DuraClone reagents are a robust solution for standardizing multicolor flow cytometry applications

Authors: Nathalie Dupas1, Snehita Sattiraju2, Neha Girish2, Emmanuel Gautherot1, Sridhar Ramanathan2, Tewfik Miloud1

Affiliation: 1 Beckman Coulter Life Sciences, Marseille, FRANCE. 2 Beckman Coulter India Pvt Limited, Bangalore Development Centre, Bangalore, INDIA

This work was originally presented as a poster at CYTO 2014 Conference, Ft. Lauderdale, Florida, USA.

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DuraClone reagents are a robust solution for standardizing multicolor flow cytometry applications

ABSTRACT

Introduction

Multicolor flow cytometry is a powerful tool to study immune responses. Standardization of sample preparation and analysis methods is crucial for being carried out at multiple sites across multiple time points. The use of Flow-Set Pro fluorospheres to set instrument settings, a standard operating procedure combined with central data analysis, contributes significantly to the standardization. However, additional parameters such as reagent handling and the degradation of the antibody conjugate cocktail due to light exposure or inappropriate storage conditions may contribute to assay variability. To circumvent these drawbacks, Beckman Coulter has developed the DuraClone technology that allows antibody conjugate stabilization in a dry reagent format and enables long term storage at room temperature.

Study

Using a 9-color dry reagent cocktail for the identification of the main white blood circulating cells, it has been demonstrated that the performance of the antibody-fluorochromes is not impacted by the drying process. Moreover, this dry reagent format is compatible with the most commonly used red blood cell lysis reagents and sample preparation protocols. A preliminary stability study was performed by exposure of the dry reagents cocktail to 37°C for 14 days then 60°C for 7 days. The performance of the dry reagents cocktail in the study was similar to its liquid counterpart which was stored at 4°C.

Conclusion

This study demonstrates the potential of DuraClone technology to generate a stable and functional dry reagent format which can be a powerful tool for the standardization of studies being conducted by multiple laboratories across multiple time points studies.

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1. DuraClone: Dry Unitized Reagents for Multicolor Applications

DuraClone technology allows antibody conjugate stabilization in a dry reagent format. As shown in Figure 1A. brightness is conserved across all dyes (including tandem conjugates) in the dry format. Using a 9-color panel liquid panel (ONE Study Immune Phenotyping panel 1) as reference, we demonstrated the recruitment of positive cells and spillover of the various fluorochrome are not affected by the drying process (Figure1B).

FITC PE ECD PC7 APC APC Alexa Fluor* 700

APC Alexa Fluor* 750

Pacific Blue*

Krome Orange

CD16 CD56 CD19 CD14 CD4 CD8 CD3 CD54 CD45

Figure 1: Representative dot plots displaying the overlayed results of whole blood samples for comparison of liquid vs DuraClone formats. (A) Single colors (B) Multicolor panel. Red blood cell lysis was performed according to Versalyse protocol.

AKappa-FITC

CD56-APC

IgM-PC7CD25-PC.5.5CD19-ECDCD7-PE

CD8-APCAlexa Fluor 700

CD45RA-APCAlexa Fluor 750

CD19Pacific Blue

CD4Krome Orange

Dry Liquid

B

Dry

Liquid

CD45-Krome Orange

CD16-FITCCD8-APC A700CD3-APC A750

CD64-Pacific BlueCD16-FITCCD14-PC7CD16-FITC

CD3-APC A750

SS IN

T

SS IN

T

SS IN

T

CD14

-PC7

CD14

-PC7

CD19

-ECD

CD56

-PE

CD4-

APC

CD56

-PE

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B Mean Fluorescence Intensity Cell RecruitmentCD56hi NK cells

CD56+ NK cells

CD8+ T cells

CD4+ T cells

CD3+ T cells

B cells

CD14+ CD64+ CD16+ Mo

CD14lo CD16+ Mo

CD14+ CD16+ Mo

CD14+ CD16- Mo

CD14+ Mo

Granulocytes

201 days 114 days

0 day

C

Mean

Fluo

resc

ence

Inten

sity

FITC+ LyPE+ LyECD+ Ly

PC7+ MoAPC+ LyAPC A700+ Ly

APC A750+PB+ MoKrO+ Ly

2. DuraClone: Long term storage at room temperature

To assess the stability of DuraClone panels stored at room temperature (18-25°C), three whole blood samples from healthy donors were stained with a freshly prepared DuraClone panel and compared to two older lots of various ages. We observed very consistent staining independent of the panel age, indicating that over a period of 200 days the DuraClone format prevents conjugate degradation (Figure 2A). The low MFI variation over time (Figure 2B) and similar cell % gated across the three donors (Figure 2C), confirms that performance of the DuraClone panels are stable over time when stored at room temperature.

Figure 2: (A) An overlay of the data shows the consistent perfomance of the DuraClone panel independent of their age. (B) The MFI of positively stained cells (among lymphocytes or monocytes) for each fluorochrome obtained with the different lots. Donor 1, shown here, is representative of the data obtained with 3 donors. (C) % gated of positive cells was evaluated applying the gating strategy shown in A according to Streitz et al1.

A

Lot A: Fresh

SS IN

T

SS IN

T

SS IN

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CD3-

APC

A750

CD56

-PE

CD8-

APC

A700

CD14

-PC7

CD64

-Pac

ific B

lue

CD16

-FIT

CCD

64-P

acific

Blue

CD45-Krome Orange CD45-Krome Orange CD14-PC7 CD16-FITC CD16-FITC

CD56-PECD4-APCCD3-APC A750CD19-ECDFS INT

Lot B: 200 day old

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3. DuraClone: Temperature Stable

DuraClone sensitivity to temperature variation, that could occur during transport and/or storage, was assessed by storing a 9-color panel for 14 days at 37°C and then 7 days at 60°C. The stressed panel performs as well as its counterpart kept at room temperature.

Figure 3: Representative dot plots displaying the overlayed results of whole blood samples for comparison of dry kept at room temperature vs dry kept in stress conditions. Red blood cell lysis was perfromed according to Versalyse protocol.

Stress (heated)

RT

CD45-KrO CD16-FITC

CD14-PC7

CD8-APC A700

CD3-APC A750

CD64-PB

CD16-FITC

CD14

-PC7

CD3-APC A750

CD14

-PC7

CD4-

APC

CD56

-PE

CD19

-ECD

SS IN

T

SS IN

T

SS IN

T

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4. DuraClone: Compatible with Intracellular staining

Simultaneous staining of intra- and extra-cellular epitopes in a 6-color DuraClone panel can be efficiently achieved using the PerFix-nc kit (Part # B31168). Figure 4A shows FoxP3 staining as well as extra-cellular gating marker staining being comparable to their liquid counterparts. Additionally, cell recruitment using the DuraClone format is also conserved (Figure 4B).

FITC PE PC7 Alexa Fluor 647 Pacific Blue Krome OrangeCD6 CD25 CD4 FoxP3 CD3 CD45

Figure 4: (A) Representative dot plots displaying the overlayed results of whole blood samples for comparison of DuraClone vs liquid. Staining and red blood cell lysis was performed according to the PerFix-nc protocol optimized for FoxP3 staining (B) Recruitment of positive cells was evaluated applying the gating strategy shown in A.

B

CD45-KO CD4-PC7

CD25-PE

CD4-PC7

CD25-PE

CD25

-PE

SS IN

TFo

xP3-

A647

CD6-

FITC

CD3-

PB

A

CD4+ T cells FoxP3+ cells FoxP3+ CD25+ cells CD4+ CD25+ cells

Dry

Liquid

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CONCLUSION

This study demonstrates that the DuraClone format generates a stable, functional reagent that can be applied to many flow cytometry applications. It can be a powerful tool for improved standardization of high content multicolor flow cytometry.

REFERENCES

1) Standardisation of whole blood immune phenotype monitoring for clinical trials: Panels and methods from “The ONE Study”. Mathias Streitz, Tewfik Miloud, Michael Kapinsky, Michael R. Reed, Robert Magari, Edward K. Geissler, James A Hutchinson, Katrin Vogt, Stephan Schlickeiser, Anders Handrup Kverneland, Christian Meisel, Hans-Dieter Volk, Birgit Sawitzki. Transplantation research. 2013 Oct 25;2(1):17

Research Use Only, Not Intended for Diagnostic Purposes

* Alexa Fluor and Pacific Blue are trademarks of Molecular Probes Inc.

DuraClone, ECD, Krome Orange, Gallios, Kaluza and Versalyse are trademarks of Beckman Coulter, Inc. Beckman Coulter and the stylized logo are trademarks of Beckman Coulter, Inc and are registered in the USPTO.

FLOW-226WP09.14-A