TITLE: Preparation, Processing and Preservation of Surgical Kidney Resections 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 living donor patient consent to tissue procurement and processing that has been extensively tested on various atlasing projects and multiple tissue types including the Human Biomolecular Atlas Program (HuBMAP) and the Kidney Precision Medicine Project (KPMP).   

[BEFORE_START]
Please review the preparation materials in each section before starting.

[STEPS]
SECTION: Preparation: Obtaining Consent and Preparing to Collect the Kidney
1. Identify the potential nephrectomy case.
SECTION: Preparation: Obtaining Consent and Preparing to Collect the Kidney
2. Request permission from the urologist to speak with the patient about possible consent.
SECTION: Preparation: Obtaining Consent and Preparing to Collect the Kidney
3. Identify an opportunity to make initial contact with the patient, whether it be over the phone, at a preop appointment, or the day of the surgery.
SECTION: Preparation: Obtaining Consent and Preparing to Collect the Kidney
4. Obtain consent from the patient.
SECTION: Preparation: Obtaining Consent and Preparing to Collect the Kidney
5. Notify the urologist of the patient’s consent so they will be aware that you will be retrieving and transporting the kidney to the gross room once it is removed.
SECTION: Preparation: Obtaining Consent and Preparing to Collect the Kidney
6. Notify pathology/gross room of the scheduled date/time of the nephrectomy.
SECTION: Preparation: Obtaining Consent and Preparing to Collect the Kidney
7. Determine what additional information or items the gross room will need to accompany the kidney, such as:

Requisition for the pathology assessment
Patient Labels
Documentation confirming that research consent has been obtained
Etc.

 


SECTION: Preparation: Day of the Nephrectomy
8. Prepare supplies (see next section Preparation: Before Kidney Arrival).
SECTION: Preparation: Day of the Nephrectomy
9. Once the surgery begins, make contact with the circulating nurse in the assigned operating room (via phone or in person):
SECTION: Preparation: Day of the Nephrectomy
9.1. Let him/her know that you will be transporting the kidney to pathology/gross room immediately upon removal.
SECTION: Preparation: Day of the Nephrectomy
9.2. Provide a list of the items you will need from the operating room to accompany the kidney to the gross room (i.e., requisition, patient labels, etc.).
SECTION: Preparation: Day of the Nephrectomy
9.3. If possible, ask the nurse to document the Clamp Time/Last Staple Time (you will need this later to calculate the warm ischemic time).
SECTION: Preparation: Day of the Nephrectomy
9.4. Make certain the nurse knows that you will be taking the kidney fresh and on ice (No formalin).
 

SECTION: Preparation: Day of the Nephrectomy
9.5. Make certain the nurse has a good contact phone number for you and request a phone call when the kidney is getting ready to be removed from the body.
 

SECTION: Preparation: Day of the Nephrectomy
9.6. Before leaving the operating room, document the following on the Worksheet (find copy of Worksheet below):

i. Clamp Time/Last Staple Time
ii. Time out of body
iii. Time on iceon ice 
iv. Left OR Time

 

SECTION: Preparation: Day of the Nephrectomy
9.7. Immediately bring the fresh kidney on ice to the gross room to begin processing.
 


SECTION: Preparation: Before Kidney Arrival
10. Prepare Worksheet with initial information (find copy of Worksheet below):

a. PPID
b. Date (of collection)
c. Laterality

 
d. 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
11. Label preliminary collection cassettes and tubes using the unique Specimen Labels prepared on the Worksheet. The table below indicates a good 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
12. 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
12.1. 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
12.2. Immediately place prelabeled 4% PFA and PBS containers in wet ice.
SECTION: Preparation: Before Kidney Arrival
13. Prepare processing area
SECTION: Preparation: Before Kidney Arrival
13.1. Clean work area, prepare as if working in sterile conditions.
SECTION: Preparation: Before Kidney Arrival
13.2. Treat any area where RNA samples will be processed with RNAse Zap to prevent RNA degradation.
SECTION: Preparation: Before Kidney Arrival
14. Gather Supplies:

a) 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)

b) Prepared Dry Ice container (break up/powder dry ice – aides in closer contact with sample cassette and keeping it frozen)

 
c) Small styrofoam cooler with embedding well (or metal block) placed inside for freezing
d) LN2 transport container (do not fill until kidney is ready)
e) Additional (unlabeled) cassettes and tubes for additional tissue (Reference Supply List section (E) Processing Tissue Containers)
f) Additional RNA & RNAse Zap for additional RNA collections
g) O.C.T
h)  Remaining autoclaved items - scalpel, long forceps and regular forceps (Reference Supply List section (B) Autoclaved Supplies)
i) Processing Tools – clipboard with Worksheet_Live 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
15. Document Arrived in Gross Room Time on Worksheet.
SECTION: Processing: Preparing Kidney
16. Work with the PA in the gross room to prep kidney for dissection:
SECTION: Processing: Preparing Kidney
16.1. Prepare cutting board to receive kidney.
SECTION: Processing: Preparing Kidney
16.2. Trim surrounding fat and large vessels to expose kidney and ureter.
SECTION: Processing: Preparing Kidney
16.3. Orient the kidney and attached ureter on cutting board.
SECTION: Processing: Preparing Kidney
16.4. Identify posterior and anterior aspects.
SECTION: Processing: Preparing Kidney
16.5. Identify upper, mid, and lower poles.
SECTION: Processing: Preparing Kidney
16.6. Identify pelvis.
SECTION: Processing: Preparing Kidney
16.7. 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
16.8. Take pictures of entire specimen - including ureter (place ruler in background if no printed measurements on cutting board)

 

 

SECTION: Processing: Preparing Kidney
16.9. 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
17. Distal Ureter:
SECTION: Processing: Kidney Dissection
17.1. Document Start Time for Distal Ureter and overall Processing Start Time on Worksheet.
SECTION: Processing: Kidney Dissection
17.2. Obtain approximately 1-3cm of distal ureter.
SECTION: Processing: Kidney Dissection
17.3. Cut into roughly 2mm cross sections and allocate for desired processing.
SECTION: Processing: Kidney Dissection
17.4. Process Distal Ureter segments following Processing Instructions for different types of preservation options found below.
SECTION: Processing: Kidney Dissection
17.5. Document Stop Time and sample types collected for distal ureter.
SECTION: Processing: Kidney Dissection
18. Proximal Ureter:
SECTION: Processing: Kidney Dissection
18.1. Document Start Time.
SECTION: Processing: Kidney Dissection
18.2. Obtain approximately 1-3cm of proximal ureter (proximal ureter boundary is at UPJ).
SECTION: Processing: Kidney Dissection
18.3. Cut into roughly 2mm cross sections and allocate for desired processing.
SECTION: Processing: Kidney Dissection
18.4. Process Proximal Ureter segments following Processing Instructions for different types of preservation options found below.
SECTION: Processing: Kidney Dissection
18.5. Document Stop Time and sample types collected for proximal ureter.
SECTION: Processing: Kidney Dissection
19. Bisect kidney on the longest axis.

 

SECTION: Processing: Kidney Dissection
19.1. Take picture of open kidney (include ruler if not on cutting board).
SECTION: Processing: Kidney Dissection
19.2. Examine kidney for any gross abnormalities and document.
SECTION: Processing: Kidney Dissection
19.3. Select healthy appearing lobe(s) for dissection (the lobe typically includes an outer cortex and underlying medullary pyramid).
SECTION: Processing: Kidney Dissection
19.4. 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
20. Region A:
SECTION: Processing: Kidney Dissection
20.1. Document Start Time, pole location, and anterior or posterior.
SECTION: Processing: Kidney Dissection
20.2. Take a picture of Region A (include ruler).
SECTION: Processing: Kidney Dissection
20.3. Cut desired sections from lobe and designate processing method for each piece.

 

SECTION: Processing: Kidney Dissection
20.4. Process Region A pieces following Processing Instructions for different types of preservation optionsfound below.
SECTION: Processing: Kidney Dissection
20.5. Document Stop Time and sample types collected for Region A.
SECTION: Processing: Kidney Dissection
21. Repeat process for Regions B, C …
SECTION: Processing: Kidney Dissection
22. 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
23. On kidney drawing, found on second page of Worksheet, document location of collected lobes as a reference for cut regions shown in picture.

 

SECTION: Processing: Final Documentation
24. Document Processing Stop Time.
SECTION: Processing: Final Documentation
25. Confirm that each sample is accounted for and correctly marked on Worksheet.
SECTION: Processing: Final Documentation
26. Calculate & document Total Transport Time in minutes (Elapsed time between Left OR Time to Arrived in Gross Room Time).
SECTION: Processing: Final Documentation
27. Calculate & document Warm Ischemic Time in minutes (Elapsed time between Clamp Time/Last Staple Time to Time on ice) .

 

SECTION: Processing: Final Documentation
28. Calculate & document Cold Ischemic Time in minutes (Elapsed time from Time on ice to Processing Start Time).

SECTION: Processing: Final Documentation
29. Calculate & document Total Ischemic Time in minutes (Total of Warm Ischemic Time and Cold Ischemic Time).
SECTION: Processing: Final Documentation
30. Calculate and document Total Processing Time in minutes (Elapsed time from Processing Start Time to Processing Stop Time).
SECTION: Processing: Final Documentation
31. Document Processor’s Initials.
SECTION: Processing: Final Documentation
32. The following day, when the 4% PFA is changed to PBS, document the Fixation Stop Date/Time on Worksheet.
SECTION: Processing: Final Documentation
33. Calculate and document Total Fixation Time in minutes (Elapsed Time from Processing Start Time to Fixation Stop Date/Time).
SECTION: Processing Instructions:
34. 
 

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

 

SECTION: Fresh Frozen Tissue (FB):
36. Using regular forceps, gently roll cut tissue selection in O.C.T. so it is completely covered.

SECTION: Fresh Frozen Tissue (FB):
37. Place O.C.T. covered tissue strategically in labeled cassette.
SECTION: Fresh Frozen Tissue (FB):
38. Squirt additional O.C.T. into cassette well until tissue is completely covered.
SECTION: Fresh Frozen Tissue (FB):
39. With large forceps, carefully place filled cassette onto embedding well or metal block within the LN2 cooler.
SECTION: Fresh Frozen Tissue (FB):
40. Cover cooler and monitor carefully.
SECTION: Fresh Frozen Tissue (FB):
41. 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):
42. 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):
43. When finished processing place in labeled resealable bag for protection.
SECTION: Fresh Frozen Tissue (FB):
44. Sample is ready for -80 °Cstorage.

 


SECTION: Flash Fresh Frozen LN2 Samples:
45. Using regular forceps, gently place cut tissue selection in empty labeled tube and place lid.
SECTION: Flash Fresh Frozen LN2 Samples:
46. With large forceps place tube directly in LN2 within the cooler.
SECTION: Flash Fresh Frozen LN2 Samples:
47. Cover cooler and monitor carefully.
SECTION: Flash Fresh Frozen LN2 Samples:
48. 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:
49. 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:
50. Sample is ready for -80 °C storage.

SECTION: RNA Samples:
51. Using regular forceps, gently place cut tissue selection in labeled tube (filled ¾ of the way with RNAlater).
SECTION: RNA Samples:
52. Place lid and temporarily store in Wet Ice container until all processing is complete.
SECTION: RNA Samples:
53. 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: RNA Samples:
54. During clean up, place RNA tubes on a rocker within cold room (4 °C) and let rock  .

SECTION: RNA Samples:
55. DAY 2: the following day, again prep work area with RNase Zap.
SECTION: RNA Samples:
56. Using a very small, tipped pipette, remove all RNAlater from tube.

 

SECTION: RNA Samples:
57. Move sample to -80 °C storage.

SECTION: 4% PFA Samples (make fresh or use within 4 days, keep cold):
58. Using regular forceps, gently place cut tissue selections in either a 2mL or 5mL tube (depending on the room needed for the number of allocated pieces).
SECTION: 4% PFA Samples (make fresh or use within 4 days, keep cold):
59. Place lid and temporarily store in Wet Ice container until all processing is complete.
SECTION: 4% PFA Samples (make fresh or use within 4 days, keep cold):
60. 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: 4% PFA Samples (make fresh or use within 4 days, keep cold):
61. During clean up, place 4% PFA tubes on a rocker within cold room (4 °C) and let incubate overnight (12-1440 min).

SECTION: 4% PFA Samples (make fresh or use within 4 days, keep cold):
62. DAY 2: Replace 4% PFA with PBS.

 

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

 

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

SECTION: 4% PFA Samples (make fresh or use within 4 days, keep cold):
63. Document Fixation Stop Date/Time (amount of time specimen was in 4% PFA) on Worksheet.
SECTION: 4% PFA Samples (make fresh or use within 4 days, keep cold):
64. Place tissue in PBS tubes on a rocker within cold room (4 °C) and let rock for at least 1440 min. Solution can be changed a few times to ensure residual PFA is washed out.

SECTION: 4% PFA Samples (make fresh or use within 4 days, keep cold):
65. 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)
66. 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)
67. Label new 2mL tube with Specimen Label and “sucrose”.
SECTION: Processing Fixed Frozen Blocks (FFB)
68. Fill tubes with prepared sucrose (reference Supply List – Section (D) Processing Liquids).
SECTION: Processing Fixed Frozen Blocks (FFB)
69. Place assigned tissue in “sucrose” tube.
SECTION: Processing Fixed Frozen Blocks (FFB)
70. Document sample type on Worksheet.
SECTION: Processing Fixed Frozen Blocks (FFB)
71. 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)
72. Once specimen sinks, freeze in O.C.T. using instructions for processing (refer section "Fresh Frozen Tissue (FB)" above).
SECTION: Processing Paraffin Blocks (PB)
73. 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)
74. Label new 2mL tube with Specimen Label and “30% EtOH”.
SECTION: Processing Paraffin Blocks (PB)
75. Fill tubes with prepared 30% EtOH (reference Supply List – Section (D) Processing Liquids).
SECTION: Processing Paraffin Blocks (PB)
76. Place assigned tissue in “30% EtOH” tube.
SECTION: Processing Paraffin Blocks (PB)
77. Document sample type on Worksheet.
SECTION: Processing Paraffin Blocks (PB)
78. 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)
79. After several hours to overnight remove 30% EtOH from tube using small pipette (leaving tissue in place).
SECTION: Processing Paraffin Blocks (PB)
80. Replace 30% EtOH with 70% EtOH (reference Supply List – Section (D) Processing Liquids for 70% EtOH recipe).
SECTION: Processing Paraffin Blocks (PB)
81. Cross out 30% EtOH and write 70% EtOH on tube.
SECTION: Processing Paraffin Blocks (PB)
82. Place new 70% EtOH tubes on a rocker within cold room (4 °C) until ready to make paraffin blocks.

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

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