Multiplex amplification and fluorimetric detection of short tandem repeats for mixed chimerism after bone marrow...

We present the clinical application of a simple method for mixed chimerism analysis after allogeneic bone marrow (BM) or peripheral blood stem cell (PBSC) transplant. A commercial kit which enables multiplex amplification of 9 highly polymorphic short tandem repeats in a single reaction tube was used. Molecular size and relative quantities of each amplified fragment are subsequently measured using an automated fluorimeter. The sensitivity and linearity were tested using whole blood or genomic DNA from two subjects, mixed in various proportions from 0.25% to 99.75%. In 70 donor-recipient pairs the median number of informative alleles for the assessment of relapse was 5.9 (range 3-11). Results showed that the linearity between the measured and expected relative quantities of amplified DNA showed a regression coefficient of 0.99 in the interval 10-90%. The mean sensitivity was 1.5% (range 0.5-2.5), greater than previously reported. A total of 70 cases of allogeneic transplant (49 with family and 21 with unrelated donors) were monitored before transplant and after 1, 2, 4, 6 or 12 months thereafter and then at 6 months intervals (range 6-36). In 18 patients mixed chimerism was observed 1 month from transplant, with donor allele percentage ranging from 1 to 6%. Fragment dimension reproducibility (CV 0.34, range 0.52-0.66) was confirmed by an internal DNA control and by amelogenin fragment length repeatability on all patients. In conclusion, the proposed method is sufficiently simple, rapid, sensible, specific and cost effective for the evaluation of mixed chimerism after BM or PBSC transplant in a clinical setting.