Use Of Cerebrospinal Fluid Research Articles

Parasitic Encephalitis in a Bitch

ABSTRACTBackground: Encephalitis is common in most inflammatory and infectious diseases of the central nervous system, and bacterial and viral pathogens, protozoa, fungi, and erythematous parasites are recognized as etiological agents. Clinical signs vary according to the anatomical location and severity of the inflammation. Some parasites may undergo aberrant migration into the brain of dogs and cats, which is are considered a rare clinical phenomenon. This case report describes the occurrence of parasitic encephalitis in a mixed breed bitch about 4 year-old.Case: A mixed breed bitch, about 4 year-old, rescued from the street with weakness of the pelvic limbs, was treated at the Veterinary Hospital of the Federal University of Piauí - UFPI. After A clinical and neurological examination, the patient was hospitalized for diagnosis, treatment, confirmation and follow-up of her clinical evolution. Complementary tests were performed to evaluate her general condition, given her unknown history because she was a rescue dog. Complete blood count (CBC), hemoparasite screening, biochemical function (urea, creatinine, ALT, alkaline phosphatase, total protein, albumin and globulin), abdominal and pelvic ultrasound, serological and parasitological tests for leishmaniasis, distemper antigen and ehrlichiosis antibody detection tests were performed, and cerebrospinal fluid (CSF) was collected for analysis. The CBC revealed normocytic hypochromic anemia and thrombocytopenia. Biochemical functions were within normal limits, except for alanine aminotransferase (ALT), which was slightly low. The ultrasound examination showed splenomegaly. The canine distemper, ehrlichiosis and leishmaniasis tests were negative. The results of the CSF analysis revealed the presence of spherical structures compatible with parasitic cysts, suggestive of Echinococcus granulosus. After the laboratory diagnosis of parasitic encephalitis, the animal was treated with prednisolone, albendazole, praziquantel and doxycycline. Three days after starting treatment for parasitic encephalitis, the patient presented significant clinical improvement, began to feed again and to move around.Discussion: The presence of parasitic cysts in cerebrospinal fluid is uncommon; however, encephalitis due to aberrant parasite migration has been described in dogs and cats. The migration and growth of the parasite causes extensive damage to the neural parenchyma. The clinical signs of worm encephalitis tend to show acute to super-acute onset, and the definitive diagnosis depends on the identification of the parasitic agent in the patient’s brain. This type of diagnosis has so far been performed only during necropsy. In the case reported here, parasitic cysts were identified in CSF, thus confirming and formulating a definitive diagnosis. It is rare to find microorganisms and tumor cells in samples of cerebrospinal fluid; however, if this occurs, a definitive diagnosis can be formulated. The use of cerebrospinal fluid is therefore an important diagnostic tool when other imaging tests such as tomography and magnetic resonance provide limited access. Praziquantel and Albendazole are recommended for the treatment of Echinococcus, prednisolone is recommended for cases of encephalitis, and prophylactic antibiotic therapy is used to prevent secondary bacterial meningoencephalitis. The recommended treatment was efficient for this patient, and could be adopted to treat other dogs with parasitic encephalitis. The main forms of prevention in dogs are periodic worming, and the ingestion of only processed or cooked foods and treated water.

Use of Cerebrospinal Fluid (1,3)-β-d-Glucan to Monitor Treatment Response in Candida albicans Meningitis in an HIV-infected Patient

Abstract Candida meningitis is rare in the setting of human immunodeficiency virus (HIV) infection and often presents a diagnostic challenge. Its clinical features typically mimic those of cryptococcal or tuberculous meningitis and Candida can be difficult to culture from cerebrospinal fluid. Furthermore, optimal treatment and monitoring parameters are not well defined. Cerebrospinal fluid levels of (1,3)-β-d-glucan, which is a component of the fungal cell wall, have been used to diagnose and monitor response to therapy in other cases of fungal meningitis, but its utility in the setting of Candida meningitis and HIV infection is less clear. Here, we present a case of spontaneous Candida albicans meningitis in a woman with advanced HIV infection and report on the clinical utility of cerebrospinal fluid (1,3)-β-d-glucan for diagnosis and monitoring of chronic infection.

New versus old: Implications of evolving diagnostic criteria for relapsing–remitting multiple sclerosis

The International Panel on Diagnosis of Multiple Sclerosis (MS) recently revised the 2010 McDonald criteria and made recommendations for revision, allowing for the earliest possible, accurate diagnosis of MS. For relapsing–remitting MS, positive, unmatched cerebrospinal fluid oligoclonal bands may substitute for dissemination in time. Symptomatic lesions, including brainstem and spinal cord, may demonstrate dissemination in space or in time if enhancing (with the exception of the optic nerve). Cortical and juxtacortical lesions are equivalent. In this retrospective analysis, we applied revised criteria to 250 patients previously diagnosed with relapsing–remitting MS according to 2010 criteria and assessed for change in diagnostic times. There was a significant improvement in time to diagnosis between 2010 and 2017 groups (p < 0.01). Median time to diagnosis according to McDonald 2010 was 7.4 months, compared with 2.3 months for McDonald 2017. Use of cerebrospinal fluid results most frequently resulted in a reduction in time to diagnosis. Symptomatic gadolinium-enhancing lesions led to earliest diagnostic times.

The use of cerebrospinal fluid C reactive protein and adenosine deaminase as diagnostic markers in differential diagnosis of meningitis

The use of cerebrospinal fluid C reactive protein and adenosine deaminase as diagnostic markers in differential diagnosis of meningitis

The Challenges of Diagnosis in Alzheimer’s Disease

Current neuropathologic examination of the brain is still the gold standard for diagnosis of Alzheimer’s disease (AD). Postmortem studies, however, have indicated that current methods for the clinical diagnosis of AD are suboptimal.1Recent research has demonstrated the clinical utility of amyloid-beta positron emission tomography (PET) scans, which detect the presence of amyloid-beta plaques in the brain. In a study presented at the Alzheimer’s Association International Conference (AAIC) in London, UK, July 2017, by Nenad Bogdanovic, MD, PhD, of the University of Oslo in Norway, amyloid PET imaging was found to be a fundamental diagnostic tool for AD, establishing a definite diagnosis or excluding AD in all 50 study participants.2 The use of cerebrospinal fluid (CSF) amyloid testing with a higher amyloid-beta plaque threshold than that traditionally used to establish a positive finding also resulted in high diagnostic accuracy, resulting in diagnosis or exclusion in 44 of 50 participants (88%) compared with only 21 individuals (42%) using traditional cutoffs.2

Chapter 1 - The use of cerebrospinal fluid in biomarker studies

Cerebrospinal fluid (CSF) is an extremely useful matrix for biomarker research for several purposes, such as diagnosis, prognosis, monitoring, and identification of prominent leads in pathways of neurologic diseases. Such biomarkers can be identified based on a priori hypotheses around prominent protein changes, but also by applying -omics technologies. Proteomics is widely used, but metabolomics and transcriptomics are rapidly revealing their potential for CSF studies. The basis of such studies is the availability of high-quality biobanks. Furthermore, profound knowledge and consequent optimization of all aspects in biomarker development are needed. Here we discuss current knowledge and recently developed protocols for successful biomarker studies, from collection of CSF by lumbar puncture, processing, and biobanking protocols, preanalytic confounding factors, and cost-efficient development and validation of assays for implementation into clinical practice or research.

Current Approaches and Clinician Attitudes to the Use of Cerebrospinal Fluid Biomarkers in Diagnostic Evaluation of Dementia in Europe.

BIOMARKAPD seeks to diminish the barriers associated with the clinical use of cerebrospinal fluid (CSF) biomarker analysis by reducing variation in CSF laboratory methodologies and generating consensus recommendations on their clinical interpretation and application for dementia diagnosis. To examine the disparity in practitioner attitudes and clinical practice relating to the use of CSF biomarkers for dementia diagnosis across Europe. Clinical dementia experts were surveyed on the prevalence of national consensus guidelines and analytical reimbursement across Europe, their biomarker platform preferences, lumbar puncture methodologies and application of reference values and cut-offs for CSF analysis. 74% of respondents (total n = 51) use CSF biomarkers in clinical practice and 69% perform lumbar punctures on an outpatient basis. Most use CSF biomarkers to diagnose atypical (84%) and early-onset cases of cognitive impairment (71%) and for the differential diagnosis of other dementias (69%). 82% state they are sufficiently informed about CSF biomarkers yet 61% report a lack of national consensus guidelines on their use for dementia diagnosis. 48% of countries represented do not reimburse clinical CSF analysis costs. 43% report using normal reference ranges derived from publications. Variations in attitude and practice relating to CSF biomarkers, widely recognised as barriers to their clinical acceptance, remain evident within and between countries across Europe, even in expert centres. These shortcomings must be addressed by developing consensus guidelines on CSF-related methodologies and their clinical application, to further their use for the diagnostic evaluation of dementia.

A dedicated lumbar puncture clinic: performance and short-term patient outcomes.

The purpose of the study was to evaluate patient outcomes, including success rates, factors associated with unsuccessful procedures and frequency of post-lumbar puncture headaches (PLPH), at a dedicated academic outpatient lumbar puncture (LP) clinic. All patients referred to our LP clinic between June 1, 2014 and May 31, 2015 were included in this consecutive observational series. We collected information about patient characteristics, operational parameters of the procedure, and complications. We also recorded rates of participation in biomedical research involving use of cerebrospinal fluid. Univariate analysis used Student's t test and Fisher's exact test. Logistic regression was used to determine independent risk factors associated with unsuccessful LP and PLPH. The mean age of patients referred to our LP clinic was 46±17years. Of the 307 referrals, 281 patients (92%) started the procedure, with successful acquisition of CSF in 267 (95%). Factors contributing to unsuccessful procedures included higher body mass index [odds ratio (OR) 1.8], older age (OR 1.9), and female sex (OR 10.3). The rate of PLPH was 5.7%. Younger age (OR 0.5), female sex (OR 6.9), high mean arterial pressure (OR 2.2), and a traumatic LP (OR 10.0) were identified as risk factors for PLPH. Notably, 202 patients (72%) consented to biomedical research. A standardized approach to outpatient LP demonstrates high procedural success rate, low PLPH rate, and high participation in biomedical research. Awareness of a group of patients at higher risk for complications including procedure failure or PLPH provides guidance for decision-making regarding referral to the outpatient LP clinic.

Diagnosis and treatment patterns for patients with leptomeningeal metastasis from solid tumors across Europe.

Leptomeningeal metastases are a late manifestation of systemic cancer which affects up to 10% of patients with solid tumors. Prognosis is poor, and overall survival at 1year is only approximately 10%. Management depends mainly on general and neurological condition, primary tumor, and patterns of metastasis, notably absence or presence of concurrent systemic or solid brain metastases. Here we set out to characterize current practice patterns of diagnosis and treatment of patients with leptomeningeal metastasis in Europe. We prepared a web-based survey including 25 simple or multiple choices questions on best practice supplemented by eight case vignettes with various diagnosis and management options. The survey was sent to the membership of the European Association of Neuro-Oncology and the European Organisation for Research and Treatment of Cancer Brain Tumor Group. Between April 7, 2016 and August 8, 2016, 224 colleagues from 26 countries initiated the survey, 115 colleagues completed the whole survey. There were major differences both in the general diagnostic and therapeutic approach, e.g., regarding the use of cerebrospinal fluid (CSF) flow studies, intra-CSF chemotherapy, various types of radiotherapy, and even more so when selecting decisions on diagnostic and therapeutic measures for single case vignettes. Diagnosis and treatment decisions for patients with leptomeningeal metastasis from solid tumors vary widely across Europe. Standardization of diagnosis and evaluation tools as well as controlled studies to improve the level of evidence for all therapeutic approaches to LM are required.

Use of cerebrospinal fluid and serum samples impregnated on FTATM Elute filter paper for the diagnosis of infections caused by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae.

BackgroundThe lack of information regarding the burden of acute bacterial meningitis in Latin America leads to a reduction in the estimated incidence rates of the disease, and impairs public health decisions on the use and follow-up of preventive interventions, particularly, the evaluation of existing vaccination policies. The use of the real-time PCR in diagnostic routine procedures has resulted in a substantial increase in confirmed bacterial meningitis cases. However, in resource-poor countries, these assays are only available in reference laboratories. Sample transportation to these laboratories is a critical constraint, as it requires specialized, high cost courier services. To overcome this barrier we evaluated the use of FTATM Elute filter paper cards for the conservation and processing of samples under normal environmental conditions, as they would be when transported from remote and under-equipped healthcare facilities to the reference centers. A total of 401 samples received in 2015 as part of Sao Paulo’s national surveillance for routine diagnosis were selected for this study.MethodsThe sensitivity and specificity of real-time PCR were evaluated using fresh serum and cerebrospinal fluid (CSF) samples processed using our laboratory’s standard DNA extraction, and processing the same samples after being dried and stored on FTATM card, and DNA extracted following the manufacturer’s instructions.ResultsThe sensitivities for detection of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from CSF dried and stored on FTATM cards were 98%, 92%, and 100%, respectively, and with serum samples were 73%, 88%, and 100%, respectively. When compared to our laboratory’s standard methodology, results showed high concordance, with Kappa index ranges of 0.9877–1.00 for CSF, and 0.8004–1.00 for serum samples.ConclusionThe use of FTATM cards for CSF and serum conservation and transport represents a rapid, reliable, and cost-effective alternative that will allow obtaining valuable epidemiological information that would otherwise be lost.

Alzheimer disease: CSF biomarkers for Alzheimer disease - approaching consensus.

A European working group has provided a new set of recommendations for the use of cerebrospinal fluid (CSF) biomarkers in the diagnostic evaluation of Alzheimer disease and mild cognitive impairment. These recommendations represent an important step towards the implementation of CSF biomarker tests in the clinic, but several challenges remain.

The Clinical Use of Cerebrospinal Fluid Biomarkers for Alzheimer's Disease Diagnosis: The Italian Selfie.

Although the use of cerebrospinal fluid (CSF) amyloid β1-42 (Aβ42), tau (T-tau), and phosphorylated tau (p-tau181) gives added diagnostic and prognostic values, the diffusion is still limited in clinical practice and only a restricted number of patients receive an integrated clinico-biological diagnosis. By a survey, we aimed to do a "selfie" of the use and diffusion of CSF biomarkers of dementia in Italy, the standardization of pre-analytical procedures, the harmonization of ranges, and the participation to Quality Control programs. An online questionnaire was sent to the members of SIBioC and SINdem-ITALPLANED and to main neurological clinics all over Italy. In Italy, 25 laboratories provide biomarkers analysis in addition to a network of 15 neighboring hospitals. In sum, 40 neurological centers require CSF analyses. 7/20 regions (35%) lack CSF laboratories. Standardization of pre-analytical procedures is present in 62.02% of the laboratories; only 56.00% of the laboratories participate in International Quality Control. There is no harmonization of cut-offs. In Italy, the use of CSF biomarkers is still limited in clinical practice. Standardization and harmonization of normal ranges are needed. To optimize and expand the use of CSF biomarkers, a cost-benefit analysis should be promoted by scientific societies and national health services.

Cerebrospinal fluid biomarkers in Alzheimer's disease: Diagnostic accuracy and prediction of dementia

IntroductionGuidelines for the use of cerebrospinal fluid (CSF) biomarkers in the diagnosis of Alzheimer's disease (AD) establish that each laboratory must use internally qualified cutoff values. We determined the concentrations of biomarkers that discriminate cases from controls and combinations that predict the progression to dementia in a Brazilian cohort. MethodsConcentrations of amyloid-beta peptide (Aβ1–42), total tau (T-tau), and 181Thr-phosphorylated-tau (P-tau) were determined in CSF samples from 184 older adults (68 mild cognitive impairment, 41 AD, 34 non-AD cognitive impairment, and 41 controls) by the INNO-BIA AlzBio3 assay. ResultsCutoff values discriminating AD from controls are as follows: Aβ1–42: 416.0 pg/mL (sensitivity [SE]: 83%, specificity (SP): 70%); T-tau: 76.7 pg/mL (SE: 82%, SP: 67%); P-tau: 36.1 pg/mL (SE: 83%, SP: 49%); Aβ1–42/P-tau <9.53 (SE: 88%, SP: 78%); and Aβ1–42/T-tau <4.13 (SE: 80%; SP: 80%). Combining values Aβ1–42 <416.5 pg/mL and Aβ1–42/P-tau <9.5 best predicted the conversion in 2 years (Cox regression: hazard ratio 7.24 [2.09–25.06], P = .002, SE: 74%, Sp: 73%). DiscussionOur findings are in line with most of the available evidence in this field; yet, our cutoff values are different from those derived from other laboratories.

Preanalytical Confounding Factors in the Analysis of Cerebrospinal Fluid Biomarkers for Alzheimer's Disease: The Issue of Diurnal Variation.

Given the growing use of cerebrospinal fluid (CSF) beta-amyloid (Aβ) and tau as biomarkers for early diagnosis of Alzheimer’s disease (AD), it is essential that the diagnostic procedures are standardized and the results comparable across different laboratories. Preanalytical factors are reported to be the cause of at least 50% of the total variability. Among them, diurnal variability is a key issue and may have an impact on the comparability of the values obtained. The available studies on this issue are not conclusive so far. Fluctuations of CSF biomarkers in young healthy volunteers have been previously reported, while subsequent studies have not confirmed those observations in older subjects, the ones most likely to receive this test. The observed differences in circadian rhythms need to be further assessed not only in classical CSF biomarkers but also in novel forthcoming biomarkers. In this review, the existing data on the issue of diurnal variations of CSF classical biomarkers for AD will be analyzed, also evaluating the available data on new possible biomarkers.

Pediatric hydrocephalus: systematic literature review and evidence-based guidelines. Part 3: Endoscopic computer-assisted electromagnetic navigation and ultrasonography as technical adjuvants for shunt placement.

This systematic review was undertaken to answer the following question: Do technical adjuvants such as ventricular endoscopic placement, computer-assisted electromagnetic guidance, or ultrasound guidance improve ventricular shunt function and survival? The US National Library of Medicine PubMed/MEDLINE database and the Cochrane Database of Systematic Reviews were queried using MeSH headings and key words specifically chosen to identify published articles detailing the use of cerebrospinal fluid shunts for the treatment of pediatric hydrocephalus. Articles meeting specific criteria that had been delineated a priori were then examined, and data were abstracted and compiled in evidentiary tables. These data were then analyzed by the Pediatric Hydrocephalus Systematic Review and Evidence-Based Guidelines Task Force to consider evidence-based treatment recommendations. The search yielded 163 abstracts, which were screened for potential relevance to the application of technical adjuvants in shunt placement. Fourteen articles were selected for full-text review. One additional article was selected during a review of literature citations. Eight of these articles were included in the final recommendations concerning the use of endoscopy, ultrasonography, and electromagnetic image guidance during shunt placement, whereas the remaining articles were excluded due to poor evidence or lack of relevance. The evidence included 1 Class I, 1 Class II, and 6 Class III papers. An evidentiary table of relevant articles was created. CONCLUSIONS/RECOMMENDATION: There is insufficient evidence to recommend the use of endoscopic guidance for routine ventricular catheter placement. Level I, high degree of clinical certainty. The routine use of ultrasound-assisted catheter placement is an option. Level III, unclear clinical certainty. The routine use of computer-assisted electromagnetic (EM) navigation is an option. Level III, unclear clinical certainty.

Cost-effectiveness of the use of biomarkers in cerebrospinal fluid for Alzheimer's disease.

The use of cerebrospinal fluid (CSF) biomarkers could facilitate early detection of Alzheimer's disease (AD) in patients with mild cognitive impairment (MCI) and the differential diagnosis between AD and non-AD dementias. To determine the cost-effectiveness of the use of amyloid-β peptide (Aβ42), total tau and phosphorylated tau proteins in CSF to diagnose AD in MCI and dementia patients. An economic evaluation was performed by means of cost-effectiveness analysis comparing two AD diagnostic alternatives: the combined determination of Aβ42 proteins, total tau and phosphorylated tau in CSF as biomarkers of AD, and the standard clinical diagnosis based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINDS-ADRDA) criteria. A decision analytic model was developed to synthesize the identified evidence and to compare the costs and effectiveness associated with each diagnostic strategy. A probabilistic sensitivity analysis using 2nd order Monte Carlo simulations was performed. Subsequently, acceptability curves were calculated and ANCOVA models were applied to the results of the Monte Carlo simulations in order to identify the parameters that led greater variability in the model outcomes. The use of CSF biomarkers as an early diagnostic strategy of AD in MCI patients is a dominant alternative (less costly and more effective strategy than standard clinical diagnostic criteria). In dementia patients, although there is a higher uncertainty, biomarkers in CSF seem a more cost-effective alternative than standard clinical diagnostic criteria. Detecting AD in MCI patients by determining Aβ42, total tau and phosphorylated tau proteins biomarkers in CSF is a cost-effective diagnostic alternative. No conclusive results were obtained on dementia patients.

Genetic discoveries in AD using CSF amyloid and tau.

The use of cerebrospinal fluid levels of Aβ42 and pTau181 as endophenotypes for genetic studies of Alzheimer's disease (AD) has led to successful identification of both rare and common AD risk variants. In addition, this approach has provided meaningful hypotheses for the biological mechanisms by which known AD risk variants modulate the disease process. In this article we discuss these successes and outline challenges to effective and continued applications of this approach. We contrast the statistical power of this approach with traditional case-control designs and discuss solutions to address challenges in quality control and data analysis for these phenotypes. Finally, we discuss the potential for the use of this approach with larger samples as well as the incorporation of next generation sequencing and for future work with other endophenotypes for AD.

Cerebrospinal fluid biomarkers for pathological processes in Alzheimer's disease

To review the rationale behind and the use of cerebrospinal fluid (CSF) biomarkers in Alzheimer's disease (AD). Established as well as new candidate biomarkers will be covered. AD is a complex disorder and the AD brain is characterized by multiple pathological processes, in addition to well-described plaque and tangle diseases. Recent studies have tried to address this by evaluating biomarkers related to features such as neuroinflammation, oxidative stress, microglial activation and synaptic degeneration, with some positive results. The CSF biomarkers total tau, phosphorylated-tau and the 42 amino acid isoform of amyloid beta reflect core elements of AD, that is, axonal degeneration, tangle disease and senile plaques, have been thoroughly tested and provide high diagnostic accuracy in the discrimination of patients with AD as compared with cognitively normal controls. They are also highly predictive of AD with dementia in patients with mild cognitive impairment, and have been included in new diagnostic criteria. New biomarkers may add to their diagnostic performance. Other potential fields of use include the monitoring of disease progression or pharmacodynamic drug effects. A common denominator for the candidate biomarkers is the need for validation in further studies to clarify their potential.

Impact of the 2008–2012 French Alzheimer Plan on the Use of Cerebrospinal Fluid Biomarkers in Research Memory Center: The PLM Study

The French Alzheimer's Disease Plan aims, in an unprecedented national effort, to develop research, promote optimal diagnosis, and take better care of patients. In order to evaluate the clinical interest and use of cerebrospinal fluid (CSF) biomarkers, a data-sharing project, the PLM (Paris-North, Lille and Montpellier) study has emerged through collaboration between these memory centers, already involved in this field. The revised Alzheimer's disease (AD) diagnosis criteria include CSF biomarkers, but little is known about their use in routine clinical practice. To evaluate their interest and diagnostic accuracy in routine AD diagnosis, a cohort of 677 patients from Montpellier was first analyzed. The results were then validated through the analysis of a second cohort of 638 patients from Lille and Paris-Nord. Diagnoses of AD and other dementias were established by multidisciplinary expert teams, based on neuropsychological exams and structural brain imaging, blinded from CSF results. CSF amyloid-β, tau, and p-tau concentrations were measured for all patients. Receiver-operating characteristic curves were used to define cut-offs and evaluate the ability of each biomarker to discriminate AD from other diagnoses. We showed that p-tau outperformed other biomarkers for discriminating AD from non-AD patients and presents a clear clinical interest. The other biomarkers also showed relevant variations especially when the differential AD diagnoses were taken into account. Altogether we could demonstrate in both mono-centric and multi-centric cohorts from memory clinics the capacity of CSF biomarkers to discriminate AD from non-AD patients in clinical routine with a high sensitivity and specificity.

The Effect of Preanalytical Factors on Stability of the Proteome and Selected Metabolites in Cerebrospinal Fluid (CSF)

To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.