Measuring Biomarker Stability in Frozen Tissue · PDF file0 10 20 30 40 50 60 70 Air - 10 Air...
Transcript of Measuring Biomarker Stability in Frozen Tissue · PDF file0 10 20 30 40 50 60 70 Air - 10 Air...
Measuring Biomarker Stability in Frozen Tissue
Post-acquisition variables:• Time at room
temperature• Temperature of room• Type of fixative• Time in fixative• Rate of freezing• Freeze / Thaw• Size of aliquots• Storage temperature• Storage duration• Storage in vacuum• Processing methods
NCI Best Practices for Biospecimen Resources
• Technical Operations Best Practices– Collection, processing, storage, retrieval, use– Collection and managing clinical data– QA/QC– Biosafety– Informatics, inventory control and tracking
• Ethical, Legal and Policy– Custodianship– Informed consent– Privacy protection– Access to biospecimens and data– Intellectual property and resource sharing
Principals of Evidence Based Biospecimen Research
• What practices affect the quality of a specimen?
• Can they be measured?• Do the measurements have meaning to
data acquired in research testing?
Frozen Tissue
• Still the pristine specimen carrying intact non-chemically altered DNA, RNA, and proteins.
• Will be needed for biomarker discover.• May be needed for future clinical
information that can’t be resolved by robust assays.
Sample Packaging and Storage
• Can the quality of frozen specimens be protected with more stringent packaging?
• What is the duration of protection?
Questions• Oxidation - RNA, DNA, &
proteins
• Dehydration• Morphological deformation
• Residual and active enzyme activity
• Storage container, temperature, length of storage
Operational Variables• Sample size• Freezing rates• Packaging analysis
– Sealer– Pouch
• Tissue container
Polymer Laminate Diagrams
Proposed Solution1. Foil laminate (high
barrier packaging)2. Embedding cassettes:
protect tissue from distortion
3. Modified atmosphere packaging: vacuum or inert gas, i.e., argon
30Embedding tray in bag (vacuum)
30Barrier bag (no vacuum)
26Barrier bag (vacuum)
3030 mil clamshell tray in bag (vacuum)
2530 mil clamshell
2320 mil clamshell
70Standard 5 ml tube
50CRE tray in standard bag
Time to Freeze (sec)Container
Based on the exhaustion of LN2 boiling
Vessel Freezing Periods
Sample Dissection for a Single Casemucosal tissue sample
200 mg pieces 200mg pieces 200mg pieces
Baseline ArgonAir (cryovial) Vacuum
(-10C -20C -80C) (-10C -20C -80C)(-10C -20C -80C)
1 2 3 6 12
Months
Months
1 2 3 6 12
1 2 3 6 12
Months
1 2 3 6 12
Months
1 2 3 6 12
Months
1 2 3 6 12
Months
Months
1 2 3 6 12
Months
1 2 3 6 12
Months
1 2 3 6 12
Sample Dissection
100 mg
3 gram tissue sample
200 mg frozen
Total mRNA RIN#
qRT-PCR Biomarkers
Histology100 mg
Immunohistochemistry
Digital Imaging & Analysis
formalin fixation
Biomarkers GAPDH - Glyceraldehyde 3-phosphate dehydrogenase
(message)– implicated in non-metabolic processes, transcription
activation, initiation of apoptosis, and ER to Golgi vesicle shuttling – house keeping gene
CEA – glycoprotein-cell adhesion (protein & message)– CEA is a tumor marker to identify recurrences– In humans, the carcinoembryonic antigen family consists of
29 genes, 18 of which are normally expressed.CDX2 (protein and message)
– CDX2 expression is maintained in the adult small and large intestinal epithelia
– CDX2 expression is induced or up-regulated in pathologic states
CK20 (protein)– cytokeratin, major cellular protein of mature enterocytes
and goblet cells and is specifically expressed in the gastric and intestinal mucosa.
Elongation ratio (Epithelial cells: red; Stromal cells: yellow)
Courtesy of Sanford Barsky, M.D., Ph.D.
CEA
CK20 CDX2
0
10
20
30
40
50
60
70
Air -10
Air -20
Air -80
Arg -10
Arg -20
Arg -80
Vac -10
Vac -20
Vac -80
CDX2 % PosCDX 2 Intensity
0
10
20
30
40
50
60
Air -10
Air -20
Air -80
Arg -10
Arg -20
Arg -80
Vac -10
Vac -20
Vac -80
CEA % PosCEA Intensity
0
10
20
30
40
50
60
Air -10
Air -20
Air -80
Arg -10
Arg -20
Arg -80
Vac -10
Vac -20
Vac -80
CK20 % PosCK20 Intensity
Degree of change between different packaging methods versus storage temperature for CDX2, CEA, and CK20
CDX2 CEA
CK20
qRT-PCR Baselines
10
15
20
25
30
1 2 3 4 5 6 7 8 9
Subjects
Cyc
le T
hres
hold
CDX2
CEA
GAPDH
# Cycles / Time point of storage - Baseline # cycles
Measurement of Change in mRNA
RIN Degradation Varying Temperature at Month 3
0
12
34
5
67
89
10
T0 T3 -10 T3 -20 T3 -80
Temperature
RN
A I
nteg
raty
Num
ber
A IR
A RGON
V a cu u m
Storage [-10C] : Packaging [Air]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2
qRT-
PCR
Cyl
ce C
hang
e
Month 1 Month 2 Month 3
Storage [-20C] : Packaging [Air]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2
qRT-
PCR
Cyc
le C
hang
es
Month 1 Month 2 Month 3
Storage [-80C] : Packaging [Air]
-6.00-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2
qRT-
PCR
Cyc
le C
hang
e
Month 1 Month 2 Month 3
Changes in Biomarker qRT-PCR
Storage [-20C] : Packaging [Air]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2qRT-
PCR
Cyc
le C
hang
Month 1
Month 2Month 3
Storage [-20C] : Packaging [Argon]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2
qRT-
PCR
Cyl
ce C
han
Month 1Month 2
Month 3
Storage [-20C] : Packaging [Vacuum]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2qRT-
PCR
Cyc
le C
hang
e
Month 1Month 2Month 3
Storage [-80] : Packaging [Air]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2qRT-
PCR
Cyc
le C
hang
e
Month 1Month 2Month 3Month 6
Storage [-80C] : Packaging [Argon]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2
qRT-
PCR
Cyc
le C
han
Month 1Month 2
Month 3
Month 6
Storage [-80C] : Packaging [Vacuum]
-4.00-2.000.002.004.006.008.00
10.0012.0014.0016.00
GAPDH CEA CDX2
qRT-
PCR
Cyc
le C
han
Month 1
Month 2
Month 3
Month 6
Biomarker Measurement Varying Temperature at Month Three
15
20
25
T0 -10 -20 -80Temperature (°C)
Cyc
le T
hres
hold
AIR GAPDH
ARGONGAPDH
VacuumGAPDH
AIR CEA
ARGON CEA
VacuumCEA
GAP-DH Cycle Threshold in Air
y = 17.345e0.054x
R2 = 0.845
y = 16.462e0.018x
R2 = 0.402
y = 16.667e0.102x
R2 = 0.868
15
16
17
18
19
20
21
22
23
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Timepoint
Cyc
le T
hres
hold
Predicted -10 Observed -10 Predicted -20 Observed -20Predicted -80 Observed -80
Air (cryovial) Packaging(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
GAPDH CEA CDX2
Argon Packaging(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
GAPDH CEA CDX2
Vacuum Packaging(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
GAPDH CEA CDX2
GAPDH(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
Air Argon Vacuum
CDX2(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Per
cent
Cha
nge
Air Argon Vacuum
CEA(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
Air Argon Vacuum
Conclusions / Future Studies• Degradation occurs at about 5% in the first 12
months according to percent change in the original quantity of message
• No difference in packaging seen at this time • Continue to measure the duration of time and
packaging for affects in tissue quality• Varying types of biomarkers need to be tested• Compare fixation methods to the frozen tissue
AcknowledgmentsDepartment of Pathology• Dr. Yufang Tang • Dr. Sanford Barsky• Dr. Kurtis Yearsley• Dan Rohrer • Susie Jones• Mary Marin • Susan Long • Nehad Mohamed
Department of Agriculture Engineering
• Dr. Gönül Kaletunç• Jason Baker• Wes Halderman*• Sara Kallio• Lily Meyer
Thank You!