Mimi Roy, PhD Senior Director & Site Head Caprion Proteomics US LLC. ISBER, Orlando May 23, 2014

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1 Controlled Analysis of Preanalytical Variables in CSF and Blood Sample Collection, Processing and Storage: Implications for Best Practices in Clinical Research Mimi Roy, PhD Senior Director & Site Head Caprion Proteomics US LLC ISBER, Orlando May 23,

2 NCI Contract Goal ( ): Aim: Test and develop highly specific quality assessment resources, tools and guidelines for the collection, manipulation and storage of human blood-derived biospecimens for protein analysis by proteomics Workflow: Computer-aided clinical sample collection Characterize protein content and stability in blood samples after collection and induction of probable sources of variation Provide recommendations on how to process samples Develop multiplexed assay to assess sample quality and history 2

3 Study Design (Cancer & Healthy; Male & Female) Blood collection tube types EDTA EDTA+PI P100 Serum Heparin Time & temperature on bench hours ⁰C 20 ⁰C 20 ⁰C 37 ⁰C Freeze-thaw cycle 1 to 5 cycles Time in freezer months ⁰C -80 ⁰C 3

4 # of Subject # of Subject Clinical sample collection Healthy Female, total 25 Breast Cancer, total Healthy Male, total 25 Age range Prostate Cancer, total Age range Study subjects samples Pilot Total

5 Controlling complex sample processing Custom on-site Tablet PC process control and monitoring 5

Tablet PC Data Analysis Variability of time on bench prior to centrifugation Very close adherence to time points on first day Second day time points are off-set from protocol and

6 Tablet PC Data Analysis Variability of time on bench prior to centrifugation Very close adherence to time points on first day Second day time points are off-set from protocol and more variable Delays are mostly caused due to overlapping patient visits Study conducted to assess compliance with pre-set time lines for sample processing by analysis of PC Tablet data 6

7 Tablet PC Data Analysis Comparison of two operators following the same SOP Operator 1 is late and more variable Difference in performance of 2 different operators 7

8 Conclusions: Software for Sample Collection The use of software to follow samples and processes aids sample collection, can guide timely processing It also enables accurate data collection on actual sample collection and processing times/variations SOPs are critical for robust and careful sample collection, operators following the same protocol may vary in performance. Operators could be qualified. The scheduling of multiple patients and overlaps in critical steps need to be planned during human specimen collection 8

, Analytical chemistry 2003 Sep 15; 75(18):4818-26 2D: Roy & Becker, Methods Mol. Biol.

9 Analytical Approach for Discovery: Label-free Differential Profiling MARS-14 depletion of abundant proteins Digestion to peptides LC-MS Plasma Or Serum 1D: Wang et al., Analytical chemistry 2003 Sep 15; 75(18): D: Roy & Becker, Methods Mol. Biol. 2007;359: D: Informatics-based Quantitative Peptide Expression Profiling Statistical Analysis Protein Identification Peptide Sequencing QExactive 9

10 MRM Discovery Protein Identification Differential Expression? Candidate Biomarkers Build Targeted Multiplexed Assay Test Independent Samples Biomarker Verification and Validation 10

11 Decreasing over incubation time plasma at 37 ⁰C Ceruloplasmin EDTA Serum Heparin Identified unique peptides : 15 11

12 Control Control Control DECREASING OVER INCUBATION TIME POST SPIN AT 20 AND 37 O C Vitronectin 0.5h 20 o C 48h 96h 37 o C 48h 96h 0.5h 20 o C 48h 96h 37 o C 48h 96h 0.5h 20 o C 48h 96h 37 o C 48h 96h 0.5h Control: 20 C, 0.5 h for Post-Spin Identified unique peptides : 11 12

13 WHOLE BLOOD CELL LYSIS MARKER Hemoglobin subunit alpha Control Pre spin 0.5h 48h Post spin Control Pre spin Post spin 48h 0.5h 48h 48h 0.5h Control: 20 C, 0.5 h for Pre & Post-Spin Control Pre spin 0.5h 48h Post spin 48h Identified unique peptides : 9 13

14 Sum of peptide intensities EDTA tubes affect calcium ion dependent leukocyte binding proteins EDTA P100 Serum Heparin 0.5h 24h 0.5h 24h 0.5h 24h 0.5h 24h EDTA tubes 0.5h Blood incubation at 20 o C Non-EDTA tubes 14

15 EDTA tubes affect calcium ion dependent leukocyte binding proteins 15

16 Verification Abundance Discovery Abundance EDTA tubes affect calcium binding proteins (e.g. Protein S100) EDTA P100 Serum Heparin (A) AUC=1 N C N C N C Control 0.5h Plasma 48h Blood 48h N C N C N C Control 0.5h Plasma 48h Blood 48h N C N C N C Control 0.5h Serum 48h Blood 48h N C N C N C Control 0.5h Plasma 48h Blood 16 48h

17 Sensitivity Sensitivity Whole blood cell lysis marker (B) Profilin AUC= Specificity Plasma/Serum incubation (C) C P B C P B C S B C P B Complement C AUC= Specificity C P B C P B C S B C P B 17

18 Freeze-thaw cycle Blood samples Centrifuge Plasma/Serum 1x to 5x F/T cycles 1 h at RT, 1 day in -80 C for each cycle 10 subjects, 4 tube types RT, 1 h -80 C, 23 h 18

19 Unique peptide Protein intensities changing over F/T cycles Intensity ratios compared to Cycle 1 2 vs 1 3 vs 1 4 vs 1 5 vs 1 FN2 VWF ACTB Thresholds: p < 0.05, q < 0.05, fold-change >1.5 Decrease Increase 19

20 Unique peptide Freeze-thaw analysis: Comparison of tube types Intensity ratios (5 vs. 1 Cycle) Serum P-100 Heparin EDTA EDTA+PI APOB APOC2 VWF FN1 ACTB TPM4 Decrease Increase Mechanical Separator thresholds p < 0.05, q < 0.05, fold-change >1.5 20

21 MRM verification FT cycle Protein degradation over freeze/thaw cycle 21

22 MRM verification FT cycle Protein degradation over freeze/thaw cycle 22

23 MRM verification FT cycle Protein degradation over freeze/thaw cycle 23

24 Discovery Verification MRM verification FT cycle Protein degradation over freeze/thaw cycle (Fibronectin) 24

25 MRM verification FT cycle Stable to Freeze/Thaw 25

26 PCA Plot: Freeze-Thaw Cycles 26

MRM PANEL Verified proteins (Freeze/Thaw) Marker Type UniProtID (Human) Protein Description Tube Types EDTA P100 Serum Heparin Change p-value # Freeze/Thaw APOC2 Apolipoprotein C-II o o o o -1.7 0.

27 MRM PANEL Verified proteins (Freeze/Thaw) Marker Type UniProtID (Human) Protein Description Tube Types EDTA P100 Serum Heparin Change p-value # Freeze/Thaw APOC2 Apolipoprotein C-II o o o o E+00 Freeze/Thaw APOC3 Apolipoprotein C-III o o o o E+00 Freeze/Thaw APOC4 Apolipoprotein C-IV o o o E-08 Freeze/Thaw APOE Apolipoprotein E o E-03 Freeze/Thaw APOM Apolipoprotein M o E-04 Freeze/Thaw VWF von Willebrand factor o E-02 Freeze/Thaw FINC Fibronectin o E+00 Fold 27

28 Conclusions: Freeze Thaw Discovery & Verification Studies Over 21 proteins identified as potential markers of degradation due to F/T. Gradual degradation with every F/T cycle. Apolipoproteins for all tube types, and fibrinogens/ clotting factors particularly for Heparin tubes, degrade over F/T. A few proteins are observed probably due to cell lysis upon F/T. P100 and Serum tube types protect better than EDTA and Heparin tubes against F/T cycles. 28

29 Study Design: Storage time in freezer Tube type EDTA P100 Serum Heparin Time in freezer -20 ⁰C 6 months 12 months 18 months -80 ⁰C 6 months 12 months 18 months Subject Cancer 10 Breast cancer 10 Prostate cancer Normal 10 healthy female 10 healthy male Control: 0-2 months in -80 ⁰C 4 tube types X 1 condition X 40 subject = 160 samples Each time in freezer: 4 tube types X 2 conditions X 40 subject = 320 samples 29

30 NCI Study 2b 6 vs. 12 vs. 18 month in freezer Greater impact of -20⁰C vs. -80⁰C on protein degradation Peptides observed in 6/12/18 month in freezer study: 4/2/1 30

31 NCI Study 2b 6 vs. 12 vs. 18 month in freezer Freezing causes cell lysis Peptides observed in 6/12/18 month in freezer study: 7/7/6 31

32 Sample integrity study Time in Freezer (Example proteins) 32

33 NCI Study 2b 6 vs. 12 vs. 18 month in freezer Stable in 6 month, but over 12 month freezing induces cell lysis Peptides observed in 6/12/18 month in freezer study: 2/1/2 33

34 Study 2b 6 vs. 12 vs. 18 month in freezer Stable over 18 months at both temperatures 34

35 Sample integrity study Time in Freezer 35

36 Verification 2 vs. 4 vs. 6 month in freezer PCA Plot (Protein Level) EDTA tube Serum tube 0m, -80 C 6m, -80 C 6m, -20 C 36

37 MRM PANEL Verified proteins (Time in Freezer) Marker Type UniProtID (Human) Protein Description Tube Types EDTA P100 Serum Heparin Change p-value # Time in Freezer SAMP Serum amyloid P-component o o o o E-10 Time in Freezer APOC4 Apolipoprotein C-IV o o o o E-03 Time in Freezer INHBE Inhibin beta E chain o o o o E+00 Time in Freezer VTDB Vitamin D-binding protein o o o o E+00 Time in Freezer APOE Apolipoprotein E o o o o E-10 Time in Freezer KAIN Kallistatin o o o o E-08 Time in Freezer SHBG Sex hormone-binding globulin o o E-04 Time in Freezer ANT3 Antithrombin-III o o o o E-08 Time in Freezer HEMO Hemopexin o o o o E-04 Time in Freezer AACT Alpha-1-antichymotrypsin o o o o E-06 Time in Freezer APOC3 Apolipoprotein C-III o o o o E-03 Time in Freezer CO3 Complement C3 o o o o E-06 Time in Freezer LG3BP Galectin-3-binding protein o o o o E-05 Time in Freezer LBP Lipopolysaccharide-binding protein o E-03 Time in Freezer PHLD Phosphatidylinositol-glycan-specific phospholipase D o o E-03 Time in Freezer APOL1 Apolipoprotein L1 o o o o E-03 Time in Freezer A1AT Alpha-1-antitrypsin o o o o E+00 Time in Freezer A1AG1 Alpha-1-acid glycoprotein 1 o o o o E-09 Fold 37

38 Conclusions: Long Term Freezer Storage Protein markers represent degradation/precipitation, cell lysis and denaturation Greater impact of protein degradation and lysis at -20 C vs. -80 C. Several proteins show changes at 6 months, with smaller changes after 18 months. A few proteins are stable at 6 months but change over 12 months. Some proteins show similar behavior in F/T and in Time in Freezer studies. 38

39 Conclusions (some evidence-based best practices) Sample preparation Operator characteristics should be recorded. Changes occur after 6 months at -80 C, more changes at -20 C. Damage due to number of F/T cycles is incremental. Apolipoproteins and coagulation proteins are markers of F/T. P100 and Serum tubes outperform when left on bench and in F/T. Samples from healthy and cancer patients show the same changes. Majority of discovered markers are successfully verified in independent samples. A sample integrity panel will be validated. 39

40 MRM SAMPLE INTEGRITY PANEL Time on Bench 40

41 MRM SAMPLE INTEGRITY PANEL Time in Freezer and Freeze/Thaw 41

42 MRM PANEL DESIGN Control Test Blood on Bench Plasma/Serum on Bench Time in Freezer Algorithm is being developed to include both internal and external references Freeze/Thaw Total unique panel proteins :32 42

43 CSF Integrity Plasma contamination effects Heatmap (All proteins z-scores ) 0 % 0.5 % 2.5 % 5 % Z-score, (x - μ) / σ Proteins x = abundance μ = mean σ = stdv. 43

44 CSF Integrity Plasma contamination effects Heatmap (All peptides intensities ) 0 % 0.5 % 2.5 % 5 % Z-score, (x - μ) / σ Peptides x = abundance μ = mean σ = stdv. 44

45 CSF Time on Bench Study Design Pooled CSF -80 ⁰C Thawed for 30 min at 4 ⁰C Time & temperature on bench -80 ⁰C 4 ⁰C 20 ⁰C 37 ⁰C 64 hours Thawed for 30 min at 4 ⁰C 1. Reduction and Digestion into Tryptic Peptides 2. Desalting 3. LC-MS/MS 45

46 CSF Integrity Temperature effects (compared to -80 ⁰C) *Differential expression (DE) thresholds: p & q-value < 0.05, fold change >

47 Schizophrenia: 2D CSF Study PCA plot (all 1241 detected proteins at 1% FDR) 47

48 Acknowledgments NCI Helen Moore Lokesh Agrawal Palo Alto Medical Foundation Menlo Park, CA Daniel Chelsky (PI) Geun-Cheol Gil Bich Nguyen Daniel Lopez-Ferrer Xiaolei Xie Fiona McAllister Sigmund Wu Howard Schulman Montreal, QC Julie Lamontagne Yiyong Zhou Funded by NCI Contract No. HHSN E & NIH Grand Opportunity 1RC2NS

49 Measuring with Perspective! 49

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