Mutational MRD screening of BM CD34⁺ cells. (A-D) ddPCR-based screening for clonal involvement of patient-specific mutations (for which HSPC VAF was >1%) in CD34-enriched cells from remission BM. (A) Five patients in whom BM MNCs were MRD-negative but purified HSPCs MRD-positive and (B) 5 patients in whom BM MNCs were MRD-positive but at lower level than in HSPCs. Blue and red indicate confidently positive and negative data, respectively, and green inconclusive as defined in the supplemental Methods. The dashed lines in CD34⁺ graphs (A-B) represent the predicted VAF (of the mutation with highest VAF) in CD34⁺ cells based on calculations from FCM and VAF data from HSPCs as described in supplemental Table 8. (C) VAF (%) for mutation with the highest VAF at indicated time points for all patients in panels A-B. The paired BM MNC, CD34⁺ cells and HSPC for each patient (blue circles) are connected by gray dashed lines. Mean (± SEM) values are also shown. P values were calculated using the Wilcoxon paired test. (D) Fold changes in VAF in BM CD34⁺ cells and HSPCs relative to MNCs (for all patients in panels A-B), in each patient using the mutation with the highest VAF. For mutations with VAFs below the LOD, the LOD value was used instead of the actual VAF. The VAFs for paired BM MNC, CD34⁺ cells and HSPCs for each patient (blue circles) are connected by gray dashed lines. Mean (± SEM) values are also shown. P values were calculated using a binomial test. Raw data can be found in source data file 1 (mutational HSPC ddPCR) and source data file 4 (mutational ddPCR in CD34⁺ fraction).