May 26, 2023
U54 SCENT Normal Lung Surgical Tissue Collection Procedure
- Carolyn Glass1,2,
- andrew.nixon3,
- Matthew Hartwig4,
- Nancy McGreal5,
- SSCRS6,
- Mary Jordan*2
- 1Department of Pathology, Duke University Hospital, Durham, NC, USA;
- 2Duke BioRepository & Precision Pathology Center, Duke University Hospital, Durham, NC, USA;
- 3Division of Medical Oncology, Department of Medicine, Duke University School of Medicine, Durham, NC, USA.;
- 4Division of Cardiovascular and Thoracic Surgery, Department of Surgery, Duke University Medical Center, Durham, NC, USA.;
- 5Department of Pediatrics, Duke University Medical Center, Durham, NC, USA.;
- 6Substrate Services Core Research Support, Duke University School of Medicine, Durham, NC, USA
- Mary Jordan*: Contact Person;
Protocol Citation: Carolyn Glass, andrew.nixon, Matthew Hartwig, Nancy McGreal, SSCRS, Mary Jordan* 2023. U54 SCENT Normal Lung Surgical Tissue Collection Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpozxl5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 25, 2023
Last Modified: May 26, 2023
Protocol Integer ID: 82483
Funders Acknowledgement:
NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director
Grant ID: U54AG075936
Abstract
This document outlines the required criteria for inclusion and collection of lung tissue obtained by surgical resection at Duke University Hospital through the Biorepository and Precision Pathology Center (BPRC) in the Department of Pathology.
Materials
Personal Protective Equipment:
• Lab coat
• Gloves
Equipment:
• Leica Autostainer XL
• Microscope
Materials:
• Bleach
• Xylene
• Alcohol, 100%
• Alcohol, 95%
• Alcohol, 80%
• Hematoxylin - Pureview Hematoxylin 660+
• Eosin - Pureview Eosin 615
• Clearing Solution - Pureview Focus-AQ
• Bluing - Pureview PH Blue
• Chemical waste container
• Wypall pads
• Slide staining rack
• Pre-cut H&E control slide
Inclusion and Exclusion Criteria
Inclusion and Exclusion Criteria
Inclusion Criteria:
• Any age
• All sexes
• Grossly normal lung obtainable through surgical pathology
Exclusion Criteria:
• No recent treatment with chemotherapy (recent <1 year)
• No recent treatment with radiation (recent <1 year)
• No history of COVID
• No recent colds (<1 year),etc
• If smoker or past smoker, check emphysematous changes in CT
Notes:
• Check recent CT scans to determine if there is evidence of acute or chronic on-going disease
After Collection:
• For quality assurance, H&Es of all grossly normal lung specimens obtained will be generated by
the BRPC and reviewed by pathologist to ensure microscopic normal lung.
Collection Protocol - Adult Tissue
Collection Protocol - Adult Tissue
U54Clinical Research Coordinator will screen for lung resection cases where normal tissue may be available. Refer to inclusion and exclusion criteria for surgical resection for
U54.
U54Clinical Research Coordinator will approach patients for consent and enroll patients in BRPC study at Duke when consent occurs.
Upon day of collection, U54Clinical Research Coordinator will be notified of tissue availability from Surgical Pathology
Receive 1 fragment of normal lung tissue (total size collected will be 3x3x3 cm; can take less if not available) from the resected site.
Process the fragment into the following samples. Make note of the weight and dimensions of each sample. Create a QC for each sample collected:
a. FFPE (3x3x0.5 cm)
b. Snap frozen: split in half
i. ½ snap freeze in cryovial
ii. ½ will be cut into “rice-size piece”
1. Wash tissue with PBS to remove the blood and epithelial lining fluid.
2. Using blade, mince tissue into approximately 20 small pieces (1-2mm).
3. Evenly distribute the rice sized pieces into 3 cryopreserved tubes with 1mL freezing media
each.
4. Cells should be frozen slowly at 1°C/min. Put tubes in the freezing container (e.g., Mr.
Frosty) with isopropyl alcohol and place them overnight in a-80'C freezer.
5. Next day, transfer tubes to liquid nitrogen
c. If tissue remaining: 3 additional cryovials of snap frozen tissue cut into rice sized pieces and
OCT.
Route FFPE samples to Research Histology Lab to:
a. Create an H&E from each FFPE sample and route to the pathologist for review.
b. After review, create 2 tissue scrolls from the tissue blocks and coordinate RNA and DNA
extraction.
c. Once RNA and DNA extraction is complete, the frozen LN2 samples will be shipped to lab for
scRNAseq
Collection Protocol - Adult Blood
Collection Protocol - Adult Blood
U54Clinical Research Coordinator will coordinate to have blood tubes dropped off prior to the procedure, if patients consented to BRPC optional blood component(s). An email will be sent to the Substrate Services Core Research Support regarding blood facilitation the day before surgery.
Drop off: (label each tube with MRN and BRPC ID)
a. 2 x 10mL EDTA tubes
b. 3 x 8.5mL ACD tubes
Make a note to prioritize EDTA tubes over ACD tubes if not all can be filled.
Once tubes are available, notify the Substrate Services Core Research Support of blood
for pickup via email and provide transport form with the available blood tubes. They will
complete further processing.
Collection Protocol - Pediatric Tissue
Collection Protocol - Pediatric Tissue
U54 Clinical Research Coordinator will screen for lung resection cases where normal tissue may be available. Refer to inclusion and exclusion criteria for surgical resection for U54.
U54 Clinical Research Coordinator or BRPC team member will approach patient’s in-person for consent and enroll patients in BRPC study at Duke when consent occurs.
Upon day of collection, U54 Clinical Research Coordinator will be notified of tissue availability from Surgical Pathology.
Receive 1 fragment of normal lung tissue (total size collected will be 3x3x3 cm; can take less if not available) from the resected site.
Process the fragment into the following samples. Make note of the weight and dimensions of each sample. Create a QC for each sample collected:
a. FFPE (3x3x0.5 cm)
b. Snap frozen: split in half
i. ½ snap freeze in cryovial as normal
ii. ½ will be cut into “rice-size piece”
1. Wash tissue with PBS to remove the blood and epithelial lining fluid.
2. Using blade, mince tissue into approximately 20 small pieces (1-2mm).
3. Evenly distribute the rice sized pieces into 3 cryopreserved tubes with 1mL freezing media
each.
4. Cells should be frozen slowly at 1°C/min. Put tubes in the freezing container (e.g., Mr.
Frosty) with isopropyl alcohol and place them overnight in a-80'C freezer.
5. Next day, transfer tubes to liquid nitrogen.
c. If tissue remaining: 3 additional cryovials of snap frozen tissue cut into rice sized pieces and
OCT.
Route FFPE samples to Research Histology Lab to:
a. Create an H&E from each FFPE sample and route to the pathologist for review.
b. After review, create 2 tissue scrolls from the tissue blocks and coordinate RNA and DNA
extraction.
c. Once RNA and DNA extraction is complete, the frozen LN2 samples will be shipped to lab for
scRNAseq.
Collection Protocol - Pediatric Blood
Collection Protocol - Pediatric Blood
U54 Clinical Research Coordinator will coordinate to have blood tubes dropped off prior to the procedure, if patients consented to BRPC optional blood component(s). An email will be sent to the Substrate Services Core Research Support regarding blood facilitation the day before surgery.
Drop off: (label each tube with MRN and BRPC ID)
a. 2 x 4mL EDTA tubes
Once tubes are available, notify the Substrate Services Core Research Support of blood for pickup via email and provide transport form with the available blood tubes. They will complete further processing.