Method Description

Genomic DNA is extracted from blood or bone marrow aspirate samples using an automated extraction machine and used in a commercial kit GlobalFiler PCR Amplification KIT following the manufacturer's instructions. Briefly, 20 different short tandem repeat (STR) marker regions are amplified in single multiplex polymerase chain reaction (PCR) using primers labeled with fluorescent tags. The products are analyzed for size and amount using capillary electrophoresis on a genetic analyzer. For the initial sample on any patient, the test is performed on 3 separate DNA samples: donor germline DNA, recipient germline DNA, and recipient posttransplant sample for chimerism determination. The STR profile of the germline samples is used to identify markers that can distinguish between the donor and recipient. Based on these profiles, the percentage of donor and recipient DNA is then determined in the post-transplant sample using the assumptions and calculations outlined.(Thiede C, Florek M, Bornhauser M, et al: Rapid of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection. Bone Marrow Transplant. 1999 May;23[10]:1055-1060). Subsequent samples for chimerism evaluation do not need to be accompanied by samples for donor and recipient germline evaluation, as the profiles from the initial testing are kept on file for comparison.(Package insert: GlobalFiler PCR Amplification Kit. User Guide. Applied Biosystems, 2019; Thiede C, Florek M, Bornhauser M, et al: Rapid quantitation of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection. Bone Marrow Transplant. 1999 May;23[10]:1055-1060)

The sensitivity of this analysis is approximately 5% in a posttransplant specimen (donor and recipient DNA mixed chimerism).