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Quantitative RT-PCR: how well do labs do?

May 2003
William Check, PhD

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 Center.



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.”