Fukushima P I, Nguyen P K, O'Grady P, Stetler-Stevenson M
Flow Cytometry Unit, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1500, USA.
Cytometry. 1996 Dec 15;26(4):243-52. doi: 10.1002/(SICI)1097-0320(19961215)26:4<243::AID-CYTO2>3.0.CO;2-D.
Analysis of light chain expression is one of the most important determinations in flow cytometric immunophenotyping of patient specimens. Numerous technical factors, such as antibody choice and cytophilic antibody artifact, impact a laboratory's ability to perform this test. There have been conflicting reports concerning the efficacy of polyclonal versus monoclonal antibodies, as well as methods of circumventing cytophilic antibodies, indicating that a consensus has not been reached on optimal methods for light chain determination. The authors have investigated methods for light chain analysis in 104 normal donors and 366 patient specimens, comparing different anti-light chain antibodies as well as strategies for analysis of specimens with low numbers of monoclonal B cells, admixed polyclonal B cells, or cytophilic antibodies. The patient specimens were either part of the initial diagnostic evaluation of patients with suspected lymphoma, or were performed for staging or assessment of treatment of patients with known B-cell neoplasia. No monoclonality was detected in control specimens, and there was no significant difference in staining with monoclonal verses polyclonal anti-light chain antibodies. In addition, cytophilic antibody did not obscure results in normal controls. Monoclonality was detected in 106 patient specimens, with 89 showing gross involvement with a predominant monoclonal B-cell process. However, in 43% of the grossly monoclonal specimens, there was failure to detect monoclonality with at least one light chain antibody set, with 8% of these cases showing failure with two anti-light chain sets. This indicates the importance of antibody choice in light chain analysis. Cytophilic antibody artifact in monoclonal specimens was easily overcome by appropriate antibody combinations, obviating the need for cytophilic antibody-shedding by incubation at 37 degrees C in fetal calf serum. In 27 patient specimens with low numbers of B cells or admixed polyclonal B cells, a clonal search based on FSC and CD19 or CD20 expression was performed. In 17 of the 27 cases (63%), a small monoclonal population was detected among admixed polyclonal B cells. The authors conclude that multiple strategies are necessary in flow cytometric analysis for B-cell monoclonality.
轻链表达分析是患者样本流式细胞术免疫表型分析中最重要的检测项目之一。许多技术因素，如抗体选择和嗜细胞抗体假象，会影响实验室进行该项检测的能力。关于多克隆抗体与单克隆抗体的功效，以及规避嗜细胞抗体的方法，一直存在相互矛盾的报道，这表明在轻链检测的最佳方法上尚未达成共识。作者对104名正常供体和366份患者样本的轻链分析方法进行了研究，比较了不同的抗轻链抗体，以及针对单克隆B细胞数量少、混合多克隆B细胞或存在嗜细胞抗体的样本的分析策略。患者样本要么是疑似淋巴瘤患者初始诊断评估的一部分，要么是对已知B细胞肿瘤患者进行分期或治疗评估时所取。对照样本中未检测到单克隆性，单克隆抗轻链抗体与多克隆抗轻链抗体染色无显著差异。此外，嗜细胞抗体在正常对照中并未掩盖结果。在106份患者样本中检测到单克隆性，其中89份显示主要为单克隆B细胞过程的广泛受累。然而，在43%的明显单克隆样本中，至少一组轻链抗体未能检测到单克隆性，其中8%的病例两组抗轻链抗体均检测失败。这表明抗体选择在轻链分析中的重要性。通过适当的抗体组合可轻松克服单克隆样本中的嗜细胞抗体假象，无需在胎牛血清中于37℃孵育以去除嗜细胞抗体。在27份B细胞数量少或混合多克隆B细胞的患者样本中，基于FSC和CD19或CD20表达进行了克隆搜索。在27例中的17例（63%）中，在混合的多克隆B细胞中检测到一小群单克隆细胞。作者得出结论，在B细胞单克隆性的流式细胞术分析中需要多种策略。
