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

Abstract

Summary: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.

Quantitative analysis of chimerism after allogeneic bone marrow transplantation using immunomagnetic selection and fluorescent microsatellite PCR

“…In contrast to Pindolia et al, 19 we have found that the presence of stutter peaks causes serious problems with both the interpretation and quantitation of chimerism. As discussed earlier, our selection criteria exclude the use of markers where donor and recipient alleles are separated by just one repeat unit.…”

Long‐term persistence of host cells detected by X‐chromosome gene‐based assay in patients undergoing gender‐mismatched hematopoietic stem cell transplantation

“…Several methods for evaluating chimerism have been developed, including cytogenetic and molecular techniques. The most widely used technique for evaluating chimerism is polymerase chain reaction (PCR) amplification of short tandem repeats (STR) [4][5][6][7][8][9][10]. However, the sensitivity of this method is only approximately 1-5%, even when fluorescent-labeled primers and an automated DNA sequencer are used [4][5][6].…”

Detection of Low Level Mixed Chimerism Using High Throughput SNP Genotyping

“…Clinical follow up of the allograft recipients requires long-term monitoring of either the malignancyspecific markers or the residual hematopoiesis of the recipient cells (mixed chimerism, MC). Minimal residual disease (MRD [1,2]) monitoring involves detection and quantification of the malignancyspecific markers, while measuring the extent of mixed chimerism [3] requires measuring the fraction of recipient cells in peripheral blood samples. MRD approaches require monitoring the malignant clone via molecular (PCR-based) or immunophenotypic methods [4].…”