SummaryMolecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.
CorrespondenceFormic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products We recently reported a new method for the extraction of DNA from paraYn wax embedded bone marrow trephine biopsies. 1 The DNA extracted from EDTA decalcified bone marrow trephine biopsies using this method was suYciently intact to allow the amplification and sequencing of relatively long polymerase chain reaction (PCR) products, including the 600 bp t(11;14) MTCA PCR product. A shorter 294 bp PCR product could only be amplified from six of 10 formic acid decalcified bone marrow trephine biopsies reported in a previous study by Provan et al. 2 These findings suggested a correlation between DNA degradation and formic acid decalcification, but required a comparative study for confirmation.We have subsequently extracted DNA from 11 formic acid decalcified bone marrow trephine biopsies using our method and determined the quality of DNA using agarose gel electrophoresis and PCR analysis, as in our initial study.The mean DNA yield from the formic acid decalcified blocks was twice that of the EDTA decalcified samples: 9.4 µg and 4.3 µg, respectively. This reflected the fact that the formic acid blocks contained approximately twice as much bone marrow trephine biopsy material as a result of the diVerences in practice between the two centres involved in the study (Exeter, EDTA decalcification; Southampton, formic acid decalcification). However, when the formic acid decalcified DNA samples were analysed by agarose gel electrophoresis, no high molecular weight DNA was detected; only a smear of degraded DNA was seen. In contrast, analysis of the EDTA decalcified bone marrow trephine biopsy DNA samples showed DNA ranging from 5 to 21 kb in length (fig 1).
Aims-To establish a robust method of extracting DNA from paraYn wax embedded bone marrow trephine (PBMT) biopsies for the amplification of relatively long polymerase chain reaction (PCR) products. Method-Xylene and ethanol were used to remove paraYn wax from eight formalin fixed, EDTA decalcified PBMT biopsies and DNA extraction was performed using a Qiagen QIAamp tissue kit. The DNA samples were amplified using nine diVerent PCR primers sets, including those used to detect chromosomal translocations t(11;14) and t(14;18), and clonal B cell populations. A t(11;14) PCR product of approximately 600 base pairs (bp) was sequenced using dye terminator cycle sequencing. Results-All eight DNA samples extracted from PBMT biopsies were amplified successfully to generate DNA fragments up to 643 bp in length. Chromosomal translocations and immunoglobulin gene rearrangements were detected by PCR in some of the samples. Sequencing of the t(11;14) PCR product demonstrated the presence of chimaeric sequences, which included both bcl-1 and immunoglobulin heavy chain (IgH) gene sequences, consistent with the presence of this translocation. Conclusions-This method enables PCR analyses of PBMT biopsies that were not previously possible, oVering the prospect of improved accuracy of diagnosis and the monitoring of patients with bone marrow disease.
Aims-To detect clonal T cell populations by high resolution polymerase chain reaction (PCR) using fluorescently labelled nucleotides and analysis on an ABI 377 DNA sequencer, and to evaluate this method using low ionic strength single strand conformation polymorphism (LIS-SSCP) analysis. Methods-DNA samples from 11 patients diagnosed with a T cell disease and 15 with no known T cell disorder were amplified using four multiplex T cell receptor (TCR ) PCR reactions containing fluorescently labelled nucleotides. PCR products were analysed using both LIS-SSCP electrophoresis and an ABI 377 DNA sequencer using Genescan TM software. A Jurkat T cell leukaemia cell line was used to determine the sensitivity of the two methods. Results-Clonal TCR populations were detected in all 11 samples from patients with a T cell disease and no clonal populations were detected in samples from patients without a T cell disorder, using both LIS-SSCP and DNA sequencer analysis. Although the sensitivity of the two methods was comparable, the data generated by the sequencer were easier to interpret than the LIS-SSCP gels, and allowed accurate size determination of every product, which was not possible using LIS-SSCP. Conclusions-The use of fluorescent labelled nucleotides provides a more flexible and economical alternative to end labelled fluorescent primers for the detection of clonal TCR gene rearrangements. This method allows clonal populations to be sized accurately and reproducibly, permitting the detection of identical clonal populations in diVerent samples, and providing a method of monitoring disease progression and response to treatment. (J Clin Pathol: Mol Pathol 2000;53:150-154)
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