doi:10.1093/ajcp/aqw052 [PMC free article] [PubMed] [CrossRef] [Google Scholar]Salem D, Stetler-Stevenson M, Yuan C, & Landgren O (2016). require a minimum of 2 million and recommend 5 million events be acquired to reach a minimum sensitivity of 10-5. As conventional immunophenotyping protocols are unable to attain these numbers, option MFC staining procedures are required. This manuscript explains two high-sensitivity MFC approaches that can be used for MM MRD testing. for 5 minutes and wash the sample once with 50 mL of FCM buffer to remove residual ACK Lysing Buffer answer. Perform absolute cell count and change cell concentration to 5 107 cells/mL using FCM buffer. Separately label sufficient 12 75 mm polystyrene tubes (e.g. Tube 1, Tube 2, and Tube 3). To Tube 1 and Tube 2, transfer 100 C 200 L (5 C 10 106 cells). To determine sample viability, transfer 10 C 20 L (5 C 10 105 cells) to Tube 3. If the sample is limiting, priority should be given to collecting a sufficient Bepotastine number of cells in Tube 1. Any remaining sample can be used for Tube 2 and Tube 3. While it is recommended that sample viability be obtained for Bepotastine each sample, Tube 3 has the lowest priority. Add mAbs for surface labeling and incubate for 30 minutes at RT in the dark. Information about the mAbs used for labeling Tube 1, Tube 2, and Tube 3 can be found in Table 1. Each of the mAb used should be titrated individually and use at saturation for optimal results. Add 2 mL of BD FACS? Lysing Treatment for each tube. Let sit for 10 minutes at RT in the dark. Centrifuge at 520 for 5 minutes and wash once with FCM buffer to remove residual lysis answer. For tubes Bepotastine to be labeled with surface antibodies only (e.g. Mouse monoclonal to CD45/CD14 (FITC/PE) Tube 1 and Tube 3), resuspend the cells in 500 L of 0.5% methanol-free formaldehyde and proceed to Step 15 for MFC data acquisition. For tubes to be labeled with intracellular antibodies to immunoglobulin light chains (e.g. Tube 2), proceed to Step 11. For intracellular staining (e.g. Tube 2), resuspend the cells in 100 L of 2% formaldehyde and incubate for 10 minutes at RT in the dark. Wash the cells once with 3 mL of FCM buffer and centrifuge at 520 for 5 minutes. Resuspend the residual volume with 100 L of diluted Permeabilization Medium B. Add saturating concentrations of anti-Kappa and anti-Lambda antibodies and incubate the cells for 30 minutes at RT in the dark. Add 3 mL of FCM buffer and let sit for 10 minutes at RT in the dark. Centrifuge the cells at 520 for 5 minutes and resuspend residual volume using 500 L of FCM buffer. If storage of samples is usually desired before data acquisition, resuspend the samples using 0.5% methanol-free formaldehyde and store at 4oC for no longer than 3 days. Setup and optimize the flow cytometers voltages and compensation for data acquisition using standardized methods as Bepotastine described by Wang et al. (Wang et al., 2017). Adjust threshold setting based on the forward scatter light characteristics to include hematogones while judiciously avoiding the recording of unwanted background noise. Analysis and Gating Strategy: 16. Identification of Total Plasma Cells: The recommended gating strategies employed for the phenotypic identification, enumeration, and characterization of normal and malignant PCs, as well as the identification of mast cells, hematogones, and erythroid precursors to assess the quality of the bone marrow samples are described below. Analyses performed in this section are not software-specific, as any commercially available software capable of generating MFC plots and associated results from millions of events can be used. On a bivariate plot of Time vs. FSC-A (Physique Bepotastine 1A), create and place a rectangular region (R1) to include all valid events acquired in chronologic homogeneity. Open in a separate window Physique 1: Detection of Total Leukocytes and Total Plasma Cells by high sensitivity MFC.(A) A rectangular region (R1) is created on a bivariate plot of Time (event chronology) vs. FSC-A to assess the compositional homogeneity of collected events. Disinterested and invalid events such as air bubbles collected during sample acquisition can be excluded using this plot. (B) A rectangular region (R2) is created on a gated (R1) bivariate plot of FSC-A vs. FSC-H to include.
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