TITLE: 616.1 URMC HTC BSL2+ Formalin-Inflated, Paraffin-Embedded Human Lung Tissue
AUTHORS: Gloria S Pryhuber, Heidie Huyck, Lisa Rogers

[DESCRIPTION]
Processing of Formalin Fixed Lung and Non-Lung Tissue for the LungMAP HTC and includes acquisition of limited Control tissue from Surgical Pathology.


[GUIDELINES]
Scientific      Principles or Validation of Procedures : 
CDC. “COVID-19 Personal Protective       Equipment (PPE) for Healthcare Personnel.” Centers for Disease       Control and Prevention, 1 Jan. 2020, www.cdc.gov/coronavirus/2019-ncov/downloads/COVID-19-PPE.pdf.
WHO. “Personal Protective Equipment for COVID-19.” World       Health Organization, World Health Organization, 5 May 2020,       www.who.int/medical_devices/priority/COVID_19_PPE/en/.
University of Rochester Medical Center, director. COVID-19       Safety Training, University of Rochester Medical Center , 15 June       2020,       rochester.csod.com/LMS/LoDetails/DetailsLo.aspx?loid=e8469bef-ae75-4d87-9ec1-f5d33e4f1519&query=%3fq%3dCOVID-19+Safety+Training&isCompletionRedirect=true&loStatus=16®num=1#t=1.

[STEPS]
SECTION: Record Process
1. Record details of procedure in Worksheet or Directly in Inventory or ELN
SECTION: Record Process
2. Keep photographic recording of inflation and all steps of sectioning and blocking of lung lobe and tissue
SECTION: Inflation Fixed (Formalin) Lung Lobe
4. Inflation fixation procedure should be accomplished in a biosafety cabinet with the operator taking appropriate fixative, blood and body fluid, including COVID-19, precautions.
SECTION: Inflation Fixed (Formalin) Lung Lobe
3. Keep the lobe / tissue cold and the surface moist at all times
SECTION: Inflation Fixed (Formalin) Lung Lobe
5. Connect a short piece of IV extension tubing, clamped with hemostat or shut off with stopcock, to a 10 or 20 ml syringe barrel (without plunger) suspended in a clamp on a ring stand so that the 5 ml syringe mark is 25 cm above the level of the main airway in the lobe to be fixed.
SECTION: Inflation Fixed (Formalin) Lung Lobe
6. Fill the syringe barrel with 10% Neutral Buffered Formalin (NBF)
SECTION: Inflation Fixed (Formalin) Lung Lobe
7. Flush the IV extension tubing through with formalin to remove air bubbles and so the NBF fluid level is at 10 or 20 ml mark on the syringe. Reclamp. prior to connection to the instillation cannulas
SECTION: Inflation Fixed (Formalin) Lung Lobe
8. Cannulate the main bronchus, identified by its cartilage containing wall, with an endotracheal tube or 16 or 18 gauge catheter. 

Fix the airway tube in place with suture material or zip tie. 

If two bronchi are apparent due to a branch-point, each can be cannulated. 

Tying in of the airway tubes is the “trickiest” part of the entire procedure and is most easily done by two people.
SECTION: Inflation Fixed (Formalin) Lung Lobe
9. Connect the IV extension tubing coming from the syringe to the airway tube(s). 

If more than one bronchi is cannulated, they can be instilled serially or simultaneously with a second syringe set up. 
SECTION: Inflation Fixed (Formalin) Lung Lobe
10. Release the clamp and allow formalin to infuse into the lung lobe that should visibly inflate, the degree of inflation varies.

 Transient, gentle pressure on the fluid head may be needed to start the flow of fixative.
SECTION: Inflation Fixed (Formalin) Lung Lobe
11. Add NBF to the syringe to maintain meniscus at 25 cm water inflation pressure, equilibrating at the 5ml syringe mark used to measure the height of the syringe barrel in step 5. Keep track of volume added.

Maintain instillation pressure for approximately 10 minutes after fluid stops flowing.
SECTION: Inflation Fixed (Formalin) Lung Lobe
12. Document volume of fixative infused and comment on any leakage (with degree -minimal, moderate, severe) 

SECTION: Inflation Fixed (Formalin) Lung Lobe
13. Once instillation is complete, pull the suture or zip tie that is on the airway and airway cannula tight while removing the cannula. 

This is best done with a second pair of hands. Also helpful to have a hemostat available as an emergency clamp. 
SECTION: Inflation Fixed (Formalin) Lung Lobe
14. Photograph anterior and posterior sides before and after inflation. 
SECTION: Inflation Fixed (Formalin) Lung Lobe
15. Place the securely tied lung lobe in a specimen container filled with 10% NBF.

For good fixation, prefer a volume ratio of 10:1 fixative:lung.
SECTION: Inflation Fixed (Formalin) Lung Lobe
16. Place in the cold room for 40-48 hours

Once tissue has been in fixative for 40-48 hours, processing may continue on grossing station or fume hood
SECTION: Inflation Fixed (Formalin) Lung Lobe
17. Forty to 48 hours after fixation begins, divide the lobe as diagrammed in attached figures into approximately   thick, 1.0 x 1.0 cm2 cubes, placing each cube/block in an appropriately labeled tissue cassette

SECTION: Inflation Fixed (Formalin) Lung Lobe
18. Place back in 10% formalin in the cold for 18-24 hours.
SECTION: Inflation Fixed (Formalin) Lung Lobe
19. The tissue cubes are then rinsed in 1xPBS and dehydrated by 20-minute  successive dwell times, with agitation on a rotating platform at room temperature, in PBS, 30% EtOH, 50% EtOH and 70% EtOH made  with diethylpyrocarbonate (DEPC) treated water.
SECTION: Inflation Fixed (Formalin) Lung Lobe
20. Tissues should be stored in 70% EtOH at 4 deg C for a minimum of overnight, or longer, until further processed into paraffin.
SECTION: Formalin Fixed Non-Lung Tissue
27. Remove excess tissues around trachea and large airways at hilum 
SECTION: Formalin Fixed Non-Lung Tissue
28. Collect lymph nodes, excess large vessels, esophagus, spleen and nerves (Best to place these back in shipping buffer and Tend to Lung First) 


SECTION: Formalin Fixed Non-Lung Tissue
28.1. Weigh each tissue collected 


SECTION: Formalin Fixed Non-Lung Tissue
28.2. The non-lung samples are sectioned into approximately 0.5-1 cm3 portions 

SECTION: Formalin Fixed Non-Lung Tissue
29. Divide these tissues between 10 % Formalin and PFAS-Cryoprotection 

Record these tissues and how processed in BRINDL database 
SECTION: Formalin Fixed Non-Lung Tissue
29.1. For 10% formalin fixation, place in labeled tissue cassette in formalin; stored at 40C to fix for 64-72 hours (consistent with lung tissue fixation time). Volume of fixative to tissue should be approximately 10:1. 

The tissue cubes are then rinsed in 1xPBS and dehydrated by 20-minute  successive dwell times, with agitation on a rotating platform at room temperature, in PBS, 30% EtOH, 50% EtOH and 70% EtOH made  with diethylpyrocarbonate (DEPC) treated water.

Tissues should be stored in 70% EtOH at 4 deg C for a minimum of overnight, or longer, until further processed into paraffin.
SECTION: Formalin Fixed Non-Lung Tissue
29.2. For Freezing in OCT: fix in 4% PFA x 20-24 hrs, cryo-protect in 30% sucrose and continue to process for freezing in OCT along with lung tissue to be frozen (See PFA fixed, sucrose cryoprotection protocol) 
SECTION: Tissue Processor
30. From 70% ethanol, place cassettes into metal basket in VIP processor’s retort (up to 150 unicassettes or 75 megacassettes) in 70% ethanol already in the retort 




SECTION: Tissue Processor
31. Use overnight program #3 on VIP Processor 
    70% Ethanol – 1 hour – 40 °C 
    80% Ethanol – 1 hour – 40 °C
    95% Ethanol – 1 hour – 40 °C 
    95% Ethanol – 1 hour – 40 °C 
    100% Ethanol – 1 hour – 40 °C 
    100% Ethanol – 1.5 hours – 40 °C
    Histological grade Xylene – 1 hour – 40 °C 
    Histological grade Xylene – 1.5 hours – 40 °C
    McCormack’s Paraplast (paraffin) – 1 hour – 60 °C 
    McCormack’s Paraplast (paraffin) – 1 hour – 60 °C 
    McCormack’s Paraplast (paraffin) – 1.5 hours – 60 °C 




SECTION: Tissue Processor
32. Remove cassettes out of processor to embed 




SECTION: Embedding Tissue
33. Place cassettes in holding tank in the embedding center until paraffin is melted (~15 minutes) 




SECTION: Embedding Tissue
34. Embed tissue in metal mold as it was in the processing cassette 




SECTION: Embedding Tissue
35. Cool and solidify blocks on cold plate on embedding center 




SECTION: Embedding Tissue
36. When blocks “pop” remove them from molds. 




SECTION: Embedding Tissue
37. Clean outside of cassettes of excess wax and trim excess wax from blocks with the paratrimmer 




SECTION: Embedding Tissue
38. Store blocks at room temperature away from heat and sunlight 




SECTION: Sectioning Paraffin Embedded Tissue
39. Cool blocks on ice
SECTION: Sectioning Paraffin Embedded Tissue
40. Using microtome, trim into block at a higher thickness (~14 microns) until a full section is  achieved.
SECTION: Sectioning Paraffin Embedded Tissue
41. Cool block on ice
SECTION: Sectioning Paraffin Embedded Tissue
42. Section blocks at 4-5 microns into a ribbon
SECTION: Sectioning Paraffin Embedded Tissue
43. Place ribbon into cool water bath
SECTION: Sectioning Paraffin Embedded Tissue
44. Place section on slide
SECTION: Sectioning Paraffin Embedded Tissue
45. Place section into hot water bath
SECTION: Sectioning Paraffin Embedded Tissue
46. Melt section onto slide
SECTION: Sectioning Paraffin Embedded Tissue
47. Dry slide standing up in rack overnight or until dry
SECTION: Sectioning Paraffin Embedded Tissue
48. Store slides in slide boxes at room temperature away from heat and sunlight
SECTION: Formalin Fixation of Lung Tissue without Instillation of Formalin
21. Place the lung slice that is to be formalin fixed in an approximately 10:1 volume of 10% buffered formalin in a leak proof container, a 200 ml specimen cup works well.
SECTION: Formalin Fixation of Lung Tissue without Instillation of Formalin
22. Shake the tightly closed container vigorously by hand for 1 minute to encourage self-inflation of the lung tissue.
SECTION: Formalin Fixation of Lung Tissue without Instillation of Formalin
23. Agitate the tissue in the container on a rotating platform for 60-120 minutes.
SECTION: Formalin Fixation of Lung Tissue without Instillation of Formalin
24. Store the tissue-containing container at 4deg C x 40-48 hrs and then cut the slice into approximately 1cm x 1cm x 0.5 cm blocks. Place the blocks in labelled tissue cassettes and place these back in the formalin for an additional 24 hours at 4 deg C 
SECTION: Formalin Fixation of Lung Tissue without Instillation of Formalin
25. The tissue cubes are then rinsed in 1xPBS and dehydrated by 20-minute  successive dwell times, with agitation on a rotating platform at room temperature, in PBS, 30% EtOH, 50% EtOH and 70% EtOH made  with diethylpyrocarbonate (DEPC) treated water.
SECTION: Formalin Fixation of Lung Tissue without Instillation of Formalin
26. Tissues should be stored in 70% EtOH at 4 deg C for a minimum of overnight, or longer, until further processed into paraffin.
SECTION: Control FFPE tissue samples from other source
49. Occasionally, completely de-identified tissue samples are obtained from the surgical pathology lab to be used as assay control or comparison tissue samples.  These could include tonsil, thymus, heart, lung and other thoracic or abdominal tissues.
50. These control tissues are received as FFPE blocks prepared by standard protocols of the surgical pathology lab with estimates of dates and times processed from patient retrieval through formalin, ethanol and embedding in paraffin and reported as metadata with datasets.  They are used for non-identifiable photomicroscopy and imaging. Nucleotide sequence is not generated.