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Letters to the Editor |
Departments of1
Laboratory Medicine, 2
Experimental Pathology, 4
and Clinical Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
3 Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
aAddress correspondence to this author at: Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic. Fax 420-543-136-721; e-mail valik{at}mou.cz.
To The Editor:
Variations in specimen collection and processing may confound analyses of protein profiles obtained by mass spectrometry (1)(2)(3)(4)(5)(6). Recently, Banks et al.(7) demonstrated that the choice of device for specimen collection affected the observed plasma proteomic profile. Extending these observations, we observed alteration of the mass spectrometric protein profile for specimens collected in clot activator– and gel-containing tubes, compared with specimens collected in the plain tubes of Banks et al. (7).
After we obtained institutional review board approval, we collected 2 fasting blood specimens from each of 20 healthy female volunteers, ages 27–60 years. Specimens were collected in 2 types of tubes: Microvette Sarstedt, type neutral (cat. no. 01.1728.001), denoted "white", and Microvette Sarstedt Serum Gel Clotting activator (cat. no. 03.1524.001), denoted "brown". The time required for complete coagulation was 
40 min with the white tubes, and 10 min with the brown tubes. To parallel conditions used for routine handling of specimens for tumor-marker testing, we centrifuged samples at 1500g for 10 min and then froze 20-µL aliquots at –25 °C. Specimens selected for analysis were thawed, and 20-µL aliquots were denatured with 30 µL of sample buffer containing 9 mol/L urea and 20 g/L 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (Fluka); the mixture was incubated at room temperature for 30 min, vortex-mixed every 10 min, and centrifuged at 14 000g for 10 min at 4 °C. We performed protein profiling of 10-µL supernatant aliquots on IMAC30-Cu arrays with a PBS IIc ProteinChip reader (Ciphergen). We measured samples in 2 series. The first 10 specimens were a test set, and the next 10 were a verification set to assess consistency of the changes observed within a period of 2 months. We analyzed spectra with ProteinChip Software 3.2 normalized to total ion current with an external normalization coefficient of 0.2, as suggested by the manufacturer, employing the baseline subtraction function.
Within the mass range analyzed (3000–20 000 m/z), 34 peak clusters were generated; of these, 26 peaks occurred in >50% of spectra, as shown in Fig 1
. The differences between proteomic profiles for the white and brown groups were considered significant if ratios of normalized peak areas derived from the respective groups were >2 and the degree of confidence was P <0.01 (Wilcoxon paired test). In the brown sample group, we detected 2 peaks, m/z 3957.3 and 4283.6, with intensities whose mean normalized peak areas were >40-fold higher than those of the white sample group. These peaks were situated next to 2 peaks with m/z 3885.2 and 4211.2, the signal of which was decreased in some spectra in the brown group. The remaining peaks did not display any statistical significance with respect to their occurrence in white and/or brown collection tubes. We observed no differences between the test set and the verification set, indicating that no detectable changes occurred during the period of specimen storage.
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Clot-activating blood-sampling devices are routinely used in clinical laboratories for serum chemistry and immunoassay testing because they provide serum within a short period of time and they may reduce turnaround times. However, it has not been sufficiently demonstrated that these tubes are free from interferences; in fact, studies carried out by Abbott and Becton Dickinson have been initiated to address this issue (8)(9). Although we did not investigate collection tubes from various manufacturers, available evidence, including our data, indicates that these problems may be common. The more rapid coagulation process in clot-activator tubes may be associated with more extensive proteolysis in the specimen, potentially leading to greater protein fragmentation that is subsequently detected by mass spectrometry. Nonbiological changes, observed repeatedly in the low-molecular-weight serum proteome profiles, raise the question of whether serum is the specimen of choice for major protein- and/or peptide-type clinical analytes such as hormones and tumor markers (8).
In summary, clot activator-containing collection tubes may lead to preanalytical artifacts in proteomic studies. In our experience, these tubes can be effectively substituted with Li-heparin plasma tubes for chemistry analytes or plain serum tubes used for immunoassay and specimen banking.
Acknowledgments
This study was supported by Internal Grant Agency of the Ministry of Health of the Czech Republic Institutional Research Grants MZO 00209805 and NR 8338-3/2005. The authors thank Dr. Lenka Dubska for comments and suggestions and Dr. Jiri Jarkovsky for advice on statistical testing. R.P., P.B., and S.B. contributed equally to this work
References
The following articles in journals at HighWire Press have cited this article:
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  F. Mannello Serum or Plasma Samples?: The "Cinderella" Role of Blood Collection Procedures Preanalytical Methodological Issues Influence the Release and Activity of Circulating Matrix Metalloproteinases and Their Tissue Inhibitors, Hampering Diagnostic Trueness and Leading to Misinterpretation Arterioscler Thromb Vasc Biol, April 1, 2008; 28(4): 611 - 614. [Full Text] [PDF]
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