Machine learning to optimize cerebrospinal fluid dilution for analysis of MRZH reaction.

OBJECTIVES: To create a supervised machine learning algorithm aimed at predicting an optimal cerebrospinal fluid (CSF) dilution when determining virus specific antibody indices to reduce the need for repeated tests. METHODS: The CatBoost model was trained, optimized, and tested on a dataset with five input variables: albumin quotient, immunoglobulin G (IgG) in CSF, IgG quotient (QIgG), intrathecal synthesis (ITS) and limes quotient (LIM IgG). Albumin and IgG concentrations in CSF and serum were performed by immunonephelometry on Atellica NEPH 630 (Siemens Healthineers, Erlangen, Germany) and ITS and LIM IgG were calculated according to Reiber. Concentrations of IgG antibodies to measles, rubella, varicella zoster and herpes simplex 1/2 viruses were analysed in CSF and serum by ELISA (Euroimmun, Lübeck, Germany). Optimal CSF dilution was defined for each virus and used as a classification variable while the standard operating procedure was set to start at 2×-dilution of CSF. RESULTS: The dataset included 571 samples with the imbalanced distribution of the optimal CSF dilutions: 2× dilution n=440, 3× dilution n=109, 4× dilution n=22. The optimized CatBoost model achieved an area under the curve (AUC) score of 0.971, and a test accuracy of 0.900. The model falsely classified 14 (9.9 %) samples of the testing set but reduced the need for repeated testing compared to the standard protocol by 42 %. The output of the CatBoost model is mostly dependant on the QIgG, ITS and CSF IgG variables. CONCLUSIONS: An accurate algorithm was achieved for predicting the optimal CSF dilution, which reduces the number of test repeats.

Determination of cystatin C reference interval for children in Croatia.

Introduction: Cystatin C is considered an early marker of kidney damage. The aim was to determine the reference interval in children since this information was not available from the test manufacturer. Materials and methods: Included were children aged 0 to 18 years undergoing routine check without history of any renal disease. Cystatin C was measured by the immunoturbidimetric method, and creatinine by the enzymatic method on a Cobas c501 analyzer (Roche Diagnostics, Manheim, Germany). Reference intervals were determined according to the CLSI C28-A3 guidelines using a robust method and a nonparametric percentile method, depending on the sample size. The Schwartz's formula was applied to estimate glomerular filtration (eGFR) from cystatin C. Results: The cystatin C reference interval for children aged 1-18 years (N = 204, median 8 years) was from 0.61 mg/L (90% CI: 0.53 to 0.64) to 1.08 mg/L (90% CI: 1.07 to 1.14). Differences according to sex were not found. For children aged 0-1 years (N = 29, median 5 months), the reference interval was from 0.60 mg/L (90% CI: 0.48 to 0.72) to 1.49 mg/L (90% CI: 1.36 to 1.61). The sample size was too small to test the difference according to sex. The eGFR was 76 (70-88) mL/min/1.73m2 for males and 83 (74-92) mL/min/1.73m2 for females. Conclusion: The cystatin C reference intervals for Croatian pediatric population according to age were determined. The cystatin C concentrations in children reach adulthood values after the first year. The cystatin C Schwartz's formula is applicable for eGFR calculation in children.

Higher MRI lesion load in multiple sclerosis is related to the N-glycosylation changes of cerebrospinal fluid immunoglobulin G.

BACKGROUND: Intrathecal clonal expansion of antibody-producing plasma cells in multiple sclerosis (MS) perpetuates central nervous system injury and is associated with active demyelination. Immunoglobulin G (IgG) effector functions are modulated by linked N-glycan structures. The aim of the study was to detect potential differences in N-glycosylation of IgG in serum and cerebrospinal fluid (CSF) and total sera proteins between people with MS and those in whom the diagnosis of MS was excluded. Furthermore, we investigated the association with standard laboratory biomarkers of intrathecal inflammation as well as clinical and neuroradiological disease activity. METHODS: This cross-sectional study included patients with suspected demyelinating disease. MS diagnosis was based on the 2017 McDonald criteria and controls were patients with excluded MS diagnosis. N-glycans were compared with Expanded Disability Status Scale (EDSS), magnetic resonance imaging (MRI) markers of disease activity and biomarkers of intrathecal inflammation (cell count, CSF-IgG concentration, percentage of intrathecal IgG, oligoclonal bands (OCB), virus-specific antibody index (MRZH reaction)). RESULTS: Differences between groups were observed only in the CSF-IgG N-glycome. In MS, the presence of bisecting N-acetylglucosamine (Padj=2.63E-05) and monogalactosylation (Padj=1.49E-06) were more abundant and associated with positive OCBs. N-glycans monogalactosylated at the α6 arm FA2[6]G1 (r = 0.56) and FA2[6]BG1 (r = 0.45) correlated with percentage of intrathecal IgG, but not total CSF-IgG. This trait was also more abundant in MRZH positive people with MS who had higher MRI lesion load (P = 0.018) but unrelated to active lesions or EDSS. CONCLUSIONS: More abundant monogalactosylation of intrathecally synthesized IgG is the most prominent trait in MS and is associated with higher MRI lesion load.

Soluble TREM2 Concentrations in the Cerebrospinal Fluid Correlate with the Severity of Neurofibrillary Degeneration, Cognitive Impairment, and Inflammasome Activation in Alzheimer's Disease.

BACKGROUND: Individuals with specific TREM2 gene variants that encode for a Triggering Receptor Expressed on Myeloid cells 2 have a higher prevalence of Alzheimer's disease (AD). By interacting with amyloid and apolipoproteins, the TREM2 receptor regulates the number of myeloid cells, phagocytosis, and the inflammatory response. Higher TREM2 expression has been suggested to protect against AD. However, it is extremely difficult to comprehend TREM2 signaling in the context of AD. Previous results are variable and show distinct effects on diverse pathological changes in AD, differences between soluble and membrane isoform signaling, and inconsistency between animal models and humans. In addition, the relationship between TREM2 and inflammasome activation pathways is not yet entirely understood. OBJECTIVE: This study aimed to determine the relationship between soluble TREM2 (sTREM2) levels in cerebrospinal fluid (CSF) and plasma samples and other indicators of AD pathology. METHODS: Using the Enzyme-Linked Immunosorbent Assay (ELISA), we analyzed 98 samples of AD plasma, 35 samples of plasma from individuals with mild cognitive impairment (MCI), and 11 samples of plasma from healthy controls (HC), as well as 155 samples of AD CSF, 90 samples of MCI CSF, and 50 samples of HC CSF. RESULTS: CSF sTREM2 levels were significantly correlated with neurofibrillary degeneration, cognitive decline, and inflammasome activity in AD patients. In contrast to plasma sTREM2, CSF sTREM2 levels in the AD group were higher than those in the MCI and HC groups. Moreover, concentrations of sTREM2 in CSF were substantially higher in the MCI group than in the HC group, indicating that CSF sTREM2 levels could be used not only to distinguish between HC and AD patients but also as a biomarker to detect earlier changes in the MCI stage. CONCLUSIONS: The results indicate CSF sTREM2 levels reliably predict neurofibrillary degeneration, cognitive decline, and inflammasome activation, and also have a high diagnostic potential for distinguishing diseased from healthy individuals. To add sTREM2 to the list of required AD biomarkers, future studies will need to include a larger number of patients and utilize a standardized methodology.

Total tau in cerebrospinal fluid detects treatment responders among spinal muscular atrophy types 1-3 patients treated with nusinersen.

AIMS: Considering the substantial variability in treatment response across patients with spinal muscular atrophy (SMA), reliable markers for monitoring response to therapy and predicting treatment responders need to be identified. The study aimed to determine if measured concentrations of disease biomarkers (total tau protein, neurofilament light chain, and S100B protein) correlate with the duration of nusinersen treatment and with scores obtained using functional scales for the assessment of motor abilities. METHODS: A total of 30 subjects with SMA treated with nusinersen between 2017 and 2021 at the Department of Pediatrics, University Hospital Centre Zagreb, Croatia, were included in this study. Cerebrospinal fluid (CSF) samples were collected by lumbar puncture prior to intrathecal application of nusinersen. Protein concentrations in CSF samples were determined by enzyme-linked immunosorbent assay in 26 subjects. The motor functions were assessed using functional motor scales. RESULTS: The main finding was significantly decreased total tau correlating with the number of nusinersen doses and motor improvement in the first 18-24 months of treatment (in all SMA patients and SMA type 1 patients). Neurofilament light chain and S100B were not significantly changed after administration of nusinersen. CONCLUSIONS: The measurement of total tau concentration in CSF is a reliable index for monitoring the biomarker and clinical response to nusinersen therapy in patients with SMA.

Serotonin Receptor Gene Polymorphisms Are Associated with Cerebrospinal Fluid, Genetic, and Neuropsychological Biomarkers of Alzheimer's Disease.

A decrease in serotonergic transmission throughout the brain is among the earliest pathological changes in Alzheimer's disease (AD). Serotonergic receptors are also affected in AD. Polymorphisms in genes of serotonin (5HT) receptors have been mostly associated with behavioral and psychological symptoms of dementia (BPSD). In this study, we examined if AD patients carrying different genotypes in 5HTR1B rs13212041, 5HTR2A rs6313 (T102C), 5HTR2C rs3813929 (-759C/T), and 5HTR6 rs1805054 (C267T) polymorphisms have a higher risk of faster disease progression (assessed by neuropsychological testing), are more prone to develop AD-related pathology (reflected by levels of cerebrospinal fluid [CSF] AD biomarkers), or have an association with an apolipoprotein E (APOE) haplotype. This study included 115 patients with AD, 53 patients with mild cognitive impairment (MCI), and 2701 healthy controls. AD biomarkers were determined in the CSF of AD and MCI patients using enzyme-linked immunosorbent assays (ELISA), while polymorphisms were determined using either TaqMan SNP Genotyping Assays or Illumina genotyping platforms. We detected a significant decrease in the CSF amyloid ß1-42 (Aß1-42) and an increase in p-tau181/Aß1-42 ratio in carriers of the T allele in the 5HTR2C rs3813929 (-759C/T) polymorphism. A significantly higher number of APOE ε4 allele carriers was observed among individuals carrying a TT genotype within the 5HTR2A T102C polymorphism, a C allele within the 5HTR1B rs13212041 polymorphism, and a T allele within the 5HTR6 rs1805054 (C267T) polymorphism. Additionally, individuals carrying the C allele within the 5HTR1B rs13212041 polymorphism were significantly more represented among AD patients and had poorer performances on the Rey-Osterrieth test. Carriers of the T allele within the 5HTR6 rs1805054 had poorer performances on the MMSE and ADAS-Cog. As all four analyzed polymorphisms of serotonin receptor genes showed an association with either genetic, CSF, or neuropsychological biomarkers of AD, they deserve further investigation as potential early genetic biomarkers of AD.

Analytical validation of 39 clinical chemistry tests and 17 immunoassays on the Alinity analytical system.

The aim of this study was to perform the analytical validation of Alinity c and i analyzers (Abbott Laboratories, Chicago, IL, USA) for 39 clinical chemistry tests and 17 immunoassays. Precision was evaluated at least at two concentration levels for 5 days in quintuplicate, following CLSI EP15-A3. Method comparison included parallel analysis of leftover routine samples on Alinity analyzers and the previously used Cobas c501 and e601 (Roche Diagnostics, Mannheim, Germany). Linearity was tested by preparing sequential sample dilutions with high analyte concentration, following the CLSI EP6 document. For clinical chemistry tests, within-run coefficients of variation (CV) were up to 6.0% (beta-2-microglobulin), while between-run CVs up to 5.4% (immunoglobulin M). Among immunoassays, the highest within-run CV was obtained for vitamin B12 (6.9%), while between-run for CA 19-9 (4.3%). Complete agreement with Roche analyzers was observed for 16 (41%) clinical chemistry assays and 6 (35%) immunoassays. Half of all evaluated assays did not meet the desirable biological variation criteria for bias, being especially exceeded for alpha1-antitrypsin, apolipoprotein A1, ceruloplasmin, complement C3 and C4, hemoglobin A1c, lipoprotein (a) and myoglobin, as well as some tumor markers (CA 125, CEA, fPSA, AFP, and ferritin), hormones (cortisol, DHEA-S, insulin) and vitamins (25-OHD). Linearity in the tested ranges was confirmed. Overall, this study revealed that precision criteria derived from manufacturer's claims were not satisfied for all assays while comparison study for some assays yielded differences that imply the need for additional assay evaluation prior to introduction into routine practice.

Heavy Metals and Essential Metals Are Associated with Cerebrospinal Fluid Biomarkers of Alzheimer's Disease.

Various metals have been associated with the pathogenesis of Alzheimer's disease (AD), principally heavy metals that are environmental pollutants (such as As, Cd, Hg, and Pb) and essential metals whose homeostasis is disturbed in AD (such as Cu, Fe, and Zn). Although there is evidence of the involvement of these metals in AD, further research is needed on their mechanisms of toxicity. To further assess the involvement of heavy and essential metals in AD pathogenesis, we compared cerebrospinal fluid (CSF) AD biomarkers to macro- and microelements measured in CSF and plasma. We tested if macro- and microelements' concentrations (heavy metals (As, Cd, Hg, Ni, Pb, and Tl), essential metals (Na, Mg, K, Ca, Fe, Co, Mn, Cu, Zn, and Mo), essential non-metals (B, P, S, and Se), and other non-essential metals (Al, Ba, Li, and Sr)) are associated with CSF AD biomarkers that reflect pathological changes in the AD brain (amyloid ß1-42, total tau, phosphorylated tau isoforms, NFL, S100B, VILIP-1, YKL-40, PAPP-A, and albumin). We used inductively coupled plasma mass spectroscopy (ICP-MS) to determine macro- and microelements in CSF and plasma, and enzyme-linked immunosorbent assays (ELISA) to determine protein biomarkers of AD in CSF. This study included 193 participants (124 with AD, 50 with mild cognitive impairment, and 19 healthy controls). Simple correlation, as well as machine learning algorithms (redescription mining and principal component analysis (PCA)), demonstrated that levels of heavy metals (As, Cd, Hg, Ni, Pb, and Tl), essential metals (Ca, Co, Cu, Fe, Mg, Mn, Mo, Na, K, and Zn), and essential non-metals (P, S, and Se) are positively associated with CSF phosphorylated tau isoforms, VILIP-1, S100B, NFL, and YKL-40 in AD.

Early biochemical markers in the assessment of acute kidney injury in children after cardiac surgery.

Our aim was to evaluate biochemical markers in plasma (NGAL, CysC) and urine (NGAL, KIM-1) in children's early onset of acute kidney injury after congenital heart defect surgery using cardiopulmonary bypass. This study prospectively included 100 children with congenital heart defects who developed AKI. Patients with acute kidney injury had significantly higher CysC levels 6 and 12 h after cardiac surgery and plasma NGAL levels 2 and 6 h after cardiac surgery. The best predictive properties for the development of acute kidney injury are the combination (+CysCpl or +NGALu) after 12 h and a combination (+CysCpl and +NGALu) 6 and 24 h after cardiac surgery. We showed that plasma CysC and urinary NGAL could reliably predict the development of acute kidney injury. Measurement of early biochemical markers in plasma and urine, individually and combination, may predict the development of cardiac surgery-associated acute kidney injury in children.

The Association of Essential Metals with APOE Genotype in Alzheimer's Disease.

BACKGROUND: The major confirmed genetic risk factor for late-onset, sporadic Alzheimer's disease (AD) is variant ɛ4 of apolipoprotein E gene (APOE). It is proposed that ApoE, a protein involved in transport of cholesterol to neurons can cause neurodegeneration in AD through interaction with metals. Previous studies mostly associated copper, iron, zinc, and calcium with ApoE4-mediated toxicity. OBJECTIVE: To test the association of essential metals with APOE genotype. METHODS: We compared plasma and cerebrospinal fluid (CSF) levels of copper, zinc, iron, sodium, magnesium, calcium, cobalt, molybdenum, manganese, boron, and chromium, and CSF ferritin levels among AD, mild cognitive impairment (MCI) patients, and healthy controls (HC) with different APOE genotype. RESULTS: Sodium, copper, and magnesium levels were increased in carriers of ɛ4 allele. Additionally, the increase in sodium, calcium and cobalt plasma levels was observed in carriers of ɛ4/ɛx genotype. The decrease in boron plasma levels was observed in carriers of ɛ4 allele and ɛ4/ɛ4 genotype. Additionally, CSF zinc levels as well as plasma sodium levels were increased in AD patients compared to HC. CONCLUSION: These results indicate that the molecular underpinnings of association of essential metals and metalloids with APOE should be further tested and clarified in vivo and in vitro.

Salivary cortisol response to psychosocial stress in patients with first-episode psychosis.

AIM: To analyze the difference in the salivary cortisol response to psychosocial stress between the patients with the first episode of psychosis (FEP) and the control group. METHODS: We performed a cross-sectional analysis of the baseline measurements of a prospective cohort study conducted from 2015 to 2018 at two Croatian psychiatric hospitals. The study consecutively enrolled 53 patients diagnosed with FEP and 63 healthy controls. The primary outcome was the difference in the changes of salivary cortisol concentration during the stress test. The secondary outcome was the difference in the baseline levels of salivary cortisol between patients with FEP and controls. The tertiary outcome were the correlations of salivary cortisol levels with the results of the Positive and Negative Syndrome Scale for Schizophrenia, Rosenberg Self-Esteem Scale, and the International Personality Item Pool. RESULTS: Patients with FEP had significantly higher baseline salivary cortisol than controls, but their salivary cortisol increased significantly less during the stress test. CONCLUSION: Patients with FEP respond differently to stressful stimuli than controls, as shown by the increased baseline salivary cortisol and blunted cortisol response, possibly indicating a greater vulnerability to psychosocial stress.

Integration of complementary biomarkers in patients with first episode psychosis: research protocol of a prospective follow up study.

In this project, we recruited a sample of 150 patients with first episode of psychosis with schizophrenia features (FEP) and 100 healthy controls. We assessed the differences between these two groups, as well as the changes between the acute phase of illness and subsequent remission among patients over 18-month longitudinal follow-up. The assessments were divided into four work packages (WP): WP1- psychopathological status, neurocognitive functioning and emotional recognition; WP2- stress response measured by saliva cortisol during a stress paradigm; cerebral blood perfusion in the resting state (with single photon emission computed tomography (SPECT) and during activation paradigm (with Transcranial Ultrasonography Doppler (TCD); WP3-post mortem analysis in histologically prepared human cortical tissue of post mortem samples of subjects with schizophrenia in the region that synaptic alteration was suggested by WP1 and WP2; WP4- pharmacogenetic analysis (single gene polymorphisms and genome wide association study (GWAS). We expect that the analysis of these data will identify a set of markers that differentiate healthy controls from patients with FEP, and serve as an additional diagnostic tool in the first episode of psychosis, and prediction tool which can be then used to help tailoring individualized treatment options. In this paper, we describe the project protocol including aims and methods and provide a brief description of planned post mortem studies and pharmacogenetic analysis.

Analytical verification of 12 most commonly used urine dipsticks in Croatia: comparability, repeatability and accuracy.

INTRODUCTION: Variability among manufacturers of urine dipsticks, respective to their accuracy and measurement range, may lead to diagnostic errors and thus create a serious risk for the patient. Our aims were to determine the level of agreement between 12 most commonly used urine dipsticks in Croatia, examine their accuracy for glucose and total protein and to test their repeatability. MATERIALS AND METHODS: A total of 75 urine samples were used to examine comparability and accuracy of 12 dipstick brands (Combur 10 TestM, ChoiceLine 10, Combur 10 TestUX, ComboStik 10M, ComboStik 11M, CombiScreen 11SYS, CombiScreen 10SL, Combina 13, Combina 11S, Combina 10M, UriGnost 11, Multistix 10SG). Agreement between each dipstick and the reference (Combur 10 TestM) was expressed as kappa coefficient (acceptable κ ≥ 0.80). Accuracy for glucose and total protein was tested by comparison with quantitative measurements on analysers: AU400 (Beckman Coulter, USA), Cobas 6000 c501 (Roche Diagnostics, Germany) and Architect plus c4000 (Abbott, USA). Repeatability was assessed on 20 replicates (acceptable > 90%). RESULTS: Best agreement was achieved for glucose, total protein and nitrite (11/11, k > 0.80) and the lowest for bilirubin (5/5, k < 0.60). Sensitivities for total protein were 41-75% (AU400) and 56-92% (Cobas and Architect); while specificities were 41-75% (AU400, Cobas, Architect). Dipsticks' sensitivity and specificity for glucose were 68-98%. Most of the dipsticks showed unacceptable repeatability (6/12, < 90%) for one parameter, most prominently for pH (3/12, < 90%). CONCLUSIONS: Most commonly used dipsticks in Croatia showed low level of agreement between each other. Moreover, their repeatability varies among manufacturers and their accuracy for glucose and proteins is poor.

Effects of Long-Term Multimodal Psychosocial Treatment on Antipsychotic-Induced Metabolic Changes in Patients With First Episode Psychosis.

Background: Antipsychotic-induced weight gain and metabolic abnormalities are one of the major challenges in the treatment of psychosis, contributing to the morbidity, mortality and treatment non-adherence. Different approaches were used to counteract these side effects but showed only limited or short-term effects. This study aims to analyse the effects of a long-term multimodal treatment program for first episode psychosis on antipsychotic-induced metabolic changes. Methods: We enrolled 71 patients with first episode psychosis treated at the Zagreb University Hospital Centre from 2016 until 2018. Participants were assigned to one of the two groups: day hospital program vs. treatment as usual (TAU). Outcomes were: body weight, blood glucose, lipids and cholesterol, psychopathology and global level of functioning during the 18-months follow-up. Results: Although the TAU group gained more weight and had higher increase of blood glucose, while the day hospital group had a higher increase in total cholesterol at 18th month follow-up, after the adjustment for age, gender and baseline measures, the type of treatment was not significantly associated with any of the primary outcome measures. Patients' psychopathology measures significantly decreased and their functional level significantly increased at month 18th in both groups. Conclusion: While both types of treatment were effective in reducing psychopathology and restoring the patients' level of functioning, both were relatively ineffective in counteracting antipsychotic-induced metabolic abnormalities and antipsychotic-induced weight gain.

Effect of sample processing and time delay on cell count and chemistry tests in cerebrospinal fluid collected from drainage systems.

INTRODUCTION: Cerebrospinal fluid (CSF) from extra-ventricular drainage (EVD) systems is routinely analysed to diagnose EVD-related bacterial meningitis. We investigated the effect of time delay and sample processing on cell count and basic biochemistry results in EVD CSF to define optimal turnaround time and whether manual and automated cell counting are comparable in such samples. MATERIALS AND METHODS: In total, 32 EVD CSF samples were analysed. Baseline testing included cell counting (Fuchs-Rosenthal chamber and Sysmex XE5000) and biochemistry analyses (glucose, lactate, proteins). Manual cell counting was also performed at intervals of 61-90 and 91-150 minutes from baseline in the residual sample. Biochemistry analyses were performed in samples before and after centrifugation at baseline and at 91-150 minutes interval. RESULTS: At 91-150 minutes total cell count (P < 0.001), large lymphocytes (P = 0.007), neutrophils (P < 0.001) and phagocytes (P = 0.006) obtained by manual counting decreased and the number of disintegrated cells count increased (P = 0.016) compared to the baseline values. Considering method comparison, proportional difference between methods for all cell (sub)groups was obtained, whereas polymorphonuclears also showed the constant difference (y = 11.21 + 1.22x). Compared to centrifuged CSF, lower concentration of glucose and lactates were obtained in uncentrifuged samples (P < 0.001) at baseline. CONCLUSIONS: Manual cell counting should be performed within 60 minutes as any delay can alter results. The same counting technique should be used to obtain longitudinally assessable results. Biochemistry tests are stable in uncentrifuged CSF up to 2.5 hours.

Event-related Potentials Improve the Efficiency of Cerebrospinal Fluid Biomarkers for Differential Diagnosis of Alzheimer's Disease.

INTRODUCTION: The pathological process of Alzheimer's disease (AD) in the brain likely begins 20-30 years earlier than the emergence of its first clinical symptoms and symptoms of AD often overlap with the symptoms of other primary causes of dementia. Therefore, it is crucially important to improve early and differential diagnosis of the disease. Event-related potentials (ERP) measured non-invasively by electroencephalography have shown diagnostic potential in AD. AIMS: The aim of this study was to compare the efficiency of P300 and N200 potentials and reaction time (RT) with commonly used protein biomarkers measured in the cerebrospinal fluid (CSF), including amyloid ß peptide (ß1-42), total tau (t-tau), tau protein phosphorylated at threonine 181 (p-tau181), tau protein phosphorylated at serine 199 (p-tau199), tau protein phosphorylated at threonine 231 (p-tau231), and visinin-like protein 1 (VILIP-1) in differential diagnosis of AD in mild cognitive impairment (MCI) and AD patients. SUBJECTS: The study involved 49 AD patients, 28 patients with MCI, 4 healthy control subjects and 16 patients with other primary causes of dementia. RESULTS: ERP (P300RT, N200RT, P300 counting and N200 counting) showed a moderate to strong correlation with protein CSF biomarkers. We confirmed previous observations of moderate to strong correlation between ERP and neuropsychological testing and showed that P300 latency and RT are shortened in AD patients on therapy with acetylcholinesterase inhibitors. Using ERP and RT, a predictive model for determination of AD likelihood in MCI patients was developed, detecting 56.3% of MCI patients with high risk for development of AD in our cohort. MCI patients with pathological levels of Aß1-42 had prolonged P300 latency, indicating that a combination of ERP and CSF protein biomarkers could improve the differential diagnosis of AD in MCI patients. Additionally, the results suggested the potential of P300 latency in differentiating AD and FTD patients. CONCLUSION: Our data provide possible solutions for improvement of differential diagnosis of AD, and reveal that the diagnostic efficiency of CSF protein biomarkers t-tau, p-tau181, p-tau199, p-tau231 and VILIP-1 could be improved by adding ERP in clinical practice.

Sex-independent expression of chloride/formate exchanger Cfex (Slc26a6) in rat pancreas, small intestine, and liver, and male-dominant expression in kidneys.

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2

Laboratory testing of extravascular body fluids in Croatia: a survey of the Working group for extravascular body fluids of the Croatian Society of Medical Biochemistry and Laboratory Medicine.

INTRODUCTION: We hypothesized that extravascular body fluid (EBF) analysis in Croatia is not harmonized and aimed to investigate preanalytical, analytical and postanalytical procedures used in EBF analysis in order to identify key aspects that should be addressed in future harmonization attempts. MATERIALS AND METHODS: An anonymous online survey created to explore laboratory testing of EBF was sent to secondary, tertiary and private health care Medical Biochemistry Laboratories (MBLs) in Croatia. Statements were designed to address preanalytical, analytical and postanalytical procedures of cerebrospinal, pleural, peritoneal (ascites), pericardial, seminal, synovial, amniotic fluid and sweat. Participants were asked to declare the strength of agreement with proposed statements using a Likert scale. Mean scores for corresponding separate statements divided according to health care setting were calculated and compared. RESULTS: The survey response rate was 0.64 (58 / 90). None of the participating private MBLs declared to analyse EBF. We report a mean score of 3.45 obtained for all statements evaluated. Deviations from desirable procedures were demonstrated in all EBF testing phases. Minor differences in procedures used for EBF analysis comparing secondary and tertiary health care MBLs were found. The lowest scores were obtained for statements regarding quality control procedures in EBF analysis, participation in proficiency testing programmes and provision of interpretative comments on EBF's test reports. CONCLUSIONS: Although good laboratory EBF practice is present in Croatia, procedures for EBF analysis should be further harmonized to improve the quality of EBF testing and patient safety.

Comparison of two commercial enzyme-linked immunosorbent assays for cerebrospinal fluid measurement of amyloid ß1-42 and total tau.

Amyloid ß1-42 (Aß1-42), total tau (t-tau), and phosphorylated tau (p-tau) are the main cerebrospinal fluid (CSF) biomarkers for early diagnosis of Alzheimer's disease (AD). Detection of AD is critically important in view of the growing number of potential new drugs that may influence the course of the disease in its early phases. However, cut-off levels for these CSF biomarkers have not yet been established. Variability in absolute concentrations of AD biomarkers is high among studies and significant differences were noticed even within the same datasets. Variability in biomarkers levels in these assays may be due to many aspects of operating procedures. Standardization of pre-analytical and analytical procedures in collection, treatment, and storage of CSF samples is crucial because differences in sample handling can drastically influence results. Multicenter studies showed that usage of ELISA kits from different manufacturers also affects outcome. So far only very few studies tested the efficiency of ELISA kits produced by different vendors. In this study, the performance of Innogenetics (Gent, Belgium) and Invitrogen (Camarillo, CA, USA) ELISA kits for t-tau and Aß1-42 was tested. Passing-Bablok analysis showed significant differences between Invitrogen and Innogenetics ELISA methods, making it impossible to use them interchangeably.

Adiponectin level and gene variability are obesity and metabolic syndrome markers in a young population.

BACKGROUND AND AIMS: Human obesity is accepted as an important risk factor for development of MetS. Adiponectin is linked to central obesity and ADIPOQ variants are promising markers for understanding the genetic base of obesity-related disorders. We performed analyses of adiponectin concentrations and ADIPOQ variants and tested their associations with obesity and MetS in young subjects of Croatian origin. METHODS: Biochemical and anthropometric parameters of MetS were obtained for 149 unrelated subjects. Adiponectin levels were measured by ELISA assay. ADIPOQ -11391G>A and -11377C>G were genotyped by real-time PCR. RESULTS: BMI and WC, TG and GLUC showed inverse correlation, whereas HDL-C showed a positive correlation with adiponectin concentrations. For central obesity, we found association with -11377C>G and with -11391G>A polymorphisms. ADIPOQ -11377GG and -11391GA significantly increased the risk for the development of central obesity (OR 5.57 and OR 3.37, respectively). Significant association was found between -11391A, -11377G allele and haplotype and increased TG. -11377C>G and -11391G>A variant were significantly associated with the incidence of MetS. C>G mutation at position -11377 significantly increased the risk of MetS development (OR = 2.93). Compared with the -11391G homozygotes, carriers of the A allele had a significantly increased risk for the development of MetS (OR = 3.15). The test of overall association showed a statistically significant correlation of MetS with -11377C>G and -11391G>A haplotypes (p = 0.008). CONCLUSIONS: Analysis of adiponectin concentration and ADIPOQ -11391G>A and -11377C>G gene variants may be clinically meaningful for estimation of MetS risk in a young population.