Name: dried blood and serum spots as a useful tool for sample storage to evaluate cancer biomarkers
Uploaded: 2018-06-11T13:00:00
Description: Watch this Scientific Journal Video about Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers at JoVE.com

Gostaríamos de agradecer Gráinne Tierney assistência editorial.

Soro e sangue periférico foram coletadas e armazenadas de doadores saudáveis e pacientes que deram consentimento escrito para participar no estudo. O protocolo de estudo foi aprovado pelo Comitê de ética local em conformidade com os padrões éticos estabelecidos na declaração de Helsinque 1964.
1. sangue armazenamento em DBS
<ol><li>
Coleta de amostra de sangue
	<ol>
<li>Colete 3 mL de amostra de sangue periférico em um tubo de 3 mL com 5,4 mg de ácido etilenodiamin...

<ol>
	<li>Bertagnolio, S., 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=HIV-1+drug+resistance+surveillance+using+dried+whole+blood+spots.">HIV-1 drug resistance surveillance using dried whole blood spots.</a> Antivir Ther. 12 (1), 107-113 (2007).</li><li>Mei, J. V., Alexander, J. R., Adam, B. W., Hannon, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+filter+paper+for+the+collection+and+analysis+of+human+whole+blood+specimens.">Use of filter paper for the collection and analysis of human whole blood specimens.</a> J Nutr. 131 (5), 1631S-1636S (2001).</li><li>Garrido, C., 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=Subtype+variability,+virological+response+and+drug+resistance+assessed+on+dried+blood+spots+collected+from+HIV+patients+on+antiretroviral+therapy+in+Angola.">Subtype variability, virological response and drug resistance assessed on dried blood spots collected from HIV patients on antiretroviral therapy in Angola.</a> J Antimicrob Chemoth. 61 (3), 694-698 (2008).</li><li>Steegen, K., 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=Feasibility+of+detecting+human+immunodeficiency+virus+type+1+drug+resistance+in+DNA+extracted+from+whole+blood+or+dried+blood+spots.">Feasibility of detecting human immunodeficiency virus type 1 drug resistance in DNA extracted from whole blood or dried blood spots.</a> J Clin Microbiol. 45 (10), 3342-3351 (2007).</li><li>Costenaro, P., 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=Viral+load+detection+using+dried+blood+spots+in+a+cohort+of+HIV-1-infected+children+in+Uganda:+correlations+with+clinical+and+immunological+criteria+for+treatment+failure.">Viral load detection using dried blood spots in a cohort of HIV-1-infected children in Uganda: correlations with clinical and immunological criteria for treatment failure.</a> J Clin Microbiol. 52 (7), 2665-2667 (2014).</li><li>Gong, Z. H., Tian, G. L., Huang, Q. W., Wang, Y. M., Xu, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+glutathione+and+glutathione+disulfide+in+the+blood+of+glucose-6-phosphate+dehydrogenase-deficient+newborns.">Reduced glutathione and glutathione disulfide in the blood of glucose-6-phosphate dehydrogenase-deficient newborns.</a> BMC Pediatr. 20 (17), 172 (2017).</li><li>Lukacs, Z., Barr, M., Hamilton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practice+in+the+measurement+and+interpretation+of+lysosomal+acid+lipase+in+dried+blood+spots+using+the+inhibitor+Lalistat+2.">Best practice in the measurement and interpretation of lysosomal acid lipase in dried blood spots using the inhibitor Lalistat 2.</a> Clin Chim Acta. 471, 201-205 (2017).</li><li>Skogstrand, P. T., Bent, N. P., Schendel, D. E., S&#248;rensen, L. C., Hougaard, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+measurement+of+25+inflammatory+markers+and+neurotrophins+in+neonatal+dried+blood+spots+by+immunoassay+with+xMAP+technology.">Simultaneous measurement of 25 inflammatory markers and neurotrophins in neonatal dried blood spots by immunoassay with xMAP technology.</a> Clin Chem. 51 (10), 1854-1866 (2005).</li><li>Amadori, D., 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=Vitamin+D+receptor+polymorphisms+or+serum+levels+as+key+drivers+of+breast+cancer+development?+The+question+of+the+vitamin+D+pathway.">Vitamin D receptor polymorphisms or serum levels as key drivers of breast cancer development? The question of the vitamin D pathway.</a> Oncotarget. 8 (8), 13142-13156 (2017).</li><li>Gupta, B. P., Jayasuryan, N., Jameel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+detection+of+hepatitis+B+virus+from+dried+blood+spots+by+polymerase+chain+reaction+amplification.">Direct detection of hepatitis B virus from dried blood spots by polymerase chain reaction amplification.</a> J Clin Microbiol. 30 (8), 1913-1916 (1992).</li><li>Colson, K. E., Potter, A., Conde-Glez, C., Hernandez, B., R&#237;osZertuche, D., Z&#250;&#241;iga-Brenes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+commercial+ELISA+for+the+detection+of+measles-specific+immunoglobulin+G+(IgG)+in+dried+blood+spots+collected+from+children+living+in+low-resource+settings.">Use of a commercial ELISA for the detection of measles-specific immunoglobulin G (IgG) in dried blood spots collected from children living in low-resource settings.</a> J Med Virol. 87 (9), 1491-1499 (2015).</li><li>Drabe, C. H., Blauenfeldt, T., Ruhwald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISA-based+assay+for+IP-10+detection+from+filter+paper+samples.">ELISA-based assay for IP-10 detection from filter paper samples.</a> Methods Mol Biol. 1172, 27-37 (2014).</li><li>St Julien, K. 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=High+quality+genome-wide+genotyping+from+archived+dried+blood+spots+without+DNA+amplification.">High quality genome-wide genotyping from archived dried blood spots without DNA amplification.</a> PLoS One. 8 (5), e64710 (2013).</li><li>Shaner, R. L., Schulze, N. D., Seymour, C., Hamelin, E. I., Thomas, J. D., Johnson, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+fentanyl+analogs+in+dried+blood+spots+by+flow-through+desorption+coupled+to+online+solid+phase+extraction+tandem+mass+spectrometry.">Quantitation of fentanyl analogs in dried blood spots by flow-through desorption coupled to online solid phase extraction tandem mass spectrometry.</a> Anal Methods. 9, 3876-3883 (2017).</li></ol>

Este protocolo investiga o potencial para armazenamento de sangue e soro em papel de filtro quando os laboratórios não têm o pessoal ou as infra-estruturas necessárias para a manipulação correta de amostras de sangue. Em particular, sangue total ou soro coletado em tubos padrão ou por picada do dedo pode ser armazenado usando este método e não há nenhuma necessidade para congelar amostras a-20 ° C ou -80 ° C imediatamente após a coleta de sangue. DBS/DSS podem permanecer em temperatura ambiente por até 14 d...

O principal objetivo da pesquisa em biomarcadores de câncer é a identificação de novos parâmetros biológicos que pode ser usada para diagnóstico, prognóstico de pacientes prever e para determinar se um paciente responderá a um tratamento específico. Esta área de investigação é fundamental para a descoberta de tratamentos inovadores e desempenha um papel fundamental no tratamento sob medido.
Os procedimentos realizados durante cada passo do biomarcador identificação e validação devem ser confiável e reprodutível. Uma pedra fundamental para o sucesso da investigação de translação é o correto armazenamento de amostras biológicas como sangue e soro. Este é o primeiro passo para obtenção de material biológico de alta qualidade que pode ser usado para realizar experimentos de biologia molecular ou ensaios da proteína.
Estudos multicêntricos são muitas vezes necessários para recrutar suficiente pacientes para obter dados robustos. Nem todos os institutos são capazes de armazenar amostras a-80 ° C ou para enviar amostras para outros centros internacionais em gelo seco. O uso de papel de filtro para coleta de sangue é um método simples para armazenar sangue e soro e não exige o imediato congelamento de amostras1,2. Uma gota de sangue ou soro pode ser vista no papel, deixada para secar durante a noite e então armazenado por até 14 dias em temperatura ambiente1,2. Isto dá a investigadores tempo para enviar as amostras de outros laboratórios. O uso de sangue seco manchas (DBS) e manchas secas soro (DSS), portanto, poderiam simplificar a colaboração entre os institutos em países desenvolvidos e em desenvolvimento.
Dada a sua facilidade de uso, amostragem de DBS é amplamente utilizada em diversos tipos de ensaios para análises a jusante serológicas ou genéticas. Por exemplo, no passado, DBS frequentemente foram usados para HIV triagem em países em desenvolvimento1,2,3,4,5. Outra vantagem deste método de armazenamento é que podem ser colhidas amostras de sangue do dedo-picas, permitindo seu uso para o recém-nascido, triagem de testes 6,7,8. Amostra de fácil manuseio e transporte são ainda mais vantagens de DBS, especialmente para amostras coletadas em locais remotos onde não há nenhum equipamento de laboratório. Em uma publicação anterior, usamos DBS e DSS para testar a vitamina D e vitamina D (DBP) proteína em uma série de pacientes africanos e caucasianos9. Nossos colegas africanas não foram capazes de obter gelo seco. Para comparar os marcadores biológicos para entender as diferenças na via da vitamina D, entre as duas populações étnicas, melhorámos ainda mais o procedimento usando combinadas amostras armazenadas sob condições normais e em papel de filtro. Depois de otimizar o procedimento DBS/DSS, fomos capazes de analisar DBP e vitamina D no soro em ambas as coortes. Avaliamos também um número de polimorfismos de nucleotídeo único (SNPs) após a extração de DNA do sangue inteiro para brancos e de DBS para africanos9. O presente protocolo permite que as amostras de sangue de alta qualidade para ser armazenado em temperatura ambiente, sem afetar diferentes tipos de análises a jusante que variam de biologia molecular para ensaios de ELISA. É recomendável para uso gerenciar materiais biológicos em estudos multicêntricos ou para centros que não têm instalações para as condições de armazenamento padrão. O protocolo seguinte representa o culminar destes procedimentos otimizados.

<table>
	<tbody>
		<tr>
			<td>Whatman protein saver cards 903 Protein saver card, 100/pk&#8206;</td>
			<td>Sigma</td>
			<td>Z761575</td>
			<td>Useful to store samples at room temperature for downstream analyses</td>
		</tr>
		<tr>
			<td>Falcon Serological Pipettes, 5 mL</td>
			<td>Stem cell</td>
			<td>#38003</td>
			<td></td>
		</tr>
		<tr>
			<td>50-200 &#181;L tips</td>
			<td>Star-Lab</td>
			<td>S1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Eppendorf</td>
			<td>4036-3204</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA microkit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td>This is a DNA extraction kit designed to isolate small quantities of DNA&#160;&#160;</td>
		</tr>
		<tr>
			<td>Buffer ATL (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Lysis Buffer 1 in the text</td>
		</tr>
		<tr>
			<td>Buffer AL (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Lysis Buffer 2 in the text</td>
		</tr>
		<tr>
			<td>QIAamp mini elute column</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as column in the text</td>
		</tr>
		<tr>
			<td>AW1&#160; (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Wash Buffer 1 IN THE TEXT</td>
		</tr>
		<tr>
			<td>AW2&#160; (included in QIAamp DNA microkit)</td>
			<td>Qiagen</td>
			<td></td>
			<td>This is reported as Wash Buffer 2 in the text</td>
		</tr>
		<tr>
			<td>Human Vitamin D BP Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>DVDBP0</td>
			<td></td>
		</tr>
	</tbody>
</table>

dried blood and serum spots as a useful tool for sample storage to evaluate cancer biomarkers

Qualidade de amostra de sangue é essencial para garantir a precisa análises a jusante como real-time PCR ou ELISA. Armazenamento correto de materiais biológicos é o ponto de partida para alcançar resultados reprodutíveis e confiáveis. Todas as amostras devem ser tratadas da mesma forma de coleta de sangue para armazenamento. Dependendo de análises a realizar, amostras de soro e sangue total devem ser armazenadas a-20 ° C ou -80 ° C até o uso. Amostras de sangue/soro também devem ser aliquotadas para evitar múltiplas de congelamento-descongelamento. Outra questão importante é as condições da amostra durante o transporte de um laboratório para outro. Se o gelo seco não está disponível ou o carregamento demora mais do que alguns dias, são necessárias abordagens alternativas. Uma opção é usar papel de filtro para coleta de sangue. Aqui, propomos um método de sangue e coleta de amostra de soro que aproveita seca manchas de sangue (DBS) e seca manchas de soro (DSS). Nós desenvolvemos o procedimento para extrair DNA de DBS para a avaliação a jusante de alguns polimorfismos de nucleotídeo único (SNPs) por PCR em tempo real. Nós também Aperfeiçoamos um ensaio de ELISA, a partir de proteínas eluídas da DSS. Esse método pode ser usado com outros ensaios de ELISA ou procedimentos de avaliação de proteínas.

Tiramos vantagem dos procedimentos de DBS e DSS para armazenar sangue e soro à temperatura ambiente, sem afetar a qualidade do material biológico. A Figura 1 mostra um exemplo do protetor de cartão de proteína sem sangue e após a coleta de sangue. A fim de confirmar que o armazenamento em papel de filtro e o procedimento para eluir sangue não interferem com a qualidade da amostra, realizamos uma comparação com métodos de armazenamento padrão. Que ex...

Watch this Scientific Journal Video about Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers at JoVE.com

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Este protocolo descreve um método simples e útil para armazenar o sangue periférico e soro/plasma para análises a jusante, tais como avaliação de nucleotídeo único (SNP) de polimorfismo e ensaio de ELISA.

Secas de sangue e soro manchas como uma ferramenta útil para o armazenamento de amostra avaliar biomarcadores de câncer

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

This method can help answer key questions in translational research such as the chance to study biomarkers in patients from developing countries. The main advantage of this technique is the possibility to store blood and serum samples for at least two weeks without freezing and provide high quality material for downstream analysis such as qPCR and ELISA. The implications of this technique extend toward diagnosis of diseases such as cancer and infectious diseases.This method can also be applied toward the diagnostic or research fields like the study of prenatal diseases or the analysis of infectious pathology such as HIV. We first had the idea for this method when we started comparing biomarkers between a Caucasian and an African case series for which frozen samples were not available. Visual demonstration of this method is critical as the blood spotting steps are difficult to learn.In this procedure, three milliliters of a blood sample is collected in a three milliliter tube with 5.4 milligrams of EDTA. Write the patient code number on the bottom right-hand corner of the saver card. Carefully re-suspend the blood with a five milliliter pipette.Next, pipette and transfer one spot of blood onto the saver card. Using a new tip for each blood aliquot, repeat the spotting to obtain a total of three to five blood spots. Leave the dried blood spots, or DBS, to dry overnight at room temperature with the cover of the saver card open in a horizontal position over an open, non-absorbent surface and avoiding direct sunlight.On the following day, store the card at room temperature in a plastic bag with desiccant until use. DNA will be extracted from three dried blood spots from the saver card. Begin by cutting each of the DBS from the card and separating them from the card.Cut the separated DBS into small pieces of about one millimeter in diameter. Place the small pieces from one DBS into a 1.5 milliliter centrifuge tube. To digest the samples, add to the tube 180 microliters of lysis buffer one from a commercially available DNA extraction kit, then add 20 microliters of proteinase K.Mix by vortexing.Place the tube in a thermo-incubator and incubate with shaking at 56 degrees celsius for 60 minutes. At the end of the incubation, briefly centrifuge the tube to remove drops from the lid. Start the wash by adding 200 microliters of lysis buffer two and vortexing for 10 seconds.Place the tube in a thermo-mixer and incubate at 70 degrees celsius for 10 minutes. Place the elusion column in a new two milliliter collection tube and discard the old one. Add 500 microliters of wash buffer and centrifuge at 6000 times gravity for one minute.Place the column in a new two milliliter collection tube and discard the old one. Add 500 microliters of wash buffer two and centrifuge at 6000 times gravity for one minute. Place the column in a new two milliliter collection tube and discard the old one.Centrifuge at 20, 000 times gravity for three minutes to dry the membrane. To elute the DNA, place the column in a new 1.5 milliliter centrifuge tube and discard the collection tube. Place 50 microliters of distilled water onto the center of the membrane and incubate at room temperature for 10 minutes.Centrifuge the tube at 20, 000 times gravity for one minute. Collection the elute and place again on the center of the membrane. Centrifuge the tube again at 20, 000 times gravity for one minute.Discard the membrane. After measuring the DNA concentration using a spectrophotometer, store the DNA at minus 20 degrees celsius until use. For this procedure, seven milliliters of peripheral blood sample is first collected in a seven milliliter tube without EDTA.Leave the blood sample to coagulate at room temperature for 30 minutes. Write the patient code number on the bottom right-hand corner of the saver card. Centrifuge the blood sample at 1100 times gravity at room temperature for 15 minutes.Remove the supernatant from the tube and transfer it to a new 15 milliliter tube. Carefully re-suspend the blood with a five milliliter pipette. Pipette and transfer one spot of serum onto the saver card.Repeat the spotting to have a total of three to five blood spots. Leave the dried sero spots to dry overnight at room temperature with the cover of the saver card open. On the following day, store the card at room temperature in a plastic bag with desiccant until use.If storage is longer than 14 days, store at minus 20 degrees celsius. Prepare for the ELISA assay by thawing the DSS for each sample if necessary. To elute the protein from the DSS, cut one DSS into small pieces of about one millimeter in diameter and put the pieces in a 1.5 milliliter tube.Then, add four microliters of PBS. Shake the samples at low speed at four degrees celsius overnight. On the following day, use an appropriate dilution of the protein elute as a serum sample for secreted protein or growth factor analysis using a commercially available kit.In this protocol, blood and serum samples were stored as dried spots on filter paper at room temperature. A representative protein saver card is shown before and after blood sampling. In order to confirm that the storage on filter paper and the procedure to elute blood do not interfere with the sample quality, ELISA assays for vitamin D binding protein, or DBP levels, were performed on eight samples of serum collected in a two milliliter tube and stored immediately at minus 80 degrees celsius or collected as dried serum spots.The coefficient of variation, or CV, between matched samples ranged from 2%to 24%and the mean CV was 9.6%Once mastered, this technique can be done in 10, 15 minutes if it is performed properly. Following this procedure, downstream methods of molecular biology or protein analysis can be performed. After its development, this technique enables research in the field of translational research and biomarker analysis to collaborate with institutes from all over the world that do not have the lab equipment to store biological samples in standard conditions.After watching this video, you should have a good understanding of how to correctly store blood samples in dried blood and serum spots. Don't forget that working with blood and chemical reagent can be extremely hazardous and precautions such as wearing gloves and a lab coat should always be taken while performing this procedure.

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