We would like to thank Gr&#225;inne Tierney for editorial assistance.

Peripheral blood and serum were collected and stored from healthy donors and patients who gave written informed consent to take part in the study. The study protocol was approved by the local Ethics Committee in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
1. Blood Storage in DBS
<ol>
	<li>Blood sample collection
	<ol>
		<li>Collect 3 mL of peripheral blood sample in a 3 mL tube with 5.4 mg of ethylenediaminetetraacetic acid (EDTA).<br...

<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>

This protocol investigates the potential for storing blood and serum on filter paper when the labs do not have the staff or infrastructures needed for the correct handling of blood samples. In particular, whole blood or serum collected in standard tubes or by finger-prick can be stored using this method and there is no need to freeze samples at -20 &#176;C or -80 &#176;C immediately after the blood collection. DBS/DSS can remain at room temperature for up to 14 days without any change in blood/serum integrity. A critical...

The main aim of the research into cancer biomarkers is the identification of new biological parameters that can be used for diagnosis, for predicting patient prognosis, and for determining whether a patient will respond to a specific treatment. This research area is fundamental for the discovery of innovative cancer treatments and plays a key role in tailored treatment.
The procedures carried out during each step of biomarker identification and validation must be reliable and reproducible. A cornerstone for the success of translational research is the correct storage of biological samples such as blood and serum. This is the first step towards obtaining high quality biological material that can be used to perform molecular biology experiments or protein assays.
Multicenter studies are often needed to recruit enough patients to obtain robust data. Not all institutes are able to store samples at -80 &#176;C or to send samples to other international centers in dry ice. The use of filter paper for blood collection is a simple method for storing blood and serum and does not require the immediate freezing of samples1,2. A drop of blood or serum can be spotted onto the paper, left to dry overnight and then stored for up to 14 days at room temperature1,2. This gives researchers time to send the samples to other laboratories. The use of dried blood spots (DBS) and dried serum spots (DSS) could thus simplify the collaboration between institutes in developed and developing countries.
Given its ease of use, DBS sampling is widely used in several types of assays for serological or genetic downstream analyses. For example, in the past, DBS were frequently used for HIV screening in developing countries1,2,3,4,5. Another advantage of this storage method is that blood samples can be collected from finger-pricks, thus enabling its use for newborn screening tests 6,7,8. Easy sample handling and transport are further advantages of DBS, especially for samples collected in remote sites where there is no laboratory equipment. In a previous publication, we used DBS and DSS to test vitamin D and vitamin D binding protein (DBP) in a series of Caucasian and African patients9. Our African colleagues were not able to obtain dry ice. To compare the biological markers to understand the differences in the vitamin D pathway between the two ethnic populations, we further improved the procedure using matched samples stored under standard conditions and on filter paper. After optimizing the DBS/DSS procedure, we were able to analyze DBP and vitamin D in patient serum in both cohorts. We also evaluated a number of single nucleotide polymorphisms (SNPs) after DNA extraction from whole blood for Caucasians and from DBS for Africans9. The present protocol permits high quality blood samples to be stored at room temperature without affecting different types of downstream analyses ranging from molecular biology to ELISA assays. It is recommended for use to manage biological materials in multicenter studies or for centers that do not have facilities for standard storage conditions. The following protocol represents the culmination of these optimized procedures.

<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

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

We took advantage of the DBS and DSS procedures to store blood and serum at room temperature without affecting the quality of the biological material. Figure 1 shows an example of the protein card saver without blood and after blood collection. In order to confirm that the storage on filter paper and the procedure to elute blood do not interfere with sample quality, we performed a comparison with standard storage methods. We extracted DNA from 3 matched sampl...

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

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

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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Research

JoVE Journal

Cancer Research

10.3K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. 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...