TITLE: Tonsil Collection and Processing Protocol
AUTHORS: Scott H. Randell, M. Leslie Fulcher, Adam J. Kimple, Kevin Byrd, Quinn T. Easter, Bruno F. Matuck, Kelly D. Chason, James S. Hagood

[DESCRIPTION]
For preserving human tonsils by formalin fixation, O.C.T. embedding and freezing, Cryostor freezing medium, and snap freezing in liquid nitrogen.

[GUIDELINES]
Illustration


 


[STEPS]
SECTION: Data Collection
1. Keep a record of the sample collection and processing to track the source and quality of the samples. Record the following data for:
SECTION: Data Collection
1.1. Sample collection:
Subject ID
Date and time collected
The number of tonsils collected
The patient position the tonsils were colleted from (left or right)
Verify that the samples are palatine tonsils
Any relevant collection notes
SECTION: Data Collection
1.2. Sample processing:
Subject ID
Date and time of processing start
Digital photograph of the tonsil(s) prior to dissection; see Section 6
The weight of each tonsil 
For the Cryostor (cryopreserved) samples:
Date and time samples were transferred to -80C  
Date and time samples were transferred to liquid nitrogen
For the Formalin Fixed (NBF) samples:  
Date and time samples were placed in NBF
Date and time samples were transferred to 70% Ethanol, or the total time in hours the samples were in NBF 
Date fixed samples were embedded in paraffin
For the O.C.T. Embedded sample:
Date and time samples were completely frozen in O.C.T compound
For the snap frozen sample:
Date and time samples were completely frozen in liquid nitrogen  
Any relevant notes for sample processing, such as whether any specimen was not processed successfully
SECTION: Sample Collection
2. Pre-collection Preparation
SECTION: Sample Collection
3. Palatine tonsils are excised by the surgeon during regularly scheduled surgical encounters.
SECTION: Sample Collection
4. Immediately after collection, mark the tonsil(s) on the cut (muscular side) with a skin marking pen and place the tonsil(s) in the specimen cup containing normal saline on ice.

SECTION: Sample Collection
5. Transport the tonsil(s) on ice, as well as the collection record, to the processing lab as soon as feasible.

SECTION: Sample Collection
2.1. Prepare a sample collection record according to step 1.1.
SECTION: Sample Collection
2.2. Patients are consented according to local and organizational requirements.
SECTION: Sample Collection
2.3. Label a sterile specimen cup with the sample type (“Tonsil”) and subject identification code (subject ID). Add enough normal saline to cover the tonsil(s).
SECTION: Preparation for Sample Processing
6. Samples should be delivered to the processing laboratory on ice, accompanied by a sample collection record.

SECTION: Preparation for Sample Processing
7. Prepare a sample processing record according to step 1.2.
SECTION: Preparation for Sample Processing
8. In the lab, assemble the following in a BSC or behind a safety shield:
SECTION: Preparation for Sample Processing
9. For cryopreservation:
SECTION: Preparation for Sample Processing
10. For O.C.T. Compound embedding, prepare a container of dry ice.
SECTION: Preparation for Sample Processing
11. For snap frozen tissue, obtain a small dewar of liquid nitrogen.
SECTION: Preparation for Sample Processing
12. Prepare the tonsils for dissection:
SECTION: Preparation for Sample Processing
8.1. Dissection board, covered first with a sterile towel drape, and optionally sterile gauze pads to absorb blood. (Option: use a Sterile 15 cm tissue culture dish with sterile gauze inside the dish).
SECTION: Preparation for Sample Processing
8.2. Sterile scissors and forceps.
SECTION: Preparation for Sample Processing
9.1. If using a Mr. Frosty or similar freezing container, add 250 mL isopropyl alcohol to the container.

SECTION: Preparation for Sample Processing
9.2. The isopropyl alcohol may be used (frozen) up to five times; replace after the fifth use.
SECTION: Preparation for Sample Processing
12.1. Transfer the tonsil(s), epithelium facing upwards, to the dissection board.

Tonsils may be marked during collection on the cut side that was adjacent to underlying muscle. This is to help differentiate it from the epithelial side.
SECTION: Preparation for Sample Processing
12.2. Digital Photograph

Place a sterile ruler beside the tonsil to measure the length. Add a note next to tissue with the subject ID and date. Take a digital photograph.
SECTION: Preparation for Sample Processing
12.3. Examine the tissue. The serosal side may have char from electrocauterization. Using a #11 scalpel, remove the char, if present.
SECTION: Preparation for Sample Processing
12.4. Tonsil Weight

Place a sterile plastic container sized to contain the tonsil on a laboratory scale; zero or tare the container. 
Place the tonsil on the container and record the weight of the tonsil.
Return the tonsil to the dissection board with the epithelium facing upwards for tissue sampling. 
SECTION: Preparation for Sample Processing
12.5. The tonsil(s) will be dissected as illustrated below, and according to sections "Procedure: Formalin Fixation in Histology Cassette", "Procedure: Cryopreservation in Cryostor freezing medium", "   
Procedure: O.C.T. Compound Embedding and Freezing", and "   
Procedure: Snap Frozen Tissue". 

It is irrelevant which parts (i.e., middle or end piece) go to which procedure.
If the tonsil is small, reduce the amount cut for O.C.T Compound and Snap Frozen tissue.

Illustration
 

SECTION: Procedure: Formalin Fixation in Histology Cassette
13. Follow your institutional guidelines for use of formalin.
SECTION: Procedure: Formalin Fixation in Histology Cassette
14. Record the information on the previously prepared processing record.
SECTION: Procedure: Formalin Fixation in Histology Cassette
15. Label a tissue cassette and specimen cup with the sample ID.; ONLY USE PENCIL on the cassette.
SECTION: Procedure: Formalin Fixation in Histology Cassette
16. Add a formaldehyde warning sticker to the cup and label it with the date, operator initials, and fixative.
SECTION: Procedure: Formalin Fixation in Histology Cassette
17. Using a #11 Scalpel cut approximately 1/4 of the tonsil as illustrated above.
SECTION: Procedure: Formalin Fixation in Histology Cassette
18. Place the tissue cut side down in the labeled cassette and close.
SECTION: Procedure: Formalin Fixation in Histology Cassette
19. Place the cassette in the labeled EMPTY specimen cup and store on ice. In the fume hood, pour enough 10% neutral buffered formalin (NBF) to cover the cassette. Cover and store at 4 °C for 1440 min.

SECTION: Procedure: Formalin Fixation in Histology Cassette
20. After fixation, aspirate the NBF into a formalin waste trap in the fume hood.
SECTION: Procedure: Formalin Fixation in Histology Cassette
21. Wash the cassette(s) with PBS, aspirate the wash, and repeat. Cross through the formaldehyde sticker and fixative label to indicate NBF is no longer present.
SECTION: Procedure: Formalin Fixation in Histology Cassette
22. Replace the last PBS wash with 70% Ethanol and store at 4 °C.

SECTION: Procedure: Formalin Fixation in Histology Cassette
23. Submit for paraffin embedding as soon as possible.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
24. Record the information on the previously prepared processing record.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
25. Label a 15 mL conical tube with the sample ID for Cryostor.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
26. Add 5 mL PBS to the tube. Store on ice.

SECTION: Procedure: Cryopreservation in Cryostor freezing medium
27. Label TWO cryovials for each tonsil with the sample ID.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
28. Using a #11 Scalpel, cut approximately 1/4 of the tonsil as illustrated above and transfer to the appropriate labeled 15 mL conical tube.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
29. Using sterile surgical scissors, finely mince the tissue to approximately 2mm pieces in the 15 mL tube.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
30. Set up 50 mL tube with 100 µM strainer on top.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
31. Pour the minced tissue in PBS onto the filter, leaving minced tissues on top of the filter.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
32. Using forceps, transfer the minced tissues to the labeled cryovials, dividing the tissue evenly between them. DO NOT ADD TISSUE HIGHER THAN the 1 mL mark on the cryovials. Discard any extra tissue remaining into a biohazard waste bin.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
33. Make sure the tissue is not stuck on the side or rim of the cryovials. Tap each cryovial on a hard surface to collect all tissue at bottom of the vial.
SECTION: Procedure: Cryopreservation in Cryostor freezing medium
34. Add 1 mL Cryostor freeze solution to each cryovial and mix thoroughly.

SECTION: Procedure: Cryopreservation in Cryostor freezing medium
35. Transfer the cryovials to a freezing container and place in a -80 °C freezer.

SECTION: Procedure: Cryopreservation in Cryostor freezing medium
36. 24-72 hours later, transfer the vials into liquid nitrogen storage.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
37. Record the information on the previously prepared processing record.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
38. Label an embedding mold with the sample ID using a permanent marker on the lip on mold.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
39. Label an aluminum foil sheet, sized to wrap around the mold. Label a small plastic bag to store the frozen sample.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
40. Place the mold and aluminum foil on the dry ice to chill.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
41. Using #11 Scalpel cut approximately 1/4 of the tonsil as illustrated above.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
42. Add a thin layer of O.C.T. Compound to mold and place the tissue, cut side down, on the O.C.T. in the center of mold. Add more O.C.T. Compound on top of the tissue.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
43. Use toothpicks or pipet tips to maintain the position of the tissue and keep it from floating to the surface. Make sure there are no bubbles around the tissue. Continue adding O.C.T. Compound until entire tissue is covered.
 

SECTION: Procedure: O.C.T. Compound Embedding and Freezing
44. Place dry ice around mold to help it freeze faster. Wrap the frozen mold with the pre-labeled aluminum foil and place it in the plastic bag.
SECTION: Procedure: O.C.T. Compound Embedding and Freezing
45. Store in -80 °C freezer.

SECTION: Procedure: Snap Frozen Tissue
46. Record the information on the previously prepared processing record.
SECTION: Procedure: Snap Frozen Tissue
47. Label a cryovial with the sample ID.
SECTION: Procedure: Snap Frozen Tissue
48. Obtain a small dewar flask of liquid nitrogen.
SECTION: Procedure: Snap Frozen Tissue
49. Using #11 Scalpel cut approximately 1/4 of the tonsil as illustrated above.
SECTION: Procedure: Snap Frozen Tissue
50. Place the tissue sample in the cryovial and cap.
SECTION: Procedure: Snap Frozen Tissue
51. Immediately don cryogenic gloves and, using forceps, submerge the vial in liquid nitrogen. Allow the vial to remain in the liquid nitrogen for at least 20 seconds to ensure freezing.
SECTION: Procedure: Snap Frozen Tissue
52. Remove the vial from the liquid nitrogen and immediately store on dry ice until transfer to liquid nitrogen storage.
SECTION: Procedure: Formalin Fixation in Histology Cassette
17.1. The slice for this section should not exceed the interior dimensions of the cassette when closed, and it should not exceed 0.5 cm to ensure complete fixation within 24 hours.
SECTION: Procedure: Formalin Fixation in Histology Cassette
19.1. If fixation for longer than 24 hours is unavoidable, tissue may remain in NBF for up to 72 hours.