Controlled Analysis of Preanalytical Variables in CSF and ... · • Over 21 proteins identified as...

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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, 2014

NCI Contract Goal (2009 – 2013):

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

Study Design (Cancer & Healthy; Male & Female)

EDTA EDTA+PI P100 Serum Heparin

0.5 1 4 24 48 96

4 ⁰C 20 ⁰C

1 to 5 cycles

0 6 12 18

-20 ⁰C -80 ⁰C

20 ⁰C 37 ⁰C

Blood collection

tube types

Time & temperature

on bench

Freeze-thaw cycle

Time in freezer

months

21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100

Age range

Healthy Female, total 25

Breast Cancer, total 25

21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100

Age range

Healthy Male, total 25

Prostate Cancer, total 25

Clinical sample collection

Study subjects samples

1 125 320

Pilot 6 500

2 100 1600

Total 231 2420

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

Study conducted to assess compliance with pre-set time

lines for sample processing by analysis of PC Tablet data

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

Difference in performance of 2 different operators

Tablet PC Data Analysis Comparison of two operators following the same SOP

Operator 1 is

late and more

variable

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

Analytical Approach for Discovery:

Label-free Differential Profiling

Plasma

MARS-14

depletion of

abundant

proteins

Digestion

peptides

Peptide Sequencing

QExactive

Protein Identification

Statistical Analysis

Informatics-based

Quantitative Peptide Expression Profiling

1D: Wang et al., Analytical chemistry

2003 Sep 15; 75(18):4818-26

2D: Roy & Becker, Methods Mol. Biol.

2007;359:87–1052D:

Protein Identification

Differential Expression?

Candidate Biomarkers

Build Targeted Multiplexed Assay

Test Independent Samples

Biomarker Verification and Validation

11 Identified unique peptides : 15

Ceruloplasmin

Decreasing over incubation time plasma at 37 ⁰C

EDTA Serum Heparin

Identified unique peptides : 11

Vitronectin

Control: 20 °C, 0.5 h for Post-Spin

48h 96h

48h 96h 0.5h

37 oC 20 oC

48h 96h

48h 96h 0.5h

37 oC 20 oC

48h 96h

48h 96h 0.5h

37 oC 20 oC

DECREASING OVER INCUBATION TIME POST SPIN AT 20 AND 37OC

WHOLE BLOOD CELL LYSIS MARKER

Identified unique peptides : 9

Hemoglobin subunit alpha

Control: 20 °C, 0.5 h for Pre & Post-Spin

Post spin Pre spin Control 48h 0.5h 48h

EDTA tubes affect calcium ion dependent leukocyte binding proteins

0.5h 24h 0.5h 24h 0.5h 24h 0.5h 24h

EDTA P100 Serum Heparin

Blood incubation at 20 oC

EDTA tubes Non-EDTA tubes

EDTA tubes affect calcium ion dependent leukocyte binding proteins

EDTA tubes affect calcium binding proteins

(e.g. Protein S100)

Control

Plasma 48h

Blood 48h

N C N C N C Control

Plasma 48h

Blood 48h

N C N C N C Control

0.5h Serum

Blood 48h

N C N C N C Control

Plasma 48h

Blood 48h

N C N C N C

ficati

P100 Serum Heparin

C P B C P B C S B C P B

AUC=0.98

AUC=0.99

0 0.2 0.4 0.6 0.8 1

Specificity

Protein S100-A9

Profilin-1

Complement C3

0 0.2 0.4 0.6 0.8 1

Specificity

AUC=0.98

0 0.2 0.4 0.6 0.8 1

Specificity

AUC=0.87

Plasma/Serum incubation

Whole blood cell lysis marker

Freeze-thaw cycle

RT, 1 h -80 °C, 23 h

• 1x to 5x F/T

cycles

• 1 h at RT, 1 day in -80°C for each

• 10 subjects, 4

tube types

Blood samples

Plasma/Serum

Centrifuge

Protein intensities changing over F/T cycles

Intensity ratios compared to Cycle 1

Thresholds: p < 0.05, q < 0.05, fold-change >1.5

2 vs 1 3 vs 1 4 vs 1 5 vs 1

Freeze-thaw analysis: Comparison of tube types

Intensity ratios (5 vs. 1 Cycle)

thresholds p < 0.05, q < 0.05, fold-change >1.5

Mechanical Separator

Serum P-100 Heparin EDTA EDTA+PI APOB

MRM verification FT cycle

Protein degradation over freeze/thaw cycle

Protein degradation over freeze/thaw cycle (Fibronectin)

ficati

Stable to Freeze/Thaw

PCA Plot: Freeze-Thaw Cycles

MRM PANEL

Verified proteins (Freeze/Thaw)

Freeze/Thaw APOC2 Apol ipoprotein C-II o o o o -1.7 0.00E+00

Freeze/Thaw APOC3 Apol ipoprotein C-III o o o o -1.5 0.00E+00

Freeze/Thaw APOC4 Apol ipoprotein C-IV o o o -1.3 1.11E-08

Freeze/Thaw APOE Apol ipoprotein E o -1.6 2.08E-03

Freeze/Thaw APOM Apol ipoprotein M o -1.3 3.35E-04

Freeze/Thaw VWF von Wi l lebrand factor o -1.3 1.16E-02

Freeze/Thaw FINC Fibronectin o -4.4 0.00E+00

Change§ p-value#Marker Type Protein Description

UniProtID

(Human)

Tube Types

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.

Study Design: Storage time in freezer

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

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

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

NCI Study 2b 6 vs. 12 vs. 18 month in freezer Freezing causes cell lysis

Sample integrity study

Time in Freezer (Example proteins)

NCI Study 2b 6 vs. 12 vs. 18 month in freezer

Stable in 6 month,

but over 12 month freezing induces cell lysis

Study 2b 6 vs. 12 vs. 18 month in freezer

Stable over 18 months at both temperatures

Sample integrity study

Time in Freezer

Verification 2 vs. 4 vs. 6 month in freezer

PCA Plot (Protein Level)

EDTA tube Serum tube

6m, -80°C

6m, -20°C

0m, -80°C

MRM PANEL

Verified proteins (Time in Freezer)

Time in Freezer SAMP Serum amyloid P-component o o o o -5.7 7.65E-10

Time in Freezer APOC4 Apol ipoprotein C-IV o o o o -4.5 2.63E-03

Time in Freezer INHBE Inhibin beta E chain o o o o -3.7 0.00E+00

Time in Freezer VTDB Vitamin D-binding protein o o o o -2.9 0.00E+00

Time in Freezer APOE Apol ipoprotein E o o o o -2.1 7.76E-10

Time in Freezer KAIN Kal l i s tatin o o o o -1.9 1.02E-08

Time in Freezer SHBG Sex hormone-binding globul in o o -1.6 9.80E-04

Time in Freezer ANT3 Antithrombin-III o o o o -1.6 9.48E-08

Time in Freezer HEMO Hemopexin o o o o -1.6 1.89E-04

Time in Freezer AACT Alpha-1-antichymotryps in o o o o -1.5 9.28E-06

Time in Freezer APOC3 Apol ipoprotein C-III o o o o -1.4 3.31E-03

Time in Freezer CO3 Complement C3 o o o o 1.8 2.68E-06

Time in Freezer LG3BP Galectin-3-binding protein o o o o 2.2 3.19E-05

Time in Freezer LBP Lipopolysaccharide-binding protein o 2.3 2.14E-03

Time in Freezer PHLD Phosphatidyl inos i tol -glycan-speci fic phosphol ipase D o o 2.4 2.47E-03

Time in Freezer APOL1 Apol ipoprotein L1 o o o o 2.4 1.01E-03

Time in Freezer A1AT Alpha-1-anti tryps in o o o o 8.5 0.00E+00

Time in Freezer A1AG1 Alpha-1-acid glycoprotein 1 o o o o 54.5 2.50E-09

Change§ p-value#Marker Type Protein Description

UniProtID

(Human)

Tube Types

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.

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.

MRM SAMPLE INTEGRITY PANEL

Time on Bench

MRM SAMPLE INTEGRITY PANEL

Time in Freezer and Freeze/Thaw

MRM PANEL DESIGN

Blood on Bench

Plasma/Serum on Bench

Time in Freezer

Freeze/Thaw

Control Test

Algorithm is

being developed

to include both

internal and

external

references

Total unique panel proteins :32

CSF Integrity

Plasma contamination effects

Heatmap (All proteins z-scores )

0 % 0.5 %

x = abundance

μ = mean

σ = stdv.

2.5 % 5 %

CSF Integrity

Plasma contamination effects

Heatmap (All peptides intensities )

0 % 0.5 %

x = abundance

μ = mean

σ = stdv.

2.5 % 5 %

CSF Time on Bench Study Design

-80 ⁰C 4 ⁰C 20 ⁰C 37 ⁰C

Pooled CSF

Time & temperature on

64 hours

-80 ⁰C

Thawed for 30 min at 4 ⁰C

1. Reduction and Digestion into Tryptic Peptides

2. Desalting

3. LC-MS/MS

CSF Integrity

Temperature effects (compared to -80 ⁰C)

*Differential expression (DE) thresholds:

p & q-value < 0.05, fold change > 1.5

Schizophrenia: 2D CSF Study

PCA plot (all 1241 detected proteins at 1% FDR)

Acknowledgments

Daniel Chelsky (PI)

Geun-Cheol Gil

Bich Nguyen

Daniel Lopez-Ferrer

Xiaolei Xie

Fiona McAllister

Sigmund Wu

Howard Schulman

Julie Lamontagne

Yiyong Zhou

Palo Alto Medical

Foundation

Menlo Park, CA Montreal, QC

Funded by NCI

Contract No.

HHSN261200800001E

NIH Grand

Opportunity

1RC2NS069502

Helen Moore

Lokesh Agrawal

Measuring with Perspective!

Controlled Analysis of Preanalytical Variables in CSF and ... · • Over 21 proteins identified as...

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