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Purpose: Deformable dose accumulation (DDA) has uncertainties which impede the implementation of DDA-based adaptive radiotherapy (ART) in clinic. The purpose of this study is to develop a multi-layer quality assurance (MLQA) program to evaluate uncertainties in DDA. Methods: A computer program is developed to generate a pseudo-inverse displacement vector field (DVF) for each deformable image registration (DIR) performed in Accuray’s PreciseART. The pseudo-inverse DVF is first used to calculate a pseudo-inverse consistency error (PICE) and then implemented in an energy and mass congruent mapping (EMCM) method to reconstruct a deformed dose. The PICE is taken as a metric to estimate DIR uncertainties. A pseudo-inverse dose agreement rate (PIDAR) is used to evaluate the consequence of the DIR uncertainties in DDA and the principle of energy conservation is used to validate the integrity of dose mappings. The developed MLQA program was tested using the data collected from five representative cancer patients treated with tomotherapy. Results: DIRs were performed in PreciseART to generate primary DVFs for the five patients. The fidelity index and PICE of these DVFs on average are equal to 0.028 mm and 0.169 mm, respectively.With the criteria of 3 mm/3% and 5 mm/5%,the PIDARs of the PreciseART-reconstructed doses are 73.9 ± 4.4% and 87.2 ± 3.3%, respectively. The PreciseART and EMCM-based dose reconstructions have their deposited energy changed by 5.6 ± 3.9% and 2.6 ± 1.5% in five GTVs, and by 9.2 ± 7.8% and 4.7 ± 3.6% in 30 OARs, respectively. Conclusions: A pseudo-inverse map-based EMCM program has been developed to evaluate DIR and dose mapping uncertainties. This program could also be used as a sanity check tool for DDA-based ART. [ABSTRACT FROM AUTHOR]