Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats (Article)

2011-09-01T00:00:00Z

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30 000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for small-scale DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.

Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: A European multicenter study (Article)

2009-03-27T00:00:00Z

A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within <0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E+04 Abelson (ABL) expression was 3.5E+03 compared with that of 2.3E+01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.

Kreyenberg, H.

Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats (Article)

Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: A European multicenter study (Article)