Laurence Lutteri

Monomeric Calgranulins Measured by SELDI-TOF Mass Spectrometry and Calprotectin Measured by ELISA as Biomarkers in Arthritis

BACKGROUND SELDI-TOF mass spectrometry (MS) is a high-throughput proteomic approach with potential for identifying novel forms of serum biomarkers of arthritis. METHODS We used SELDI-TOF MS to analyze serum samples from patients with various forms of inflammatory arthritis. Several protein profiles were collected on different Bio-Rad Laboratories ProteinChip arrays (CM10 and IMAC-Cu(2+)) and were evaluated statistically to select potential biomarkers. RESULTS SELDI-TOF MS analyses identified several calgranulin proteins [S100A8 (calgranulin A), S100A9 (calgranulin B), S100A9*, and S100A12 (calgranulin C)], serum amyloid A (SAA), SAA des-Arg (SAA-R), and SAA des-Arg/des-Ser (SAA-RS) as biomarkers and confirmed the results with other techniques, such as western blotting, immunoprecipitation, and nano-LC-MS/MS. The S100 proteins were all able to significantly differentiate samples from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) from those of patients with inflammatory bowel diseases used as an inflammatory control (IC) group, whereas the SAA, SAA-R, and SAA-RS proteins were not, with the exception of AS. The 4 S100 proteins were coproduced in all of the pathologies and were significantly correlated with the plasma calprotectin concentration; however, these S100 proteins were correlated with the SAA peak intensities only in the RA and IC patient groups. In RA, these S100 proteins (except for S100A12) were significantly correlated with the serum concentrations of C-reactive protein, matrix metalloproteinase 3, and anti-cyclic citrullinated peptide and with the Disease Activity Score (DAS(28)). CONCLUSIONS The SELDI-TOF MS technology is a powerful approach for analyzing the status of monomeric, truncated, or posttranslationally modified forms of arthritis biomarkers, such as the S100A8, S100A9, S100A12, and SAA proteins. The fact that the SELDI-TOF MS data were correlated with results obtained with the classic calprotectin ELISA test supports the reliability of this new proteomic technique.

Proteomics for prediction and characterization of response to infliximab in Crohn's disease: A pilot study

OBJECTIVES Infliximab is the first anti-TNFalpha accepted by the Food and Drug Administration for use in inflammatory bowel disease treatment. Few clinical, biological and genetic factors tend to predict response in Crohns disease (CD) patient subcategories, none widely predicting response to infliximab. DESIGN AND METHODS Twenty CD patients showing clinical response or non response to infliximab were used for serum proteomic profiling on Surface Enhanced Lazer Desorption Ionisation-Time of Flight-Mass Spectrometry (SELDI-TOF-MS), each before and after treatment. Univariate and multivariate data analysis were performed for prediction and characterization of response to infliximab. RESULTS We obtained a model of classification predicting response to treatment and selected relevant potential biomarkers, among which platelet aggregation factor 4 (PF4). We quantified PF4, sCD40L and IL-6 by ELISA for correlation studies. CONCLUSIONS This first proteomic pilot study on response to infliximab in CD suggests association between platelet metabolism and response to infliximab and requires validation studies on a larger cohort of patients.

Analytical study of three cystatin C assays and their impact on cystatin C-based GFR-prediction equations.

BACKGROUND Cystatin C-based equations are used to estimate GFR. However, three cystatin C immunoassays are on the market. Difference in cystatin C assays could have strong consequences on the accuracy and precision of cystatin C-based equations. We have performed an analytical study of these three assays and studied potential differences between assays on the precision of cystatin C-based equations. METHODS We have studied imprecision, recovery, linearity and interferences of the three immunoassays (nephelometric assay from Siemens and turbidimetric assays from Dako and Gentian). The impact of differences in cystatin C assays has been studied for the equations published by Levey (Siemens assay) and Grubb (Dako assay). RESULTS Analytical performance of the Dako assay is slightly less high. For cystatin C values below 2.5 mg/L, no statistical difference is found between results given by the Dako and the Gentian assays. So, both assays can be used in the Grubb equation. Cystatin C results are different with the Siemens assay. The Levey equation, built with the Siemens assay, can only be used with cystatin C values measured with this assay. Using the Dako or Gentian assay results in the Levey equation can lead to differences in estimating GFR up to 6 mL/min/1.73 m2. Differences can reach 9.5 mL/min/1.73 m2 if the Siemens assay is used in the Grubb equation. CONCLUSION The Siemens and Gentian assays seem analytically more valid than the Dako assay for cystatin C determination. Differences in cystatin C assays can lead to significant differences in cystatin C-based equations. However, these differences seem less important than the differences observed with creatinine and creatinine-based equations.

Discrepancies between creatinine-based and cystatin C-based equations in estimating prevalence of stage 3 chronic kidney disease in an elderly population

Background. The prevalence of stage 3 chronic kidney disease (CKD) is increasing, calculated using the modification of diet in renal disease (MDRD) study equation for estimating glomerular filtration rate (GFR). Cystatin C‐based equations are also being used to estimate GFR. Using creatinine‐based and cystatin C‐based equations, the aim of our study was to measure the difference in prevalence of stage 3 CKD in a population. Methods. CKD screening is organized in the Province of Liège, Belgium. On a voluntary basis, people aged between 45 and 75 years are invited for screening. GFR is estimated using the MDRD study equation and by the three recent cystatin C‐based equations proposed by Leveys group. The Levey 1 equation is based on cystatin C only and the Levey 2 equation on cystatin C corrected for age and sex. The Levey 3 equation combines cystatin C, creatinine, age and sex. Results. The population screened comprised 754 people. Cystatin C is highly correlated with creatinine (r = 0.6196, p<0.0001). Prevalence of stage 3 CKD when GFR is estimated by the MDRD equation study is 17.2 %, which is significantly and much higher than the prevalence obtained when cystatin C‐based equations are used. Indeed, prevalence is 2 %, 3.3 % and 5.8 % with the Levey 1, 2 and 3 equations, respectively. Conclusions. The prevalence of stage 3 CKD varies strongly following the method used for estimating GFR, creatinine‐based or cystatin C‐based equations. Such discrepancies must be confirmed and explained in additional studies using GFR measured with a reference method.

Apolipoprotein-A1 as a Damage-Associated Molecular Patterns Protein in Osteoarthritis: Ex Vivo and In Vitro Pro-Inflammatory Properties

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.

Anticorps anti-gliadines déamidées et maladie cœliaque : données actuelles et évaluation des faux positifs de cinq trousses de dosage

Recently, anti-deamidated gliadin antibodies were proposed for the serological diagnosis of celiac disease. We evaluate the specificity of different anti-deamidated gliadin antibodies ELISA in comparison with conventional anti-native gliadin kits. Serum samples from 46 non celiac patients were analyzed by five different quantitative ELISA for anti-native gliadin, anti-deamidated gliadin and anti-transglutaminase neo-epitope antibodies together with a screening ELISA. Twenty-four percent of the patients demonstrated anti-native gliadin IgA and 63% IgG antibodies. Using anti-deamidated gliadin antibodies, the number of false positive IgA and, particularly, IgG results, markedly decreased in the non celiac patients: 21 and 24% respectively with anti-Gliadin (GAF-3X) Euroimmun kit, 7 and 26% with Bindazyme Human Anti-Gliadin (MGP) The Binding Site kit and 0 and 41% with Celiac G+ Immco kit. The new assay which makes use of the physiological complex of tissue transglutaminase cross-linked with deamidated gliadin peptides, called neo-epitope, did not improve the differential diagnosis of celiac disease with 30% of false positive results in IgG (2% in IgA). Using the Inova screening kit, a positive result for IgA and/or IgG anti-deamidated gliadin and/or anti-tissue transglutaminase antibodies was obtained in 24% of the non celiac patients. In conclusion, our study assessed the superiority, in terms of specificity, of anti-deamidated gliadin antibodies, over the conventional anti-gliadin antibodies for the differential diagnosis of celiac disease.

Reference ranges of the Sebia free light chain ratio in patients with chronic kidney disease

*Corresponding author: Joannes F.M. Jacobs, PhD, MD, Department of Laboratory Medicine, Radboud University Medical Center, Laboratory Medical Immunology (route 469), Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands, Phone: +31 (0)24-3617414, Fax: +31 (0)24-3619415, E-mail: H.Jacobs@Radboudumc.nl Laurence Lutteri: Laboratory of Clinical Chemistry, CHU Hospital of Liege, Liege, Belgium Letter to the Editor

Evaluation of the new Sebia free light chain assay using the AP22 ELITE instrument

BACKGROUND Serum kappa and lambda free light chains (FLC) are useful in the diagnosis, prognosis and monitoring of patients with monoclonal gammopathies. The Binding Site and Siemens were the only suppliers of kits for these analyses until recently, when Sebia introduced an enzyme-linked immunosorbent assay (ELISA) on an automated instrument, DAS AP22 ELITE. METHOD Samples from routine analysis, controls and chronic kidney disease (CKD) patients were tested using the automated version of Sebia FLC ELISA with the AP22 ELITE and results were compared with Freelite on the SPA PLUS (The Binding Site). RESULTS Sebia FLC ELISA showed a good performance using the AP22 ELITE. A concordance of 82% was found with the results obtained with Freelite. Sebia FLC is a reproducible assay, requiring less retesting than Freelite thanks to a broader range. Earlier findings that the results obtained are closer to the FLC monoclonal band measured by electrophoresis were confirmed. Higher kappa and lambda values obtained in CKD individuals were also shown, confirming that a kappa/lambda FLC ratio should be introduced by Sebia for CKD patients, as with The Binding Site. CONCLUSIONS Sebia put forward new technology that automatically measures free light chains. This technique is suitable for routine use; however, the results cannot be used interchangeably with Freelite kits.

Adipsic diabetes insipidus revealing a bifocal intracranial germinoma

Adipsic diabetes insipidus is a rare complication of intracranial tumors in which impaired antidiuretic hormone secretion is associated with the loss of thirst sensation. Here, we present the case of a patient with bifocal intracranial germinoma, diagnosed due to symptoms mainly caused by adipsic diabetes insipidus. This is, to our knowledge, the first case of adipsic diabetes insipidus revealing an intracranial germinoma reported in the literature. We describe the diagnostic procedures and the three-year follow-up of this patient. Management of intracranial germ-cell tumors is made complex by the wide range of histological features. Although germinomas have a generally better prognosis than most nongerminomatous tumors, they can have severe or even life-threatening presentations. Adipsic diabetes insipidus is one such severe presentation and its rarity can make it difficult to recognize and manage. Awareness of this potential entity is therefore important for clinical practice.

Hypothyroïdie infraclinique non autoimmune et statut iodé : étude prospective d’intervention

Objectifs.– La déficience iodée chronique s’associe à une élévation de la TSH comme dans l’hypothyroïdie infraclinique. Nous étudions la réponse biologique et immunitaire à une supplémentation de 100 g/j d’iodide de potassium chez des patients adressés pour suspicion d’hypothyroïdie. Patients et méthodes.– Une série de 31 patients (24 F/7 H), âgés de 43 ± 18 ans sont adressés pour suspicion d’hypothyroïdie, avec une TSH > 2,5 mUI/mL et sans autoimmunité thyroïdienne. Avant l’administration de 100 g d’iode/j (Iodid