VENIPUNCTURE TECHNIQUE AND SPECIMEN COLLECTION GENERAL INSTRUCTIONS

WEAR GLOVES DURING VENIPUNCTURE AND WHEN HANDLING BLOOD COLLECTION TUBES TO MINIMIZE EXPOSURE HAZARD

• • Select a PPT-I tube.
  • Gently tap tubes containing additives to dislodge any material that may have adhered to the stopper.
  • Place tube into holder of blood collection device. Note: Do not puncture stopper.
  • Select site for venipuncture.
  • Apply tourniquet. Prepare venipuncture site with an appropriate antiseptic. DO NOT PALPATE VENIPUNCTURE AREA AFTER CLEANSING.
  • Place patient’s arm in a downward position.
  • Remove needle shield. Perform venipuncture WITH ARM DOWNWARD AND TUBE STOPPER UPPERMOST.
  • Place tube into holder of the blood collection device. Push tube onto needle, puncturing stopper diaphragm. Center tube in holder when penetrating the stopper to prevent sidewall penetration and resultant premature vacuum loss.
  • REMOVE TOURNIQUET AS SOON AS BLOOD APPEARS IN TUBE. DO NOT ALLOW CONTENTS OF TUBE TO CONTACT THE STOPPER OR END OF THE NEEDLE DURING PROCEDURE.
    Note: Blood may occasionally leak from the needle sleeve. Practice Universal Precautions to minimize exposure hazard. If no blood flows into tube or if blood ceases to flow before an adequate specimen is collected, the following steps are suggested to complete satisfactory collection:

a. Push the tube forward until tube stopper has been penetrated, if necessary, hold in place to ensure complete vacuum draw.

b. Confirm correct position of needle cannula in vein.

c. If second tube does not draw, remove needle and discard. Repeat procedure from step 1.

• Once the tube has been filled to its stated volume and blood flow ceases, remove the tube from holder. Repeat this step for all successive tubes. For each filled tube, immediately perform inversions in order to mix the anticoagulant, as described in (15).
• Remove needle from vein, applying pressure to puncture site with dry sterile swab until bleeding stops.
• Once clotting has occurred, apply bandage if desired.
• After venipuncture, the top of the stopper may contain residual blood. Take proper precautions when handling tubes to avoid contact with this blood. Any needle holder that becomes contaminated with blood is considered hazardous and should be decontaminated with bleach or disposed of.
• Dispose of the used needle using an appropriate disposal device. DO NOT RESHIELD. Re-shielding of needles increases the risk of needle-stick injury and blood exposure.
• Turn the filled tube upside-down and return it to an upright position. That is one complete inversion. For proper additive performance, invert tube 5 times. Do not shake. Vigorous mixing may cause foaming or hemolysis. In tubes with anticoagulants, inadequate mixing may result in platelet clumping, clotting and/or incorrect test results.

 

CENTRIFUGATION

• Place the tubes in a centrifuge according to the instructions below.
• Centrifuge the tubes at 1500 RCF for 10 minutes, braking 30-45 seconds according to the instructions and precautions below:

Caution:

• Do not centrifuge glass tubes at forces above 1550 RCF in a horizontal head (swinging bucket) centrifuge as breakage may occur. Follow manufacturer instruction when using centrifuge. Glass tubes may break if centrifuged above 1550 RCF in fixed angle centrifuge heads.
• Use of tubes with cracks or chips or excessive centrifugation speed may cause tube breakage, with release of sample, droplets, and aerosols into the centrifuge bowl. Release of these potentially hazardous materials can be avoided by using specially designed sealed containers in which tubes are held during centrifugation.
• Centrifuge carriers and inserts should be of the size specific to the tubes used. Use of carriers too large or too small may result in breakage.
• Ensure that tubes are properly seated in the centrifuge carrier. Incomplete seating could result in separation of the stopper closure from the tube or extension of the tube above the carrier. Tubes extending above the carrier could be caught on centrifuge head, resulting in breakage.
• Balance tubes according to centrifuge instructions to minimize the chance of glass breakage.
• Always allow centrifuge to come to a complete stop before attempting to remove tubes. When centrifuge head has stopped, open the lid and examine for possible broken tubes. If breakage is indicated, use mechanical device, such as forceps or hemostat to remove tubes.
• Do not remove broken tubes by hand. Reference centrifuge instruction manual for disinfection instructions.

RCF is related to centrifuge speed setting (rpm) using either of the following equations:

The flow properties of the barrier material are temperature-related. Flow may be impeded if chilled before or during centrifugation. Gel separation tubes should be centrifuged no later than 2 hours after collection.
Tubes should not be re-centrifuged once barrier has formed.

OPTION 1 – Concentrated PRP – an open system

BALANCE

When processing an odd number of PRP tubes, place contra balance tube filled with water with the same weight directly opposite from the platelet preparation tube in the centrifuge. When processing even number of PRP tubes, place tubes directly opposite from each other in the centrifuge. Note: Balance instructions may vary based on centrifuge. Please check centrifuge instructions for use.

PRP COLLECTION

After centrifugation, the tube should contain an upper clear yellowish fraction, separation gel barrier, and a lower red-coloured fraction. The platelets reside on the top of the separation gel. Important: Avoid agitating the tube as it may result in preliminary re-suspension of platelets and/or platelet activation.

Footnotes
PRP – Platelet-Rich Plasma
PPP – Platelet Poor Plasma
PRIF – Platelet-Rich Injectable PRP

Symbols & Mark Key

OPTION 1 – Concentrated PRP – an open system

1. Once centrifugation step is complete, remove cap from tube. Important: Avoid agitating the tube as it may result in preliminary re-suspension of platelets and/or platelet activation.

2. Removal of PPP: Place tube in tube rack. Attach aspiration needle to 10 mL syringe and insert needle into upper surface of plasma fraction. From the surface, draw about 50% to 80% of plasma fraction. Caution: Do not insert needle too deep from plasma surface.

3. PRP Withdrawal: Close tube with cap. Suspend platelets situated on gel by gently inverting tube horizontally 10 times. Place tube in tube rack. Caution: Do not shake. Do not completely invert tube.

4. Attach sharp aspiration needle to disc filter. Connect disk filter to 10 mL syringe.

5. Place tube in the rack. Remove tube cap, insert needle into surface of PRP fraction and carefully draw PRP into syringe. Alternatively, hold tube in one hand and syringe in the other and pull PRP into syringe with thumb. It is recommended to gently press the needle tip against the tube wall for better control. Important: Place needle tip just above separation gel, while turning tube slightly on its side to help with PRP collection. Do not touch the gel with needle tip as it may result in clogging the needle.

OPTION 2 – Concentrated PRP – a closed system

1. Once centrifugation step is complete, gently transfer tube to tube rack. Important: Avoid agitating the tube as it may result in preliminary re-suspension of platelets and/or platelet activation.

2. Aseptically clean tube cap by gently wiping with alcohol swap. Avoid agitating as indicated in step 1.

3. Insert vented needle through tube cap to equalize pressure in vacuum tube. Caution: Do not touch the plasma.

4. Attach sharp aspiration needle to disc filter. Connect disk filter to 10 mL syringe.

5. Removal of PPP: Insert sharp needle into upper surface of plasma fraction. From the surface, draw about 50% to 80% of plasma fraction. Caution: Do not insert needle too deep from plasma surface. Important: Take care to perform step while tube is in tube rack. Support tube in one hand while applying pressure and piercing the cap with the other hand.

6. PRP Withdrawal: Suspend platelets situated on gel by gently inverting tube horizontally 10 times. Perform inversions while holding tube in one hand and syringe in the other. Caution: Do not shake. Do not completely invert tube.

7. Place tube in tube rack, holding the filter, remove syringe and replace with new 10 mL syringe. Important: performing this step while tube is not supported may result in disconnection of the cap from the tube.

8. Insert needle into upper surface of PRP fraction. From the surface carefully draw PRP tube in tube rack. Alternatively, hold tube in one hand and syringe in the other and pull PRP into syringe with thumb. It is recommended to gently press the needle tip against the tube wall for better control. Important: Place needle tip just above separation gel, while turning tube slightly on its side to help with PRP collection. Do not touch the gel with needle tip as it may result in clogging the needle.

9. Gently disconnect PRP collection syringe from disk filter. The PRP is now ready for use.

OPTION 3 – PRP collection for specific application in which the whole plasma is needed – a closed system

1. Once centrifugation step is complete, suspend platelets situated on gel by gently inverting tube horizontally 10 times. Caution: Do not shake. Do not completely invert tube.

2. Aseptically clean tube cap by gently wiping with alcohol swap.

3. PRP Withdrawal: Attach sharp aspiration needle to disc filter. Connect disk filter to 10 mL syringe. Insert needle through tube cap.

4. Invert tube so plasma touches tube cap. Holding tube in one hand and syringe in the other, carefully draw the PRP into syringe with thumb until all plasma liquid is collected. Note: If needed, rotate the needle by small and gentile movements to collect the residual PRP on the cap.

5. Gently disconnect PRP collection syringe from disk filter. The PRP is now ready for use.

OPTION 4 – Heating Process Method – PLATELET-RICH INJECTABLE GEL

Reaching 100 °C may take up to 30 minutes for some heating blocks. It is recommended to have heating device at 100 °C to keep it ready for the rest of the day by following the next 3 steps:

1. Turn ON heater by pressing the power switch on back of device.

2. Use PMG buttons to select the “S1” program and press START/STOP button.

3. Device will audibly signal when 100 °C is reached.

Device will maintain temperature (100 °C) until turned off.

Inserting sample into heating block:
A. Insert sample, press and hold START/STOP button for 2 seconds.
B. Use PMG buttons to select the “S2” program and press START/STOP button.
C. Device will recalibrate temperature and audibly signal when calibration is complete. Keep sample in heating block, heating time (10 minutes) will run automatically.
D. Device will audibly signal when heating time completes.
E. Sample is now ready.
F. To maintain device at 100 °C, press and hold START/STOP button for 2 seconds, use PGM buttons to change program to “S1” and press START/STOP button. Device will be ready for next procedure. To stop heating, press and hold START/STOP button for 2 seconds.
Note: To start new heating cycle (when inserting new sample), start from step A. If heating device has been turned off, follow instructions from step 1.

If cooling module is used, it is recommended to turn on the device before the PPP is ready to be cooled (step 7) following the next 3 steps:

1. Fill bath with distilled water until syringe contents will be below water surface (~80% full).

2. Place filled bath in device base.

3. Turn ON cooler by pressing START/STOP button on front of base. LED displays blue when cooling is active.

Device will maintain temperature until START/STOP button is pressed again.

1. Once centrifugation step is complete, gently transfer tube to tube rack. Important: Avoid agitating the tube as it may result in preliminary re-suspension of platelets and/or platelet activation.

2. Aseptically clean tube cap by gently wiping with alcohol swap. Avoid agitating as indicated in step 1.

3. Removal of PPP: Insert vented needle through tube cap to equalize pressure in vacuum tube. Caution: Do not touch the plasma.

4. Attach sharp aspiration needle to disc filter. Connect disk filter to 10 mL syringe.

5. Insert sharp needle into upper surface of plasma fraction. Carefully draw up to a maximum of 5 mL of PPP if using 11 mL tube or 9-10 mL if using 22 mL tube. Note: Density of final PRP Gel can be increased by drawing more PPP or decreased by drawing more. Caution: Do not insert needle too deep from plasma surface. Important: Take care to perform step while tube is in tube rack. Support tube in one hand while applying pressure and piercing the cap with the other hand.

6. Gently disconnect the PPP collection syringe from the disk filter, ensure no air inside syringe and immediately connect it to syringe stopper. Leave disk filter with sharp needle inserted into tube and replace the PPP collection syringe with new 10 mL syringe.

7. PPP Preparation: Place PPP collection syringe with stopper into heating block (make sure block has reached 100 °C). Select 10 minute program “S2” and press START. Follow heating instructions. When program is complete, remove syringe from block and allow it to cool to around body temperature or less. Alternatively, a cooling module or cold storage may be used to speed up the cooling process, refer to instructions included with the accessory, cool to around body temperature or less.

8. PRP Withdrawal: While step 7 is being processed, suspend platelets situated on gel by gently inverting tube horizontally 10 times. Perform inversions while holding tube in one hand and syringe in the other. Caution: Do not shake. Do not completely invert tube.

9. Insert needle of assembled accessory further to reach surface of PRP fraction. From the surface carefully draw PRP tube in tube rack. Alternatively, hold tube in one hand and syringe in the other and pull PRP into syringe with thumb. It is recommended to gently press the needle tip against the tube wall for better control. Important: Place needle tip just above separation gel, while turning tube slightly on its side to help with PRP collection. Do not touch the gel with needle tip as it may result in clogging the needle.

10. Disconnect PRP collection syringe from disk filter and attach to one side of female-to-female luer lock connector, carefully advance PRP through to the end of luer to avoid air gaps.

11. PRP Gel Mixing: Insert cooled processed PPP syringe into second side of female-to-female luer lock connector, ensure no air bubbles trapped inside. When both syringes are safely fastened to the luer lock and free of air bubbles, transfer the PRP to the PPP syringe by pushing the PRP syringe plunger completely. Repeat transfer gently from one syringe to the other until both biomaterials are thoroughly mixed by pushing syringe plungers one at a time.

12. When mixture is homogeneous, transfer desired of PRP Gel from 10 mL syringe to 1 mL syringe to divide into desired aliquots to perform procedure and enable better control of extrusion force.

13. PRP Gel is now ready to inject (with 21G to 25G needles/cannulas). Note: Keep syringes closed with syringe cap while they are not in use.