Hayrettin Tumani

A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in CSF are being used in clinical practice. One of the most critical factors in CSF biomarker research is the inadequate powering of studies because of the lack of sufficient samples that can be obtained in single-center studies. Therefore, collaboration between investigators is needed to establish large biobanks of well-defined samples. Standardized protocols for biobanking are a prerequisite to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by preanalytical factors. Here, a consensus report on recommendations for CSF collection and biobanking is presented, formed by the BioMS-eu network for CSF biomarker research in multiple sclerosis. We focus on CSF collection procedures, preanalytical factors, and high-quality clinical and paraclinical information. The biobanking protocols are applicable for CSF biobanks for research targeting any neurologic disease.

Elevated levels of tau-protein in cerebrospinal fluid of patients with Creutzfeldt–Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare, fatal, neurodegenerative disease caused by a transmissible agent designated as proteinaceous infectious agent (prion). Searching for biochemical markers of CJD, we analysed cerebrospinal fluid (CSF) samples of 53 patients for tau-protein using an enzyme linked immunoassay (ELISA). In a group of 21 patients with definite CJD seen in the German case control study for CJD, tau-protein concentrations in CSF were significantly higher than in two control-groups of patients with other diseases (median 13,153 pg/ml, range 1,533-27,648 pg/ml; P = 0.0001). One group comprised 19 patients who were seen in the same study and were diagnosed as having other dementing diseases (tau concentration: median 558 pg/ml, range 233-1,769 pg/ml). The second control group comprised 13 patients from our hospital with no dementing disease (tau concentration: median 296 pg/ml, range 109-640 pg/ml). We conclude that determination of tau protein levels in CSF is a useful marker for laboratory diagnosis of CJD.

The Alzheimer's Association external quality control program for cerebrospinal fluid biomarkers.

The cerebrospinal fluid (CSF) biomarkers amyloid β (Aβ)‐42, total‐tau (T‐tau), and phosphorylated‐tau (P‐tau) demonstrate good diagnostic accuracy for Alzheimers disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimers Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch‐to‐batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program.

Axonal damage markers in cerebrospinal fluid are increased in ALS

Objective: To test whether biomarkers for axonal degeneration correlated with clinical subtypes and were of use in predicting progression of ALS. Methods: Patients with ALS (n = 69), patients with Alzheimer disease (AD; n = 73), and age-matched controls (n = 33) were included in this prospective study. CSF levels of tau protein and neurofilaments (NfHSMI35) were measured using ELISA. In 49 patients with ALS, follow-up data were available (median follow-up 7 months). Results: CSF levels of NfHSMI35 were five times higher in patients with ALS (1.7 ng/mL) than in controls (0.3 ng/mL, p < 0.001) and 10 times higher than in patients with AD (0.14 ng/mL, p < 0.001). NfHSMI35 values were also higher in patients with upper motor neuron–dominant ALS than in patients with typical ALS (upper motor neuron + lower motor neuron) at p = 0.02. Values of NfHSMI35 were higher in ALS of more rapid progression. The values of NfH and tau did not correlate with CSF protein content. Conclusions: The authors propose that axonal damage markers in CSF may discriminate between subtypes of ALS and that they could be used as markers for therapeutic trials. CSF NfH was superior to tau in these discriminations.

TDP-43 in cerebrospinal fluid of patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

BACKGROUND Recently, TAR DNA-binding protein 43 (TDP-43) was identified as the major component of ubiquitin-positive tau-negative neuronal and glial inclusions in the most common form of frontotemporal lobar degeneration (FTLD) and in amyotrophic lateral sclerosis (ALS). It was demonstrated that different TDP-43 profiles correspond to clinical phenotypes of FTLD or ALS subgroups, and the differential diagnostic potential of TDP-43 was suggested. OBJECTIVES To examine TDP-43 in cerebrospinal fluid (CSF) and to analyze whether it could serve as a diagnostic marker. DESIGN We characterized CSF TDP-43 by immunoblot using different TDP-43 antibodies and determined the relative TDP-43 levels in CSF samples from patients. SETTING Academic research. PATIENTS Twelve patients with FTLD, 15 patients with ALS, 9 patients with ALS plus FTLD, 3 patients with ALS plus additional signs of frontal disinhibition, and 13 control subjects. MAIN OUTCOME MEASURES Results of TDP-43 immunoblot. RESULTS Polyclonal TDP-43 antibodies recognized a 45-kDa band in all analyzed samples. Two monoclonal and N-terminus-specific antibodies did not detect any specific bands, but C-terminus-specific antibodies detected a 45-kDa band and additional bands at approximately 20 kDa in all CSF samples. Relative quantification of 45-kDa bands revealed significant differences among the diagnostic groups (P =.046). Specifically, patients with ALS (P =.03) and FTLD (P =.02) had higher TDP-43 levels than controls but with a prominent overlap of values. CONCLUSION Although there is no evidence of pathologically altered TDP-43 proteins in CSF, TDP-43 levels in CSF might aid in characterizing subgroups of patients across the ALS and FTLD disease spectrum.

Cerebrospinal fluid analysis in affective and schizophrenic spectrum disorders: Identification of subgroups with immune responses and blood–CSF barrier dysfunction

Immune and inflammatory mechanisms are detected in a subgroup of treatment resistant hospitalized affective and schizophrenic spectrum disorder patients. We analysed albumin, IgG, IgA, IgM, oligoclonal IgG and specific antibodies in paired cerebrospinal fluid (CSF) and serum samples. Numerical and graphical interpretation of CSF protein data was performed by Reibergrams with a new CSF statistics tool for nonlinear group analysis with reference to a large control group (n=4100). In 41% of the psychiatric patients (n=63) we observed CSF pathologies: 14% displayed intrathecal humoral immune responses, 10% slightly increased CSF cell counts (5-8/microL) and 29% had moderate blood-CSF barrier dysfunctions, in 24% as the only pathological sign with normal IgG, IgA and IgM concentrations in CSF (p=0.9 testing the null hypothesis for intrathecal synthesis with reference to Qmean of the reference group). In the group of affective (n=24) spectrum disorders 20% displayed a systemic immune reaction as detected by oligoclonal IgG. CSF analysis and interdisciplinary clinical approach revealed 6% of psychiatric patients likely to represent a virusspecific, bacterial or autoimmune associated disorder with CNS involvement. Elevated CSF neopterin concentration in 34% of the patients was interpreted as an increased release from astrocytes or from other glia cells. The low level immune response and barrier dysfunctions are discussed on the base of a mild encephalitis pathomechanism in subgroups of psychiatric patients. CSF analysis is shown to be a useful diagnostic tool for differential diagnosis in psychiatric diseases.

Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis

We describe the specificity and sensitivity of measuring a combination of basic CSF variables and Borrelia burgdorferi (Bb)-specific IgG and IgM antibody index (AI) values for the diagnosis of early neuroborreliosis. Basic CSF variables included total cell count, quantitation of activated B cells (IgG, IgA, and IgM classes), CSF/serum quotient diagrams for IgG, IgA, and IgM (to quantitate brain-derived immunoglobulin fractions in CSF), and CSF/serum albumin ratio as a measure of blood-CSF barrier function. The Bb-specific component of immunoglobulins in CSF and serum was quantitated by ELISA. Results are based on data from CSF and serum of 24 patients with definite neuroborreliosis, 45 patients with other neurologic diseases, and 28 control individuals. Combined evidence of an elevated CSF cell count, IgM-class dominance in both the cellular and intrathecal humoral immune response, and blood-CSF barrier dysfunction yielded 70% diagnostic sensitivity and 98% diagnostic specificity for detection of neuroborreliosis. Intrathecal production of Bb-specific IgM, evaluated as Bb-specific IgM antibody index (Bb-IgM-AI; pathologic value more than 1.4) showed 79% diagnostic sensitivity and 96% diagnostic specificity. Correspondingly, elevated Bb-specific IgG antibody index (Bb-IgG-AI; pathologic value more than 1.4) displayed 63% diagnostic sensitivity and 89% diagnostic specificity. Combined analysis of Bb-specific AI values and basic CSF variables gave the highest sensitivity (80%) and specificity (98%). Analysis of CSF variables over a disease course showed that acute versus past disease could be discriminated by a combination of basic CSF variables and Bb-specific AI. The presence of activated B cells (IgM class), indicative of acute disease with 82% diagnostic sensitivity and 94% diagnostic specificity, is the earliest marker to disappear following effective therapy. NEUROLOGY 1995;45: 1663-1670

International quality control survey of neurochemical dementia diagnostics.

UNLABELLED Currently, neurochemical dementia diagnostics (NDD) are increasingly entering routine clinical neurochemistry, offering improved early and differential diagnosis of dementias. However, there is an obvious lack of standardization in pre-analytical sample handling and systematic quality surveys. Therefore, in this study, 14 laboratories in Germany, Austria, and Switzerland were given aliquots of a human cerebrospinal fluid (CSF) sample, and were asked to measure Alzheimers disease (AD) biomarkers (amyloid beta (Abeta) peptides, total Tau protein, and phosphorylated Tau protein (P-tau(181P))) according to their routine protocols. RESULTS The inter-laboratory coefficients of variation of the results obtained by the laboratories participating in this study were in the range of 20-30%. Although the results of this quality control survey are promising, the quality of measurements has to be further optimized.

Cerebrospinal fluid biomarkers in multiple sclerosis

In patients with multiple sclerosis (MS) intensive efforts are directed at identifying biomarkers in bodily fluids related to underlying disease mechanisms, disease activity and progression, and therapeutic response. Besides MR imaging parameters cerebrospinal fluid (CSF) biomarkers provide important and specific information since changes in the CSF composition may reflect disease mechanisms inherent to MS. The different cellular and protein-analytical methods of the CSF and the recommended standard of the diagnostic CSF profile in MS are described. A brief update on possible CSF biomarkers that might reflect key pathological processes of MS such as inflammation, demyelination, neuroaxonal loss, gliosis and regeneration is provided.

IgG antibodies against measles, rubella, and varicella zoster virus predict conversion to multiple sclerosis in clinically isolated syndrome.

Background Multiple sclerosis (MS) is characterized by a polyspecific B-cell response to neurotropic viruses such as measles, rubella and varicella zoster, with the corresponding antibodies measurable in CSF as the so-called “MRZ reaction” (MRZR). We aimed to evaluate the relevance of MRZR to predict conversion of patients with clinically isolated syndrome (CIS) to MS, and to compare it to oligoclonal bands (OCB) and MRI. Methodology/Principal Findings MRZR was determined in a prospective study over 2 years including 40 patients that remained CIS over follow-up (CIS-CIS) and 49 patients that developed MS (CIS-RRMS) using ELISA. Using logistic regression, a score (MRZS) balancing the predictive value of the antibody indices included in MRZR was defined (9 points measles, 8 points rubella, 1 point varicella zoster, cutpoint: sum of scores greater 10). MRZR and MRZS were significantly more frequent in CIS-RRMS as compared to CIS-CIS (p = 0.04 and p = 0.02). MRZS showed the best positive predictive value (PPV) of all parameters investigated (79%, 95%-CI: 54–94%), which could be further increased by combination with MRI (91%, 95%-CI: 59–99%). Conclusions/Significance Our data indicate the relevance of MRZR to predict conversion to MS. It furthermore shows the importance of weighting the different antibody indices included in MRZR and suggest that patients with positive MRZR are candidates for an early begin of immunomodulatory therapy.