I REAGENTS, SUPPLIES AND EQUIPMENT

1 Reagents

• Human serum albumin, 5%, USP
• Dextran 40 in Sodium Chloride Injection, USP (10% Low Molecular Weight Dextran in 0.9% Sodium Chloride Injection)
• Sterile water
• 70% Isopropyl Alcohol (for cleaning)

2 Supplies

• Bacterial culture bottles, aerobic and anaerobic [or other supplies for performing sterility testing]
• Bag spike with tubing (2)
• 300 ml transfer pack (2)
• 60 ml sterile disposable syringe with tubing (4)
• 10 ml sterile disposable syringe (2)
• 3 ml sterile disposable syringe with tubing (1)
• 16-gauge needles
• Blunt end needle (Emergency Product Recovery)
• 2 ml cryogenic vial
• Tubes for cell counts, progenitor assays (optional)
• Alcohol prep pads
• Sterile plastic zip-lock bags
• Product labels
• Refrigerated gel/ice pack
• Hemostat or Hoffman clamp
• Absorbent wipes
• Canister opening tool
• Scissors
• Personal protective equipment:    gloves
                                                        protective cryogloves
                                                        safety goggles or face shield

3 Equipment

• Biosafety Cabinet
• Liquid nitrogen freezer
• Refrigerated centrifuge
• Water bath
• Sterile tubing connecting device
• Tube sealer, compatible with polyvinyl chloride (PVC)
• Automated cell counter
  

4 Forms

• Cell Product Shipment Integrity Form
• National Marrow Donor Program (NMDP) Receipt of Cord Blood Unit Form

II PRODUCT RECEIPT AND IDENTITY VERIFICATION

HPC, Cord Blood is shipped frozen at or below -150°C using a liquid nitrogen (LN2) dry shipper.  The unit is packed for transport in a stainless steel canister contained within an insulated transport sleeve. Keep HPC, Cord Blood at or below -150°C, either inside the dry shipper container used for shipping or in a liquid nitrogen (LN2) cooled device at the transplant center (recommended).  If the HPC, Cord Blood will be stored inside the dry shipper, add liquid nitrogen to the vessel to ensure the unit will be kept at or below -150°C.

Upon receipt of the shipment perform the following steps:

1. Using scissors, cut the zip ties used to secure the outer lid of the liquid nitrogen dry shipper. 
2. Open the outer lid of the liquid nitrogen dry shipper and remove shipment documentation packet from inside of lid.
3. Remove the Cell Product Shipment Integrity form from the shipment documentation packet and complete the following information:
   
   3.1  Record date and time of receipt
   3.2  Inspect shipper for damage or tampering, verify shipper inner lid is secured with tie tag
   3.3  Record temperature from the temperature data logger attached to the inner lid on the
          Cell Product Shipment Integrity form.  Check for alarms.
          NOTE:  Do not stop the logging process or download data when recording temperature from data logger.
   
   
4. Cut the tie tag securing the inner lid assembly and remove inner lid.
   NOTE: The lid assembly should be held by another person or placed, black side down, on a stable surface to protect the temperature probe (see Figure 1).
   
   
   
5. Wearing cryogloves, remove the HPC, Cord Blood product and accompanying samples from the shipper and transfer to vapor phase of liquid nitrogen and maintain temperature at or below -150°C for label and product integrity verification.
   5.1  Remove the canister from the insulated transport sleeve.
   5.2  Remove the canister opening tool from the shipment documentation packet.
   5.3  Using the canister opening tool, carefully pry open the canister at the top and bottom, 
          working carefully to avoid damage to the frozen plastic product bag (See Figure 2).
          NOTE: Use of other methods for opening the canister could result in damage to the cryopreserved HPC, Cord Blood product.
   
   
   
   
   
   
6. Verify that the product and any accompanying samples have remained frozen and document
   on the Cell Product Shipment Integrity form.
   
7. Verify the physical integrity of the cryobag for visible cracks, holes or leaks; document
   cryobag integrity or any damage on the Cell Product Shipment Integrity form.
8. Verify patient/donor name, medical record number and unit number information match the
   information on the bag’s label(s), shipment documentation and your records. Document match
   on the Cell Product Shipment Integrity form.
   
   NOTE:  If there is any error or ambiguity with the HPC, Cord Blood product, accompanying
                samples, or product documentation, keep the product in the canister and store at or
                below -150°C.  Report the discrepancy immediately to the MD Anderson Cord Blood
                Bank at 1-713-563-8000. Do not proceed until the problem is resolved.
   
   
9. Close the canister and transfer the HPC, Cord Blood cryobag and accompanying samples to
   storage vessels capable of maintaining HPC, Cord Blood at or below -150°C and
   accompanying samples at or below -70°C. 
    
   9.1  If the HPC, Cord Blood cryobag and accompanying samples are to be stored in the dry
   shipper until time of thaw, add additional liquid nitrogen to the dry shipper to ensure the
   product will be maintained at or below -150°C.
   
   NOTE:  Aside from the segment, ancillary samples are not intended to represent the cell 
                count or potency of the cryopreserved HPC, Cord Blood product.
   
   
10. Fax the completed Cell Product Shipment Integrity form to the MD Anderson Cord Blood
    Bank at 1-713-563-3468.
11. Once use of the dry shipper is complete, prepare for shipment and return to MD Anderson
    Cord Blood bank per instructions provided in the shipment documentation packet.
    

III PREPARATION

1. General Preparation Information
   1.1.   Every attempt should be made to ensure HPC, Cord Blood is thawed rapidly, kept cool after thawing, and
            immediately washed as detailed below in order to limit exposure of thawed cells to DMSO.
   1.2.   Use aseptic techniques in a biological safety cabinet (BSC) for processing steps,
            including spiking of the product bag ports and post-wash product sampling.
   1.3    Use only sterile, single use supplies and reagents.
   1.4    Record the manufacturer information, lot number and expiration date (if applicable) of all reagents and
            supplies.
   1.5    Review Section 9: Emergency Product Recovery in the Event of Primary Container 
            Failure prior to thawing product. Always thaw the cryobag inside a sterile zip-lock bag
            so product can be recovered if leakage occurs. If product leaks from the cryobag into the
            sterile zip-lock bag, follow the Emergency Product Recovery procedure described below
            in Section 9.
   
   
2. Prepare 200 ml Dextran 40/Albumin Wash Solution  
   2.1.   Aseptically withdraw 100 ml of Human Serum Albumin and 100ml of Dextran 40 and
            combine in a single 300 ml transfer pack (Dextran/Albumin wash solution) labelled with the
            HPC, Cord Blood identifier.
   2.2.   Mix contents of the transfer pack. 
            NOTE:    The transfer bag now contains a total of 200 ml of solution containing both
                           Dextran 40 and human serum albumin and will be referred to as the wash solution
   
   2.3.  Using a sterile tubing connecting device, sterile connect a 60ml syringe with tubing to the
           transfer pack containing the Dextran/Albumin wash solution.  Withdraw 20 ml of the wash
           solution for diluting the thawed HPC, Cord Blood product.
   2.4.   Using a heat sealer, remove the 60ml syringe containing the 20ml aliquot of wash solution
            from the bag of wash solution ensuring to leave enough of the transfer line for additional
            connections.
   2.5.   Repeat steps 2.3 through 2.4 to create a second 20ml aliquot of wash solution for rinsing
            the HPC, Cord Blood bag.
   2.6.   Using a sterile tubing connecting device, sterile connect a 60ml syringe with tubing to the
            transfer pack containing the Dextran/Albumin wash solution.  Withdraw 50 ml of the wash
            solution for resuspending the washed HPC, Cord Blood product/cells.
   2.7    Using a heat sealer, remove the 60ml syringe containing the 50ml aliquot of wash
            solution from the bag of wash solution, ensuring to leave enough of the transfer line for
            connection to the cryobag.
   2.8    Place the transfer pack containing the remaining wash solution and the 60ml syringes into
            a zip-lock bag and store at 2 - 8°C until ready to use.
   
   
3. Thawing
   3.1.    Fill an empty, clean water bath with sterile water.  Verify the set point of water bath is 37±1°C
   3.2.    Prior to removal of HPC, Cord Blood from storage, confirm water bath temperature is
             37± 1°C.  If the water bath temperature is not within range, wait and/or adjust it such that
             it reaches the desired range.
   3.3.    Retrieve canister containing HPC, Cord Blood from storage and place into vapor phase of
             the LN2 freezer or other vessel capable of maintaining temperatures at or below -150°C.
             NOTE:  Handle HPC, Cord Blood with extreme caution when removing it from the liquid
                          nitrogen, metal cassette and the protective overwrap. Cryobags are very fragile.
   
   3.4.   Working in the vapor phase LN2, remove HPC, Cord Blood cryobag from the metal 
            canister.
            3.4.1    Verify label information with a second technologist against transport
                        documentation and confirm unit identity and intended recipient.
                        NOTE:  Labels on HPC, Cord Blood cryobag should match the labels on the
                                     metal cassette, the tie tag, and on the accompanying paperwork.
   
            3.4.2     Visually inspect the physical integrity of the cryobag for visible cracks, holes or
                         leaks.
   3.5.    Using scissors, remove and place the remaining segment in a labeled 2ml cryovial (See
             Figures 3a and 3b). Store the segment in liquid or vapor phase of liquid nitrogen at or
             below -150°C.
   
   
             
   3.6.    Gently, wipe the external surface of the cryobag with an absorbent wipe soaked with
             70% isopropyl alcohol.
   3.7.    Place the cryobag inside a sterile zip-lock bag, let out the air, and seal the zip-lock bag.
             NOTE:  Use of the sterile zip-lock bag allows for recovery of product in the case of an 
                          unexpected leak or container failure during thawing or centrifugation
   3.8.    Place zip-lock bag into 37±1°C water bath.
   3.9.    Gently massage the cryobag to promote uniform thawing (See Figure 4).  Observe 
             cryobag for any sign of leakage.
   
   
   
   3.10.  Continue to massage the cryobag until the HPC, Cord Blood reaches a slushy
             consistency.  This generally takes about 1-2 minutes.
   3.11.  Carefully remove the thawed HPC, Cord Blood cryobag from the zip-lock bag and dry
             off moisture using an absorbent wipe(s).
   3.12   Place the thawed cryobag on ice or cool pack in the BSC.
   
   
4. Cell Extraction
   4.1.   Using scissors cleaned with an alcohol prep pad, aseptically cut open the port protector 
            of the 20% fraction and insert the bag spike with tubing (See Figures 5a, 5b, and 5c).
            NOTE: Ensure clamp on bag spike with tubing is closed prior to spiking bag ports.
   
   
   
   4.2.   Using scissors cleaned with alcohol, repeat the process in 4.1 above and aseptically open
            the port protector of the 80% fraction and insert bag spike with tubing (See Figures 6a
            and 6b).
   
   
   
   4.3.   With the cryobag on the ice or cool pack, aseptically connect one of the 60ml
            syringes with tubing containing 20 ml of wash solution onto the tubing from the 80% fraction of
            the unit using a sterile connection device.
   4.4.   Slowly inject half of the wash media (approx. 10 ml) from the 60ml syringe into the
            HPC, Cord Blood cryobag; gently mix, and then remove the same amount of solution
            added (See Figure 7).
   
   
   
   
   4.5.   Repeat once more by adding half the contents of the 60ml syringe into the HPC, Cord
            Blood cryobag gently mix, and then empty the cryobag compartment by drawing the
            entire contents into the syringe.
   4.6.   Using a heat sealer, seal the line attached to the bag spike, ensuring enough tubing is left
            for connection of the second 20ml aliquot of wash solution.
   4.7.   Using a sterile tubing connecting device, sterile connect the syringe removed from the
            80% fraction to the 20% fraction and repeat steps 4.5 through 4.8.  Seal the line and
            sterile connect the 60ml syringe containing the cells and wash solution to the
            Dextran/Albumin wash transfer pack and add the entire contents of the syringe to the
            300ml transfer pack containing the remainder wash solution; this is now the product
            bag. Mix well.
   
   
5. Cryobag Rinse
   5.1.   Using a sterile tubing connecting device, sterile connect the second 60ml syringe
            containing 20 ml of wash solution onto the tubing from the 80% fraction of the cryobag.         
   5.2.   Rinse the 80% compartment with wash solution and collect the contents.
   5.3    Seal the line and remove the syringe.
   5.4    Repeat steps 5.1 through 5.3 for the 20% fraction.
   5.5    Using a sterile tubing connecting device, sterile connect the 60ml syringe containing the
            rinse solution to Dextran/Albumin wash transfer pack and add the entire contents of the
            syringe.  Mix well.
   5.6    Clamp the tubing between the syringe and 300ml transfer pack now containing the
            product using a hemostat.
   5.7    Using a sterile tubing connecting device, sterile connect a second 300ml transfer pack to
            the product bag.  Do not open weld.  This bag will be used to collect the wash
            supernatant (See Figure 8).
   
   
   
   
   
6. Centrifugation and Supernatant Removal
   6.1.   Place the 300ml transfer pack containing the HPC, Cord Blood product into a sterile zip-lock
            bag supported to sit in the middle of the centrifuge bucket.
   6.2.   Balance and centrifuge at 800g for 15 minutes at 4°C.
   6.3.   After centrifugation, carefully remove bags from centrifuge bucket ensuring not to
            disturb the cell pellet.  
   6.4.   Place the bag containing the washed HPC, Cord Blood upright in the plasma expresser.
   6.5.   Open the sterile weld to supernatant bag and using a hemostat or Hoffman clamp to
            control flow rate, and carefully express supernatant into the empty transfer pack using
            the plasma expressor until approximately 50 ml remains in the product bag.  Carefully
            avoid passage of cells into supernatant (See Figures 9a and 9b).
   
   
   
   6.6.   Seal line and remove transfer pack containing supernatant.
   6.7.   Resuspend cell pellet in remaining supernatant.
   6.8.   Using a sterile tubing connecting device, sterile connect a 60ml syringe with tubing onto
            HPC, Cord Blood product bag and measure the volume.
   6.9.   Push the resuspended cells back into the transfer pack.
   6.10. Using a sterile tubing connecting device, sterile connect the 60ml syringe containing 50 ml
            of wash solution to the transfer pack and adjust the volume of the cell suspension in
            the transfer pack to a maximum volume of 100 ml.
   6.11. Record the date/time of completion of the wash procedure.
   6.12. Proceed to Section 7.
   
   
7. Preparation of HPC, Cord Blood for Transplantation
   7.1.   Using a sterile tubing connecting device, sterile connect a 3ml syringe with tubing to the
            transfer line of the transfer pack containing the washed product (See Figure 10).
   
   
   
   
   7.2.   Working in a BSC, using aseptic technique, aseptically remove a 1 ml sample from the
            washed product using the 3ml syringe with tubing.
            NOTE: Use this sample to perform viability analysis, total nucleated cell count and
                        other testing as recommended in Section 8: Quality Control Testing.
   7.3.   Using a heat sealer, seal the line between the washed product bag and the 3ml syringe. 
            Separate the 3ml syringe from the line by cutting through the seal using scissors.
   7.4.   Working in a BSC, using aseptic technique, use two 10ml syringes to
            obtain two separate 8 ml aliquots of wash supernatant from the supernatant transfer pack for
            sterility testing.
   7.5.   Calculate viable cell recovery using the following formula:
            (Total Viable Nucleated Cells on the Infusion Ready Product ÷ Total Viable Nucleated
            Cells of Cryopreserved Unit Provided on Shipment Report) x 100%
   7.6    Label the infusion bag with patient and donor information per institutional standard
            operating procedures.
   7.7    Label the infusion bag with the washed product expiration date/time.  The expiration 
           date/time of the washed product is four hours from completion of wash procedure
           described in Section 6.
   7.8   Confirm and document, with a second technologist or designee, the cord blood identifier
           is affixed to infusion bag.
   7.9   Complete appropriate site-specific laboratory forms to document the thawing process
           and final volumes.
   7.10 Transport infusion bag to the site of administration to the patient in a transport container
           capable of maintaining the unit temperature between 2 - 22°C.
   7.11 Infuse within four hours of completion of wash procedure.
   7.12 Infuse the product through a 170-260 micron filter at the bedside. Do not use
           leukoreduction filters.
   
   
8. Quality Control Testing
   The following tests are recommended:
   8.1.   Cell Counts: perform on final product to be infused.
   8.2.   Cell Viability: perform on final product to be infused (Viable CD45+ determination by
            flow cytometric analysis recommended).
   8.3.   Colony assay for CFU-GM, CFU-GEMM, and BFU-E: perform on final product to be
            infused.
   8.4.   Viable CD34+ determination by flow cytometric analysis: perform on final product to
            be infused.
   8.5.   Sterility test: perform on wash supernatant.
   
   
9. Emergency Product Recovery in Event of Primary Container Failure
   9.1    If the cryobag is compromised, move further handling into BSC after thawing.
            NOTE: It is the transplant physician’s (or designee’s) responsibility to determine
                        whether the product will be used or discarded if the container is
                        compromised at any step of the procedure.
   9.2    If the transplant physician (or designee) decides that the product should be used,
            recovery of the product may be attempted as described below.
   9.3    Use a long (3-5 inch) blunt needle (a sterile spinal needle with trochar removed if
            available).
   9.4    Attach the blunt needle to a sterile 60 mL syringe.
   9.5    Aspirate the product from the cryobag and inject into a sterile transfer bag.
   9.6    Continue to the wash step for the transferred product.
   9.7    Start and complete the wash process for the transferred product.
   9.8    Perform sterility test on a sample of the recovered wash product.
            NOTE: Alert the clinical team at the transplant center that the product could potentially
                        be contaminated.
   9.9    Contact the MD Anderson Cord Blood Bank at 1-713-563-8000 or 713-794-1908 if
            during thawing, any portion of the product container appears to be damaged or
            compromised. Save the ruptured cryobag if possible.  The Cord Blood Bank will
            provide further instructions for return of the container, if required for investigation. 
   

Distributed by:
MD Anderson Cord Blood Bank
1841 Old Spanish Trail
Houston, TX 77054
713-563-8000
U.S. License # 2072