HuBMAP | VU TMC Kidney | Human Kidney Processing

The International Institute for the Advancement of Medicine will identify patient kidneys for research with the following criteria:

Basic Requirements:

Age: 18-55 years

DCD - WIT < 15 minutes

Any Downtime

CIT < 36 hrs

Will accept CMV, EBV, HSV 1 or 2, TOXO & HBsAb (+) w/ confirmed vaccination only

Will accept DCD

Will accept IVDA (current use/ past use)

Will accept Chagas & Strongyloidiasis

Will accept Donor that previously tested COVID-19 (+) & now COVID-19 (-) on Case-By-Case Basis

Will accept Kidneys on a pump

Will accept Biopsy - Fibrosis on CASE-BY-CASE BASIS

Exclusionary Criteria:

Diabetes Type 1 or 2

Hepatitis B/C positive serology results

HIV positive serology results

Meningitis Bacterial/Viral positive

MRSA - any

RPR/Syphilis positive serology

Sepsis

Contraindications:

Biopsy - Necrosis

Kidney Disease (past/current)

DM Type 1 or 2

Donor on dialysis

Cancer (current/past hx)

Bacterial Viral meningitis

MRSA in any culture

Sepsis

Ensure that 1 liter of 2.6% CMC is prepared (requires overnight preparation)* Label embedding molds with Date-Anterior/Posterior-Block Location 5.g. 05/26/23

              Ant - A1

• Label 2 paraffin fixation cassettes, one for anterior and one for posterior

• Clear lab benches and safety hood

• Transfer 2.6% CMC into 6 squeeze bottles and refrigerate overnight

• Gather materials and place on appropriate benches: Dry Ice Pans with Lids iPad Stand

    Labeled embedding molds                 Foil squares
  
    Face shields                                        Lab Gowns
  
    Gloves                                                  Styrofoam box
  

• Clean biosafety hood items with 70% Ethanol. Place in a cleaned (70% EtOH) plastic container in a 4^oC refrigerator to cool overnight. 2 steel pans Large knife

    teflon mold                                          styrene divider sheets
  
    disposable scalpel                              surgical scissors
  
    forceps                                                 labeled rulers
  

Clean biosafety hood with 70% Ethanol* Print "Sample Mold Chart" with Kidney ID and side for notetaker and freezing station

• Set up stations Picture station: iPad stand, iPhone, aluminum tray lid for transporting samples

    Placement station: Dry ice pan with ice, disposable aluminum pan, labeled embedding molds,         
  
              forceps, plastic jar with 10% formalin    
  
    Freezing Station:  2 dry ice pans with ice, isopentane, chilled 2.6% CMC in squeeze bottles,
  
                foil squares, sharpie markers, freezer boxes with labels (kidney ID & date), timers, dry ice    
  
                box for holding frozen/wrapped samples 
  
    Note Taker:  place chair next to biosafety hood, notebook, pens
  

• Label rulers, foil trays, embedding molds, and wrapping foil with block names.

Remove kidney from shipping container and place on cold dissection tray. Remove as much outer fat as possible.

Dissect the kidney by carefully cutting it in half and remove any visible fat. Work on one half of the kidney while the other half is left on an ice tray to keep cool.

Once the fat is removed, place the kidney half into the millotome. The kidney is first cut longitudinally, after each cut dividers are put down to hold the kidney in its place. Next, the kidney is cut latitudinally. After each latitudinal cut, that row of tissue block is collected and transferred to labelled ruler. The ruler is transferred at a photo stage where an iPad/iPhone is used to take a quick snapshot before each block is transferred to a labelled foil tray on an isopentane slurry.

Note: if the kidney doesn't fill out in the mold, place dividers into the edge of the mold to make it fit.

Note: remove any extra visible fat from the tissue blocks

Collect 1 sample from each side for FFPE.

Choose block and cut a thin nickel thick section. Place in labeled tissue cassette and then into a container of 10% neutral buffered formalin. Follow Freezing and Formalin Fixation of Tissue V.3 for processing the formalin samples.

Place aluminum pan on top of filled dry ice pan. After taking a picture of the sample ruler, place each tissue into the corresponding labeled mold. After two rows are placed, move entire sample pan to the freezing station.

Prepare isopentane slurry. Place a layer of dry ice into an insulated, lidded dry ice pan and add enough isopentane to cover the dry ice.

Place each tissue mold into the isopentane slurry. Cover the dry ice pan with lid and allow tissue to completely freeze.

Once the tissue is completely frozen, slowly add CMC to the mold until the tissue is completely covered. Keep the lid closed over the slurry as much as possible. When completely frozen, wrap mold in prelabeled square of aluminum foil.

Note time that the CMC is completely frozen for each sample on the Sample Mold chart.

Place all wrapped samples in prelabeled freezer boxes and store at -80°C