VENIPUNCTURE TECHNIQUE AND SPECIMEN COLLECTION GENERAL INSTRUCTIONS

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

• 1. Select a PPT-I tube.
• 2. Gently tap tubes containing additives to dislodge any material that may have adhered to the stopper.
• 3. Place tube into holder of blood collection device. Note: Do not puncture stopper.
• 4. Select site for venipuncture.
• 5. Apply tourniquet. Prepare venipuncture site with an appropriate antiseptic. DO NOT PALPATEVENI PUNCTURE AREA AFTER CLEANSING.
• 6. Place patient’s arm in a downward position.
• 7. Remove needle shield. Perform venipuncture WITH ARM DOWNWARD AND TUBE STOPPER UPPERMOST.
• 8. Push tube onto needle, puncturing stopper diaphragm. Center tubes in holder when penetrating the stopper to prevent sidewall penetration and resultant premature vacuum loss.
• 9. 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 in to 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 procedurefromStep1
• 10. 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).
• 11. Remove needle from vein, applying pressure to puncture site with dry sterile swab until bleeding stops.
• 12. Once clotting has occurred, apply bandage if desired.
• 13. 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.
• 14. 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.
• 15. 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

• Lace 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.
• Balancetubesaccordingtocentrifugeinstructionstominimizethechanceofglassbreakage.
• 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:

CENTRIFUGATION SPEED AND TIME
PRODUCT	rpm	TIME (min) / Brake
PPT-I Vacuum Tube – Model 642VES PPT-I Vacuum Tube – Model 755VES	3500 2950	10 / 30-45 seconds 10 / 30-45 seconds
Choose Speed and Time based on Centrifuge
RCF = Relative Centrifugal Force, g’, rpm = rotations per minute

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

• Once centrifugation step is completed, remove cap from tube.
• Removal of PPP phase. Place the tube in the rack. Attach the 65mm aspiration needle to 10 mL syringe and insert needle until you reach the upper surface of plasma fraction. Then Carefully draw plasma from the surface for about 50% to 80%. You are now removing the PPP phase. Do not insert needle too deep from the surface of plasma.
• PRP harvesting: Close the tube with the cap and invert it(in half turn movements) 10 times to mix the plasma with the platelets situated on the gel.
• 4.Placethetubeinthe rack. Remove the cap and take the sleeve filter, peel sterile cover to a halfway from the filter side to expose bottom side of sleeve. Hold cap with sterile cover on and insert filter into the tube until filter gently touches gel surface. PRP now enters inside the sleeve chamber.
• Insert 65mm aspiration needle into sleeve chamber through Mono Cap® hole to collect the PRP. The PRP is now ready to use

OPTION 2 –Concentrated PRP-a closed system

Removal of Platelet Poor Plasma(PPP)fraction.

• Once centrifugation step is completed, gently place the tube in the rack. Important: Avoid agitating the tube as it may result in preliminary re-suspension of platelets and/or platelet activation. After centrifugation the tube should contain upper clear yellowish PPP fraction, separation gel barrier and the lower red colored fraction. The platelets reside on top of the separation gel.
• Insert a Vented Needle with filter in hub into the tube, for equalizing pressure in vacuum tube. Caution: Do not touch the PPP.
• Attach the Sharp Aspiration Needle to the filter disc. Connect the filter disc toa 10 mL syringe
• Aseptically clean the cap of the tube by wiping it gently with an alcohol swab (not supplied). Avoid agitating the tube, as indicated in step1.
• Insert the needle until you reach the upper surface of plasma fraction. Perform piercing while supporting the tube in one hand and holding the syringe in another hand. Then carefully draw plasma from the surface for about 50% to 80%. You are now removing the PPP phase. Do not insert needle too deep from the surface of plasma.
  Important: Take care to perform this step while the tube is placed in a rack. Hold the tube in one hand while applying pressure and piercing the cap with another hand.
• Platelet Rich Plasma (PRP) Withdrawal: Suspend platelets to prepare PRP by inverting the whole device horizontally (tube+ vacutainer) gently 10 times. Perform this step while holding the tube in one hand and the syringe in the other hand horizontally and rotate the device to an upward position.
• Place the tube in the rack, hold the assembled accessory by its filter, remove the syringe and replace it with a new one. Important: Performing this step while the tube is not supported may result in disconnection of the tube from the cap.
• Insert the needle of the assembled accessory lower in to the tube in order to reach the PRP surface. Important: Place the needle tip just above the separation gel, while turning the tube slightly on its side to help with the PRP collection. Avoid touching the gel with a needle tip as it may result in clogging the needle.
• Draw PRP into the PRP collection syringe while placing the tube in the rack, or alternatively, hold the tube in one hand and the syringe in the other hand and pull the PRP into the syringe with your thumb in order to collect all the PRP. It is recommended to gently press the tip of the needle against the walls of the tube for better control of the needle.
• Gently disconnect the PRP collection syringe from vacutainer. The PRP is now ready for use.

OPTION3–PRP collection for specific application in which the whole plasma is needed–a closed system

• Once centrifugation step is completed, take the tube and invert it (in half turn movements) 10 times to mix the plasma with the platelets sited on the gel.
• Aseptically clean the cap of the tube by wiping it gently with an alcohol swab(not supplied).
• Platelet Rich Plasma (PRP) Withdrawal: Assemble required accessory number 4 (Holder, luer adapter with female port (termedVACU10Sor VACU20S), or other device for drawing PRP), to a syringe. Place the tube into the tube holder. Push tube forward until tube stopper has been penetrated
• Invert the tube (upside down) so the plasma will touch the cap. Hold the inverted tube in one hand and the syringe in your other hand.
• Carefully draw the PRP into the syringe with your thumb until all plasma liquid is collected. Important: If needed, rotate the holder’s needle by small and gentle movements to collect the residual PRP on the cap.
• Notice that due to the safety one-way valve, no fluid from the syringe will enter back into the tube
• Gently disconnect PRP collection syringe from the assembled accessory. The PRP is now ready for use.

CAUTION: Federal law (USA) restricts this device to sell by or on the order of a physician.

It is the laboratory’s ultimate responsibility to determine reference intervals for all analytes based upon

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

PRP COLLECTION

After centrifugation, the tube should contain upper clear yellowish fraction, separation gel barrier, and a lower red colored 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

Symbols & Mark Key