Estimation of serum M-protein concentration from polyclonal immunoglobulins: an alternative to serum protein electrophoresis and standard immunochemical procedures

2008 ◽  
Vol 46 (8) ◽  
Author(s):  
Enrique Bergón ◽  
Elena Miravalles
Keyword(s):  
Serum Protein ◽  
Protein Concentration ◽  
Protein Electrophoresis ◽  
Serum Protein Electrophoresis ◽  
M Protein

scholarly journals An international multi-center serum protein electrophoresis accuracy and M-protein isotyping study. Part II: limit of detection and follow-up of patients with small M-proteins

2020 ◽  
Vol 58 (4) ◽  
pp. 547-559 ◽  
Author(s):  
Joannes F.M. Jacobs ◽  
Katherine A. Turner ◽  
Maria Stella Graziani ◽  
Jody L. Frinack ◽  
Michael W. Ettore ◽  
...  
Keyword(s):  
Serum Protein ◽  
Protein Concentration ◽  
Limit Of Detection ◽  
Negative Impact ◽  
Protein Electrophoresis ◽  
Protein Quantification ◽  
Serum Protein Electrophoresis ◽  
M Protein ◽  
M Proteins

AbstractBackgroundElectrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories.MethodsSera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia).ResultsAll M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L.ConclusionsIn this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

Underestimation of monoclonal proteins by serum protein electrophoresis on cellulose acetate.

1987 ◽  
Vol 33 (1) ◽  
pp. 182-184 ◽  
Author(s):  
F J Liu ◽  
H A Fritsche ◽  
J M Trujillo
Keyword(s):  
Cellulose Acetate ◽  
Serum Sample ◽  
Serum Protein ◽  
Protein Concentration ◽  
Protein Electrophoresis ◽  
Response To Therapy ◽  
Serum Protein Electrophoresis ◽  
M Protein ◽  
Albumin Concentration ◽  
Undiluted Serum

Abstract Our study of 95 serum samples from 37 patients with monoclonal gammopathy revealed distorted irregular monoclonal (M) protein bands after serum protein electrophoresis (SPE) on cellulose acetate membrane. In 71 (75%) of the 95 sera, the M-protein was underestimated and the albumin concentration overestimated. Dilution of the serum sample before SPE eliminated the abnormality of the M-protein bands. By SPE, the mean albumin concentration in these 71 undiluted sera was 45.8 (SD 7.4) g/L vs 37.9 (SD 5.8) g/L for the diluted sera; moreover, this was true of individual samples: measured albumin concentration in each diluted serum sample was always less than in the undiluted serum. As measured by the bromcresol green dye-binding method, the albumin concentration was 32.8 (SD 5.9) g/L. Similarly, the M-protein concentration in SPE was 49.5 (SD 12.3) g/L for the undiluted sera vs 61.8 (SD 15.1) g/L for the diluted sera, and the M-protein concentration in each diluted serum sample always exceeded that in the undiluted serum. Underestimation of M-protein limits the usefulness of M-protein measurement in evaluating the patient's response to therapy and for early detection of disease progression. SPE strips should be carefully inspected visually, and sera with M-protein band abnormalities should be diluted and re-assayed if SPE is to quantify concentrations of M-protein and albumin accurately.

Measurement of Monoclonal Immunoglobulin Protein Concentration in Serum Protein Electrophoresis: Comparison of Automated vs Manual/Human Readings

2019 ◽  
Vol 51 (3) ◽  
pp. 252-258 ◽  
Author(s):  
Alex Clavijo ◽  
Nathan Ryan ◽  
Hongyan Xu ◽  
Gurmukh Singh
Keyword(s):  
Serum Protein ◽  
Protein Concentration ◽  
Protein Electrophoresis ◽  
Serum Protein Electrophoresis ◽  
Response To Treatment ◽  
Computer Assisted ◽  
Positive Bias ◽  
Monoclonal Immunoglobulin ◽  
Monoclonal Immunoglobulins ◽  
Immunofixation Electrophoresis

Abstract Background Protein concentration of monoclonal immunoglobulin in plasma-cell myeloma/multiple myeloma provides an estimate of the tumor mass and allows for monitoring of the response to treatment. Accurate and reproducible estimates of the monoclonal immunoglobulin concentration are important for patient care. Objective To address the optimum method for estimation of the concentration of monoclonal immunoglobulins. Methods Serum protein electrophoresis and immunofixation electrophoresis were conducted by using the Helena SPIFE Touch instrument. Estimation of the protein concentration of monoclonal immunoglobulin in the gamma region by computer-assisted reading was compared with the reading by technologists and pathology residents, in 300 gels. The data were compared using t-testing and analysis of variance. Results Computer-generated readings had a consistent positive bias. The correlation coefficient of the average reading by technologists and residents with the computer generated value was 0.997. The average positive bias by the computer reading was 0.29 g per dL. The intercept on the regression analysis was 0.22 g per dL. The reading by the computer was significantly higher than each of the human-interpreted readings. The readings by the 3 human groups were not significantly different amongst them. The main reason for the higher reading by the computer was inclusion of a greater area on the anodal size of the peak on the densitometric scan. Conclusions Human- and computer-interpreted readings of the protein concentration of monoclonal immunoglobulin have a high degree of correlation. The consistent positive bias by the computer reading occurred due to inclusion of a greater area of the densitometric scan on the anodal side of the peak. We suggest that vendors should adjust such computer programs to provide readings comparable to those generated by expert humans. We recommend manual delineation of the monoclonal peaks for measuring the concentration of monoclonal immunoglobulins.

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

2020 ◽  
Vol 58 (4) ◽  
pp. 533-546 ◽  
Author(s):  
Katherine A. Turner ◽  
Jody L. Frinack ◽  
Michael W. Ettore ◽  
Jillian R. Tate ◽  
Maria Stella Graziani ◽  
...  
Keyword(s):  
Serum Protein ◽  
Protein Electrophoresis ◽  
Serum Protein Electrophoresis ◽  
M Protein ◽  
Accuracy And Precision ◽  
Low Concentrations ◽  
Percent Recovery ◽  
Limit Of Quantitation ◽  
M Proteins ◽  
Adequate Accuracy

AbstractBackgroundSerum 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.

scholarly journals Abnormal Heavy/Light Chain Ratio after Treatment Is a Robust Predictor of Shorter Survival in Patients with IgA Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3254-3254
Author(s):  
Yoshiaki Abe ◽  
Yasuhito Suehara ◽  
Hiroyuki Takamatsu ◽  
Yoshiaki Usui ◽  
Kentaro Narita ◽  
...  
Keyword(s):  
Overall Survival ◽  
Serum Protein ◽  
Light Chain ◽  
Protein Electrophoresis ◽  
Serum Protein Electrophoresis ◽  
M Protein ◽  
Lower Sensitivity ◽  
Prognostic Impact ◽  
Monoclonal Protein ◽  
Significant Difference

Abstract Accurate quantification of monoclonal protein (M-protein) is essential for response assessment, management, and prediction of prognosis in patients with multiple myeloma (MM). Serum protein electrophoresis (SPEP) is commonly used to determine the degree of M-protein reduction in intact immunoglobulin (Ig) MM. However, SPEP has limitations when M-protein comigrates into the beta-fraction or M-protein fails to show a distinct sharp spike on densitometry. Ig heavy/light chain (HLC) assay enables separate quantification of the different light chain types of each Ig class. Although HLC assay quantifies different light chain subtypes of Ig classes, the sensitivity for detecting M-protein clonality and its impact on outcome may differ between IgA and IgG myeloma. To investigate the clinical and prognostic impact of HLC assay, we retrospectively analyzed the correlation of heavy/light chain ratio (HLCR) with clinical status and its impact on outcome. A total of 402 frozen serum samples from 120 patients with MM (41 for IgA and 79 for IgG) treated at Kameda Medical Center (Kamogawa-shi, Chiba, Japan) and Kanazawa University Hospital (Kanazawa-shi, Ishikawa, Japan) at the times of various IMWG responses were collected. Samples were analyzed using the HLC assay, and the results were compared with serum protein electrophoresis (SPEP), free light chain ratio (FLCR), immunofixation, total IgG, IgA, and overall survival (OS). Percentages of samples with normal HLCR at presentation, PR, VGPR, CR, and sCR were 0%, 0%, 27.6%, 100%, and 88.9%, respectively, for IgA MM and 0%, 12.5%, 64.3%, 100%, and 84.3%, respectively, for IgG MM. Normalization of HLCR at VGPR was more frequent in IgG MM compared to IgA MM (PR; 12.5% vs. 0%, respectively, P = 0.169, VGPR; 64.3% vs. 27.6%, respectively, P = 0.004), which suggests the lower sensitivity of detecting clonality in patients with IgG MM than those with IgA MM. Abnormal HLCR was seen more frequently in patients with poorer myeloma response, and it appeared to be more sensitive for detecting clonality in IgA myeloma compared to IgG myeloma after treatment. No significant difference in OS was observed between patients with or without uninvolved Ig suppression and OS if they obtained ≥ VGPR. Among the 85 patients that achieved ≥ VGPR, those that remained HLCR abnormal showed significantly shorter overall survival (OS) compared to those achieving normal HLCR (not reached vs. 55.5 months, P = 0.032). This correlation was seen in IgA myeloma patients (not reached vs. 30.1 months, P = 0.014), but not in IgG myeloma patients when analyzed separately. Univariate and multivariate analyses of factors that may affect survival identified abnormal HLCR at the best response as the only independent risk factor (hazard ratio, 4.7; 95% confidence interval, 1.4 - 15.26; P = 0.012) for shorter OS in this subset of patients. In conclusion, HLC assay is useful for accurate monitoring of monoclonal protein in patients with myeloma. The results suggested that obtaining normal HLCR indicated a more favorable prognosis in patients with IgA myeloma, but not IgG myeloma, that achieved VGPR or better response. Disclosures Takamatsu: Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria.

Methodologic Differences in Values for M-Proteins in Serum, as Measured by Three Techniques

1975 ◽  
Vol 21 (2) ◽  
pp. 243-248 ◽  
Author(s):  
Jerry C Daniels ◽  
Theona M Vyvial ◽  
William C Levin ◽  
Stephan E Ritzmann
Keyword(s):  
Electrophoretic Mobility ◽  
Serum Protein ◽  
Monoclonal Gammopathy ◽  
Protein Concentration ◽  
Protein Electrophoresis ◽  
Serum Protein Electrophoresis ◽  
Radial Immunodiffusion ◽  
Protein Abundance ◽  
M Proteins ◽  
Chain Type

Abstract Accurate and reproducible measurement of M-proteins is essential for managing patients with monoclonal gammopathies, but serum protein electrophoresis, radial immunodiffusion, and electroimmunodiffusion yield comparatively divergent results. We have studied these differences and their causes. Sera from cases of IgG-monoclonal gammopathy, IgA-monoclonal gammopathy, and IgM-monoclonal gammopathy were assayed by each of the three techniques. Results indicated intermethod discrepancies as great as fivefold for all proteins studied. For IgG-monoclonal gammopathy, radial immunodiffusion values were uniformly higher; electroimmunodiffusion values were less consistently so. For IgA-monoclonal gammopathy, both radial immunodiffusion and electroimmunodiffusion gave lower results than did serum protein electrophoresis. For IgM-monoclonal gammopathy, results were variable, but values by radial immunodiffusion tended to be higher than, and electroimmunodiffusion comparable to, those for serum protein electrophoresis. The differences were not correlated with protein abundance, serum freshness, immunoglobulin class, light-chain type, ultracentrifugal characteristics, or electrophoretic mobility. Clearly, values for M-protein concentration depend on the techniques used to obtain them. We postulate that subclass differences may contribute to the diversity of radial immunodiffusion results, and that for electroimmunodiffusion the fixed electrophoretic mobility of M-proteins leads to unpredictable results. We conclude that serum protein electrophoresis is the best of the three assay techniques for M-proteins.

Integrating Serum Protein Electrophoresis with Mass Spectrometry, A New Workflow for M-Protein Detection and Quantification

2020 ◽  
Vol 19 (7) ◽  
pp. 2845-2853 ◽  
Author(s):  
Marina Zajec ◽  
Joannes F. M. Jacobs ◽  
Corrie M. de Kat Angelino ◽  
Lennard J. M. Dekker ◽  
Christoph Stingl ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Serum Protein ◽  
Protein Detection ◽  
Protein Electrophoresis ◽  
Serum Protein Electrophoresis ◽  
M Protein ◽  
Detection And Quantification

scholarly journals Diagnosing Paraproteinemic Keratopathy: A Case Report

2021 ◽  
pp. 337-343
Author(s):  
Eugenie Mok ◽  
Ka Wai Kam ◽  
Anthony J. Aldave ◽  
Alvin L. Young
Keyword(s):  
Optical Coherence Tomography ◽  
Genetic Testing ◽  
Serum Protein ◽  
Molecular Genetic ◽  
Anterior Segment ◽  
Protein Electrophoresis ◽  
Serum Protein Electrophoresis ◽  
Optical Coherence ◽  
Molecular Genetic Testing ◽  
Corneal Opacities

A 65-year-old man presented with bilateral, painless, progressive blurring of vision over 9 years. Slit-lamp examination revealed bilateral subepithelial corneal opacities in clusters located at the mid-periphery. Anterior segment optical coherence tomography, in vivo confocal microscopy (IVCM), serum protein electrophoresis, and molecular genetic testing were performed to evaluate the cause of corneal opacities. Anterior segment optical coherence tomography revealed a band-like, hyperreflective lesion in the Bowman layer and anterior stroma of both corneas. IVCM revealed hyperreflective deposits in the epithelium, anterior stroma, and endothelium. Serum protein electrophoresis identified the presence of paraproteins (immunoglobulin kappa), and molecular genetic testing revealed absence of mutations in the transforming growth factor beta-induced gene (<i>TGFBI</i>) and collagen type XVII alpha 1 gene (<i>COL17A1</i>). The ocular diagnosis of paraproteinemic keratopathy eventually led to a systemic diagnosis of monoclonal gammopathy of undetermined significance by our hematologist/oncologist. Paraproteinemic keratopathy is a rare differential diagnosis in patients with bilateral corneal opacities and therefore may be misdiagnosed as corneal dystrophy or neglected as scars. In patients with bilateral corneal opacities of unknown cause, serological examination, adjunct anterior segment imaging, and molecular genetic testing play a role in establishing the diagnosis.

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