Effect of Different Integration Approaches to the M-Spike Quantification in Serum Protein Electrophoresis and How It Correlates to the New Heavy/Light Chain Assay
Authors: Jiménez Jiménez Juana , Pais Tiago , Campos Luisa , Hernando de Larramendi Carmen 
Published: October 2019

Abstract
Introduction
To measure the Monoclonal Protein (MP) is essential in monoclonal gammopathies (MG) for both diagnosis and response assessment. However, the M-spike quantification may be affected by the inherent subjectivity and by the opted integration method to determine the area under the M-spike corresponding to the MP in a serum protein electrophoresis (SPE). Co-migration with other serum proteins and strong polyclonal background may also affect the MP accurate measurement. Due to its pivotal role in managing MG, and since new automatized techniques have been developed for the MP quantification, this study aims at compare different M-spike integration methods on the measurement of MP, and compare it to the new Heavy/Light chain assay (HLC/ Hevylite®), which allows to separately quantify, by automated nephelo/turbidimetry, the serum levels of IgG-K, IgG-L, IgA-K, IgA-L, IgM-K and IgM-L.
Material and Methods
147 samples from 143 MGUS and MM patients were included. All the samples were analyzed by SPE (SEBIA- HYDRASYS 2) and two integration methods were used for quantifying the MP: MP1: peak defined until baseline; MP2: peak defined excluding the polyclonal part. The size of the paraprotein measured by the MP1 method was used to divide the samples in MP<10g/L and MP>10g/L. Statistical analysis included Passing-Bablok (PB) and Bland-Altman (Analyse-It®) and Mann-Whitney test (GraphPad Prism).
Results
The MP quantification by the MP1 and MP2 methods was significantly different (medians: 7.4 g/L vs 4.1 g/L, P<0.0001), with a relative difference of -64.36%. When dividing samples according to the MP size, the mean relative difference found was higher in samples with MP<10g/L than MP>10g/L (MP1:-85.02% vs MP2:-28.23%; medians: 5.4g/L vs 2.2g/L, P<0.0001; and 18.45g/L vs 14.65g/L, P=0.0253; respectively). Likewise, the correlation between MP1 and MP2 was good (r=0.991) but it weakened to r=0.864 in samples with MP< 10g/L. Therefore, the two integration methods diverged more in the quantification of small MP.
In all analysis, MP1 correlated better than the MP2 method with the involved HLC (iHLC) (r=0.886 and r=0.87, respectively) and the total Ig (r=0.924 and r=0.9, respectively), with lower mean relative differences. The MP1 correlation with iHLC was considerably better in samples with MP>10g/L in respect to MP<10g/L (r=0.837 and r=0.55, respectively). PB analysis further revealed an agreement between MP1 and iHLC quantification in samples with MP>10g/L: iHLC=2.096+1.045PM1 (95%CI: Intercept -3.978 to 5.719; Slope: 0.8568 to 1.320), confirming that Hevylite is a useful tool for the MP quantification. Moreover, PB analysis on the summation of Heavy/Light pairs of the same immunoglobulin isotype resulted in an agreement with the levels of total Immunoglobulin (tIg) (r=0.857. PB: ∑HLC=0.1819+0.9211tIg; 95%CI: Intercept -0.9733 to 1.331; Slope: 0.8454 to 1.006), being slightly better in MP<10g/L than >10g/L, indicating the HLC analysis is valid for small MP measurements.
HLC analysis also allows measuring the uninvolved-HLC pair (uHLC), a new immunoparesis parameter than has been suggested to have prognostic value. In this study, 32/34 MM and 49/109 MGUS samples had suppressed levels of uHLC. Interestingly, the MP1 and MP2 correlation with iHLC was better in MM than in GMSI samples (r=0,864 and r=0,857, versus, r=0,678 and r=0,606, respectively), which may be due both to generally higher levels of MP or lower polyclonal background in MM samples. Furthermore, 2/109 MGUS samples resulted in a different risk-of-progression category depending on the method of M-spike integration, with the iHLC being in agreement with the MP1 in one of the samples and discordant with the other.
Conclusions
The MP measurement by M-spike integration until baseline correlated with the iHLC and total immunoglobulin measurement better than when the M-spike integration excludes the polyclonal part. The good agreement between the iHLC and SPE in the quantification of the monoclonal protein puts this assay as an alternative method especially relevant at low concentration levels, which may help correcting for methodology associated variability within clinical laboratories. Finally, HLC may be easier to standardize than the SPE analysis and could eliminate the need for total Ig quantification.

Disclosures
Pais: The Binding Site Spain: Employment. Campos:The Binding Site Spain: Employment.