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

Traditional qualitative gel electrophoresis approaches lack accurate and quantitative 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 automated DNA sequencer. This allows for more precise interpretation of mixed chimerism with a detection level approximating 1%, To evaluate this approach to quantitative assessment of chimeric populations we mixed varying proportions 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 mixing varying proportions of cells together prior to DNA isolation. The allelic-peak area ratios were identical to allelic-peak height ratios and corresponded to the proportion of mixed DNA, regardless of the method used to create 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-laboratory concordance of results was also obtained with an eight marker panel using 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.