Monoclonal protein reference change value as determined by gel-based serum protein electrophoresis

Abstract Serum free light chain measurements have been shown to be useful in the diagnosis and monitoring of patients with monoclonal gammopathies. The present study was undertaken to evaluate the effect of adding the measurement of serum free light chain kappa to lambda ratios to the serum protein electrophoresis evaluation that we typically use as an initial screen for the detection of monoclonal proteins. We retrospectively tested 347 consecutive samples from individuals who had no previous history of plasma cell dyscrasia and had not previously had a serum or urine electrophoresis or immunofixation electrophoresis test at our institution. The quantitative serum protein electrophoresis test that was ordered was performed using Hydragel Beta 1- Beta 2 gels and Hydrasis instrument (Sebia, Inc., Norcross, GA). The protein content of the electrophoresis zones were quantitated by scanning densitometry and the electrophoresis pattern of each sample was qualitatively examined for abnormal bands and suspicious findings by a single, experienced observer. Serum free light chain concentrations and the serum free light chain kappa to lambda ratios were determined using the Freelite Human Kappa and Lambda Kits (The Binding Site Ltd, Birmingham, UK) and the Immage analyzer (Beckman Coulter Inc., Brea, CA). The serum free light chain kappa to lambda ratios were outside the reference interval (0.25 to1.65) in 23 of the samples. Ten abnormal ratios were observed among a group of 57 samples that had either positive or suspicious qualitative evaluations for the presence of a restriction or that demonstrated hypo-gammaglobulinemia. Both abnormalities led to recommendations for follow-up testing, which confirmed the presence of a monoclonal protein in 21 of the samples. Six abnormal ratios were observed among a group of 159 specimens that had quantitative abnormalities in albumin or one or more of globulin fractions (hypo-gammaglobulinemia excepted) and normal qualitative evaluations. Seven abnormal ratios were observed among a group of 131 samples that had normal quantitative results and normal qualitative evaluations. Follow-up testing is not usually recommended for serum protein electrophoresis results like those in the latter two groups. We found that the addition of the serum free light chain kappa to lambda ratio to the serum protein electrophoresis test increased the number of abnormal screens that would have required further clinical and/or laboratory evaluation by 23%(i.e. from 57 to 70). Given the high specificity of the serum free light chain kappa to lambda ratio for monoclonal light chains, the additional 13 abnormal samples identified by this test are expected to have a high likelihood of harboring a monoclonal protein that would have otherwise eluded detection. Pending a definitive prospective study, we estimate that the addition of a serum free light chain kappa to lambda ratio to the serum protein electrophoresis screen would increase the rate of detection of serum monoclonal proteins by as much as 1.6-fold.

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Usefulness of Serum-Free-Light-Chains-Ratio (SFLCR) and Serum Heavy-Light-Chains-Ratio (SHLCR) in Multiple Myeloma in the Context of Three GEM/Pethema Clinical Trials

Blood ◽

10.1182/blood.v126.23.2962.2962 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2962-2962

Author(s):

Lucia Lopez-Anglada ◽

Cecilia Cueto-Felgueroso ◽

Maria-Victoria Mateos ◽

Laura Rosiñol ◽

Albert Oriol ◽

...

Keyword(s):

Clinical Trials ◽

Serum Protein ◽

Progression Free Survival ◽

High Sensitivity ◽

Protein Electrophoresis ◽

Serum Protein Electrophoresis ◽

Light Chains ◽

Advisory Committees ◽

Free Survival ◽

Monoclonal Protein

Abstract Introduction: The sFLC assay was introduced in 2006 for diagnosis and monitoring of gammopathies, being currently the normalization of its ratio one of the requirements for defining stringent complete response (sCR). The new sHLC assay allows identifying the different light chain types bound to each heavy chain (i.e. separate the amount of IgG Κ from IgG λ) and may have greater prognostic value than sFLC assay. However, its advantages over immunofixation (IFE) have to be demonstrated and the sHLC assay does not work well for every light chain secretors MM. We wanted to investigate the utility and prognostic impact of serum free-light chains (sFLC) and heavy-light chains (sHLC) studies in multiple myeloma (MM) in the context of three GEM clinical trials (GEM05>65y,GEM05>65y and GEM2010>65y). Methods and Patients: Frozen sera from patients were retrospectively analyzed for sFLC and sHLC (623 and 183 patients at diagnosis, respectively) in the context of three GEM/PETHEMA clinical trials. In addition, sHLC measurements performed in 30 healthy individuals were used for control values. After induction, regardless of the achieved response, serum samples were available in 308 cases for sFLC and 89 for sHLC. All patients in which sHLC assay was analyzed were IgG or IgA-MM. Results and discussion: In our series, around 95% of the patients had abnormal values of sFLC and sHLC ratios at the time of diagnosis. Normal values did not impact in prognosis, and even when we considered Òvery pathologicalÓ (VP) sFLCr values (0.03-32), no prognostic differences were observed [Figure 1A]. Conversely, after establishing several arbitrary cut-offs, we note that very pathological sHLC values of the ratios (<0.29 or >73) at diagnosis had greater risk of progression (p=0.006) [Figure 1B], confirmed by multivariate analysis ― age [p=0,003; OR 1.04 (1.01-1.06)]; LDH [p= 0.03; OR 0.4 (0.26-0.94)]; VP-sHLCr [p=0.01; OR 1.78 (1.14-2.78)]; high vs low risk FISH [p=0.02; OR 1.75 (1.11-2.74)]―. sHLC non-involved pair suppression (described as a 50% reduction under de lower range limit) at diagnosis wasnÕt related to worse prognosis in our series. The HLC-involved-Ig values show a strict linear correlation with values of monoclonal protein (MP) by serum protein electrophoresis (sPE) (p= 0,000; Pearson's r=0.676) [Figure 2] and thus the high sensitivity of sHLC for IgA MP should prove useful for monitoring MP migrating within the β fractions. After treatment, as we might expect, there was a clear association between quality of response and normalization of the sFLC ratio (Pearson's χ2 p<0.001). Concerning the sFLCr, among the 130 patients in CR after induction, no prognostic difference was observed between patients with a normal (0.26-1.65) vs pathological sFLCr (OS p=0.137; PFS p=0.359). No conclusive results were obtained from the normalization of sHLC in CR due to a low number of samples available for these studies. Conclusions: Very pathological (<0.29 or >73) sHLC ratios at diagnosis have bad prognosis impact. However sFLC did not show any prognosis impact. Regarding response measurement sFLC and sHLC did not show any advantages over conventional methods. Additionally, sHLC values are highly related to the M component levels, thus the high sensitivity of sHLC for IgA should prove very useful for monitoring MP migrating within the β fractions. Figure 1. Impact of "very pathological" (VP) sFLC and sHLC ratios on progression free survival (PFS) Figure 1. Impact of "very pathological" (VP) sFLC and sHLC ratios on progression free survival (PFS) Figure 2. Correlation of monoclonal protein (MP) quantification by serum protein electrophoresis (sPE) and HLC-involved-Ig values Figure 2. Correlation of monoclonal protein (MP) quantification by serum protein electrophoresis (sPE) and HLC-involved-Ig values Disclosures San Miguel: Millennium, Celgene, Novartis, Janssen, Onyx, BMS, MSD: Membership on an entity's Board of Directors or advisory committees. Lahuerta:Janssen Cilag, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Use of isoelectric focusing to discriminate transient oligoclonal bands from monoclonal protein in treated myeloma

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2015-0642 ◽

2016 ◽

Vol 54 (6) ◽

Author(s):

Robyn Henry ◽

Debra Glegg

Keyword(s):

Prostate Cancer ◽

Serum Protein ◽

Isoelectric Focusing ◽

Clinical Laboratory ◽

Normal Serum ◽

Protein Electrophoresis ◽

Serum Protein Electrophoresis ◽

Oligoclonal Bands ◽

Abnormal Protein ◽

Monoclonal Protein

AbstractA 62-year-old diabetic man with prostate cancer first presented to our clinical laboratory in 2003 with a normal serum protein electrophoresis and immunofixation. In March 2009 he was diagnosed with an IgG κ myeloma. He underwent treatment and went into remission with the original paraprotein band being undetectable. Over the following 5 years, he developed oligoclonal bands and then eventually relapsed. Serum protein electrophoresis and immunofixation were inconclusive, however, isoelectrofocusing identified the oligoclonal pattern then the return of the original band, indicating relapse. This case illustrates the usefulness of an isoelectric focusing method to correctly determine clonality of small abnormal protein bands. It also highlights the need for appropriate commenting on reported results so that they are not confusing for clinicians.

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

Blood ◽

10.1182/blood.v128.22.3254.3254 ◽

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.

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Electrophoretic patterns post daratumumab

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/0004563217710489 ◽

2017 ◽

Vol 55 (2) ◽

pp. 299-301 ◽

Cited By ~ 1

Author(s):

Joanna Sheldon ◽

Rachel D Wheeler ◽

Ray Powles

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Serum Protein ◽

Protein Electrophoresis ◽

Serum Protein Electrophoresis ◽

Serum Samples ◽

Monoclonal Protein ◽

Electrophoretic Patterns ◽

Small Band ◽

Human Igg1

Background Daratumumab (Darzalex) is a human IgG1 kappa monoclonal antibody targeting CD38 that has been recently approved for the treatment of refractory multiple myeloma. As it is a monoclonal protein, it can be detected on routine serum protein electrophoresis and by immunofixation. Methods Serum samples from four patients were analysed by serum protein electrophoresis immediately pre- and post-treatment with daratumumab. Results For all four patients, daratumumab was visible on serum protein electrophoresis as an additional small band (approximately 1 g/L) in the slow gamma region. Conclusion Diagnostic laboratories should be aware that daratumumab can be detected on routine serum protein electrophoresis of myeloma patients and should liaise closely with clinicians to ensure the presence of daratumumab is not misinterpreted as development of a new monoclonal protein.

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Vertical cutoff methods in serum protein electrophoresis for the measurement of monoclonal protein concentrations: Which is best?

Clinica Chimica Acta ◽

10.1016/j.cca.2020.07.051 ◽

2020 ◽

Vol 510 ◽

pp. 573-580

Author(s):

MC. Cárdenas Fernández ◽

D. Pérez Surribas ◽

R. Pérez Garay ◽

J. Jiménez Jiménez ◽

A. Gella Concustell ◽

...

Keyword(s):

Serum Protein ◽

Protein Electrophoresis ◽

Serum Protein Electrophoresis ◽

Monoclonal Protein

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Comparison of Kappa & Lambda Freelite to Total Kappa & Lambda Immunoassays for the Detection of Monoclonal Gammopathies, Both As Standalone Tests and Alongside Serum Protein Electrophoresis

Blood ◽

10.1182/blood.v124.21.5705.5705 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5705-5705 ◽

Cited By ~ 1

Author(s):

Vania T M Hungria ◽

Petros Kampanis ◽

Mark T Drayson ◽

Tim Plant ◽

Edvan de Queiroz Crusoe ◽

...

Keyword(s):

B Cell ◽

Serum Protein ◽

Light Chain ◽

Protein Electrophoresis ◽

Immunoglobulin M ◽

Serum Protein Electrophoresis ◽

Monoclonal Protein ◽

Monoclonal Gammopathies ◽

Total Light ◽

Age Range

Abstract Introduction Monoclonal gammopathies are a disparate group of diseases from benign to malignant which are characterised by the proliferation of a single B cell clone that produces a homogeneous monoclonal immunoglobulin (M-Ig). The method of detection and quantification of the M-Ig will depend upon whether it is an intact immunoglobulin or present as serum free light chain only. Historically serum (SPEP) and urine (UPEP) electrophoresis were considered the gold standard for identifying intact M-Ig and FLC respectively. In 2001 the introduction of the Freelite test changed the diagnostic and monitoring paradigm. The assay is now recommended as a tool to diagnose and monitor patients with B cell disorders. However, the assay is sometimes confused with monospecific immunoassays for measuring total kappa and total lambda. Here we compare kappa & lambda Freelite with total kappa & lambda immunoassays alongside SPEP as tools to identify patients with monoclonal gammopathies. Materials and Methods Sera from 102 blood donors (55 males and 47 females, age range 18-67 years) and 103 patients with light chain associated gammopathies (44 males and 59 females, age range 38 to 88 years, 60 kappa / 43 lambda)taken during the course of their treatment were available. The sera was analysed retrospectively with FreeliteTM (The Binding Site Ltd, Birmingham, UK) on a SPAPLUSand Total Kappa &Lambda nephelometricassays (Beckman Coulter, USA) on an Immage.Monoclonality was identified by results falling outside of manufacturers normal ratio ranges (Freelite 0.256-1.65, Total light chain 1.53-3.29). Serum protein electrophoresis was performed and unexpectedly positive or negative results were assessed using immunofixation on the Hydrasys electrophoretic system (Sebia, France). Results Monoclonal production was identified in 80/103 light chain associated gammopathiesby Freelite, negative IFE confirmed the absence of monoclonal protein in 22/23 patients with normal FLC kappa/lambda ratios and 1/23 patients had an IgG lambda intact immunoglobulin. SPEP was positive in 30/103 patients, with total kappa/lambda immunoassays detecting monoclonal protein in just 26/103 samples. Freelite was positive in 6/102, SPEP in 2/102 and total kappa/lambda in 8/102 normal blood donor sera. Interestingly, in 1 patient with an abnormal FLC ratio and total kappa/lambda result had a lambda light chain identified using IFE.Comparisons between the performances of Freelite, Freelite + SPEP, Total kappa/lambda and total kappa/lambda + SPEP are shown in table 1). Conclusion In keeping with Kyle et al (1999) our study confirms the limitations of total kappa / lambda assays as tools to identify M-Igs. This is the first study looking to apply the recommended algorithm of Freelite + SPEP to the total kappa/lambda assays. The addition of SPEP to total kappa/lambda assays improved the performance to detect abnormalities, but even combined they were neither as sensitive, specific or accurate as the Freelite assay. Given the limitations of the total light chain assays identified in our study, it is important that physicians are aware of which assay is being utilised; one easy method to discriminate would be to look at the normal range of the assay being reported. Table 1: Comparison of Freelite, Freelite + SPEP, Total kappa/lambda, Total kappa/lambda + SPEP Freelite Freelite + SPEP Total Total + SPEP Sensitivity 77.67 81.55 25.24 43.69 Specificity 94.12 92.16 92.16 91.18 PPV 93.02 91.30 76.47 83.33 NPV 80.67 83.19 54.97 61.59 Accuracy 85.85 86.83 58.54 67.32 Disclosures No relevant conflicts of interest to declare.

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Diagnosing Paraproteinemic Keratopathy: A Case Report

Case Reports in Ophthalmology ◽

10.1159/000514375 ◽

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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