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...
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Measuring Biomarker Stability in Frozen Tissue
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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
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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
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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?
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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.
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Sample Packaging and Storage
• Can the quality of frozen specimens be protected with more stringent packaging?
• What is the duration of protection?
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Questions• Oxidation - RNA, DNA, &
proteins
• Dehydration• Morphological deformation
• Residual and active enzyme activity
• Storage container, temperature, length of storage
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Operational Variables• Sample size• Freezing rates• Packaging analysis
– Sealer– Pouch
• Tissue container
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Polymer Laminate Diagrams
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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
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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
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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
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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
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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.
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Elongation ratio (Epithelial cells: red; Stromal cells: yellow)
Courtesy of Sanford Barsky, M.D., Ph.D.
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CEA
CK20 CDX2
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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
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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
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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
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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
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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
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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
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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
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Air (cryovial) Packaging(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
GAPDH CEA CDX2
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Argon Packaging(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
GAPDH CEA CDX2
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Vacuum Packaging(-80C)
-10
-5
0
5
10
15
1 2 3 6 12
Perc
ent C
hang
e
GAPDH CEA CDX2
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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
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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
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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
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Thank You!