Application of a chemiluminescent methodology for detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Analysis of minimal residual disease (MRD) can predict outcome in childhood acute lymphoblastic leukemia (ALL). We applied a chemiluminescent methodology in 20 children with ALL. We detected MRD at different time-points throughout the follow-up of our patients, concluding that chemilumi-nescent detection of MRD is a reliable, safe and sensitive method. Recent prospective studies clearly demonstrate the prognos-tic value of MRD in children with ALL. 1,2 Several methodologies are available for MRD analyses in ALL patients. 1-5 In order to test a safe, sensitive and reliable method for those laboratories in which neither radioactive analysis nor the TaqMan strategy can be carried out, 3 we present here a report on application of digox-igenin (DIG)-labeled patient-specific probes for a chemilumi-nescent detection of MRD in children with ALL. Twenty children with ALL diagnosed at our institution and treated according to the ongoing protocol of the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP-ALL 95) were included in this study. The childrens' characteristics are listed in Table 1. We collected samples at 5 time-points (TP): after 43 days (TP1), after three months (TP2), five (TP3), seven (TP4) and 24 months (TP5) of therapy. T-cell receptor (TCR) γ and TCR δ gene rearrangements were identified and characterized by performing diagnostic polymerase chain reactions (PCR), het-eroduplex and sequencing analyses using standardized techniques. 6,7 PCR of follow-up were performed using different protocols in order to amplify TCRγ and TCRδ rearrangements. 7 Seven microliters of the products were spotted onto positively charged nylon membranes (NYTRAN N+, Roche Boehringer). Next, 200 pmol of each oligonucleotide were 3'-end labeled with DIG-ddUTP using a DIG oligonucleotide 3'-end labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) according to manufacturer's instructions. The membranes were prehy-bridized at 68°C for 2 hours using 25 mL hybridization solution (Roche Molecular Biochemicals) and then hybridized in a sealed plastic bag with 3 mL hybridization solution and 20 µL of the labeling probe (200 pmol). The membranes were incubated overnight at 54°C and then washed twice in 2 × SSC, 0.1% SDS at room temperature for 5 min. The membranes were soaked with blocking solution (Roche Molecular Biochemicals) for 30 min at room temperature. Twenty-four milliliters of blocking solution containing 2.4 µL of anti-DIG-alkaline phosphatase Fab fragments (Roche Molecular Biochemicals) were added, followed by incubation for 30 min. Finally, the membranes were washed twice with washing buffer (Roche Molecular Biochemicals) for 15 min and incubated with detection buffer (Roche …