The relationship between atherosclerosis and dementia.

OBJECTIVE: The main objective is to confirm a hypothesis that atherosclerosis, through various mechanisms, considerably influences cognitive impairment and significantly increases the risk for developing dementia. Complete sample should be 920 individuals. The present study aimed to analyse epidemiological data from a questionnaire survey. METHODS: The work was carried out in the form of an epidemiological case control study. Subjects are enrolled in the study based on results of the following examinations carried out in neurology departments and outpatient centres during the project NU20-09-00119 from 2020 to 2023. Respondents were divided into four research groups according to the results of clinical examination for the presence of atherosclerosis and dementia. The survey was mainly concerned with risk factors for both atherosclerosis and dementia. It contained questions on lifestyle factors, cardiovascular risk factors, leisure activities, and hobbies. RESULTS: Analysis of the as yet incomplete sample of 877 subjects has yielded the following selected results: on average, 16% of subjects without dementia had primary education while the proportion was 45.2% in the group with both dementia and atherosclerosis. Subjects with dementia did mainly physical work. Low physical activity was more frequently noted in dementia groups (Group 2 - 54.4% and Group 3 - 47.2%) than in subjects without dementia (Group 1 - 19.6% and Group 4 - 25.8%). Coronary heart disease was more frequently reported by dementia patients (33.95%) than those without dementia (16.05%). CONCLUSION: Cognitively impaired individuals, in particular those with vascular cognitive impairment, have poorer quality of life and shorter survival. Risk factors contributing to such impairment are similar to those for ischaemic or haemorrhagic stroke. It may be concluded that most of the analysed risk factors play a role in the development of both atherosclerosis and dementia.

Detection of monoclonal free light chains by immunofixation electrophoresis and isoelectric focusing - comparison with the quantitative method of determination.

The study aimed to investigate free light chain (FLC) monoclonality in patients with an abnormal free kappa/lambda ratio (FLC ratio). Seventy serum samples with abnormal FLC ratio were examined using an immunoturbidimetry (Binding Site, SPA) and the two different enzyme-linked immunosorbent assays (1. Sebia diagnostic kit; 2. in house methods), the monoclonal or oligoclonal bands of (FLC) by immunofixation electrophoresis (IE) and isoelectric focusing followed by affinity immunoblotting (IEF/AIB). The reference interval was calculated by non-parametric percentile method. 5.7% of samples examined by IE were suspected of monoclonal character of FLCs, but subsequently monoclonality was refuted by more sensitive IEF/AIB method; 7%, resp. 2.9% of samples showed FLC kappa, resp. FLC lambda oligoclonal character of bands. A statistically significant dependence was found between FLC ratio (Sebia) and FLC ratio (SPA), rs = 0.510, p = .001. Kappa statistic evaluated a fair conformity between the FLC ratio (Sebia) and IEF/AIB (kappa = 0.468) and between FLC ratio (in house) and IEF/AIB (kappa = 0.300). The verified reference interval for FLC ratio (Binding Site) is between 0.35 and 2.18. The IEF/AIB is the most sensitive method to discriminate between monoclonal and oligoclonal bands of FLC. The Binding Site and Sebia diagnostic kits do not give consistent results. The Binding Site diagnostic kit provides more results above reference interval of FLC ratios. For routine decision on monoclonality of the FLC ratio (SPA) it is advisable to use a verified reference interval.

Neurofilament levels in patients with neurological diseases: A comparison of neurofilament light and heavy chain levels.

BACKGROUND: Neurofilaments are the major cytoskeletal components of neurons, and cell injury leads to their release into the surrounding area. The aim of this study was to compare the cerebrospinal fluid (CSF) and serum (S) concentrations of neurofilament light chains (NFLs) and phosphorylated neurofilament heavy chains (pNFHs). METHODS: Neurofilament concentrations were measured in CSF and S samples from 172 patients using three enzyme-linked immunosorbent assays. Excel, Stata version 13, MedCal version 17.9.7., and NCSS 2007 software were used for the statistical analysis. RESULTS: There was a statistically significant correlation between the concentrations of CSF NFL and CSF pNFH (rs  = 0.748; n = 89; P < 0.001), but Passing-Bablok regression showed systematic deviation between the values obtained using the two assays. This indicates that the assays were not interchangeable. CSF pNFH and S pNFH concentrations showed low correlation. The kappa statistic showed moderate conformity between CSF pNFH and CSF NFL concentrations (κ = 0.556). CONCLUSIONS: The CSF NFL and CSF pNFH assays gave clinically consistent results that reflected the degree of axonal damage, independent of any particular neurological diagnosis. The S pNFH assays had a lower predictive value due to the low correlation coefficient and the kappa index of the CSF pNFH method.

Quantitation of IgG kappa and IgG lambda in the cerebrospinal fluid by sandwich ELISA method.

IgG kappa and IgG lambda concentrations were quantified in 96 paired CSF and sera using Hevylite™ antibodies in an in-house developed sandwich ELISA method. In 56 of these samples, the results were compared with a qualitative isoelectric focusing/affinity-mediated immunoblotting assay for oligoclonal IgG kappa and IgG lambda. Normal IgG kappa/lambda ratio in the CSF was the same as in serum. In 19/33 patients with intrathecal oligoclonal IgG synthesis, skewed IgG kappa/lambda ratio was observed (increased in 16 and decreased in 3 cases). The analysis of light chain composition of intrathecally synthesised immunoglobulins could contribute to our understanding of intrathecal humoral immune response, although its diagnostic utility is limited.

Reference intervals of plasma matrix metalloproteinases 2, 3, and 9 and serum asymmetric dimethylarginine levels.

OBJECTIVE: The present study aimed to verify the reference intervals of plasma matrix metalloproteinases (MMPs) 2, 3, and 9 and serum asymmetric dimethylarginine (ADMA) in a healthy population with an average age corresponding to that of patients with cardiovascular diseases. METHODS: The study included 180 healthy volunteers. Plasma MMP-2, MMP-3, MMP-9, and serum ADMA levels were determined using an enzyme-linked immunosorbent assay. These levels were analyzed for association with age and gender. The Cbstat5, R software, and NCSS 2007 programs were used for statistical analysis. RESULTS: The average volunteer age was 47.4 years in the group in which MMP-3 and ADMA were analyzed, 40.3 years in the MMP-9 group, and 47.8 years for the MMP-2 group. Serum ADMA levels were determined to be independent of age and gender. Plasma MMP-2 levels were significantly correlated with age (p = 0.001), with lower levels detected in persons ≤ 49 years of age. Plasma MMP-3 was significantly associated with both age (p < 0.0001) and gender, with lower levels detected in persons of ≤ 47 years of age and among women. Plasma MMP-9 levels were not age dependent, but were associated with gender (p = 0.014), showing lower levels in women. CONCLUSIONS: Reference intervals of heparin-plasma MMP-2, MMP-3, and MMP-9 and serum ADMA levels were determined. MMP-2 and MMP-3 levels were found to be age dependent, and MMP-3 and MMP-9 levels were gender dependent.

A comparison of measles-rubella-zoster reaction, oligoclonal IgG bands, oligoclonal kappa free light chains and kappa index in multiple sclerosis.

BACKGROUND AND OBJECTIVES: To evaluate the diagnostic performance of the measles-rubella-zoster reaction (MRZR) in a large real-world multiple sclerosis (MS) cohort. Second, to compare MRZR with the determination of oligoclonal IgG bands (OCB), oligoclonal kappa free light chain bands (oKFLC), and the KFLC index. METHODS: A single-center retrospective study was conducted at the University Hospital Ostrava (Czech Republic). Patients were eligible if aged ≥18 years with a determined clinical diagnosis. IgG antibodies against measles (M), rubella (R), and varicella zoster (Z) viruses were determined in paired CSF and serum using ELISA and MRZR indicated as positive if at least two components had an antibody index >1.4. OCB and oKFLC were detected by means of isoelectric focusing, and KFLC CSF and serum concentrations for calculation of the KFLC index were determined immunochemically. RESULTS: A total of 1,751 patients were included in the analyzed data set, which comprised 379 MS patients and 1,372 non-MS controls. The frequency of positive MRZR was higher in MS than in non-MS cases (MS 32.2 % vs non-MS 2.8 %; p < 0.001). This corresponded to a specificity of 97.2 % (95 % CI 96.1-98.0) and sensitivity of 32.2 % (95 % CI 27.5-37.2) and overall accuracy of 83.1 % (95 % CI 81.3-84.8). In comparison, the highest sensitivity of 95.6% (95 % CI 93.0-97.5) was for OCB with specificity of 86.9 % (95 % CI 84.9-88.7), followed by oKFLC with sensitivity and specificity of 94.7 % (95 % CI 91.5-96.9) and 78.4% (95 % CI 75.7-80.8), respectively, and the KFLC index with sensitivity of 92.5 % (95 % CI 86.6-96.3) and specificity of 93.5 % (95 % CI 90.5-95.9). DISCUSSION: MRZR remains a very specific test for the diagnosis of MS but has low sensitivity, which disallows its independent use. In contrast, OCB showed the highest sensitivity and thus remains the gold standard for the diagnosis of MS.

Serum neurofilament light chain levels in patients with cognitive deficits and movement disorders: comparison of cerebrospinal and serum neurofilament light chain levels with other biomarkers.

Background: Serum neurofilament light chain (S NfL) is a non-specific marker of neuronal damage, including Alzheimer's disease (AD). We aimed to verify the reference interval (RI) of serum NfL using a highly sensitive ELISA, and to estimate the optimal cut-off value for neuronal damage. Our second objective was to compare NfL in cerebrospinal fluid (CSF) and serum (S) with the routine neurodegeneration biomarkers used in AD, and to assess their concentrations relative to the degree of cognitive deficit. Methods: Samples from 124 healthy volunteers were used to estimate the S NfL RI. For the comparison study, we used CSF and S samples from 112 patients with cognitive disorders. Cognitive functions were assessed using the mini-mental state examination. ELISA assays were used to determine the CSF and S NfL levels, CSF ß-amyloid peptide42 (Aß42), CSF ß-amyloid peptide40 (Aß40), CSF total tau protein (tTau), CSF phosphorylated tau protein (pTau), and CSF alpha-synuclein (αS). Results: The estimated RI of S NfL were 2.25-9.19 ng.L-1. The cut-off value of S NfL for assessing the degree of neuronal impairment was 10.5 ng.L-1. We found a moderate statistically significant correlation between S NfL and CSF Aß42 in the group with movement disorders, without dementia (rs = 0.631; p = 0.016); between S NfL and CSF Aß40 in the group with movement disorder plus dementia (rs = -0.750; p = 0.052); between S NfL and CSF tTau in the control group (rs = 0.689; p = 0.009); and between S NfL and CSF pTau in the control group (rs = 0.749; p = 0.003). The non-parametric Kruskal-Wallis test revealed statistically significant differences between S NfL, CSF NfL, CSF Aß42, CSF tTau, and CSF pTau and diagnosis within groups. The highest kappa coefficients were found between the concentrations of S NfL and CSF NfL (κ = 0.480) and between CSF NfL and CSF tTau (κ = 0.351). Conclusion: Our results suggested that NfL and tTau in CSF of patients with cognitive decline could be replaced by the less-invasive determination of S NfL using a highly sensitive ELISA method. S NfL reflected the severity of cognitive deficits assessed by mini-mental state examination (MMSE). However, S NfL is not specific to AD and does not appear to be a suitable biomarker for early diagnosis of AD.

Analysis of cerebrospinal fluid cells by flow cytometry: Comparison to conventional cytology.

AIMS: This study compared the results obtained by basic immunophenotyping of cerebrospinal fluid (CSF) cells by flow cytometry (FC) to the results of conventional cytology and evaluated the possibility of detailed analyses of CSF B-cell subpopulations. METHODS: Samples from 42 patients were examined by conventional cytology (native and/or pre-centrifuged CSF) and FC. The results from 15 patients without evidence of organic neurological disease were used to estimate reference ranges. RESULTS: Pre-centrifugated CSF had significantly higher cell yield on the cytologic slide, but cell subpopulation percentages were altered; the percentage of lymphocytes was significantly higher and monocytes significantly lower compared to both native CSF slides and FC. The percentage of granulocytes was higher in FC compared to cytology. For leukocyte count, the following reference ranges were estimated for Fuchs-Rosenthal chamber (FR) counting and FC, respectively: leukocytes ≤4.7/µL and ≤2.5/µL, lymphocytes ≤4.1/µL and ≤1.8/µL, monocytes ≤1.2/µL and ≤0.9/µL, and granulocytes 0/µL and ≤0.2/µL. The following reference ranges were estimated for basic subpopulations: T-lymphocytes 84.1-100%, B lymphocytes 0.0-1.5%, NK cells 0.0-6.3%, NKT cells 0-9.5%, and CD3+CD4+/CD3+CD8+ 0.8-4.9. Using a volume of 1.2-2.4 mL, the number of B lymphocytes was too low (<20) in samples with ≤2.7 cells/µL in the FR. CONCLUSIONS: Even normal CSF samples are amenable to basic mononuclear cell subpopulation analysis by FC. However, analysis of the B-cell subpopulations requires either a larger sample volume or selection of samples with ≥ 3 cells/µL.

Cerebrospinal fluid neurofilament light chains and CXCL13 as predictive factors for clinical course of multiple sclerosis.

AIM: The aim of this study was to identify whether NfL and CXCL13 cerebrospinal fluid (CSF) concentrations at diagnostic lumbar puncture can predict the course of multiple sclerosis (MS) in terms of relapses, higher expanded disability status scale (EDSS) and magnetic resonance imaging (MRI) activity. METHODS: We conducted a single-centre prospective observational cohort study at the MS center, University Hospital Ostrava, Czech Republic. CSF NfL (cNfL) and CXCL13 concentrations were examined (ELISA method) in patients with clinically isolated syndrome (CIS) and relapsing-remitting MS (RRMS) at the time of diagnostic lumbar puncture. RESULTS: A total of 44 patients with CIS or early RRMS were enrolled, 31 (70.5%) of whom were women. The median age at the time of CSF sampling was 31.21 years (IQR 25.43-39.32), and the follow-up period was 54.6 months (IQR 44.03-59.48). In the simple and multiple logistic regression models, CXCL13 levels did not predict relapses, MRI activity or EDSS > 2.5. Similarly, cNfL concentrations did not predict relapses or MRI activity in either model. In the multiple regression, higher cNfL levels were associated with reaching EDSS > 2.5 (odds ratio [OR] 1.002, 95% confidence interval [CI] 1.000 to 1.003). CONCLUSIONS: Our data did not confirm cNfL and/or CXCL13 CSF levels were predictive factors for disease activity such as relapses and MRI activity at the time of diagnostic lumbar puncture in patients with RRMS. While cNfL CSF levels predicted higher disability only after adjustment for other known risk factors, elevated CSF CXCL13 did not predict higher disability at all.

Prognostic value of intrathecal IgM synthesis determined by various laboratory methods in patients with early multiple sclerosis - a prospective observational study.

BACKGROUND: Intrathecal IgM synthesis has been identified as an adverse prognostic factor in patients with multiple sclerosis (MS); however, some studies have not confirmed this association. The objective of this study was to evaluate the clinical utility of intrathecal IgM synthesis for prediction of disease activity and disability in patients after the first demyelinating event of MS. METHODS: We conducted a single-centre prospective observational cohort study at the Department of Neurology, University Hospital Ostrava, Czech Republic. Intrathecal IgM synthesis was demonstrated by the presence of cerebrospinal fluid-restricted oligoclonal IgM bands and calculated using the Reiber, Auer, and Öhman formula and IgM index. RESULTS: A total of 61 patients with a clinically isolated syndrome or early relapsing-remitting MS were enrolled into the analysis of which 37 (61 %) were women. The median age at the disease onset was 32 years (interquartile range [IQR] 25 - 42), and the median disease duration was 2.8 years (IQR 2.4 - 3.5). Thirty-eight (62 %) patients experienced a second relapse of MS with a median of 312 days (IQR 192 - 424), and 29 (47.5 %) developed magnetic resonance imaging (MRI) activity during the follow-up. Intrathecal IgM synthesis did not affect the risk of a second relapse or evidence of MRI activity in univariate and multivariate Cox regression analysis. There was no significant difference in disability using the Expanded Disability Status Scale and progression index in patients with or without intrathecal IgM synthesis. CONCLUSION: This prospective cohort study did not demonstrate that intrathecal IgM synthesis is a risk factor for a second relapse or MRI activity. It was not associated with higher disability in patients after the first demyelinating event.

Alpha-defensins determination in different types of synovial fluid and parallel collected serum samples by ELISA.

AIMS: The study aims were to verify the serum (S) and synovial fluid (SF) reference intervals (RIs) for human neutrophil defensins (HNP1-3); measure S and SF defensin concentrations in different types of SF, including non-inflammatory, inflammatory non-pyogenic, inflammatory pyogenic, and hemorrhagic; and to compare the HNP1-3 concentrations in SF and S with those of other inflammatory biomarkers. METHODS: SF and S samples were collected from 92 patients. HNP1-3 concentrations were determined using enzyme-linked immunosorbent assays; glucose, lactate, interleukin-6, and procalcitonin using an automatic analyzer; and presepsin using a Pathfast system. There were 61 non-inflammatory, 11 inflammatory non-pyogenic, 11 inflammatory pyogenic, and 9 hemorrhagic SF. Non-inflammatory SF was divided into non-inflammatory normal and non-inflammatory osteoarthritis. The former was used to estimate the HNP1-3 RI in SF and S. RESULTS: The estimated HNP1-3 RIs of SF and S were 12.47-437.42 mg/L and 5.45-44.75 µg/L, respectively. HNP1-3 differed significantly between S and SF and individual groups of SF (P<0.001 and P=0.001, respectively). There were significant relationships between SF HNP1-3 and S HNP1-3 (P<0.001), S C-reactive protein (P<0.001), and S interleukin-6 (P=0.007), and between SF HNP1-3 and SF C-reactive protein (P=0.004) and SF interleukin-6 (P<0.001). The highest kappa coefficient was between SF HNP1-3 and SF interleukin-6 (κ=0.507). CONCLUSIONS: We validated the SF HNP1-3 diagnostic kit and demonstrated that SF and S HNP1-3 are promising biomarkers for distinguishing inflammatory from non-inflammatory joint diseases.

Determination of chitinase 3-like 1 in cerebrospinal fluid in multiple sclerosis and other neurological diseases.

OBJECTIVES: Chitinase 3-like 1 (CHI3L1) is an extracellular monomeric single-chain glycoprotein expressed by many types of cells. Its elevated levels were found in cerebrospinal fluid in central nervous system (CNS) inflammatory diseases patients. The aim of the study was 1) to validate the reference interval of cerebrospinal fluid (CSF) CHI3L1 in a control group; 2) to measure the CHI3L1 concentration in different diagnosis groups .including multiple sclerosis (MS); and 3) to correlate those values with other biomarkers of axonal damage or neuroinflammation in different grous. METHODS: The study included 132 CSF samples sent to the Department of Clinical Biochemistry, Institute of Laboratory Diagnostics, University Hospital Ostrava. Concentrations of CHI3L1, CXCL13 chemokine, neurofilament light chains, and phosphorylated neurofilament heavy chains were determined by enzyme-linked immunosorbent assays. IgG oligoclonal bands were detected by isoelectric focusing in agarose gels followed by immunofixation. IgM and FLC oligoclonal bands were analyzed by IEF followed by affinity immunoblotting. The group consisted of 42 patients with multiple sclerosis, 14 with clinically isolated syndrome, 11 with other central nervous system inflammatory diseases, 46 with non-inflammatory diseases of the central nervous system, 4 with inflammatory diseases of the peripheral nervous system, and 15 controls. RESULTS: The estimated reference values of CHI3L1 were 28.6-182.5 µg.L-1. Statistically significant differences of CSF CHI3L1 concentrations were found among diagnosis groups (p < 0.0001), after age adjustment (p = 0.002). There was a statistically significant relationship between CHI3L1 and NFL in the MS group (rs = 0.460; P = 0.002), and between CHI3L1 and pNFH in the MS group (rs = 0.691; P < 0.001). No statistically significant difference was found in the categorical comparison of CHI3L1 in the MS group and other diagnostic groups as well as when using the Mann-Whitney U test for CHI3L1 with additional parameters with and without oligoclonal bands present. CONCLUSIONS: CSF CHI3L1 values differ depending on diagnosis and correlate significantly with concentrations of the axonal damage markers CSF neurofilament light chains, and CSF phosphorylated neurofilament heavy chains, but not with CSF concentrations of the inflammatory marker CXCL13. Thus, CSF CHI3L1 could be another promising prognostic, albeit probably etiologically nonspecific, biomarker of MS.

Measurement of novel adipokine visfatin in young patients with acute myocardial infarction. Clinical testing of a new ELISA.

OBJECTIVES: Adipose tissue produces a number of adipokines that have metabolic effect. Visfatin is a recently discovered adipokine whose concentration in plasma increases in obesity. It is also a proinflammatory mediator that promotes atherosclerosis and plays a role in plaque destabilization. The aim of this study was to evaluate an assay for the determination of visfatin in human plasma and to investigate its clinical relevance as a marker of acute coronary syndrome (ACS) in a young population (Men under 45 y, Women under 55 y). DESIGN AND METHODS: We clinically tested a sandwich ELISA assay in young individuals with acute myocardial infarction (n=36) vs. a control group (n=21). The control sample was a healthy proband without inflammation, hepatic or renal injury and under 55 years of age. RESULTS: Visfatin in plasma was able to differentiate the control group from young patients with acute myocardial infarction (5 vs. 27 ng/L). Visfatin in the plasma of acute myocardial infarction (AMI) probands, correlated in individuals with acute coronary syndrome was related to plasma glucose (r=0.47; P=0.01), type 2 diabetes mellitus (r=0.65; P=0.01), plasma creatinine concentration (r=0.3, P=0.02), hsCRP (r=0.29; P=0.03), BMI values (r=0.18; P=0.04), triglycerides (r=0.5; P=0.01) and NT-proBNP (r=0.21; P=0.04). In healthy subjects, these relations were not found. ROC analysis: visfatin cut-off concentration was 20 ng/L with a sensitivity of 84% and a specificity of 90%. The area under the curve (AUC) of cTNI was 0.96, the AUC of visfatin was 0.96. Thus, there was no difference. CONCLUSION: We conclude that visfatin in serum may be a new independent potential marker of AMI.

Cerebrospinal fluid oligoclonal IgM test in routine practice: Comparison with quantitative assessment of intrathecal IgM synthesis.

BACKGROUND: Intrathecal IgM synthesis demonstrated either as cerebrospinal fluid (CSF)-restricted oligoclonal (o-) IgM bands or calculated using various formulas has been linked to more aggressive multiple sclerosis (MS) course. However, the proportion of MS patients showing intrathecal IgM synthesis varies largely between studies. We aimed to explore the relation between different formulas and results of o-IgM, and to assess the frequency of o-IgM bands in an unselected series of samples. METHODS: 432 samples were analyzed for o-IgM, o-IgG and quantitative measures of IgM and IgG synthesis. IgM index and formulas of Reiber, Auer and Öhman were compared to the result of the o-IgM test. RESULTS: At the cut-off commonly used, the non-linear formulas for intrathecal synthesis were specific (>94%) but rather insensitive (<40% even at a cut-off of 4 CSF-restricted bands) compared to o-IgM. No significant difference was noted in the performance of different formulas. At a cut-off of 4 bands, 61% of MS patients, but none of the controls were positive for o-IgM. CONCLUSIONS: Formulas for intrathecal IgM synthesis are insensitive compared to o-IgM. We propose to evaluate samples with 2 or 3 extra-CSF IgM bands as borderline and only samples with 4 or more as definitely positive.

Diagnostic relevance of free light chains in cerebrospinal fluid - The hyperbolic reference range for reliable data interpretation in quotient diagrams.

BACKGROUND: Free light chains, type kappa (FLC-K), in cerebrospinal fluid (CSF) were compared to oligoclonal IgG in many studies for sensitive detection of immune reactions in brain. The missing consensus about CSF data interpretation prevents reliable conclusions. This can be overcome by a theory-based hyperbolic reference range in CSF/serum quotient diagrams. METHODS: Mean Quotients for FLC-K, QKappa, and albumin, QAlb, of grouped, biochemically defined controls (N = 433) are fitted with the hyperbolic function QKappa(mean) = a/b (QAlb2 + b2)0.5 - c by a generally applicable procedure excluding outliers. RESULTS: With QKappa(mean), the coefficient of variation CV (22.5%) and the reference range (QKappa(mean) ±â€¯3 CV) we got the discrimination line QKappa(lim) = (3.27(QAlb2 + 33)0.5-8.2) ×10-3 in a FLC-K Reibergram. Intrathecal FLC-K was found in 8% of another control group without OCB (N = 388) but was missed in 7% of patients with definite Multiple sclerosis (N = 95). In MS the mean intrathecal fraction was threefold larger for FLC-K (95%) compared to total IgG (36%). Similar mean quantities of intrathecal FLC-K contradict an immunological conversion between a Clinically isolated syndrome and MS. DISCUSSION: The hyperbolic reference range is superior to linear FLC-K Index (10 to 15% false negatives) and exponential curves (30% false positive interpretations for controls) in the analytical range of MS data, with excellent data fit for up to ten-fold larger QAlb values. Dynamics of the small molecule FLC-K contribute to the understanding of molecular size dependent barrier functions.

Chemiluminescent detection of oligoclonal immunoglobulins after isoelectric focusing and affinity-mediated immunoblotting.

BACKGROUND AND AIMS: Detection of oligoclonal IgG (o-IgG) in the cerebrospinal fluid (CSF) not found in serum is the principal laboratory test to support a diagnosis of multiple sclerosis. The aim of this study was to compare chemiluminescent and chromogenic detection of oligoclonal immunoglobulins in the cerebrospinal fluid and serum after their separation by means of isoelectric focusing followed by immunoblotting. METHODS: A set of experiments was designed to detect oligoclonal immunoglobulins by means of alkaline phosphatase BCIP/NBT substrate and chemiluminescent peroxidase substrate. RESULTS: Based on visual evaluation of signals, chemiluminescent detection requires about a 4 times lower amount of applied protein than very sensitive BCIP/NBT chromogenic detection. Very good correlation between methods has been shown for oligoclonal IgG. Antigen-specific oligoclonal IgG could be demonstrated by both methods although the pattern was clearer using chemiluminescence. In one patient, oligoclonal IgD bands barely visible by BCIP/NBT were convincingly demonstrated by chemiluminescence. CONCLUSION: Chemiluminescent detection is a feasible option for oligoclonal immunoglobulin detection and could be used in cases when the sensitivity needs to be improved. Further studies and method optimisation are warranted.

Comparison of the Efficacy of Paclitaxel-Eluting Balloon Catheters and Everolimus-Eluting Stents in the Treatment of Coronary In-Stent Restenosis: The Treatment of In-Stent Restenosis Study.

BACKGROUND: The aim of this prospective randomized noninferiority study was to compare the efficacy of paclitaxel-eluting balloon (PEB) catheters and everolimus-eluting stents (EES) in the treatment of bare metal stent restenosis. METHODS AND RESULTS: A total of 136 patients were enrolled in the study. Each treatment group included 68 patients with 74 in-stent restenotic lesions. The primary end point was in-segment late lumen loss (LLL) at 12 months. Secondary end points were the incidence of binary in-stent restenosis and 12-month major adverse cardiac events. The 2-sided 95% confidence interval of LLL difference between treatments (0.149-0.558) was greater than noninferiority margin (0.12), which demonstrates both noninferiority and superiority of PEB treatment. Furthermore, the PEB group had significantly less 12-month LLL than the EES group (0.02 versus 0.19 mm; P=0.0004). The difference in the incidence of repeated binary restenosis (8.7% versus 19.12%; P=0.078) and 12-month major adverse cardiac events (10.29% versus 19.12%; P=0.213) was not significant. The 12-month LLL was significantly less in the PEB group and also in subgroups with in-stent restenosis >10 mm (0.05 versus 0.26 mm; P=0.0002) and artery diameter <3 mm (0.05 versus 0.16 mm; P=0.003) compared with the EES groups, but not in the subgroup of patients with diabetes mellitus (P=0.254). In the EES group, repetitive binary restenosis had a significantly greater chance of occurring (odds ratio=3.132; 95% confidence interval, 1.058-9.269; P=0.039), even when adjusting for other risk factors. CONCLUSIONS: Treatment of bare metal stent restenosis using PEB led to significantly less 12-month LLL than the implantation of second-generation EES. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01735825.

Quantitation of free light chains in the cerebrospinal fluid reliably predicts their intrathecal synthesis.

BACKGROUND: The results of free light chains quantitation in the cerebrospinal fluid were recently compared with the presence of cerebrospinal fluid-restricted oligoclonal IgG, but not oligoclonal free kappa light chains and oligoclonal free lambda light chains. We therefore aimed to compare the performance of the quantitative tests with the qualitative one for the same molecule. METHODS: Seventy-five paired cerebrospinal fluid and serum samples were analysed for oligoclonal IgG, oligoclonal free kappa light chains and oligoclonal free lambda light chains. Cerebrospinal fluid and serum free kappa and lambda light chains were quantified using Freelite™ kits on SPA Plus analyzer. ROC curves were analysed for the prediction of intrathecal synthesis and compared for cerebrospinal fluid concentration, cerebrospinal fluid/serum quotient (QfLC) and index (QfLC/QAlbumin). The presence of cerebrospinal fluid-restricted oligoclonal free kappa light chains and oligoclonal free lambda light chains bands was used as reference. RESULTS: No statistically significant differences were observed among cerebrospinal fluid concentration, QfLC and index for the prediction of free light chain intrathecal synthesis. Each parameter was able to predict the occurrence of cerebrospinal fluid-restricted oligoclonal free light chain bands (AUCs 0.932-0.999). However, we noted elevated cerebrospinal fluid free light chain concentrations in the absence of cerebrospinal fluid-restricted oligoclonal free light chain bands in two patients with very high serum free light chain values. CONCLUSIONS: Quantitation of cerebrospinal fluid free light chains reliably predicts their intrathecal synthesis. Yet, cerebrospinal fluid/serum quotient may still be preferred to correct for high serum free light chain concentrations. An appropriate formula should be sought to correct for blood-cerebrospinal fluid barrier status.

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting.

OBJECTIVES: We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. METHODS: We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. RESULTS: Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. CONCLUSIONS: Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.

Increased levels of MMP-3, MMP-9 and MPO represent predictors of in-stent restenosis, while increased levels of ADMA, LCAT, ApoE and ApoD predict bare metal stent patency.

AIMS: We sought to identify biochemical predictors that indicate susceptibility to in-stent restenosis (ISR) after coronary artery bare-metal stenting. METHODS: A total of 111 consecutive patients with post-percutaneous coronary intervention (PCI) in-stent restenosis of a target lesion within 12 months were matched for age, sex, vessel diameter, and diabetes with 111 controls without post-PCI ISR. Plasma or serum levels of biochemical markers were measured: matrix metalloproteinases (MMP) 2, 3, 9; myeloperoxidase (MPO); asymmetric dimethylarginine (ADMA); lipoprotein (a) (Lp[a]); apolipoproteins E and D (ApoE and D); and lecitin-cholesterol acyltransferase (LCAT). Multivariable logistic regression association tests were performed. RESULTS: Increased plasma MMP-3 (OR: 1.013; 95% CI: 1.004-1.023; P = 0.005), MMP-9 (OR: 1.014; 95% CI: 1.008-1.020; P < 0.0001) or MPO (OR: 1,003; 95% CI: 1.001-1.005; P = 0.002) was significantly associated with increased risk of ISR. Increased levels of ADMA (OR: 0.212; 95% CI: 0.054-0.827; P = 0.026), ApoE (OR: 0.924; 95% CI: 0.899-0.951; P < 0.0001), ApoD (OR: 0.919; 95% CI: 0.880-0.959; P = 0.0001), or LCAT (OR: 0.927; 95% CI: 0.902-0.952; P < 0.0001) was associated with risk reduction. No correlation was found between plasma MMP-2 or Lp (a) and ISR risk. CONCLUSIONS: Increased levels of MMP-3, MMP-9, and MPO represent predictors of ISR after bare-metal stent implantation. In contrast, increased ADMA, LCAT, and Apo E and D indicate a decreased in-stent restenosis occurrence.