ORIGINAL ARTICLE Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats M van der Burg 1 , H Kreyenberg 2 , A Willasch 2 , BH Barendregt 1,3 , S Preuner 4 , F Watzinger 4 , T Lion 4 , E Roosnek 5 , J Harvey 6 , M Alcoceba 7 , MG Dı ´az 7 , P Bader 2 and JJM van Dongen 1 on behalf of the EU-supported EuroChimerism Consortium (project QLRT-2001-01485) 1 Department of Immunology, Erasmus MC, Rotterdam, The Netherlands; 2 Department of Pediatrics, Frankfurt, Germany; 3 Department of Pediatrics, Erasmus MC, Rotterdam, The Netherlands; 4 Children’s Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria; 5 Division of Hematology, Department of Internal Medicine, Geneva University Hospitals and University of Geneva, Switzerland; 6 H & I Laboratory, National Health Service Blood and Transplantation, Filton, Bristol, UK and 7 Department of Hematology, Hospital Clinico Universitario, Salamanca, Spain 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 labora- tories 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 standar- dized DNA isolation method well suited for chimerism studies on low cell numbers. Leukemia (2011) 25, 1467–1470; doi:10.1038/leu.2011.118; published online 17 June 2011 Keywords: chimerism; DNA isolation; short tandem repeats Introduction Stem cell transplantation is the treatment of choice for several subgroups of hematological disorders and for some primary immunodeficiencies. 1,2 To monitor donor engraftment or relapse, the chimeric status of leukocytes after stem cell transplantation can be analyzed. 3–8 Several techniques have been used for this purpose, including cytogenetic analyses 9 and analysis of restriction fragment length polymorphisms. 10 PCR analysis of short tandem repeats (STR), however, is now routinely used, because it is a quantitative, reliable and rapid screening method requiring only small amounts of DNA. 3,5,6,11–13 In some situations, it is necessary to isolate specific leukocyte sub-populations for chimerism analysis. 14,15 Depending on the clinical situation of the patient, the number of isolated cells can be very low. Collection of larger patient samples may enlarge the number of sorted leukocyte sub-populations, 16 but for routine diagnostics, handling of large volumes is not suitable, especially in the treatment of children. Commercial kits are available for isolation of good quality DNA from high numbers of cells (4500 000 cells), 17 but no standardized protocols have been developed for DNA isolation from low cell numbers (o50 000 cells). The objective of this study was to establish a standardized DNA isolation protocol for genetic profiling of low cell numbers by PCR analysis of STR, such as in case of chimerism diagnostics after stem cell transplantation. In Work Package 3 of the EU-supported EuroChimerism project (QLRT-2001-01485), four different DNA isolation techniques were selected and tested for their ability to recover reproducible amounts of good quality DNA from low cell numbers. Subsequently, the sensitivity of the best-performing DNA isolation protocol was determined. The isolated DNA was tested in PowerPlex16 mulitplex and singleplex PCR assays, as well as in PCR assays using the primer sets of the EuroChimerism Consortium (Lion et al., manuscript submitted). Materials and methods Preparation of cell samples For testing the four DNA protocols, DNA was isolated from different cell numbers (that is, 1000, 3000, 10 000, 30 000) of post-Ficoll mononuclear cells from 16 different donors by six laboratories. To obtain the appropriate cell numbers, the mononuclear cells were brought to a concentration of 10  10 6 mononuclear cells per ml of phosphate-buffered saline, subsequently diluted 20-fold to 0.5  10 6 cells/ml. From this suspension, 2, 6, 20 and 60 ml were used. For the donor–recipient dilution series (n ¼ 5), 4 ml of 1  10 5 post-Ficoll mononuclear cells/ml from the donor and 400 ml of 1  10 5 cells/ml from the recipient were used to prepare four donor/recipient ‘master dilutions’ of 1 ml in the ratios of 80:20, 90:10, 97:3 and 99:1. From each master dilution, 10, 30 and 100 ml were taken to obtain three dilution series containing 1000, 3000 and 10 000 cells, respectively. DNA isolation protocol A: direct lysis without proteinase K Cells were spun down in a microcentrifuge tube and the pellet was resuspended in 20 ml of phosphate-buffered saline and covered with 20 ml PCR-grade oil (only needed when Received 18 March 2011; accepted 31 March 2011; published online 17 June 2011 Correspondence: Dr M van der Burg, Department of Immunology, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: m.vanderburg@erasmusmc.nl Leukemia (2011) 25, 1467–1470 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu