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
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
Definition of the Preanalytical Phase 9 tips for successful presentations 3
Definition of the Preanalytical Phase 4
Blood Collection Tube Comparison 5 Yi et al., Proteome Res. 2007 May;6(5):1768-81.
LC/MALDI: Separation Enables Visualization of More Proteins 2 1 0 10 4 1537.001 1896.312 1060.802 1465.965 1211.958 882.674 1348.998 1778.279 2021.388 1606.140 before after chromatography time 6 Mass
EDTA = Unstable Protein Sample: A Two-frame Movie EDTA 240 min 7
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
P100 = Stable Protein Sample: Two-frame Movie P100 240 min 9 Analytical Letters, 2009
Pro BNP Cleavages In blood, BNP levels increase due to ventricular wall stretch and pressure overload observed in cardiac disease. 10
Enhanced Measurement of B-type Natriuretic Peptide Through the Use of P100 11
Veterinary Tube for NT-proBNP 12
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 Glucagon like peptide 1 (GLP 1) Gastric inhibitory polypeptide (GIP), Glucagon Oxyntomodulin * For Research Use Only Not for Use in Diagnostic Procedures 14
Incretin Peptide Degradation 15
GLP-1 Degradation by DPP-IV P800 Stabilization vs EDTA by MS Yi et al., PLoS One. 2015 July;10(7) 16
GLP-1 Degradation by DPP-IV P800 Stabilization at the Point of Collection by ELISA Yi et al., PLoS One. 2015 July;10(7) 17
What are ELISA and MS measuring? 18
GIP Mass Spectrometry Measurement EDTA vs P800 Yi et al., PLoS One. 2015 July;10(7) 19
GIP Mass Spectrometry vs ELISA Eli Lilly built n-terminal sensitive ELISA and used P800 - Troutt et al., Clinical Chemistry 2011 57:6:849-855. Yi et al., PLoS One. 2015 July;10(7) 20
Oxyntomodulin Degradation by DPP-IV in EDTA and Stabilization in P800 Yi et al., PLoS One. 2015 July;10(7) 21
Oxyntomodulin Degradation by proteases in EDTA and Stabilization in P800 Yi et al., PLoS One. 2015 July;10(7) 22
Oxyntomodulin ELISA not sensitive to DPP-IV degradation Yi et al., PLoS One. 2015 July;10(7) 23
Glucagon stabilization in P800 vs EDTA Glucagon (D1) 3.5 3 Ln (Ratio) [G/aG] 2.5 2 1.5 1 EDTA: T 1/2 = 20.4 h P800: T 1/2 = 45.6 h 0.5 0 0 10 20 30 40 50 60 70 80 Time [h] Yi et al., PLoS One. 2015 July;10(7) 24
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):3262-77. Yi et al., J Proteome Res. 2007 May;6(5):1768-81. Yi et al., J Proteome Res. 2008 Dec;7(12):5112-8. O Mullan et al., Clin. Chem. Lab. Med. 2009; 47(6): 985-693. Craft et al, Anal. Lett. 2009; 42:1398-1406. 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. 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 2009 62(10): 1161 78
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 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 100 80 60 40 20 % CD63 + Events 100 80 60 40 20 Resting ADP TRAP CD62P 0 EDTA (Improved) 0 0 05 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. 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
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
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 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 15 20 minutes Enables shipment of isolated mononuclear cells in autologous plasma 32
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 18-25 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
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
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
Thank You bdbiosciences.com/us/cbp 36