Interference of bone marrow CD56+ mesenchymal stromal cells in minimal residual disease investigation of neuroblastoma and other CD45−/CD56+ pediatric malignancies using flow cytometry

Abstract Background Bone marrow (BM) samples obtained from minimal residual disease (MRD)‐negative children with B‐cell acute lymphoblastic leukemia (B‐ALL) were used in our laboratory as negative biological controls for the development of a neuroblastoma (NBL) flow‐cytometric (FC) protocol. The accidental, but systematic, identification of rare cell populations (RCP) mimicking NBL cells (CD45 − /CD56 + ) in these samples indicated the need for their thorough immunophenotypic identification, in order to elucidate their possible interference in NBL‐MRD assessment. Procedure RCP observed in BM samples from 14 children recovering from BM aplasia due to intensive chemotherapy for B‐ALL were investigated with the following markers: CD81, CD200, CD24, GD2, CD73, CD13, CD90, CD146, CD9, CD117, CD10, CD99, and NG2. BM samples from six newly diagnosed patients with NBL and an NBL cell line were simultaneously investigated as positive controls. Results The frequency of RCP in B‐ALL BM samples was < 1/1 × 10 4 cells (bulky lysis), and their immunophenotypic profile was indicative of CD56 + mesenchymal stromal cells (MSCs) (CD45 − , CD90 + , CD146 + , CD73 + ). Also, RCP expressed CD81 and CD200, simulating NBL cells. The most useful discriminative markers for CD56 + MSCs were CD13 and CD73. An appropriate protocol consisting of two tubes with seven color combinations was further proposed: SYTO‐16, GD2 (first tube) or CD73 (second tube)‐PE, CD24‐ECD, CD13‐PC5.5, CD45‐PC7, CD81‐APC, and CD56‐APC700. Conclusions RCP that were immunophenotypically similar to NBL were identified as CD56 + MSCs. As these cells might pose an obstacle to accurate NBL disease assessment by FC, especially MRD, an enhanced NBL‐FC protocol is proposed for prospective evaluation.