Troubleshooting and FAQ P a g e 1

Transcription

1 Troubleshooting and FAQ P a g e 1 Table of Contents: MediVet PRP Kit Page 2 Small Adipose Processing Pages 3-5 Large Kit Adipose Processing Pages 6-10 Adipose Stem Cell Injections Page 11 MediVet Hot Water Bath Page 12 MediVet CellCycle 360 Centrifuge Page 13 MediVet Vacuum Pump Page 14 Technician Notes Page 15

2 Troubleshooting and FAQ P a g e 2 PRP Kit What happens if I cannot get the recommended amount of venous blood from the animal? Get as much blood as you can safely draw. MediVet recommends at least 2 tubes (16 mls) of blood for the PRP Processing. Any tubes that are not completely filled with blood can be filled with normal saline. This is important for spin separation, tube balancing, and ACD-A solution distribution. I only filled one tube with blood and do not have 3cc of Plasma. Add saline solution to 3cc and continue with addition of activator solution. The PRP is not forming a gel. Be patient minutes is average gel formation time but the physiology of the animal is ultimately going to determine clot formation kinetics. You may place the 15mL conical tube in a nonshaking MediVet Hot Water Bath at 37*C or place in a beaker of warm water to speed up the process. The gel is not retracting. Can I speed it up? You may initiate the retraction of the PRP clot by gently separating the gel from the side of the tube using a syringe (in sterile packaging) attached to a spinal needle or 1.5 needle. Use a swirling circular motion starting at the top of the gel and continuing downwards to remove the clot. It will stick to the needle. You may discard the gel portion. Only use the liquid portion for injection with the ADSC kit. I do not see a large platelet pellet. Don t worry in the typical dog there are platelets per ul (microliter). They are only 2-4 microns in size! You have enough for significant growth factor concentration with the amount of blood you have collected. I do not have a MediVet Centrifuge. You may use plastic Red-Top (without serum separator gel!!) tubes in place of the 15 ml Conical tubes. What is the best way to use the PRP? For joint/soft tissue injections: Direct injection into the site. Place the animal on a 7-10 day course of oral antibiotics or give an IM injection of a long-acting antibiotic such as Convenia.

3 Troubleshooting and FAQ P a g e 3 Small Adipose Kit Processing I am missing kit components or some of the components were damaged. Please call and we will send you replacement consumables. Why do we use Gentamicin during Adipose Stem Cell Processing and is it safe? MediVet America uses Gentamicin in its processing due to its broad-spectrum activity. When developing the Adipose Stem Cell Kit, MediVet looked both at current research and did its own internal testing to ensure that Gentamicin did not inhibit proliferation of ADSCs. The recommended intraarticular flush behind stem cell injections with Gentamicin using the Medivet system is no more than 2 mg/kg split up between all joints and no more than 24 mg total (whichever is lower). This usually brings up questions regarding Renal compromised patients. The processing protocol includes a dilute (20 mg in 25 ml diluent buffer) Gentamicin solution during incubation at 37ºC and an even more dilute solution (5 ml of 20mg/25mL in diluent buffer) during washing and filtering of the cells. Additionally, all the Wash solution is removed before adding PRP for injection. In conclusion, the amount of Gentamicin used will not compromise the health of any animal undergoing intra-articular injection. What antibiotic can be used as a replacement for Gentamicin in the procedure? Data does not support the use of any other antibiotic as they can decrease and stop stem cell proliferation in vitro and in vivo. Please contact MediVet Labs for more information. I do not have 40 mg/ml Gentamicin. That s fine. You probably found that 100 mg/ml Gentamicin was much easier to source. No matter what concentration you have, we want to add 20 mg to Solution D (See Stage 2, Preparation of Solutions, Step 2). So if you have 100 mg/ml you will only add 0.2 cc to Solution D. I am having some difficulty obtaining 15 ml of venous blood from my animal. Get as much blood as you can safely draw. MediVet recommends at least 2 tubes (12 ml) of blood for the Small Kit processing. Any tubes that are not completely filled with blood can be filled with normal saline. This is important for spin separation, tube balancing, and ACD-A solution distribution. I only filled one tube with blood and do not have 3cc of Plasma. Add saline solution to 3 cc and continue with addition of activator solution. The PRP is not forming a gel. Be patient minutes is average gel formation time but the physiology of the animal is ultimately going to determine clot formation kinetics. You may place the 15mL conical tube in a nonshaking MediVet Hot Water Bath at 37ºC or place in a beaker of warm water to speed up the process. The gel is not retracting. Can I speed it up? You may initiate the retraction of the PRP clot by gently separating the gel from the side of the tube using a syringe (in sterile packaging) attached to a spinal needle or 1.5 needle. Use a swirling circular motion starting at the top of the gel and continuing downwards to remove the clot. It will stick

4 Troubleshooting and FAQ P a g e 4 to the needle. You may discard the gel portion. Only use the liquid portion for injection with the ADSC kit. I do not see a large platelet pellet. Don t worry in the typical dog there are platelets per µl (microliter). They are only 2-4 microns in size! You have enough for significant growth factor concentration with the amount of blood you have collected. I am on Step 8 and there is white fiber floating in the red (bottom) layer. Have I done something wrong? Absolutely not! Adipose tissue from animal to animal varies in fibrin content. Be sure to remove the WHOLE bottom layer (as much as you can!!) because there are probably stem cells mixed in with the fibrin. We will filter twice during the procedure so there is no worry about ending up with it when your cells are ready for injection. My 100 µm SteriFlip has clogged. What do I do? When the vacuum pump starts to resist movement, go ahead and stop. Do not force it it WILL break! Detach the flexible hose from the SteriFlip. Detach the whole SteriFlip unit, filter and all, and place it face down (it has a lip on it and is safe to place face down) on the benchtop. Turn the SteriFlip so that the contents have settled to the bottom of the tube. Detach the filter unit and pour the liquid portion (may contain some fibrin) to the filtered portion. (Using a Sterile Transfer Pipette (there are two extra in your Small Adipose Kit), remove any large clumps of undigested fat. Place the lid on the 50 ml conical tube and continue with step 11. I see wisps of cells or fibrous material floating after my first wash and spin (Step 13). When in doubt spin again! You may spin for 5-10 minutes at 2300 rpm. How much supernatant (top layer) do I remove in Step 13? MediVet suggests removing as much as you are comfortable. We would rather you have MORE supernatant than accidently remove too much and discard part of your cell fraction during this step! What can I do if I see wisps of cells floating after my final spin? Spin again for 5-10 minutes at 2300 rpm. You want a very clearly defined cell pellet in the bottom of your tube! What is the best use of the remaining PRP after mixing what I need with the cells? It is up to you: 1. Use the PRP topically on the surgical site 2. Use it to inject an additional joint for 4-6 weeks pain relief 3. Freeze for a follow-up injection or additional wound healing if needed. It is good for 14 days frozen. How long can the PRP sit before it is mixed with the cells? The PRP is fine to sit at room temperature or in the MediVet Hot Water Bath until the procedure is over.

5 Troubleshooting and FAQ P a g e 5 How do I administer cells IV? It is best to use the IV Transfusion set that comes with the Small Adipose Kit. You may mix the cells with normal saline solution or lactated ringers solution. Do not mix with dextrose-containing products. Closely monitor the animal during administration to ensure catheter patency. We do not recommend an IV push with the regenerative cell fraction due to possible catheter issues especially in older animals with delicate veins. What if I wanted to count cells using my own equipment, such as a CBC machine or hemocytometer? Yes. Keep in mind that this is an alternative method and will not give exceptionally accurate results. The protocol for using a CBC machine can be found at the end of the Small Adipose protocol directly after Step 23. MediVet recommends diluting 0.1 cc of the cell fraction in 0.4 cc normal saline for counting with a CBC machine. Remember to take the dilution factor of 4 into account when calculating your cells! Please contact MediVet Labs for instructions and materials needed for counting your regenerative cell fraction using a hemocytometer.

6 Troubleshooting and FAQ P a g e 6 Large Adipose Kit Processing I am missing kit components or some of the components were damaged. Please call and we will send you replacement consumables. Why do we use Gentamicin during Adipose Stem Cell Processing and is it safe? MediVet Biologics uses Gentamicin in its processing due to its broad-spectrum activity. When developing the Adipose Stem Cell Kit, MediVet looked both at current research and did its own internal testing to ensure that Gentamicin did not inhibit proliferation of ADSCs. The recommended intraarticular flush behind stem cell injections with Gentamicin using the Medivet system is no more than 2 mg/kg split up between all joints and no more than 24 mg total (whichever is lower). This usually brings up questions regarding Renal compromised patients. The processing protocol includes a dilute (20 mg in 25 ml diluent buffer) Gentamicin solution during incubation at 37ºC and an even more dilute solution (5 ml of 20mg/25mL in diluent buffer) during washing and filtering of the cells. Additionally, all the Wash solution is removed before adding PRP for injection. In conclusion, the amount of Gentamicin used will not compromise the health of any animal undergoing intra-articular injection. What antibiotic can be used as a replacement for Gentamicin in the procedure? Data does not support the use of any other antibiotic as they can decrease and stop stem cell proliferation in vitro and in vivo. Please contact MediVet Labs for more information. I do not have 40 mg/ml Gentamicin. That s fine. You probably found that 100 mg/ml Gentamicin was much easier to source. No matter what concentration you have we want to add 20 mg to Solution D (See Stage 2, Preparation of Solutions, Step 3). So if you have 100 mg/ml you will only add 0.2 cc to Solution D. I am using a Large Kit for canine or feline use. What happens if I cannot get 28 ml of venous blood from the animal? Get as much blood as you can safely draw. MediVet recommends at least 2 tubes (16 ml) of blood for the Large Kit processing. Any tubes that are not completely filled with blood can be filled with normal saline. This is important for spin separation, tube balancing, and ACD-A solution distribution. I only filled one tube with blood and do not have 3cc of Plasma. Add saline solution to 3cc and continue with addition of activator solution. The PRP is not forming a gel. Be patient minutes is average gel formation time but the physiology of the animal is ultimately going to determine clot formation kinetics. You may place the 15 ml conical tube in a nonshaking MediVet Hot Water Bath at 37ºC or place in a beaker of warm water to speed up the process. The gel is not retracting. Can I speed it up? You may initiate the retraction of the PRP clot by gently separating the gel from the side of the tube using a syringe (in sterile packaging) attached to a spinal needle or 1.5 needle. Use a swirling circular motion starting at the top of the gel and continuing downwards to remove the clot. It will stick

7 Troubleshooting and FAQ P a g e 7 to the needle. You may discard the gel portion. Only use the liquid portion for injection with the ADSC kit. I do not see a large platelet pellet. Don t worry in the typical dog there are platelets per µl (microliter). They are only 2-4 microns in size! You have enough for significant growth factor concentration with the amount of blood you have collected. I am on Step 9 and there is white fiber floating in the red (bottom) layer. Have I done something wrong? Absolutely not! Adipose tissue from animal to animal varies in fibrin content. Be sure to remove the WHOLE bottom layer (as much as you can!!) because there are probably stem cells mixed in with the fibrin. We will filter twice during the procedure so there is no worry about ending up with it when your cells are ready for injection. My 100 µm SteriFlip has clogged. What do I do? When the vacuum pump starts to resist movement, go ahead and stop. Do not force it it WILL break! Detach the flexible hose from the SteriFlip. Detach the whole SteriFlip unit, Filter and all, and place it face down (it has a lip on it and is safe to place face down) on the benchtop. Turn the SteriFlip so that the contents have settled to the bottom of the tube. Detach the filter unit and pour the liquid portion (may contain some fibrin) to the filtered portion. Using a Sterile Transfer Pipette (there are two extra in your Large Adipose Kit), remove any large clumps of undigested fat. Place the lid on the 50 ml conical tube and continue with Step 12. I see wisps of cells of fiber floating after my first wash and spin (Step 15). When in doubt spin again! You may spin for 5-10 minutes at 2300 rpm. How much supernatant (top layer) do I remove in Step 16? MediVet suggests removing as much as you are comfortable. We would rather you have MORE supernatant than accidently remove too much and discard part of your regenerative cell fraction during this step! What can I do if I see wisps of cells floating after my final spin? Spin again for 5-10 minutes at 2300 rpm. You want a very clearly defined cell pellet in the bottom of your tube! What is the best use of the remaining PRP after mixing what I need with the cells? It is up to you: 1. Use the PRP topically on the surgical site 2. Use it to inject an additional joint for 4-6 weeks pain relief 3. Freeze for a follow-up injection or additional wound healing if needed. It is good for 14 days frozen.

8 Troubleshooting and FAQ P a g e 8 How long can the PRP sit before it is mixed with the cells? The PRP is fine to sit at room temperature or in the MediVet Hot Water Bath until the procedure is over. How do I administer cells IV? It is best to use the IV Transfusion set that comes with the Small Adipose Kit. You may mix the cells with normal saline solution or lactated ringers solution. Do not mix with dextrose-containing products. Closely monitor the animal during administration to ensure catheter patency. We do not recommend an IV push with the regenerative cell fraction due to possible catheter issues especially in older animals with delicate veins. What if I wanted to count cells using my own equipment, such as a CBC machine or hemocytometer? Yes. Keep in mind that this is an alternative method and will not give exceptionally accurate results. The protocol for using a CBC machine can be found at the end of the Small Adipose protocol directly after Step 23. MediVet recommends diluting 0.1 cc of the cell fraction in 0.4 cc normal saline for counting with a CBC machine. Remember to take the dilution factor of 4 into account when calculating your cells! Please contact MediVet Labs for instructions and materials needed for counting your regenerative cell fraction using a hemocytometer. What is the best way to get the final volume my vet wants?

9 Troubleshooting and FAQ P a g e 9 How do I get the cells out of the large cryovial? Use a spinal needle (included) to draw cells out of the Cryovial using the syringe that you are using for injection. Your veterinarian may prefer to this using sterile gloves just before injection.

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11 Troubleshooting and FAQ P a g e 11 Adipose Stem Cell Injections What is my final volume going to be for injection? This depends on how many sites there are to be injected. Reference the chart below if you unsure: Size of Animal Hip Carpus/Hock Shoulder Knee Elbow IV <25 lbs cc cc cc cc cc Any Amount lbs cc cc cc cc cc Any Amount >45 lbs cc cc cc cc cc Any Amount How do I fill the syringes for injection with the SVF? You may use a needle to fill the syringes for injection. Should I use a separate syringe for each planned injection? Yes. Be sure to label all doses clearly. How much Gentamicin should I inject intra-articular after the SVF? MediVet recommends no more than 2 mg/kg of Gentocin split up between all the joints to be injected with a maximum of 24 mg total used (whichever is lower). If you are using 100 mg/ml Gentocin it is fine for the Veterinarian to dilute it in normal saline. A volume of 0.1 cc to 0.2 cc is appropriate for a flush into a joint. What is the time frame for injection after the procedure is complete? The cells are best if injected right away for maximum proliferation potential. The cells may be stored in a cool, dark place for up to 6 hours. Next Day injections using In-Clinic processing are not recommended. BE sure to mix the cells well before injection they will settle to the bottom of syringes and cryovials! Do I need to process PRP to inject cells that have come from a dose banked at MediVet Labs? Yes. A PRP kit is included in the Banked Dose Recovery Fee and will be shipped with the animal s cells. What is the protocol for reconstituting my cells from a banked dose? Please call MediVet Labs at , Ext. 308 to request an advance copy of this protocol. You will receive a copy with every Cell Count and Viability Report performed on your requested thawed dose, and we also include a hard copy with every banked dose cell shipment. Do I need to process PRP if I am administering a thawed dose IV only? Yes. MediVet recommends PRP with any ADSC stem cell delivery.

12 Troubleshooting and FAQ P a g e 12 MediVet Hot Water Bath What kind of water do I fill my bath with? Tap water or distilled water. We recommend distilled water as it is less likely to leave mineral deposits or other residues inside the bath. How much water should I but in my MediVet Hot Water Bath? Fill the water bath just until it just covers the wire bath rack(s). The MediVet Hot Water Bath is not heating up. First check to see if the heating element light is on. If it is not, turn the unit off and unplug. There is a button on the left-hand back side of the unit labeled Over Temp Reset. Press it and observe the bath. If this still did not fix the problem please contact the lab at , Ext My MediVet Hot Water Bath is either not shaking at 3.5 or shakes intermittently. Try using a commercial mechanical lubricant on the arm of the instrument while the unit is completely off. If this does not fix the problem, contact the lab at , Ext My MediVet Hot Water Bath is at 36.5ºC or 38.0ºC. This is normal. The heating element is very sensitive in your bath and the temperature may fluctuate during processing. Also, the water bath will overshoot slightly every time the water is heated from room temperature. How often should I clean my bath? Clean inside and out after every use with 70% Isopropyl Alcohol. Do not use any other commercial cleaner that could leave a residue (i.e. Roccal, Tek-Trol).

13 Troubleshooting and FAQ P a g e 13 MediVet CellCycle 360 Centrifuge My centrifuge is giving me an error. What does it mean? Please see the MediVet CellCycle 360 Manual for specific error codes. Call the lab with any concerns at , Ext My centrifuge makes a sound when it is speeding up and slowing down. Ensure your tubes are very well balanced when spinning at any speed. This includes ensuring that the rubber inserts are seated at the bottom of the metal buckets. While the unit is considerably forgiving for slightly off-balanced tubes, you may experience slight vibration between ~ rpm. How do I change the time, speed, etc? Pushing the Menu button and using the Up/Down arrows will allow you to change the Speed, Time, Acceleration, Deceleration and Rotor. The settings should always be: Acceleration-9, Deceleration- 1, Rotor-3. Be sure to press the Enter Key to save your settings. The Enter key looks like this:

14 Troubleshooting and FAQ P a g e 14 MediVet Vacuum Pump What is the best way to attach the vacuum pump to the SteriFlip? Attach the flexible tubing to the spigot on the SteriFlip and apply a few pumps of pressure before turning the cells to be filtered over. This will ensure that no cells are backed up in the tubing. Continue to gently apply pressure to the Vacuum Pump handle until the entire sample has passed through the filter. My vacuum pump has drawn cells up through the tubing. Stop pumping your vacuum. Remove the flexible tubing from the SteriFlip. You will need to get a new Filter. Follow the instructions in the question What is the best way to attach the vacuum pump to the SteriFlip? The handle on my vacuum pump gets very hard to move and no more cells are filtering. Do not force the SteriFlip as you could break the handle. Follow the steps in either the Small or Large Adipose Protocol on what to do if your filter clogs. Oops! I have broken my MediVet Vacuum Pump. I am in the middle of a procedure!!! 1. Do Not Panic. 2. You will need a new SteriFlip unit. MediVet recommends having a few extra on hand. If not, remove one from another kit and call , Ext. 308 to order a replacement (a SteriFlip unit costs around $15, a new Vacuum Pump is around $80). 3. Aseptically transfer (you may pour) all the cells, filtered and unfiltered, to one 50 ml Conical tube. Recap immediately. Throw away the compromised filter. 4. Remove the flexible tubing from the hard plastic part of the Air Reservoir. (See below) 5. You will need a catheter tip syringe (60 cc is best) and two people. 6. Connect the closed Catheter Tip syringe to the free end of the tubing. 7. Connect the tubing with the syringe on the end of it to the SteriFlip. 8. Flip the unit over, being very careful not to disconnect either the syringe or the tubing from the SteriFlip unit. 9. Pull back on the Catheter Tip syringe. 10. Have the second person crimp the tubing to preserve the vacuum when no more air can be pulled from the syringe. 11. Disconnect the syringe from the tubing. 12. Close the syringe. 13. Place the closed syringe back on the tubing. 14. Repeat 9-13 until all your cell suspension has filtered through. This may seem tedious but once the vacuum is created it will begin to flow at a steady pace.

15 Troubleshooting and FAQ P a g e 15 Technician Notes: