Journal
CLINICAL CHEMISTRY AND LABORATORY MEDICINE
Volume 50, Issue 2, Pages 301-310Publisher
WALTER DE GRUYTER GMBH
DOI: 10.1515/CCLM.2011.762
Keywords
acute myeloid leukemia; agarose gel electrophoresis; capillary electrophoresis; fms-like tyrosine kinase 3 (FLT3); internal tandem duplication (ITD); polyacrylamide gel electrophoresis
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Funding
- TAMOP [4.2.1/B-09/1/KONV-2010-0007]
- European Social Fund
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Background: In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. Methods: A complex evaluation of the analytical properties of the three most frequently used detection methods - PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) - was performed on 95 DNA samples obtained from 73 AML patients. Results: All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%-2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes. Conclusions: This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.
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