a Beckman Coulter Life Sciences: White Paper

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1 a Beckman Coulter Life Sciences: White Paper DuraClone improves standardization of compensation workflow for high content multicolor applications by flow cytometry Authors: Neha Girish 2, Sudharsan Sathyamurthy 2, Riddhi Bhide 2, Neema Jayadas 2,Valerie Mallet 1, Tewfik Miloud 1, Nathalie Dupas 1, Mike Carlson 3, Michael Kapinsky 1, Julie Vernier 1, Sylvain Monseaux 1, Soraya Kadour 1, Frederic Monsonis 1, James Tung 1, Vishakha Mangale 2, William Godfrey 3, Sridhar Ramanathan 2, Emmanuel Gautherot 1, Affiliation: 1 Beckman Coulter Life Sciences, Marseille, FRANCE., 2 Beckman Coulter India Pvt Limited, Bangalore Development Centre, Bangalore, INDIA 3 Beckman Coulter Life Sciences, Miami, FL, USA. This work was originally presented as a poster at ESCCA Conference, 2014, Lisbon.

2 DuraClone improves standardization of compensation workflow for high content multicolor applications by flow cytometry ABSTRACT Introduction: Multiparametric flow cytometry is a valuable tool for clinical research as it allows the use of multiple dyes, including tandems, to monitor several markers simultaneously. However, degradation of tandem dyes over time as a result of inconsistent reagent handling, light exposure, inappropriate storage conditions and/or manufacturing lot-to-lot variability can severely affect their spectral spillover and result in frequent adjustment in compensation. Beckman Coulter recently introduced DuraClone, a new proprietary format that allows antibody conjugate stabilization in a dry, unitized format enabling long-term storage at room temperature. The DuraClone format accommodates both multicolor panel and single color conjugate formats to enable easy compensation set-up while also improving standardization of compensation workflow in high content multicolor applications by flow cytometry. Study: The instrument was set-up using the Beckman Coulter Navios flow cytometer Autosetup scheduler to generate a robust and testable compensation matrix for routine use in multicolor panels, including those containing PE- and APC- tandems. The compensation matrix was set-up using a whole blood sample stained with DuraClone single color conjugates. The PMT settings are established by setting the X-Median of each single stain at 0.3 ± Positively stained cells are used by the Autosetup scheduler to estimate the spill-over and generate the compensation matrix. When the antigen is rarely or dimly expressed in biological samples, VersaComp positive beads will be spiked in the biological samples to obtain sufficient positive signal. PMT settings can be recorded using Flow-Set Pro Fluorosphere beads, allowing recurrent use of the established settings. Results: Taken together, the use of Beckman Coulter s proprietary DuraClone reagent technology and application of workflow tools such as the AutoSetup Scheduler, using the Navios software platform, enabled automatic compensation matrices to be established as applicable to multicolor panel design. These touch points reduce, or in some cases eliminate altogether, the need for reiterative fine adjustments in compensation outcomes. Conclusions: This study demonstrates the potential of our novel DuraClone reagent technology to deliver stable reproducible results for high content multicolor flow acquisition and analysis. BECKMAN COULTER WHITE PAPER 2

3 MATERIAL AND METHODS To demonstrate the compensation set-up and standardization (Fig 1) of the multicolor panels, two panels were used, DuraClone IM B cell (Table 1a) and DuraClone IM Dendritic cell panels (Table 2a). Generic single colors, with CD4 or CD8 specificities were used to set compensation for Non-Tandem dyes and Lot-matched conjugates from the panels were used for tandem dyes (Table 1b, 2b). The study was conducted using fresh human blood. Samples were processed according to a Stain-Lyse-Wash protocol using 2 ml VersaLyse lysing buffer for singles and DuraClone IM Dendritic Cell panel. One drop of positive VersaComp Beads was added to the CD123-APCA700 single tube with blood for staining. For the DuraClone IM B Cell panel, Wash-Stain-Lyse-Wash protocol was used. Processed samples were analyzed on a Navios flow cytometer equipped with 3 lasers capable of detecting 12 parameters at 20-bit resolution. These data were analyzed using Kaluza Software. Table 1 DuraClone IM B Cell Panel q Compensation kit for B Cell Panel q DuraClone IM B Cell Panel * Dye FITC PE ECD PC7 APC APC A750 (1) Pacific Blue* Marker IgD CD21 CD19 CD27 CD24 CD38 IgM CD45 Compensation Kit - B Cells (single color tubes) * Dye FITC PE ECD PC7 APC APC A750 (1) Pacific Blue* Marker CD4 CD4 CD19 CD27 CD4 CD38 CD4 CD8 Table 2 DuraClone IM Dendritic Cell Panel q DuraClone IM Dendritic Cell Panel * (coming soon) Dye FITC PE PC5.5 PC7 APC Marker CD16 Lineage (CD3/CD19/ CD20/CD14/ CD56) Compensation kit for Dendritic Cell panel q APC Pacific A700 (2) Blue* CD1c CD11c Clec9A CD123 HLA-DR CD45 Compensation Kit - Dendritic Cells (single color tubes) * Dye FITC PE PC5.5 PC7 APC APC Pacific A700 (2) Blue* Marker CD4 CD4 CD1c CD11c CD4 CD123 CD4 CD8 (1) APC-Alexa Fluor* 750 (2) APC-Alexa Fluor* 700 * For Research Use Only. Not for use in diagnostic procedures. BECKMAN COULTER WHITE PAPER 3

4 Fig 1: Instrument setup for PMT (Photomultiplier Tube) and Compensation Standardization on Navios Flow Cytometer (a) Initial setup (b) Setup for routine use (a) INITIAL SETUP (b) ROUTINE USE Set PMT Adjust PMT voltage to place negative populations of the single color stained conjugates in each channel at X-Median=0.3 ± 0.01 Record PMT for Standardized use Standardize PMTs Run Flow-Set PRO beads. Target intensities match requirements? NO Adjust PMT of Flow-Set Pro Protocol Adjust PMT voltage on Flow-Set Pro Standardized protocol to match target value requirements. Record New PMT settings. Flow-Set Pro Fluorospheres are run to acquire target intensity values for the established PMT settings. Settings saved as Flow Set Pro Standardized protocol. YES Adjust PMT settings of DuraClone Panel Protocol to match Flow-Set Pro Standardized protocol. Do not adjust compensation. Generate Compensation Matrix Use compensation tubes to generate matrix on the Auto Setup Scheduler for the Navios Flow Cytometer. Save Protocol as DuraClone Panel Protocol. Acquire Sample on DuraClone Panel Protocol Acquire Sample on DuraClone Panel Protocol BECKMAN COULTER WHITE PAPER 4

5 RESULTS Fig 2: Use of VersaComp Beads for dimly or rarely expressed antigen Example shown for compensation setup of DuraClone IM Dendritic cell panel, CD123-APC Alexa700 is dimly stained in blood. Therefore, VersaComp beads, spiked during staining of sample, were used as positive stained population during compensation setup. For PMT setup, negative of the Lymphocyte population was used to set X-Median at 0.3 Fig 3: DuraClone B Cell panel on Automated Compensation setup Figure 3 depicts typical gating strategy for the DuraClone IM B cell panel. All major B cell subtypes are identified with no adjustment in compensation. BECKMAN COULTER WHITE PAPER 5

6 Fig 4: DuraClone IM Dendritic cell panel on Automated Compensation Setup Figure 4 depicts DuraClone IM Dendritic cell panel when Autosetup is used to generate the compensation matrix. All circulating Dendritic cells are identified with no compensation adjustment required. Fig 5: Overlay of DuraClone IM B cell Panel across two Navios flow Cytometers Figure 5 depicts that the multicolor cocktail shows similar performance in terms of recruitment (i.e. % gated), across both of the flow cytometers, when the flow cytometers are setup using the DuraClone Standardized AutoSetup. BECKMAN COULTER WHITE PAPER 6

7 SUMMARY The use of the DuraClone reagent technology with the Navios AutoSetup Scheduler, or similar work tools, enables easy and accurate generation of a compensation matrix. The use of Flow-Set-Pro Fluorospheres and AutoSetup tools with the dry DuraClone reagent format enables standardization of high content multicolor flow cytometry. ORDERING INFORMATION Premixed, dry reagent cocktails to standardize and streamline flow cytometry workflow in clinical research studies involving the identification of principal populations of the human immune system. PN Panels FITC PE ECD PC5.5 PC7 APC A700 (1) APC- A700 (2) APC- A750 (3) Pacific Blue B53309 DuraClone IM Phenotyping Basic Tube CD16 CD56 CD19 CD14 CD4 CD8 CD3 CD45 B53318 DuraClone IM B cell Tube IgD CD21 CD19 CD27 CD24 CD38 IgM CD45 B53328* DuraClone IM T cell subsets Tube CD45RA CCR7 CD28 PD1 CD27 CD4 CD8 CD3 CD57 CD45 B53351* DuraClone IM Dendritic cell Tube CD16 Lineage (CD3/CD19/ CD20/CD14/CD56) CD1c CD11c Clec9A CD123 HLA-DR CD45 (1) Alexa Fluor*700 (2) APC-Alexa Fluor* 700 (3) APC-Alexa Fluor* 750 * PN B53328 and B53351 are available from December Available in convenient 25 test package. Each package includes three complete compensation kits with single color set of compensation reagents specific for each Panel. For Research Use Only. Not for use in diagnostic procedures. Navios is CE marked for 10-color in-vitro diagnostic use. In the U.S., Navios is intended for use as an in-vitro diagnostic device for immunophenotyping with Navios tetra software and CYTOSTAT tetrachrome reagents. All other uses are for research use only. * Pacific Blue and Alexa Fluor are registered trademarks of Molecular Probes, Inc. DuraClone, Navios, Kaluza, Flow-Set PRO Fluorospheres and are trademarks of Beckman Coulter, Inc. Beckman Coulter; the stylized logo are registered trademarks of Beckman Coulter, Inc and are registered in the USPTO. BECKMAN COULTER WHITE PAPER 7 FLOW-520WP10.14-A 2014 Beckman Coulter, Inc. All rights reserved.