Quantitative RT-PCR: how well do labs do?
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Since quantitative RT-PCR is an important element in monitoring imatinib
therapy, a survey was done to compare the performance of this test
across laboratories as well as to gather information on how different
laboratories do the test and on their reporting strategies.
“Using the Association for Molecular Pathology listserve,
we recruited laboratories offering quantitative RT-PCR for the BCR-ABL
translocation as a clinical test,” reports Karen Mann, MD,
PhD, director of molecular hematopathology at Emory University Medical
Twelve laboratories participated: four reference laboratories and
eight associated with academic medical centers. Eight were performing
the test clinically, while four were in the validation phase. “The
longest anyone was doing it was three years,” Dr. Mann says,
“so clearly this test is still new.”
Controls included two cell lines positive for the translocation
(used at several dilutions) and one that was negative. Nine patient
samples were used. RNA was extracted in Dr. Mann’s laboratory
and the extracts sent.
“Everyone got the negative samples correct, and all laboratories
appropriately detected the translocation in the most concentrated
positive samples,” Dr. Mann reports. Some participating laboratories
used Taqman chemistry on the ABI Prism and others used the Roche
LightCycler. “Both worked and gave equivalent sensitivity,”
Dr. Mann says.
On the negative side, it was difficult to compare results among
laboratories because of different reporting practices. “If
I had CML and had one sample run at one laboratory, then a later
sample at another laboratory, it would be impossible to compare
the two,” Dr. Mann says. “This is not a surprise, but
it is something that needs to be addressed. Some degree of national
consensus is important for good patient care.”
Also, some laboratories did not reliably detect the translocation
in the most dilute samples—one per 100,000 or one per million.
However, all had much lower than a one percent limit of detection,
and the best laboratories were routinely detecting down to one in
100,000. “RT-PCR is still the most sensitive test,”
Dr. Mann concludes.
Results from this survey, and other information, will be used to
develop reporting guidelines.
“Barring any new development in CML testing,” Dr. Mann
says, “I would imagine that any medical center that has molecular
hematopathology testing would want to do this test.” Driving
this is the increasing use of imatinib. “Previously, minimal
residual disease testing was used primarily post-bone marrow transplant,”
she notes. “Now it is less restricted.”