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Background: Albuminocytological dissociation (ACD) of the cerebrospinal fluid (CSF) is defined as an increased total protein concentration with normal total nucleated cell count. It is suspected to occur in diseases that alter the blood-brain barrier, increase the production of protein or obstruct the flow of CSF. The purposes of this study were to review the CSF analysis results of a large cohort of dogs with neurological conditions, to analyse the total prevalence of ACD and to describe which diseases have a higher prevalence of ACD., Study Design and Methods: Medical records were retrospectively searched for dogs whom CSF was sampled from 2012-2019. Data collected included signalment, body weight, site of collection of the CSF, CSF analysis results, and final diagnosis., Results: A total of 497 dogs met the inclusion criteria. ACD was identified in 16.5% (82/497) of dogs. The diseases with higher proportion of ACD were cranial nerve neuropathy (6/10; 60.0%), brain tumour (10/24; 41.7%), idiopathic vestibular disease (7/17; 41.2%) and brain vascular disease (4/13; 30.8%)., Clinical Significance: This study describes the CSF patterns of the most common neurological conditions in dogs, also characterizing, for the first time in dogs, the prevalence and causes of ACD, which was identified in 16.5% of the samples. The diseases with highest proportions of ACD were cranial nerve neuropathy, brain tumour, idiopathic vestibular disease and brain vascular disease., (© 2021 British Veterinary Association.)

Background: Laboratory tests to assist in the diagnosis and monitoring of neuroinflammatory diseases are scarce. The soluble form of the CD27 molecule (sCD27) is shed in high concentrations by activated T cells and can be detected in the cerebrospinal fluid. The aim of this study was to investigate whether CSF quantitation of sCD27 could discriminate between inflammatory and non-inflammatory neurological diseases., Methods: The concentration of sCD27 was measured using a commercially available ELISA in 803 well-defined subjects from a study cohort comprised of 338 patients with neuroinflammatory disease, 338 with non-inflammatory neurological disease and 127 controls without neurological disease., Results: The median value of cerebrospinal fluid sCD27 was 64 pg/mL (IQR 0-200) in controls, 58 pg/mL (IQR 0-130) in patients with non-inflammatory disease and 740 pg/mL (IQR 230-1800) in patients with inflammatory disease. The likelihood ratio of having an inflammatory disease was 10 (sensitivity 74% and specificity 93%) if the sCD27 concentration was >250 pg/mL. In patients with a known inflammatory condition, the likelihood ratio of having an infection was 10 (sensitivity 40% and specificity 96%) if the sCD27 concentration was >2500 pg/mL., Conclusions: The likelihood of having an inflammatory neurological condition is increased with elevated concentrations of sCD27 in cerebrospinal fluid. Rapid tests of sCD27 should be developed to assist clinicians in diagnosis of neuroinflammatory disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Cerebrospinal fluid total protein (CSF-TP) is measured in the diagnosis of a range of immune or infectious disorders of the nervous system. Most laboratories and the medical literature use an antiquated, age-independent upper limit of 0.45 g/L. Therefore, we performed a systematic review of reference studies in the medical literature, with the primary objective of determining the CSF total protein upper reference limit (URL). Secondary objectives were to assess the effects of age, gender, laboratory methods, and methodological quality., Methods: A pre-planned and peer-reviewed electronic search strategy was used to search Ovid Medline and EMBASE for 1960-2017. All records underwent title/abstract review, and potentially relevant records underwent independent full-text review by two researchers. The remaining studies underwent quality assessment using a modification of the QUADAS2 revised tool. CSF-TP upper reference limits extracted from these studies were used to compute weighted means., Results: Twenty-two articles were retained for qualitative analysis and 20 for quantitative analysis. The weighted average of CSF-TP URL was 0.55 g/L, in studies with high methodological quality. Studies that examined the effect of age reported consistent correlations with advancing age, and CSF-TP URL values incrementally exceeded 0.60 g/L after age 50. There were no meaningful differences according to gender, laboratory method, or quality assessment score., Conclusions: There is concordance in available literature to recommend increasing CSF total protein upper reference limits, and to consider implementing age-adjusted values above 0.60 g/L starting at age 50. This information merits worldwide dissemination, to reduce the risk of over-diagnosis.

The field of neurological diseases strongly needs biomarkers for early diagnosis and optimal stratification of patients in clinical trials or to monitor disease progression. Cerebrospinal fluid (CSF) is one of the main sources for the identification of novel protein biomarkers for neurological diseases. Despite the enormous efforts employed to identify novel CSF biomarkers, the high variability observed across different studies has hampered their validation and implementation in clinical practice. Such variability is partly caused by the effect of different pre-analytical confounding factors on protein stability, highlighting the importance to develop and comply with standardized operating procedures. In this chapter, we describe the international consensus pre-analytical guidelines for CSF processing and biobanking that have been established during the last decade, with a special focus on the influence of pre-analytical confounders on the global CSF proteome. In addition, we provide novel results on the influence of different delayed storage and freeze/thaw conditions on the CSF proteome using two novel large multiplex protein arrays (SOMAscan and Olink). Compliance to consensus guidelines will likely facilitate the successful development and implementation of CSF protein biomarkers in both research and clinical settings, ultimately facilitating the successful development of disease-modifying therapies.

Cerebrospinal fluid is investigated in biomarker studies for various neurological disorders of the central nervous system due to its proximity to the brain. Currently, only a limited number of biomarkers have been validated in independent studies. The high variability in the protein composition and protein abundance of cerebrospinal fluid between as well as within individuals might be an important reason for this phenomenon. To evaluate this possibility, we investigated the inter- and intraindividual variability in the cerebrospinal fluid proteome globally, with a specific focus on disease biomarkers described in the literature. Cerebrospinal fluid from a longitudinal study group including 12 healthy control subjects was analyzed by label-free quantification (LFQ) via LC-MS/MS. Data were quantified via MaxQuant. Then, the intra- and interindividual variability and the reference change value were calculated for every protein. We identified and quantified 791 proteins, and 216 of these proteins were abundant in all samples and were selected for further analysis. For these proteins, we found an interindividual coefficient of variation of up to 101.5% and an intraindividual coefficient of variation of up to 29.3%. Remarkably, these values were comparably high for both proteins that were published as disease biomarkers and other proteins. Our results support the hypothesis that natural variability greatly impacts cerebrospinal fluid protein biomarkers because high variability can lead to unreliable results. Thus, we suggest controlling the variability of each protein to distinguish between good and bad biomarker candidates, e.g., by utilizing reference change values to improve the process of evaluating potential biomarkers in future studies., Competing Interests: The authors have declared that no competing interests exist.

The physiological and biophysical knowledge base for interpretations of cerebrospinal fluid (CSF) data and reference ranges are essential for the clinical pathologist and neurochemist. With the popular description of the CSF flow dependent barrier function, the dynamics and concentration gradients of blood-derived, brain-derived and leptomeningeal proteins in CSF or the specificity-independent functions of B-lymphocytes in brain also the neurologist, psychiatrist, neurosurgeon as well as the neuropharmacologist may find essentials for diagnosis, research or development of therapies. This review may help to replace the outdated ideas like "leakage" models of the barriers, linear immunoglobulin Index Interpretations or CSF electrophoresis. Calculations, Interpretations and analytical pitfalls are described for albumin quotients, quantitation of immunoglobulin synthesis in Reibergrams, oligoclonal IgG, IgM analysis, the polyspecific ( MRZ- ) antibody reaction, the statistical treatment of CSF data and general quality assessment in the CSF laboratory. The diagnostic relevance is documented in an accompaning review.

Cerebrospinal fluid (CSF) can be considered the most promising biosample for the discovery and analysis of biomarkers in neuroscience, an area of great medical need. CSF is a body fluid that surrounds the brain and provides a rich pool of biochemical markers, both proteomic and metabolomic, that reflect the state of neurological processes. Such biomarkers can either serve as diagnostic or prognostic biomarkers to improve the characterization of patients and preclinical disease models, or can be used to demonstrate drug-related exposure and efficacy. Here, we describe the proteomic toolbox for studying CSF from a drug-discovery perspective, and the trends and challenges that lie ahead.

2D DIGE is a promising approach to comparative proteome analysis known for a high sensitivity and high reproducibility compared with classical 2DE techniques. It offers new possibilities for the detection of cerebrospinal fluid (CSF) biomarkers in neurological diseases, such as dementia, amyotrophic lateral sclerosis or multiple sclerosis. We review the first studies using 2D DIGE for analysis of the CSF proteome in neurological diseases and discuss advantages, as well as drawbacks, of the methodological approach with special emphasis on CSF-related aspects.

Background and Purpose: In recent years, different approaches have been used to investigate changes of cerebrospinal fluid (CSF) proteome in patients affected by multiple sclerosis (MS) with the aim to identify protein markers with potential diagnostic or prognostic value. Because of the lack of standardization of current proteomic techniques, contrasting results were achieved until now in different laboratories. In this study, we compare CSF proteome of 10 relapsing-remitting MS (RR-MS) patients, 11 patients with clinically isolated syndrome (CIS), and 10 control subjects without neurological or systemic diseases., Methods: The differential expression of CSF proteins amongst these cohorts of patients was investigated by using two-dimensional electrophoresis and mass spectrometry., Results and Conclusions: We found an overexpression of IgG free kappa light chain protein in both CIS and RR-MS patients, compared with control subjects and an increased expression of an apolipoprotein E isoform in RR-MS patients, compared with CIS and control groups. Our results confirm the presence of CSF proteome changes in MS patients. Future research should be aimed to investigate the role of these candidate CSF markers in larger cohorts of CIS and MS patients.

Biomarkers in the cerebrospinal fluid (CSF) may be important for the diagnosis of chronic degenerative disorders in the central nervous system including dementia. Existing CSF biomarkers for dementia, however, are relatively nonspecific. More specific markers may be found by targeting investigations based on knowledge of the molecular pathology of the disease in question. In Alzheimer's disease, hyperphosphorylation of the tau protein is a characteristic feature and thus a comprehensive characterization of the phosphoproteome of the CSF may be pursued to obtain a complete picture of phosphorylation aberrations in health and disease. Toward that goal we here describe a method for a comprehensive isolation and identification of phosphorylated tryptic peptides derived from CSF proteins using a simple sample preparation step and titanium dioxide-affinity chromatography followed by MALDI-TOF or LC-MS/MS linear ion-trap-FT mass spectrometry. Whereas not all previously reported phosphoproteins were found in normal CSF, we detected 56 putative novel phosphorylation sites in 38 proteins in addition to known sites. The approach seems to be a promising foundation for the discovery of new biomarkers embedded in the CSF phosphoproteome.

A total of 164 cerebrospinal fluid (CSF) samples taken from neurological patients were classed into four groups according to the clinical diagnosis: multiple sclerosis (MScl, n = 44), clinically isolated syndrome of demyelination (CIS, n = 40), other inflammatory neurological disease (OIND, n = 26) and other neurological disease (OND, n = 54). After tryptic digestion, the samples were measured by MALDI-TOF MS. Spectra were analyzed using the R-project software package, in which a peak detection algorithm was developed. Subsequently, the peak lists were compared based on ranked data (non-parametric). Significant differences were observed in the comparisons of MScl vs. OND and CIS vs. OND. The comparisons of MScl vs. OIND, and CIS vs. OIND showed fewer significant differences. No significant differences were found in comparisons MScl vs. CIS and OIND vs. OND. MScl and CIS had strikingly similar profiles, probably a reflection of common pathological mechanisms. Three differentially expressed proteins in the comparison of MScl vs. OND were identified: chromogranin A, a potential marker for neurodegeneration; and two important factors in complement-mediated inflammatory reaction, clusterin and complement C3. CSF chromogranin A levels were confirmed to be significantly elevated in the MScl group using an ELISA.

This study evaluates levels of cerebrospinal fluid (CSF) brain-specific proteins (BSP) in subjects with optic neuritis (ON) who are at high risk of progression to multiple sclerosis (MS). Forty-one subjects had acute ON and 17 subjects with other neurological diseases (OND) served as controls. Twenty-one subjects with ON had white matter lesions on magnetic resonance imaging (MRI) and intrathecal synthesis of oligoclonal IgG bands (OB) consistent with being at high risk of progression to MS; eight of whom later were diagnosed with clinically definite MS (CDMS). Levels of S100B, ferritin and two neurofilament heavy chain phosphoforms (NfH(SM134) and NfH(SM135)) were analysed using ELISA technique. A putative index of 'axonal health' was expressed as a ratio of NfH(SM134) to NfH(S135). NfH(SM134) and the NfH(SM134:SM135) were significantly elevated in subjects with ON compared to controls. No significant differences in levels of CSF BSP were seen between ON subjects with CDMS plus those at high risk of progression to MS and ON subjects with normal MRI and negative CSF analysis. In conclusion, there is evidence of axonal damage in subjects who present with ON, which is independent of the diagnosis of CDMS.

The measurement of protein concentration in the cerebrospinal fluid is a basic analytical method in neurology. In this study, a pyrogallol red technique using a human albumin calibrator previously validated in human medicine was tested for canine samples, and the results were compared with those obtained using urine test strips. Pyrogallol red significantly (P<0.05) but moderately underestimated purified dog albumin and globulins. The imprecision of the technique was low: intra- and between-series coefficients of variation were 1.6 and 4.3 per cent at protein concentrations of about 0.3 g/litre. Over 49 samples, there was good agreement between the pyrogallol red and test strip results (r=0.63), especially for low and high protein concentrations, but misclassifications were observed with '+' test strip readings.

Capillary zone electrophoresis and high-resolution agarose gel electrophoresis were compared to detect protein components in serum and cerebrospinal fluid of patients. Both electrophoretic methods proved to be useful for detection of protein abnormalities (e.g., mono- and oligoclonal bands) in biological fluids, but capillary electrophoresis offered several important advantages, such as sample application without preliminary concentration, lack of staining procedures, and on-line evaluation of patterns. Furthermore, capillary electrophoresis exhibits shorter analysis time and high resolution with low baseline noise. The results were proven to be powerful in diagnosis and monitoring of dyscrasias in routine laboratory practice.

One-step capillary isoelectric focusing (cIEF), which uses reduced but non-zero electroosmosis flow to mobilize the focused proteins, was applied to the analysis of proteins in cerebrospinal fluid (CSF) and serum of patients with various neurological disorders. Under the conditions employed, pathological changes in the CSF proteins were clearly detected on the electropherograms within 25 min, although the serum proteins did not vary significantly between samples. The present one-step cIEF system seems to be useful in routine laboratory examinations of a large number of CSF samples as an aid in neurological diagnosis.

Cerebrospinal fluid (CSF) analysis is a basic tool for diagnosis of neurological diseases. Knowledge regarding blood-CSF barrier function (molecular flux/CSF flow theory) and neuroimmunology is reviewed to aid understanding and evaluation of CSF data. Disease-related immunoglobulin patterns (IgG, IgA, IgM with reference to albumin) are described in CSF/serum quotient diagrams with the hyperbolic reference range for blood-derived protein fractions in CSF. Clinical relevance of complementary analyses (cytology, PCR, oligoclonal IgG, antibody detection and brain-derived proteins) is briefly discussed. Integrated CSF data reports are shown with numerical and graphical data representation, reference range-related interpretation and diagnosis-related comments. The principles and rationale of general CSF analysis reported in this review should enable the reader to accurately interpret CSF data profiles, and to plan a proper evaluation of new brain- or blood-derived analytes in CSF.

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.

Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in HIV-associated neurological diseases. The activity of the 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) was detected by zymography in the cerebrospinal fluid (CSF) of 138 HIV-infected patients (40 with AIDS dementia, 83 with brain opportunistic infections and 15 neurologically asymptomatic), 26 HIV-seronegative individuals with inflammatory neurological diseases (IND) and 12 HIV-seronegative subjects with noninflammatory neurological diseases (NIND). MMP-2 was present in all CSF samples from HIV-seropositive and HIV-seronegative individuals, including those of subjects with NIND. On the contrary, MMP-9 was absent in the CSF of NIND controls, whereas the activity of this MMP was found in the 77 - 100% of CSF samples from HIV-infected patients, including those with HIV dementia, central nervous system (CNS) opportunistic infections or neurologically asymptomatic subjects. The highest levels of MMP-9 were found in the CSF of patients with cryptococcosis, cytomegalovirus encephalitis and tuberculous meningitis and were comparable with those found in the CSF of HIV-negative patients with multiple sclerosis or meningitis. A significant correlation between CSF MMP-9 activity and CSF cell count was found only in patients with HIV dementia. The increased CSF activity of MMPs capable to degrade components of the extracellular matrix of blood-brain barrier may contribute to the transendothelial migration of virus-infected cells into the CNS and development of HIV-associated neurologic damage.

The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39-42-amino-acid protein, amyloid beta protein (A beta). This study examined whether the measurement of A beta levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and A beta concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean A beta value for the AD group (15.9 +/- 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 +/- 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. A beta values did not correlate with age (r = -0.05, p = 0.59), severity of cognitive impairment (r = 0.22, p = 0.21), or duration of AD symptoms (r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of A beta in CSF. A beta immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of A beta reactivity. In conclusion, the total CSF A beta level is not a useful marker for current diagnosis of AD.

Previous studies in patients receiving interferon-alpha (IFN-alpha) therapy and patients with systemic lupus erythematosus have demonstrated that elevated cerebrospinal fluid (CSF) levels of IFN-alpha are associated with cognitive dysfunction. We measured IFN-alpha levels in CSF and blood by ELISA in human immunodeficiency virus (HIV)-positive patients with (n = 21) and without (n = 23) dementia and HIV-negative controls (n = 48). IFN-alpha was significantly elevated in the CSF of HIV-positive patients with dementia compared to those without dementia and controls. An increasing amount of IFN-alpha in the CSF was correlated with the clinical parameter of increasing Memorial Sloan Kettering scores; although these correlations were not statistically significant, they further suggest an association of increased CSF IFN-alpha with neurocognitive dysfunction in AIDS. Immunocytochemical staining of brains demonstrated IFN-alpha-positive macrophages and astrocytes in frontal cortex and white matter and IFN-alpha mRNA was detected by reverse transcriptase-polymerase chain reaction, further indicating that IFN-alpha is made by cells within the brain and suggesting that the significant increases of IFN-alpha protein found in the CSF of patients with HIV-associated dementia complex are derived from intrinsic brain cells such as macrophages and astrocytes. Increased local production of IFN-alpha during HIV infection may contribute directly or indirectly to the pathogenesis of HIV-associated dementia.

A method for the analysis of proteins in cerebrospinal fluid (CSF) using free solution capillary electrophoresis (FSCE) was developed, in which the adsorption of proteins to the fused silica capillary wall was virtually eliminated. FSCE was carried out on 30 randomly selected CSF samples. Good sensitivity and resolution was obtained with minimal baseline noise. These preliminary results clearly demonstrated the potential of FSCE to analyse proteins in CSF, both qualitatively and quantitatively more rapidly than has been possible hitherto. A total run time of approximately 30 min allowed between 20 to 25 peaks to be separated. Many peaks with long migration times were detected in CSF that appeared to be specific since they were not detectable in corresponding serum samples. The separation patterns for most of the CSF samples selected showed relatively similar patterns but there were often minor qualitative and quantitative differences. Four CSF samples showed patterns that were significantly different from the others, primarily in the peaks with long migration times. A larger clinical study is now being undertaken.

D-dimer assay was performed on 145 cerebrospinal fluid (CSF) samples from patients with or without neoplastic diseases. Levels of D-dimers were significantly higher in carcinoma and lymphoid malignancies with clinical or biological evidence of central nervous system (CNS) involvement than in diseases without such complications. In one patient, serial determinations of D-dimers were well correlated with the appearance and disappearance of CNS involvement. Although this test is not specific for neoplastic affections, our data suggest that the measurement of D-dimers in CSF may be useful in the diagnosis of CNS involvement of neoplastic cells and in monitoring intrathecal therapy in patients with lymphoma, acute lymphoblastic leukaemia or carcinoma. In this study, the D-dimer assay was also positive in some non neoplastic diseases, but failed to differentiate subarachnoid haemorrhage from traumatic lumbar puncture.

Variations in either the polypeptide sequence or the carbohydrate moieties of transferrin may result in altered electrophoretic mobility of this molecule. We report a case of an allelic (polypeptide) variant of transferrin with mobility similar to that of the beta 2 (sialic acid-depleted) transferrin found in cerebrospinal fluid (CSF) and a few other body fluids. Allelic variation and other transferrin anomalies may be mistaken for the CSF isoform, resulting in false diagnoses of CSF fistulae.

The assessment of proteins intrathecal synthesis (ITS) is an essential step in the CSF analysis. It can be established qualitatively by different ratios and quantitatively by empirical formulae. Schuller and Sagar's formula was proposed 10 years ago for the calculation of IgG ITS. From this calculation, the antibody specific activity of intrathecal immunoglobulins may be also evaluated. The same principle may be used for complement components and for different other CSF proteins. Two examples (concerning Fibronectin and prealbumin ITS) demonstrate the usefulness of this approach, which can be programmed by a computer.

Oligoclonal IgG bands were analyzed in matching pairs of cerebrospinal fluid (CSF) and serum from 12 subacute sclerosing panencephalitis (SSPE) patients, using isoelectric focusing and immunofixation. Each patient was given isoprinosine, and four of the 12 patients were given alpha-interferon in addition. Two to 4 serial CSF and serum samples were collected from each SSPE patient during periods ranging from 1 to 16 months. In 3 SSPE patients a small number of new oligoclonal bands were seen in the follow-up CSF samples. In the other 9 SSPE patients there was no change in CSF band patterns between initial and follow-up specimens. Band patterns in serum remained unchanged between initial and follow-up samples. Although all 12 SSPE cases had higher IgG indices and increased rate of intra blood-brain barrier (BBB) IgG synthesis in comparison to patients with other neurological diseases, the values did not significantly differ between the first and follow-up specimens. We conclude that treatment of SSPE patients with isoprinosine or with isoprinosine and alpha-interferon had no significant effect on the CSF oligoclonal band profiles or IgG synthesis within the central nervous system.

We detected immunoreactive interferon-gamma (IFN-gamma) both in cerebrospinal fluid (CSF) and in serum of 5 patients with scrub typhus, one with meningitis and 4 other cases with neither CSF pleocytosis nor blood-CSF barrier dysfunction. Our data suggest intrathecal synthesis of IFN-gamma without pleocytosis which implies occult cerebral involvement in scrub typhus.

Isoelectric focusing (IEF) and agarose gel electrophoresis were used to detect oligoclonal IgM bands in cerebrospinal fluid and serum samples from 850 patients with diverse neurological diseases. Oligoclonal IgM bands in cerebrospinal fluid were mainly detected in patients with infectious and inflammatory disorders of the nervous system. Both IEF and agarose electrophoresis revealed similar frequencies of oligoclonal IgM bands. Bands detected by IEF were mainly seen in the anodal range. Despite higher resolving capacity, IEF was less specific than agarose gel electrophoresis. It is concluded that oligoclonal IgM bands have important diagnostic significance and that agarose gel electrophoresis is more suitable for their detection in routine clinical work and use in differential diagnosis.

Two-dimensional electrophoresis (2-DE) of cerebrospinal fluid (CSF) samples--from 347 patients with various psychiatric and neurological disorders--and subsequent silver staining revealed two additional polypeptides (Mr 40,000) in 49% of 111 schizophrenics, 46% of 43 schizoaffective patients, 36% of 41 patients with affective disorders, 43% of 28 patients with multiple sclerosis, but not in 25 patients without neurological symptomatology, nor in 9 patients with Lues, and in only 2 of 25 patients with AIDS. The two polypeptides, as detected by 2-DE, eluted after size exclusion chromatography in fractions containing proteins with Mr greater than 200,000. After 2-DE of CSF samples, enriched by gel chromatography, the polypeptides were immobilized by blotting onto glass-fiber membranes and subjected to N-terminal sequencing. Polypeptide A was identified as beta-chain remnant (beta 2), derived from plasmin cleavage of fibrin(ogen). After size exclusion chromatography, 2-DE, and Western blotting, polypeptide A and B, as well as several other spots, reacted with fibrinogen antibodies, suggesting that the polypeptides are subunits of a fibrin degradation complex.

Apolipoprotein E was measured in paired sera and cerebrospinal fluid samples from 483 neurological patients. The average apolipoprotein E concentration was 7.5 (+/- 3.1) mg/l in cerebrospinal fluid and 93.5 (+/- 29.7) mg/l in serum. Mean apolipoprotein B concentrations in 88 patients were 0.77 +/- 3.4 mg/l in cerebrospinal fluid and 1.06 +/- 0.31 g/l in serum. The apolipoprotein E concentration in cerebrospinal fluid was much greater than expected for passive diffusion from serum. By contrast to apolipoprotein B, there was no correlation between the apolipoprotein E cerebrospinal fluid/serum concentration quotient and the albumin concentration quotient. This suggests that apolipoprotein E in cerebrospinal fluid, unlike apolipoprotein B, is locally synthesized and that the use of a cerebrospinal fluid/serum concentration quotient is unnecessary. In a control group (n = 64) the mean cerebrospinal fluid apolipoprotein E value (+/- SD) was 5.9 (1.6) mg/l. Elevated cerebrospinal fluid apolipoprotein E concentrations were observed in acute (n = 22, 12.5 +/- 6.2 mg/l) and chronic inflammatory central nervous system diseases (n = 15, 10.4 +/- 2.4 mg/l).

We analyzed 352 paired CSF and serum samples with isoelectric focusing and immobilized pH gradients (IPG-IEF) using three different gradients. All MS patients (n = 35) had several (from 5 to 30) oligoclonal bands (OBs) in the CSF and most of them had few (from 1-8) OBs in serum. 54.5% patients with CNS infections (n = 12) had oligoclocal bands. OBs in these patients were fewer in number and located at the more acidic pH region compared with the MS samples. Also, corresponding serum bands were more often recognized. We suggest that IPG-IEF improves the diagnostic evaluation of the oligoclonal band pattern. The possibility of the use of extremely narrow gradients makes it a valuable tool for the analysis of selective proteins in various disorders.

Beta-2-microglobulin was measured in specimens of cerebrospinal fluid (CSF) collected from 167 patients classified in 14 diagnostic categories at an outpatient Department of Neurology. In the control group of 29 subjects without any obvious disease of the nervous system, the concentration of beta-2-microglobulin was 1.15 +/- 0.37 mg/1 (M +/- s.d.). The concentration was almost significantly elevated in the groups with fresh brain infarct, central nervous system infection, and polyneuropathy. The serum concentrations of beta-2-microglobulin did not differ significantly among these diagnostic categories. The mean ratio between CSF and serum beta-2-microglobulin was 0.79 +/- 0.32 in the control group and more than 1.0 in the patients with brain infarcts, CNS infections and spinal paresis, but the differences were not statistically significant.

Cerebrospinal fluid (CSF) protein values were measured in 652 children between the ages of 1 day and 17 years, allowing the authors to define the dynamics of the blood-brain barrier under normal conditions and during inflammation of the nervous system. The ratio of CSF albumin/serum albumin (whose upper limit was 0.65 in the study) was the best sign of alteration of blood-brain barrier permeability. The CSF IgG level, whose upper limit was 0.85 (for serum IgG between 10 and 14 g/l) is the most useful criterion for detecting an intra-thecal synthesis of IgG. Six patterns of CSF proteins are defined on the basis of immunochemical and electrophoretic studies. The ratio of CSF albumin/serum albumin and the CSF IgG level must be compared to the electrophoretic pattern of CSF proteins in order to better characterize one aspect of the blood-brain barrier under normal and pathologic conditions of the central nervous system.

CSF and serum was obtained from 216 patients with neurological or psychoneurotic symptoms and the concentrations of albumin and IgG were immunologically determined. The IgG/albumin index, calculated as the quotient of the CSF/serum ratios of IgG and albumin was compared with electrophoresis on agar gel. In "normal" cases, the IgG/albumin index was between 0.26-0.66. Pathological electrophoresis, i.e. with two or more IgG bands in the gamma globulin region was found in 85 per cent of the MS patients; in 29 per cent of the patients with a possible demyelinating disease; in 41 per cent of patients with CNS infection; and in 4 per cent of patients with other neurological disorders; whereas an increased IgG/albumin index ( greater than 0.66) was found in 88 per cent of the MS patients; in 43 per cent of the patients with a possible demyelinating disease; in 50 per cent of the patients with CNS infection; in 11 per cent of patients with immunological disorders; and in 18 per cent of patients with other neurological diseases. The increase of the IgG/albumin index was sometimes moderate (0.67-0.90), except in patients with MS, syphilis and other CNS infections, where a pathological electrophoresis combined with an IgG/albumin index above 1.0 was found to be a valuable support for the clinical diagnosis.

Isoelectric focusing (IEF) and isotachophoresis (ITP), two methods with excellent separation capacities, have been adapted during recent years for the analysis of CSF proteins. The fractions separated by these techniques can be further studied by e.g. immunological methods. ITP has besides its high separation capacity several valuable advantages: very small samples are needed, unconcentrated CSF can be examined, the analyses are quickly performed and the results are immediately obtained on a recorder. Examinations by thin-layer IEF in a series of about 2,000 patients have afforded much new information about the CSF and serum proteins in many neurological diseases. Different complex CSF protein aberrations have been found in the gammaglobulin range as well as in more anodal positions in MS, infectious neurological diseases and Guillain-Barré syndromes. These aberrations are probably the result of several interacting factors, e.g. the temporal and spatial characteristics of the disease, the release of decomposition products from destroyed tissues, the genetically determined reactivity of the individual and the type of etiological agent.

The CSF and serum proteins of 120 patients with known or probable infectious neurological diseases or the Guillain-Barré syndrome were examined with thin-layer IEF. All but two of these patients exhibited one or combinations of different CSF-protein aberrations in the acidic and alkaline range. Aberrant non-Ig fractions (including transferrin, the tau-fraction and gamma-trace protein) were found in frequencies varying between 4 and 48%. CSF Ig components of restricted heterogeneity, i.e. oligoclonal bands and/or regional increases of gamma-globulins, were more frequent in patients with (meningo-)encephalitic or (meningo-)-myelitis/radiculitic disorders (respectively 69 and 48%) than in subjects with meningitis or Guillain-Barré syndrome (17%). The occurrence of such Ig abnormalities was higher in subacute or chronic than in acute disease and in subjects examined greater than 4 weeks after the onset rather than earlier. Ig-band spectra with marked anodal extension were found predominantly in (meningo-)encephalitic disorders with infratentorial symptoms. Age and sex were not found to influence the occurrence of abnormal Ig fractions. Such components could be detected in spite of pronounced blood-CSF barrier defects.

The author studied the fractional content of proteins in the CSF in 105 children with different disorders of the nervous system and 35 children of a control group (aged 2-13 years) with the aid of disc-electrophoresis in polyacrylamide gel. The proteins of the CSF were studied in encephalitis, polyradiculoneuritis, cerebral arachnoiditis, brain tumors and epilepsy. The content of total proteins in the CSF in 42% of the studied children did not differ significantly from the usual age norm. More distinct changes were found in proteinogramms of the CSF where 14 protein fractions were singled out. It was established that a change in the content of separate fractions, their intercorrelation depended on the character of the pathological process.

The cerebrospinal fluid (CSF) IgG index, the CSF to serum albumin ratio, and electrophoresis on agarose gel of CSF and serum were evaluated retrospectively for their usefulness in the differential diagnosis of multiple sclerosis (MS). Standardized procedures were adopted for grading the intensity of oligoclonal banding and for the certainty of diagnosis of MS. One hundred and forty-nine patients were studied, including 23 with definite multiple sclerosis (MS), 12 with probable MS, 20 with possible MS, 20 with inflammatory neurologic disease, 65 with noninflammatory neurologic disease, and nine with no neurologic disease. The CSF IgG index and the CSF to serum albumin ratio were calculated from nephelometric measurements of serum and CSF IgG and albumin. The intensity of oligoclonal banding was graded relative to the density of the prealbumin band. Eighty-eight per cent of cases of definite MS had distinct oligoclonal bands, and an equal number had an elevated IgG index. These tests were not specific for MS, however, since 50% of cases of inflammatory neurologic disease and 5% of those with noninflammatory neurologic disease had an elevated IgG index. Similarly, 48% of cases with inflammatory disease and 25% with noninflammatory disease had oligoclonal bands. However, only patients with definite MS (21%) or possible MS (4%) had prominent oligoclonal bands whose density was greater than or equal to that of prealbumin, together with a CSF IgG index greater than 1.50. This combination of findings therefore may enhance the level of suspicion of MS. By contrast, an isolated increase in the CSF to serum albumin ratio may suggest a diagnosis other than MS.

The clinical data and the CSF and serum findings on isoelectric focusing (IEF) and electrophoresis were studied in nine patients with neurological disorders combined with proliferations of reticulocytes, lymphocytes or plasmocytes. Paraneoplastic neurological manifestations were considered in some of the subjects. CSF findings compatible with intrathecal synthesis of oligoclonal immunoglobulins were observed in two patients with lymphoreticular neoplasms. One of these had an IEF band spectrum differing from those found with multiple sclerosis (MS), while the other had changes indistingusihable from those with MS. On IEF the CSF and serum M components of the patients with plasma cell dyscrasias, as well as the serum M components of nine other patients with myelomatosis, exhibited considerable microheterogeneity with 3-18 abnormal bands. The isoelectric point (pI) of the individual bands was 5.8-9.2, mostly greater than 6.4; the band spectra exhibited pI ranges of 0.4-2.5 pH units, most frequently 0.4-1.2. The CSF findings, possible modified by the blood-CSF barrier, reflected the serum changes of plasma cell dyscrasias. The immunoglobulins produced in such disorders gave IEF band spectra differing from those of intrathecally produced immunoglobulins in MS.

The purpose of this investigation was to examine the diagnostic value of nine specific proteins in differentiating multiple sclerosis from other neurological disorders. The quantitative determination of albumin, transferrin, haptoglobin, IgG, IgA, IgM, orosomucoid, complement-C3 and prealbumin was carried out using an automated immunonephelometric method. Using a combination of IgG-index ((Sp-IgG/S-IgG)/(Sp-albumin/S-albumin)) and haptoglobin-index ((Sp-haptoglobin/S-haptoglobin)/(Sp-albumin/S-albumin)) it is possible to increase the diagnostic value. Using a modified index it is possible to obtain the same diagnostic value by combining modified IgG-index ((Sp-IgG/S-IgG)/Sp-albumin) and modified haptoglobin-index ((Sp-haptoglobin/S-haptoglobin)/Sp-albumin). The diagnostic specificity and sensitivity for the modified combined index was 0.60 and 1.00, respectively.

Isoelectric focusing in immobilized pH gradients, supplemented with 0.5% w/v carrier ampholytes was applied for studies of native proteins, especially immunoglobulin G, in cerebrospinal fluid and serum. All 72 paired samples were run on pH 4-10 gels; 25 of them were also examined in pH 7-10 gels. Silver staining and nitrocellulose blotting with amplified immunoperoxidase detection of immunoglobulin G were used for protein visualization. Intrathecally produced immunoglobulin G was resolved into sharply focused, straight and easily identifiable fractions. The pH gradients were stable and the inter-gel reproducibilities of individual immunoglobulin G patterns were good.

Using radial immunodiffusion, albumin and immunoglobulin G were determined in non-preconcentrated cerebrospinal fluid from 127 controls and from 239 patients. In controls the concentrations of albumin and immunoglobulin G followed normal distribution. The two variables were correlated linearly (r = 0.60). The elliptic bivariate normal range was calculated, and was found to contain 95% of the paired values. As a clinical limit, this range discriminated more effectively between normal and altered pairs than the two one-dimensional normal ranges X+/-2 s, thus improving the evaluation of laboratory findings in the single case. Likewise in clinically defined groups of patients, bivariate evaluation of results provided additional evidence. In many distinct clinical syndromes, e.g. bacterial encephalomeningitis, polyneuropathy, amyotrophic lateral sclerosis, albumin and immunoglobulin G concentrations exhibited an especially close correlation, probably resulting from damage to the blood-cerebrospinal fluid barrier. However, no correlation of these two variables was detected in acute encephalomeningitis due to virus infection, and in multiple sclerosis: in these groups, immunoglobulin G concentrations were elevated independently of albumin. Since evidence is lacking as to the pathogenesis of multiple sclerosis, it seems noteworthy that the same phenomenon was observed in a well-defined group of viral infections.