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Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin

Leukemia volume 17, pages 268–270 (2003)Cite this article

TO THE EDITOR

Real-time quantitative PCR (RQ-PCR) of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements is widely employed as a method for the detection of minimal residual disease (MRD) in the bone marrow (BM) or peripheral blood (PB) of children with acute lymphoblastic leukemia (ALL). The main advantages of RQ-PCR over other methodologies for the detection of MRD are its ability to reproducibly and sensitively quantify the level of MRD. Accuracy of this quantification is entirely dependent upon knowledge of the quantity and amplifiability of target DNA. The amount of DNA added is initially assessed by spectrophotometry, and subsequently checked by use of a suitable control PCR, which also assesses the quality (amplifiability) of the DNA. In the European pre-BMT MRD consortium and I-BFM-SG MRD Task Force, 500 ng of target DNA is subjected to an albumin gene control RQ-PCR. In the majority of samples, the albumin gene RQ-PCR indicates that amplifiable DNA is present within an acceptable experimental range (250–1000 ng). However, upon examination of 316 samples from 15 patients, we found that the albumin RQ-PCR gave discrepant quantification compared to spectrophotometry (RQ-PCR value <50% of that suggested by spectrophotometry) in 18% of samples. Agarose gel-based analysis of DNA integrity in these samples confirmed that the DNA was not degraded. Therefore, the most likely explanation for this phenomenon was the presence of PCR inhibitors. As such inhibitors may hamper quantitative MRD analysis, we investigated this in more detail.

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Authors and Affiliations

  1. Department of Paediatric Oncology, Bristol Royal Hospital for Children, Bristol, UK

    J Moppett, J Hancock & N Goulden

  2. Department of Immunology, Erasmus University Medical Center Rotterdam, The Netherlands

    V H J van der Velden, A J M Wijkhuijs & J J M van Dongen

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  1. J Moppett
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  2. V H J van der Velden
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  3. A J M Wijkhuijs
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  4. J Hancock
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  5. J J M van Dongen
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  6. N Goulden
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Moppett, J., van der Velden, V., Wijkhuijs, A. et al. Inhibition affecting RQ-PCR-based assessment of minimal residual disease in acute lymphoblastic leukemia: reversal by addition of bovine serum albumin. Leukemia 17, 268–270 (2003). https://doi.org/10.1038/sj.leu.2402751

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