Schroeder Lee F, Huls Forest, Williams Christopher L, Li Shih-Hon, Keren David F
Department of Pathology, University of Michigan, Ann Arbor, MI.
Department of Pathology, University of Oklahoma Health Sciences Center, Norman, OK.
J Appl Lab Med. 2018 May 1;2(6):914-919. doi: 10.1373/jalm.2017.024950.
M-protein quantification is routinely performed by demarcating serum protein electrophoresis (SPE) regions. However, quantification of β-migrating M-protein is hindered by overlapping nonimmunoglobulin protein. Immunosubtraction (ISUB) on capillary electrophoresis is a method currently used qualitatively to subtract out (and therefore highlight) immunoglobulin isotypes in serum, thus reducing the masking effect of normal serum proteins. This study expands on traditional ISUB by developing a quantitative immunosubtraction (qIS) methodology.
qIS is achieved by estimating the monoclonal class-specific immunoglobulin contribution to the SPE region containing the M-protein. We conducted a recovery study by use of serial dilutions from 3 patients with β-region M-proteins (n = 22), performing SPE and ISUB on each dilution. We visualized the difference between the ISUB electrophoresis trace and the involved ISUB isotype-subtracted trace to distinguish M-protein and background polyclonal immunoglobulins, which was demarcated independently by 3 pathologists. The M-protein contribution to the β-region was calculated and applied to the β-region protein concentration producing the quantitative M-protein concentration, while minimizing contamination by nonimmunoglobulin or polyclonal immunoglobulin proteins.
Using a quality target of 25% error, we determined that our analytical measurable range spanned the maximum concentration tested (0.81 g/dL) to 0.05 g/dL. Passing-Bablok regression between qIS and the expected M-protein produced a slope of 1.04 (95% CI, 0.94-1.09), r = 0.99. Total CV was 4.8% and intraclass correlation between pathologists was 0.998.
qIS promises quantification of β-migrating M-proteins at concentrations an order of magnitude lower than traditional SPE methodology, allowing earlier detection of increasing or decreasing M-protein.
M蛋白定量通常通过划定血清蛋白电泳（SPE）区域来进行。然而，β迁移M蛋白的定量受到重叠的非免疫球蛋白蛋白的阻碍。毛细管电泳上的免疫扣除（ISUB）是目前定性用于扣除血清中免疫球蛋白同种型（从而突出显示）的一种方法，从而降低正常血清蛋白的掩盖效应。本研究通过开发一种定量免疫扣除（qIS）方法对传统ISUB进行了扩展。
qIS通过估计单克隆类别特异性免疫球蛋白对包含M蛋白的SPE区域的贡献来实现。我们对3例β区域M蛋白患者（n = 22）的系列稀释样本进行了回收率研究，对每个稀释样本进行了SPE和ISUB。我们可视化了ISUB电泳图谱与相关的ISUB同种型扣除图谱之间的差异，以区分M蛋白和背景多克隆免疫球蛋白，由3名病理学家独立划定。计算M蛋白对β区域的贡献，并将其应用于β区域蛋白浓度，得出定量的M蛋白浓度，同时尽量减少非免疫球蛋白或多克隆免疫球蛋白蛋白的污染。
使用25%误差的质量目标，我们确定我们的分析可测量范围涵盖了测试的最大浓度（0.81 g/dL）至0.05 g/dL。qIS与预期M蛋白之间的Passing-Bablok回归产生的斜率为1.04（95%CI，0.94 - 1.09），r = 0.99。总CV为4.8%，病理学家之间的组内相关性为0.998。
qIS有望对β迁移M蛋白进行定量，其浓度比传统SPE方法低一个数量级，从而能够更早地检测到M蛋白的增加或减少。
