Systemic complement activation levels in Stargardt disease.

PURPOSE: Preclinical research provides evidence for the complement system as a potential common pathway in Stargardt disease (STGD1) and age-related macular degeneration (AMD) leading to retinal pigment epithelium (RPE) loss. However, systemic complement activation has not yet been assessed in STGD1 patients. We conducted a cross-sectional case-control study to assess systemic complement activation in STGD1 patients and its association with disease severity. METHODS: Systemic concentrations of complement component C3 and its degradation product C3d were compared between 80 STGD1 patients and 80 controls that were frequency matched for age and sex. The C3d/C3 ratio was used as parameter of systemic complement activation. Within the STGD1 cohort, we additionally examined the association between the C3d/C3 ratio, demographic and behavioural factors (age, sex, smoking and BMI), and measures of disease severity (age at onset, visual acuity, and area of atrophy). RESULTS: The C3d/C3 ratio did not significantly differ between patients (mean C3d/C3 ratio 3.5±1.4) and controls (mean C3d/C3 ratio 3.6±1.0), mean difference -0.156 (p = 0.804, independent samples t-test). The overall effect size was 8% (95% confidence interval, 3-15%). Elevated C3d/C3 ratios (>8.1) were found in three patients who all had a concomitant inflammatory condition at the time of blood draw. Within the patient cohort, C3 levels were associated with sex (mean difference -134, p = 0.001, independent samples t-test) and BMI (correlation coefficient 0.463, p<0.001, Spearman's Correlation). CONCLUSIONS: Systemic complement levels were not elevated in STGD1 patients compared to age and sex matched controls and was not associated with STGD1 severity. Considering the continued absent proof of a systemic contribution of the complement system to RPE loss in STGD1 patients, we hypothesize that complement activation in STGD1 is more likely a local process. In light of upcoming complement-targeted therapies, further studies are needed that measure complement levels in the eye of STGD1 patients.

External quality assessment of M-protein diagnostics: a realistic impression of the accuracy and precision of M-protein quantification.

OBJECTIVES: Studies that investigate the accuracy and precision of M-protein quantification are scarce. These studies are prone to give a biased view, since they are exclusively performed by institutions with international top-expertise on M-protein diagnostics. To obtain a realistic impression of the accuracy and precision of M-protein quantification, we studied results of 73 laboratories participating in the Dutch External Quality Assessment (EQA) program for M-protein diagnostics. METHODS: To measure accuracy, healthy serum was spiked with respectively 1 and 5 g/L human IgG-kappa monoclonal antibody daratumumab. To measure precision, five sera were selected to be repeatedly send to all blinded EQA-participants. RESULTS: The reported concentrations for the EQA-sample spiked with 5 g/L daratumumab ranged from 2.6 to 8.0 g/L (mean 4.9 g/L, between-laboratory CV = 23%). 98% of the participants detected and correctly characterized the 1 g/L daratumumab band. Both the accuracy (mean 1.7 g/L) and precision (between-laboratory CV = 46%) of this 1 g/L M-protein was poor. In the five EQA-samples that were repeatedly send to the same 73 participating laboratories, between-laboratory precision (mean CV = 25%) was significantly different than the within-laboratory precision (mean CV = 12%). Relatively poor precision was observed in sera with small M-proteins. CONCLUSIONS: The EQA-data reveal a large variation in reported M-protein concentrations between different laboratories. In contrast, a satisfactory within-laboratory precision was observed when the same sample was repeatedly analyzed. The M-protein concentration is correlated with both accuracy and precision. These data indicate that M-protein quantification to monitor patients is appropriate, when subsequent testing is performed within the same laboratory.

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

Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are standard tools for multiple myeloma (MM) routine diagnostics. M-protein is a biomarker for MM that can be quantified with SPE and characterized with IFE. We have investigated combining SPE/IFE with targeted mass spectrometry (MS) to detect and quantify the M-protein. SPE-MS assay offers the possibility to detect M-protein with higher sensitivity than SPE/IFE, which could lead to better analysis of minimal residual disease in clinical laboratories. In addition, analysis of archived SPE gels could be used for retrospective MM studies. We have investigated two different approaches of measuring M-protein and therapeutic monoclonal antibodies (t-mAbs) from SPE/IFE gels. After extracting proteotypic peptides from the gel, they can be quantified using stable isotope labeled (SIL) peptides and measured by Orbitrap mass spectrometry. Alternatively, extracted peptides can be labeled with tandem mass tags (TMT). Both approaches are not hampered by the presence of t-mAbs. Using SIL peptides, limit of detection of the M-protein is approximately 100-fold better than with routine SPE/IFE. Using TMT labeling, M-protein can be compared in different samples from the same patient. We have successfully measured M-protein proteotypic peptides extracted from the SPE/IFE gels utilizing SIL peptides and TMT.

Method comparison of four clinically available assays for serum free light chain analysis.

Background Serum free light chain (sFLC) measurements are increasingly important in the context of screening for monoclonal gammopathies, prognostic stratification and monitoring of therapy responses. In this study we have performed a method comparison of four sFLC assays that are currently available for routine clinical use. Methods In a retrospective study, sFLC analyses were performed on a cohort that included 139 patients with various monoclonal gammopathies and 54 control sera without an M-protein. Method comparisons of the following four FLC assays were performed: Freelite (Binding Site), N-Latex FLC (Siemens), Seralite (Abingdon Health) and Sebia FLC (Sebia). Results Bland-Altman agreement analysis showed biases varying between -0.1 and 16.2 mg/L for κFLC, -6.0 and 6.8 mg/L for λFLC and -0.04 and 0.38 for the ratio of the involved to uninvolved FLC. Strong agreements were observed for FLC-concentrations below 100 mg/L. The clinical concordance of the κ/λFLC-ratio of the four methods varied between 86% and 92%. Significant quantitative differences were observed between the different methods, mainly in sera with high FLC concentrations. Most assays consistently overestimated FLC concentrations compared to SPE. Conclusions Good overall clinical concordances were observed between the four sFLC assays that were compared in this study. Although good agreements were observed between the FLC assays, significant absolute differences in FLC concentrations in individual patients can be seen, particularly at higher FLC concentrations. Because of inequivalent absolute sFLC values between the methods in individual patients, none of the four sFLC assays can be used interchangeably.

Development of a Targeted Mass-Spectrometry Serum Assay To Quantify M-Protein in the Presence of Therapeutic Monoclonal Antibodies.

M-protein diagnostics can be compromised for patients receiving therapeutic monoclonal antibodies as treatment in multiple myeloma. Conventional techniques are often not able to distinguish between M-proteins and therapeutic monoclonal antibodies administered to the patient. This may prevent correct response assessment and can lead to overtreatment. We have developed a serum-based targeted mass-spectrometry assay to detect M-proteins, even in the presence of three therapeutic monoclonal antibodies (daratumumab, ipilimumab, and nivolumab). This assay can target proteotypic M-protein peptides as well as unique peptides derived from therapeutic monoclonal antibodies. We address the sensitivity in M-protein diagnostics and show that our mass-spectrometry assay is more than two orders of magnitude more sensitive than conventional M-protein diagnostics. The use of stable isotope-labeled peptides allows absolute quantification of the M-protein and increases the potential of assay standardization across multiple laboratories. Finally, we discuss the position of mass-spectrometry assays in monitoring minimal residual disease in multiple myeloma, which is currently dominated by molecular techniques based on plasma cell assessment that requires invasive bone marrow aspirations or biopsies.

Analytical validation of the Hevylite assays for M-protein quantification.

BACKGROUND: The heavy/light chain (HLC) immunoassay quantifies the different heavy chain/light chain combinations of each immunoglobulin (Ig) class. This makes the HLC assay suited to quantify monoclonal immunoglobulins (M-protein) and for monitoring of patients with monoclonal gammopathies. This method is particularly advantageous for those samples in which electrophoretic quantification of the M-protein is not possible. METHODS: In this study we tested the analytical performance of the HLC assay in 166 routine clinical samples and in 27 samples derived from the Dutch external quality assessment (EQA) for M-protein diagnostics (74 participating laboratories). Analytical accuracy was assessed by verification that the sum of the HLC-pairs equaled total Ig concentration. Sensitivity of the HLC assay was determined in a direct method comparison with immunofixation electrophoresis (IFE). RESULTS: Comparison of HLC data with routine Ig diagnostics in 27 EQA samples showed very good correlation for both the quantification of polyclonal and monoclonal IgG, IgA and IgM (Pearson correlations [r] were 0.94, 0.99 and 0.99, respectively; slopes were 0.94, 1.07 and 0.98, respectively). The overall concordance between IFE and the HLC ratio was high (93%) with a Cohen κ coefficient of 0.84. Discrepancies between both assays were mainly caused by the higher sensitivity of IFE to detect monoclonality. CONCLUSIONS: We conclude that the HLC assay is an accurate method to quantify M-proteins that can improve monitoring of M-proteins in the beta fraction that cannot be quantified using electrophoretic techniques.

Evaluation of a new free light chain ELISA assay: bringing coherence with electrophoretic methods.

BACKGROUND: Serum free light chain (sFLC) measurements are increasingly important in the context of screening for monoclonal gammopathies, prognostic stratification, and monitoring of therapy responses. At the same time, analytical limitations have been reported with the currently available nephelometric and turbidimetric sFLC assays. We have evaluated a new quantitative sFLC ELISA for its suitability in routine clinical use. METHODS: Reference ranges of the Sebia FLC assay were calculated from 208 controls. Assay interference, reproducibility, lot-to-lot variability, and linearity were assessed. Method comparison to the Freelite assay (Binding Site) was conducted by retrospective analysis of 501 patient sera. RESULTS: Reference ranges of the Sebia κ/λFLC-ratio were 0.37-1.44. We observed good sensitivity (1.5 mg/L) and linearity in both polyclonal and monoclonal sFLC samples and never experienced antigen excess. Sebia FLC reproducibility varied between 6.7% and 8.1% with good lot-to-lot consistency. Method comparison with Freelite showed the following correlations: κFLC R=0.94, λFLC R=0.92 and κ/λFLC-ratio R=0.96. The clinical concordance of the κ/λFLC-ratio of both methods was 94%. Significant quantitative differences were observed between both methods, mainly in sera with high FLC concentrations. The Sebia monoclonal FLC concentrations were coherent with those obtained by serum protein electrophoresis (SPE). Freelite monoclonal FLC concentrations were consistently higher, with a mean 12-fold overestimation compared to SPE. CONCLUSIONS: The Sebia FLC assay provides a novel platform for sensitive and accurate sFLC measurements. The Sebia FLC showed good clinical concordance with Freelite. Further studies are warranted to confirm the clinical value of this assay.