**2.2 Methods**

The PCR-based STR (PCR-STR) DNA markers were amplified using fluorescently labelled allele-specific primers, and different amplicons were separated by capillary electrophoresis on Genetic analyser. The direct quantification of donor and recipient PCR-STR DNA markers was provided by fragment analysis (FA) of GeneMapper software (Applied Biosystems) [20, 24–28].

## **2.3 Sample collection**

Whole peripheral blood samples were collected for DNA extraction from both the donor and recipient before transplantation in order to determine an informative STR marker. The samples were collected at weekly or monthly intervals during the first 100 days, and monthly or every 2–3 months thereafter during the first year according to the transplantation centre. During the second year, the frequency was reduced to twice a year, only if the clinical situation warranted, more frequent chimerism analyses were performed.

## **2.4 DNA extraction**

Genomic DNA was extracted from 200 μl of fresh or frozen peripheral blood using a column-based DNA isolation technique (Qiagene DNA Blood mini kit, QIAGEN, Hilden, Germany) according to the manufacturer's instructions. DNA quantification was performed using standard UV absorption at 260 nm, and DNA samples were stored until use at −80°C. To obtain an informative STR system for chimerism analysis, we performed a donor/recipient genotyping using a commercially available STR multiplex amplification kit PowerPlex 16 (Promega, Madison, WI, USA) that contains tetranucleotide STR markers, e.g. D18S51, D21S11, TH01 and D3S1358, as well as pentanucleotide STRs Penta E and Penta D and the primers specific for the Amelogenin locus [27, 28].

#### **2.5 PCR amplification**

About 10–50 ng of genomic DNA and BioThermStar DNA polymerase (GeneCraft, Lüdinghausen, Germany) instead of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) in some reactions. The denaturation,

annealing and extension cycles were programmed in Techgene thermal cycler (Techne Inc., Burlington, NJ, USA) as follows: preincubation 95°C for 10 min, 96°C for 1 min, 10 cycles with 96°C/30 s, 60°C/30 s, 70°C/45 s and 14 and 18 cycles with 96°C/30 s, 60°C/30 s, 70°C/45 s for monoplex and multiplex kits, respectively, and final elongation step performed at 60°C for 30 min.
