Myeloid Leukemia

52 Picard, Silvy, and Gabert
sition and processing, especially as multicenter protocols are currently being
developed.
Nucleic acid amplification techniques are prone to variation in results,
because the sensitivity of these assays makes them very susceptible to small
changes in assay conditions, leading to false-positive or false-negative results.
Standardization and validation of biological assays are necessary before routine
use in order to control inter- and intra-laboratory variations in results, to
reduce errors, and to detect critical loss of assay sensitivity.
The reference material should resemble, as closely as possible, the test material,
which in this case is cellular material from patients undergoing therapy
or not. Thus, the use of cellular material as a reference material would be preferable
to cDNA or plasmid, as this would allow control of the extraction step,
which is probably the most variable part of a PCR assay.
For example, reference reagents and World Health Organization (WHO)
international standards have been established for nucleic acid amplification
assays for the detection of several viruses in human plasma. These reagents
consist of virus diluted in pooled plasma, and closely resemble the samples routinely
tested by blood banks and blood product manufacturers. These reagents
are lyophilized for greater long-term stability and ease of transport (35–37).
Collaborative work between the National Institute for Biological Controls
and Standards (NIBSC; http//:www.nibsc.ac.uk) and a study group of Professor
Gabert has been performed to develop international standards for real-time
BCR-ABL transcript PCR dosage using lyophilized cells. This procedure permits
the control of the extraction phase, the reverse transcription, and the RQ-
PCR. In 2004, a reference value was established for BCR-ABL expression in a
lyophilized K562 cell line. Experiments in accelerated aging of lyophilized
cells have shown that the material, transported at room temperature, is stable
for 3 yr for RQ-PCR application. A French patent was obtained by the University
of the Méditerranée and the NIBSC in March 2003, and a European Patent
Cooperation Treaty (PCT) extension was applied for a year later.
Fusion gene-positive cell line RNA, or negative cell line RNA spiked with
in vitro synthesized fusion gene, RNA, or plasmid DNA (EAC plasmid,
Ipsogen, France) can be used as controls for cDNA synthesis and most importantly
for amplification using the TaqMan primer sets. In the same way,
Roboscreen has created a standard, the “intelligent reaction tube.” In this
system, common glass capillaries or plastic tubes required for either standardized
nucleic acid purification or quantification are precoated with defined
amounts of distinct control DNA or RNA, respectively (38). However, these
controls suffer from the limitation that they do not reliably evaluate sample
handling and RNA extraction steps, which may also have an important bearing
on assay sensitivity. Another method of RNA conservation is the use of