Flow Cytometry Services at QPS

Flow Cytometry is a laser‐based technology, capable of detecting

fluorescent markers on cells, bead, and other microparticls.

Combined with our strong biomarker, pharmacogenomics, and

cell‐based assay development capabilities, QPS’ flow cytometry

offerings can provide excellent support for both research and

clinical studies, particularly in the oncology and anti‐inflammatory

therapeutic areas. QPS can customize staining panels up to 8

colors (parameters) to analyzecomplex cellular processes.

Flow Cytometry Applications and Techniques

In flow cytometrysingle particles, most often cells, are analyzed

for size, complexity and fluorescence properties as they flow

through a beam of light. Multiple lasers are used to detect

different fluorescent properties (colors). This analysis can be used

to measure cell surface and intracellular biomarkers on cells and

to determine cell status and viability.

Instrumentation

The Becton Dickinson (BD) FACSCanto II Flow Cytometer

provides a flexible work platform, configured with three lasers to

detect up to eightcolors, maximizing the amount of information

acquired from individual samples.Innovations in the fluidics

system include a fixed alignment flow cell to minimize startup time

and improve reproducibility.The optical system has been

designed to maximize sensitivity and resolution for each color.

Two Flow Cytometry software packages provide resources and

flexibility for acquisition and analysis. The BD FACSCanto™

Clinical Software is used for FDA‐approved, In‐Vitro Diagnostics

assays including BD’s TBNK analysis kits. The TBNK kits are

capable of providing a complete immune panel (T‐, B‐ and NK

lymphocyte subsets) in a single tube, saving time and resources.

Additionally, when used with BD Trucount™ Tubes, the software

is able to determine absolute counts of cells per µL.

Immunophenotyping using BD Trucount™ Beads

Whole blood was collected with K2EDTA, and stained for cell surface

markers specific to neutrophils, monocytes and T cells in the presence

of BD Trucount beads. Each BD Trucount tube contains a lyophilized

pellet containing a known number of fluorescent beads. During sample

preparation, the beads are released into the sample. BD FACSCanto

clinical software was used to calculate absolute cell counts for each

defined cell population. Calculation to determine absolute cell counts

is: (# of events in region containing cell / # of events in absolute count

bead region) X (# of beads per test / test volume).

Trucount™Beads

monocytes

Neutrophils

RBCs

Granulocytes

Applications and Techniques

Flow Cytometry DescriptionApplication

Cell phenotyping Quantitation of cell populations (eg., neutrophils, T‐cells, B‐cells) and subpopulations (eg., cytotoxic T‐cells, regulatory T‐cells (Treg cells)) as percentages or as number of cell/µL.

Cell Viability Exclusion of dead cells from analyses.

Cell surface Expression levels of key receptors andexpression other cell surface biomarkers.

Intracellular Permeabilization of the cell membraneexpression allows for intracellular staining. May be performed in combination with cell surface staining.

Absolute cell counts Numbers of cell populations of interestusing BD Trucount™ are expressed in #cells/µL.Tubes

Receptor Occupancy To measure levels of drug‐target bindingStudies

CytometricBead Bead‐Based Immunoassays of solubleArray (CBA) biomarkers in individual samples (plasma or other biological matrix). Assays can be multiplexed to allow for panel ofseveral biomarkers to be analyzed at once within a small sample volume.

Cell Cycle Analysis Measures DNA content of cells to determine cell cycle status.

Cell Proliferation Capable of measuring cell divisions usingAssays cell tracer dyes.

Apoptosis Assays Measurement of regulated cell death using Annexin V Staining

CD3+ Tcells

CD4 + T helper cells

CD8 + Cytotoxic Tcells

In addition to use of our in‐house FACSCanto II instrumentation,

QPS also partners with Medical Centers using their on‐site flow

cytometry systems.

Support of Clinical Trials

QPS scientists are available to train or assist clinical sites in on‐site

stimulations or preparations of whole blood or cells.

Flow Cytometry PD Biomarker Analysis Supporting

Clinical Trials

Expression levels of two key receptors were determined on both

monocytes and neutrophils. Before bioanalysis of clinical trial

samples, the flow cytometry method was qualified according to a

fit-to-purpose approach including the following items: titration of

antibodies, precision and robustness. Figure 1 shows typical

populations of monocytes and neutrophils that were recovered

during the method qualification and the clinical trial. The neutrophils

were visualized by CD16-PE/Cγ7 antibodies and the monocytes

were visualized by CD14-APC antibodies. Figure 2 shows the effect

of the study drug on the percentage of monocytes positive or

negative for two different receptors. The qualification samples and

clinical trial samples were analyzed on a MACSQuant® Analyzer

(Miltenyi Biotec) located in the Flow Cytometry Centre of the

University Medical Centre Groningen (UMCG).

Figure 2: Percentage of monocytes positive or negative for two

different receptors in the absence (A) or presence (B) of study drug.

The expression of Receptor 2 decreases in the presence of the

drug, while Receptor 1 expression remains largely unchanged.

# of Events % of All % of T Absolute # of Events cells cells/µL of whole blood

All Events 3,619,377 NA NA NA

Lymphocytes 9,272 0.3 NA 2,494

T cells 7,434 0.2 NA 1,999

T helper cells 3,863 NA 52.0 1,039

CytotoxicT cells 2,311 NA 31.1 622

Granulocytes 11,893 0.3 NA 3,198

Neutrophils 6,120 0.2 NA 1,646

Beads 3,712 0.1 NA NA

Unlysed RBCs 3,590, 386 99.2 NA NA

Monocytes 2,406 0.1 NA 647

49,914 beads in TruCount pellet added to 50 µL of whole blood

Figure 1: Typical populations of monocytes and neutrophils in human heparin blood.