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Blood, 1 August 2002, Vol. 100, No. 3, pp. 1094-1095
CORRESPONDENCE
To the editor:
Gene expression changes in blood after phlebotomy: implications
for gene expression profiling
Rapidly developing technologies such as quantitative
real-time reverse transcriptase-polymerase chain reaction
(RT-PCR) and microarrays enable characterization and
quantification of nucleic acids in clinical samples. Identification of
DNA from pathogens such as bacteria or viruses is already widely used
in molecular diagnostics.1 The use of RNA as a diagnostic
tool is currently being investigated. Gene expression profiling of
tumor samples using microarray technology has become the prototypic
application for this new type of molecular diagnostics. Cluster
analysis of large-scale microarrays revealed that characterization of
only a few genes is sufficient to distinguish different
tumors.2 However, levels of gene expression may change
between sample acquisition and the beginning of analysis, and these
changes are not well understood. Blood is frequently used for diagnostic sampling and processing.
However, differences in blood collection and preparation techniques may
cause changes in gene expression levels ex vivo and consequently affect
the resulting mRNA expression profiles. For example, several
investigators have shown that anticoagulants affect leukocyte viability
and cause ex vivo changes in cytokine production.3,4 The extent to which events after blood collection influence gene
expression is not known. To our knowledge, changes in gene expression
between phlebotomy and the beginning of analysis have not been studied.
We evaluated the effect of ex vivo incubation of blood on the gene
expression status of human peripheral blood. After informed consent,
blood was obtained from healthy donors. Whole blood collected
in EDTA (ethylenediaminetetraacetic acid) tubes was stored under
sterile conditions at room temperature for different periods of time.
Gene expression was monitored by quantitative real-time RT-PCR using
TaqMan assays on an ABI PRISM 7700 (Applied Biosystems,
Weiterstadt, Germany). Primer and probe sequences are
available upon request. mRNA quantity was calculated using the
 CT method (PE Applied Biosystems User Bulletin #2; ABI PRISM 7700 Sequence Detection System, 1997). We studied the changes in transcript levels of proinflammatory
genes known to be sensitive to extracellular stimuli in whole blood
after phlebotomy. Figure 1 shows the
changes in levels of IL-1 , IL-6, and TNF transcripts up to 7 days. Levels of IL-6 and TNF transcripts increased over the entire
observation period, reaching maximum levels of about 20-fold higher
than levels measured immediately after blood collection. IL-1
transcripts showed a different pattern. After 6 hours a decrease in
expression was observed, followed by an increase after 1 and 3 days.
Seven days after phlebotomy, IL-1 mRNA levels again decreased to
below the original level. Since changes in mRNA levels after blood
collection affect accurate measurement of gene expression, we tested 2 methods to stabilize RNA after phlebotomy. First we tried addition of a
mixture of acidic phenol and guanidine isothiocyanate to blood immediately after collection. Then we used the PAXgene blood RNA tube
(PreAnalytix, Hombrechtikon, Switzerland), a blood collection tube containing an additive that stabilizes cellular RNA. RNA was
prepared at different time points from these stabilized samples and
compared with whole blood samples collected in conventional EDTA tubes.
Both stabilization methods preserved the RNA of all 3 cytokines for up
to 7 days. No significant changes in mRNA levels compared to levels at
phlebotomy were observed.
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| Figure 1.
Induction of proinflammatory cytokines in whole blood.
Fresh blood samples were incubated at ambient temperature for up to 7 days. Levels of TNF ( ), IL-1 ( ), and IL-6 ( ) mRNA were
determined using real-time RT-PCR. Data are expressed as arbitrary
units normalized to -actin to correct for RNA quantity and
integrity. RNA levels at the time of phlebotomy were used as a
reference and set to 1. Data points are mean ± SE (mean of 10 donors). *P < .05, **P < .01 compared with
mRNA levels at phlebotomy (day 0).
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These results highlight the need for standardization and stabilization
of patient samples after phlebotomy. In blood, cells are
exposed to a variety of extracellular agents, which may influence their
activation status. The change of environment after phlebotomy and the
exposure to other surfaces during and after blood collection is likely
to affect gene expression. Both methods tested for stabilization of RNA
worked equally well in preserving the transcript levels of the genes
analyzed. However, immediate addition of a toxic solution to blood is
impracticable in the clinic. The use of a collection device containing
stabilizing reagents, which preserves gene expression profiles at the
time of phlebotomy, is preferable. Studies using gene expression profiling of clinical samples must be
aware of these events and implement routines to standardize and
stabilize clinical samples before RNA isolation and analysis. Neglecting nucleic acid stabilization may lead to artifactual results
in gene expression profiling.
Andreas Pahl and Kay Brune
Correspondence: Andreas Pahl, Institute of Pharmacology,
University of Erlangen, Fahrstr 17, D-91054 Erlangen, Germany; e-mail:
pahl{at}pharmakologie.uni-erlangen.de
Acknowledgments
This work was supported by the Bundesministerium für
Bildung und Forschung (BMBF) (grant no. 312242).
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