Inhibition of the PCR by genomic DNA.

Aims: qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6.

Sensitive Measurement of Minimal Residual Disease in Blood by HAT-PCR.

PCR is widely used to measure minimal residual disease (MRD) in lymphoid neoplasms, but its sensitivity is limited. High Adenine/Thymine PCR and High Annealing Temperature PCR (HAT-PCR) is a modified PCR designed to minimize nonspecificity and hence increase sensitivity. It was evaluated in the laboratory and the clinic, using samples from 58 patients.

Patient-Specific Minimal Residual Disease Primers Amplify with Uniformly High Efficiency.

The widespread use of PCR to quantify minimal residual disease has been hampered by the apparently wide variation in amplification efficiency (AE) of PCR primers. A new method to measure AE was developed on the basis of the Ct results of PCR amplification of single copies of a target molecule placed by limiting dilution into wells of a microplate. The mean one copy Ct of a population of primers or of a reference primer was calibrated against the AE determined by the standard method of regression analysis.

Sources of error in measurement of minimal residual disease in childhood acute lymphoblastic leukemia.

Introduction: The level of minimal residual disease (MRD) in marrow predicts outcome and guides treatment in childhood acute lymphoblastic leukemia (ALL) but accurate prediction depends on accurate measurement.

Methods: Forty-one children with ALL were studied at the end of induction. Two samples were obtained from each iliac spine and each sample was assayed twice.

BCR-ABL1 expression, RT-qPCR and treatment decisions in chronic myeloid leukaemia.

Aims: RT-qPCR is used to quantify minimal residual disease (MRD) in chronic myeloid leukaemia (CML) in order to make decisions on treatment, but its results depend on the level of BCR-ABL1 expression as well as leukaemic cell number. The aims of the study were to quantify inter-individual differences in expression level, to determine the relationship between expression level and response to treatment, and to investigate the effect of expression level on interpretation of the RT-qPCR result.

Methods: BCR-ABL1 expression was studied in 248 samples from 65 patients with CML by determining the difference between MRD quantified by RT-qPCR and DNA-qPCR.

A DNA real-time quantitative PCR method suitable for routine monitoring of low levels of minimal residual disease in chronic myeloid leukemia.

The BCR-ABL1 sequence has advantages over the BCR-ABL1 transcript as a molecular marker in chronic myeloid leukemia and has been used in research studies. We developed a DNA real-time quantitative PCR (qPCR) method for quantification of BCR-ABL1 sequences, which is also potentially suitable for routine use. The BCR-ABL1 breakpoint was sequenced after isolation by nested short-range PCR of DNA from blood, marrow, and cells on slides, obtained either at diagnosis or during treatment, or from artificial mixtures.

Rapid isolation of translocation breakpoints in chronic myeloid and acute promyelocytic leukaemia.

Isolation and sequencing of the translocation breakpoint in chronic myeloid leukaemia-(CML) and acute promyelocytic leukaemia (APML) may help to elucidate the mechanism of translocation and provide a molecular marker for monitoring of minimal residual disease. Amplification across the translocation breakpoint was performed in samples from 91 patients with CML and 15 patients with APML using single-tube multiplex polymerase chain reaction (PCR) involving 308 primers for CML and 40 primers for APML. Nonspecific amplification was removed by a modification of PCR, termed sequential hybrid primer PCR (SHP-PCR), which involved two sequential rounds of PCR, each of which used a low concentration of a specially designed hybrid primer.

Determining the repertoire of IGH gene rearrangements to develop molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia.

Molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia were identified by determining, at the time of diagnosis, the repertoire of rearrangements of the immunoglobulin heavy chain (IGH) gene using segment-specific variable (V), diversity (D), and junctional (J) primers in two different studies that involved a total study population of 75 children and 18 adults. This strategy, termed repertoire analysis, was compared with the conventional strategy of identifying markers using family-specific V, D, and J primers for a variety of antigen receptor genes. Repertoire analysis detected significantly more markers for the major leukemic clone than did the conventional strategy, and one or more IgH rearrangements that were suitable for monitoring the major clone were detected in 96% of children and 94% of adults.

Sensitive and specific measurement of minimal residual disease in acute lymphoblastic leukemia.

A sensitive and specific quantitative real-time polymerase chain reaction method, involving three rounds of amplification with two allele-specific oligonucleotide primers directed against an rearrangement, was developed to quantify minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL). For a single sample containing 10 microg of good quality DNA, MRD was quantifiable down to approximately 10(-6), which is at least 1 log more sensitive than current methods. Nonspecific amplification was rarely observed.