We would like to thank Henriette Mair-Meijers for invaluable attention to detail and hours devoted to processing the blood collections. We are grateful for the graphic design expertise of Natalie Jameson Kiesling. We also appreciate the approval of the manufacturers (Qiagen (Valencia, CA), BD Biosciences (San Jose, CA), Life Technologies (Grand Island, NY)) mentioned herein to publish the use of their products as described. Funding for this work was generously provided by the National Institutes of Health award numbers DA022720, RC1MH088283, and DA022720-05-S1.

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E., Wildman, D., de los Santos, R., Galea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+and+inflammatory+marker+profiles+associated+with+depression+in+a+community-based+epidemiologic+sample.">Epigenetic and inflammatory marker profiles associated with depression in a community-based epidemiologic sample.</a> Psychol Med. 41, 997-1007 (2011).</li><li>Uddin, 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=Post-traumatic+stress+disorder+is+associated+with+immunosenescent+T+cell+phenotypes+in+the+Detroit+Neighborhood+Health+Study.">Post-traumatic stress disorder is associated with immunosenescent T cell phenotypes in the Detroit Neighborhood Health Study.</a> , (2011).</li><li>Uddin, 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=Post-traumatic+stress+disorder+is+associated+with+immunosenescent+T+cell+phenotypes+in+the+Detroit+Neighborhood+Health+Study.">Post-traumatic stress disorder is associated with immunosenescent T cell phenotypes in the Detroit Neighborhood Health Study.</a> , (2011).</li><li>Uddin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+signatures+of+post-traumatic+stress+disorder+in+the+Detroit+Neighborhood+Health+Study.">Biological signatures of post-traumatic stress disorder in the Detroit Neighborhood Health Study.</a> , (2010).</li><li>Aiello, A. 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The authors declare that they have no competing interests.

We have described a streamlined protocol that has been successfully applied to process more than 1,600 whole blood samples in the Detroit Neighborhood Health Study. Although many of these techniques are available in the existing literature, our step-by-step compilation, including precisely timed alterations between each step, reflects an optimized, efficient protocol that successfully produces a variety of biologic specimens with a wide range of downstream applications, including DNA methylation, mRNA expression and immunological analysis. These specimens have already been tested in a variety of experiments, results of which have been published in peer-reviewed literature and/or presented at national meetings11,20,21,23,24,29. This protocol should thus be of interest to other investigators seeking to collect biological specimens in population-based studies similar to the DNHS.
When processing samples in such a high-throughput manner, it is of utmost importance to maintain accurate records at all stages. We recommend developing a database to store all of the cryovial information at the outset. This database should include all aspects of the sample within each cryovial including the volume, concentration, quality, barcode, and storage location (storage box number and location within the box). We recommend preparing pre-labeled cryovials and storage boxes which contain a barcode. Further, we have found it convenient to have a &#8220;master&#8221; document containing the barcodes for each tube, specific to each participant (Table S3). This enables rapid input of the cryovial data into the database without excessive handling of the samples, introducing unnecessary freeze/thaw cycles to the samples and eliminates the potential for human error in entering in the barcodes manually. It is also essential that the labels adhere at extreme (e.g., vapor phase of liquid nitrogen -178 to -150 &#186;C) temperatures. The documentation can be time consuming, and with the necessity of efficient processing upon delivery, we have found that having at least two technicians divide the processes produces the greatest efficiency.
A limitation of this method is the proximity of the laboratory to the collection site. For PBMC isolation in particular, the samples must be processed within 2 hr of collection to avoid significant increases in red blood cell contamination and decreases in viable mononuclear cells. As such, the laboratory should be no more than 30 min&#160;from a collection site to account for any traffic related issues that may arise. In addition, any laboratory proposing to undertake the protocol outlined here will require two technicians on hand to ensure optimal sample processing. Furthermore, each laboratory must have access to the requisite equipment for each step outlined above. Thus, laboratories with fewer staff and/or limited equipment would likely be unable to undertake this protocol.
An advantage of this protocol is the ability to collect specimens directly in the homes of consenting individuals. This allows the study to reach individuals with mental or other health issues who would not typically seek medical help, perhaps because of a lack of insurance or transportation. It also the enables comparison of affected and unaffected individuals living within the same community who have experienced similar triggers, but differ in terms of their mental health symptoms. Obtaining specimens in this manner requires precise timing and coordination of phlebotomists in the field. Because our laboratory was located within the community we were studying, a phlebotomist could typically collect samples from two-three homes and deliver the samples to the laboratory within the 2 hr window of the first collection. Efficiency is key to our sample processing, and as such, we had multiple phlebotomists in the field, increasing the need for precise coordination. The laboratory received multiple blood deliveries with up to eight participants per delivery spaced 2 hr apart. The phlebotomists met at a designated location and combined their collections with only one phlebotomist delivering the specimens to the laboratory as a single batch. The methods described here can easily be adapted to study many other phenotypes and the biologic specimens collected can be used in a multitude of downstream assays.

Multiple studies have characterized differences in gene expression, DNA methylation and cell subset in blood among individuals with and without mental (or other) illnesses1-4. These studies, however, have been obtained from clinical settings in which disease-associated differences may be magnified due to the generally more severe nature of the illnesses for which patients are seeking treatment. Due to advances in &#8220;omics&#8221; approaches, the past decade has seen an explosion of interest in obtaining biologic samples from community and/or epidemiologic settings5-7, in order to provide population-based estimates of disease prevalence and a broader picture of the environmental determinants of these mental and/or physical illnesses.
A key challenge in this regard is the requirement for rapid processing of the collected specimens. Degradation of mononuclear cells, key immune system components that are frequently used to assess the health of an individual, begins immediately upon blood draw with a significant decrease in recovery after 2 hr of collection8-10. To address this challenge, we present an optimized protocol in which multiple components of human whole blood are simultaneously isolated from samples obtained in homes of subjects living in a large metropolitan area. The protocol is based upon our compilation and modification of current techniques, including storage of all &#8220;extra&#8221; fractions in the event future techniques allow for further isolation/analyses. While alternative methods or kits may be employed in place of the individual methods described here, those outlined have proven to be a reliable and efficient means for processing samples in a high-throughput manner. High-quality fractions (PBMCs, DNA, serum, and RNA) of fresh blood can be produced within 2 hr of collection and all assay-ready specimens can be available within 2 days (Figure 1).
This protocol was developed to enable the efficient processing of samples collected from community-dwelling, adult residents of the city of Detroit for testing in the Detroit Neighborhood Health Study (DNHS; DA022720, RC1MH088283, DA022720-05-S1), a population-based study of the social and biological determinants of post-traumatic stress disorder (PTSD) and other mental illnesses. The prevalence of PTSD in Detroit is more than twice the national average11,12. Identifying biological determinants of PTSD in this population may help to develop appropriate pharmacologic and/or cognitive-behavioral interventions to aid those suffering from the disorder, both in this urban population, and in other high-risk populations (e.g., returning military veterans). Our laboratory, previously located at Wayne State University in Detroit, Michigan, was selected for processing based on our expertise in handling fresh tissue samples derived from a variety of sources, the necessity to begin processing the samples within 2 hr of collection, and our proximity to the collection sites. With this unique opportunity at hand, our goal was to optimize the processing for greatest yield of DNA, RNA, serum and peripheral blood mononuclear cells (PBMCs) from each specimen (a total of N=1,639 samples over 5 waves of specimen collection). The procedures outlined here can be performed simultaneously in a non-clinical setting, thus producing starting material (see Table 1 for average yields) for a multitude of downstream applications including microarray, epigenetic, real-time RT-PCR, and flow cytometry analyses.
<img alt="Figure 1" src="/files/ftp_upload/52227/52227fig1highres.jpg" width="700" /> 
Figure 1. Overall work flow. The overall process depicted here includes the logistics of obtaining the blood specimens from identifying consenting participants to the blood draw itself. High-quality, fractions (peripheral blood mononuclear cells; PBMCs, DNA, serum, and RNA) of fresh whole blood can be produced within 2 hr of collection and all assay-ready specimens can be available within 2 days. Moreover, the fractions prepared through this method are suitable for long-term storage if samples are not to be tested immediately. The entire timeline outlined here could be completed in a single day (~5 hr total). However, such a day would be extremely labor intensive especially for a single technician with substantial experience with the techniques. Thus, we recommend dividing the procedures on Day 1 between at least two technicians and completing the RNA processing on Day 2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/52227fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>QIAamp DNA Blood Mini Kit</td><td>Qiagen</td><td>51104</td><td>Day 1: DNA isolation</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma</td><td>P5493-1L</td><td>Day 1: PBMC isolation</td></tr><tr><td>5 &amp;frac34;&amp;rdquo; Pasteur pipets</td><td>Fisher</td><td>13-678-6A</td><td>Day 1: PBMC isolation</td></tr><tr><td>Fetal Bovine Serum (FBS), heat inactivated</td><td>Life Technologies</td><td>10082147</td><td>Day 1: PBMC isolation</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma</td><td>D8418-500ml</td><td>Day 1: PBMC isolation</td></tr><tr><td>RPMI Medium 1640, liquid</td><td>Invitrogen</td><td>11875119</td><td>Day 1: PBMC isolation</td></tr><tr><td>0.4% trypan blue stain&amp;nbsp;</td><td>Invitrogen</td><td>T10282</td><td>Day 1: PBMC isolation</td></tr><tr><td>Countess Cell Counting Chamber</td><td>Invitrogen</td><td>C10283</td><td>Day 1: PBMC isolation</td></tr><tr><td>Countess Automated Cell Counter or cell counting device such as a microscope and hemocytometer</td><td>Invitrogen</td><td>C10281</td><td>Day 1: PBMC isolation</td></tr><tr><td>LeukoLOCK Fractionation &amp;amp; Stabilization Kit&amp;nbsp;</td><td>Ambion</td><td>1933</td><td>Day 1: Leukocyte RNA isolation</td></tr><tr><td>25&amp;nbsp;G x 5/8 in. needles</td><td>Becton Dickinson</td><td>305122</td><td>Day 1: Leukocyte RNA isolation</td></tr><tr><td>Syringes (5&amp;nbsp;ml)</td><td>Becton Dickinson</td><td>309646</td><td>Days 1 and 2: Leukocyte RNA isolation</td></tr><tr><td>Denaturing Lysis Solution&amp;nbsp;</td><td>Ambion</td><td>8540G</td><td>Day 2: Leukocyte RNA isolation</td></tr><tr><td>5&amp;nbsp;M NaCl</td><td>Life Technologies</td><td>24740011</td><td>Day 2: Leukocyte RNA isolation</td></tr><tr><td>TRI Reagent</td><td>Ambion</td><td>9738</td><td>Day 2: Leukocyte RNA isolation</td></tr><tr><td>Bromo-3-chloro-propane (BCP)</td><td>Sigma</td><td>B-9673</td><td>Day 2: Leukocyte RNA isolation</td></tr><tr><td>spin cartridges&amp;nbsp;</td><td>Ambion</td><td>10051G</td><td>Day 2: Leukocyte RNA isolation</td></tr><tr><td>0.1&amp;nbsp;mM EDTA</td><td>Ambion</td><td>9912</td><td>Day 2: Leukocyte RNA isolation</td></tr><tr><td>DNA-free Kit</td><td>Ambion</td><td>AM1960</td><td>Day 2: DNase treament</td></tr><tr><td>RNA 6000 Ladder&amp;nbsp;</td><td>Agilent</td><td>5067-1529</td><td>Day 2: Bioanalyzer analysis</td></tr><tr><td>RNA 6000 Nano Series II Kit&amp;nbsp;</td><td>Agilent</td><td>5067-1511</td><td>Day 2: Bioanalyzer analysis</td></tr><tr><td>RNaseZAP</td><td>Ambion</td><td>AM8782</td><td>Day 2: Bioanalyzer analysis</td></tr><tr><td>Ethanol &gt;99%</td><td>Sigma</td><td>E7023-500ml</td><td></td></tr><tr><td>Isopropanol &gt;99%&amp;nbsp;</td><td>Sigma</td><td>I9516-500ml</td><td></td></tr><tr><td>Nuclease-free ultra pure water&amp;nbsp;</td><td>Invitrogen</td><td>9938</td><td></td></tr><tr><td>Pipette tips (nuclease-free)</td><td>Eppendorf</td><td>22491253</td><td></td></tr><tr><td>Pipetter (serological)</td><td>Eppendorf</td><td>2223020-4</td><td></td></tr><tr><td>Pipetters (for volumes under 1&amp;nbsp;ml)</td><td>Eppendorf</td><td>3120000-054</td><td></td></tr><tr><td>Pipettes (serological)</td><td>Fisher</td><td>13-678-27E</td><td></td></tr><tr><td>Controlled rate freezing containers</td><td>Nalgene</td><td>5100-0001</td><td></td></tr><tr><td>Cryoboxes (to hold 2&amp;nbsp;ml and 5&amp;nbsp;ml cryovials and 1.5&amp;nbsp;ml microcentrifuge tubes)</td><td>Fisher</td><td>03-395-464</td><td></td></tr><tr><td>Test tube rack</td><td>Thermo Scientific</td><td>14-804-134</td><td></td></tr><tr><td>15&amp;nbsp;ml polypropylene tubes</td><td>Fisher</td><td>14-959-49D</td><td></td></tr><tr><td>1.5&amp;nbsp;ml and 0.65 microcentrifuge tubes</td><td>Fisher</td><td>07-200-534 and 07-200-185</td><td></td></tr><tr><td>2&amp;nbsp;ml and 5&amp;nbsp;ml cryovials</td><td>Fisher</td><td>10-500-26 and 10-269-88F&amp;nbsp;</td><td></td></tr><tr><td>8&amp;nbsp;ml CPT vacutainer</td><td>BD Biosciences</td><td>362761</td><td>2 tubes</td></tr><tr><td>6&amp;nbsp;ml K&lt;sub&gt;2&lt;/sub&gt; EDTA vacutainer</td><td>BD Biosciences</td><td>367863</td><td>2 tubes</td></tr><tr><td>8.5&amp;nbsp;ml SST vacutainer</td><td>BD Biosciences</td><td>367988</td><td>1 tube</td></tr><tr><td>Vortexer</td><td>Fisher</td><td>2215365</td><td></td></tr><tr><td>Dry bath incubator with heating block for microcentrifuge tubes</td><td>Fisher</td><td>11-715-1250</td><td></td></tr><tr><td>Filtration/vacuum system for use within the cell culture hood</td><td>Fisher</td><td>01-257-87</td><td></td></tr><tr><td>Fixed-angle rotor for microcentrifuge tubes with aerosol-tight lid</td><td>Eppendorf</td><td>22637002</td><td></td></tr><tr><td>Refrigerated centrifuge with a swing-bucket rotor and aerosol-tight caps for 16 x 125&amp;nbsp;mm vacutainers and 15&amp;nbsp;ml polypropylene tubes</td><td>Eppendorf</td><td>22628157</td><td>2, one does not need to be refrigerated</td></tr><tr><td>Nanodrop 2000 (recommended for accuracy of small volumes) or other spectrophotometric device</td><td>Fisher</td><td>13-400-411</td><td></td></tr><tr><td>Agilent Bioanalyzer</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>Liquid nitrogen tank</td><td>Thermo Scientific</td><td>11-676-56</td><td></td></tr><tr><td>Sharps container</td><td>Fisher</td><td>22-037-970</td><td></td></tr><tr><td>Biological waste container</td><td>Thermo Scientific</td><td>1223P52</td><td></td></tr><tr><td>Biosafety Level 2&amp;nbsp; certified cell culture hood</td><td>Thermo Scientific</td><td>13-261-315</td><td></td></tr></tbody></table>

rapid fractionation and isolation of whole blood components in samples obtained from a community-based setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

It is essential that the overall procedure produce high quality material for analysis in a multitude of downstream applications including gene expression via microarray and RT-PCR analysis, detection of epigenetic modifications, and cell subset variations. Table 1 indicates the average yield and quality of materials from each of the processes. Figure 3 provides an example of the quality output of the leukocyte RNA isolation and filter processing methods. The image on the upper left of Figure 3 is the gel image resulting from the capillary electrophoresis. Each lane should produce two distinct bands with minimal shadowing which would indicate degradation. The chromatograms below the gel provide an additional look at the level and type of degradation that can be determined based upon the location and size of the peaks. The RNA Integrity Number (RIN) is another quality measure that ranges from 1 (low; degraded) to 10 (high; pure, good quality RNA).
Such a high-throughput methodology lends itself to an occasional error, but there are several checkpoints throughout the various stages of processing to ensure quality control. Figure 2 displays the proper separation following centrifugation of the Ficoll containing vacutainer. There are several reasons a vacutainer may not display this separation including an error in centrifugation speed, low collection volume, or lack of a fasting participant as indicated in Table 2.
Subsets (n=100) of the specimens isolated using this procedure have been analyzed with success by HumanMethylation27 (HM27) DNA Analysis BeadChips including validation of those results by pyrosequencing11. Genotyping, targeted bisulfite pyrosequencing, and real-time RT-PCR has been successfully performed in the Wildman and Uddin laboratories20,21,22,23. Serum, isolated in parallel with the DNA isolation for both the Beadchip and genotyping analyses, was successfully analyzed for IL-6 and C-reactive protein activity24. T-cell subsets from the isolated PBMCs have been successfully analyzed by flow cytometry25-28. Additionally, RNA from the modified leukocyte procedure has been subjected to Genome Wide gene expression profiling 29.
<img alt="Figure 2" src="/files/ftp_upload/52227/52227fig2highres.jpg" width="300" /> 
Figure 2. Layer separation following centrifugation of Ficoll containing vacutainer. A visualization of the separation of multiple blood components following centrifugation. The mononuclear layer is further purified while the other layers are stored at -80 &#176;C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/52227fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52227/52227fig3highres.jpg" width="700" /> 
Figure 3. Expected results from leukocyte RNA processing. Bioanalyzer results displaying two distinct bands representing 18S and 28S ribosomal RNA with RNA integrity numbers (RINs) above 8 isolated with the described leukocyte RNA isolation and filter processing methods (see protocol 3.6 and 5.3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/52227fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Total Average Yield (N&#8776;500)</td>
			<td>Average Quality</td>
		</tr>
		<tr>
			<td>Serum</td>
			<td>2.30 ml</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>DNA from whole blood</td>
			<td>39.97 &#181;g</td>
			<td>Absorbance at 260/280 = 1.74</td>
		</tr>
		<tr>
			<td>PBMC</td>
			<td>22.25 million viable cells</td>
			<td>At least 95% viability of overall isolation</td>
		</tr>
		<tr>
			<td>RNA from Leukocytes</td>
			<td>44.09 &#181;g</td>
			<td>Absorbance at 260/280 = 2.01 
			RNA Integrity Number (RIN) = 6.48</td>
		</tr>
	</tbody>
</table>
Table 1. Expected yields. Average quantity and quality of samples processed using the described methods.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Potential Solution</td>
		</tr>
		<tr>
			<td rowspan="2">No separation after Ficoll containing vacutainer centrifugation</td>
			<td>Vacutainer is not filled to capacity &#8212; the minimum collection volume for adequate processing is 6 ml.</td>
		</tr>
		<tr>
			<td>Check centrifuge settings, be sure the speed is in g-force. If incorrect, set to g-force and respin.</td>
		</tr>
		<tr>
			<td rowspan="3">Low PBMC count</td>
			<td>Volume of blood draw too low.</td>
		</tr>
		<tr>
			<td>Aspirated too close to the pellet in step 3.7.8</td>
		</tr>
		<tr>
			<td>Non-fasting participant.</td>
		</tr>
		<tr>
			<td>No separation after serum vacutainer centrifugation</td>
			<td>Check centrifuge settings, be sure the speed is in g-force. If incorrect, set to g-force and respin.</td>
		</tr>
		<tr>
			<td>Unexpected concentration using the Nanodrop 1000</td>
			<td>Make sure the measurement is for the appropriate sample type (e.g., DNA or RNA).</td>
		</tr>
	</tbody>
</table>
Table 2. Troubleshooting. Common problems encountered with the described methods and potential solutions.
<img alt="Table S1" src="/files/ftp_upload/52227/52227tableS1.jpg" /> 
Supplementary Table 1. Tracking sheet. An example of tracking the vacutainers from blood collection to laboratory delivery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/Table_S1.xlsx" target="_blank">Please click here to view this table.</a>
<img alt="Table S2" src="/files/ftp_upload/52227/52227tableS2.jpg" /> 
Supplementary Table 2. Specimen log. An example of the document used to document the processing of the vacutainers upon delivery to the lab. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/Table_S2.xlsx" target="_blank">Please click here to view this table.</a>
<img alt="Table S3" src="/files/ftp_upload/52227/52227tableS3.jpg" /> 
Supplementary Table 3. Cryovial Numbering System. An example of the numbering system used for each participant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/Table_S3.xlsx" target="_blank">Please click here to view this table.</a>
Supplementary Information 1. Preprocessing details. Details the duties of the Study Coordinator, Courier and Phlebotomist. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/SI_1.docx" target="_blank">Please click here to view supplementary information 1.</a>
<img alt="Figure S2" src="/files/ftp_upload/52227/52227supfig2.jpg" /> 
Supplementary Information 2. Recipes. A list of recipes for the necessary reagents. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52227/SI_2.docx" target="_blank">Please click here to view supplementary information 2.</a>

Watch this Scientific Journal Video about Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting at JoVE.com

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

rapid-fractionation-isolation-whole-blood-components-samples-obtained

Nanyang Technological University

Research

JoVE Journal

Medicine

16.0K Views.  University of Illinois at Urbana-Champaign. We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Video: Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Immunology and Infection

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Cancer Research

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood.
The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x&#160;g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at &#8805;93% of viable cells for both types.
This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.

Here, we provide a reliable approach for isolating low- and normal-density neutrophils from whole blood using magnetic isolation (negative selection) and discontinuous density gradient medium. It ensures untouched isolation of high-purity cells (&#8805;93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Biology

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

High-Throughput PBMC Isolation Using Immunomagnetic Beads

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

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