TITLE: 603.3 & 604.5_URMC_HTC_Whole Lung and Lobe Processing 
AUTHORS: Gloria Pryhuber, Heidie Huyck

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-weight:bold;">Purpose and Scope of the Procedure</span></div><style>
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							</style></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Standardize process for processing lung donations into components for storage and distribution</li><li style = "counter-reset:ol0;">Scope:       coordination of receipt and gross dissection of tissue</li></ol></div></div>

[STEPS]
?. i.CT Scan
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							    ii.Separation of Lobes
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							    iii.Tissue Dissociation
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							    iv.Formalin Inflation
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							    v.OCT /CMC Inflation
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							    vi.Paraformaldehyde Inflation
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?. Record details of procedures in Worksheets.
?. [Unpacking]
Shipped package is opened on arrival to remove a blood sample, if provided, as it should be kept at room temperature. The remainder of the package is to be kept in cold room until processing begins to maintain approximately  temperature of tissue.
4 °C
?. [Unpacking]
Determine BRINDL PID to assign to tissues and images by clicking the “arrived” button in BRINDL Screening log
?. [Unpacking]
Record Information on Shipping Labels in Database or worksheet for entry ASAP    i.UNOS #; Referring Company #    ii.Courier and Tracking Numbers
?. [Unpacking]
Remove Shipping Labels from package; place aside to be added to Sample File
?. [Unpacking]
Open Shipping Box and Remove Styrofoam Coveri.Note Condition of Packing in Database (ice sufficient, layered packaging, no leakage, etc.)
?. [Unpacking]
6.Open container containing tissue and move lung tissue to a clean plastic bag on ice.    i.Note Condition of Lung Tissue in Database        a.Is tissue submerged (usually in plastic bag, inside a hard plastic container)        b.Are Trachea and both lungs included and intact        c.Is Trachea Occluded, if so how        d.Are Thymus, Spleen, Blood Sample, Lymph Nodes included?
?. [CT Scan]
See separate protocol for CT Scan of Air Inflated Lung procedures.
?. [Grossing of Tissues]
Maintain tissues in transplant buffer on ice. Prevent open Trachea from being submerged , +/- ETT removal
?. [Grossing of Tissues]
Weigh intact organ as sent; Continue to Record all measurements and procedures in Worksheet or directly in BRINDL database
?. [Grossing of Tissues]
Determine displacement volume without inflation
?. [Grossing of Tissues]
Determine plan for organ lobes ie which to be inflated or infused and which to be dissociated
?. [Grossing of Tissues]
Remove excess tissues around trachea and large airways at hilum      Collect lymph nodes, excess large vessels, esophagus and nerves         (Best to place these back in storage buffer and Tend to Lung First)          a.     Weigh each tissue collected          b.     The non-lung samples are sectioned into approximately 0.5-1 cm3 portions          c.     Divide these tissues between                                                                                                                   i.    10% formalin, place in labeled tissue cassette in formalin                    Store at  to fix for 44-48 hrs,                     Section at mid-time.                      Volume of fixative to tissue should be approximately 10:1.              ii.     PFA: place in 4%PFA stored at  to fix for 20-28 hours before moving to 30%Sucrose          d.     Record these tissues and how processed in BRINDL database
4 °C
4 °C
?. [Grossing of Tissues]
Dissect free the bronchial branches, identify pulmonary arteries and veins
?. [Grossing of Tissues]
Divide airways and vessels to obtain 5 independent lung lobes
?. [Grossing of Tissues]
Weigh each lobe once isolated
?. [Grossing of Tissues]
Proceed with processing for each lobe as Outlined in Table 1 using appropriate Protocols
?. [Grossing of Tissues]
Prepare accompanying tissue such as thymus, spleen, esophagus, blood, heart, in similar fashion with specific as outlined in the appropriate Protocol / SOP.
?. [Grossing of Tissues]
ABCDE1  Organ
    Starting Tissue
    Prep
    Process
  BioSpecimen Type Collected from Organ2  Trachea and large
  bronchi
    Intact
    Paraffin and OCT
  Frozen
  Bisect longitudinally; Section into   Formalin Fixed,
  Paraffin embedded
  PFASOCT embedded
  and frozen
  3  Alternate Trachea
  and large bronchi
    Intact
    Cell Isolates
    Enzymatic
  digestion and cell isolation
  Frozen isolated cell aliquots4Lung: Right Upper Lobe +/- Right Middle Lobe  Fresh Tissue Blocks
    Mixed Dissociated
  Cells
    Dissociate cells
  by mechanical and enzymatic digestion procedure
  Cryopreserve mixed cell aliquots5  Sorted Dissociated
  Cells
  Flow Activated Cell Sorting dissociated cellsCryopreserve sorted cell aliquots “ex. Endothelial, Epithelial, Fibroblast, Bone Marrow Derived (CD45+) Separation”6Cryo-preserved QICryo-preserved
  cellsRecover frozen
  cell aliquots, test purity by PCR, culture for viability and proliferative/ differentiation potential7Add’l Right Middle Lobe  Intact - Biopsies
    Flash Frozen
    Stored at -80
  Protein Homogenates, RNA and/or DNA Isolation8Alternate Right Middle Lobe  Intact
    Formalin
  Inflation, Paraffin
  Inflation fixed
  with 10% buffered formalin x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Dehydrated to 70% Ethanol  Inflation fixed,
  paraffin embedded ready for sectioning
  9Alternate Right Middle Lobe  Fresh Tissue
  Blocks
    Fresh Frozen
  Embedded in OCT/CMC/none
  Vertical 0.1 cm slices, Section each slice by grid into 1 cm2 blocks, alternate slices to embed blocks in OCT or approx. 5% CMC (see protocol) or freeze without embedding material  Non-inflated, non-
  fixed flash frozen tissue blocks, mapped to originating location
  10Right Lower Lobe  Intact
    Formalin
  Inflation, Paraffin
  Inflation fix with
  10% buffered formalin x 24 hr (Intact Lobe)
  Sectioned by grid into 0.5-1 cm3 pieces
  Dehydrated to 70% Ethanol  Inflation fixed,
  paraffin embedded ready for sectioning
  11  Left Upper Lobe
    Intact
    PFA Fixed, Sucrose
  Cryopreserved, OCT embedded
  Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe);  Sectioned by grid into 0.5-1 cm3 pieces;  Cryopreserved in 10% sucrose; Block in OCT  Inflation fixed,
  cryo-preserved and frozen for cryo-sectioning
  12Alternate Left Upper Lobe  Intact
    OCT/PBS
    Inflate with 50:50
  OCT:10%PBS
    Frozen for
  cryo-sectioning
   
  1314Alternate Left Upper Lobe  Intact
    Air Inflated,
  Vascular Contrast
    To be determined
    To be determined
  15  Left Lower Lobe
   
    Intact
    PFA Fixed, Sucrose Cryopreserved, OCT embedded
  Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Cryopreserved in 10% sucrose; Block in OCT  Inflation fixed, cryo-preserved and frozen for
  cryo-sectioning
  16  Additional Tissue
    Intact
    Similar to Lung, see specific SOPs
  These include thymus, spleen, esophagus, heart, nerve, etc; Block as appropriate for tissueFormalin Fixed,
  Paraffin embedded,
  PFA/OCT embedded-frozen
  Single cell dissociation17  Peripheral Blood
    Whole
    Cryopreserved, see specific SOPs
  PBMC isolation, Plasma and Serum Storage  Cryopreserved
ABCDE1  Organ
    Starting Tissue
    Prep
    Process
  BioSpecimen Type Collected from Organ2  Trachea and large
  bronchi
    Intact
    Paraffin and OCT
  Frozen
  Bisect longitudinally; Section into   Formalin Fixed,
  Paraffin embedded
  PFASOCT embedded
  and frozen
  3  Alternate Trachea
  and large bronchi
    Intact
    Cell Isolates
    Enzymatic
  digestion and cell isolation
  Frozen isolated cell aliquots4Lung: Right Upper Lobe +/- Right Middle Lobe  Fresh Tissue Blocks
    Mixed Dissociated
  Cells
    Dissociate cells
  by mechanical and enzymatic digestion procedure
  Cryopreserve mixed cell aliquots5  Sorted Dissociated
  Cells
  Flow Activated Cell Sorting dissociated cellsCryopreserve sorted cell aliquots “ex. Endothelial, Epithelial, Fibroblast, Bone Marrow Derived (CD45+) Separation”6Cryo-preserved QICryo-preserved
  cellsRecover frozen
  cell aliquots, test purity by PCR, culture for viability and proliferative/ differentiation potential7Add’l Right Middle Lobe  Intact - Biopsies
    Flash Frozen
    Stored at -80
  Protein Homogenates, RNA and/or DNA Isolation8Alternate Right Middle Lobe  Intact
    Formalin
  Inflation, Paraffin
  Inflation fixed
  with 10% buffered formalin x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Dehydrated to 70% Ethanol  Inflation fixed,
  paraffin embedded ready for sectioning
  9Alternate Right Middle Lobe  Fresh Tissue
  Blocks
    Fresh Frozen
  Embedded in OCT/CMC/none
  Vertical 0.1 cm slices, Section each slice by grid into 1 cm2 blocks, alternate slices to embed blocks in OCT or approx. 5% CMC (see protocol) or freeze without embedding material  Non-inflated, non-
  fixed flash frozen tissue blocks, mapped to originating location
  10Right Lower Lobe  Intact
    Formalin
  Inflation, Paraffin
  Inflation fix with
  10% buffered formalin x 24 hr (Intact Lobe)
  Sectioned by grid into 0.5-1 cm3 pieces
  Dehydrated to 70% Ethanol  Inflation fixed,
  paraffin embedded ready for sectioning
  11  Left Upper Lobe
    Intact
    PFA Fixed, Sucrose
  Cryopreserved, OCT embedded
  Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe);  Sectioned by grid into 0.5-1 cm3 pieces;  Cryopreserved in 10% sucrose; Block in OCT  Inflation fixed,
  cryo-preserved and frozen for cryo-sectioning
  12Alternate Left Upper Lobe  Intact
    OCT/PBS
    Inflate with 50:50
  OCT:10%PBS
    Frozen for
  cryo-sectioning
   
  1314Alternate Left Upper Lobe  Intact
    Air Inflated,
  Vascular Contrast
    To be determined
    To be determined
  15  Left Lower Lobe
   
    Intact
    PFA Fixed, Sucrose Cryopreserved, OCT embedded
  Inflation fixed with 4% paraformaldehyde x 24 hr (Intact Lobe); Sectioned by grid into 0.5-1 cm3 pieces; Cryopreserved in 10% sucrose; Block in OCT  Inflation fixed, cryo-preserved and frozen for
  cryo-sectioning
  16  Additional Tissue
    Intact
    Similar to Lung, see specific SOPs
  These include thymus, spleen, esophagus, heart, nerve, etc; Block as appropriate for tissueFormalin Fixed,
  Paraffin embedded,
  PFA/OCT embedded-frozen
  Single cell dissociation17  Peripheral Blood
    Whole
    Cryopreserved, see specific SOPs
  PBMC isolation, Plasma and Serum Storage  Cryopreserved
?. [Lobe Sectioning Diagrams]
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