We thank Jessica Jones, Erica Clark, Constance Ducar, Donna Smith, Roy Lewis, Lily Apedaile, Joanne Wiesner, Devin Adams, Corey McBain and Stephen Voght for their assistance in the development of this method, manuscript and video.
This work was supported by the Bill and Melinda Gates Foundation CAVD grant 38645 (M.J.M.) and National Institutes of Health grants UM1 AI068618 and U01 AI069481 (M.J.M.). E.A-N. is supported by NIH Grant T32 AI007140. We thank the James B. Pendleton Charitable Trust for their generous equipment donation.

Note: To protect the fluorophore-conjugated antibodies from light, perform all steps in a bio-safety cabinet with the light off.
1. Antibody Staining Panel Preparation
<ol>
 <li> The antibody staining panel can be found in Table 1. Antibody concentration should be defined by titration with whole blood and using the same flow cytometry equipment and procedures that will be used to acquire the stained phenotyping samples. </li>
 <li> Once appropriate staining titers are determined, combine all antibodies into a single mixture in a lock-cap tube. Add flow wash buffer (Dulbecco's PBS with 2% heat inactivated fetal bovine serum) to bring the total volume to 100 &mu;l. Scale the mixture for the number of samples being stained. This mixture can be stored at 4 &deg;C for up to eight weeks. </li>
</ol>
2. Staining
<ol>
 <li> If the blood collected is to be used for other purposes in addition to this assay, set an aliquot aside while more time-sensitive procedures are performed on the remaining blood. The aliquot can be stored at room temperature for up to 4 hr after venipuncture without significant cell loss. </li>
 <li> Verify that there is an intact bead pellet at the bottom of the Trucount tube and label the tube to identify the sample being stained. In HVTN clinical trials, the Laboratory Data Management System (Frontier Science and Technology Research Foundation; Amherst, NY) is used to label and track stained samples. </li>
 <li> Record the lot numbers and expiration dates of all reagents. Record the Trucount tube bead count number provided by the manufacturer on the bag of tubes; make sure that the lot number on the bag matches the lot number on the tube. </li>
 <li> Use reverse pipetting to accurately pipette 100 &mu;l of whole blood into the Trucount tube, just above the metal retainer. Avoid smearing blood down the side of the tube. </li>
 <li> Using regular (forward) pipetting technique, pipette 100 &mu;l of the mixed antibody staining panel (See Table 1) into the Trucount tube. Cap the tube and vortex at low speed for approximately 15 sec to mix. Visually inspect the tube to ensure that the bead pellet is fully dissolved. </li>
 <li> Incubate the Trucount tube for 15 minutes at room temperature (15-30 &deg;C) in the dark. </li>
 <li> If necessary, dilute an aliquot of 10&times; FACS Lysing solution to 1X using diH2O. Add 900 &mu;l 1&times; FACS Lysing solution to the tube. </li>
 <li> Cap the tube and vortex thoroughly at low speed for approximately 15 sec to mix. Push the cap down firmly into locking position on the tube and seal with laboratory film. </li>
 <li> Store the tube at -65 &deg;C to -95 &deg;C until the sample is ready for shipment to the central analysis lab or for analysis in house. Samples are stable at this stage for at least four weeks. If shipping or assaying immediately, this step can be omitted. </li>
</ol>
3. Shipping
Note: The following instructions utilize an insulated shipping system from Saf-T-Pak, Inc. specifically designed for shipping category B exempt biological substances according to International Air and Transport Association (IATA) regulations. If analyzing the samples in the same location as the staining occurred, go to section 4.
<ol>
 <li> Samples can be shipped immediately after staining or once they are frozen at -65 &deg;C to -95 &deg;C. Wrap each tube completely in foil and place in the stained specimen box. Place the stained specimen box inside a leakproof poly-bag with absorbent material. </li>
 <li> Place the leakproof poly-bag and contents into a Tyvek bag and seal with as little air as possible in the bag.</li>
 <li> Place the specimen package (specimen inside secondary packaging) inside the inner brown box. </li>
 <li> Place the inner brown box inside the Styrofoam chest, securing it into the indentation to prevent shifting. </li>
 <li> Fill the Styrofoam chest with dry ice (approximately 8 kg) and place the lid tightly on the chest. </li>
 <li> Securely tape the shipping box and ship as Biological Substance, Category B (UN3373) with the proper Dry Ice (UN1845) markings; follow IATA PI-650 instructions. </li>
 <li> Upon receipt, samples are stored at -65 &deg;C to -95 &deg;C until they are analyzed. </li>
</ol>
4. Thawing and Flow Cytometry Analysis
<ol>
 <li> Remove the stained sample from the freezer to thaw at room temperature in the dark before collecting on the flow cytometer. If collecting data on more than one sample, standardize the process for all tubes by staggering thawing so that the tubes are not sitting at room temperature for more than 1 hr each. </li>
 <li> Samples should be acquired using a four laser flow cytometer equipped with appropriate filters, such as the BD LSR II. Use standard cytometer calibration and fluorescence compensation methods for data collection 4.</li>
 </ol>
 Note: Do not set forward scatter or side scatter thresholds during collection 5. Trucount beads can fall below the lowest possible threshold setting for these parameters causing a subset of beads to not be accounted for during analysis. If required by the instrument to set a threshold, set the lowest possible Am Cyan channel threshold. Because CD45+ leukocytes stained with the panel will be Am Cyan positive, and Trucount beads also fluoresce in the Am Cyan channel, this should allow for all relevant data to be appropriately collected. 
 <ol start="3">
 <li> Vortex the sample tube for 5 sec before loading on the flow cytometer. Trucount beads fluoresce highly in many channels. Gate the beads during collection by finding the population that is highly double-positive for PE Cy5 and APC (the two colors that most easily distinguish the beads from the cells may vary depending on instrumentation). Select the bead gate as your stopping gate, and record data until at least 20,000 beads are acquired. </li>
 <li> Analyze the data using appropriate software, such as FlowJo (Treestar; Ashland, OR). Figure 1 shows the gating scheme utilized for analysis of different leukocyte populations from a representative control blood draw. </li>
</ol>
5. Trucount Calculations
<ol>
 <li> Each Trucount tube contains a lyophilized pellet of fluorescent beads. After adding liquid to the tube and vortexing, the beads should become equally distributed throughout the sample. The number of beads in the pellet varies slightly by lot number and can be found on the storage bag for the tubes. </li>
 <li> Gate the Trucount beads and cell populations as shown in Figure 1 to determine event counts for each population. Comparing the number of events in the bead gate to the total number of beads originally in the tube will allow you to determine the ratio of sample collected, which can then be used to determine the absolute concentration (i.e., cells/&mu;l) for each population. The following equation can be used for this purpose: Cell concentration (# cells/&mu;l whole blood)= [# population events/(# bead events/# total beads in pellet)]/100 &mu;l. </li>
</ol>
6. Representative Results
<img src="/files/ftp_upload/4302/4302fig1.jpg" alt="Figure 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> 
Figure 1. Gating scheme utilized for analysis of major leukocyte populations showing representative data from a healthy volunteer. A) Trucount beads (i) are gated and excluded from cells. Granulocytes (ii) are delineated and lympohcytes and monocytes are divided into 3 popluations: CD14 negative lymphocytes (iii), all CD14 negative cells (iv), and non-lymphocytes (v). B) CD14 negative lymphocytes are gated to distinguish CD4+ T cells (vi), CD8+ T cells (vii), B cells (viii), CD56 bright NK cells (ix), CD56 dim NK cells (x), and CD56 negative NK cells (xi). C) All CD14 negative cells are gated to distinguish myeloid (xii) and plasmacytoid (xiii) dendritic cells. D) Non-lympocytes are gated to distinguish non-classical (xiv), intermediate (xv), and classical (xvi) monocytes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4302/4302fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
 More specific cell subsets that are not shown in Figure 1 (e.g., NKT cells or neutrophils) can also be distinguished using the panel we present, and the gating scheme can be expanded or modified to meet specific study needs. Certain gating steps shown are unique to this method. Of particular note, an inclusion gate and exclusion gate are drawn around the Trucount beads and placed on top of one another, one to gate the beads for counting, and one to exclude the beads from the cellular analysis (Figure 1A). Also, because lymphocytes, monocytes, and granulocytes are not as easily distinguished in whole blood as they are in peripheral blood mononuclear cells by forward scatter and side scatter, gating these cells using CD45 expression and side scatter is often necessary (Figure 1A). Contaminating granulocytes (circled) that could not be separated from lymphocytes and monocytes using CD45 and side scatter are distinguishable in some plots by their high CD16 expression (Figure 1C and Figure 1D). The number of contaminating granulocytes is usually small, and they do not interfere with monocyte and NK cell gating.

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	<li>Taylor, M. J., London, N. J., Thirdborough, S. M., Lake, S. P., James, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryobiology+of+rat+and+human+dendritic+cells:+preservation+and+destruction+of+membrane+integrity+by+freezing.">The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing.</a> Cryobiology. 27, 269-278 (1990).</li><li>Reimann, K. A., Chernoff, M., Wilkening, C. L., Nickerson, C. E., Landay, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+lymphocyte+immunophenotype+and+proliferative+responses+in+cryopreserved+peripheral+blood+mononuclear+cells+from+human+immunodeficiency+virus+type+1-infected+donors:+implications+for+multicenter+clinical+trials.+The+ACTG+Immunology+Advanced+Technology+Laboratories.">Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials. The ACTG Immunology Advanced Technology Laboratories.</a> Clin Diagn Lab Immunol. 7, 352-359 (2000).</li><li>Boonlayangoor, P., Telischi, M., Boonlayangoor, S., Sinclair, T. F., Millhouse, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+granulocytes:+study+of+granulocyte+function+and+ultrastructure.">Cryopreservation of human granulocytes: study of granulocyte function and ultrastructure.</a> Blood. 56, 237-245 (1980).</li><li>Perfetto, S. P., Ambrozak, D., Nguyen, R., Chattopadhyay, P., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+assurance+for+polychromatic+flow+cytometry.">Quality assurance for polychromatic flow cytometry.</a> Nat Protoc. 1, 1522-1530 (2006).</li><li>Brando, B., Barnett, D., Janossy, G., Mandy, F., Autran, B., Rothe, G., Scarpati, B., D'Avanzo, G., D'Hautcourt, J. L., Lenkei, R., Schmitz, G., Kunkl, A., Chianese, R., Papa, S., Gratama, J. 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B., Roederer, M., Herzenberg, L., Blackwell, C., Weir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The handbook of Experimental Immunology. 43, 1-49 (1996).</li><li>Hulspas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+the+stability+of+phycoerythrin-tandem+dye+conjugates.">Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.</a> Cytometry A. 75, 966-972 (2009).</li><li>Le Roy, C., Varin-Blank, N., Ajchenbaum-Cymbalista, F., Letestu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+APC-tandem+dyes+are+degraded+through+a+cell-dependent+mechanism.">Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.</a> Cytometry A. 75, 882-890 (2009).</li><li>Mandy, F., Brando, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+absolute+cell+counts+using+immunophenotypic+techniques.">Enumeration of absolute cell counts using immunophenotypic techniques.</a> Curr. 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No conflicts of interest declared.

In this report, we present a bead-based method for enumerating leukocyte populations in fresh whole blood by flow cytometry and cover the parameters necessary for its use in a multicenter clinical trial with centralized sample analysis. This method builds on and optimizes the BD Trucount protocol and enables its reliable use in a multicenter clinical trial setting. The staining assay is simple and takes approximately 45 minutes to perform, making it feasible for blood processing laboratory technicians to perform it and to freeze and ship the samples to a central assay lab for analysis. The assay requires only 100 &mu;l of whole blood, which eliminates the need for time- and cost-intensive PBMC processing and uses far less volume than standard phenotyping assays. In addition, by using fresh whole blood, this assay more accurately depicts the in vivo state of the cells and eliminates the detrimental effects of cryopreservation observed on some cell types 1-3. This staining method produces accurate measurement of cell concentrations, but with the added benefit of distinguishing significantly more cell types than CBC and other counting methods. The key to obtaining accurate and consistent results is to carefully standardize critical steps in the protocol and to utilize a documentation system that keeps track of all the steps so that any anomalies that are discovered can be traced to the source of the error.
There are multiple critical steps throughout the process that influence the quality of the data acquired from this assay. First, standardization of the treatment and handling of blood from the time it is drawn to the time it is stained is important, similar to what has been previously reported for PBMC processing 6. We obtain consistent data when blood is stored in the collection tubes at the clinical site and transported to the processing labs at ambient temperature, staining is performed within 4 hr of blood draw, and the stained samples are then shipped on dry ice to the central assay lab. In addition, by making the antibody cocktail at the central lab and sending it to the site processing labs, we ensure consistency between sites and can test each cocktail on a control blood draw before it is used to stain trial samples. It is important that the central assay lab standardize this process. Therefore we recommend that once the constituent antibodies are titrated to optimize the separation between the positive and negative populations 7, the same lot of antibody be used for the staining of all samples in a clinical trial to maintain consistent staining levels for each marker. If this is not possible (e.g., lack of availability from an antibody manufacturer), each new lot of antibody should be appropriately titrated to ensure that the flow cytometry data will be comparable to that obtained using the previous lot. The central lab and the site processing lab must take great care to ship and store the antibody cocktail at 4 &deg;C and avoid light exposure to minimize degradation that can occur, especially with tandem dyes 8-9.
The accuracy of the data acquired with this assay depends on several critical steps during the staining procedure and sample acquisition on the flow cytometer. Of particular note, accurate reverse pipetting of the blood into the Trucount tube is essential; a small difference in blood volume could have a large effect on the calculated concentrations of cell subsets. Vortexing at low speed after adding the antibody cocktail and BD FACS lyse to thoroughly mix the reagents is also important. Similarly, vortexing thoroughly to dispense the beads evenly throughout the sample before acquisition is essential for accurate counting 10. Slight variations in analysis settings on the flow cytometer can also have a large impact on the data acquired, so careful attention should be paid to the calibration and setup of the cytometer with daily use4.
It should also be noted that while preliminary data suggests that freezing the stained samples does not appear to affect the cell concentrations obtained (data not shown), this has yet to be tested thoroughly. Although this is a potential concern, comparisons between samples can still be made with confidence as long as all samples are handled in a consistent manner. 
 Careful sample tracking throughout staining and analysis is particularly important, especially when hundreds of samples are processed in the context of a clinical trial. Once the data is acquired and analysis begun, it is critical to standardize analysis gates as much as possible between samples. Some variability in the fluorescence of certain populations may occur, especially between different volunteers, but standard gate locations should be set that are applicable to as many samples as possible. Any shifting of the gates from the standard location should be documented and taken into account when determining if changes in cell count occur between samples.
This staining method was optimized to enumerate the major leukocyte subsets using one antibody staining panel, but the procedure and panel could be easily modified to meet specific study needs. Contrary to the Trucount technical data sheet procedure, which suggests using 50 &mu;l of blood, the method we present uses 100 &mu;l. By using a larger blood volume, more events can be acquired during analysis, which is useful when studying cell populations with relatively low concentrations in the peripheral blood, such as dendritic cells. Although previous studies have enumerated dendritic cell populations via Trucount using 50 &mu;l and 100 &mu;l blood volumes 11-12, reports indicate that reasonable precision is dependent on acquiring at least 400 positive population events, and this can often be difficult with dendritic cells when using only 50 &mu;l of blood 13. The blood volume used can be further adjusted to meet the analysis requirements for specific cell types or staining panels. Similarly, the staining panel could be modified to focus on certain cell types, thus allowing for the enumeration of more specific cell subsets not currently distinguished by the panel shown here (e.g., additional NK cell subsets or regulatory T cells). The use of a single panel simplifies the staining protocol and reduces errors associated with multiple panels, such as mislabeling of stained samples. Although the use of multiple panels increases the costs and potential for error, separate panels tailored to specific cell types allow for more detailed phenotyping and/or the inclusion of activation markers, and their use could be easily implemented in multicenter clinical trials, taking into account the considerations previously discussed.
<table border="1">
<tbody>
 <tr>
 <td>Marker</td>
 <td>Fluorophore</td>
 <td>Company</td>
 <td>Clone</td>
 </tr>
 <tr>
 <td>CD45</td>
 <td> Am Cyan</td>
 <td> BD Biosciences</td>
 <td> 2D1</td>
 </tr>
 <tr>
 <td>CD3</td>
 <td> FITC</td>
 <td> BD Biosciences</td>
 <td> SK7</td>
 </tr>
 <tr>
 <td>CD8</td>
 <td> PerCP Cy5.5</td>
 <td> BD Biosciences</td>
 <td> SK1</td>
 </tr>
 <tr>
 <td>CD4</td>
 <td> Ax700</td>
 <td>BD Biosciences</td>
 <td>RPA-T4</td>
 </tr>
 <tr>
 <td>HLA-DR</td>
 <td> ECD </td>
 <td>Beckman Coulter</td>
 <td> Immu-357</td>
 </tr>
 <tr>
 <td>CD14</td>
 <td> v450</td>
 <td> BD Biosciences</td>
 <td> M&#x03C6;P9</td>
 </tr>
 <tr> 
 <td>CD19</td>
 <td> PE</td>
 <td> BD Biosciences</td>
 <td> HIB19</td>
 </tr>
 <tr>
 <td>CD16</td>
 <td> APC-H7</td>
 <td> BD Biosciences</td>
 <td> 3G8</td>
 </tr>
 <tr>
 <td> CD56</td>
 <td> PE Cy7</td>
 <td> BD Biosciences</td>
 <td> NCAM16.2</td>
 </tr>
 <tr>
 <td>CD11c</td>
 <td> APC </td>
 <td>BD Biosciences</td>
 <td> B-ly6</td>
 </tr>
 <tr>
 <td>CD123</td>
 <td> PE Cy5</td>
 <td> BD Biosciences</td>
 <td> 9F5</td>
 </tr>
 </tbody>
 </table>
Table 1. Antibody staining panel to determine all major peripheral blood leukocyte populations.

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>	Company	</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
		<td>Trucount Absolute Counting Tubes</td>
 <td>	BD Biosciences</td>
 <td>	340334</td>
 </tr>
 <tr>
 	<td>10X FACS Lysing Solution</td>
 <td>	BD Biosciences</td>
 <td>	349202</td>
 </tr>
 <tr>
 	<td>Category B &amp; Exempt Shipping System, Insulated</td>
 <td>Saf-T-Pak</td>
 <td>	STP-320</td>
 </tr>
 <tr>
 	<td>CD45 AmCyan monoclonal antibody	</td>
 <td>BD Biosciences</td>
 <td>	339192</td>
 </tr>
 <tr>
 	<td>CD3 FITC monoclonal antibody</td>
 <td>	BD Biosciences	</td>
 <td>349201</td>
 </tr>
 <tr>
 	<td>CD8 PerCp-Cy 5.5 monoclonal antibody</td>
 <td>BD Biosciences</td>
 <td>341051</td>
 </tr>
 <tr>
 	<td>CD4 Alexa Fluor 700 monoclonal antibody	</td>
 <td>BD Biosciences</td>
 <td>557922</td>
 </tr>
 <tr>
 	<td>HLA-DR ECD monoclonal antibody	</td>
 <td>Beckman Coulter</td>
 <td>IM3636</td>
 </tr>
 <tr>
 	<td>CD14 v450 monoclonal antibody	</td>
 <td>BD Biosciences</td>
 <td>560349</td>
 </tr>
 <tr>
 	<td>CD19 PE monoclonal antibody	</td>
 <td>BD Biosciences</td>
 <td>555413</td>
 </tr>
 <tr>
 	<td>CD16 APC-H7 monoclonal antibody	</td>
 <td>BD Biosciences</td>
 <td>560195</td>
 </tr>
 <tr>
		<td> CD56 PE-Cy7 monoclonal antibody</td>
 <td>	BD Biosciences	</td>
 <td>335791</td>
 </tr>
 <tr>
 	<td>CD11c APC monoclonal antibody	</td>
 <td>BD Biosciences</td>
 <td>	559877</td>
 </tr>
 <tr>
 	<td>CD123 PE-Cy5 monoclonal antibody	</td>
 <td>BD Biosciences	</td>
 <td>551065</td>
 </tr>
 </tbody>
</table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

enumeration-major-peripheral-blood-leukocyte-populations-for

Nanyang Technological University

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Immunology and Infection

14.9K Views.  Fred Hutchinson Cancer Research Center. In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Video: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

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