Cerebrospinal Fluid Contamination Research Articles

Subarachnoid haemorrhage or traumatic lumbar puncture. Differentiation by cerebrospinal fluid parameters in a multivariable approach

Lumbar puncture (LP) is recommended in patients with thunderclap headache and negative computed tomography to rule out spontaneous subarachnoid haemorrhage (SAH). Blood contamination of cerebrospinal fluid (CSF) due to traumatic LP poses a diagnostic dilemma. Therefore, routine CSF parameters were investigated to distinguish between SAH and a traumatic LP. CSF red blood cell (RBC), white blood cell (WBC) count, total protein, CSF colour and supernatant were used for group comparisons of patients with SAH and ‘symptomatic controls’. Due to variable time intervals between bleeding onset and LP in SAH patients in contrast to patients with traumatic LP, where blood contamination of CSF occurs at the time of LP, CSF variables were adjusted for decay in time to allow comparability. Logistic regression analysis identified bloody CSF [odds ratio (OR) 32.6], xanthochromic supernatant [OR 15.5] and WBCadjusted [OR 4.5 (per increase of 100/µl)] as predictors of SAH, while age, sex and CSF total proteinadjusted were no predictors. Optimal cut-point of RBCadjusted (determined at day 1 after bleeding) was > 3667/µl to identify SAH patients with a 97% sensitivity and 94% specificity. Combination of low RBC and clear CSF supernatant was found in none of SAH patients. Combined CSF RBC count and CSF supernatant reliably distinguished traumatic LP from SAH.

Quantitative perfusion and water transport time model from multi b-value diffusion magnetic resonance imaging validated against neutron capture microspheres.

Quantification of perfusion in ml/100 g/min, rather than comparing relative values side-to-side, is critical at the clinical and research levels for large longitudinal and multi-center trials. Intravoxel incoherent motion (IVIM) is a non-contrast magnetic resonance imaging diffusion-based scan that uses a multitude of -values to measure various speeds of molecular perfusion and diffusion, sidestepping inaccuracy of arterial input functions or bolus kinetics. Questions remain as to the original of the signal and whether IVIM returns quantitative and accurate perfusion in a pathology setting. This study tests a novel method of IVIM perfusion quantification compared with neutron capture microspheres. We derive an expression for the quantification of capillary blood flow in ml/100 g/min by solving the three-dimensional Gaussian probability distribution and defining water transport time (WTT) as when 50% of the original water remains in the tissue of interest. Calculations were verified in a six-subject pre-clinical canine model of normocapnia, induced hypercapnia, and middle cerebral artery occlusion (ischemic stroke) and compared with quantitative microsphere perfusion. Linear regression analysis of IVIM and microsphere perfusion showed agreement (slope = 0.55, intercept = 52.5, ) with a Bland-Altman mean difference of . Linear regression between dynamic susceptibility contrast mean transit time and IVIM WTT asymmetry in infarcted tissue was excellent (, intercept = 0.3, ). Strong linear agreement was found between IVIM and reference standard infarct volume (slope = 1.01, ). The simulation of cerebrospinal fluid (CSF) suppression via inversion recovery returned a blood signal reduced by 82% from combined T1 and T2 effects. The accuracy and sensitivity of IVIM provides evidence that observed signal changes reflect cytotoxic edema and tissue perfusion and can be quantified with WTT. Partial volume contamination of CSF may be better removed during post-processing rather than with inversion recovery.

Patterns and Prognostic Impact of CNS Infiltration in Adults with Newly Diagnosed Acute Lymphoblastic Leukemia

Acute lymphoid leukemia (ALL) is a subtype of acute leukemia which portends a high risk of central nervous system (CNS) infiltration. At the diagnosis, 5-10% of ALL patients have CNS involvement, which has been demonstrated to decrease overall survival (OS). Based on pediatric trials, conventional cytology (CC) has been the standard for CNS disease assessment, although, more recently, flow cytometry (FC) has gained space across several centers. In this study, we intend to: i) access the impact of CNS involvement (defined by CC or FC) on OS, event-free survival (EFS) and relapse of ALL patients treated in a public health center; ii) find variables associated with CNS infiltration in this cohort. Methods: This retrospective study encompassed 105 cases of ALL newly diagnosed at Instituto do Cancer de Sao Paulo (ICESP). Clinical data were collected from Leukemia clinic database, already approved by Local Ethics Committee. Variables related to cerebrospinal fluid (CSF) were collected from the first lumbar puncture (LP). Traumatic LP was defined as more than 10 red blood cells/µL. CNS status was assessed through the COG's classification and divided into positive/negative for analysis purposes. Patients were treated with different regimens, but intensive intrathecal chemotherapy was given in CNS2 or 3 cases, while cranial irradiation was scheduled for CNS3 patients, only. Results: A total of 105 cases were diagnosed from 2014 to 2020. Only one was excluded (death before the first LP). Median age at diagnosis was 37 years (range, 15-79) with a male majority (58%). Most cases had a B-cell phenotype (76%), followed by T (19%) and ambiguous (5%). Philadelphia chromosome was found in 39%. Eighty-nine percent received corticosteroid at the time of LP with a median time of 7 days. Traumatic LP was reported in 50%. CSF resulted positive for disease in 15% (16/104) and 26% (19/72), by CC and FC, respectively. Among those with a positive CC, 56% (9/16) had a traumatic LP. By COG's classification, CNS1, CNS2 and CNS3 were found in 84%, 14% and 2% of cases, respectively. At the time of PL, 44% of patients presented blasts in peripheral blood. By comparing CC and FC, we noticed only one case with CCpos/FCneg whereas 9/19 with FCpos did not have CCpos. Sensitivity and specificity of CC for CNS disease were 53% and 98%. Traumatic LP and positive CC were not statistically correlated (p=0.8) and prior chemotherapy was associated to higher traumatic LP rates (56 vs 31%, p=0.019). Higher white-blood cell (WBC) counts were found in subjects with positive CC or FC (Mann-Whitney test, p=0.007 for CC). Furthermore, the blast percentage was also relevant for CNS positivity (p=0.027). Genetic subgroup, age, CD34 expression and phenotype were not correlated with CNS invasion, among others. Most patients received adapted versions of BFM (n=43), GRAAPH (n=24) and GRAALL-Elderly (n=18) regimens. Five patients received cranial irradiation. Median OS for the whole cohort was 16.4 months, with a 3y OS of 42.5% (95% CI 33.5-54). For BFM subset (Philadelhpia-negative, below 50 years-old), OS and EFS were 53.5% and 37.3%. We did not find correlation between CNS disease with OS or EFS in univariate analysis. Three-year relapse rate was 38.5% (95% CI 29-49) whereas non-relapse mortality (NRM) was 33%. In multivariate analysis, a positive CC increased relapse rate (HR= 2.3 [95% CI 1.1-4.9, p 0.029]). Initial WBC was also correlated with relapse in our cohort. Among relapses, only one was isolated in CNS, while 13 had concomitant bone marrow and CNS involvement. Eighteen patients proceeded to allo-stem cell transplantation so far. Conclusion: Several studies have demonstrated correlation between CNS infiltration and shorter OS in ALL, even though it relies on how these patients are managed and how CNS is assessed upfront. Our data showed that those who had a positive CSF cytology had a higher risk of relapse, whereas FC was not able to discriminate this risk. Survival rates seem to be increasing over the last years by the adoption of a stratified risk strategy. CSF contamination do not represent a major concern according to our report, as it did not increase CNS involvement or relapse rates. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Establishing pre-analytical requirements and maximizing peptide recovery in the analytical phase for mass spectrometric quantification of amyloid-β peptides 1–42 and 1–40 in CSF

Abstract Objectives Amyloid-β (Aβ) peptides in cerebrospinal fluid (CSF), including Aβ42 (residues 1–42) and Aβ40 (residues 1–40), are utilized as biomarkers in the diagnostic workup of Alzheimer’s disease. Careful consideration has been given to the pre-analytical and analytical factors associated with measurement of these peptides via immunoassays; however, far less information is available for mass spectrometric methods. As such, we performed a comprehensive evaluation of pre-analytical and analytical factors specific to Aβ quantification using mass spectrometry. Methods Using our quantitative mass spectrometry assay for Aβ42 and Aβ40 in CSF, we investigated the potential for interference from hemolysate, bilirubin, lipids, and anti-Aβ-antibodies. We also optimized the composition of the calibrator surrogate matrix and Aβ recovery during and after solid phase extraction (SPE). Results There was no interreference observed with total protein up to 12 g/L, hemolysate up to 10% (v/v), bilirubin up to 0.5% (v/v), intralipid up to 1% (v/v), or anti-Aβ-antibodies at expected therapeutic concentrations. For hemolysate, bilirubin and lipids, visual CSF contamination thresholds were established. In the analytical phase, Aβ recovery was increased by ∼50% via SPE solvent modifications and by over 150% via modification of the SPE collection plate, which also extended analyte stability in the autosampler. Conclusions Attention to mass spectrometric-specific pre-analytical and analytical considerations improved analytical sensitivity and reproducibility, as well as, established CSF specimen acceptance and rejection criteria for use by the clinical laboratory.

PATTERNS AND PROGNOSTIC IMPACT OF CNS INFILTRATION IN ADULTS WITH NEWLY DIAGNOSED ACUTE LYMPHOBLASTIC LEUKEMIA

Acute lymphoid leukemia (ALL) is a subtype of acute leukemia which portends a high risk of central nervous system (CNS) infiltration, which decreases overall survival (OS). Conventional cytology (CC) has been the standard for CNS disease assessment, although flow cytometry (FC) has gained space across several centers. In this study, we intend to: i) access the impact of CNS involvement (defined by CC or FC) on OS, event-free survival (EFS) and relapse of ALL patients treated in a public health center; ii) find variables associated with CNS infiltration. This retrospective study encompassed 105 cases of newly diagnosed ALL at Instituto do Cancer de São Paulo. Data were collected from Leukemia clinic database, approved by Local Ethics Committee. Variables related to cerebrospinal fluid (CSF) were collected from the first lumbar puncture (LP). Traumatic LP was defined as more than 10 red blood cells/μL. CNS status was assessed through the COG's classification and divided into positive/negative. Intensive intrathecal chemotherapy was given in CNS2 or 3 cases, while cranial irradiation was scheduled for CNS3 patients. A total of 105 cases were diagnosed from 2014 to 2020. Median age at diagnosis was 37 years (range, 15-79) with a male majority (58%). Most cases had a B-cell phenotype (76%), followed by T (19%) and ambiguous (5%). Philadelphia chromosome was found in 39%. Eighty-nine percent received corticosteroid at the time of LP with a median time of 7 days. Traumatic LP was reported in 50%. CSF was positive for disease in 15% (16/104) and 26% (19/72),by CC and FC, respectively. Among those with a positive CC, 56% (9/16) had a traumatic LP,but they were not statistically correlated (p = 0.8). Prior chemotherapy was associated to higher traumatic LP rates (56 vs 31%, p = 0.019). CNS1, CNS2 and CNS3 were found in 84%, 14% and 2% of cases, respectively. At the time of PL,44% of patients presented blasts in peripheral blood. By comparing CC and FC, we found one case with CC pos /FC neg whereas 9/19 with FC pos did not have CC pos . Sensitivity and specificity of CC for CNS disease were 53% and 98%. Higher white-blood cell (WBC) counts were found in subjects with positive CC or FC (Mann-Whitney test, p = 0.007 for CC). Also, the blast percentage was relevant for CNS positivity (p = 0.027). Genetic subgroup, age, CD34 expression and phenotype were not correlated with CNS invasion. Most patients received adapted versions of BFM (n = 43), GRAAPH (n = 24) and GRAALL-Elderly (n = 18) regimens. Median OS for the whole cohort was 16.4 months, with a 3y OS of 42.5% (95% CI 33.5-54). For BFM subset, OS and EFS were 53.5% and 37.3%. We did not find correlation between CNS disease with OS or EFS in univariate analysis. Three-year relapse rate was 38.5% (95% CI 29-49) whereas non-relapse mortality (NRM) was 33%. In multivariate analysis, a positive CC increased relapse rate (HR = 2.3 [95% CI 1.1-4.9, p 0.029]), aswell as initial WBC. Among relapses, only one was isolated in CNS and 13 had also concomitant bone marrow involvement. Several studies have demonstrated correlation between CNS infiltration and shorter OS in ALL, even though it relies on how these patients are managed and how CNS is assessed upfront. Our data showed that those with a positive CSF cytology had a higher risk of relapse, whereas FC was not able to discriminate this risk. Survival rates are increasing over the last years by the adoption of a stratified risk strategy. CSF contamination did not increase CNS involvement or relapse rates.

Kappa Free Light Chains in the Context of Blood Contamination, and Other IgA- and IgM-Related Cerebrospinal Fluid Disease Pattern.

In this retrospective, monocentric cohort study, we tested if an intrathecal free light chain kappa (FLC-k) synthesis reflects not only an IgG but also IgA and IgM synthesis. We also analysed if FLC-k can help to distinguish between an inflammatory process and a blood contamination of cerebrospinal fluid (CSF). A total of 296 patient samples were identified and acquired from patients of the department of Neurology, University Medicine Greifswald (Germany). FLC-k were analysed in paired CSF and serum samples using the Siemens FLC-k kit. To determine an intrathecal FLC-k and immunoglobulin (Ig) A/-M-synthesis we analysed CSF/serum quotients in quotient diagrams, according to Reiber et al. Patient samples were grouped into three cohorts: cohort I (n = 41), intrathecal IgA and/or IgM synthesis; cohort II (n = 16), artificial blood contamination; and the control group (n = 239), no intrathecal immunoglobulin synthesis. None of the samples had intrathecal IgG synthesis, as evaluated with quotient diagrams or oligoclonal band analysis. In cohort I, 98% of patient samples presented an intrathecal synthesis of FLC-k. In cohort II, all patients lacked intrathecal FLC-k synthesis. In the control group, 6.5% presented an intrathecal synthesis of FLC-k. The data support the concept that an intrathecal FLC-k synthesis is independent of the antibody class produced. In patients with an artificial intrathecal Ig synthesis due to blood contamination, FLC-k synthesis is lacking. Thus, additional determination of FLC-k in quotient diagrams helps to discriminate an inflammatory process from a blood contamination of CSF.

Cerebrospinal Fluid (CSF) Analysis of Tumor-Specific Cell-Free DNA (cfDNA) As a Diagnostic and Prognostic Tool for Central Nervous System (CNS) Invasion in Lymphoma

Background: CNS invasion in aggressive lymphoma (including diffuse large B-cell [DLBCL], double-hit [DHL], Burkitt [BL] and plasmablastic lymphoma [PBL]) remains a major diagnostic and therapeutic challenge. Parenchymal CNS invasion requires a brain biopsy to diagnose, and conventional CSF evaluations lack sensitivity. Furthermore, determining which patients (pts) without CNS disease at diagnosis might be at high risk of future CNS relapse, and thus would most benefit from aggressive CNS prophylaxis, is difficult. Predictors that use clinical variables (e.g. the CNS-IPI) can select a high-risk group with ~10% risk of CNS relapse, but ~50% of events occur in low/intermediate-risk groups. We evaluated a novel CSF assay that detects clonotype-specific cell-free DNA (cfDNA) by next-generation sequencing (NGS) combined with multiplex PCR (Chang T et al, BMC Cancer 2020). This assay can identify minimal residual disease (MRD) in the plasma of pts with DLCBL and can predict systemic relapse (Roschewski et al, Lancet Oncol 2015). Methods: To examine the diagnostic sensitivity of the NGS-MRD assay in the CSF, we prospectively collected CSF from 6 pts with lymphomas who had known parenchymal or leptomeningeal CNS invasion. We also tested stored DNA from historical CSF samples of 8 pts with CNS lymphoma who tested negative by conventional IGH-PCR. We then enrolled 19 pts with newly diagnosed high-risk lymphomas without CNS invasion to examine the prognostic value of the NGS-MRD assay in the CSF collected at diagnosis. In the two-step assay, tumor-specific clonotype sequences were first identified from genomic DNA in primary tumor specimens using NGS of rearranged IGK, IGH (VJ or DJ), and IGL loci. Tumor-specific clonotypes were then selected for tracking in the CSF (split into acellular CSF fluid and cell pellet) and in paired plasma samples. The assay quantitated copy numbers of each tracked sequence per mL of acellular CSF or per 106 cells in the pellet, as well as tumor clonotype frequency (%) per all B-cells. Results: The NGS-MRD detected tumor-specific clonotype in 100% of CSF samples from pts with known CNS lymphoma (n=6; Fig A), including pts with isolated parenchymal-only CNS disease that was not detected by CSF cytology, flow cytometry, or IGH-PCR. For pts with CNS parenchymal disease, median cfDNA copy count in the CSF fluid was 2 /mL (range, 0.4-929) and median clonotype frequency was 9.0% (range, 0.03-68.9%). For pts with leptomeningeal disease, these values were 2233 /mL (1.2-5620) and 37% (1.7-98.4%), respectively (Fig. B). Furthermore, the NGS-MRD assay detected tumor clonotypes in DNA isolates from 6 historical CSF samples (collected in 2014-2018) of pts with known CNS disease, in which lymphoma was not detectable by conventional IGH-PCR (2 additional historical samples failed DNA quality check). Among 19 newly diagnosed pts with no known CNS invasion (median age 57, 42% women, median LDH 352 IU/L), 12 (63%) had DLBCL with high risk of CNS relapse by virtue of high CNS-IPI or epidural tumor, and 7 had other high-risk histologies (2 DHL, 2 BL, HIV+ PBL, and 2 lymphoblastic leukemias). The CSF NGS-MRD assay was positive for tumor-specific clonotype in 8 pts (42%). Median cfDNA copy count in the CSF fluid was 3 /mL (range, 0.9-15.6) and median clonotype frequency was 18.6% (range, 1.4-28.5%). We observed no significant correlation between the amount of cfDNA and number of red cells /mL of CSF (a measure of potential CSF contamination by blood during collection procedure; Fig. C) or between the amount of cfDNA in plasma and in CSF (Fig. D). With median follow-up of 11 months, no pts with negative baseline NGS-MRD assay in CSF relapsed, whereas 2 of 10 with a positive assay had a CNS relapse (12-month incidence, 29%; Fig. E), despite both pts having negative CNS imaging, CSF cytology, flow cytometry, and IGH-PCR at diagnosis. Conclusions: In this proof-of-concept study, the NGS-MRD CSF assay showed 100% sensitivity for diagnosing intraparenchymal CNS invasion in aggressive lymphomas that were not detected in the CSF using conventional methods. In a prospective cohort of newly diagnosed pts without baseline CNS invasion but at high clinical risk for CNS relapse, the assay identified lymphoma-derived cfDNA in 42% of cases. The 100% sensitivity and 100% negative predictive value of the assay for subsequent CNS relapse warrants further prospective evaluation as a potential tool for personalizing CNS-directed prophylaxis. Disclosures Olszewski: Spectrum Pharmaceuticals: Research Funding; TG Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Genentech, Inc.: Research Funding. Mullins:Adaptive Biotechnologies: Current Employment, Other: shareholder.

Blood contamination in cerebrospinal fluid and its effect on biogenic amines and vitamins

Cerebrospinal fluid (CSF) analysis offers the most innocuous analytical tool for assessing the cellular and biochemical environment of CNS and hence aids in studying neurometabolomic conditions. The aim of this study was to assess the effect of blood contamination of CSF on the levels of selected biogenic amines and vitamins. CSF samples were spiked with increasing volumes of whole fresh blood (2.5%, 5%, 10% and 20%) under conditions: (a) spiking of pooled CSF samples followed by freezing to cause red blood cell (RBC) lysis; (b) spiking of pooled CSF samples followed by centrifugation to remove RBCs. CSF concentrations of two biogenic amines Homovanillic acid (HVA) and 5-hydroxyindoleaceticacid (5-HIAA); and three vitamins Pyridoxal 5’-phosphate (PLP),5-methyltetrahydrofolate (MTHF) and Thiamine were analysed by HPLC coupled with electrochemical detection. HVA and 5-HIAA illustrated lower values when RBC lysis was caused. However, PLP, 5-MTHF, and thiamine depicted higher concentrations on RBC lysis in contrast to RBC removal. CSF metabolomic investigations involving biogenic amines and vitamins as biomarkers is possible even when remarkable RBC CSF contamination occurs provided that CSF is centrifuged for the removal of RBC prior to freezing.

Bowel perforation of ventriculoperitoneal shunt catheter: endoscopically treated two cases.

Many techniques were used for the treatment of hydrocephalus, and ventriculoperitoneal shunt surgery is a widely used procedure. Ventriculoperitoneal shunt surgery has been associated with several complications like obstruction of the tube, infection, cerebrospinal fluid loculation, intestinal obstruction, migration of the shunt, and perforation of the intestinal organs. Perforation of the bowel owing to protrusion of ventriculoperitoneal shunt catheter from the anus is an extremely rare complication. Mini or exploratory laparotomy and revision of peritoneal part of shunt and repair of bowel perforation, or pulling out the ventriculoperitoneal shunt catheter and using external ventricular drainage and antibiotics, or colonoscopic removal of ventriculoperitoneal shunt catheter and repair of the bowel can be performed. Retrograde contamination of cerebrospinal fluid and meningitis is a very important part of the treatment in these cases. We aimed to present two cases with bowel perforation who treated with endoscopically. We report the cases of 2 patients with transanal protrusion of VPS catheter and the management via endoscopic therapeutic options. Successful treatment of the patients was achieved by endoscopic removal of the catheter and endoscopic repair of the bowel perforation. If peritonitis, bowel obstruction, or abscess does not occur, endoscopic removal of shunt and bowel repairing with endoclips may be enough.

MTE-NODDI: Multi-TE NODDI for disentangling non-T2-weighted signal fractions from compartment-specific T2 relaxation times

Neurite orientation dispersion and density imaging (NODDI) has become a popular diffusion MRI technique for investigating microstructural alternations during brain development, maturation and aging in health and disease. However, the NODDI model of diffusion does not explicitly account for compartment-specific T2 relaxation and its model parameters are usually estimated from data acquired with a single echo time (TE). Thus, the NODDI-derived measures, such as the intra-neurite signal fraction, also known as the neurite density index, could be T2-weighted and TE-dependent. This may confound the interpretation of studies as one cannot disentangle differences in diffusion from those in T2 relaxation. To address this challenge, we propose a multi-TE NODDI (MTE-NODDI) technique, inspired by recent studies exploiting the synergy between diffusion and T2 relaxation. MTE-NODDI could give robust estimates of the non-T2-weighted signal fractions and compartment-specific T2 values, as demonstrated by both simulation and in vivo data experiments. Results showed that the estimated non-T2 weighted intra-neurite fraction and compartment-specific T2 values in white matter were consistent with previous studies. The T2-weighted intra-neurite fractions from the original NODDI were found to be overestimated compared to their non-T2-weighted estimates; the overestimation increases with TE, consistent with the reported intra-neurite T2 being larger than extra-neurite T2. Finally, the inclusion of the free water compartment reduces the estimation error in intra-neurite T2 in the presence of cerebrospinal fluid contamination. With the ability to disentangle non-T2-weighted signal fractions from compartment-specific T2 relaxation, MTE-NODDI could help improve the interpretability of future neuroimaging studies, especially those in brain development, maturation and aging.

The Impact of Immunomodulatory Treatment on Kappa Free Light Chains as Biomarker in Neuroinflammation.

Background: Kappa free light chains (KFLC) are a promising new biomarker to detect neuroinflammation. Still, the impact of pre-analytical effects on KFLC concentrations was not investigated. Methods: KFLC concentrations were measured in serum and cerebrospinal fluid (CSF) of patients with a newly diagnosed multiple sclerosis (MS) or clinically isolated syndrome (CIS) before (n = 42) or after therapy with high-dose methylprednisolone (n = 65). In prospective experiments, KFLC concentrations were analyzed in the same patients in serum before and after treatment with high-dose methylprednisolone (n = 16), plasma exchange (n = 12), immunoadsorption (n = 10), or intravenous immunoglobulins (n = 10). In addition, the influence of storage time, sample method, and contamination of CSF with blood were investigated. Results: Patients diagnosed with MS/CIS and treated with methylprednisolone showed significantly lower KFLC concentrations in serum as untreated patients. Repeated longitudinal investigations revealed that serum KFLC concentrations continuously decreased after each application of methylprednisolone. In contrast, other immune therapies and further pre-analytical conditions did not influence KFLC concentrations. Conclusion: Our results show prominent effects of steroids on KFLC concentrations. In contrast, various other pre-analytical conditions did not influence KFLC concentrations, indicating the stability of this biomarker.

Fit-for-purpose biomarker LC-MS/MS qualification for the quantitation of very long chain fatty acids in human cerebrospinal fluid.

Aim: Very long chain fatty acids (VLCFAs) have been identified as biomarkers for several peroxisomal disorders necessitating the need for reliable biomarker assays in particular C20, C22, C24, C26 in cerebrospinal fluid (CSF). Until now no absolute quantitation assay for total VLCFAs in CSF has been successfully developed and qualified for clinical use. Methodology: A quantitative LC-MS/MS assay for total VLCFA in human CSF was developed. Derivatization tag and coupling chemistry were optimized for sensitivity. CSF contamination by blood, non-specific binding of VLCFA to surfaces and exogenous VLCFA contamination was minimized. Discussion/conclusion: This fit for purpose biomarker assay was used to measure baseline healthy human VLCFA levels across multiple subjects to establish an understanding of concentration ranges and feasibility.

Effect of blood contamination of cerebrospinal fluid on amino acids, biogenic amines, pterins and vitamins

BackgroundCerebrospinal fluid (CSF) metabolomic investigations are a powerful tool for studying neurometabolic diseases. We aimed to assess the effect of CSF contamination with blood on the concentrations of selected biomarkers.MethodsCSF samples were spiked in duplicate with increasing volumes of whole blood under two conditions: (A) pooled CSF spiked with fresh whole blood and frozen to cause red blood cell (RBC) lysis; (B) pooled CSF spiked with fresh blood and centrifuged (the supernatant with no RBCs was frozen until the moment of analysis). CSF concentrations of amino acids, biogenic amines, pterins, and vitamins were analysed by HPLC coupled with tandem mass spectrometry, electrochemical and fluorescence detection.ResultsAspartate, glutamate, taurine, ornithine, glycine, citrulline, pyridoxal 5´-phosphate, 5-methyltetrahydrofolate, and thiamine showed higher values when RBCs were lysed when compared with those of CSF with no RBC, while arginine, 5-hydroxyindoleacetic and homovanillic acids showed lower values. When RBCs were removed from CSF, only some amino acids, thiamine and pyridoxal 5´-phosphate showed moderately higher values when compared with the non-spiked CSF sample.ConclusionsCSF-targeted metabolomic analysis is feasible even when substantial RBC contamination of CSF has occurred since CSF centrifugation to remove RBC prior to freezing eliminated most of the interferences observed.

Resection of a Neuroenteric Cyst Using a Far Lateral Approach.

This operative video highlights a rare case of a neuroenteric cyst at the ventral craniocervical junction. The case involved a 30-year-old man who initially presented 13 years earlier with acute onset of headache and visual changes. At that time, he was found to have a small, enhancing ventral intradural extramedullary mass at the rostral aspect of C1 thought to be a meningioma. The lesion was managed conservatively, and surveillance imaging tracked its slow progressive enlargement to a size of 1.4 cm ( Fig. 1A, B ). Although he remained asymptomatic, nonurgent elective resection was recommended because of his age and mass progression. The patient underwent a left far lateral approach to the craniocervical junction for resection of the mass. This involved dissection of the suboccipital musculature to expose the C1 transverse process in the suboccipital triangle and ultimately the vertebral artery. After a small craniectomy and C1 hemilaminectomy, the dura was opened and a cystic lesion encountered ( Fig. 2 ). The cystic contents were debulked and the capsule resected. Histopathologic examination revealed abundant goblet cells consistent with a neuroenteric cyst. Dural closure was bolstered with fascia lata and autologous fat graft. Postoperative magnetic resonance imaging (MRI) was consistent with gross total resection ( Fig. 1C, D ). The patient tolerated the procedure well with no new postoperative neurological deficits and was discharged home on postoperative day 2. On completing a 3-day decadron taper, he developed steroid-responsive symptoms consistent with aseptic meningitis, possibly related to cerebrospinal fluid contamination with the cyst contents during resection. The link to the video can be found at: https://youtu.be/SskETPe5PXQ .

The Influence of Blood Contamination on Cerebrospinal Fluid Diagnostics.

Background: Blood contamination due to traumatic lumbar puncture presents a diagnostic pitfall in cerebrospinal fluid (CSF) analysis. It is controversially discussed if phagocytosis of erythrocytes which can be found in the CSF after subarachnoid hemorrhage can also develop in vitro in the presence of artificial blood contamination. Furthermore, there is no consensus about the acceptable amount of artificial blood contamination on CSF protein results.Methods: Two measurement series were performed in order to investigate the role of artificial blood contamination on the possible development of erythrophages and siderophages in the CSF: (1) blood contamination was simulated in vitro by adding blood into the CSF. (2) CSF was investigated when blood contamination occurred during a traumatic lumbar puncture. In both types of experiments, CSF including blood was incubated for 24 h and for 72 h at room temperature or at 4°C. In the third measurement series, the effects of artificial blood contamination on CSF protein results were investigated. Blood contamination was simulated in vitro by adding different amounts of blood ending up with five different samples containing erythrocyte counts of 2,500, 5,000, 7,500, 10,000, and 20,000 per μl CSF.Results: Cytological examination revealed no evidence of erythrophages or siderophages in vitro. In contrast, already a low blood contamination (2,500 erythrocytes/μl CSF) led to false pathological results of total protein and albumin. Along with increasing amounts of blood, the frequency of false pathological protein results increased. A blood contamination of 5,000 erythrocytes/μl CSF resulted in a false positive intrathecal IgM production in nearly every fifth patient. In contrast, blood contamination with 5,000 erythrocytes/μl CSF was the acceptable amount of blood which did not lead to a false positive intrathecal synthesis of IgG and IgA.Conclusion: Erythrophages and siderophages do not develop in vitro. An extensive diagnostic work up for the source of blood in the CSF should be performed when erythrophages or siderophages are found in the CSF. The contamination of CSF with increasing volume of blood resulted in falsely elevated CSF protein concentrations. Hence, the amount of blood contamination has to be taken into consideration when interpreting CSF protein measurement results.

Preliminary Results of a New Experimental Model for Intratympanic Treatment.

Corticosteroids have been applied via transtympanic route for a long time to treat the inner ear disorders. A few animal models were used to answer the questions, "How much drug goes into the inner ear?" and "How far does the drug reach through the scala tympani and/or scala vestibuli?" However, the cerebrospinal fluid contamination poses a major problem. The aims of this study were to create a new sampling model showing the dexamethasone distribution in the inner ear and to provide more reliable data about drug concentrations. Ten Hartley strain albino guinea pigs that weighted between 400 and 600 g were used. After dexamethasone application to the left ear, they were sacrificed at two time points: after 0.5 hours (Exp 1) and after 2 hours (Exp 2). The temporal bones were immediately dissected and put into liquid nitrogen for freezing. The apex, second turn, and basal turns of the cochlea and vestibule were separated, while the bone was in the frozen state. The samples were prepared and measured with ultraviolet (UV) spectroscopy. The total amount of dexamethasone was statistically higher in the left ear than the control ear. Although the basal turn and vestibule were the most prominent parts, there was no statistical difference between the different parts of the inner ear at 0.5 hours. The vestibule and the apex showed the highest level of dexamethasone at 2 hours. Although the model has some limitations, it can measure dexamethasone concentrations and show the time variability in the inner ear.

Estimation of diffusion, perfusion and fractional volumes using a multi-compartment relaxation-compensated intravoxel incoherent motion (IVIM) signal model

Compartmental diffusion MRI models that account for intravoxel incoherent motion (IVIM) of blood perfusion allow for estimation of the fractional volume of the microvascular compartment. Conventional IVIM models are known to be biased by not accounting for partial volume effects caused by free water and cerebrospinal fluid (CSF), or for tissue-dependent relaxation effects. In this work, a three-compartment model (tissue, free water and blood) that includes relaxation terms is introduced. To estimate the model parameters, in vivo human data were collected with multiple echo times (TE), inversion times (TI) and b-values, which allowed a direct relaxation estimate alongside estimation of perfusion, diffusion and fractional volume parameters. Compared to conventional two-compartment models (with and without relaxation compensation), the three-compartment model showed less effects of CSF contamination. The proposed model yielded significantly different volume fractions of blood and tissue compared to the non-relaxation-compensated model, as well as to the conventional two-compartment model, suggesting that previously reported parameter ranges, using models that do not account for relaxation, should be reconsidered.

Quantitative MR spectroscopy reveals metabolic changes in the dorsolateral prefrontal cortex of patients with temporal lobe epilepsy.

To characterize possible metabolic changes of the dorsolateral prefrontal cortex (DLPFC) in patients with temporal lobe epilepsy (TLE). Quantitative proton magnetic resonance spectroscopy (1H-MRS) studies were performed on 24 TLE patients and 22 healthy controls. Metabolite concentrations were calculated using a linear combination model (LCModel) and corrected for cerebrospinal fluid contamination. Comparisons were performed between the TLE patients and the controls and between the left DLPFC and right DLPFC in each group. Pearson correlation coefficients were calculated between the metabolite concentrations and epilepsy duration and between the metabolite concentrations and voxel tissue composition: [gray matter (GM)/(GM+white matter (WM))]. Metabolic asymmetry was found in controls between the left and right DLPFC, i.e., the NAA concentration of the left DLPFC was significantly higher than that of the right. However, such metabolic asymmetry was not observed in TLE patients. Compared with the controls, TLE patients showed significantly decreased NAA and Ins, and the reductions were greater in the left DLPFC. No significant correlation was found between the metabolite concentrations and epilepsy duration or between the metabolite concentrations and voxel tissue composition [GM/(GM+WM)]. This study suggests that TLE can produce metabolic changes to DLPFC that is remote from the seizure focus. • Magnetic resonance spectroscopy probes the brain metabolism noninvasively. • Dorsolateral prefrontal reductions in NAA (a neuronal marker) and Ins are observed in TLE. • Temporal lobe epilepsy can result in metabolic changes remote from the seizure focus.

Traumatic Neonatal Lumbar Punctures: Experience at a Large Pediatric Tertiary Care Center in Canada.

Contamination of cerebrospinal fluid (CSF) by blood during neonatal lumbar puncture (LP) is common and poses diagnostic difficulties. Our objectives were to determine the number of traumatic LPs performed at the BC Children's Hospital over 9 years and whether there was an association between traumatic LPs and demographic variables, hospital location, or time of the procedure. This study was a retrospective review of neonatal CSF samples from May 2006 to March 2015. The data were analyzed to establish the rate of traumatic samples and whether there was an association between traumatic LPs and demographic variables (age, gender), location of procedure, positive CSF culture, and/or timing of the procedure. A total of 1,263 LPs were reviewed, 47.7% (n = 602) were contaminated with >400 red blood cells/high-power field. The median age of neonates whose samples were uncontaminated was 10.580 days compared with 6.535 days in the group with contaminated samples (z = - 2.884, p = 0.004). None of the other factors studied was associated with traumatic taps. Detected organisms included Escherichia coli (n = 12), coagulase-negative Staphylococcus (n = 7), Enterococcus faecalis (n = 3), and group B Streptococcus (n = 2). Nearly half of all CSF samples in the study period were contaminated. Traumatic samples were more common in younger neonates.

USEFULNESS AND STABILITY OF MULTICENTER DIFFUSION TENSOR IMAGING AS AN EARLY MARKER FOR SUBJECTIVE COGNITIVE DECLINE AND AMNESTIC MILD COGNITIVE IMPAIRMENT: FIRST RESULTS FROM THE PROSPECTIVE DZNE DELCODE STUDY

Diffusion tensor imaging (DTI) is a useful diagnostic marker for differentiating patients with mild cognitive impairment (MCI) and Alzheimer's disease dementia (AD) from cognitively normal controls, as reported in various monocentric studies. Few monocentric studies performed DTI in individuals with subjective cognitive decline (SCD), who may be at risk of future AD. Compared to normal controls, most of these studies showed decreased fractional anisotropy (FA) and increased mean diffusivity (MD). However, multicenter studies using retrospective data reported a high vulnerability of DTI indices to nuisance variance, introduced by differences in acquisition parameters and study procedures. In the present study, we examined the stability of DTI in a large prospective multicenter cohort using identical scanning protocols and high-level standardized procedures. Furthermore, we assessed the usefulness of multicenter DTI to differentiate between individuals with SCD, MCI patients, AD patients and normal controls. N = 198 subjects from the DZNE Longitudinal Cognitive Impairment and Dementia (DELCODE) study were included in the analysis. We calculated the FA and MD indices for the baseline DTI data that was acquired across seven sites and analyzed them using tract-based spatial statistics (TBSS) and voxel-based analysis. For the latter approach, we applied partial volume correction to reduce the effect of cerebrospinal fluid contamination on grey matter MD estimates. Results will be presented at the conference. We expect to find reduced inter-site variability compared to a multicenter DTI study without harmonized acquisition parameters (European DTI study on Dementia; EDSD). Comparing the diagnostic groups, we expect to find decreased FA and increased MD indices in SCD subjects compared to normal controls, as well as in MCI and AD patients compared to SCD subjects and normal controls. The results from this study will contribute to the evaluation of DTI as a diagnostic marker at prodromal and at-risk stages of AD.