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Technical Briefs |
1 Kerckhoff Heart Center, Department of Cardiology, Bad Nauheim, Germany; 2 Klinikum Grosshadern, Ludwig-Maximilians University, Munich, Germany;
aaddress correspondence to this author at: Kerckhoff Heart Center, Department of Cardiology, Benekestrasse 2–8, 61231 Bad Nauheim, Germany; fax 49-6032-9962313, e-mail M.Weber{at}kerckhoff-klinik.de)
Abstract
Background: Several studies have consistently shown that soluble CD40 ligand (sCD40L) concentrations are increased in patients with acute coronary syndromes and can serve as a biomarker for risk stratification. However, few data are available on preanalytic conditions that impact sCD40L values. Thus, the aim of our prospective study was to evaluate the impact of sampling techniques and storage conditions on sCD40L concentrations.
Methods: We included a total of 30 patients with no, stable, or unstable coronary heart disease. Blood samples were collected in gel-filled tubes without additives, in EDTA-filled tubes, and in citrate-filled tubes and were kept at various storage conditions.
Results: Median (interquartile range) sCD40L values at baseline were higher in serum samples [5.29 (3.89–6.33) µg/L] than in either EDTA plasma [0.78 (0.39–1.12) µg/L; P <0.001] or citrate plasma [0.37 (0.22–0.51) µg/L; P <0.001]. Serum values increased with delayed processing [7.94 (5.97–9.62) µg/L after 1.5 h (P <0.001) vs baseline; 10.55 (7.58–11.55) µg/L after 3 h (P <0.001) vs baseline]. However, after centrifugation, sCD40L values remained stable for all 3 sample types.
Conclusion: Plasma, but not serum, samples are appropriate for sCD40L measurements. In general, preanalytic conditions are critical in the assessment of sCD40L concentrations and thus should be carefully considered for future studies.
CD40 ligand (CD40L) is expressed on a variety of cell types, including activated platelets. After expression on the cell surface, CD40L is partly cleaved by proteases and subsequently released into the circulation as soluble CD40L (sCD40L), which can be detected in serum and plasma (1)(2)(3)(4). Several studies have consistently shown that sCD40L measured in serum placed on ice immediately after blood drawing, in citrate plasma, or in heparin plasma is increased in patients with acute coronary syndromes (ACS) and that it provides prognostic information with therapeutic implications independent of established cardiac markers, e.g., cardiac troponins (5)(6)(7). Thus, there is great hope that sCD40L may serve as a clinically useful biomarker for risk stratification and for therapeutic decision making in the setting of ACS (8). However, few data are available on the preanalytic conditions, including blood-sampling technique and storage conditions, that may have a significant impact on sCD40L measurements. Thus, the aim of our prospective study was to evaluate the impact of sampling techniques and storage conditions on sCD40L concentrations.
A total of 30 patients, who were referred to our institution for elective or immediate coronary angiography, were included in this study. All patients gave written informed consent, and the study was approved by the local ethics board.
Venous blood was collected from all patients the day after coronary angiography. For blood sampling, we used commercially available plastic tubes (S-Monovette®; Sarstedt Ag) and collected blood from each patient in 3 different types of tubes: gel-filled tubes without any additives; tubes containing 0.3 mL of 0.106 mol/L trisodium citrate solution; and tubes containing 1.6 mg of tripotassium EDTA per mL of blood. In all samples, sCD40L was determined by a prototypic developmental electrochemiluminescence immunoassay (Elecsys®; Roche Diagnostics; lot no. A1). Intraassay imprecision (CV) was determined by measuring samples from 4 different citrated plasma pools 21 times in one run, and interassay imprecision (CV) was evaluated from the analysis of one sample from each plasma pool at 6 days. Intraassay CVs for sCD40L were 8.9% at 1.8 µg/L, 2.1% at 1.1 µg/L, 1.9% at 0.4 µg/L, and 1.9% at 0.08 µg/L, and interassay CV were 4.8% at 1.6 µg/L, 1.7% at 1.1 µg/L, 2.0% at 0.4 µg/L, and 2.3% at 0.08 µg/L. Preliminary assessment of the upper reference limit, defined as the 95th percentile derived from 20 healthy volunteers, gave a value of 0.24 µg/L.
All samples were centrifuged at 1000g for 10 min. Baseline sCD40L values were measured in all sample types immediately after centrifugation. From all patients, 10 specimens of each tube type were stored under various storage conditions: 1.5 and 3 h at room temperature before centrifugation; 1.5 and 3 h at 4–8 °C before centrifugation; 1.5, 3, and 24 h at room temperature after centrifugation; and 1.5, 3, and 24 h at 4–8 °C after centrifugation. All samples were incubated at room temperature for 30 min before centrifugation, and samples were analyzed immediately after centrifugation after the respective storage periods according to the study protocol. Samples were not frozen.
Values for sCD40L are given as the median (interquartile range); all other variables are given as the mean (SD). For pairwise comparisons, we used the Wilcoxon rank test (2 groups) and the Friedman test (n groups). For the correlation of sCD40L values at baseline assessed by the different sample types, we calculated the Spearman correlation coefficient. All tests were performed two sided, and P values <0.05 were considered to indicate statistical significance. For all statistical analyses, the statistical software SPSS 10.0 for Windows was used.
Thirty patients were included [21 males; mean (SD) age, 61 (10) years]. No relevant coronary artery disease was present in 11 patients. The measured sCD40L values obtained for the various storage conditions are shown in detail in Table 1
. Baseline sCD40L values measured in serum samples were 6.8- and 14.3-fold higher than in EDTA-plasma and citrate-plasma samples, respectively. In samples stored at room temperature or at 4–8 °C in tubes without additives and without being centrifuged, sCD40L values were increased at 1.5 and at 3 h. In contrast, sCD40L values from EDTA-filled tubes kept at room temperature were below baseline values at 1.5 h but had returned to baseline values by 3 h (Fig. 1
). If the EDTA samples were kept at 4–8 °C, the values decreased continually. Similarly, sCD40L values in samples kept at room temperature in citrate-filled tubes before centrifugation were below baseline values at 1.5 h and returned back to baseline values at 3 h. However, if citrate samples were kept at 4–8 °C, values remained unchanged at both time points. After centrifugation, sCD40L values in serum samples remained unchanged if stored at room temperature or at 4–8 °C over 1.5 and 3 h. After an extended period of 24 h, sCD40L values had decreased. Values measured in EDTA plasma remained unchanged over the entire 24-h study period after centrifugation if kept at room temperature. For the corresponding samples stored at 4–8 °C, sCD40L values remained stable for 3 h but were decreased after 24 h. In citrate plasma, we observed a slight increase of sCD40L values, which reached statistical significance after 24 h of storage at room temperature and after 1.5 and 3 h of storage at 4–8 °C. Serum sCD40L values at baseline did not correlate with sCD40L values measured in citrate or EDTA plasma, but the values measured in citrate plasma were significantly correlated with values obtained in EDTA plasma (r = 0.706; P <0.001).
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In the present study we investigated the impact of preanalytic factors, sample type, and storage conditions on sCD40L values. Our investigation yielded two major findings: (a) sCD40L values in serum were significantly higher than in plasma, either citrate plasma or EDTA plasma. Serum values increased further according to the delay between blood sampling and processing. In contrast to serum samples, plasma values, either EDTA or citrate, decreased after 1.5 h of preprocessing time and returned to baseline values after 3 h. (b) After centrifugation, the values measured from all sample types were stable over a time period of at least 3 h at room temperature and at 4–8 °C.
Our findings are consistent with those reported by Ahn et al. (9), who found higher values for sCD40L in serum kept at room temperature compared with values measured in citrate plasma and in serum placed on ice immediately after blood drawing. Furthermore, they found that serum sCD40L values were associated with the platelet count, whereas plasma values were independent of the platelet count. In another, brief report, Halldorsdottir et al. (10) found, in a single-person study, that serum sCD40L was higher than plasma sCD40L and that serum sCD40L increased with time duration from sampling to processing, whereas in EDTA plasma, the sCD40L concentration remained unchanged over time. Our findings together with previously reported findings indicate that in vitro platelet activation and subsequent sCD40L release contribute considerably to increased sCD40L values in serum and limits their diagnostic utility. EDTA or citrate plasma should be the preferred sample type. Values determined from EDTA plasma were higher than those determined from citrate plasma. Thus, our results suggest that different decision limits need to be defined depending on the sample type used. Whether the difference in sCD40L values measured in citrate and EDTA plasma has an impact on the diagnostic and prognostic information obtained remains to be investigated in future studies. However, even with those sample types, attention must be paid to processing times. Furthermore, other preanalytic conditions, such as centrifugation force and the circadian rhythm, need to be evaluated in future studies.
In several recent studies evaluating the diagnostic and prognostic potential of sCD40L in patients with an ACS, different sample types were used. For example, Aukrust et al. (5) used serum placed on ice immediately after blood drawing, whereas Varo et al. (7) used citrate plasma. According to our findings and the findings of Ahn et al. (9), the sampling techniques applied in those studies were appropriate. However, the obtained sCD40L values in the different studies are not comparable, which underlines the need for standardization of preanalytic conditions.
Acknowledgments
This study was sponsored by Roche Diagnostics (Mannheim, Germany), and assay reagents were provided free of charge.
References
The following articles in journals at HighWire Press have cited this article:
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  D. A. Morrow, M. S. Sabatine, M.-L. Brennan, J. A. de Lemos, S. A. Murphy, C. T. Ruff, N. Rifai, C. P. Cannon, and S. L. Hazen Concurrent evaluation of novel cardiac biomarkers in acute coronary syndrome: myeloperoxidase and soluble CD40 ligand and the risk of recurrent ischaemic events in TACTICS-TIMI 18 Eur. Heart J., May 1, 2008; 29(9): 1096 - 1102. [Abstract] [Full Text] [PDF]
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M. Weber and C. Hamm Novel biomarkers--the long march from bench to bedside Eur. Heart J., May 1, 2008; 29(9): 1079 - 1081. [Full Text] [PDF]
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B. T. Ivandic, E. Spanuth, D. Haase, H.-G. Lestin, and H. A. Katus Increased Plasma Concentrations of Soluble CD40 Ligand in Acute Coronary Syndrome Depend on in Vitro Platelet Activation Clin. Chem., July 1, 2007; 53(7): 1231 - 1234. [Abstract] [Full Text] [PDF]
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F. S. Apple, L. A. Pearce, A. Chung, R. Ler, and M. M. Murakami Multiple Biomarker Use for Detection of Adverse Events in Patients Presenting with Symptoms Suggestive of Acute Coronary Syndrome Clin. Chem., May 1, 2007; 53(5): 874 - 881. [Abstract] [Full Text] [PDF]
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