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Technical Briefs |
Istituto di Chimica Microscopia Clinica, Dipartimento di Scienze Morfologico-Biomediche, Università degli Studi di Verona, Verona, Italy;
aaddress correspondence to this author at: Istituto di Chimica e Microscopia Clinica, Dipartimento di Scienze Morfologico-Biomediche, Ospedale Policlinico G.B. Rossi, Piazzale Scuro, 10, 37134 Verona, Italy; fax 39-45-8201889, e-mail ulippi{at}tin.it
Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and D-dimer assays are part of the conventional routine coagulation panel. Accurate standardization of both the preanalytical and analytical phases is pivotal to achieving accuracy and precision of results. Routine blood coagulation assays and D-dimer testing strongly influence clinical decision-making because they represent crucial steps in the diagnostic approach to thromboembolic and hemorrhagic disorders and in the monitoring of anticoagulant therapy with heparin or oral anticoagulants.
Among major determinants of preanalytical variability, sample collection exerts considerable influence on the reliability of results (1); problems arising from cumbersome blood withdrawal, inadequate filling or mixing of the tube, and inappropriate treatment of specimens are important sources of imprecision. In particular, it has been suggested that the precision of fibrinogen measurements might be influenced by procedures used for specimen collection, leading to the suggestion that the first tube of blood collected be discarded (2). To establish the potential impact of sample collection on imprecision of routine coagulation assays, we measured PT, aPTT, fibrinogen, and D-dimer in 30 consecutive patients on oral anticoagulant therapy.
The study was performed according to the following protocol: Three independent samples were successively collected from each patient. Sample A was collected as the first specimen immediately after venipuncture of the median cubital or basilic vein of the left arm; sample B was collected directly after sample A; and sample C was collected as the first specimen after a second venipuncture of the median cubital or basilic vein of the right arm. All blood collections were performed on the morning of the same day by a single practiced phlebotomist, with patients fasting before venipuncture. All phases of sample collection were standardized, including time of tourniquet placement (<30 s), the use of 20-gauge needles, and use of evacuated tubes from the same lot (Becton Dickinson).
After collection into evacuated silicon tubes containing 0.123 mol/L sodium citrate, samples were gently mixed by inverting the tubes 4–6 times and were centrifuged at 3000g for 10 min at 10 °C. Plasma was separated and stored in aliquots at –70 °C until measurement. In cases in which either the above criteria were not fulfilled or attempts to collect one or more of the patients’ samples were unsatisfactory (difficulty in locating easily accessed veins, missing the vein with the needle, or hemolyzed or lipemic specimens), all results for samples A, B, and C were excluded from the statistical evaluation. On the basis of these criteria, data for two patients originally enrolled were excluded, and the final study population consisted of 28 individuals (16 women and 12 men; mean age, 52 years). PT, aPTT, and fibrinogen measurements were performed on the Dade-Behring Coagulation System (BCS) with use of proprietary reagents. Plasma D-dimer was measured with the Vidas DD, a rapid, quantitative automated ELISA with fluorescent detection, on the Mini Vidas Immunoanalyzer (bioMerieux). Calibrations were performed according to the instructions provided by the manufacturers. All measurements were performed in duplicate within a single analytical session, and final results were averaged. Analytical imprecision, expressed in terms of mean interassay CV, was quoted by the manufacturers as being 2–5%. Significance of differences between samples was assessed by paired Student t-test, and the level of statistical significance was set at P <0.05. Bland–Altman plots were used to compare the results of the independent measurements on samples A, B, and C; plot differences were reported as percentage of means.
The results of the evaluation are given in Table 1
. We found no statistically significant differences among samples A, B, and C. The substantial agreement among specimens collected during separate consecutive venipunctures or multiple subsequent blood samples collected on the same phlebotomy was confirmed by use of linear regression analysis, which yielded satisfactory correlation coefficients, and the satisfactory precision of repeated measurements is demonstrated further in the Bland–Altman plots (Fig. 1
).
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Among routine coagulation assays, fibrinogen and D-dimer measurements are thought to be more susceptible to variations introduced in the preanalytical phase (2)(3)(4). The term "D-dimer" usually refers to a heterogeneous class of end-stage degradation products of cross-linked fibrin that occur in vivo with a wide range of molecular weights and contain various numbers of the D-dimer motif (5). The strong heterogeneity of D-dimer in plasma might be reflected by most immunoassays (the monoclonal antibodies show variable reactivity with different molecular forms) because the various forms occur naturally in the plasma of patients. This might be particularly true when evaluating D-dimer after continuous blood-sampling because it is conceivable that different samples from the same patients might contain heterogeneous D-dimer isoforms. Some recommended procedures for acquiring samples for coagulation analysis, especially those involving the measurement of D-dimer and fibrinogen by the Clauss method, mandate that the first sample be discarded because tissue thromboplastin released by the trauma of venipuncture could interfere with coagulation assays by activating intrinsic pathway (2). However, in agreement with the earlier report by Rosenson et al. (6), our results demonstrate that a standardized procedure for blood collection does not influence the reliability of in vitro routine coagulation testing. This is particularly true for fibrinogen and D-dimer measurements, as it had been speculated previously that discrepancies might arise from different specimens collected simultaneously from the same patient (2)(6).
References
The following articles in journals at HighWire Press have cited this article:
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  G. Lippi, G. L. Salvagno, M. Montagnana, G. Poli, and G. C. Guidi Influence of centrifuge temperature on routine coagulation testing. Clin. Chem., March 1, 2006; 52(3): 537 - 538. [Full Text] [PDF]
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