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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, 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
16
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
18
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
19
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
20
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
21
MRM verification FT cycle
Protein degradation over freeze/thaw cycle
22
MRM verification FT cycle
Protein degradation over freeze/thaw cycle
23
MRM verification FT cycle
Protein degradation over freeze/thaw cycle
24
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
26
PCA Plot: Freeze-Thaw Cycles
27
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
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.
29
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
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
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
32
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.
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.
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!