TITLE: U54 SCENT Normal Lung Surgical Tissue Collection Procedure
AUTHORS: Carolyn Glass, andrew.nixon, Matthew Hartwig, Nancy McGreal, SSCRS, Mary Jordan*

[DESCRIPTION]
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.

[STEPS]
SECTION: Inclusion and Exclusion Criteria
1. 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. 
SECTION: Collection Protocol - Adult Tissue
2. 
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.
SECTION: Collection Protocol - Adult Tissue
3. U54Clinical Research Coordinator will approach patients for consent and enroll patients in BRPC study at Duke when consent occurs.
SECTION: Collection Protocol - Adult Tissue
4. Upon day of collection, U54Clinical Research Coordinator will be notified of tissue availability from Surgical Pathology
SECTION: Collection Protocol - Adult Tissue
5. Receive 1 fragment of normal lung tissue (total size collected will be 3x3x3 cm; can take less if not available) from the resected site.
SECTION: Collection Protocol - Adult Tissue
6. 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. 
SECTION: Collection Protocol - Adult Tissue
7. 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
SECTION: Collection Protocol - Adult Blood
8. 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.
SECTION: Collection Protocol - Adult Blood
9. Drop off: (label each tube with MRN and BRPC ID)
    a. 2 x 10mL EDTA tubes
    b. 3 x 8.5mL ACD tubes
SECTION: Collection Protocol - Adult Blood
10. Make a note to prioritize EDTA tubes over ACD tubes if not all can be filled.
SECTION: Collection Protocol - Adult Blood
11. 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.
SECTION: Collection Protocol - Pediatric Tissue
12. 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.
SECTION: Collection Protocol - Pediatric Tissue
13. 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.
SECTION: Collection Protocol - Pediatric Tissue
14. Upon day of collection, U54 Clinical Research Coordinator will be notified of tissue availability from Surgical Pathology.
SECTION: Collection Protocol - Pediatric Tissue
15. Receive 1 fragment of normal lung tissue (total size collected will be 3x3x3 cm; can take less if not available) from the resected site.
SECTION: Collection Protocol - Pediatric Tissue
16. 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.
SECTION: Collection Protocol - Pediatric Tissue
17. 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.
SECTION: Collection Protocol - Pediatric Blood
18. 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.
SECTION: Collection Protocol - Pediatric Blood
19. Drop off: (label each tube with MRN and BRPC ID)
    a. 2 x 4mL EDTA tubes
SECTION: Collection Protocol - Pediatric Blood
20. 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.