Cells were collected from the patient at diagnosis (PB), 20 days postdiagnosis of MDS (BM), and 8 days post-AML recurrence (PB and BM). Ficoll-Paque density gradient or hypotonic ammonium chloride solution (ACK Lysing Buffer; Gibco) were used to deplete BM and PB of red blood cells (RBCs). Post-RBC depletion, ∼2 × 10⁵ mononuclear cells (MNCs) were set aside for VCN analysis (unsorted BM sample). For PB, the remaining cells were stained with anti-CD34 BV421 antibody (BD Biosciences) and separated into CD34⁺ and CD34⁻ fractions using fluorescence-activated cell sorting (SH800 cell sorter; Sony Biotechnology). For BM, remaining MNCs were stained with an anti–CD34-allophycocyanin, washed, and sorted via a Beckman Coulter MoFlo Astrios EQ cell sorter to isolate CD34⁻ (CD34⁻ selected) and CD34⁺ (with malignant blasts as main contributors) cells. Purity of sorted cell populations was subsequently determined. Additionally, sorted cells were counted by trypan blue exclusion on a hemacytometer and cells from each population were used for VCN analysis, conducted as previously described.³²  For post-MDS diagnosis VCN, mean ± standard deviation is shown.

LOQ, limit of quantitation; n/a, not applicable; nd, not determined.

After haploidentical transplant.