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

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).

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product

“…To measure the number of T reg cells present in the leukapheresis and the respective fractions derived from the immunoselection procedure (starting material, postdepletion, and postselection samples), we used a singleplatform technology based on an internal bead standard, a 6-color flow cytometer, and a sequential gating strategy to determine CD45 + CD4 + CD25 + absolute counts, similar to that described previously [53,54]. Briefly, 100 μL of whole leukapheresis blood was transferred to Becton Dickinson (BD) Trucount tubes and stained as detailed in Additional file 1: Table S2.…”

Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay

“…Standard flow cytometry-based assays, such as TruCount [4] on the other hand, can yield phenotypic and cell subset-specific information, but require resources and specialized expertise that is not readily accessible at many field sites, especially in resource-constrained settings. Development of alternative, bead-based methods that allow batched analysis of stained and cryopreserved WB samples have reduced workload at field sites, improved the cost-effectiveness and minimized the variability of the results, especially in studies involving multiple sites [5].…”

“…Standard flow cytometry‐based assays, such as TruCount [4] on the other hand, can yield phenotypic and cell subset‐specific information, but require resources and specialized expertise that is not readily accessible at many field sites, especially in resource‐constrained settings. Development of alternative, bead‐based methods that allow batched analysis of stained and cryopreserved WB samples have reduced workload at field sites, improved the cost‐effectiveness and minimized the variability of the results, especially in studies involving multiple sites [5]. However, real‐time staining of fresh cells followed by fixation still constitutes a potential source of variability, does not enable measurement of the expression of intracellular markers and relies on pre‐defined antibody panels that cannot be modified once the samples have been stored.…”