A non-isotopic nested polymerase chain reaction method to quantitate minimal residual disease in patients with non-Hodgkin's lymphoma

The polymerase chain reaction (PCR) is a highly sensitive technique for the detection of lymphoma cells presenting specific rearrangements or translocations, like the t(14;18). However, few methods are available for the quantitative evaluation of clinical samples, especially using nested PCR. In this study, we describe a method for the semiquantitative estimation of low numbers of lymphoma cells using the nested PCR methodology. Samples evaluated consisted of RL cells, a non-Hodgkin's lymphoma cell line harbouring a bcl-2 translocation, mixed with normal mononuclear cells (MC) obtained from bone marrow or peripheral blood. DNA was extracted from samples with RL:MC ratios ranging from 5 x 10(-2) to 5 x 10(-7), and amplified for detection of this translocation. The product of this first amplification reaction was serially diluted 10-fold from 10(0) to 10(-7). Then each fraction was PCR-amplified with nested oligonucleotide primers. Aliquots of the second amplification were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Within the range tested, there was a log-linear relationship between the number of PCR positive bands and the number of translocated cells in each sample. This procedure was highly reproducible in experiments evaluating the interrun and intrarun variability. In addition, there was no interaction with normal cells obtained from peripheral blood or bone marrow, or from different individuals. Furthermore, results were identical with two different cell lines and consistent with patient lymphoma cells obtained from a lymph node biopsy.(ABSTRACT TRUNCATED AT 250 WORDS)