Feb 02, 2023
Version 2
616.1 URMC HTC BSL2+ Formalin-Inflated, Paraffin-Embedded Human Lung Tissue V.2
Version 1 is forked from 611.2 URMC HTC Formalin-Inflated, Paraffin-Embedded Human Lung Tissue
- Gloria S Pryhuber1,
- Heidie Huyck1,
- Lisa Rogers2
- 1University of Rochester Medical Center;
- 2University of Rochester
Protocol Citation: Gloria S Pryhuber, Heidie Huyck, Lisa Rogers 2023. 616.1 URMC HTC BSL2+ Formalin-Inflated, Paraffin-Embedded Human Lung Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxejwdv8j/v2Version created by Gloria S Pryhuber
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 01, 2023
Last Modified: February 02, 2023
Protocol Integer ID: 76236
Keywords: Inflation fixation, lung, FFPE, COVID-19, SARS CoV2,
Funders Acknowledgement:
Lung Molecular Atlas Program
Grant ID: U01HL148861
Human Biomolecular Atlas Program
Grant ID: U54HL165443
Abstract
Processing of Formalin Fixed Lung and Non-Lung Tissue for the LungMAP HTC
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.
Materials
Worksheet 603.A.2 HTC_Whole_or_Partial_Lung_Processing
Biosafe surface and environment for manipulation of lung
Grossing Station, Biosafety Cabinet
- N95 masks, face shields, double gloves, shoe covers, hair nets, sleeve covers, and lab coat for PPE, all consistent with recommended PPE for COVID-19
- Biohazards Disposal Bag
- Red Sharps Container
- Tissue mega-cassettes (optional) and uni-cassettes
- Scalpels and Trimming Blades with Handles; Forceps; Other Dissection Equip
- Labeling pencil
- 10% neutral buffered formalin – at least one liter, with more on hand if needed for larger lungs
- 1xPhosphate Buffered Sodium
- 30% Ethanol; 50% Ethanol and 70% Ethanol. All solutions are made with Diethylpyrocarbonate (DEPC) treated water or directly treated with DEPC to minimize RNA degradation
- Small and Medium Gauze Pads – multi-pack
- Standard Balance and Weigh Boats: Medium and Large
- Airway Cannulas – Selection of sizes
(i) 16 and 18 gauge angiocatheters (needles removed and discarded)
(ii) tracheal cannulas 2.0-4.0 OD
(iii) endotracheal tubes 2.5 – 8 mm OD; tubes >/= 4.0 cuffed
14. IV extension set tubing with clamp
15. 20 ml syringe barrels for fixative
16. Needle free suture material
17. Bleech
18. Oxivir Tb or simlar anti-viral
Safety warnings
Working with potentially or known SARS CoV2/COVID-19+ human tissue: Personnel will adhere to safe work processes all consistent with recommended PPE for COIVD-19 work. PPE will be used including N95 mask, face shield, lab coat, closed shoes and double gloves, shoe covers, hair net, and sleeve covers. All activity will be behind shield of biosafety cabinet and/or with mask and safety glasses. Biosafety level 2 + practices will be followed, and the work performed in the designated lab space that is covered by annually updated IBC approved protocol. All institutional biosafety measures are followed in any manipulation of these human tissues.
Record Process
Record Process
Record details of procedure in Worksheet or Directly in Inventory or ELN
Keep photographic recording of inflation and all steps of sectioning and blocking of lung lobe and tissue
Inflation Fixed (Formalin) Lung Lobe
Inflation Fixed (Formalin) Lung Lobe
Keep the lobe / tissue cold and the surface moist at all times
Inflation fixation procedure should be accomplished in a biosafety cabinet with the operator taking appropriate fixative, blood and body fluid, including COVID-19, precautions.
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.
Fill the syringe barrel with 10% Neutral Buffered Formalin (NBF)
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
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.
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.
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.
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.
Document volume of fixative infused and comment on any leakage (with degree -minimal, moderate, severe)
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.
Photograph anterior and posterior sides before and after inflation.
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.
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
Forty to 48 hours after fixation begins, divide the lobe as diagrammed in attached figures into approximately 0.5 cm thick, 1.0 x 1.0 cm2 cubes, placing each cube/block in an appropriately labeled tissue cassette
Place back in 10% formalin in the cold for 18-24 hours.
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.
Formalin Fixation of Lung Tissue without Instillation of Formalin
Formalin Fixation of Lung Tissue without Instillation of Formalin
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.
Shake the tightly closed container vigorously by hand for 1 minute to encourage self-inflation of the lung tissue.
Agitate the tissue in the container on a rotating platform for 60-120 minutes.
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
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.
Formalin Fixed Non-Lung Tissue
Formalin Fixed Non-Lung Tissue
Remove excess tissues around trachea and large airways at hilum
Collect lymph nodes, excess large vessels, esophagus, spleen and nerves (Best to place these back in shipping buffer and Tend to Lung First)
Weigh each tissue collected
The non-lung samples are sectioned into approximately 0.5-1 cm3 portions
Divide these tissues between 10 % Formalin and PFAS-Cryoprotection
Record these tissues and how processed in BRINDL database
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.
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)
Tissue Processor
Tissue Processor
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
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
Remove cassettes out of processor to embed
Embedding Tissue
Embedding Tissue
Place cassettes in holding tank in the embedding center until paraffin is melted (~15 minutes)
Embed tissue in metal mold as it was in the processing cassette
Cool and solidify blocks on cold plate on embedding center
When blocks “pop” remove them from molds.
Clean outside of cassettes of excess wax and trim excess wax from blocks with the paratrimmer
Store blocks at room temperature away from heat and sunlight
Sectioning Paraffin Embedded Tissue
Sectioning Paraffin Embedded Tissue
Cool blocks on ice
Using microtome, trim into block at a higher thickness (~14 microns) until a full section is achieved.
Cool block on ice
Section blocks at 4-5 microns into a ribbon
Place ribbon into cool water bath
Place section on slide
Place section into hot water bath
Melt section onto slide
Dry slide standing up in rack overnight or until dry
Store slides in slide boxes at room temperature away from heat and sunlight