Dr. Priscilla Yeung, Instructor, Department of Pathology, Stanford University
Dr. Ruben Y. Luo, Assistant Professor, Department of Pathology, Stanford University
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 as OPEX-MS, to directly determine the clonality of FLCs. OPEX immunocapture was performed using microprobes loaded with anti-kappa or anti-lambda light chain antibodies, as shown in Figure 1. Captured proteins were separated by reversed-phase LC and analyzed by HR-MS.
Four cohorts of samples (50 negative, 49 kappa FLC elevated, 45 lambda FLC elevated, 100 both kappa FLC and lambda FLC elevated) 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 accurate masses of FLC monomers and dimers for each clone, as shown in Figure 2. The study discovered that 16 out of 49 (33%) samples with elevated kappa FLC levels and 83 out of 100 (83%) samples with elevated levels of both kappa and lambda FLCs did not have monoclonal FLCs. This finding aligns with the understanding that samples showing mildly elevated FLC immunoassay results often do not have a clonal population. In summary, the OPEX-MS method is able to serve as a complementary approach to directly determine clonality in patients with difficult-to-interpret FLC immunoassay results.
Figure 1. Schematic of the on-probe-extraction coupled with high-resolution mass spectrometry (OPEX-MS) method to identify monoclonal serum free light chains.
Figure 2. Example deconvoluted mass spectra and chromatography traces of serum samples from an OPEX-MS positive IgG lambda MM patient (left), an OPEX-MS positive IgG kappa MM patient (middle), and a patient that is negative by OPEX-MS (right). The monoclonal FLC peaks are chromatographically separated from IgG, albumin, and transferrin.
使用探针提取结合液相色谱-高分辨率质谱法检测单克隆血清游离轻链
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量化总血清游离轻链，其中包括单克隆游离轻链以及背景中的多克隆游离轻链。患有慢性疾病、炎症性疾病或肾功能障碍的患者的总游离轻链可能会升高，从而导致结果不明确。这些患者可能受益于直接测量单克隆游离轻链。本研究的目的是开发一种将探针提取(OPEX)与液相色谱-高分辨率质谱(LC-HR-MS)相结合的方法，简称为 OPEX-MS，以直接确定游离轻链的克隆性。 如图 1 所示, OPEX是通过载有抗 kappa 或抗 lambda 轻链抗体的微探针免疫捕获游离轻链，而被捕获的轻链经过反相液相色谱分离之后进行高分辨率质谱分析。
本研究根据免疫测定游离轻链结果对四组样本进行了测试， 包括阴性样本组 (50)，kappa游离轻链升高的样本组 (49)，lambda游离轻链升高的样本组(45)，kappa和lambda游离轻链均升高的样本组(100)。如图 2 所示，OPEX-MS方法中的液相色谱-高分辨率质谱分析为每个克隆提供了独特的保留时间以及游离轻链单体和二聚体的精确分子量。研究发现，33% kappa游离轻链升高的样本(16 /49)以及83%kappa和lambda游离轻链均升高的样本(83/100)没有检测到单克隆的游离轻链，这与游离轻链免疫测定结果轻度升高的样本中通常没有单克隆免疫球蛋白的认识一致。综上，OPEX-MS方法可以作为常规方法的补充，用于直接测定常规免疫分析无法确定的患者样品中是否存在单克隆游离轻链。
Reference
[1] Yeung PSW, Liu Y, Yang S, Ruan A, Kerr CR, Wong C, Shi RZ, Iberri DJ, Luo RY. Clonality Determination by Detecting Unmodified Monoclonal Serum Free Light Chains Using On-Probe Extraction Coupled with Liquid Chromatography-High-Resolution Mass Spectrometry. Clin Chem, DOI: 10.1093/clinchem/hvae130.
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