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

Summary

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.

Abstract

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).

Protocol

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

1. 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.
2. 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....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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