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Technical Briefs |
Institute of Clinical Chemistry, University of Munich, Munich, Germany;
aaddress correspondence to this author at: University Hospital of Munich-Grosshadern, Institute of Clinical Chemistry, Marchioninistrasse 15, D-81377 Munich, Germany; fax 49-89-7095-6298, e-mail Stefan.Holdenrieder{at}.med.uni-muenchen.de
Circulating DNA is increased in various benign and malignant pathologic conditions, including cancers, sepsis, and graft-vs-host and autoimmune diseases as well as after trauma or ischemia (1)(2)(3)(4)(5)(6)(7)(8). Changes in circulating DNA correlate with the response to antitumor therapy and with tumor recurrence (9)(10)(11). Furthermore, DNA concentration reportedly has predictive and prognostic relevance in cancer (11)(12). Despite the nonspecific nature of circulating DNA, it might have considerable potential for monitoring cancer and management of therapy (9)(10)(11)(12).
In serum and plasma, DNA is thought to exist predominantly as mono- and oligonucleosomes (13)(14), which are formed by a core particle of a double set of the histones H2A, H2B, H3, and H4 wrapped by 146 bp of DNA on the outside (15). By this composition they seem to be protected against rapid digestion by endonucleases (16). Circulating nucleosomes can be quantified by real-time PCR of the DNA but also by immunologic assays, which are particularly well suited for serial measurements (17).
Achieving reliable results in these immunochemical assays requires adherence to a strict preanalytical protocol that includes careful venipuncture, centrifugation of the sample within 1–2 h after venipuncture, addition of EDTA for stabilization of nucleosomes, and storage at –70 °C if measurement is to be delayed. This procedure is based on our earlier studies on preanalytical factors that could influence the nucleosome concentrations between venipuncture and centrifugation, between centrifugation and EDTA addition, between EDTA addition and freezing, during long-term storage, and between thawing and test performance (17). Our results indicated that a delay between venipuncture and centrifugation can lead to a time-dependent increase in nucleosome concentrations, which was most pronounced at 37 °C, whereas a delay in EDTA addition after centrifugation was associated with a time-dependent decrease in results (17).
In many instances, blood samples are transported to the laboratory by mail; we therefore investigated various additional preanalytical conditions that might influence the stability of nucleosomes in serum during shipping: Sera from 5 volunteers were exposed to prolonged time of transportation, different temperatures, shaking and rolling, several freeze–thaw cycles, and measurements with various delays after thawing. In all experiments, the samples were centrifuged, within 30 min after venipuncture, at 3000g for 15 min and stabilized with 10 mmol/L EDTA (pH 8) immediately after centrifugation. Subsequently, they were aliquoted, and methodical experiments were performed. They were then stored at –70 °C and analyzed in batches containing all samples from a single patient.
The nucleosome ELISA (Cell Death Detection ELISAplus; Roche Diagnostics) is based on a quantitative sandwich enzyme immunoassay principle: Monoclonal mouse antibodies directed against DNA (single- and double-stranded DNA) and histones (H1, H2A, H2B, H3, and H4) detect specifically mono- and oligonucleosomes. The anti-histone antibody is bound to the microtiter plate, whereas the anti-DNA antibody labeled with peroxidase reacts with 2,2'-azino-di(3-ethylbenzthiazoline-sulfonate). The amount of captured nucleosomes is proportional to the resulting color development and enables spectrophotometric quantification in arbitrary units (17).
In the first experiment, we varied the time between stabilization of the sera with EDTA and storage at –70 °C (1, 2, 3, 4, 6, and 24 h and 2, 3, and 7 days), and samples were stored at various temperatures during that time (4, 25, and 37 °C) to simulate potential stressful transportation conditions. Prolonged "transportation time" clearly did not influence the values in those stabilized sera that were stored at 4 and 25 °C. The median SD for all time points was <10%. At 37 °C, however, the values decreased continually, and after 24 h, only approximately one-half of the initial concentration remained (median SD, 50.2%; Fig. 1
, A–F).
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In the second experiment, we investigated the influence of agitation by vortex-mixing the samples for 5, 10, and 30 s or, alternatively, by rolling them in a slow overhead roller for 15 and 30 min. Subsequently, all of the samples were incubated at 25 or 37 °C for 4 h before they were frozen at –70 °C. Neither after shaking nor after rolling the samples did we observe major changes in the concentrations of stabilized sera, particularly if they were incubated at 25 °C for 4 h (median SD <5%). However, after additional incubation at 37 °C for 4 h, the values tended to be lower (median SD up to 20%; Fig. 1
, G and H).
We then analyzed the influence of freezing and thawing on the stabilized sera. Repeated refreezing (up to 3 times at –70 °C) led to only minor changes in the concentrations (median SD <10%). Thawing the samples at various time points before measurements (2 and 12 h) and storage at various temperatures (4 and 25 °C) also had no impact on the values (median SD <10%; Fig. 1
, I and J).
Our results indicate that the concentration of nucleosomes in sera stabilized with 10 mmol/L EDTA is not influenced by preanalytical conditions such as time of transportation, moderate temperature (4–25 °C), shaking, rolling, and several freeze–thaw cycles. However, long-term exposure to high temperatures (37 °C) should be avoided as it can cause a notable decrease in the measured nucleosome concentration. This might be attributable to enhanced activation of serum nucleases or by direct thermal damage of the nucleosomes.
When these precautions are taken and the preanalytical protocol is followed, including early centrifugation and subsequent stabilization of the sera with EDTA, samples can be shipped by mail without adverse effects on the results of nucleosome measurements.
Acknowledgments
The nucleosome assays were provided by Roche Diagnostics, Germany.
References
The following articles in journals at HighWire Press have cited this article:
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  U. Deligezer, E. E. Akisik, N. Erten, and N. Dalay Sequence-Specific Histone Methylation Is Detectable on Circulating Nucleosomes in Plasma Clin. Chem., July 1, 2008; 54(7): 1125 - 1131. [Abstract] [Full Text] [PDF]
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