BACKGROUND Serum free light chains (FLCs) are an essential clinical biomarker for the diagnosis and monitoring of patients with plasma cell neoplasms. The current widely used immunoassay methods quantify total serum FLCs, which include monoclonal FLCs as well as FLCs in the polyclonal background. Patients with chronic diseases, inflammatory disorders, or renal dysfunction can have elevated total FLCs that lead to ambiguous results. These patients may benefit from a direct measurement of monoclonal FLCs. The purpose of this study was to develop a method that couples on-probe extraction (OPEX) with liquid chromatography-high-resolution mass spectrometry (LC-HR-MS), abbreviated to OPEX-MS, to directly determine the clonality of FLCs. METHODS OPEX immunocapture was performed using microprobes loaded with anti-kappa or anti-lambda light chain antibodies. Captured proteins were separated by reversed-phase LC and analyzed by HR-MS. RESULTS Four cohorts of samples from unique patients were tested based on immunoassay FLC results. The LC-HR-MS analysis in the OPEX-MS method provides both a unique retention time along with deconvoluted masses of FLC monomers and dimers for each clone. The study found that 16 out of 49 (33%) kappa FLC elevated samples as well as 83 out of 100 (83%) dual kappa and lambda FLC elevated samples did not have monoclonal FLCs, which is consistent with the knowledge that there is often no clonal population in samples with mildly elevated FLC immunoassay results. CONCLUSIONS The OPEX-MS method can serve as a complementary approach to directly determine clonality in patients with difficult-to-interpret FLC immunoassay results.

中文翻译：

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通过使用探针上萃取结合液相色谱-高分辨率质谱法检测未修饰的单克隆血清游离轻链来确定克隆性。


背景 血清游离轻链 （FLC） 是诊断和监测浆细胞肿瘤患者的重要临床生物标志物。目前广泛使用的免疫测定方法可定量总血清 FLC，包括单克隆 FLC 以及多克隆背景中的 FLC。患有慢性病、炎症性疾病或肾功能不全的患者总 FLC 可能升高，从而导致结果不明确。这些患者可能受益于单克隆 FLC 的直接测量。本研究的目的是开发一种将探针上提取 （OPEX） 与液相色谱-高分辨率质谱 （LC-HR-MS）（简称 OPEX-MS）耦合以直接确定 FLCs 克隆性的方法。方法 使用载有抗 κ 或抗 λ 轻链抗体的微探针进行 OPEX 免疫捕获。通过反相 LC 分离捕获的蛋白质，并通过 HR-MS 进行分析。结果 根据免疫测定 FLC 结果对来自独特患者的 4 组样本进行了测试。OPEX-MS 方法中的 LC-HR-MS 分析为每个克隆提供了独特的保留时间以及 FLC 单体和二聚体的去卷积质量。研究发现，49 个 kappa FLC 升高样品中有 16 个 （33%） 以及 100 个 （83%） 双重 kappa 和 lambda FLC 升高样品中有 83 个 （83%） 没有单克隆 FLC，这与 FLC 免疫测定结果轻度升高的样品中通常没有克隆种群的信息一致。结论 OPEX-MS 方法可以作为一种补充方法，直接确定 FLC 免疫测定结果难以解释的患者的克隆性。