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Clinical Chemistry 49: 1458-1466, 2003; 10.1373/49.9.1458
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(Clinical Chemistry. 2003;49:1458-1466.)
© 2003 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Numeric Definition of the Clinical Performance of the Nested Reverse Transcription-PCR for Detection of Hematogenous Epithelial Cells and Correction for Specific mRNA of Non-Target Cell Origin as Evaluated for Prostate Cancer Cells

Denis Schamhart1,a, Johannes Swinnen2, Karl-Heinz Kurth1, Alex Westerhof1, Ron Kusters3, Holger Borchers4 and Cora Sternberg5

1 Department of Urology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

2 LEGENDO, University of Leuven, B-3000 Leuven, Belgium.

3 Hieronymus Bosch Hospital (Jeroen Bosch Ziekenhuis), 5211 NL Den Bosch, The Netherlands.

4 Department of Urology, University of Aachen, D-52057 Aachen, Germany.

5 Foundation Vincenzo Pansadoro, 00165 Rome, Italy.

aAddress correspondence to this author at: Department of Urology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Fax 31-20-6919647; e-mail d.h.schamhart{at}amc.uva.nl.

Background: Inappropriate quality management of reverse transcription-PCR (RT-PCR) assays for the detection of blood-borne prostate cancer (PCa) cells hampers clinical conclusions. Improvement of the RT-PCR methodology for prostate-specific antigen (PSA) mRNA should focus on an appropriate numeric definition of the performance of the assay and correction for PSA mRNA that is not associated with PCa cells.

Methods and Results: Repeated (RT-)PCR tests for PSA mRNA in single blood specimens from PCa patients and PCa-free controls, performed by four international institutions, showed a large percentage ({approx}50%) of divergent test results. The best estimates of the mean, {lambda} (SD), of the expected Poisson frequency distributions of the number of positive tests among five replicate assays of samples from PCa-free individuals were 1.0 (0.2) for 2 x 35 PCR cycles and 0.2 (0.1) for 2 x 25 PCR cycles. Assessment of the numeric value of the mean can be considered as a new indicator of the performance of a RT-PCR assay for PSA mRNA under clinical conditions. Moreover, it determines the required number of positive test repetitions to differentiate between true and false positives for circulating prostate cells. At a predefined diagnostic specificity of >=98%, repeated PCRs with {lambda} of either 1.0 or 0.2 require, respectively, more than three or more than one positive tests to support the conclusion that PSA mRNA-containing cells are present.

Conclusions: Repeated nested PCR tests for PSA and appropriate handling of the data allow numeric quantification of the performance of the assay and differentiation between analytical false and true positives at a predefined accuracy. This new approach may contribute to introduction of PSA RT-PCR assays in clinical practice.




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