The Impact of Preanalytical Variables in Blood: Enabling High Quality Protein Analysis

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1 The Impact of Preanalytical Variables in Blood: Enabling High Quality Protein Analysis David Craft, PhD Senior Manager Biological Sciences R&D BD Life Sciences Preanalytical Systems 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. 1

2 Outline for Today s Webinar Brief definition of the preanalytical phase General protein degradation and stabilization solutions Incretin peptide instability and stabilization solutions Platelet instability inducing α granule release Brief overview of other cell and biomarker stabilization solutions 2

3 Definition of the Preanalytical Phase 9 tips for successful presentations 3

4 Definition of the Preanalytical Phase 4

5 Blood Collection Tube Comparison 5 Yi et al., Proteome Res May;6(5):

LC/MALDI: Separation Enables Visualization of More Proteins 2 1 0 10 4 1537.001 1896.312 1060.

6 LC/MALDI: Separation Enables Visualization of More Proteins before after chromatography time 6 Mass

7 EDTA = Unstable Protein Sample: A Two-frame Movie EDTA 240 min 7

5mL System Components: - P100 Plasma Tubes (12) - Single Use Tube

8 BD TM P100 Blood Collection System for your Clinical Proteomics Research * ~3.5mL Plasma ~8.5mL System Components: - P100 Plasma Tubes (12) - Single Use Tube Holders - Blood Collection Sets Patents issued * For Research Use Only Not for Use in Diagnostic Procedures 8

9 P100 = Stable Protein Sample: Two-frame Movie P min 9 Analytical Letters, 2009

10 Pro BNP Cleavages In blood, BNP levels increase due to ventricular wall stretch and pressure overload observed in cardiac disease. 10

11 Enhanced Measurement of B-type Natriuretic Peptide Through the Use of P100 11

12 Veterinary Tube for NT-proBNP 12

13 Centrifugation Study: Benefits of Mechanical Separation 13

BD TM P800 for Metabolic Peptide Biomarkers Collaborators/Customers request a tube optimized for stabilization of common metabolic disease peptides

14 BD TM P800 for Metabolic Peptide Biomarkers Collaborators/Customers request a tube optimized for stabilization of common metabolic disease peptides Glucagon like peptide 1 (GLP 1) Gastric inhibitory polypeptide (GIP), Glucagon Oxyntomodulin * For Research Use Only Not for Use in Diagnostic Procedures 14

15 Incretin Peptide Degradation 15

16 GLP-1 Degradation by DPP-IV P800 Stabilization vs EDTA by MS Yi et al., PLoS One July;10(7) 16

17 GLP-1 Degradation by DPP-IV P800 Stabilization at the Point of Collection by ELISA Yi et al., PLoS One July;10(7) 17

18 What are ELISA and MS measuring? 18

19 GIP Mass Spectrometry Measurement EDTA vs P800 Yi et al., PLoS One July;10(7) 19

20 GIP Mass Spectrometry vs ELISA Eli Lilly built n-terminal sensitive ELISA and used P800 - Troutt et al., Clinical Chemistry :6: Yi et al., PLoS One July;10(7) 20

21 Oxyntomodulin Degradation by DPP-IV in EDTA and Stabilization in P800 Yi et al., PLoS One July;10(7) 21

22 Oxyntomodulin Degradation by proteases in EDTA and Stabilization in P800 Yi et al., PLoS One July;10(7) 22

23 Oxyntomodulin ELISA not sensitive to DPP-IV degradation Yi et al., PLoS One July;10(7) 23

24 Glucagon stabilization in P800 vs EDTA Glucagon (D1) Ln (Ratio) [G/aG] EDTA: T 1/2 = 20.4 h P800: T 1/2 = 45.6 h Time [h] Yi et al., PLoS One July;10(7) 24

25 Current Solutions to your Biomarker Stabilization Needs P100 Broad Spectrum Protection P800 Preservation of GLP-1, GIP, glucagon, and oxyntomodulin Publication list Rai et al., Proteomics 2005 Aug;5(13): Yi et al., J Proteome Res May;6(5): Yi et al., J Proteome Res Dec;7(12): O Mullan et al., Clin. Chem. Lab. Med. 2009; 47(6): Craft et al, Anal. Lett. 2009; 42: Yi et al, PLoS One 2015 July;10(7) bdbiosciences.com/us/cbp * For Research Use Only Not for Use in Diagnostic Procedures 25

Platelets are a Rich Source of Chemical Signaling Molecules Platelet storage granules possess a wealth of chemical signals that modulate thrombosis, inflammation, angiogenesis, and wound healing.

26 Platelets are a Rich Source of Chemical Signaling Molecules Platelet storage granules possess a wealth of chemical signals that modulate thrombosis, inflammation, angiogenesis, and wound healing. Activation triggers selective or bulk degranulation which results in both paracrine and autocrine signal transduction. Preanalytically induced platelet degranulation can result in falsely elevated biomarker concentrations in plasma. 26 Rev Esp Cardiol (10):

Novel Chemistries lmproved EDTA for Biomarker/Platelet Stabilization Problem: How can we balance the benefits of EDTA with the challenge of platelet degranulation?

27 Novel Chemistries lmproved EDTA for Biomarker/Platelet Stabilization Problem: How can we balance the benefits of EDTA with the challenge of platelet degranulation? EDTA: Provides a lower protease activity sample environment Compatibility with nucleic acid testing No plasma dilution Stable shelf life in plastic tubes of multiple dimension But Results in robust platelet α granule release CTAD: Provides strong platelet inhibition against a citrate background Designed for compatibility with coagulation testing But Additive is light sensitive Generates a 10% plasma dilution Requires a glass tube for maximum shelf life 27

Improved EDTA is a Potent Platelet Stabilizing Formula Flow cytometry was used to evaluate platelet degranulation via CD62P and CD63 expression in

Improved EDTA inhibits combined EDTA and agonist (ADP or TRAP) induced platelet release.

28 Improved EDTA is a Potent Platelet Stabilizing Formula Flow cytometry was used to evaluate platelet degranulation via CD62P and CD63 expression in whole blood. EDTA whole blood samples were stimulated with ADP or TRAP to induce further degranulation. Improved EDTA inhibits combined EDTA and agonist (ADP or TRAP) induced platelet release. EDTA Resting ADP TRAP Granule Release (Mean ±SD, n = 3) Dense Granule/Lysosome Release (Mean ±SD, n = 3) EDTA (Improved) % CD62P + Events % CD63 + Events Resting ADP TRAP CD62P 0 EDTA (Improved) EDTA EDTA EDTA (Improved) CD63 28

Improved EDTA Reduces α Granule Release Over Time Flow cytometry was used to evaluate platelet CD62P expression over 24 hours of whole blood dwell.

29 Improved EDTA Reduces α Granule Release Over Time Flow cytometry was used to evaluate platelet CD62P expression over 24 hours of whole blood dwell. EDTA (Improved) provided superior CD62P levels compared to EDTA alone. No statistically significant differences in %CD62P + platelets were observed between CTAD and EDTA (Improved) until 24 hours (P < 0.05, two-way ANOVA). No statistically significant differences in MFI were observed between CTAD and EDTA (Improved) throughout 24 hours (P > 0.05, two-way ANOVA). 0 hrs 2 hrs 8 hrs 24 hrs CD62P 29

30 Improved EDTA Provides Superior Background Levels Current Status Initial feasibility testing complete. Define key customer and product requirements. Expand to additional markers? Optimize for post-24 hour stability? Limited shelf-life and sterilization activity characterization 30

31 Technical Summary: Anticoagulation with EDTA induces rapid and robust platelet α granule release, the extent of which varies across individuals and is unpredictable. Platelet degranulation results in non-physiological background levels of various biomarkers such as PDGF a/a, TGF β1, VEGF, and RANTES. Broad spectrum platelet signal transduction suppression mitigates EDTA mediated platelet α granule release as measured by flow cytometry and ELISA. Improved EDTA formula enables consistent and predictable background concentrations of platelet released biomarkers across a 24 hour time period of whole blood sample dwell and provides similar performance to CTAD. 31

BD Vacutainer CPT System Features & Benefits Sterile, closed-system method for isolating mononuclear cells from venous blood For diagnostic Use CE, 510(k) Available with Sodium Citrate (4 ml and 8

32 BD Vacutainer CPT System Features & Benefits Sterile, closed-system method for isolating mononuclear cells from venous blood For diagnostic Use CE, 510(k) Available with Sodium Citrate (4 ml and 8 ml) or Sodium Heparin (8 ml) anticoagulant FICOLL TM Hypaque TM density fluid facilitates mononuclear cell separation Polyester gel provides eliminates need for tedious blood layering and prevents accidental mixing of separated cells Centrifugation time is minutes Enables shipment of isolated mononuclear cells in autologous plasma 32

33 PAXgene Blood RNA Tube Features & Benefits 2.5 ml draw volume Preservation of cellular RNA at point of collection Sample stabilization during transport and storage o up to 3 days at C o up to 5 days at 2-8 C o 7 years storage at -20 C or -80 C (studies ongoing ) RNA is stable for 3 freeze/thaw cycles For diagnostic Use CE, 510(k) in 2005 System integrated manual and automated RNA and mirna isolation kits for use on QIAcube, QIAsymphony or BioRobot Universal for research studies 33

34 PAXgene Blood ccfdna Tube Features & Benefits 10.0 ml draw volume Stabilization of circulating, cell-free DNA (ccfdna) Sample stabilization during transport and storage o Up to 7 days at room temperature (15 25 C) o Up to 24 hours at 35 C For Research Use Only Designed for use with QIAamp Circulating Nucleic Acid Kit 34

35 BD Vacutainer Specialist Blood Collection Tubes PAXgene Blood ccfdna System Stabilization of circulating, cellfree DNA (ccfdna) from plasma PAXgene Tissue System (RUO) bdbiosciences.com/us/cbp 35

36 Thank You bdbiosciences.com/us/cbp 36

Circulating Cell-Free DNA Pre-analytics: Importance of ccfdna Stabilization and Extraction for Liquid Biopsy Applications

Circulating Cell-Free DNA Pre-analytics: Importance of ccfdna Stabilization and Extraction for Liquid Biopsy Applications Martin Schlumpberger, Ass.Director R&D, QIAGEN GmbH IQNpath 2017 - Circulating