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, 2014

2

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

3

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

hours

months

4

0

2

4

6

8

10

12

14

16

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

# o

f S

ub

jec

t

Age range

Healthy Female, total 25

Breast Cancer, total 25

0

2

4

6

8

10

12

14

16

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

# o

f S

ub

jec

t

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

5

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

6

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

7

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

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

9

Analytical Approach for Discovery:

Label-free Differential Profiling

Plasma

Or

Serum

MARS-14

depletion of

abundant

proteins

Digestion

to

peptides

Peptide Sequencing

QExactive

Protein Identification

Statistical Analysis

Informatics-based

Quantitative Peptide Expression Profiling

LC-MS

1D: Wang et al., Analytical chemistry

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

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

2007;359:87–1052D:

10

Protein Identification

Differential Expression?

Candidate Biomarkers

Build Targeted Multiplexed Assay

Test Independent Samples

Biomarker Verification and Validation

MR

M D

isco

very

11 Identified unique peptides : 15

Ceruloplasmin

Decreasing over incubation time plasma at 37 ⁰C

EDTA Serum Heparin

12

0.5h

Identified unique peptides : 11

Vitronectin

Control: 20 °C, 0.5 h for Post-Spin

48h 96h

Co

ntr

ol

48h 96h 0.5h

37 oC 20 oC

48h 96h

Co

ntr

ol

48h 96h 0.5h

37 oC 20 oC

48h 96h

Co

ntr

ol

48h 96h 0.5h

37 oC 20 oC

DECREASING OVER INCUBATION TIME POST SPIN AT 20 AND 37OC

13

WHOLE BLOOD CELL LYSIS MARKER

0.5h

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

Post spin Pre spin Control 48h 0.5h 48h

Post spin Pre spin Control 48h 0.5h 48h

14

EDTA tubes affect calcium ion dependent leukocyte binding proteins

0.5h

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

EDTA P100 Serum Heparin

Blood incubation at 20 oC

Su

m o

f p

ep

tid

e in

ten

sit

ies

EDTA tubes Non-EDTA tubes

15

EDTA tubes affect calcium ion dependent leukocyte binding proteins

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EDTA tubes affect calcium binding proteins

(e.g. Protein S100)

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 48h

N C N C N C

EDTA

Dis

co

very

V

eri

ficati

on

P100 Serum Heparin

Ab

un

dan

ce

A

bu

nd

an

ce

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

(A)

AUC=1

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

(B)

AUC=0.98

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

(C)

AUC=0.99

17

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

Sens

itiv

ity

Specificity

Protein S100-A9

Profilin-1

Complement C3

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

(A)

AUC=1

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

(B)

(C)

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

Sens

itiv

ity

Specificity

AUC=0.98

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1

Sens

itiv

ity

Specificity

AUC=0.87

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

Plasma/Serum incubation

Whole blood cell lysis marker

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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

cycle

• 10 subjects, 4

tube types

Blood samples

Plasma/Serum

Centrifuge

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Protein intensities changing over F/T cycles

Intensity ratios compared to Cycle 1

De

cre

as

e

Inc

rea

se

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

Un

iqu

e p

ep

tid

e

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

FN2

VWF

ACTB

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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

De

cre

ase

In

cre

as

e

Un

iqu

e p

ep

tid

e

Serum P-100 Heparin EDTA EDTA+PI APOB

APOC2

VWF

FN1

ACTB

TPM4

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MRM verification FT cycle

Protein degradation over freeze/thaw cycle

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MRM verification FT cycle

Protein degradation over freeze/thaw cycle

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MRM verification FT cycle

Protein degradation over freeze/thaw cycle

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MRM verification FT cycle

Protein degradation over freeze/thaw cycle (Fibronectin)

Dis

co

very

V

eri

ficati

on

25

MRM verification FT cycle

Stable to Freeze/Thaw

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PCA Plot: Freeze-Thaw Cycles

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MRM PANEL

Verified proteins (Freeze/Thaw)

EDTA P100 Serum Heparin

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

Fold

Change§ p-value#Marker Type Protein Description

UniProtID

(Human)

Tube Types

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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.

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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

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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

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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

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Sample integrity study

Time in Freezer (Example proteins)

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

34

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

Stable over 18 months at both temperatures

35

Sample integrity study

Time in Freezer

36

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

37

MRM PANEL

Verified proteins (Time in Freezer)

EDTA P100 Serum Heparin

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

Fold

Change§ p-value#Marker Type Protein Description

UniProtID

(Human)

Tube Types

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.

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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.

40

MRM SAMPLE INTEGRITY PANEL

Time on Bench

41

MRM SAMPLE INTEGRITY PANEL

Time in Freezer and Freeze/Thaw

42

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

43

CSF Integrity

Plasma contamination effects

Heatmap (All proteins z-scores )

0 % 0.5 %

Z-s

co

re, (

x -

μ)

/ σ

x = abundance

μ = mean

σ = stdv.

Pro

tein

s

2.5 % 5 %

44

CSF Integrity

Plasma contamination effects

Heatmap (All peptides intensities )

0 % 0.5 %

Z-s

co

re, (

x -

μ)

/ σ

x = abundance

μ = mean

σ = stdv.

Pe

ptid

es

2.5 % 5 %

45

CSF Time on Bench Study Design

-80 ⁰C 4 ⁰C 20 ⁰C 37 ⁰C

Pooled CSF

Time & temperature on

bench

64 hours

-80 ⁰C

Thawed for 30 min at 4 ⁰C

Thawed for 30 min at 4 ⁰C

1. Reduction and Digestion into Tryptic Peptides

2. Desalting

3. LC-MS/MS

46

CSF Integrity

Temperature effects (compared to -80 ⁰C)

*Differential expression (DE) thresholds:

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

47

Schizophrenia: 2D CSF Study

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

48

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

NCI

49

Measuring with Perspective!