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Technical Briefs |
1 Institute for Experimental Haematology and Transfusion Medicine, University of Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
2 Department of Haemostaseology and Transfusion Medicine, Kerckhoff-Klinik, Bad Nauheim, Germany
aauthor for correspondence: fax 49-228-2879090, e-mail bernd.poetzsch{at}ukb.uni-bonn.de
Up-regulation of tissue factor (TF) expression in circulating blood cells, especially monocytes, plays a key role in the pathogenesis of various thromboembolic diseases (1)(2)(3)(4). Measurement of TF expression in monocytes therefore might be helpful in the diagnosis of a hypercoagulable state (5)(6)(7). Monocytic TF expression can be measured on both the protein and RNA levels. Testing on the RNA level seems to be more appropriate than antigen testing to detect a procoagulant state because monocytes that already express functionally active TF on their membrane surfaces are rapidly cleared from the circulation (8).
The aim of the present study was to develop a protocol for quantitative determination of TF mRNA transcripts in monocytes and other circulating blood cells. The method should be rapid, robust, and applicable in whole blood without the need for cell isolation. We developed a one-step quantitative reverse transcription (qRT)-PCR assay based on the real-time TaqMan® technology and evaluated the preanalytical conditions required for the use of TF mRNA measurements on a routine clinical basis as well as several normalization strategies, including normalization based on CD14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene expression. Finally, we measured baseline TF expression in healthy individuals and in thrombophilic patients.
RNA calibrators for quantification were prepared by in vitro transcription of DNA hybrids that combined the T7 promoter sequence with the following sequences of TF, CD14, or GAPDH cDNA: for TF, bp 265–848 (GenBank accession no. M16553); for CD14, bp 18–564 (GenBank accession no. M86511); and for GAPDH, bp 8–525 (GenBank accession no. M33197). Generated RNAs were treated with DNase I (Roche) to remove the added DNA templates and purified by use of the RNeasy Mini Kit (Qiagen). RNA was quantified by photometric measurement (A260 reading of 1 = 44 mg/L). Oligonucleotide primers and probes for TF and CD14 qRT-PCR were designed by use of Primer Express software, Ver. 1.5 (Perkin-Elmer), and were purchased from Eurogentec. All sequences shown in Table 1
were chosen to prevent amplification of genomic DNA. Primer and probe sequences for amplification of GAPDH mRNA were taken from the "TaqMan Gold RT-PCR Kit" protocol (Perkin-Elmer).
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PCRs were performed in a final volume of 20 µL using the QuantiTect Probe RT-PCR-Mix (Qiagen). Optimum reaction conditions were as follows: 1x Mastermix (including PCR buffer, deoxynucleotide triphosphates, 4 mM MgCl2, and Rox reference dye); TF or CD14 forward and reverse primers (200 and 150 nM, respectively); GAPDH forward and reverse primers (100 nM); TF or CD14 probe (200 nM); GAPDH probe (100 nM); 1 U of QT Probe RT Mix; and 5 µL (TF/GAPDH PCR) or 1 µL (CD14/GAPDH PCR) of calibrator or sample preparation. Calibrators and samples were run in duplicate.
Thermal cycling was performed in a 96-well spectrofluorometric thermal cycler (Prism SDS 7700; Applied Biosystems) with the following profile: 50 °C for 20 min for the reverse transcription reaction; 95 °C for 15 min; and 40 cycles of denaturation for 20 s at 95 °C and annealing/extension for 60 s at 60 °C. The TF qRT-PCR performed in this way exhibited a linear range extending from 102 to 107 copies/reaction. The amplification efficacy (E) of 0.99 as determined from the slope (s) of the calibration curve according to the equation E = 10-1/s - 1 was not influenced by coamplification of GAPDH or CD14 mRNA, making multiplex approaches possible. CV were calculated based on concentrations as determined by additional processed calibration curves. Intraassay CVs were <19%. Including the RNA isolation process, interassay CVs ranged between 3.1% and 21%. Variations of this magnitude are regular for quantitative PCR, and our results were well within accepted values for such assays.
Accurate analysis of in vivo gene expression might be complicated by unintended ex vivo gene expression or degradation of gene transcripts. This is especially a problem when working with monocytes because monocytes are highly reactive and TF expression can be induced by contact with foreign surfaces and extended blood storage (9). To minimize these ex vivo changes, we tested a blood sampling system (PAXgeneTM Blood RNA tubes; PreAnalytiX) that includes a stabilizing additive in the blood collection tube (10). Whole blood from one healthy donor was drawn in parallel into PAXgene and EDTA tubes. Tubes were stored at room temperature or 4 °C, and total RNA was isolated after 0, 1, 3, 5, and 7 days of storage. TF, CD14, and GAPDH RNA concentrations remained stable up to 5 days of storage in PAXgene tubes at 4 °C. Storage at room temperature reduced this time span to 24 h. In EDTA-anticoagulated blood, the RNA expression profile dramatically changed within the first hours independent of the storage temperature (for details see Fig. 1
in the Data Supplement that accompanies the online version of this Technical Brief athttp://www.clinchem.org/content/vol50/issue1/ ). On the basis of these findings, we conclude that quantification of TF mRNA is reliable and reproducible only if the cellular RNA profile undergoes immediate stabilization after blood sampling. Methods that require isolation of peripheral blood mononuclear cells before RNA determination do not necessarily reflect the in vivo situation.
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Induction of monocytic TF expression by lipopolysaccharide (LPS) plays an important role in the pathogenesis of sepsis-associated thrombotic complications and has been investigated extensively in several in vitro studies (8)(11)(12)(13). We therefore used the LPS model to evaluate the newly established PCR methods. As shown in Fig. 1A
, increasing amounts of TF mRNA were quickly generated, and peak concentrations were reached between 1 and 2 h after exposure to LPS, followed by a progressive decrease thereafter. This pattern of TF mRNA expression agrees well with previously published data (11)(12)(13). The LPS receptor CD14 has been proposed to be a suitable marker for the number of monocytic cells in blood (14). This requires that CD14 mRNA expression is relatively constant and not regulated by different triggers. Studying CD14 expression patterns in the LPS model, we found LPS-independent but time-dependent changes in CD14 mRNA concentrations (Fig. 1B
). Moreover, our data obtained on blood samples from healthy individuals demonstrate high interindividual variability of CD14 mRNA concentrations when normalized to the number of monocytes (range, 215–684 copies/monocyte), indicating that CD14 cannot be used as a marker for the number of monocytes.
The use of monocytic TF mRNA as a diagnostic marker requires the definition of a reference interval. We established a reference interval from TF mRNA and monocyte measurements of 50 healthy blood volunteers (mean age, 33.0 years; range, 19–65 years). The percentage of monocytes in whole-blood samples was determined by flow cytometry using a FACScan and CellQuest software, Ver. 3.3 (Becton Dickinson). Monocytes were identified by their specific forward and side scatter patterns and CD14 expression. The mean (SD) transcription rate of 0.0041 (0.0021) TF mRNA copies/monocyte indicates very low basal TF expression that is 5 orders of magnitude lower than CD14 expression. The data showed a log-normal distribution, and a 95% reference interval of 0.0018–0.0095 TF transcripts/monocyte was calculated according to IFCC guidelines (Fig. 1C
) (15). As a consequence, a patient will be scored positive if monocytic TF expression exceeds 0.0115 transcripts/monocyte (upper limit of the reference interval + 2 SD).
Monocytes are the major, but may not be the only source of TF mRNA in whole blood (1)(4). At present it is a matter of discussion whether platelets contain TF mRNA and whether granulocytes can be induced to express TF mRNA (16)(17). To consider these potential TF mRNA sources, we also normalized TF mRNA results to the volume of whole blood. Using this mode of normalization, we calculated a 95% reference interval of 685-3155 TF transcripts/mL. On the basis of these data, a patient will be scored positive if TF expression exceeds 4745 TF transcripts/mL.
The TF transcription profile was also analyzed in blood samples taken from 27 thrombophilic patients (16 females and 11 males; mean age, 49.5 years; range, 18–83 years). All patients had a positive history of at least one idiopathic deep venous thrombosis but showed no acute event in the last 3 months. The results shown in panels C and D of Fig. 1
show that these patients had statistically significant higher TF expression rates than the reference group. These findings suggest that increased TF expression in whole blood contributes to the procoagulant state in thrombophilic patients and support the findings of other groups (6)(18). Further studies on larger series of patients, however, are required to clarify whether increased TF mRNA concentrations in circulating blood cells are a thrombophilic risk factor.
In summary, the TF multiplex approach described here allows rapid and quantitative determination of TF mRNA concentrations in whole blood without the need for cell isolation. Ex vivo changes in the expression pattern of blood cells were avoided by the use of blood-sampling tubes that contain RNA preservation and cell-fixing additives. Together with the established reference interval, this easy-to-perform method could provide a useful basis for further studies in clinical settings in which TF expression plays an essential role.
References
The following articles in journals at HighWire Press have cited this article:
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  H. P.Y. Fan, C. Di Liao, B. Y. Fu, L. C.W. Lam, and N. L.S. Tang Interindividual and Interethnic Variation in Genomewide Gene Expression: Insights into the Biological Variation of Gene Expression and Clinical Implications Clin. Chem., April 1, 2009; 55(4): 774 - 785. [Abstract] [Full Text] [PDF]
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, results obtained with unstimulated samples (n = 3). Results are expressed as the mean ± SD (error bars). TF mRNA was measured in blood samples obtained from 50 healthy blood donors and 27 thrombophilic patients. Results were normalized to the number of monocytes (C) or to the volume of whole blood (D). Boxes represent the interquartile range, with the median represented by the line inside the box. Whiskers indicate the highest and lowest values, excluding outliers (