Este trabalho foi apoiado pelo Trabalho, Família e Rede de Saúde (www.WorkFamilyHealthNetwork.org), que é financiado por um acordo de cooperação com os Institutos Nacionais de Saúde e os Centros de Controle e Prevenção de Doenças: Eunice Kennedy Shriver National Institute of Child Health e Desenvolvimento Humano (Grant # U01HD051217, U01HD051218, U01HD051256, U01HD051276), do Instituto Nacional do Envelhecimento (Grant # U01AG027669), Secretaria de Ciências Comportamentais e Science Research, National Heart, Lung and Blood Institute (R01HL107240) e Instituto Nacional de Segurança Segurança e Saúde (Grant # U01OH008788, U01HD059773). Bolsas do William T. Grant Foundation, Alfred Sloan Foundation P, ea Administração para Crianças e Famílias têm fornecido financiamento adicional. O conteúdo desta publicação é de responsabilidade exclusiva de seus autores e não representam necessariamente a posição oficial dos institutos e escritórios.

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Orfeu Buxton recebeu uma subvenção iniciado pelo investigador de Sepracor Inc (agora Sunovion; ESRC-0977, ClinicalTrials.gov Identificador NCT00900159); atua como consultor e perito para Dinsmore LLC, no Conselho Consultivo da Matsutani Scientific America, e recebeu consultoria honorários da Universidade Florestal Centro Médico Wake.

Biomarcadores são ferramentas extremamente úteis em várias disciplinas, como medicina, biologia celular, genética, psicologia, sociologia, demografia e epidemiologia social. Dentro de cada disciplina, validade e confiabilidade fase inicial de pesquisa de biomarcadores foi usado para descobrir tanto o risco de doença oculta e fornecer previsão longitudinal dos resultados de saúde. Nestes casos, os biomarcadores que são precisas e durável (isto é, podem sobreviver ao desfasamento entre a recolha da amostra e a execução do ensaio de laboratório para extrair o biomarcador), e versátil (isto é, útil em uma variedade de modelos) são necessários. Enquanto coleção DBS é um procedimento relativamente simples, várias técnicas podem ajudar a maximizar o volume de pontos coletados. Antes da coleta começa, garantindo a mão de um participante é quente e, portanto, que os vasos sanguíneos se dilatam, vai ajudar o fluxo de sangue uma vez que o dedo está perfurado. Usando aquecedores de mão ou ter umparticipante executar suas mãos em água morna pode ajudar com este processo. Ordenhar o dedo antes da coleta de sangue vai puxar a ponta do dedo e aquecer a mão para ajudar o fluxo sanguíneo. A ordenha é um processo que melhora com a prática. Os coletores devem usar qualquer técnica que achar mais confortável e eficaz, mas a abordagem mais comum envolve a ordenha manual do participante com a mão em frente ao colecionador. Este método permitirá que o polegar do coletor de trabalhar em profundidade palma do participante, enquanto a mão oposta pode estabilizar o pulso do participante. Como exemplo, se um estavam coletando sangue de um dedo na mão esquerda do participante, o coletor seria estabilizar o pulso do participante com a sua mão esquerda e leite com a mão direita. Erros comuns cometidos durante a ordenha não incluem o uso de um aperto firme, puxando a pele acima da mão, em vez de trabalho para os músculos para puxar o sangue para a ponta dos dedos. É também importante para evitar pinoching o dedo com a mão ordenha ou segurando firmemente o pulso do participante com a mão nonmilking, os quais impedem o fluxo sanguíneo. Ao perfurar a dedo, fazer a punção o mais profundamente possível, pressionando firmemente a lanceta no dedo antes da alta. Uma punção superficial resulta em menor fluxo de sangue e de coagulação mais rápido. Após a perfuração do dedo, trabalhando rapidamente é fundamental. Fina sangue leva a mais fácil coleta e manchas maiores. Após a punção, o sangue torna-se mais e mais viscoso até coagulação. Quanto menos tempo necessário para coletar as manchas de sangue no cartão, maior o seu volume. O ideal é o sangue do participante será suficiente fina que cai vai cair de seu próprio peso, mas isso não será o caso para todos os participantes, como o sangue de algumas pessoas é mais grosso ou coágulos mais rápido do que outros. Se o sangue é muito grosso para cair do dedo, o cartão de filtro deve ser trazido para o sangue para a coleção. When trazer o cartão para o sangue é importante para manter o cartão em contacto com a gota, enquanto ele está pendurado a partir do dedo durante o tempo possível, em vez de apenas limpando o sangue para o cartão. Isto irá ajudar a puxar o sangue do dedo para maximizar o tamanho da mancha. Esta técnica requer mãos firmes para manter contato entre o sangue eo cartão. Com as duas mãos, uma para segurar a mão participantes e um para segurar o cartão, e mantendo as duas mãos em contacto uns com os outros ajuda a estabilizar o cartão eo sangue juntos. Áreas de pesquisa avançadas, tais como a avaliação de risco cardiometabólico, há muito tempo invocado biomarcadores, como a proteína C-reativa (PCR), insulina e hemoglobina glicosilada (HbA1C) para a detecção precoce dos riscos e os esforços de prevenção, bem como biomarcadores de &quot;endpoints rígidos&quot; para a doença cardiovascular (DCV) e mortalidade associada à doença cardiovascular 12. Epidemiologia clínica freqüentemente avalia biomarcadores para prospectively prever resultados de risco cardiometabólico, como doença cardíaca e diabetes tipo II. Talvez o mais utilizado de todos os biomarcadores é o escore de risco de Framingham, um composto que inclui a pressão arterial, os níveis de colesterol HDL e os níveis totais de cholesterols que demonstrou utilidade na previsão de DCV 13. No entanto, o escore de Framingham tem experimentado algumas controvérsias técnicas, e tem limitações em estudos voltados para ligar os fatores sociais e ambientais para a saúde, o que sugere a necessidade de biomarcadores mais precisos e específicos relacionados com a investigação cardiovascular. Há um número de kits disponíveis comercialmente utilizando técnicas de ELISA ou similares disponíveis para o ensaio de amostras de DBS. Necessária para o ensaio de amostras de DBS são o uso de etapas específicas e optimizadas de eluição para levar as amostras secas em solução 2. Esta é uma área em expansão de investigação como ensaios para mais analitos estão sendo desenvolvidos atualmente 1. Entre ªe ensaios já validados e sendo amplamente utilizados são o colesterol 2, CRP 10,14,15, IgE16, EBV 9,15, a leptina 17 e cyotkines 18. Há uma série de laboratórios especializados na análise de DBS, incluindo a Universidade de Washington Departamento de Medicina Laboratorial (UW Lab Med) Biomarcador Laboratory (Diretor: Mark Wener). Tendo em conta que um painel de biomarcadores, ao invés de um biomarcador solitário, é mais útil nas áreas de pesquisa cardiovascular e diabetes, os biomarcadores devem ser considerados, juntamente com uma variedade de fatores contextuais, a fim de maximizar a sua utilidade. Pesquisa de biomarcadores pode ser caro no entanto, tanto financeiramente como no tempo de coleta de dados. Assim, embora seja vital para escolher biomarcadores que são precisas, durável e versátil, o trabalho ea família investigação biomarcador deve estar radicado na praticidade, selecionando biomarcadores que são de baixo custo, apropriado e pode ser recolhida rapidamente wiª facilidade. Além disso, considerações de ordem prática, muitas vezes resultar em uma incapacidade de maximizar simultaneamente preço, qualidade e velocidade dentro de um procedimento. Aqui encontram-se os próximos passos na pesquisa de biomarcadores de saúde dentro de estudos de campo e da população. O campo de pesquisa de biomarcadores está atualmente em um período de forte crescimento, no entanto, a maior parte do trabalho ainda está por vir.

Biomarcadores podem fornecer informações valiosas sobre os processos biológicos que variam de normal a patogênica que pode contribuir para a doença clinicamente identificável 1. Em ambientes de pesquisa, manchas de sangue seco (DBS) estão se tornando cada vez mais preferido para punção venosa como um método para coleta de biomarcadores no sangue total. Recolha DBS é simples, relativamente fácil, menos invasiva, em seguida, a punção venosa, e requer o mínimo de requisitos de armazenamento (isto é, as amostras não precisam de ser imediatamente congelados e pode ser armazenado durante um longo período de tempo em um ambiente estável congelador antes do ensaio). Amostras DBS pode ser testado para uma variedade de diferentes analitos, incluindo o colesterol, a proteína C-reactiva, a hemoglobina glicosilada, anticorpos contra o vírus de Epstein-Barr, e diversas citoquinas. Punção venosa tem sido o padrão-ouro para a coleção de biomarcadores no sangue. No entanto, a obtenção, armazenamento e transporte de amostras de sangue requer aderênciasce a uma série de protocolos e códigos de saúde, que pode não ser possível em certos protocolos de pesquisa. Flebotomistas deve ser oficialmente certificadas para a punção venosa, enquanto procedimentos de amostragem de DBS são mais simples e não necessitam de certificação oficial. Coleta de sangue venoso também exige mais equipamentos do que a coleção DBS (ou seja, agulhas, tubos, torniquetes), e mais esforço de pós-colheita. Punção venosa devem ser processados ​​(por exemplo, soro ou plasma extraído a partir da amostra de sangue através de centrifugação ou de repousar à temperatura ambiente durante um determinado período de tempo) e congelados entre -20 ° C e -70 ° C imediatamente a fim de evitar a degradação dos analitos , enquanto que as amostras de DBS deve simplesmente ser deixada secar ao ar e, em seguida, pode ser armazenada à temperatura ambiente durante uma semana ou mais, dependendo de o analito, antes da transferência para um congelador 2. Amostras DBS são menos caros do que para coletar amostras de punção venosa, por razões descritas acima. Amostras DBS tambémocupam menos espaço, reduzindo os custos de transporte e armazenamento antes do ensaio. O uso de saliva como biomarcador tornou-se fundamental em estudos biobehavioral. Saliva oferece um método simples, não invasivo para coletar biomarcadores e tornou-se o padrão para o trabalho de campo que visa medir o cortisol ou cotinina 3-5. A urina é outra amostra que podem ser recolhidos de forma não invasiva e podem ser utilizados em estudos de nível de população para ensaios de melatonina ou de toxicologia. Embora ambos urina e saliva oferecer para a frente os procedimentos de coleta que são capazes de ser concluída pelos próprios participantes, nem todos os analitos pode ser medida em estes meios. DBS oferece uma simples e de baixo custo, alternativa para punção venosa e estão se tornando cada vez mais uma ferramenta de pesquisa implementado no campo e estudos populacionais 6.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Asepti II Microbial Wipes</td>
			<td style="width:71px;">EcoLab</td>
			<td style="width:119px;">D12330345</td>
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			<td height="84" style="height:84px;width:216px;">Bio-hazard waste bags</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">14220-092</td>
			<td style="width:167px;">High Strength Autoclavable Polypropylene Biohazard Bags, 2 mil</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bio-hazard waste barrel</td>
			<td style="width:71px;">Tyco</td>
			<td style="width:119px;">58578-092</td>
			<td style="width:167px;">8g. Slide lid</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sharps Container</td>
			<td style="width:71px;">BD Medical</td>
			<td style="width:119px;">BD305487</td>
			<td style="width:167px;">1.4 qt.</td>
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			<td height="42" style="height:42px;width:216px;">Chux</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td style="width:167px;">Absorbent bench underpads&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Gloves</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">XSmall: 89038-267</td>
			<td style="width:167px;">Powder-free, latex-free nitrile gloves</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;">Small: 89038-268</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;"></td>
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			<td style="width:119px;">Medium: 89038-270</td>
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			<td style="width:119px;">Large: 89038-272</td>
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			<td height="42" style="height:42px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;">XLarge: 89038-274</td>
			<td style="width:167px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hand sanitizer (e.g., Purell)</td>
			<td style="width:71px;">Johnson &#38; Johnson</td>
			<td style="width:119px;">89040-210</td>
			<td style="width:167px;">3rd party shipping</td>
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			<td height="63" style="height:63px;width:216px;">Alcohol wipes</td>
			<td style="width:71px;">WEBCOL and CURITY</td>
			<td style="width:119px;">82003-856</td>
			<td style="width:167px;">Prep pads</td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
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			<td height="42" style="height:42px;width:216px;">Gauze</td>
			<td style="width:71px;">VERSALON</td>
			<td style="width:119px;">82004-826</td>
			<td style="width:167px;">Non-woven sponges</td>
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		<tr height="21">
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			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="63" style="height:63px;width:216px;">Band-Aids</td>
			<td style="width:71px;">Nutramax Stat Strips</td>
			<td style="width:119px;">15431-475</td>
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			<td height="42" style="height:42px;width:216px;">Lancets</td>
			<td style="width:71px;">Owen Mumford</td>
			<td style="width:119px;">82028-060</td>
			<td style="width:167px;">3.0 mm, 21 ga.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protein paper collection cards</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">Custom-build</td>
			<td style="width:167px;">903 Protein Saver Collection paper</td>
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			<td height="21" style="height:21px;width:216px;">Desiccant</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">61161-319</td>
			<td style="width:167px;">Humidity sponges</td>
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			<td height="168" style="height:168px;width:216px;">Specimen bags</td>
			<td style="width:71px;">Lab Guard Reclosable Biohazard Bags, ITW Minigrip</td>
			<td style="width:119px;">56766-370</td>
			<td style="width:167px;">6&#8221; x 9&#8221;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Specimen storage cooler</td>
			<td style="width:71px;">Igloo</td>
			<td style="width:119px;">Playmate Mini</td>
			<td style="width:167px;">4 qt.; direct from manufacturer</td>
		</tr>
	</tbody>
</table>

dried blood spot collection of health biomarkers to maximize participation in population studies

Biomarcadores são indicadores biológicos diretamente-medidos de doença, saúde, exposições, ou outra informação biológica. Na população e as ciências sociais, os biomarcadores precisa ser de fácil obtenção, transporte e analisar. Manchas de sangue seco atender a essa necessidade, e podem ser coletadas no campo com altas taxas de resposta. Estes elementos são particularmente importantes em projetos de estudo longitudinal, incluindo intervenções onde o atrito é fundamental para evitar, e altas taxas de resposta melhorar a interpretação dos resultados. Coleção secas amostra mancha de sangue é simples, rápido e relativamente indolor, menos invasiva, em seguida, a punção venosa, e exige requisitos de armazenamento campo mínimos (ou seja, as amostras não precisam ser imediatamente congelado e pode ser armazenado por um longo período de tempo em um ambiente estável congelador antes ensaio). As amostras podem ser analisadas para uma variedade de diferentes analitos, incluindo o colesterol, a proteína C-reactiva, a hemoglobina glicosilada, numerosas citocinas, e outras analitos, bem como fornecer material genético. Recolha DBS é descrito como empregada em diversos estudos recentes.

<img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50973/50973fig1highres.jpg" src="/files/ftp_upload/50973/50973fig1.jpg" /> Figura 1. Secas amostra mancha de sangue. Uma placa de coleção é mostrada com quatro, válido pontos coletados no processo de secagem. <a href="https://www.jove.com/files/ftp_upload/50973/50973fig1highres.jpg" target="_blank">Clique aqui para ver imagem ampliada.</a> <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/50973/50973fig2highres.jpg" src="/files/ftp_upload/50973/50973fig2.jpg" /> Figura 2. Boa e má amostras de sangue seco. Resultados de sucesso (à direita) e sem sucesso (à esquerda) coleção DBS são mostrados. <a href="https://www.jove.com/files/ftp_upload/50973/50973fig2highres.jpg" target="_blank">Clique aqui para ver maior im</a>idade. <img alt="Figura 3" fo:content-width="5in" fo:src="/files/ftp_upload/50973/50973fig3highres.jpg" src="/files/ftp_upload/50973/50973fig3.jpg" /> Figura 3. Gestores atitudes e práticas associadas com o risco de doença cardiometabólico empregado 6. Este valor foi modificado a partir do Berkman, LF, Buxton, OM, Ertel, K. &amp; Okechukwu, práticas C. do Gestor relacionadas ao trabalho familiar equilíbrio prever cardiovascular empregado risco e duração do sono em ambientes de cuidados prolongados. Jornal de Psicologia da Saúde Ocupacional 115, 316-329 (2010). Trabalhadores expostos a gestores com dezenas trabalho-família meados ou baixos de abertura e criatividade para ajudar com o trabalho de seus funcionários e família precisam, têm chances aumentadas estatisticamente significativas de ter dois ou mais riscos cardiovasculares (fumante atual, obesidade, pressão alta ou campanha publicitáriartension, colesterol alto e alto de hemoglobina glicosilada (Hb A1C) ou diagnóstico de diabetes). Painéis superior e médio representam valores médios ± desvios-padrão para o colesterol total avaliado pela mancha de sangue seco e Hb A1C avaliada por dispositivo de ponto de atendimento (que também pode ser mensurado de forma confiável por manchas de sangue seco). Painel inferior mostra o risco de doença cardiovascular pelo escore gerente. Análise restrita aos enfermeiros e outros funcionários que prestam atendimento direto indicam que a exposição para os gerentes com baixa criatividade e abertura questões relacionadas ao trabalho-família pode ser particularmente importante para estes indivíduos (OR para baixa pontuação WF = 6,4, p = 0,01). Todos os modelos de controle de idade, sexo, salário, educação, raça / etnia e local de trabalho. <a href="https://www.jove.com/files/ftp_upload/50973/50973fig3highres.jpg" target="_blank">Clique aqui para ver imagem ampliada.</a>

Watch this Scientific Journal Video about Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies at JoVE.com

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarcadores são indicadores biológicos diretamente-medidos de doença ou de saúde. Na população e as ciências sociais, os biomarcadores precisa ser de fácil obtenção, transporte e analisar. Secas mancha de sangue (DBS) coleção atende a essa necessidade, podem ser coletadas no campo com altas taxas de resposta e analisados ​​para uma variedade de biomarcadores.

Secas Coleta de Sangue Mancha de Biomarcadores de saúde para maximizar a participação em Estudos Populacionais

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

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Research

JoVE Journal

Medicine

36.1K visualizações.  Harvard School of Public Health.  Biomarcadores são indicadores biológicos diretamente-medidos de doença ou de saúde. Na população e as ciências sociais, os biomarcadores precisa ser de fácil obtenção, transporte e analisar. Secas mancha de sangue (DBS) coleção atende a essa necessidade, podem ser coletadas no campo com altas taxas de resposta e analisados ​​para uma variedade de biomarcadores. 

Vídeo: Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicina

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

High Throughput ELISA sequencial para a validação dos biomarcadores da doença enxerto-versus-hospedeiro aguda

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

Um método de monitoramento acessível HIV-1 Resistência às Drogas para recursos configurações limitadas

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

Quantificação do imunossupressor tacrolimus em manchas de sangue seco utilizando LC-MS / MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In Vivo e Ex Vivo Abordagens para o Estudo do cancro do ovário metastático Colonização de Estruturas Láctea natural em Peritoneal adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

A Method of Trigonometric Modelling of Seasonal Variation Demonstrated with Multiple Sclerosis Relapse Data

This report describes a novel Stata-based application of trigonometric regression modelling to 55 years of multiple sclerosis relapse data from 46 clinical centers across 20 countries located in both hemispheres. Central to the success of this method was the strategic use of plot analysis to guide and corroborate the statistical regression modelling. Initial plot analysis was necessary for establishing realistic hypotheses regarding the presence and structural form of seasonal and latitudinal influences on relapse probability and then testing the performance of the resultant models. Trigonometric regression was then necessary to quantify these relationships, adjust for important confounders and provide a measure of certainty as to how plausible these associations were. Synchronization of graphing techniques with regression modelling permitted a systematic refinement of models until best-fit convergence was achieved, enabling novel inferences to be made regarding the independent influence of both season and latitude in predicting relapse onset timing in MS. These methods have the potential for application across other complex disease and epidemiological phenomena suspected or known to vary systematically with season and/or geographic location.

Combining plot analysis with trigonometric regression is a robust method for exploring complex, cyclical phenomena such as relapse onset timing in multiple sclerosis (MS). This method enabled unbiased characterisation of seasonal trends in relapse onset permitting novel inferences around the influence of seasonal variation, ultraviolet radiation (UVR) and latitude.

Um Método de Trigonometria Modelagem da Variação Sazonal demonstrado com esclerose múltipla Relapse Dados

This report describes a novel Stata-based application of trigonometric regression modelling to  55 years of multiple sclerosis relapse data from 46 ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

A ressecção apical mouse modelo para estudar precoce Regeneração Mammalian Coração

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...

Assessment of Child Anthropometry in a Large Epidemiologic Study

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to understand health effects of child growth and adiposity. Gold standard methods of measuring adiposity, such as dual X-ray absorptiometry (DXA), may not be feasible in large field studies. Research staff can, however, complete anthropometric measurements, such as body circumferences and skinfold measurements, using inexpensive portable equipment. In this protocol we detail how to obtain manual anthropometric measurements from children, including standing and sitting height, weight, waist circumference, hip circumference, mid-upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness, and procedures to assess the quality of these measurements. To demonstrate accuracy of these measurements, among 1,110 school-aged children in the pre-birth cohort Project Viva we calculated Spearman correlation coefficients comparing manual anthropometric measurements with a gold standard measure of body fat, DXA fat mass1. To address reliability, we evaluate intra-rater technical error of measurement at a quality control session conducted on adult female volunteers.

In epidemiologic studies of children, well-trained research staff can accurately and precisely assess weight, height, sitting height, skinfold thicknesses, and body circumferences.

Avaliação da Criança Antropometria Em um grande estudo epidemiológico

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to ...

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

Repetiu a coleta de sangue da veia da cauda de ratos não anestesiados com um sistema de coleta de sangue do vácuo

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

Punção da veia subclávia como um método alternativo de coleta de amostra de sangue em ratos

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...