Preanalytical protocol for fresh solid tumor biospecimens

Abstract Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. 1–4 These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. 2,5 Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Sterilize the solution using a 500 mL capacity vacuum filter.Store 50 mL aliquots at 4°C; stable for up to 12 months; reusable.

Total 50 mL
Sterilize the solution using a 150 mL capacity vacuum filter into a 50 mL conical tube.Store at 4°C; stable for up to 12 months; reusable.

Total 561 mL
Sterilize the solution using a 500 mL capacity vacuum filter.Store 50 mL aliquots at 4°C; stable for up to 12 months; reusable.

Reagent or resource Amount
1.5 mL centrifuge tube with 4% PFA 1 mL 5 mL conical tube with transport media 3.5 mL Page: 5 of 10

Reusable ice pack 3
Tissue specimen collection form 1 Transport box 1

Packaging and shipping tissue procurement kits to collection facility
This first segment of the protocol describes the preanalytical parameters involved in sending tissue procurement kits to the collection facility.Note: To ensure consistency in packaging materials and procedures, fully assembled tissue procurement kits containing all materials needed for biospecimen storage and transportation are shipped from the CerFlux-Lab to the tissue collection facility.
Caution: Strict aseptic techniques must be implemented when preparing and handling media and other reagents.Preparing and packaging tissue procurement kits for shipment to collection facility must be done in a sterile environment.
1. Prepare transport media in a sterile biosafety cabinet.Note: At a minimum, biosafety hood must be sterilized by exposure to UV for about 15 minutes followed by wiping down with 70% ethanol.
Caution: Ensure biologic and reagent substances are not present in the biosafety cabinet during sterilization.
a. Prepare transport media as detailed in Materials and equipment.
b. Use a pipette aid and sterile 10 mL serological pipette to transfer 3.5 mL into a sterile, labeled 5 mL conical tube.
c. Aliquot remaining transport media into sterile 50 mL conical tubes using a pipette aid and sterile 50 mL serological pipettes.Label and store in a sterile fridge at 4°C for future use.
2. Use 1250uL electronic single channel ClipTip pipette with sterile 1000uL ClipTip tips to dispense 1 mL of sterile 4% PFA into a sterile, labeled 1.5 mL centrifuge tube.
3. Spray exteriors of both the 5 mL conical tube, containing transport media, and the 1.5 mL centrifuge tube, containing PFA, with 70% ethanol; wipe down with Kimwipes; 4. Place both tubes in a sterile biohazard specimen bag and seal shut.

Receiving tissue procurement kit at collection facility
This second step of the protocol focuses on the preanalytical parameters involved in receiving and storing the contents of the tissue procurement kits.

Caution:
The handling of the tissue procurement kit must be done in a sterile environment.
1. Spray the exterior of the transport box with 70% ethanol and open it in a sterile environment.
2. Retrieve biohazard bag containing tissue specimen collection form and spray exterior with 70% ethanol before opening.Note: This form should be completed at the time of biospecimen collection.
3. Remove the foam container and spray its exterior with 70% ethanol before opening.
4. Remove the ice packs and sterilize with 70% ethanol before storing in freezer at -20°C for reuse.
5. Retrieve biohazard specimen bag containing media and PFA tubes; spray the exterior of the bag with 70% ethanol before unsealing.
6. Spray the exteriors of both tubes with 70% ethanol before examining for damage or contamination.
7. Store 5 mL conical tube containing transport media in a sterile environment at 4°C until use.
8. Store 1.5 mL centrifuge tube containing 4% PFA in a sterile environment at ambient room temperature until use.9. Sterilize transport box and Govee Wi-Fi Thermo-Hygrometer with 70% ethanol and retain for shipping back to CerFlux-Lab.
11. Ensure return shipping label has been received electronically.
10. Handle fresh solid tissue biospecimens in a sterile biosafety cabinet.Note: At a minimum, biosafety hood must be sterilized by exposure to UV for about 15 minutes followed by wiping down with 70% ethanol.
Caution: Ensure biologic and reagent substances are not present in the biosafety cabinet during sterilization.
a. Sterilize a glass Petri dish with 70% ethanol; once dry, sterilize in a UV hood for 10 minutes.
Bring the dish into the biosafety cabinet and keep cold using sterile ice packs.
b. Prepare 1X PBS solution as detailed in Materials and equipment.
c. Remove the biospecimen from the 5 mL tube using a pipette aid and sterile 10 mL serological pipette.
d. Use a pipette aid and sterile 10 mL serological pipettes to thoroughly wash biospecimen five times with 5 mL of ice-cold 1X PBS to remove debris.
e. Transfer specimen into a sterile, plastic, lidded Petri dish.
f. Place Petri dish with biospecimen on a sterile analytical balance to accurately determine initial biospecimen weight.Note: Ensure balance has been tared to display 0.000 g using an empty Petri dish prior to weighing sample.Compare data provided in the tissue specimen collection form and document for laboratory records.
g. Move Petri dish with biospecimen on a scale to image and ascertain proper dimensions.
Note: Compare data provided in the tissue specimen collection form and document for laboratory records.
h. Use forceps to transfer biospecimen back into the cold glass Petri dish.Note: Forceps should be sterilized in a glass bead sterilizer at 260°C then acclimated to ambient temperatures before use.
i. Prepare washing media as detailed in Materials and equipment.Add 6 mL into the glass Petri dish.j.Hold biospecimen in place using forceps; section off a sample from the outside of the specimen using a scalpel and 10 blade.Note: Forceps, scalpel, and blade should be sterilized in a glass bead sterilizer at 260°C then acclimated to ambient temperatures before use.
i. Place sectioned sample into a sterile, labeled 1.5 mL centrifuge tube containing 1 mL of 4% PFA.Note: This sample will be fixed for histological and correlative studies.
ii. Follow steps 9a-g above for this specimen as well.
The remainder of the fresh solid tumor tissue biospecimen can be processed for analytical laboratory testing.

Figures
Figure 1 Unnumbered image in the Materials and equipment section.

5 .
Prepare tissue procurement kit in a sterile environment.a. Open the foam container and spray the interior with 70% ethanol.b.Spray one reusable ice pack with 70% ethanol and place it on the bottom of the box.c.Spray biohazard specimen bag containing both tubes and Govee Wi-Fi Thermo-Hygrometer with 70% ethanol; place them side-by-side onto ice pack.Page: 6 of 10 d.Spray two reusable ice packs with 70% ethanol and insert them on either side of the biohazard bag and Govee; angle ice packs to cover the top.e. Close the lid of the foam container and spray the exterior with 70% ethanol.f.Seal foam container using clear packaging tape.g.Spray interior of transport box with 70% ethanol and place foam container inside.h.Insert the tissue specimen collection form inside a biohazard bag and place on top of the foam container.i. Seal transport box using clear packaging tape and attach shipping label to the outside.6. Notify the collection facility of package shipment.7. Send return shipping label to collection facility electronically.