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][2][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). Video LinkThe video component of this article can be found at http://www.jove.com/video/4302/ Protocol Note: To protect the fluorophore-conjugated antibodies from light, perform all steps in a bio-safety cabinet with the light off.

Optimization of a whole blood phenotyping assay for enumeration of peripheral blood leukocyte populations in multicenter clinical trials

“…Blood was collected from volunteers in ACD anticoagulant; 100µl of whole blood was transferred to Becton Dickinson (BD) Trucount tubes as detailed previously (Hensley et al, 2012) and stained using the 11 color TP1 antibody staining panel, containing fluorescently-labeled monoclonal antibodies specific for the major cellular populations in blood (Table 1). Staining parameters were varied to determine their effect on staining stability as described in section 2.4.…”

“…After staining, erythrocytes were lysed, the remaining cells were fixed using BD FACSLyse (BD Bioscience), and variations in the storage and shipping conditions were tested as described in section 2.4. The Trucount tubes were then analyzed using a BD LSRII flow cytometer with a configuration as previously published (De Rosa et al, 2012), and the number of cells per microliter of whole blood was calculated as previously described (Hensley et al, 2012). …”

“…The gating strategy to distinguish subpopulations follows, with slight modifications, the previously published strategy (Hensley et al, 2012) (Figure 1). Briefly, our gating strategy first used the APC and PE-Cy5 channels to draw an inclusion gate around the Trucount beads to gate them for counting, and an exclusion gate to exclude them from subsequent cellular analysis.…”

“…We present data to quantify the variability of the assay and demonstrate the high correlation between counts obtained using TP1 or a CBC in a vaccine trial setting. Together, the definition of these conditions led to an optimized protocol that we are currently implementing in multicenter clinical trials in the HIV Vaccine Trials Network (Hensley et al, 2012). This protocol is adaptable for use in different experimental medicine trials and represents a promising tool to better elucidate the early immune responses by assessing trafficking and activation state of key innate cells such as dendritic cells, monocytes and NK cells.…”

Initial Diagnostic Workup of Acute Leukemia: Guideline From the College of American Pathologists and the American Society of Hematology

“…These latter, detrimental effects appear to depend, in part, on the protocols used for cryopreservation and cell storage and, perhaps, on the cell lineage. [205][206][207][208][209][210][211][212][213][214][215][216] Cryopreservation procedures require mononuclear cells be suspended in a solution containing a cryoprotective agent (usually dimethyl sulfoxide), cooled in a cooling device to the storage temperature, and then stored in liquid nitrogen (for use in studies in which viable cells are necessary) or a À808 freezer, which is satisfactory for most molecular studies. Cryopreservation does require specialized equipment and storage facilities, which may not be available in all laboratories.…”