ThermoBrite Elite. Feasibility/Time Savings Study. Blood/Bone Marrow Protocol

Transcription

1 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-14q 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.

2 MATERIALS 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 IGH/MAF IGH/MYC IGH/FGFR3 IGH/CCND1/FGFR3 IGH/MALT1 IGH/CCND1 Use Detects rearrangements of IGH with translocation partner MAF Detects rearrangements of IGH with translocation partner MYC Detects rearrangements of IGH with translocation partner FGFR3 Detects rearrangements of IGH with translocation partners CCND1 and FGFR3 Detects rearrangements of IGH with translocation partner MALT1 Detects rearrangements of IGH with translocation partner CCND1 Test sample U266 myeloma cancer cell line processed with EpiSep HS and Anti-CD138 paramagnetic antibodies

3 METHODS 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 % 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 % Fill with 85% Ethanol, Incubate 2 minutes, Drain % 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 % 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)

4 METHODS 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) Average Temperature= 69.7

5 RESULTS 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 IGH/MAF Green/Orange Bright Minimal 2 1A IGH/MAF Green/Orange Bright Minimal 3 1B IGH/MYC Orange/Green Bright Minimal 4 1B IGH/MYC Orange/Green Bright Minimal 5 2A IGH/FGFR3 Green/Orange Bright Minimal 6 2A IGH/FGFR3 Green/Orange Bright Minimal 7 2B IGH/CCND1/FGFR3 Green/Orange/Aqua Bright Minimal 8 2B IGH/CCND1/FGFR3 Green/Orange/Aqua Bright Minimal 9 3A IGH/MALT1 Green/Orange Bright Minimal 10 3A IGH/MALT1 Green/Orange Bright Minimal 11 3B IGH/CCND1 Green/Orange Bright Minimal 12 3B IGH/CCND1 Green/Orange Bright Minimal

6 RESULTS Hands Off Time (Minutes) Manual ThermoBrite Elite Pretreatment 0 40 Probe Application 0 0 Denature and Hybridization Post Wash 0 17 Hands On Time (Minutes) Manual ThermoBrite Elite Pretreatment 53 3 Probe Application Denature and Hybridization 0 0 Post Wash Sub Total Assay Time (Minutes) Manual ThermoBrite Elite Pretreatment Probe Application Denature and Hybridization Post Wash Summary Manual ThermoBrite Elite Total Assay Time (Minutes) Total Assay Time (Hours) 18 hours, 21 minutes 17 hours, 44 minutes Total Hands On Time 2 hours, 12 minutes 36 minutes Time Savings Reduced assay time by 37 minutes Reduced hands on time by 96 minutes

7 IMAGES Slide 1 Probe: IGH/MAF Signals: 2 Green, 14q32.33, 2 Orange 16q23.1-q23.2 Slide 3 Probe: IGH/MYC Signals: 2 Orange 14q32.33, 2 Green 8q24.21 Slide 5 Probe: IGH/FGFR3 Signals: 2 Green 14q32.33, 2 Orange 4p16.6 Slide 7 Probe: IGH/CCND1/FGFR3 Signals: 2 Green 14q32.33, 2 Orange 11q13.3, 2 Aqua 4p16.3 Slide 10 Probe: IGH/MALT1 Signals: 2 Green 18q21.2, 2 Orange 14q32.33 Slide 11 Probe: IGH/CCND1 Signals: 2 Green 14q32.33, 2 Orange 11q13.3

8 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 Rev B 6/2013 Copyright by Leica Biosystems, Richmond, IL., Subject to modifications. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.