Serum free light chain quantitative assays: Dilemma of a biomarker.

BACKGROUND: Serum free light chains detection assays are consistently meeting greater interest for the diagnosis and monitoring of monoclonal gammopathies and plasma cell dyscrasias. Nowadays, there are neither standardized methods nor reference material for the determination of free light chains; for this reason, it is important to compare two different assays used in clinical laboratory. METHODS: We evaluated 300 serum samples from patients with B-cell disorders and compared the analytical performances of both assay. Each test was assayed on both testing platforms (Siemens Dade Behring BN II Nephelometer and SPAPLUS by The Binding Site). κ/λ ratios were determined and compared. Results were analyzed by Passing-Bablok and Bland-Altman plots to evaluate comparability of the two techniques and to determine bias. RESULTS: The reproducibility of both assays is acceptable, reaching minimum and desirable analytical goals derived from biological variability. However, values are not interchangeable between systems. This study shows that the two systems do not allow results to be transferred from one method to the other even if they display good agreement. CONCLUSION: Our study highlights the importance of elaborating an international standard for free light chains quantification in order to offer homogeneous results as well as guarantee harmonization of values among laboratories. Moreover, the assays should be validated in specific patient groups to determine that they are clinically fit for purpose.

Comparison of Fully Automated and Semiautomated Systems for Protein Immunofixation Electrophoresis.

BACKGROUND: In order to establish a diagnosis of monoclonal gammopathy, it is necessary to detect and identify monoclonal components. To confirm the immunological nature of the proteins, the next step is to define their composition in heavy and light chains using immunofixation. The purpose of this study was to compare two different instruments, one semiautomated and the other fully automated for serum and urine immunofixation. METHODS: We selected 150 sera and 100 urines from patients admitted for routine analysis, which were analyzed by immunofixation to characterize monoclonal components. RESULTS AND CONCLUSION: Comparison study showed a difference in the identification of small monoclonal components and hypogammaglobulinemia, in serum and urine, between the two analyzers. We also observed a difference in the length of the electrophoretic pattern that is of considerable importance as it leads to a better resolution of the gamma region, allowing to identify even the smallest monoclonal component that can be easily hide in an oligoclonal pattern. For this reason, there is need to ameliorate commercial immunofixation assays. It is essential to improve data harmonization and standardize measurement procedures in order to guarantee a correct diagnosis for the right patient care.

Reactive oxygen metabolites (ROMs) are associated with cardiovascular disease in chronic hemodialysis patients.

BACKGROUND: The aim of our study was to measure reactive oxygen metabolites (ROMs) in chronic hemodialysis (HD) patients and evaluate the possible association with cardiovascular disease (CVD) and mortality. METHODS: We measured ROMs in 76 HD patients and correlated with CVD, cardiovascular (CV) events in the follow-up and all-cause and CVD-related mortality. RESULTS: The levels of ROMs presented a median value of 270 (238.2-303.2) CARR U (interquartile range). We created a ROC curve (ROMs levels vs. CVD) and we identified a cut-off point of 273 CARR U. Patients with ROMs levels ≥273 CARR U were significantly older, had higher C-reactive protein levels and lower creatinine concentrations. The prevalence of CVD was higher in patients with ROMs levels ≥273 (87.1%) than in those with ROMs levels <273 CARR U (17.7%; p<0.0001). ROMs levels were significantly higher in patients with CVD (317±63.8) than in those without (242.7±49.1; p<0.0001). At multiple regression analysis, age, creatinine and C-reactive protein were independent factors associated with ROMs. At multiple logistic regression analysis the association between ROMs and CVD was independent (OR: 1.02, 95% CI: 1.00-1.05; p=0.03). Twenty six patients developed cardiovascular (CV) events during the follow-up. Of these, seven were in the group with ROMs levels <273 CARR U and 19 in the group with ROMs levels ≥273 CARR U. The logistic regression analysis showed that both age (OR: 1.06, 95% CI: 1.01-1.12; p=0.013) and ROMs levels (OR: 1.10, 95% CI: 1.00-1.02; p=0.045) were independently associated with CV events in the follow-up. CONCLUSIONS: ROMs are independently associated with CVD and predict CV events in chronic HD patients.

Anorexia and plasma levels of free tryptophan, branched chain amino acids, and ghrelin in hemodialysis patients.

OBJECTIVE: The aim of the present cross-sectional study was to assess appetite and to examine at the same time the associations between self-reported appetite and orexigen (ghrelin) and anorexigen (free tryptophan, free tryptophan/large neutral amino acid ratios, low branched chain amino acid levels) substances in chronic hemodialysis patients. DESIGN: Cross-sectional study. SETTING: Patients were recruited from the Catholic University Outpatient Dialysis Clinic. PATIENTS: A total of 59 patients (32 men and 27 women) were included in this study. The mean age was 63.7 +/- 13.9 years, and the mean dialytic age was 6.6 +/- 5.1 years. Their mean body mass index of the study population was 25.1 +/- 4.1 kg/m(2). METHODS: The first question of the Hemodialysis (HEMO) Study Appetite questionnaire was used to assess the appetite of the hemodialysis patients. The multiple-choice answers for the first question, "During the past week, how would you rate your appetite?" were (1) very good, (2) good, (3) fair, (4) poor, or (5) very poor. Plasma amino acid concentrations were measured with the use of liquid chromatography. Ghrelin levels were measured with Ghrelin-RIA (Mediagnost). RESULTS: According to the questionnaire, in 16 of 59 (27.1%) hemodialysis patients, their appetite was very good (group 1); in 15 (25.4%), it was good (group 2); in another 15 (25.4%), it was fair (group 3); in 10 (16.9%), it was poor; and in 3 (5%), it was very poor. For statistical purposes, patients with a poor or very poor appetite were pooled together into a single group (group 4). Body mass index and serum albumin were significantly lower in patients with a fair and poor/very poor appetite than in patients with a very good or good appetite. According to the Subjective Global Assessment, all patients in groups A and B were well-nourished, whereas most patients in groups C (60%) and D (68%) were severely malnourished. Most of the comorbid conditions were significantly higher in patients of groups C and D. Branched chain amino acids were significantly lower in patients with a fair or poor/very poor appetite with respect to patients with a very good or good appetite. Free tryptophan levels were similar in the four groups of patients. The molar sum in plasma of the other large neutral amino acids (valine, leucine, isoleucine, tyrosin, phenylalanine) (large neutral amino acids) tended to be lower in patients with a fair and poor/very poor appetite than in patients with a very good or good appetite. However, the free tryptophan/large neutral amino acid ratio did not change significantly according to the appetite reported by the patients. Mean ghrelin levels were significantly higher in patients of group D than in other groups and in patients of groups B and C than in patients of group A. CONCLUSIONS: The present study shows that poor appetite is associated with significantly lower branched chain amino acid levels but not with higher free tryptophan levels and higher free tryptophan/large neutral amino acid ratios in hemodialysis patients. In addition, significantly higher levels of ghrelin have been observed in patients with a poor/very poor appetite.

Autoimmunity and lymphoproliferation markers in naïve HCV-RNA positive patients without clinical evidences of autoimmune/lymphoproliferative disorders.

BACKGROUND: HCV can lead to both chronic liver disease and B-cell lymphoproliferative disorders. A strong association exists between HCV and mixed cryoglobulinaemia (MC). METHODS: Anti-nuclear antibodies (ANA), rheumatoid factor Ig-G (RF-IgG), free light chain κ and λ (FLC-κ, FLC-λ) levels and κ/λ ratio were evaluated in 50/420 subjects unexpectedly resulted anti-HCV positive after routine screenings for non-hepathological procedures. RESULTS: Three/fifty patients had HCV-RNA undetectable in the serum and were excluded from the analysis. Thirty-nine/fifty patients had laboratory evidence of circulating cryoglobulins without liver disease and MC-related symptoms. Among them, 17 resulted ANA-positive. The mean cryocrit was higher in ANA-positive patients, while no other demographic/clinical differences were observed between the groups. Significantly higher levels of RF-IgG were observed in ANA-positive vs ANA-negative patients. κ and λ FLC were higher in ANA-positive patients. A ROC analysis, based on ANA-positivity vs ANA-negativity, confirmed a high sensitivity and specificity of RF-IgG test. CONCLUSIONS: Published data concerning MC come mostly from symptomatic vasculitis. We analyzed HCV-patients without MC symptoms, founding cryoglobulins in the majority of them. The increased levels of FR-IgG and FLC in CGs-ANA-positive patients, suggest these test could be used to identify a state of silent autoimmune and/or lymphoproliferative condition before the transition to a frank disease in naïve HCV-patients without symptoms of extrahepatic manifestations.

Anti-nuclear antibody detection in cryoprecipitates: distinctive patterns in hepatitis C virus-infected patients.

BACKGROUND: Anti-nuclear antibodies are immunoglobulins directed against nuclear antigens. They are associated with many autoimmune disorders, but are frequently found in patients infected with hepatitis C virus, possibly indicating an underlying common origin. Likewise, mixed cryoglobulinemia often accompanies autoimmune diseases and hepatitis C infection. AIM: To compare anti-nuclear antibodies and immunoglobulin content of cryoprecipitates from hepatitis C virus-positive patients in order to assess their predictive value in the onset of hepatitis C virus-driven extrahepatic disorders. METHODS: Serum from 40 hepatitis C virus-positive patients and 50 controls with rheumatoid arthritis was processed for cryoglobulin detection: all subjects presented with Type III mixed cryoglobulinemia. Immunoglobulin content and immunoglobulin subclasses of cryoprecipitates were assessed by immunofixation and tested by ELISA for rheumatoid factor. Cryoprecipitates were also analysed for anti-nuclear antibodies by indirect immuno-fluorescence to identify specific patterns typical of each condition. RESULTS: Anti-nuclear antibody patterns differed significantly; 26 infected subjects (65%) were IgG3 positive: of these, 25 were also anti-nuclear antibody-positive (96.1%). CONCLUSIONS: IgG3 are autoreactive clones unrelated to viral recognition and possibly involved in autoimmune disorders. Altogether, these results may represent useful diagnostic device for early detection of hepatitis C virus-induced autoimmune diseases.

Relationship between circulating syndecan-1 levels (CD138s) and serum free light chains in monoclonal gammopathies.

BACKGROUND: Monoclonal gammopathies encompass a wide range of diseases characterized by the monoclonal expansion of a B-cell clone. Despite emerging therapeutic strategies, chances of survival of patients who are affected are still scarce, which implies that new tools are necessary not only for the diagnosis but also for the follow-up of patients affected by such diseases. In this context, the use of free light chains (FLCs) has been incorporated into many guidelines. Likewise, tumor microenvironment is consistently gaining importance as role player in tumor pathogenesis. Specifically, Syndecan-1 (CD138), a heparan-sulfate proteoglycan is attracting interests as it is highly expressed and shed by myeloma plasma-cells. The aim of our study was to analyze CD138 levels in the serum of patients affected by multiple myeloma or light chain only disease, and to compare the values obtained with free light chain (FLC) kappa, lambda and FLC ratio in both groups of patients. METHODS: 84 patients affected by Multiple Myeloma and Light Chain Myeloma were recruited for this study. Serum CD138 was assessed by ELISA (Diaclone Research, France) and FLC values were quantified by nephelometry (Freelite TM Human Kappa and Lambda Free Kits, The Binding Site, UK). Data was analyzed by GraphPad Prism software and Statgraph. RESULTS: We observed higher CD138 mean values in myeloma patients compared to the light chain only myeloma group. A positive linear regression of CD138 and FLC was observed in the light chain only cohort as opposed to myeloma patients which show an inverse trend. CONCLUSIONS: The study highlighted an existing relationship between FLCs and CD138 and wishes to seek also a correlation in order to rapidly and efficiently perform diagnosis and different diagnostic schemes.