BACKGROUND Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post-treatment. To achieve the high sensitivity (one abnormal cell in 105 -106 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, "Pre-lysis" method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the "Pooled-tube" and "Dextran Sedimentation" staining procedures and correlated them with the "Pre-lysis" method as potential alternative approaches. METHODS A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the "Pre-lysis," "Pooled-tube," and "Dextran Sedimentation" techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments. RESULTS The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the "Pooled-tube" method when compared to "Pre-lysis." "Pre-lysis" was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by "Pooled-tube" when alternative CD56 PE clones were used. "Dextran sedimentation" yielded similar staining results when compared to "Pre-lysis" for all the markers analyzed. The "Pooled-tube" method, when normalized to "Pre-lysis," recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between "Pre-lysis" versus "Dextran Sedimentation." All three flow cytometric assays achieved a minimum sensitivity of 10-5 and approached that of 10-6 for detecting rare events. CONCLUSION Both "Pooled-tube" and "Dextran Sedimentation" staining procedures were comparable to the "Pre-lysis" method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing.

中文翻译：

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高灵敏度检测多发性骨髓瘤可测量残留疾病的方法学考虑。

背景技术治疗干预的最新进展显着提高了多发性骨髓瘤（MM）患者的完全缓解率。识别残留骨髓瘤细胞（例如，可测量的残留疾病[MRD]）的能力可以提供有关患者对治疗的反应深度和复发风险的有价值的信息。多参数流式细胞术是监测MRD的出色技术，并已证明与治疗后患者的预后相关。为了获得MRD评估所需的高灵敏度（105个-106个细胞中有一个异常细胞），必须获取数百万个细胞，并且常规的免疫表型方案无法获得这些数量，这表明需要其他流式细胞仪染色程序。大量的“预裂解” 该方法是对大量细胞进行染色的共识方法，需要两个红细胞裂解步骤，并且可能对表位密度产生不利影响。在这项研究中，我们测试了“管式”和“ Dextran沉淀法”染色程序，并将它们与“预裂解”法相关联作为潜在的替代方法。方法使用“预裂解”，“池管”和“ Dextran沉淀法”技术并行处理来自浆膜细胞（PC）异常型患者的22份骨髓抽吸物。计算并比较染色指数，以评估其对共识面板中使用的每种抗体的染色性能的影响。正常和异常PC，肥大细胞，计数B细胞前体并用荧光珠将其计数归一化后进行比较。使用系列稀释实验确定空白限，检测限和定量下限。结果与“预裂解”相比，使用“管式”方法改善了CD19 PECy7，CD27 BV510，CD81 APCH7和CD138 BV421的染色性能。“预裂解”在使用克隆C5.9分离CD56方面更好，但是我们的结果表明，当使用其他CD56 PE克隆时，“池管”也可以实现类似的改进。与“ Pre-lysis”相比，所有标记物的“ Dextran沉降”结果均相似。当标准化为“预裂解”时，“管式”方法 恢复的总PC数量更高（1.2±0.2倍; p = .049），正常PC（1.4±0.26; p = .007），肥大细胞（1.46±0.27; p = .003）和B细胞前体（ 1.42±0.3; p = .011），但不是异常PC（1.09±0.2; p = .352）。没有证据表明“ Pre-lysis”和“ Dextran Sedimentation”之间的细胞回收率有所不同。所有三种流式细胞仪检测的最低灵敏度为10-5，接近10-6的罕见事件检测灵敏度。结论“管式”和“ Dextran沉淀法”染色程序均与“预裂解”法相当，并且是适用于处理骨髓样本进行MM MRD测试的高灵敏度流式细胞仪方法。p = .007），肥大细胞（1.46±0.27; p = .003）和B细胞前体（1.42±0.3; p = .011），但不是异常PC（1.09±0.2; p = .352）。没有证据表明“ Pre-lysis”和“ Dextran Sedimentation”之间的细胞回收率有所不同。所有三种流式细胞仪检测的最低灵敏度为10-5，接近10-6的罕见事件检测灵敏度。结论“管式”和“ Dextran沉淀法”染色程序均与“预裂解”法相当，是适用于处理骨髓样本进行MM MRD测试的高灵敏度流式细胞仪方法。p = .007），肥大细胞（1.46±0.27; p = .003）和B细胞前体（1.42±0.3; p = .011），但不是异常PC（1.09±0.2; p = .352）。没有证据表明“ Pre-lysis”和“ Dextran Sedimentation”之间的细胞回收率有所不同。所有三种流式细胞仪检测的最低灵敏度为10-5，接近10-6的罕见事件检测灵敏度。结论“管式”和“ Dextran沉淀法”染色程序均与“预裂解”法相当，并且是适用于处理骨髓样本进行MM MRD测试的高灵敏度流式细胞仪方法。没有证据表明“ Pre-lysis”和“ Dextran Sedimentation”之间的细胞回收率有所不同。所有三种流式细胞仪检测的最低灵敏度为10-5，接近10-6的罕见事件检测灵敏度。结论“管式”和“ Dextran沉淀法”染色程序均与“预裂解”法相当，是适用于处理骨髓样本进行MM MRD测试的高灵敏度流式细胞仪方法。没有证据表明“ Pre-lysis”和“ Dextran Sedimentation”之间的细胞回收率有所不同。所有三种流式细胞仪检测的最低灵敏度为10-5，接近10-6的罕见事件检测灵敏度。结论“管式”和“ Dextran沉淀法”染色程序均与“预裂解”法相当，并且是适用于处理骨髓样本进行MM MRD测试的高灵敏度流式细胞仪方法。