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1Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center
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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.
Hensley, T. R., Easter, A. B., Gerdts, S. E., De Rosa, S. C., Heit, A., McElrath, M. J., et al. Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay. J. Vis. Exp. (67), e4302, doi:10.3791/4302 (2012).
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 μl 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 (version 8/2010). 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).
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
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.
4. Thawing and Flow Cytometry Analysis
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.
5. Trucount Calculations
6. Representative Results
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. Click here to view larger figure.
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.
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 μl 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 °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 μl of blood, the method we present uses 100 μl. 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 μl and 100 μl 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 μl 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.
|CD45||Am Cyan||BD Biosciences||2D1|
|CD8||PerCP Cy5.5||BD Biosciences||SK1|
|CD56||PE Cy7||BD Biosciences||NCAM16.2|
|CD123||PE Cy5||BD Biosciences||9F5|
Table 1. Antibody staining panel to determine all major peripheral blood leukocyte populations.
No conflicts of interest declared.
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.
|Trucount Absolute Counting Tubes||BD Biosciences||340334|
|10X FACS Lysing Solution||BD Biosciences||349202|
|Category B & Exempt Shipping System, Insulated||Saf-T-Pak||STP-320|
|CD45 AmCyan monoclonal antibody||BD Biosciences||339192|
|CD3 FITC monoclonal antibody||BD Biosciences||349201|
|CD8 PerCp-Cy 5.5 monoclonal antibody||BD Biosciences||341051|
|CD4 Alexa Fluor 700 monoclonal antibody||BD Biosciences||557922|
|HLA-DR ECD monoclonal antibody||Beckman Coulter||IM3636|
|CD14 v450 monoclonal antibody||BD Biosciences||560349|
|CD19 PE monoclonal antibody||BD Biosciences||555413|
|CD16 APC-H7 monoclonal antibody||BD Biosciences||560195|
|CD56 PE-Cy7 monoclonal antibody||BD Biosciences||335791|
|CD11c APC monoclonal antibody||BD Biosciences||559877|
|CD123 PE-Cy5 monoclonal antibody||BD Biosciences||551065|
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