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Preparing PPP

From Rebecca Jones, Trinity Medical Center, Birmingham:

Hey George, love the website! If only all disciplines had access to all this knowledge! Thanks for all you do.

I have another question for you to ponder…or not. What is your feeling about performing a “hard spin” on coagulation specimens? We do a “quick spin” for 2–3 minutes and then do prothrombin times (PT), partial thromboplastin times (PTT), fibrinogens, and D-dimers. The only reference in CLSI documents (specifically D-dimer) states that the recommendation is a slower spin to have platelet poor plasma. These small stat centrifuges are giving platelet poor plasma, however I need some reassurance. Especially if I start adding more special coagulation to my in house menu. Thanks again!

Also an update on the Plavix question. We are still using the VerifyNow. We gave the physicians a percent inhibition guide when releasing the new PRU value. We’ve received only three phone calls so far with physicians needing help interpreting. We are considering moving Plavix to the TEG and adding PFA and other testing as well. We have a TEG on site and are currently monitoring heparin in surgery with it. More to come on that project later.

Hi, Rebecca and thank you for the compliment. Rebecca is a graduate of our UAB Masters in Clinical Laboratory Science program, and I had the pleasure of working with her in the graduate program and in UAB Hematology laboratory. Thank you for the update on the VerifyNow report, it may be that others would like to learn how you are managing your reports during the switch-over to PRUs. Also, the Thromboelastograph (TEG) appears to be a good substitute and is getting a lot of attention.

The small desk-top centrifuges, such as the Stat-Spin Express, do a fine job of producing platelet-poor plasma (PPP, plasma whose platelet count is <10,000/μL). There is one caveat. Because they are angle-head centrifuges, the platelets tend to adhere to the side of the tube and slowly release back into the plasma. So, you can’t let them stand more than an hour or so before testing. You can determine the exact time limit with a simple experiment: just count the plasma at regular intervals to record a rise.

The Stat-Spin centrifuges have limited capacity, however, so you may want to have a larger centrifuge for big batches. Choose a centrifuge with swinging heads that leave behind a level interface. If you have a centrifuge capable of reaching 8000 RPM, you can prepare PPP in 2–3 minutes that is adequate for your routine testing.

For special coagulation testing, for example, factor assays or lupus anticoagulant profiles, most lab directors require double-spinning in accordance with CLSI H21. Spin for 10 minutes at 2500 g (g-force, not RPM), separate the plasma, and spin the plasma at 2500g for an additional 10 minutes. With this technique, the plasma platelet count ends up usually at <5,000/µL. You should also choose this approach if you plan to freeze the specimen. I hope this helps!

Comments (2)
Specimen Management
Sep 13, 2012 4:20am

Thank you to “jlow” for the comment and especially the refer
Thank you to “jlow” for the comment and especially the reference, which updates my original comment and illustrates the limitations of the small bench-top centrifuge. The single high-speed spin works well at UAB without PF4 release, but of course, that is an n of 1. I also received this in an email from Ali Sadeghi-Khomami, PhD, Sr. Research Scientist at Precision BioLogic Inc.: Based on a recommendation provided in JTH 2009, 1737: Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. “In order to achieve <10,000 platelet/micL (PPP), first we need to do 2000 g 15 min followed by >2500 g for 10 min. Surprisingly, they referenced CLSI H21-A5 but these numbers are not actually recommended in that document.

Sep 10, 2012 6:24pm

Dear George,
I have 2 comments:
1. I am not sure because t

Dear George,
I have 2 comments:
1. I am not sure because the studies might not be there, and I have not had time to look, what effect PF4 release from platelets centrifuged at high speed might have on the APTT of heparinized specimens.
2. The fast spin with small numbers of tubes approach might be suitable for a dedicated stat lab where one person can be responsible for the centrifugation and then making sure that testing is done within a certain time to prevent platelets sliding back into the plasma – 10 min in paper by Kao (J Biomed Lab Sci 2010 22, 23.). It might not work so well logistically in a lab where one operator is responsible for stats and routine and special haemostasis processing.

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