Name: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay (Video) | JoVE Journal
Uploaded: 2012-09-16T14:00:00.000+00:00
Duration: 14 min 45 s
Description: Learn a standardized whole blood phenotyping assay for enumerating major leukocyte populations in multicenter clinical trials using flow cytometry. This method enables accurate tracking of immune responses over time.

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5 minutes

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 μl. Scale the mixture for the number of samples being stained. This mixture can be stored at 4 °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 μl 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 μl 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 °C) in the dark. </li>
 <li> If necessary, dilute an aliquot of 10× FACS Lysing solution to 1X using diH2O. Add 900 μl 1× 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 °C to -95 °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 °C to -95 °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 °C to -95 °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/μl) for each population. The following equation can be used for this purpose: Cell concentration (# cells/μl whole blood)= [# population events/(# bead events/# total beads in pellet)]/100 μl. </li>
</ol>
6. Representative Results
<img src="/files/ftp_upload/4302/4302fig1.jpg" alt="Flow cytometry plots showing immune cell subset gating strategy. Panels A-D, CD markers visualization." fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
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/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>Boonlayangoor, P., Telischi, M., Boonlayangoor, S., Sinclair, T. F., Millhouse, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryopreservation+of+human+granulocytes%3A+study+of+granulocyte+function+and+ultrastructure%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Quality+assurance+for+polychromatic+flow+cytometry%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytofluorometric+methods+for+assessing+absolute+numbers+of+cell+subsets+in+blood.+European+Working+Group+on+Clinical+Cell+Analysis%5BTitle%5D)+AND+%22Cytometry%22%5BJournal%5D)">Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.</a> Cytometry. 42, 327-346 (2000).</li>
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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 t...

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

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

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).

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 ...

This video describes The procedure developed by the HIV Vaccine Trials Network at Fred Hutchinson Cancer Research Center for staining fresh whole blood in multicenter clinical trials to be analyzed by a centralized laboratory. The procedure utilizes tru count tubes made by Becton Dickenson biosciences, each of which contains a lyophilized pellet that dissolves during sample preparation to release a known number of fluorescent beads. Fluorescent antibodies are used to stain for surface markers that are specific to different cell populations.The number of cells in these different populations can be compared to the number of fluorescent beads during flow cytometry analysis and absolute concentrations of different leukocyte populations in whole blood can be tracked over time in relation to intervention. Hello, my name is Erica Anderson Nissan and I'm a staff scientist in the HIV Vaccine Trials Network. The procedure that you're about to watch is one that we've developed for use in our multicenter clinical trials.In these trials, blood processing laboratory technicians receive fresh hole blood specimens, stain them with fluorescently labeled antibodies, and then ship the specimens to a centralized laboratory that's capable of analyzing them on the flow. Cytometer fresh hole blood is often not analyzed in the context of clinical trial settings due to cost and feasibility issues, but we've developed a single antibody staining panel that can identify the major leukocyte populations in the peripheral blood and staining for these populations takes under 45 minutes making it feasible for use in clinical trials in the HVTN, we're using this panel to understand the early events that occur after vaccination with candidate HIV vaccines, but this PI panel could be further modified to focus in on cell types of interest to the individual investigator. Thank you very much for watching and we hope that this will be of use to you in the future.Table One shows an 11 color antibody staining panel developed to phenotype and enumerate the major leukocyte populations within the peripheral blood. The antibody cocktail is made by the central assay lab and sent to the processing labs to ensure consistency between sites and to allow for cocktails to be tested on a control blood draw before they're used to stain trial samples. Each fluorescent conjugated antibody is titrated separately and then the antibodies are combined using appropriate titers into a single mixture with flow wash buffer containing delcos PBS with 2%heat and activated fetal bovine serum.The antibody cocktail is labeled with a batch number and expiration date and shipped from the central assay lab to the processing labs using a packaging system that maintains a temperature of four degrees Celsius during shipping. Once received, the antibody staining panel can be stored at four degrees Celsius in the dark for up to eight weeks. BD fax slicing solution is used after staining to lice the red blood cells and fix the antibody stains while preserving the leukocytes.The solution is received from the manufacturer at a 10 x concentration and is diluted one to 10 by the central assay lab with deionized water. The diluted solution is then labeled with a lot number and expiration date and shipped at ambient temperature to the processing labs once received. Fax lacing solution is stored at room temperature between 15 and 30 degrees Celsius prior to drawing blood For true count staining.The processing lab must make sure that all of the appropriate reagents have been obtained from the central assay lab. These include BD TRU count tubes, the combined antibody staining panel, and one x fax licensing solution. The HVTN utilizes a laboratory data management system to record all relevant information for each sample.A unique identifying code is printed on a label which is placed on the true count tube. To track the specimen throughout the process, label a cryo vial to identify the sample being stained. This will usually include a patient or volunteer identifier, A visit identifier and the date of blood collection aliquot the required amount of blood into the labeled cryo vial using aseptic technique.The amount of blood used for staining depends on the staining panel being used, but is usually 50 or 100 microliters per panel. The cryo vial can then be set aside for up to four hours at room temperature before staining. One more time sensitive procedures are performed on the remaining blood.Verify that there is an intact bead pellet in the bottom of the tube and label the tru count tube. With the relevant specimen information record the lot numbers and expiration dates of all reagents. Worksheets are useful for tracking this information throughout a clinical trial.The tru count tube bead count number and lot number can be found on The front of the bag of tru count tubes. When you are ready to begin the tru Count staining process, make sure that you have all the materials and reagents ready for use in the biological safety cabinet. These include the blood aliquot labeled tru count tube combined antibody staining panel tube, one x fax lacing solution, 200 microliter and 1000 microliter micro pipettes and pipette tips, foil bleach, laboratory film, and a timer after staining.You'll also need a stained specimen box, but this does not need to be in the hood. The amount of blood and antibody to use varies depending on which cells are distinguished by the panel and how many cells are needed for proper analysis. Each panel is tested on control samples to determine the optimal amount of blood for staining and the amount of antibody used is equal to the amount of blood used for the panel shown in this video.100 microliters of blood and 100 microliters of antibody will be used before pipetting. Mix the blood by gently inverting the tube several times and gently pipette up and down several times. To further ensure that the blood is well mixed.Then use reverse pipetting to accurately pipette whole blood from the aliquot into the tru count tube just above the metal retainer. Avoid smearing blood down the side of the tube. Replace the pipette tip between each tru count tube.Now it is time to stain the blood with the antibody staining panel before pipetting. Mix the staining panel by gently pipetting up and down several times. Add 100 microliters of the antibody mixture to the blood in the true count tube.Do not reverse pipette in this step as it will waste valuable reagent. Cap the tube and vortex at low speed for approximately 15 seconds to mix. Make sure the bead pellet is dissolved completely.Cover the tube with foil and set a timer to stain the blood for 15 minutes. The amount of one x fax slicing solution needed is nine times the amount of blood used in the assay, so in this example, 900 microliters of one x fax slicing solution is used. After the 15 minute incubation, add 900 microliters of the one x fax slicing solution to the stain blood to fix the stain and lice the red blood cells.Reverse pipetting is not necessary in this step. Cap the tube and vortex at low speed for approximately 15 seconds to mix. Push the cap down firmly into locking position and seal.With laboratory film. Store the tubes in a dark box or completely wrapped in foil at minus 65 to minus 95 degrees Celsius until they're shipped to the central assay.Lab. To date testing has shown Samples to be stable for up to four weeks at this temperature, stain samples must be shipped As category B biological substances following International Air and Transport Association or I ATA.Regulations at the HVTN samples are shipped weekly from the processing labs to the central assay lab using an insulated shipping system from safety pack ink that is specifically designed to meet these requirements. When using this system, place the inner brown box inside the styrofoam chest, securing it to the indentation to prevent shifting. Fill the chest completely with dry ice and wait five to 10 minutes to allow the box to cool down.To prepare specimens for shipping, completely wrap each tube and foil and place in the stained specimen box. Place absorbent material in a leakproof poly bag with the stained specimen box. Remove excess air from the bag and tightly seal.Place the leakproof poly bag inside of a Tyvek bag. Again, removing all of the air from the bag before ceiling. Place the specimen package inside the inner brown box and place the lid tightly on the styrofoam chest.Securely tape the shipping box and ship as biological substance category B with the proper dry ice markings. Follow I ATA PI six 50 instructions in multicenter clinical trials. It is important to track specimens throughout the short-term storage, shipping, and long-term storage stages.The data management system used by the HVTN allows for the creation of a shipping manifest that can be imported into the specimen database at the central assay lab where the samples will be stored and analyzed. This creates a record of when the samples were shipped and received, where they were terminally stored And when they were removed. For analysis, remove the stain sample from the Freezer to thaw room temperature in a dark box.No more than one hour before collecting on the flow cytometer. If collecting data on more than one sample stagger thawing so that the tubes are not sitting at room temperature for more than one hour each samples should be acquired using a four laser flow cytometer equipped with appropriate filters such as the BD LSR two, use standard cytometer calibration and fluorescence compensation methods for data collection if required by the instrument. To set a threshold, set the lowest possible am cyan channel threshold vortex is sample tube for five seconds before loading on the flow.Cytometer tru count beads fluoresce highly in many channels. Gate the beads during collection by finding the population that is highly double positive. For PE SCI five and a PC, note that the two colors that most easily distinguish the beads from the cells may vary depending on instrumentation.Select the tru count bead gate as your stopping gate and record data until at least 20, 000 beads are acquired. Analyze the data using appropriate software such as FlowJo. Now we will go through the gating scheme utilized for analysis of major leukocyte populations using data from a representative control blood draw.More specific cell subsets can also be distinguished using the panel such as NKT cells or neutrophils, but these will not be shown in this example. First, an inclusion gate and exclusion gate are drawn around the tru count beads and placed on top of one another one to get the beads for counting and one to exclude the beads from the cellular analysis. Next, the cellular data excluding the beads is gated to include only single cells using forward scatter height versus forward scatter area.This gate also excludes small debris. The CD 45 positive cells are then gated, separating the granulocytes from the lymphocytes and monocytes using side scatter. The lymphocytes and monocytes are then separated into three distinct groups using CD 14 and side scatter.These include CD 14 negative lymphocytes, all CD 14 negative cells and non lymphocytes from the CD 14 Negative lymphocyte gate cells are first divided into three groups, CD three positive CD 19 negative CD three negative CD 19 positive and CD three negative CD 19 negative T cells are gated from the CD three positive CD 19 negative gait. After excluding CD 56 positive and CD 16 positive cells and CD four positive and CD eight positive T cells can be distinguished. B cells are gated from the CD three negative CD 19 positive gait after excluding CD 56 positive and CD 16 positive cells and NK cells are gated from the CD three negative CD 19 negative gait.After excluding possible contaminating monocytes using CD 11 C and CD four, the NK cells are divided into CD 56 brite, CD 56 DM and CD 56 negative populations. To distinguish dendritic cell population, all CD 14 negative cells are gated to exclude CD three positive, CD 19 positive, CD 56 positive and CD 16 positive cells. From there, the HL A DR positive cells are gated and myeloid dendritic cells and plasmacytoid dendritic cells can be distinguished by their CD 11 C and CD 1 23 expression.Lastly, from the non lymphocyte gait, monocyte subsets are gated after excluding possible contaminating CD 56 positive CD three positive and CD 19 positive cells. Non-classical, intermediate and classical monocytes are distinguished based on their CD 16 and CD 14 expression. Once the populations of interest are gated concentrations can be calculated.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. Our cells per microliter for each population. The equation shown here can be used for this Purpose.In this video, We demonstrated the procedure used by blood processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stain samples at a central assay lab. In the context of a multicenter clinical trial, we have demonstrated that the assay is simple and relatively shortened duration, making it feasible for blood processing laboratory technicians to perform it and to freeze and shift the samples to essential assay lab for analysis. 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 of the steps so that any anomalies that are discovered can be traced to the source of the air.The data generated from this staining panel can be used to track changes in leukocyte concentrations over time and could easily be further developed to assess activation states of specific cell types of interest.

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JoVE Journal

Immunology and Infection

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Source: Xu, C., et al. An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection. J. Vis. Exp. (2018).This video demonstrates the phenotyping of EBV-infected T and NK cell lines using flow cytometry, highlighting the preparation, antibody staining, and analysis steps to identify lineage-specific surface markers. It shows how marker profiles confirm distinct T-cell and NK-cell populations.

Phenotyping EBV-Infected Cell Lines Using Flow Cytometry

This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, γδ T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating strategy.

A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

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.

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

A protocol to measure peripheral blood leukocytes using a POCT card-based leukocyte analyzer is presented here. Same blood samples were tested by two automated hematology analyzers to evaluate the consistency and accuracy of the results. The results showed that the evaluated analyzer had a good correlation with the reference system.

Evaluation of a Point-of-Care Testing Analyzer for Measuring Peripheral Blood Leukocytes

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.

Depletion of Specific Cell Populations by Complement Depletion

Learn a standardized whole blood phenotyping assay for enumerating major leukocyte populations in multicenter clinical trials using flow cytometry. This method enables accurate tracking of immune responses over time.

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay (Video) | JoVE Journal

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

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