TITLE: Preparation, Processing and Preservation of Deceased Donor Kidney Tissue for Multiomic Studies
AUTHORS: Sanjay Jain, Amanda Knoten, Kristy Conlon

[DESCRIPTION]
Multiomic technologies are increasingly being used on human samples and generating multidimensional data. Preanalytical and tissue procurement factors can have a lasting and negative effect on omic results.  For these data to be biologically and clinically useful, it is essential that high quality samples are obtained with rigor and reproducibility.   By using kidney tissue procurement as an example, we present an end-to-end detailed pipeline from deceased donor patients from consent to tissue procurement, processing and preservation.   This approach has been extensively tested on various atlasi projects and multiple tissue types including the Human Biomolecular Atlas Program (HuBMAP) and the Kidney Precision Medicine Project (KPMP).  

[BEFORE_START]
Please review the entire protocol for necessary preparation and items needed including lines of communications before starting

[STEPS]
SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
1. Check patient file for the following information:
Consent to donate organs
Downtime, if any
Was kidney on pump?
BUN/Cr
Kidney biopsy report/any underlying disease
Medical history for comorbidities
General good health before the current event
Cold ischemia time


 

SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
2. Arrange time of boxed organ pickup.
SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
3. Upon arrival to OPO:
SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
3.1. Confirm case number matches package labeling.
SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
3.2. Confirm documentation is packaged with organ.
SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
3.3. Document Left OPO time on Worksheet (find copy of Worksheet below):
SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
4. Transport kidney to lab for processing.
SECTION: Procurement: Procuring Kidney from organ procurement organization (OPO)
4.1. Document Arrived in Lab time on Worksheet.
SECTION: Preparation: Before Kidney Arrival
5. Prepare Worksheet with initial information (find copy of Worksheet below):
PPID
Date (of collection)
Laterality
Date/Time of Extubation
Date/Time of Perfusion

 
Specimen Labels (i.e., “K23-00001” - We use “K” for KTRC + the year followed by the next consecutive specimen number. Each new year begins at 00001, and so on.)

 

SECTION: Preparation: Before Kidney Arrival
6. Label preliminary collection cassettes and tubes using the unique Specimen Labels prepared on the Worksheet. The table below indicates an estimate of the number of prelabeled cassettes and tubes that may be needed at the time of collection. (Have extra available for additional desired tissue).

 

SECTION: Preparation: Before Kidney Arrival
7. Prefill the pre-labeled tubes (3/4 full) with 4%PFA, PBS or RNA for faster processing once kidney arrives
SECTION: Preparation: Before Kidney Arrival
7.1. Reference Supply List_Processing Kidney Tissue Section (D) Processing Liquids for information on liquids needed and how to prepare solutions.
SECTION: Preparation: Before Kidney Arrival
7.2. Immediately place prelabeled 4% PFA and PBS containers in wet ice.
SECTION: Preparation: Before Kidney Arrival
8. Prepare processing area
SECTION: Preparation: Before Kidney Arrival
8.1. Clean work area, prepare as if working in sterile conditions
SECTION: Preparation: Before Kidney Arrival
8.2. Treat any area where RNA samples will be processed with RNAse Zap to prevent RNA degradation
SECTION: Preparation: Before Kidney Arrival
9. Gather Supplies:
SECTION: Preparation: Before Kidney Arrival
9.1. Prepared Wet Ice container - include:
i. Pre-labeled 4% PFA and PBS tubes
ii. Additional PBS
iii. Additional 4% PFA
iv. Glass Petri dish(es) (Petri dishes should be chilled to keep dissected tissue section cold before processing and preservation)
SECTION: Preparation: Before Kidney Arrival
9.2. Prepared Dry Ice container (break up/powder dry ice – aides in closer contact with sample cassette and keeping it frozen)

 

SECTION: Preparation: Before Kidney Arrival
9.3. Small styrofoam cooler with embedding well (or metal block) placed inside for freezing
SECTION: Preparation: Before Kidney Arrival
9.4. LN2 transport container (do not fill until kidney is ready)
SECTION: Preparation: Before Kidney Arrival
9.5. Additional (unlabeled) cassettes and tubes for additional tissue (Reference Supply List section (E) Processing Tissue Containers)
SECTION: Preparation: Before Kidney Arrival
9.6. Additional RNA & RNAse Zap for additional RNA collections
SECTION: Preparation: Before Kidney Arrival
9.7. O.C.T
SECTION: Preparation: Before Kidney Arrival
9.8. Remaining autoclaved items - scalpel, long forceps, and regular forceps (Reference Supply List section (B) Autoclaved Supplies)
SECTION: Preparation: Before Kidney Arrival
9.9. Processing Tools – clipboard with Worksheet_Deceased Donor Kidney Specimens, pencil, Sharpie, scalpel blades, cutting board, (2) 4” x 4” squares of parafilm sheets, ruler, and camera (Reference Supply List section (C) Processing Tools)
SECTION: Processing: Preparing Kidney
10. Fill LN2 Transport Container.
SECTION: Processing: Preparing Kidney
11. Prep kidney for dissection:
SECTION: Processing: Preparing Kidney
11.1. Prepare cutting board to receive kidney.
SECTION: Processing: Preparing Kidney
11.2. Trim surrounding fat and large vessels to expose kidney and ureter.
SECTION: Processing: Preparing Kidney
11.3. Orient the kidney and attached ureter on cutting board.
SECTION: Processing: Preparing Kidney
11.4. Identify posterior and anterior aspects.
SECTION: Processing: Preparing Kidney
11.5. Identify upper, mid, and lower poles.
SECTION: Processing: Preparing Kidney
11.6. Identify pelvis.
SECTION: Processing: Preparing Kidney
11.7. In centimeters measure and document on Worksheet:
(i) Kidney (cm) - (L x W x D)
(ii) Ureter (cm) - from ureteropelvic junction (UPJ) to distal end
SECTION: Processing: Preparing Kidney
11.8. Take pictures of entire specimen - including ureter (place ruler in background if no printed measurements on cutting board)

 

 

SECTION: Processing: Preparing Kidney
11.9. Pour LN2 in cooler up to the top of metal embedding well (or block). Do not cover top surface of block. LN2 may need to be replenished several times while processing.
SECTION: Processing: Kidney Dissection
12. Distal Ureter:
SECTION: Processing: Kidney Dissection
12.1. Document Start Time for Distal Ureter and overall Processing Start Time on Worksheet.
SECTION: Processing: Kidney Dissection
12.2. Obtain approximately 1-3cm of distal ureter.
SECTION: Processing: Kidney Dissection
12.3. Cut into roughly 2mm cross sections and allocate for desired processing.
SECTION: Processing: Kidney Dissection
12.4. Process Distal Ureter segments following Processing Instructions for different preservation methods found below.
SECTION: Processing: Kidney Dissection
12.5. Document Stop Time and sample types collected for distal ureter.
SECTION: Processing: Kidney Dissection
13. Proximal Ureter:
SECTION: Processing: Kidney Dissection
13.1. Document Start Time.
SECTION: Processing: Kidney Dissection
13.2. Obtain approximately 1-3cm of proximal ureter (proximal ureter boundary is at UPJ).
SECTION: Processing: Kidney Dissection
13.3. Cut into roughly 2mm cross sections and allocate for desired processing.
SECTION: Processing: Kidney Dissection
13.4. Process Proximal Ureter segments following Processing Instructions for different preservation methods  found below.
SECTION: Processing: Kidney Dissection
13.5. Document Stop Time and sample types collected for proximal ureter.
SECTION: Processing: Kidney Dissection
14. Bisect kidney on the longest axis.

 

SECTION: Processing: Kidney Dissection
14.1. Take picture of open kidney (include ruler if not on cutting board).
SECTION: Processing: Kidney Dissection
14.2. Examine kidney for any gross abnormalities and document.
SECTION: Processing: Kidney Dissection
14.3. Select healthy appearing lobe(s) for dissection (the lobe typically includes an outer cortex and underlying medullary pyramid).
SECTION: Processing: Kidney Dissection
14.4. Carefully cut out first selected lobe containing cortex and medulla, and assign as Region A (“Region A” is shown as “Section A” in examples below).

 

SECTION: Processing: Kidney Dissection
15. Region A:
SECTION: Processing: Kidney Dissection
15.1. Document Start Time, pole location, and anterior or posterior.
SECTION: Processing: Kidney Dissection
15.2. Take a picture of Region A (include ruler).
SECTION: Processing: Kidney Dissection
15.3. Cut desired sections from lobe and designate processing method for each piece.

 

SECTION: Processing: Kidney Dissection
15.4. Process Region A pieces following Processing Instructions for different preservation methods  found below.
SECTION: Processing: Kidney Dissection
15.5. Document Stop Time and sample types collected for Region A.
SECTION: Processing: Kidney Dissection
16. Repeat process for Regions B, C …
SECTION: Processing: Kidney Dissection
17. If possible, after all lobes have been collected, take an additional picture of bisected kidney to aid in identifying the locations from where the tissue was dissected (see picture below).
SECTION: Processing: Kidney Dissection
18. On kidney drawing, document location of collected lobes as a reference for cut sections shown in picture.

 

SECTION: Processing: Final Documentation
19. Document Processing Stop Time.
SECTION: Processing: Final Documentation
20. Confirm that each sample is accounted for and correctly marked on Worksheet.
SECTION: Processing: Final Documentation
21. Calculate & document Total Transport Time in minutes (Elapsed time from Left OPO time to Arrived in Lab time).
SECTION: Processing: Final Documentation
22. Calculate & document Warm Ischemic Time in minutes (Elapsed time from Date/Time of Extubation to Date/Time of Perfusion).
SECTION: Processing: Final Documentation
23. Calculate & document Cold Ischemic Time in minutes (Elapsed time from Date/Time of Perfusion to Processing Start Time).
SECTION: Processing: Final Documentation
24. Calculate & document Total Ischemic Time in minutes (Total of Warm Ischemic Time and Cold Ischemic Time).
SECTION: Processing: Final Documentation
25. Calculate and document Total Processing Time in minutes (Elapsed time from Processing Start Time to Processing Stop Time).
SECTION: Processing: Final Documentation
26. Document Processor’s Initials
SECTION: Processing: Final Documentation
27. The following day, when the 4% PFA is changed to PBS, document the Fixation Stop Date/Time on Worksheet.
SECTION: Processing: Final Documentation
28. Calculate and document Total Fixation Time in minutes (Elapsed Time from Processing Start Time to Fixation Stop Date/Time).
SECTION: Processing Instructions: Different Preservation Methods
29. 
 

SECTION: Fresh Frozen Tissue (FB): OCT embedding
30. Squirt a quarter sized amount of O.C.T on a 4” x 4” square of paraffin film.

 

SECTION: Fresh Frozen Tissue (FB): OCT embedding
31. Using regular forceps, gently roll cut tissue selection in O.C.T. so it is completely covered.
SECTION: Fresh Frozen Tissue (FB): OCT embedding
32. Place O.C.T. covered tissue strategically in labeled cassette.
SECTION: Fresh Frozen Tissue (FB): OCT embedding
33. Squirt additional O.C.T. into cassette well until tissue is completely covered.
SECTION: Fresh Frozen Tissue (FB): OCT embedding
34. With large forceps, carefully place filled cassette onto embedding well or metal block within the LN2 cooler.
NOTE: The metal block or plate should be prechilled in LN2.  After pacing the metal block in the cooler or styrofoam container, fill the container with LN2 to upto half the depth of the metal block.  Initially the LN2 will bubble but then will settle.  Replenish if the LN2 evaporates to keep the block chilled.  This usually takes about 5 -10 min.  Wear protective gear to protect yourself from accidental LN2 splashes or spills.    
SECTION: Fresh Frozen Tissue (FB): OCT embedding
35. Cover cooler and monitor carefully.
SECTION: Fresh Frozen Tissue (FB): OCT embedding
36. Once block is completely frozen, use large forceps to remove frozen cassette from cooler and place on bed of powdered dry ice.

 

SECTION: Fresh Frozen Tissue (FB): OCT embedding
37. Document sample type and number – using our nomenclature, circle “1” under correct tissue column to indicate unique specimen label (i.e., K2300003_1FB - This is your first piece of fresh frozen tissue collected. Your second piece of fresh frozen tissue under this parent number would be K2300003_2FB); see example Worksheet at the beginning of this section.
SECTION: Fresh Frozen Tissue (FB): OCT embedding
38. When finished processing place in labeled resealable bag for protection.
SECTION: Fresh Frozen Tissue (FB): OCT embedding
39. Sample is ready for -80 °C storage.

 

SECTION: Flash Fresh Frozen LN2 Samples:
40. Using regular forceps, gently place cut tissue selection in empty labeled tube and place lid.
SECTION: Flash Fresh Frozen LN2 Samples:
41. With large forceps place tube directly in LN2 within the cooler.
SECTION: Flash Fresh Frozen LN2 Samples:
42. Cover cooler and monitor carefully.
SECTION: Flash Fresh Frozen LN2 Samples:
43. Once completely frozen, use large forceps to remove tube from cooler and place on bed of powdered dry ice.
SECTION: Flash Fresh Frozen LN2 Samples:
44. Document sample type and number – using our nomenclature, circle “17” under correct tissue column to indicate unique specimen label (i.e., K2300003_17 - This is your first piece of flash frozen tissue collected. Your second piece of flash frozen tissue under this parent number would be K2300003_18); see example Worksheet at the beginning of this section.
SECTION: Flash Fresh Frozen LN2 Samples:
45. Sample is ready for -80 °C storage.

SECTION: Preservation in RNAlater for RNA:
46. Using regular forceps, gently place cut tissue selection in labeled tube (filled ¾ of the way with RNAlater).
SECTION: Preservation in RNAlater for RNA:
47. Place lid and temporarily store in Wet Ice container until all processing is complete.
SECTION: Preservation in RNAlater for RNA:
48. Document sample type and number – using our nomenclature, circle “19” under correct tissue column to indicate unique specimen label (i.e., K2300003_19 - This is your first piece of RNA treated tissue collected. Your second piece of RNA treated tissue under this parent number would be K2300003_20); see example Worksheet at the beginning of this section.
SECTION: Preservation in RNAlater for RNA:
49. During clean up, place RNA tubes on a rocker within cold room (4 °C) and let rock  

SECTION: Preservation in RNAlater for RNA:
50. DAY 2: the following day, again prep work area with RNase Zap.
SECTION: Preservation in RNAlater for RNA:
51. Using a very small, tipped pipette, remove all RNAlater from tube.
 

SECTION: Preservation in RNAlater for RNA:
52. Move sample to -80 °C storage.

SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
53. Using regular forceps, gently place cut tissue selections in either a 2mL or 5mL tube with the fixative(depending on the room needed for the number of allocated pieces).
NOTE: As an alternative, neutral buffered formalin can also be used  for fixation and in this case fixation is at room temperature,overnght
SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
54. Place lid and temporarily store in Wet Ice container until all processing is complete.
SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
55. Document sample types and number of pieces per tube (if using more than one tube, make sure to document tube number on both tube and worksheet).
SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
56. During clean up, place 4% PFA tubes on a rocker within cold room (4 °C) and let incubate  (12-1440 min).

SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
57. DAY 2: Replace 4% PFA with PBS.
 

SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
57.1. Gently remove each piece of tissue using forceps and rinse thoroughly in a small dish of PBS.
 

SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
57.2. Gently place rinsed tissue back in tube prefilled with PBS.
 

SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
58. Document Fixation Stop Date/Time (amount of time specimen was in 4% PFA) on Worksheet.
SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
59. Place tissue in PBS tubes on a rocker within cold room (4 °C) and let rock for at least 24 hours. Solution can be changed a few times to ensure residual PFA is washed out.

SECTION: Preservation in 4% paraformaldehyde (PFA) (make fresh or use within 4 days, keep cold): Fixation
60. DAY 3: After specimens have rinsed in PBS they can be processed for either cryopreservation using sucrose to make a fixed frozen block (FFB) or prepared for paraffin embedding to make a paraffin block (PB).
SECTION: Processing Fixed Frozen Blocks (FFB)
61. Assign unique Specimen Label (derivative of parent label) for each FFB – using our nomenclature, on the Worksheet, put a square around the next consecutive number after the O.C.T. blocks to indicate “Fixed Frozen Block” (i.e., K2300003_4FFB - This is your first piece of fixed frozen tissue. Your
second piece of fixed frozen tissue under this parent number would be K2300003_5FFB and so on); see example Worksheet at the beginning of this section.
SECTION: Processing Fixed Frozen Blocks (FFB)
62. Label new 2mL tube with Specimen Label and “sucrose”.
SECTION: Processing Fixed Frozen Blocks (FFB)
63. Fill tubes with prepared sucrose (reference Supply List – Section (D) Processing Liquids).
SECTION: Processing Fixed Frozen Blocks (FFB)
64. Place assigned tissue in “sucrose” tube.
SECTION: Processing Fixed Frozen Blocks (FFB)
65. Document sample type on Worksheet.
SECTION: Processing Fixed Frozen Blocks (FFB)
66. Place new sucrose tubes on a rocker within cold room (4 °C) and let rock for around 1-5760 min until specimen sinks to bottom of tube.

SECTION: Processing Fixed Frozen Blocks (FFB)
67. Once specimen sinks, freeze in O.C.T. using instructions for processing (refer section "Fresh Frozen Tissue (FB)".
SECTION: Processing Paraffin Blocks (PB)
68. Assign unique Specimen Label (derivative of parent label) for each PB – using our nomenclature, on  the Worksheet, put a triangle around the next consecutive number after the FFB blocks to indicate “Paraffin Block” (i.e., K2300003_6PB - This is your first piece of fixed paraffin tissue. Your second piece of fixed paraffin tissue under this parent number would be K2300003_7PB and so on.); see example Worksheet at the beginning of this section (_7PB not shown).
SECTION: Processing Paraffin Blocks (PB)
69. Label new 2mL tube with Specimen Label and “30% EtOH”.
SECTION: Processing Paraffin Blocks (PB)
70. Fill tubes with prepared 30% EtOH (reference Supply List –Section (D) Processing Liquids).
SECTION: Processing Paraffin Blocks (PB)
71. Place assigned tissue in “30% EtOH” tube.
SECTION: Processing Paraffin Blocks (PB)
72. Document sample type on Worksheet.
SECTION: Processing Paraffin Blocks (PB)
73. Place new 30% EtOH tubes on a rocker within cold room (4 °C) and let rock for several hours to 1440 min.

SECTION: Processing Paraffin Blocks (PB)
74. After several hours to 1440 min remove 30% EtOH from tube using small pipette (leaving tissue in place).

SECTION: Processing Paraffin Blocks (PB)
75. Replace 30% EtOH with 70% EtOH (reference Supply List –Section (D) Processing Liquids for 70% EtOH recipe).
SECTION: Processing Paraffin Blocks (PB)
76. Cross out 30% EtOH and write 70% EtOH on tube.
SECTION: Processing Paraffin Blocks (PB)
77. Place new 70% EtOH tubes on a rocker within cold room (4 °C) until ready to make paraffin blocks.

SECTION: Processing Paraffin Blocks (PB)
78. Place tissue in labeled paraffin cassettes.
SECTION: Processing Paraffin Blocks (PB)
79. Store cassettes in beaker of 70% EtOH at 4 °C.

SECTION: Processing Paraffin Blocks (PB)
80. Submit labeled cassettes for paraffin embedding to the histology core facility.