Cd138 magnetic beads

Bone marrow samples were collected from 15 patients with MM and 10 patients with non-haematological diseases at Shengjing Hospital of China Medical University from February, 2015 to November, 2017. The basis clinical information of the study subjects is presented in Table SI. Samples were extracted using CD138 magnetic beads (Miltenyi Biotec GmbH). The purity of the CD138⁺ plasma cells was at least 90% (data not shown). Mononuclear cells were extracted from bone marrow using Ficoll-Hypaque (lymphocyte separation fluid; Beijing Solarbio Science & Technology Co., Ltd.) density gradient centrifugation. The present study was approved by the Research Ethics Committee of 0Shengjing Hospital of China Medical University (approval no. 2019PS270K) and all patients provided informed consent.

The iFISH technique used in this study has been previously described. Bone marrow (BM) aspirate samples anticoagulated with EDTA were collected, and CD138⁺ plasma cells (PC) were isolated using CD138⁺ magnetic beads (Miltenyi Biotec, Paris, France). iFISH analysis for CA included del(13q), del(17p), gain/amp(1q), t(11;14)(q13;q32), t(4;14)(p16.3;q32), and t(14;16)(q32;q23) in 200 interphase nuclei. The cut-off values for del(17p), gain/amp(1q), del(13q), and translocations were previously reported to be 50%, 20%, 10%, and 10%, respectively.^{13 (link)} Patients with del(17p), t(4;14), or t(14;16) were categorized as having high-risk CA,^{10 (link)} whereas those without these CA were considered standard-risk.

Samples were initially collected from patients at the first diagnosis and at the required follow-up time points (once every 3 months); 5 ml blood was collected in serum collection tubes. Whole blood was allowed to stand for ~1 h at room temperature, before centrifugation at 1,200 × g for 10 min at 4°C, followed by the separation of serum. Successive centrifugation was performed for 10 min at 10,000 × g at 4°C to remove the cellular debris. The resulting serum was aliquoted into Eppendorf tubes and stored at −80°C to determine IL-6 and circulating miR-451a levels.
BM samples collected from both patients and controls were anticoagulated with EDTA-K₂ (BD Biosciences) at room temperature. Some BM samples were diluted 1:1 with RPMI-1640 basic medium (Gibco; Thermo Fisher Scientific, Inc.). Mononuclear cells were isolated with Ficoll cell isolation medium at room temperature (density 1.077 g/ml; Stemcell Technologies, Inc.). Then, plasma cells were isolated using CD138 magnetic beads at room temperature (Miltenyi Biotec, Inc.) for RNA extraction to analyze miR-451a expression and proteins were extracted to measure IL-6R levels. The remaining BM was used for MFC.