Coag Specimens, Delayed Analysis, Centrifugation

Coag Specimens, Delayed Analysis, Centrifugation
Sep 13, 2021 8:42am

From a colleague: Hi George, I was having a discussion with a colleague regarding the proper or best way to handle a coagulation sample if the processing was going to be delayed [~4  hrs]. Your name popped up, so hence my email:

Option 1: the sample is spun when received and then analyzed later. Our concern was whether the platelets would go back into plasma during the waiting period on the counter, not being transported and having any jostling of the sample.
Option 2: the sample is spun right before analysis. Is there a concern that the platelets in the plasma would interfere with phospholipids, etc, and spinning at receipt would minimize that effect?
Of course, I realize that the platelets are still in the presence of plasma, whether there’s less effect having them spun down to a ‘buffy coat’ level.
Which got me thinking about why have they not created a ‘gel’ tube so that the centrifugation could separate the plasma from the interfering platelets?
I appreciate your time and any thoughts you may have on this.


Hello, and thank you for your questions. Laboratory managers and blood collectors reference the Clinical and Laboratory Standards Institute [CLSI]: Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays, Approved Guideline--Fifth Edition, 2007. CLSI document H21-A5 [ISBN 1-56238-000-0], prepared by Adcock DM, Hoefner DM, Kottke-Marchant K, Marlar RA, Szamosi DI, and Warunek DJ. The guideline is also summarized in Fritsma GA. Chapter 41. Laboratory evaluation of hemostasis. In: Keohane EM, Otto CN, Walenga JM, Rodak's Hematology: Clinical Principles and Applications. Elsevier 2020.

In short, specimens for PTs may be held uncentrifuged for up to 24 hours, specimens for PTT for up to 4 hours, and specimens collected to monitor therapeutic unfractionated heparin for up to one hour [four if collected in a CTAD tube]. Tubes must remain stoppered and specimens are maintained at ambient temperatures, 15°–25° C, never chilled. If specimens are to be held for periods exceeding these limits, they are centrifuged at a speed and force that produces platelet-poor plasma; plasma whose platelet count does not exceed 10,000/uL. The supernatant plasma is transferred to a plastic freezer tube, labeled, sealed, and frozen. If left on the cells, platelets drift back into the supernatant plasma where they may become partially activated, secreting von Willebrand factor, factor V [5], and platelet factor 4, which neutralizes heparin.

I know of no efforts to develop a gel tube, and I assume tube manufacturers would consider such developments as proprietary. They may be reluctant because the CLSI document discourages filtration through a 0.2 um filter. the filter removes, along with platelets, fibrinogen, and factors V, VIII, IX, XII, and VWF , thus factitiously prolonging PT and PTT results.

I hope this answers your questions. Guidelines for valid coagulation specimen collection create limitations that restrict clinics, transporters, and reference laboratories. I often wonder whether the guidelines are being carefully enforced.

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From a colleague: Hi George, I was having a discussion with a colleague regarding the proper or best way to handle a coagulation sample if the processing was going to be delayed [~4  hrs]. Your name popped up, so hence my email:

Option 1: the sample is spun when received and then analyzed later. Our concern was whether the platelets would go back into plasma during the waiting period on the counter, not being transported and having any jostling of the sample.
Option 2: the sample is spun right before analysis. Is there a concern that the platelets in the plasma would interfere with phospholipids, etc, and spinning at receipt would minimize that effect?
Of course, I realize that the platelets are still in the presence of plasma, whether there’s less effect having them spun down to a ‘buffy coat’ level.
Which got me thinking about why have they not created a ‘gel’ tube so that the centrifugation could separate the plasma from the interfering platelets?
I appreciate your time and any thoughts you may have on this.


Hello, and thank you for your questions. Laboratory managers and blood collectors reference the Clinical and Laboratory Standards Institute [CLSI]: Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays, Approved Guideline--Fifth Edition, 2007. CLSI document H21-A5 [ISBN 1-56238-000-0], prepared by Adcock DM, Hoefner DM, Kottke-Marchant K, Marlar RA, Szamosi DI, and Warunek DJ. The guideline is also summarized in Fritsma GA. Chapter 41. Laboratory evaluation of hemostasis. In: Keohane EM, Otto CN, Walenga JM, Rodak's Hematology: Clinical Principles and Applications. Elsevier 2020.

In short, specimens for PTs may be held uncentrifuged for up to 24 hours, specimens for PTT for up to 4 hours, and specimens collected to monitor therapeutic unfractionated heparin for up to one hour [four if collected in a CTAD tube]. Tubes must remain stoppered and specimens are maintained at ambient temperatures, 15°–25° C, never chilled. If specimens are to be held for periods exceeding these limits, they are centrifuged at a speed and force that produces platelet-poor plasma; plasma whose platelet count does not exceed 10,000/uL. The supernatant plasma is transferred to a plastic freezer tube, labeled, sealed, and frozen. If left on the cells, platelets drift back into the supernatant plasma where they may become partially activated, secreting von Willebrand factor, factor V [5], and platelet factor 4, which neutralizes heparin.

I know of no efforts to develop a gel tube, and I assume tube manufacturers would consider such developments as proprietary. They may be reluctant because the CLSI document discourages filtration through a 0.2 um filter. the filter removes, along with platelets, fibrinogen, and factors V, VIII, IX, XII, and VWF , thus factitiously prolonging PT and PTT results.

I hope this answers your questions. Guidelines for valid coagulation specimen collection create limitations that restrict clinics, transporters, and reference laboratories. I often wonder whether the guidelines are being carefully enforced.

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