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Technical Briefs |
Departments of
1
Internal Medicine and
2 Clinical Chemistry, St. Antonius Hospital Koekoekslaan 1, PO Box 2500, 3430 EM Nieuwegein, The Netherlands;
3
Central Laboratory Clinical Chemistry, Klinikum Innenstadt, Munich University Hospital, 80336 Munich, Germany
aauthor for correspondence: fax 31-30-6056357, e-mail schutgensvos{at}hetnet.nl
D-Dimer, a classic coagulation marker, is increasingly used in the exclusion of venous thromboembolism and the evaluation of coagulopathies. As with all other coagulation tests, the sample material is citrate plasma. When the D-dimer test is performed on a clinical chemistry analyzer, however, the use of citrate plasma is a burden. Depending on whether other coagulation tests are requested, either an extra tube for the D-dimer determination or sample splitting is needed. Furthermore, because citrate plasma used for coagulation tests is centrifuged differently from heparin plasma and serum, it disturbs the clinical chemistry workflow. Because D-dimer will be used in an emergency setting, where a short turnaround time (TAT) is mandatory, it would be advantageous if D-dimers could also be determined in heparin plasma.
Although many investigators have studied D-dimer assays mainly in search of such clinical outcomes as sensitivity and negative predictive value (1)(2)(3), it is possible that differences in the (pre)analytic processes can lead to different results in the D-dimer assay. There are reports on the effect of freezing on D-dimer concentration (4)(5)(6)(7), but the effects of transport and type of analyzer have been less studied (8)(9).
The first objective of this study is the validation of D-dimer determinations using heparin plasma. The second objective of this study is to investigate whether the results of a D-dimer assay are influenced by different (pre)analytic processes, such as transportation, time of measurement, freezing, and the type of analyzer used.
We used the Tina-quant® (Roche) quantitative latex assay for determination of D-dimer concentrations. Samples were collected into S-Monovette® 9NC tubes (0.106 mol/L sodium citrate; Sarstedt) and S-Monovette lithium heparin tubes (15 IU/mL lithium heparin). Citrate samples were centrifuged according to the standard protocol for coagulation samples (3000g for 15 min without brake), and lithium-heparin samples were centrifuged according to the protocol for clinical chemistry samples (2200g for 10 min with brake). Measurements were performed on a Roche Hitachi 917 analyzer (Roche Diagnostics) and a Cobas Integra 700 analyzer (Roche Diagnostics). For comparison of D-dimer results, the Pearson and Kendall correlation coefficient and the Passing–Bablok regression were used. The Student t-test was used to determine the influence of simulated transport on the D-dimer concentration and to determine the sample stability of D-dimer concentration.
We randomly obtained citrate- and heparin-plasma samples from 190 patients (139 in Munich and 51 in Nieuwegein). The samples in Munich were analyzed on a Hitachi 917, and the samples in Nieuwegein were analyzed on an Integra 700 analyzer. We found that the results obtained for heparin plasma were higher than results for citrate plasma: mean, 2.51 vs 2.06 mg/L. There was a high correlation between the citrate- and heparin-plasma samples and between the two analyzers [r = 1.0; slope=1.196; Kendall 
= 0.959, md(95) = 0.18; Fig. 1
].
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To investigate the effect of transport conditions, we collected two tubes of heparin plasma from 25 patients. One of the heparin-plasma samples was sent to the laboratory by pneumatic mail (a distance of 
100 m); the other sample was carried by hand. We found no difference between the two sample groups [mean, 1.57 vs 1.59 mg/L; r = 0.999; slope = 0.993; Kendall = 0.967; md(95) = 0.09; P = 0.381]. In another experiment, transport was simulated by placing sample tubes on an oscillating roller. One tube of citrate plasma and two tubes of lithium-heparin plasma from three healthy donors were obtained. The citrate sample and one of the lithium-heparin samples were centrifuged immediately. The citrate sample was left standing at room temperature. The centrifuged lithium-heparin plasma was split into two tubes; one was left standing at room temperature, the other tube was placed on a roller. The second lithium-heparin tube was placed directly on the roller (anticoagulated whole blood) and centrifuged afterward. The results are given in Table 1
; there was no difference in D-dimer concentrations among the samples.
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To determine sample stability, we collected random citrate (n = 15) and heparin (n = 17) plasma samples from patients. The D-dimer concentration was measured directly and measured again after the samples had stood at room temperature for 16 and 24 h and after having been frozen once (snap-frozen in liquid nitrogen). In addition, five citrate plasma samples from a 2-year-old clinical study, which had been stored at -70 °C, were measured. No differences were found between the different times of measurement (P = 0.352 for 16 h and 0.557 for 24 h in citrate plasma; P = 0.332 for 16 h and 0.315 for 24 h in heparin plasma). There was no effect of freezing (P = 0.319 for citrate-plasma samples frozen once; P = 0.116 for citrate-plasma samples stored frozen for 2 years; P = 0.340 for heparin-plasma samples frozen once).
Our data confirm and extend the previous report of Vukovich et al. (10) on the validity of performing the Tina-quant D-dimer test with heparin plasma rather than the standard measurements in citrate plasma. This finding substantially decreases the TAT of the D-dimer test by reducing preanalytic processing. We estimate that, in our hospital, this TAT has been reduced from 60 to 30 min. This indicates that a D-dimer test performed on a chemical analyzer can compete with the newer point-of-care D-dimer tests. The higher D-dimer concentration in heparin plasma compared with citrate plasma is attributable to the dilution factor introduced by the citrate solution. Because all reports in the literature are based on the standard citrate solution, we therefore multiply the results for the heparin samples by a factor of 0.84 (1/1.19) to avoid the need for changes in reference and cutoff values. This correction factor is in accordance with the value that has been found by Vukovich et al. (10) and corresponds with what one would theoretically expect when 1 part citrate solution and 9 parts blood with a hematocrit of 42% are taken together (11).
The D-dimer concentration in lithium-heparin plasma in our study is unaffected by transport by pneumatic mail or by rough handling, simulated by placing the sample on an oscillating roller. This opens the possibility of pneumatic dispatch of the sample tubes and thus a gain in time at the emergency department. It also indicates that samples obtained by general practitioners or local hospitals with limited capacity can be transported to a central laboratory for D-dimer measurements without decreasing their quality. That means that patients do not necessarily have to go to the emergency room for D-dimer determinations.
We found that the D-dimer concentrations in citrate and heparin plasma were stable regardless of the time of measurement or freezing. The results obtained with different types of analyzers were comparable. This indicates that it is valid to perform studies on frozen material and that test results of the Tina-quant assay, obtained under different (pre)analytic conditions, can be compared without loss of reliability.
In conclusion, measurement of D-dimer concentrations with the Tina-quant D-dimer test using heparin plasma is valid and provides a reduction in TAT. The D-dimer assay remains valid under different (pre)analytic conditions.
References
The following articles in journals at HighWire Press have cited this article:
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  G. Lippi and G. C. Guidi Effect of Specimen Collection on Routine Coagulation Assays and D-Dimer Measurement Clin. Chem., November 1, 2004; 50(11): 2150 - 2152. [Full Text] [PDF]
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) and Hitachi 917 (
) analyzers (n = 190).