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 titer...

<ol>
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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 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

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JoVE Journal

Immunology and Infection

14.9K Views.  Fred Hutchinson Cancer Research Center. In questo rapporto, dimostriamo la procedura di colorazione e l'analisi di un test di fenotipizzazione effettuato su sangue intero fresco per enumerare le principali popolazioni innata e adattiva dei leucociti. Sottolineiamo considerazioni per l'esecuzione di queste procedure nel contesto di uno studio clinico multicentrico.