An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part I: factors impacting limit of quantitation of serum protein electrophoresis

Abstract

BackgroundSerum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents.MethodsSera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%–120% was set as acceptable. The inter-laboratory %CV was calculated.ResultsGamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations.ConclusionsThis study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

Refining amyloid complete hematological response: Quantitative serum free light chains superior to ratio

“…The serum IFE assessed migration patterns for γ, α, μ, κ, and λ immunoglobulin chains (Hydrasys and Hydragel; Sebia) with a sensitivity of approximately 0.5 g/L 10,11 . Urine IFE assays were performed on the same day as receipt of the sample.…”

A Comparison of gel (Hydragel 30) and capillary (Capillarys III Tera) electrophoresis for the characterization of human serum proteins

“…Previous studies have compared gel to capillary electrophoresis [ 8 , [13] , [14] , [15] ] and shown excellent agreement (>95%) in determining the concentration of the five serum protein fractions (albumin, alpha-1, alpha-2, beta, and gamma). Goal of the present study was to compare the performance of gel electrophoresis to that of the new capillary electrophoresis apparatus Capillarys III Tera (Sebia, Lisses, France) in a routine clinical setting at a tertiary referral hospital, using a large patient population and focusing on the characterization of the M-spike.…”

Our Laboratory Experience: Comparison of Capillary Electrophoresis/Immunosubtraction and Agarose Gel/Immunofixation

“…The abnormality of monoclonal protein may be the alert for undercovered lymphoid malignancy or other pathologic complications [3]. The monoclonal protein serves as a tumor (or clonal) marker in these patients and needs to be monitored to determine whether the clone of plasma cells is expanding [4,5]. The role in the diagnosis, classification, and management of this disorder is committed by quantifying and immunotyping of serum proteins.…”