and hypotension. In these hemodynamically unstable trauma patients, the FAST exam permits rapid and noninvasive screening for free pericardial or peritoneal fluid, the latter of which implicates intra-abdominal injury as a likely contributor to the hypotension and justifies emergent abdominal surgical exploration. Further, the abdominal portion of the FAST exam can also be used outside of the trauma setting to screen for free peritoneal fluid in patients who become hemodynamically unstable in any context, including after procedures that may inadvertently injure abdominal organs. These "non-trauma" situations of hemodynamic instability are often triaged by providers from specialties other than emergency medicine or trauma surgery who are not familiar with the FAST exam. Therefore, there is a need to promulgate knowledge about the FAST exam to all clinicians caring for critically ill patients. Toward this end, this article describes FAST exam image acquisition: patient positioning, transducer selection, image optimization, and exam limitations. Since the free fluid is likely to be found in specific anatomic locations that are unique for each canonical FAST exam view, this work centers on the unique image acquisition considerations for each window: subcostal, right upper quadrant, left upper quadrant, and pelvis.

Focused Assessment with Sonography for Trauma FAST Exam: Image Acquisition

The Focused Assessment with Sonography for Trauma (FAST) exam is a diagnostic point-of-care ultrasound examination used to screen for the presence of free fluid in the pericardium and peritoneum. Indications, techniques, and pitfalls of the procedure are discussed in this article.