CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Q. We have validated that our centrifuge produces platelet-poor plasma (<10 × 109/L) for coagulation samples at a time of 10 minutes. The Clinical and Laboratory Standards Institute (CLSI) recommendation is 1,500g for no less than 15 minutes. Is the time and speed centrifuge-specific? Have studies been performed on StatSpins? Can StatSpin-type centrifuges be used to spin coagulation samples?
A. CLSI document H21-A5: “Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays; approved guideline—5th edition,” as correctly pointed out, provides a recommendation for the relative centrifugal force (RCF) and time of centrifugation necessary to obtain sodium citrate platelet-poor plasma (PPP) samples.1 PPP is generally defined as post-centrifugation plasma that contains less than 10 × 109/L platelets. Specifically, H21-A5 states that the most common condition under which to obtain PPP is 1,500g for no less than 15 minutes at room temperature, but that centrifugal speed and duration must be established by each laboratory. The RCF and duration required to consistently produce PPP will vary depending on the brand and model of centrifuge used. This is because RCF is dependent on the speed (revolutions per minute, or RPM) and distance of the specimen from the axis, or the rotating radius. Furthermore, to prevent remixing of plasma and reintroduction of cellular elements, it is recommended that a swing-out bucket (angle) rotor be used and that the brake not be applied at the end of centrifugation.It has been documented, however, that routine coagulation assays, such as APTT, PT/INR, and thrombin time, are not affected by platelet counts up to 200 × 109/L (200,000/μL) when testing is performed on fresh samples.2,3 Shorter centrifuge times at 1,500g therefore are acceptable for routine coagulation assays, if testing is performed on fresh samples immediately post-centrifugation and only when there are no subsequent test requirements, thereby ensuring that plasma will not be frozen or processed for additional assays.4
Another means to reduce the time needed for centrifugation, but still achieve PPP, is to increase the RCF. Using centrifugal forces greater than 1,500g is generally discouraged as this may induce platelet activation and lysis of red blood cells.4 To the contrary, a number of studies have reported no adverse effect on routine coagulation testing, such as APTT, PT, and fibrinogen, if centrifuged at high speed (for example, 11,000g) for short (for example, two-minute) durations.5,6 H21-A5 also states that higher-speed and shorter-duration centrifuges (also known as “Statfuge”) can be used as long as speed and duration of centrifugation are tested to determine optimum conditions for producing PPP.1 It has been cautioned, however, that samples spun in this manner should be tested within about 10 minutes if sampled from the primary tube or promptly aliquoted to a secondary tube, to prevent the drift of platelets, which cling to the side of the tube at high RCF, back into the plasma.7 Also of potential relevance here is the recommendation from the International Society on Thrombosis and Haemostasis Scientific Standardisation Committee on Lupus Anticoagulants (LA) that samples destined for LA testing after freezing be first processed by double centrifugation, with recommended speeds of 2,000g and “>2,500g,” respectively.8
In summary, the most important considerations are that any deviation from recommended practice such as CLSI1 should be validated by the individual laboratory and that PPP should contain less than 10 × 109/L platelets if the sample is not tested immediately and instead needs to be frozen for subsequent testing (for routine coagulation and specialized hemostasis tests).
References
1. Clinical and Laboratory Standards Institute. Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays; approved guideline—5th edition. CLSI document H21-A5. Wayne, Pa.: CLSI;2008.
2. Carroll WE, Wollitzer AO, Harris L, et al. The significance of platelet counts in coagulation studies. J Med. 2001;32(1–2):83–96.
3. Barnes PW, Eby CS, Lukoszyk M. Residual platelet counts in plasma prepared for routine coagulation testing with the Beckman Coulter power processor. Lab Hematol. 2002;8(4):205–209.
4. Lippi G, Salvagno GL, Montagnana M, et al. Influence of the centrifuge time of primary plasma tubes on routine coagulation testing. Blood Coagul Fibrinolysis. 2007;18(5):525–528.
5. Nelson S, Pritt A, Marlar RA. Rapid preparation of plasma for ‘stat’ coagulation testing. Arch Pathol Lab Med. 1994;118(2):175–176.
6. Pappas AA, Palmer SK, Meece D, et al. Rapid preparation of plasma for coagulation testing. Arch Pathol Lab Med. 1991;115(8):816–817.
7. Kao CH, Shu LC, Yen WH. Evaluation of a high-speed centrifuge with rapid preparation of plasma for coagulation testing to improve turnaround time. J Biomed Lab Sci. 2010;22(1):23–27.
8. Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009;7(10):1737–1740.
Dorothy M. (Adcock) Funk, MD
Medical/Laboratory Director
Esoterix Coagulation Inc.
Englewood, Colo.
Member, CAP Coagulation Resource Committee
Giuseppe Lippi, MD
Laboratory of Clinical Chemistry and Hematology
Department of Pathology and Laboratory Medicine
Academic Hospital of Parma
Parma, Italy
Emmanuel J. Favaloro, PhD, FFSc (RCPA)
Department of Haematology
Institute of Clinical Pathology and
Medical Research
Westmead Hospital
Westmead NSW
Australia
Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla. Use the reader service card to submit your inquiries, or address them to Sherrie Rice, CAP TODAY, 325 Waukegan Road, Northfield, IL 60093; srice@cap.org.