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High-speed Centrifugation and PPP

An interesting question about centrifugation from Warren Varden, Tech Specialist of UAB’s special coagulation laboratory.

Hey George:

We are currently validating a stat centrifuge for routine coagulation testing. This centrifuge is marketed specifically for producing PPP in coagulation. It spins at 8,000 rpm with an RCF of 6,153. One of the techs questioned if such a high rate would potentially damage the platelets releasing platelet factor 4 (PF4), thus compromising the plasma. Any thoughts or information on this subject?

Thanks, Warren Varden, UAB Coagulation Department, Birmingham, Al

Hi, Warren,

Your concern for release of PF4 probably has to do with its ability to neutralize heparin, thereby shortening activated partial thromboplastin time (PTT, APTT) results. This would cause you to report a factitiously low heparin level when using the PTT to monitor unfractionated heparin therapy.

I’ve dug through literature and can find no study that specifically addresses PF4 release. However, this publication indirectly gives us an answer:

Nelson S, Pritt A, Marlar RA. Rapid preparation of plasma for ‘Stat’ coagulation testing. Arch Pathol Lab Med 1994;118:175-6. “We undertook to confirm and extend previous work on the use of microcentrifugation (2 minutes at 11,000 g) to prepare platelet-poor plasma for assay of the prothrombin time, partial thromboplastin time, fibrinogen level, D-dimer, antithrombin, and dilute Russell viper venom time. We compared results of routinely submitted blood samples by both high-speed and routine (15 minutes at 1800g) centrifugation. We found no significant differences in assay results and concluded that the high-speed technique is a reliable and useful option for minimizing turnaround times for these coagulation assays.”

Given there is no difference in the PTT result, we can probably conclude no in vitro PF4 release. Likewise, the dilute Russell viper venom (DRVVT) results imply no release of platelet membrane phospholipids, which could otherwise neutralize lupus anticoagulant. While these are reasonable conclusions, they are a slight stretch. It might be smart to repeat this study using heparin therapy and lupus anticoagulant specimens.

Dave McGlasson alerted me to a white paper published by StatSpinÒ, Inc. Norwood, MA: Peerschke EIB. Residual platelet counts of plasma prepared for coagulation studies using the StatSpin Express®. 2002. (StatSpin is now owned by Iris Corp). The paper addresses only platelet counts and contains this statement: “Preliminary studies suggest that samples should be analyzed or the supernatant plasma removed within 10 min of centrifugation to avoid redistribution of platelets at the plasma blood interface.” High-speed centrifuges employ angle heads, which force a number of platelets to adhere to the side of the tube. Upon standing, the platelets drift back into the plasma, raising the platelet count to above 10,000/uL. This is an important limitation that affects specimen management.

I may not have answered your question completely, and would like to hear from others. I know that StatSpin and its competitors are popular and are used in many coagulation laboratories.

Comments (1)
Aug 2, 2009 8:43pm

Hi George! Ah, but does it have to be PPP for routine testi
Hi George! Ah, but does it have to be PPP for routine testing? Remember this: “…2008 revision of Clinical and Laboratory Standards Institute (CLSI) Standard H21-A5, Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays. One interesting comment she made is that it is not necessary to prepare platelet-poor plasma (PPP; plasma with a platelet count less than 10,000/uL) for routine coagulation testing on fresh plasma.”

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