U54 SCENT Normal Lung Surgical Tissue Collection Procedure

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. 

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

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.

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.

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.