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ThermoBrite Elite
®
Feasibility/Time Savings Study
Blood/Bone Marrow Protocol
For this study, WaveSense, Inc. provided twelve U266 myeloma
cell line slides prepared with their patented EpiSep cell enrichment
technology using Anti-CD138 paramagnetic antibodies. The following
will describe the adaptation of a blood and bone marrow FISH
protocol using commercially available probes and EpiSep HS® slides
for automation on the ThermoBrite® Elite. Twelve slides were probed
with various IGH probes, having 2-3 targets, each corresponding to a
unique fluorophore. Results from the slides will be discussed.
Plasma cell captured using EpiSep HS ® slide using
Anti-CD138 antibodies
Probe: IGH/MAF
Signals: 2 Green-14q32.33-2 Orange 16q23.1-q23.2
Important Note: This is a recommended protocol for FISH testing based on commercially available probes. Due to the nature
of FISH testing, some protocols may have to be adapted to achieve optimal performance.
MATERIALS
ThermoBrite Elite Adapted Protocol
Equipment/Supplies
ThermoBrite Elite S/N 003, Software version 1.5.8F
Fluorescence microscope
X-Cite 120Q® light source
Commercially available FISH imaging software
DAPI, Spectrum Green, Spectrum Orange, DEAC, Cy3.5v1 filter sets
Elmer’s rubber cement
18x18mm glass coverslips
Reagents
Use
2XSSC Wash Buffer
Wash between steps
0.4XSSC/0.3% NP-40
Stringency wash to remove unbound probe
2XSSC /0.1%NP-40
Wash buffer
DAPI II
Nucleic acid stain (blue)
70% Ethanol
Dehydration
85% Ethanol
Dehydration
100% Ethanol
Dehydration
Probes
Use
IGH/MAF
Detects rearrangements of IGH with translocation partner MAF
IGH/MYC
Detects rearrangements of IGH with translocation partner MYC
IGH/FGFR3
Detects rearrangements of IGH with translocation partner FGFR3
IGH/CCND1/FGFR3
Detects rearrangements of IGH with translocation partners CCND1 and FGFR3
IGH/MALT1
Detects rearrangements of IGH with translocation partner MALT1
IGH/CCND1
Detects rearrangements of IGH with translocation partner CCND1
Test sample
U266 myeloma cancer cell line processed with EpiSep HS and Anti-CD138 paramagnetic antibodies
METHODS
ThermoBrite Elite Adapted Protocol
Notes
•
All steps rocked medium (12 cycles/min)
•
BC= base camp temperature
•
100% Fill= 30mL
•
50% Fill= 15mL
•
“Quotes” indicate pause step programmed in protocol
Day 1: Pretreatment / Denaturation & Hybridization
1.
Drain
2.
100% Fill of 2XSSC, BC Temp 62°C Incubate 18 seconds, Temp 73°C Incubate 2 minutes, BC Temp 60°C, Temp 25°C No wait, Drain
3.
Temp 25°C No wait, 50% Fill of dH20, Incubate 1 minute, Drain
4.
Temp 25°C No wait, 100% of Fill of 70% Ethanol, Incubate 2 minutes, Drain
5.
100% Fill with 85% Ethanol, Incubate 2 minutes, Drain
6.
100% Fill with 100% Ethanol, Incubate 2 minutes, Drain
7.
Pause “remove slides, air dry, add probe & cement, reinsert slides face up” (offline)
8.
50% Fill of dH20, BC Temp 63°C Incubate 18 seconds, Temp 73°C Incubate 2 minutes, Temp 60°C, Temp 37°C No wait, Drain
9.
Temp 37°C No wait, 50% Fill of dH20, Temp 37°C Incubate 16 hours, Pause “Hold Hybe”, Drain
Day 2: Post Wash
1.
Drain
2.
100% Fill of 0.4XSSC/0.3% NP-40, BC Temp 62°C Incubate 18 seconds, Temp 70°C
Incubate 2 minutes, Temp 60°C, Temp 25°C No wait, Drain
3.
Temp 25°C No wait, 100% Fill of 2XSSC/0.1% NP-40, Incubate 2 minutes, Drain
4.
Dry slides completely. (offline)
5.
Apply mounting medium (DAPI II) to target area and apply coverslip. (offline)
METHODS
ThermoBrite Elite Adapted Protocol
Protocol Adaptations
•
Following the 73°C 2XSSC aging step, a 50% Fill of dH20 step was added. This allows the sample chambers to cool prior to
filling with ethanol.
•
The post wash step calls for a 73°C (±1°C) wash for 2 minutes. When running manually in a 73°C coplin jar, the addition of four slides
decreases the wash buffer temperature for the entire duration of the wash. See chart below. The post wash was performed at 70°C.
Coplin Jar Temperature Test
Time Point (seconds)
Temperature (°C)
15
68.0
30
69.4
45
69.8
60
69.9
75
70.0
90
70.1
105
70.2
120
70.3
Average Temperature= 69.7
RESULTS
ThermoBrite Elite Adapted Protocol
Slides were viewed with a fluorescence microscope using FITC/Spectrum Green, CY3/Spectrum
Orange, DEAC/Aqua and DAPI filters. Images were captured using commercially available FISH
imaging software.
All 12 slides produced bright signals with minimal background and could be easily spot counted/
analyzed. In conclusion, paramagnetic antibody recovered cell slides processed on the ThermoBrite
Elite produced quality FISH signals while providing time savings and walk away convenience.
See Images section for corresponding images.
Slide #
Module
Probe
Fluorochrome Color
Signal Intensity
Background
1
1A
1A
1B
1B
2A
2A
2B
2B
3A
3A
3B
3B
IGH/MAF
Green/Orange
Bright
Minimal
IGH/MAF
Green/Orange
Bright
Minimal
IGH/MYC
Orange/Green
Bright
Minimal
IGH/MYC
Orange/Green
Bright
Minimal
IGH/FGFR3
Green/Orange
Bright
Minimal
IGH/FGFR3
Green/Orange
Bright
Minimal
IGH/CCND1/FGFR3
Green/Orange/Aqua
Bright
Minimal
IGH/CCND1/FGFR3
Green/Orange/Aqua
Bright
Minimal
IGH/MALT1
Green/Orange
Bright
Minimal
IGH/MALT1
Green/Orange
Bright
Minimal
IGH/CCND1
Green/Orange
Bright
Minimal
IGH/CCND1
Green/Orange
Bright
Minimal
2
3
4
5
6
7
8
9
10
11
12
RESULTS
ThermoBrite Elite Adapted Protocol
Hands Off Time (Minutes)
Manual
ThermoBrite Elite
Pretreatment
0
40
Probe Application
0
0
969
972
0
17
Manual
ThermoBrite Elite
Pretreatment
53
3
Probe Application
16
16
Denature and Hybridization
0
0
Post Wash
64
17
Manual
ThermoBrite Elite
Pretreatment
53
43
Probe Application
16
16
Denature and Hybridization
969
972
Post Wash
64
33
Manual
ThermoBrite Elite
1101.71
1064.46
Total Assay Time (Hours)
18 hours, 21 minutes
17 hours, 44 minutes
Total Hands On Time
2 hours, 12 minutes
36 minutes
Denature and Hybridization
Post Wash
Hands On Time (Minutes)
Sub Total Assay Time (Minutes)
Summary
Total Assay Time (Minutes)
Time Savings
• Reduced assay time by 37 minutes
• Reduced hands on time by 96 minutes
IMAGES
ThermoBrite Elite Adapted Protocol
Slide 1
Slide 3
Slide 5
Probe: IGH/MAF
Probe: IGH/MYC
Probe: IGH/FGFR3
Signals: 2 Green, 14q32.33, 2 Orange 16q23.1-q23.2
Signals: 2 Orange 14q32.33, 2 Green 8q24.21
Signals: 2 Green 14q32.33, 2 Orange 4p16.6
Slide 7
Slide 10
Slide 11
Probe: IGH/CCND1/FGFR3
Probe: IGH/MALT1
Probe: IGH/CCND1
Signals: 2 Green 14q32.33, 2 Orange 11q13.3,
2 Aqua 4p16.3
Signals: 2 Green 18q21.2, 2 Orange 14q32.33
Signals: 2 Green 14q32.33, 2 Orange 11q13.3
www.LeicaBiosystems.com
ThermoBrite Elite is a Class I Medical Device, ETL, CSA and CE Marked for in vitro diagnostic use.
ThermoBrite® Elite is a registered trademark of Leica Biosystems. EpiSep HS® is a
registered trademark of WaveSense, Inc.
95.12057 Rev B ∙ 6/2013 ∙ Copyright © by Leica Biosystems, Richmond, IL., 2013. Subject to
modifications. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.