Enhanced assessment of allogeneic bone marrow transplant engraftment usingautomated fluorescent-based typing

Traditional qualitative gel electrophoresis approaches lack accurate and qu antitative assessment of mixed chimerism in BMT patients. The likelihood of informative markers is greatly increased using simultaneous amplification of 10 highly polymorphic loci with fluorescent-labeled primers in an automa ted DNA sequencer. This allows for more precise interpretation of mixed chi merism with a detection level approximating 1%, To evaluate this approach t o quantitative assessment of chimeric populations we mixed varying proporti ons of samples from two unrelated donors, by either mixing aliquots of DNA isolated from whole blood, or by first counting the white blood cells and m ixing varying proportions of cells together prior to DNA isolation. The all elic-peak area ratios were identical to allelic-peak height ratios and corr esponded to the proportion of mixed DNA, regardless of the method used to c reate the mixture. Formulas to provide routine, consistent and quantitative interpretation of mixed chimerism are presented, We analyzed 14 allograft recipients and one autologous BMT patient with transfusion-induced GVHD, In all cases, at least four out of nine markers were informative. Inter-labor atory concordance of results was also obtained with an eight marker panel u sing an automated Alf-Express. In conclusion, the automated DNA fluorescent -labeled primer approach using an eight to 10 marker panel is quantitative and informative in assessing chimerism.