Becton, Dickinson and Company · NYSE:BDX
Becton, Dickinson and — Technical Presentation (2019)
35 slides from Becton, Dickinson and's technical presentation, 2019
Slide 1: CLL phenotyping with BD Rhapsody Targeted Panels and BD AbSeq Assays
CLL phenotyping with BD Rhapsody™ Targeted Panels and BD® AbSeq Assays BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respective owners. © 2019 BD. All rights reserved. 1
Slide 2: Background
Background • Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults and is characterized by a progressive accumulation of functionally incompetent lymphocytes in blood and bone marrow. • Immunophenotyping is the standard diagnostic tool for CLL. • Recent bulk RNA sequencing
Slide 3: Experimental design and workflow
Experimental design and workflow BD® Single-Cell Multiplexing Kit Healthy (8 tags) BD FACSMelody™ Sample Cell Sorter BD Rhapsody™ (4 samples) (sorting for B cells) Single-Cell Analysis System BD® AbSeq Assays BD Rhapsody™ (32 markers ) Targeted Panel (Human Immune Response Panel + Supplemental Panel
Slide 4: Experimental design and workflow
Experimental design and workflow BD® Single-Cell Multiplexing Kit Healthy (8 tags) BD FACSMelody™ Sample Cell Sorter (4 samples) (sorting for B cells) BD Rhapsody™ Single-Cell Analysis System BD® AbSeq BD Rhapsody™ Assays Targeted Panel (32 markers ) (Human Immune Response PBMCs Panel + Supplemental
Slide 5: Panel design
Panel design B-cell Sorting 32-Plex AbSeq Panel Marker Fluorochrome CD5 CD24 CD69 CD127 CD3 APC CD7 CD27 CD80 CD133 CD14 APC CD8 CD33 CD81 CD184 CD16 APC CD9 CD34 CD86 CD275 CD19 PE CD10 CD38 CD90 CD279 CD41a APC CD11c CD40 CD103 CD294 CD56 APC CD19 CD45 CD117 IgD CD235a APC CD20 CD45RA CD123 IgG •
Slide 6: Experimental design and workflow
Experimental design and workflow BD® Single-Cell Multiplexing Kit Healthy (8 tags) BD FACSMelody™ Sample Cell Sorter BD Rhapsody™ (4 samples) (sorting for B cells) Single-Cell Analysis System BD® AbSeq BD Rhapsody™ Assays Targeted Panel (32 markers ) (Human Immune Response PBMCs Sort and purity Pane
Slide 7: Confirming the purity of the sorted cells
Confirming the purity of the sorted cells Sort Gate of CD19 Cells Post Sort Purity Check • Purity of the sorted CD19+ B-cells were confirmed using a BD FACSMelody cell sorter. • On the left, the gate was set to sort on CD19+ cells. The chart on the right shows that the majority of the sorted cells a
Slide 8: Experimental design and workflow
Experimental design and workflow BD® Single-Cell Multiplexing Kit Healthy (8 tags) BD FACSMelody™ BD Rhapsody™ Sample Cell Sorter Single-Cell Analysis System (4 samples) (sorting for B cells) QC tool BD® AbSeq BD Rhapsody™ Assays Targeted Panel (32 markers ) (Human Immune Response PBMCs Panel + Supp
Slide 9: BD Rhapsody Scanner as a QC tool
BD Rhapsody Scanner as a QC tool • The viability and concentration of the sorted cells were calculated on the BD Rhapsody™ Scanner after cells were stained with viability dyes, Calcein and DRAQ7TM. • One of the healthy samples had poor viability and a low cell concentration and was excluded from the
Slide 10: Experimental design and workflow
Experimental design and workflow BD® Single-Cell Multiplexing Kit Healthy (8 tags) BD FACSMelody™ Sample Cell Sorter BD Rhapsody™ (4 samples) (sorting for B cells) Single-Cell Analysis System BD® AbSeq BD Rhapsody™ Assays Targeted Panel (32 markers ) (Human Immune Response PBMCs Panel + Supplemental
Slide 11: Flexible workflow
Flexible workflow • Post cDNA synthesis, the cell capture beads can be stored up to 12 weeks before proceeding to library preparation workflow. • In our case, this gave us the flexibility of waiting for more clinical research samples to arrive for single-cell capture on BD Rhapsody beads so that we
Slide 12: Experimental design and workflow
Experimental design and workflow BD® Single-Cell Multiplexing Kit Healthy (8 tags) BD FACSMelody™ Sample Cell Sorter (sorting for B cells) BD Rhapsody™ (4 samples) Single-Cell Analysis System BD® AbSeq BD Rhapsody™ Assays Targeted Panel (32 markers ) (Human Immune Response Panel + Supplemental panel
Slide 13: Targeted NGS library preparation
Targeted NGS library preparation • Following cDNA synthesis, we used a targeted approach for library preparation. • BD Rhapsody™ Immune Response Targeted Panel (Human) and a custom, CLL- specific, BD Rhapsody™ Supplemental Panel were used. 13 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq ar
Slide 14: Experimental design and workflow
Experimental design and workflow BD® Single-Cell Multiplexing Kit Healthy (8 tags) BD FACSMelody™ Sample Cell Sorter BD Rhapsody™ (4 samples) (sorting for B cells) Single-Cell Analysis System BD® AbSeq BD Rhapsody™ Assays Targeted Panel (32 markers ) (Human Immune Response PBMCs Panel + Supplemental
Slide 15: Clusters are well defined using mRNA and protein analysis
Clusters are well defined using mRNA and protein analysis t-SNE 15 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respective owners. © 2019 BD. All rights reserved.
Slide 16: Samples from healthy donors cluster similarly
Samples from healthy donors cluster similarly Healthy Samples t-SNE 16 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respective owners. © 2019 BD. All rights reserved.
Slide 17: CLL samples cluster separately and uniquely
CLL samples cluster separately and uniquely CLL Samples t-SNE 17 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respective owners. © 2019 BD. All rights reserved.
Slide 18: Expression of CD5 (AbSeq) and CD20 (AbSeq) correlates with disease-state
Expression of CD5 (AbSeq) and CD20 (AbSeq) correlates with disease-state CD5 (AbSeq) CD20 (AbSeq) Healthy vs CLL Samples t-SNE t-SNE t-SNE • Using BD AbSeq assays, the expression profile of CD5 is higher in the CLL samples while the expression profile of CD20 is higher in the healthy samples, consis
Slide 19: Expression of CLL markers is similar using AbSeq reagents and fluorochrome-conjugated antibodies
Expression of CLL markers is similar when determined using BD® AbSeq reagents and fluorochrome-conjugated antibodies Healthy CLL CD19 ++ ++ CD20 ++ + CD81 ++ - High expression: ++ CD10 + - Expression: + t-SNE t-SNE t-SNE CD11c - +/-* No expression: – CD38 + +/-* * Indicates heterogeneous CD5 - ++ po
Slide 20: Results using the complete AbSeq panel
Results using the complete AbSeq panel CD10 CD103 CD117 CD11c CD123 CD33 CD34 CD38 CD4 CD40 Healthy vs CLL Samples CD127 CD133 CD184 CD19 CD20 CD45 CD45RA CD5 CD69 CD7 t-SNE CD24 CD27 CD275 CD279 CD294 CD8 CD80 CD81 CD86 CD9 CD90 IgD IgG 20 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are
Slide 21: Evaluation of the top highly differentially expressed genes across donors
Evaluation of the top 5 highly differentially expressed genes across donors The heat map shows the top 5 highly expressed genes in each sample 21 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the pro
Slide 22: Evaluation of the top highly differentially expressed genes (DEGs) across donors
Evaluation of the top 5 highly differentially expressed genes (DEGs) across donors The heat map shows the top 5 highly expressed genes in each sample DEGs between CLL01 and CLL03 are similar. 22 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or
Slide 23: Evaluation of top highly differentially expressed genes across donors
Evaluation of top 5 highly differentially expressed genes across donors The heat map shows the top 5 highly expressed genes in each sample IGLC2 and IGLC3 expression is higher in healthy donors. 23 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company
Slide 24: Evaluation of unique protein expression across donors
Evaluation of unique protein expression across donors 24 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respective owners. © 2019 BD. All rights reserved.
Slide 25: Evaluation of the top differentially expressed features across CLL samples
Evaluation of the top 5 differentially expressed features across CLL samples CTLA4 CD38 t-SNE t-SNE CTLA4: • Associated with a higher number of cells in G0-G1 phase; may delay cell cycle progression • Potential useful immunotherapy strategy for patients with cancer • Important marker for determining
Slide 26: CLL04 has a CD79+ population detected by flow analysis
CLL04 has a CD79+ population detected by flow analysis CLL01 CLL02 CLL03 CLL04 CD79b CD79b CD79b CD79b CD23 CD23 CD23 CD23 • CLL01, CLL02 and CLL03 are examples of the flow analysis profiles typical for CLL patients. • CLL04 flow analysis reveals the presence of two populations. The lower population
Slide 27: CLL04 sample has 20% more prolymphocytes than a typical CLL sample
CLL04 sample has 20% more prolymphocytes than a typical CLL sample Prolymphocytes Healthy vs CLL Samples Typical CLL t-SNE 27 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respe
Slide 28: Further identification of sub-clusters across the different samples
Further identification of sub-clusters across the different samples Naïve NS Memory S Memory B Effector-memory Switch (S) memory cells don’t express IgD, instead they express IgG, IgA or IgE. • B cells in seven samples were characterized using the BD AbSeq markers for CD27 and IgD. • The samples clu
Slide 29: CLL04 sample contains S and NS memory cells
CLL04 sample contains S and NS memory cells Healthy vs CLL Samples B Effector-memory S Memory NS Memory Naïve t-SNE t-SNE • The three samples from healthy donors are primarily naïve memory cells. The two small populations near the bottom of the t-SNE plots also contain switched memory cells. • In co
Slide 30: CLL04 has similar percentages of S and NS memory cells
CLL04 has similar percentages of S and NS memory cells % Per Sample CD27 vs IgD 120 100 80 60 40 B Effector-memory 20 S Memory 0 NS Memory Naïve • The majority (>70%) of the populations for CLL01, CLL02, and CLL03, are switched memory cells. • However, for CLL04, about 55% of the cells are non-switc
Slide 31: Differential expression of analysis within the CLL04 cluster
Differential expression of analysis within the CLL04 cluster NS Memory Cells S Memory Cells IgD present in NS cells t-SNE CLL04 Cluster t-SNE_1 31 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the pr
Slide 32: NS and S cells equally express genes involved in B-cell differentiation and maintenance
NS and S cells equally express genes involved in B-cell differentiation and maintenance • IL4R: regulates IgE production; involved in the B-cell development pathways and cytokine signaling in the immune system • FCER2: regulates IgE production; plays essential role in B-cell growth and differentiati
Slide 33: Markers from T-cell lineage detected in higher levels
Markers from T-cell lineage detected in higher levels NS Memory S Memory CLL04 Cluster t-SNE_1 33 BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respective owners. © 2019 BD. All
Slide 34: Summary
Summary • Investigated differences between 4 CLL and 3 healthy samples with ~500 targeted genes and 32 BD AbSeq markers. • Multiplexing samples using the BD Single-Cell Multiplexing Kit allowed the investigation of up to 4 different samples simultaneously. • Significant heterogeneity was observed ac
Slide 35: Thank you!
Thank you! BD FACSMelody cell sorter is a Class 1 Laser Product. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, DRAQ, FACSMelody, Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. 35 All other trademarks are the prope
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