Donor origin of leukemia demonstrated by 2 different molecular methods. (A) Fluorescence in situ hybridization examination of the bone marrow demonstrating 85% of the cells to exhibit a XX chromosomal pattern (2 red signals per nucleus), all of them were morphologic with immature aspect, suggesting the leukemic cells of donor origin. Note the few residual cells of the recipient with XY chromosomal pattern (1 red and 1 green signal per nucleus). FISH was performed using the directly labeled, dual-color (red/green) X- and Y-chromosome probe mix (Xp11.1-q11.1/Yq12) Z-2016 (ZytoVision, Bremerhaven, Germany). The sections were processed with a paraffin pretreatment reagent kit (Abbott/Vysis, Baar, Switzerland), and hybridization was performed according to the manufacturer’s specifications. Denaturation was conducted for 10 minutes at 73°C, and the FISH probes were incubated overnight at 37°C in Hybrite (Abott/Vysis). Counterstaining was performed with 4,6-diamidino-2-phenylindole. The FISH signals were visualized on a Olympus BX43 fluorescence microscope equipped with double bandpass filters for simultaneous visualization of green and red signals. (B) Short tandem repeat profile of the 2 representative chromosomal loci vWA and TH01. Left: Recipient bone marrow cells at diagnosis of AML. Middle: Recipient buccal cells. Right: Donor white blood cells, demonstrating mixed chimerism and donor origin of cells. Taking all 8 discriminative chromosomal loci and the Amelogenin system into calculation, a donor chimerism of 86% can be demonstrated. This is in perfect agreement with the >80% blast infiltration of the marrow and 85% of cells exhibiting an XX chromosomal pattern.