Centrifuging for PPP

Centrifuging for PPP
Sep 2, 2020 8:46am

Hi, I used to work in special coag at VCU, it was over 10 years ago. When we were freezing specimens we had to double spin them at 3500 rpm for 15 minutes. Our quick-spin centrifuges for PT/PTT could cause the platelets to leak out their activation factors. Where I now work, we called Mayo and UVA who claim to have never heard of this. I only process for send outs occasionally but when I do I cannot bring myself to use the quick centrifuges since I was trained differently, however, if this is an outdated practice, I can change but I don't want to do so without verification from someone I trust. Thank you, Anne.

Hello, Anne, thank you for being a Fritsma Factor participant and thank you for your question. You are correct, and for confirmation, I discussed this with colleagues Dave McGlasson and Dennis Ernst.

If you pardon my self-reference, here is the section entitled "Platelet-poor Plasma for Clot-based Testing" from Fritsma, GA. Chapter 41: Laboratory Evaluation of Hemostasis. In Keohane EM, Otto CN, Walenga JM: Rodak's Hematology: Clinical Principles and Applications. Elsevier, 2020:

"Clot-based plasma coagulation tests require platelet-poor plasma [PPP ] with a platelet count of less than 10,000/uL. Sodium citrate–anticoagulated whole blood is centrifuged at 1500 xg relative centrifugal force [RCF] for 15 minutes in a horizontal-head centrifuge to produce supernatant PPP. Alternatively, a HemoCue StatSpin type of centrifuge that generates 4400 xg RCF can produce PPP within 3 minutes. The advantage of the horizontal centrifuge head is that it produces a straight, level plasma–blood cell interface, making it possible for automated coagulometers to sample from the supernatant plasma of the blood collection tube [the “primary” tube]. Angled centrifuge heads cause platelets to adhere to the side of the tube. If the “angle-spun” tube is allowed to stand, the adherent platelets drift back into the plasma and release granule contents. Each hemostasis laboratory manager establishes the correct centrifugation speed and times locally.

In the special hemostasis laboratory the manager may choose a double-spin approach. The primary tube is centrifuged and the plasma is transferred to a secondary plastic tube, which is labeled and centrifuged again. The double-spin approach may be used to produce PPP with a plasma platelet count of less than 5000/uL, which some laboratory directors prefer for lupus anticoagulant (LAC ) testing and for freezing.

A plasma platelet count greater than 10,000/uL affects clot-based test results. Platelets become activated in vitro and release microparticles and the membrane phospholipid phosphatidylserine, which trigger plasma coagulation and interfere with LAC testing. Platelets also secrete fibrinogen, factors V and VIII, and VWF. These may desensitize PT and PTT assays and interfere with clot-based coagulation assays. In addition, platelets release PF4 , a protein that binds and neutralizes therapeutic heparin in vitro, falsely shortening the PTT and interfering with heparin management."

You may also wish to employ the formula for calculating RCF: RCF = 11.2 × r (RPM/1000)2, where r = the radius of the centrifuge head, available from the manufacturer's package insert. The lab manager typically confirms the effectiveness of the centrifugation by periodically running a platelet count on a randomly selected specimen.

 

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Hi, I used to work in special coag at VCU, it was over 10 years ago. When we were freezing specimens we had to double spin them at 3500 rpm for 15 minutes. Our quick-spin centrifuges for PT/PTT could cause the platelets to leak out their activation factors. Where I now work, we called Mayo and UVA who claim to have never heard of this. I only process for send outs occasionally but when I do I cannot bring myself to use the quick centrifuges since I was trained differently, however, if this is an outdated practice, I can change but I don't want to do so without verification from someone I trust. Thank you, Anne.

Hello, Anne, thank you for being a Fritsma Factor participant and thank you for your question. You are correct, and for confirmation, I discussed this with colleagues Dave McGlasson and Dennis Ernst.

If you pardon my self-reference, here is the section entitled "Platelet-poor Plasma for Clot-based Testing" from Fritsma, GA. Chapter 41: Laboratory Evaluation of Hemostasis. In Keohane EM, Otto CN, Walenga JM: Rodak's Hematology: Clinical Principles and Applications. Elsevier, 2020:

"Clot-based plasma coagulation tests require platelet-poor plasma [PPP ] with a platelet count of less than 10,000/uL. Sodium citrate–anticoagulated whole blood is centrifuged at 1500 xg relative centrifugal force [RCF] for 15 minutes in a horizontal-head centrifuge to produce supernatant PPP. Alternatively, a HemoCue StatSpin type of centrifuge that generates 4400 xg RCF can produce PPP within 3 minutes. The advantage of the horizontal centrifuge head is that it produces a straight, level plasma–blood cell interface, making it possible for automated coagulometers to sample from the supernatant plasma of the blood collection tube [the “primary” tube]. Angled centrifuge heads cause platelets to adhere to the side of the tube. If the “angle-spun” tube is allowed to stand, the adherent platelets drift back into the plasma and release granule contents. Each hemostasis laboratory manager establishes the correct centrifugation speed and times locally.

In the special hemostasis laboratory the manager may choose a double-spin approach. The primary tube is centrifuged and the plasma is transferred to a secondary plastic tube, which is labeled and centrifuged again. The double-spin approach may be used to produce PPP with a plasma platelet count of less than 5000/uL, which some laboratory directors prefer for lupus anticoagulant (LAC ) testing and for freezing.

A plasma platelet count greater than 10,000/uL affects clot-based test results. Platelets become activated in vitro and release microparticles and the membrane phospholipid phosphatidylserine, which trigger plasma coagulation and interfere with LAC testing. Platelets also secrete fibrinogen, factors V and VIII, and VWF. These may desensitize PT and PTT assays and interfere with clot-based coagulation assays. In addition, platelets release PF4 , a protein that binds and neutralizes therapeutic heparin in vitro, falsely shortening the PTT and interfering with heparin management."

You may also wish to employ the formula for calculating RCF: RCF = 11.2 × r (RPM/1000)2, where r = the radius of the centrifuge head, available from the manufacturer's package insert. The lab manager typically confirms the effectiveness of the centrifugation by periodically running a platelet count on a randomly selected specimen.

 

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