Klin Padiatr 2004; 216 - 33
DOI: 10.1055/s-2004-828578

Combined use of 4-color flow cytometry and real-time PCR to detect minimal residual disease (MRD) in childhood acute lymphoblastic leukemia

G Kerst 1, H Kreyenberg 1, E Koscielniak 2, G Dückers 1, C Niemeyer 3, I Müller 1, K Schilbach 1, R Handgretinger 4, PG Schlegel 5, D Niethammer 1, P Bader 1
  • 1University Children's Hospital Tübingen, Germany
  • 2Olgahospital Stuttgart, Germany
  • 3University Children's Hospital Freiburg, Germany
  • 4St. Jude Children's Research Hospital, Memphis, USA
  • 5University Children's Hospital Würzburg, Germany

Introduction: MRD detection in ALL patients is commonly performed by PCR amplification of clone-specific immunoglobulin and T-cell receptor gene rearrangements. Despite its high sensitivity, PCR amplification carries the risk of false-negative results due to clonal evolution and can not yet be applied to all patients. Therefore, the additional use of a second method seems feasible.

Methods: Flow cytometric analysis of MRD was performed according to the protocols invented by E. Coustan-Smith and D. Campana (St. Jude Children's Research Hospital, Memphis, TN). Real-time PCR was performed by the use of Taqman® technology. Both techniques can detect 1 leukemic cell among 10000 or more normal bone marrow mononuclear cells.

Results: Targets for PCR amplification were identified in 85% (28 out of 33) of diagnostic samples. Leukemia-specific immunophenotypes were detected in 90% (38 out of 42) of cases. Either method allowed MRD detection in those cases that could not be followed by the other method alone. Both methods quantified MRD accurately (flow cytometry: r=0.999, n=26, PCR: r=0.953, n=17) and correlated well (r=0,924, n=35).

Conclusion: These results indicate that the combined use of 4-color flow cytometry and real-time PCR might be able to detect MRD in virtually all patients. Furthermore, the combined use might prevent false-negative results due to clonal evolution or phenotypic shifts.

This work was supported by the fortüne-Programm of the University of Tübingen (G.K.), by the Deutsche Krebshilfe (P.B.) and the BMBF IZKF IIA (P.B.)