Fresh Cerebrospinal Fluid Research Articles

Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. The advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. Challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

A structured evaluation of cryopreservation in generating single-cell transcriptomes from cerebrospinal fluid

Single-cell transcriptomics allows characterization of cerebrospinal fluid (CSF) cells at an unprecedented level. Here, we report a robust cryopreservation protocol adapted for the characterization of fragile CSFcells by single-cell RNA sequencing (RNA-seq) in moderate- to large-scale studies. Fresh CSF was collected from twenty-one participants at two independent sites. Each CSF sample was split into two fractions: one was processed fresh, while the second was cryopreserved for months and profiled after thawing. B and Tcell receptor sequencing was also performed. Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types.We find no significant difference in cell type proportions and cellular transcriptomes between freshandcryopreserved cells. Results were comparable at both sites and with different single-cell sequencing chemistries. Cryopreservation did not affect recovery of T and B cell clonotype diversity. Our CSF cell cryopreservation protocol provides an important alternative to fresh processing of fragile CSF cells.

Extracellular RNA splice events in cerebrospinal fluid as candidate biomarkers of myotonic dystrophy type 1 (N4.004)

<h3>Objective:</h3> To evaluate extracellular RNA (exRNA) in cerebrospinal fluid (CSF) as a source of CNS-derived splice events that may serve as biomarker of DM1 disease activity. <h3>Background:</h3> Alternative splicing is mis-regulated in post-mortem DM1 central nervous system (CNS) tissue. In past few years Cerebrospinal Fluid (CSF) biomarkers have been used to characterize disease severity and prognosis of the disease and helpful tool for to measure changes after treatment. exRNA in CSF provides a source of CNS-derived splice events in living individuals. <h3>Design/Methods:</h3> We examined fresh CSF samples from unaffected controls (N=14) from the MGH CSF biobank and DM1 individuals with mild CNS involvement (N=2). To separate EVs from cells contained in CSF, we used low speed centrifugation followed by filtration of the supernatant. EV size and concentration were measured using microscopy and particle tracking analysis software (NanoSight). We extracted exRNA from the EV pellet, produced cDNA, and quantified gene expression and splice events (% exon inclusion) by droplet digital PCR. <h3>Results:</h3> Mean particle diameter is about 175 nm in CSF and serum vs. 220 nm in urine. In CSF cells, total RNA content is 10 – 100-fold higher than exRNA. Normalized expression of both <i>DMPK</i> and <i>CNBP</i> was 50 – 60% higher in CSF exRNA than in CSF cells. Splicing is significantly different in CSF exRNA vs CSF cells. Alternative splicing of GOLGA4 exon 23 inclusion was 65% in DM1 and 40% in UA (P&lt;0.01). Differential splicing of NUMA1, NCOR2 and MBNL2 was non-significant between groups. <h3>Conclusions:</h3> Quantification of CNS-derived alternative splice products in CSF exRNA is feasible. Removal of leukocytes and erythrocytes normally found in CSF will enhance accurate quantification of CNS-derived splice events. GOLGA4 is a candidate biomarker of early CNS involvement in DM1, while NUMA1, NCOR2, and MBNL2 splice events are candidate biomarkers of disease severity. <b>Disclosure:</b> Dr. Kumari has nothing to disclose. Mr. Hu has nothing to disclose. Mr. Sizemore has nothing to disclose. Ms. Sullivan has nothing to disclose. Mr. McKee has nothing to disclose. Mr. Conquest has nothing to disclose. Dr. Wheeler has nothing to disclose.

Transitioning to Elecsys automated immunoassay for fresh CSF biomarkers to support the diagnosis of Alzheimer’s disease in Australia

AbstractBackgroundAlzheimer’s disease (AD) biomarker proteins, amyloid β1‐42 (Aβ42) and Tau (Phospho‐tau 181 [pTau] or Total‐tau [tTau]), in the cerebrospinal fluid (CSF) are used to support the clinical diagnosis of AD, and for enrolment of patients into therapeutic trials. Currently Roche Elecsys Generation I (GI) for frozen CSF is used, which will be replaced by Generation II (GII) for routine diagnostics use. This study examined the correlation of the biomarkers obtained from the automated Elecsys GI vs the manual INNOTEST immunoassays and the concordance of diagnostics outcome between the two methods. In addition, we also examined performance of GII on fresh CSF.MethodCerebrospinal fluid (CSF) samples were collected from local hospitals/clinics and from the study cohort of the Australian Imaging Biomarkers and Lifestyle study of Aging (AIBL). Aβ42, pTau and tTau were measured with both Elecsys and INNOTEST. Most recent collected CSF with a paired frozen and fresh CSF were assayed in parallel of both GI and GII.ResultIn the cohort of 34 collected frozen CSF from the AIBL cohort, the Spearman r correlations between INNOTEST and Elecsys methods were 0.91, 0.98, and 0.97 for Aβ42, pTau181, tTau, respectively. From 166 clinical diagnostic CSF samples (from local hospitals and clinics), the correlation was 0.89, 0.93, 0.88, respectively. Using the Aβ42 and pTau as primary parameters, the diagnostic outcome is highly concordant between the two methods in these two groups. Initial data from the paired frozen and fresh CSF showed GI and GII correlated well (r = 0.9643). Assessments on the GII cohort are ongoing.ConclusionThe automated Elecsys is highly correlated with the INNOTEST and offers high throughput and excellent precision with lower interassay/interlaboratory variation. This supports the use of Elecsys for CSF AD diagnostics in routine practice. The availability of fresh CSF analysis on GII will make the test more easily accepted into routine pathology setting.

Second‐generation fully automated Elecsys cerebrospinal fluid immunoassays demonstrate high precision, reproducibility, and sample stability suitable for clinical use to aid Alzheimer’s disease diagnosis

AbstractBackgroundBeta‐amyloid(1–42) (Abeta42) and phosphorylated tau 181 (pTau) cerebrospinal fluid (CSF) biomarker analysis is recommended alongside cognitive evaluation to aid Alzheimer’s disease (AD) diagnosis. For clinical use, CSF biomarker assays should demonstrate high precision/reproducibility and sample stability. This study evaluated precision, reproducibility, and sample stability of second‐generation fully automated Elecsys® CSF immunoassays.MethodFrozen/pooled CSF and PreciControl samples were analyzed using Elecsys β‐Amyloid(1–42) CSF II/Phospho‐Tau (181P) CSF immunoassays on cobas e 601 (Roche Diagnostics International Ltd). pTau/Abeta42 ratios were calculated. Precision was evaluated at one site per Clinical and Laboratory Standards Institute (CLSI) EP02‐A3 (two runs/day in duplicate over 21 days; n=84). Reproducibility was determined per CLSI EP05‐A3 over five days using a single lot across three sites (site‐to‐site)/three lots at one site (lot‐to‐lot). Coefficients of variation (CVs)/standard deviations (SDs) for repeatability/intermediate precision/reproducibility were calculated using variance component analysis. Sample stability was determined from fresh CSF, collected per manufacturer's instructions. Baseline measurements were taken &lt;6 hours after lumbar puncture and follow‐up measurements (from stored aliquots) at defined intervals for ≤8 days at room temperature (RT; 25°C; n=13), ≤15 days at 6°C (n=14), and ≤13 weeks at ‐20°C (n=25). Storage stability was determined from percent recoveries/ordinary linear regression.ResultRepeatability CVs were &lt;2.4% and &lt;1.9% for Abeta42 and pTau immunoassays, respectively, and &lt;2.4% for pTau/Abeta42 ratio. Intermediate precision CVs were &lt;3.3% and &lt;3.7% for Abeta42 and pTau immunoassays, respectively, and &lt;3.9% for pTau/Abeta42 ratio. Site‐to‐site reproducibility CVs were &lt;5.1% and &lt;3.6% for Abeta42 and pTau immunoassays, respectively, and &lt;6.5% for pTau/Abeta42 ratio (Table 1). For the Abeta42 immunoassay, lot‐to‐lot reproducibility CVs were &lt;5.4%, except for one sample pool (mean concentration: 322 pg/mL; SD: 34.1 pg/mL; Table 2). Lot‐to‐lot reproducibility CVs were &lt;3.6% for the pTau immunoassay and &lt;6.0% for pTau/Abeta42 ratio. At maximum sample storage duration (8 days at RT/15 days at 2−8°C), median recoveries of 95%/98% were observed for Abeta42 and 97%/99% for pTau immunoassays. Stability assessment at ‐20°C is ongoing.ConclusionThe Elecsys immunoassays demonstrated high precision/reproducibility, supporting clinical use in AD diagnosis. Conservative recommendations for sample storage are 5 days at RT/14 days at 2−8°C.

Stabilization of leukocytes from cerebrospinal fluid for central immunophenotypic evaluation in multicenter clinical trials

Analysis of cerebrospinal fluid (CSF) represents a valuable window into the pathogenesis of neuroinflammatory diseases, such as multiple sclerosis (MS). However, analysis of the cellular fraction of CSF is often neglected because CSF cells die rapidly ex vivo. Immunophenotyping of CSF cells in multicenter clinical trials requires sample preservation and shipping to a centralized lab. Yet, there is no consensus on the best method to preserve intact CSF cells and no detailed evaluation of subset-specific cell loss.We used flow cytometry to compare major leukocyte populations in fresh CSF (processed within 2 h) to cells fixed for 48 h with TransFix-EDTA® or cryopreserved and thawed after 96 h. We observed a statistically significant loss of total mononuclear cells, total T cells, CD3+ CD8- T cells, and CD3+ CD8+ T cells after cryopreservation compared to fresh or fixed (p < 0.001), with no significant difference between fresh and fixed. Thus, our results demonstrate that TransFix-EDTA® was superior to cryopreservation for preserving intact CSF T cells. Surprisingly, neither cryopreservation nor fixation had a significant effect on recovery of low frequency cell subsets in CSF, including B cells, NK cells, NKT-like cells, CD14+ monocytes, or CD123+ DCs, versus fresh CSF.To determine the effect of prolonged fixation on cell recovery, we analyzed major CSF cell subsets by flow cytometry after 24, 48, or 72 h of fixation with TransFix-EDTA®. We observed a consistent and progressive loss in the absolute counts of all subsets over time, although this effect was not statistically significant.We conclude that for immunophenotyping of major CSF cell subsets by flow cytometry, fixation with TransFix-EDTA®, shipment to a central lab, and analysis within 48 h is a feasible method to ensure stability of both absolute cell number and relative frequency. This method is a valuable alternative to fresh CSF analysis and can be implemented in multicenter clinical trials.

P362: MINIMAL-LOSS-CYTOMETRY FOR EVALUATION OF CEREBROSPINAL FLUID IN PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA. SINGLE CENTER STUDY.

Background: Central nervous system (CNS) involvement at diagnosis of acute lymphoblastic leukemia (ALL) represents a risk factor for CNS relapse. Currently, examination of the cerebrospinal fluid (CSF) is performed and evaluated quantitatively using a counting chamber and qualitatively using cytomorphological analysis of cytospin. Recently, flow cytometry (FC) evaluation of CSF was shown to provide more reliable results and to predict relapse risk (de Haas et al., 2021, Thastrup et al., 2020). Independently of these studies, we applied a lyse-no wash technique aiming at minimizing cell loss during preparation and at improved cell concentration estimate. Aims: Our aim was to compare our findings with morphological criteria and published data and correlate them with clinical outcome in pediatric patients with ALL. Methods: We included pediatric patients with newly diagnosed ALL (04/2014 – 01/2022), whose CSF examination was performed in CLIP laboratory (n=208). Patients were classified as B cell precursor ALL (BCP ALL; 85%) and T ALL (15%) and treated according to following protocols: AIEOP-BFM ALL 2009 (n=118), AIEOP-BFM ALL 2017 (n=71), EsPhALL(n=8), Interfant06 (n=5), off-protocol (n=6). During follow up, 14 patients relapsed (5 CNS incl. combined, 9 non-CNS). CSF (drawn into tube with no fixative agent and processed within 2 hours) was concentrated by centrifugation and incubated with combination of antibodies (CD20/CD10/CD45/CD34/CD19/CD3/Syto41 in BCP ALL and CD4/CD99/CD5/CD3/CD7/CD16 + 56/CD8/CD45/Syto41 in T ALL). Residual erythrocytes were lysed with NH4Cl solution, followed by immediate acquisition on BD FACS Lyric or BD LSRII cytometers. Blasts were quantified to initial CSF volume. Cluster of 20 events was required to assign samples as FC positive (FC+). In parallel, CNS status was concluded according to morphological criteria, being classified as CNS1 (no blasts), CNS2a/b (≤5 WBC/μL with blasts), CNS2c/CNS3 (≥5 WBC/μL with blasts) according to treatment protocol guidelines. Continuous variables were compared with the Mann–Whitney test, survival data were analyzed using Mantel-Cox test. P values<0.05 were considered as significant. Results: Using FC, we identified atypical blasts in 50 out of the 208 analyzed samples (24%). Median of analyzed CSF volume was 367µL (range 143-1033µL), which enabled median sensitivity 0.054 events/µL (range 0.02-0.14ev/µL). Mean blast concentration was 0.47ev/µL (range 0-25ev/µL) in all samples and 2 ev/µL (range 0.01-25ev/µL, median 0.53ev/µL) in FC+ samples. We observed significantly higher blast concentration in patients with T ALL, in patients with CNS2 and CNS3 and those who subsequently relapsed. Patients with FC+ had significantly lower relapse-free survival than FC- patients (55% vs 94%), which provided better separation than CNS status (68% vs.85% for CNS2/3 vs. CNS1, respectively). Image:Summary/Conclusion: Using fresh CSF sample with lyse-no wash protocol enabled us to detect approximately 10x higher blast count (0.24ev/µL in FC+CNS1 and 1.25ev/µL in FC+CNS2/3) than was recently published (0.015ev/µL in FC+CNS1 and 0.073ev/µL in FC+CNS2/3 in Thastrup et al., 2020), and thus it better correlates to cytomorphological data. Interestingly, the proportion of FC+ patients in our cohort is comparable to (24% vs 25%), showing that although in our method we lose fewer cells, we identify the same proportion of FC+ patients. The presented study shows a higher impact of FC positivity on relapse risk than described previously. The disadvantage of our method is a need for a fresh sample, which is investigated locally. Supported by UNCE/MED/015, NU20J-07-00028.

Characterization of Antigen-Induced CD4+ T-Cell Senescence in Multiple Sclerosis.

Antigen-induced T-cell exhaustion and T-cell senescence are peripheral regulatory mechanisms that control effector T-cell responses. Markers of exhaustion and senescence on T Cells indicate the previous activation by repetitive stimulation with specific antigens. Malignant tumors are accompanied by enhanced T-cell exhaustion and T-cell senescence resulting in immune evasion, while these control mechanisms might be diminished in autoimmune diseases including multiple sclerosis (MS). To better understand the involvement of antigen-induced T-cell senescence in controlling CD4+ T-cell-mediated autoimmune responses in MS, we have analyzed the re-expression of CD45RA and the downregulation of CD28 and CD27 molecules as markers of antigen-induced T-cell senescence in fresh cerebrospinal fluid (CSF)-infiltrating and paired circulating T cells from patients with MS. Patients with different levels of CD4+ T-cell senescence were identified and characterized regarding demographical and clinical features as well as intrathecal markers of neurodegeneration. CD4+ T-cell senescence was also analyzed in control patients to explore a putative deficit of this regulatory mechanism in MS. This study shows heterogeneity of markers of CD4+ T-cell senescence in patients with MS. Patients with high levels of CD4+ T-cell senescence in peripheral blood showed increased frequencies of CSF-infiltrating CD28+ CD27-EM CD4+ T cells with a proinflammatory Th1 functional phenotype. The correlation of these cells with the intrathecal levels of neurofilament light chain, a marker of neurodegeneration, suggests their relevance in disease pathogenesis and the involvement of T-cell senescence in their regulation. Markers of antigen-induced T-senescence, therefore, show promise as a tool to identify pathogenic CD4+ T cells in patients with MS.

Establishment of updated biomarker cut‐off values for the second‐generation Elecsys β‐amyloid(1–42), pTau and tTau CSF immunoassays

AbstractBackgroundCompared with the first‐generation (Gen1), second‐generation (Gen2) Elecsys® cerebrospinal fluid (CSF) immunoassays (currently under development, Roche Diagnostics International Ltd) for detection of β‐amyloid(1–42) (Abeta42), phospho‐tau(181P) (pTau) and total‐tau (tTau) have higher thresholds for biotin interference. Abeta42 Gen2 is re‐standardized to Abeta42 certified reference materials ERM‐DA480/IFCC, ERM‐DA481/IFCC and ERM‐DA482/IFCC and has an extended measurement range. Additionally, the newly developed pre‐analytical ‘Routine‐Use’ protocol provides more reliable analyte recovery for Abeta42 against comparators (Hansson et al., Alzheimers Dement. 2020;16(S3):e047394). Here, we establish adjusted cut‐off values for concordance of Abeta42, pTau/Abeta42 and tTau/Abeta42 Gen2 results with amyloid‐PET based on those previously established for Gen1, accounting for different pre‐analytical protocols.MethodCSF samples from 26 patients with normal pressure hydrocephalus were collected per the Routine‐Use protocol for fresh CSF (n=3 aliquots/patient) and two clinical trial protocols (BioFINDER and Roche‐Trial) for frozen CSF (&lt;–60°C; n=4 aliquots/patient). Abeta42, pTau and tTau were quantified using Elecsys Gen1/Gen2 immunoassays (cobas e 601 analyzer; Roche Diagnostics International Ltd). Bias at medical decision point was estimated for comparison of Gen1 and Gen2 immunoassays (Roche‐Trial protocol) by weighted‐Deming regression. Original cut‐off values were determined in the BioFINDER cohort using Gen1 immunoassays (Hansson et al.). Adjusted cut‐off values for Gen2 immunoassays with both Routine‐Use and Roche‐Trial protocols were determined. Concentration bias between Routine‐Use and Roche‐Trial protocols was also investigated, measured using Gen2 immunoassays. Mean percentage differences provided estimates of overall bias between protocols and protocol‐assay combinations.ResultNo clinically meaningful differences between assay generations and pre‐analytical protocols were obtained for pTau or tTau. Bias at medical decision point for Abeta42 Gen2 immunoassay versus Gen1 was –20% (95% confidence interval [CI]: –22%, –18%). Bias between concentrations of Abeta42 measured using BioFINDER/Gen1 versus Routine‐Use/Gen2 was –6.3% (95% CI: –8.7%, –3.9%). Cut‐off values for Routine‐Use/Gen2 were adjusted based on this result (). Bias between Routine‐Use versus Roche‐Trial protocols for Abeta42 Gen2 was –22% (95% CI: –24%, –19%). Cut‐off values for Roche‐Trial/Gen2 were adjusted based on this result.ConclusionElecsys CSF Abeta42 results were independently affected by immunoassay generation and pre‐analytical protocol, while tau results were unaffected. Appropriate adjusted cut‐off values have been established.

Spinal Fluid Myeloid Microvesicles Predict Disease Course in Multiple Sclerosis.

In vivo measures of myeloid activity are promising biomarkers in multiple sclerosis. We previously demonstrated that cerebrospinal fluid (CSF) myeloid microvesicles are markers of microglial/macrophage activity and neuroinflammation in multiple sclerosis. Here, we aimed at investigating the diagnostic and prognostic value of myeloid microvesicles in a clinical setting. Six hundred one patients discharged with a diagnosis of neuroinflammatory, neurodegenerative, or no neurological disease were enrolled. Myeloid microvesicles were measured with flow cytometry as isolectin B4-positive events in fresh CSF. Clinical, demographical, and magnetic resonance imaging (MRI) data were collected at diagnosis (all patients) and during follow-up (n=176). CSF myeloid microvesicles were elevated in neuroinflammatory patients compared to the neurodegenerative and control groups. In multiple sclerosis, microvesicles were higher in patients with MRI disease activity and their concentration increased along with the number of enhancing lesions (p < 0.0001, Jonckheere-Terpstra test). CSF myeloid microvesicles were also higher in patients with higher disease activity in the month and year preceding diagnosis. Microvesicles excellently discriminated between the relapsing-remitting and control groups (receiver operator characteristic curve, area under the curve=0.939, p < 0.0001) and between radiologically isolated syndrome and unspecific brain lesions (0.942, p < 0.0001). Furthermore, microvesicles were independent predictors of prognosis for both the relapsing-remitting and progressive groups. Microvesicles independently predicted future disease activity in relapsing-remitting patients (hazard ratio [HR]=1.967, 95% confidence interval [CI] = 1.147-3.372), correcting for prognostic factors of standard clinical use. In the progressive group, microvesicles were independent predictors of disability accrual (HR=10.767, 95% CI=1.335-86.812). Our results confirm that CSF myeloid microvesicles are a clinically meaningful biomarker of neuroinflammation and microglial/macrophage activity in vivo. These findings may support a possible use in clinical practice during diagnostic workup and prognostic assessment. ANN NEUROL 2021;90:253-265.

Design of an Innovative Methodology for Cerebrospinal Fluid Analysis: Preliminary Results.

Cerebrospinal fluid (CSF) analysis supports diagnosis of neurodegenerative diseases (NDs), however a number of issues limits its potentialities in clinical practice. Here, a newly developed technique for fluid voltammetry, relying on a simple sensor (BIOsensor-based multisensorial system for mimicking Nose, Tongue and Eyes, BIONOTE), was used to test the applicability for CSF analysis. BIONOTE was initially calibrated on an artificial CSF-like solution and then applied on human CSF, either immediately after collection or after refrigerated storage. Following optimization, it was used to evaluate 11 CSF samples correlating the electrochemical dataset with CSF routine parameters and biomarkers of neurodegeneration. Multivariate data analysis was performed for model elaboration and calibration using principal component analysis and partial least squares discriminant analysis. BIONOTE presented a high capacity to predict both physiological and pathological constituents of artificial CSF. It differentiated distinct fresh human CSF samples well but lost accuracy after refrigerated storage. The electrochemical analysis-derived data correlated with either CSF routine cytochemical indexes or a biomarker of neurodegeneration. BIONOTE resulted as being a reliable system for electrochemical analysis of CSF. The CSF fingerprint provided by the sensor has shown itself to be sensitive to CSF modification, thus it is potentially representative of CSF alteration. This result opens the way to its testing in further study addressed at assessing the clinical relevance of the methodology. Because of its advantages due to the ease and rapidity of the methodology, a validation study is now required to translate the technique into clinical practice and improve diagnostic workup of NDs.

A Structured Evaluation of Cryopreservation in Generating Single Cell Transcriptomes From Cerebrospinal Fluid

Abstract Importance A robust cerebrospinal fluid (CSF) cell cryopreservation protocol using high resolution single-cell (sc) transcriptomic data would enable the deployment of this important modality in multi-center translational research studies and clinical trials in which many sites do not have the expertise or resources to produce data from fresh samples. It would also serve to reduce technical variability in larger projects. Objective To test a reliable cryopreservation protocol adapted for CSF cells, facilitating the characterization of these rare, fragile cells in moderate to large scale studies. Design Diagnostic lumbar punctures were performed on twenty-one patients at two independent sites. Excess CSF was collected and cells were isolated. Each cell sample was split into two fractions for single cell analysis using one of two possible chemistries: 3’ sc-RNA-Sequencing or 5’sc-RNA-Sequencing. One cell fraction was processed fresh while the second sample was cryopreserved and profiled at a later time after thawing. Setting The research protocol was deployed at two academic medical centers taking care of multiple sclerosis and other neurological conditions. Participants 21 subjects (age 24 – 72) were recruited from individuals undergoing a diagnostic lumbar puncture for suspected neuroinflammatory disease or another neurologic illness; they donated excess CSF. Findings Our comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. The proportion of all cell types was similar between the fresh and the cryopreserved cells processed, and RNA expression was not significantly different. Results were comparable at both performance sites, and with different single cell sequencing chemistries. Cryopreservation also did not affect recovery of T and B cell clonotype diversity. Conclusion and relevance Our cryopreservation protocol for CSF-cells provides an important alternative to fresh processing of fragile CSF cells: cryopreservation enables the involvement of sites with limited capacity for experimental manipulation and reduces technical variation by enabling batch processing and pooling of samples. Key points Question How efficient is CSF cryopreservation for single-cell transcriptome analysis and can it be implemented in large multi-center translational and clinical trial settings? Findings We compared single-cell transcriptomes of paired fresh and cryopreserved CSF from 21 patients at two independent sites. We validate the efficacy of a simple and cost effective CSF cryopreservation method that preserves the composition and the transcriptomes of CSF cells stored for weeks-months. The protocol is deployed in a large multicenter Phase 4 MS clinical trial. Meaning A validated CSF cryopreservation method that would significantly advance basic science and biomarker research in neurological disorders by implementing single-cell transcriptome analyses in multi-center research and clinical trials.

Stability of canine and feline cerebrospinal fluid samples regarding total cell count and cell populations stored in \u201cTransFix\xae/EDTA CSF sample storage tubes\u201d

BackgroundBecause of fast leucocyte degeneration in cerebrospinal fluid (CSF) laboratory examinations of CSF samples should be performed approximately within 30 min after withdrawal. This study examines the storage of canine and feline CSF samples in “TransFix®/EDTA CSF Sample Storage Tubes” (Cytomark, Buckingham, UK) for preventing leucocytes from degeneration, so that routine and flow cytometry examinations are feasible up to 3 days after sampling.ResultsAfter storage in TransFix® tubes, leukocytes could not be adequately stained with Türk’s solution and differentiating between erythrocytes and leukocytes was cumbersome. In addition, the cell morphology could not be sufficiently assessed on cytospin preparations because of shrunken leukocytes and indistinct cell nuclei. In contrast, by flow cytometry, a significantly higher cell count was measured over the entire study period in the samples stored in TransFix® tubes compared to the untreated samples. The antibodies (AB) against CD3, CD4 and CD21, against CD11b and against CD45 showed a good binding strength and thus enabled a good differentiation of cell populations. However, after storage in the TransFix® tubes, monocytes were no longer detectable using an AB against CD14.ConclusionBased on these results, “TransFix®/EDTA CSF Sample Storage Tubes” can be used for extended storage prior to flow cytometric analysis of lymphocytes and granulocytes in CSF samples but not for detecting monocytes. However, standard examinations, such as microscopic cell counting and morphological cell assessment should be performed on fresh CSF samples.

Pre-analytical protocol for measuring Alzheimer's disease biomarkers in fresh CSF.

IntroductionWe aimed to establish a standardized, routine‐use pre‐analytical protocol for measuring Alzheimer's disease (AD) biomarkers in cerebrospinal fluid (CSF).MethodsThe effect of pre‐analytical factors (sample collection/handling/storage/transportation) on biomarker levels was assessed using freshly collected CSF. Tube type/sterilization was assessed using previously frozen samples. A low‐bind false‐bottom tube (FBT, Sarstedt) was used for all experiments, except tube types/sterilization experiments. Biomarkers were measured using Elecsys CSF assays.ResultsAmyloid beta (Aβ)1‐42 levels varied by tube type, using a low‐bind FBT reduced variation. Aβ1‐42 levels were higher with no mixing versus roller/inversion mixing. Aβ1‐42 levels were lower with horizontal versus upright transportation; this was resolved by maximal tube filling and storage at 2°C to 8°C. Aβ1‐40 levels were less strongly affected. Phospho‐tau and total‐tau levels were largely unaffected.DiscussionWe propose an easy‐to‐use, standardized, routine‐use pre‐analytical protocol, using low‐bind FBTs, for measuring AD CSF biomarkers in clinical practice.

Characteristics of Diffuse Leptomeningeal Glioneuronal Tumor With First-Episode Headache and Rapid Blindness Misdiagnosed as Viral Meningoencephalitis

Diffuse leptomeningeal glioneuronal tumor has a high degree of malignancy and high mortality. The purpose of this paper is to describe the characteristics of atypical diffuse leptomeningeal glioneuronal tumor and analyze the causes of misdiagnosis as viral meningoencephalitis. An adolescent female patient presented with headache, nausea, vomiting, sharp vision loss and cognitive dysfunction. After poor therapeutic effect of standard antiviral treatment, further inspection found that malignant cells were detected by cerebrospinal fluid (CSF) cytology; and enhanced magnetic resonance imaging (MRI) showed extensive enhancement of the leptomeningeal. In conclusions, when patient with unexplained high intracranial pressure, it is necessary to be alert to the diagnosis of diffuse leptomeningeal glioneuronal tumor. Multiple examinations of fresh CSF are helpful to increase the positive detection rate of tumor cells. Early diagnosis and active treatment are conducive to improving survival rate. J Med Cases. 2019;10(8):241-245 doi: https://doi.org/10.14740/jmc3347

Towards a unified protocol for handling of CSF before \u03b2-amyloid measurements

BackgroundWidespread implementation of Alzheimer’s disease biomarkers in routine clinical practice requires the establishment of standard operating procedures for pre-analytical handling of cerebrospinal fluid (CSF).MethodsHere, CSF collection and storage protocols were optimized for measurements of β-amyloid (Aβ). We investigated the effects of (1) storage temperature, (2) storage time, (3) centrifugation, (4) sample mixing, (5) blood contamination, and (6) collection gradient on CSF levels of Aβ. For each study participant, we used fresh CSF directly collected into a protein low binding (LoB) tube that was analyzed within hours after lumbar puncture (LP) as standard of truth. Aβ42 and Aβ40 were measured in de-identified CSF samples using EUROIMMUN and Mesoscale discovery assays.ResultsCSF Aβ42 and Aβ40 were stable for at least 72 h at room temperature (RT), 1 week at 4 °C, and 2 weeks at − 20 °C and − 80 °C. Centrifugation of non-blood-contaminated CSF or mixing of samples before the analysis did not affect Aβ levels. Addition of 0.1–10% blood to CSF that was stored at RT without centrifugation led to a dose- and time-dependent decrease in Aβ42 and Aβ40, while Aβ42/Aβ40 did not change. The effects of blood contamination were mitigated by centrifugation and/or storage at 4 °C or − 20 °C. Aβ levels did not differ between the first to fourth 5-ml portions of CSF.ConclusionsCSF can be stored for up to 72 h at RT, 1 week at 4 °C, or at least 2 weeks at either − 20 °C or − 80 °C before Aβ measurements. Centrifugation of fresh non-blood-contaminated CSF after LP, or mixing before analysis, is not required. In case of visible blood contamination, centrifugation and storage at 4 °C or − 20 °C is recommended. After discarding the first 2 ml, any portion of up to 20 ml of CSF is suitable for Aβ analysis. These findings will be important for the development of a clinical routine protocol for pre-analytical handling of CSF.

Clinical Features of Diffuse Leptomeningeal Glioneuronal Tumor with Rapid Blindness Misdiagnosed as NMOSD and Literature Review

Diffuse leptomeningeal glioneuronal tumor has various clinical manifestations and is easily misdiagnosed. This article aims to describe the clinical features of a patient with diffuse leptomeningeal glioneuronal tumor misdiagnosed as optic neuromyelitis lineage disease (NMOSD), analyze the causes of misdiagnosis, and identify the disease with similar clinical manifestations. Adolescent women with decreased vision and headache, nausea, and vomiting were initially diagnosed as NMOSD. Immunotherapy was ineffective; enhanced MRI and cerebrospinal fluid cytology examination were completed and compared with classic meningeal carcinoma, NMOSD, multiple sclerosis, and Vogt-Koyanagi-Harada (VKH) syndrome. Diffuse leptomeningeal glioneuronal tumor was finally diagnosed in adolescent women with no history of tumor. Compared with NMOSD: no history of infection, no AQP4, MOG antibody, no spinal segmental abnormalities, immunotherapy was ineffective; compared with multiple sclerosis: no history of infection, no plaque, no oligoclonal antibody, immunotherapy was ineffective; compared with VKH syndrome: no history of infection, no fever, no treatment for immunotherapy; and compared with classic meningeal carcinoma: adolescents, no extracranial tumor lesions, super high protein, more different sugar excess. Patients with acute visual impairment should consider not only NMOSD, but also diffuse leptomeningeal glioneuronal tumor (DLGNT) and other diseases. DLGNT could not be excluded when CSF cytology and MRI plain scan were negative. The positive rate of malignant cells can only be increased by repeatedly sending fresh cerebrospinal fluid for examination; DLGNT needs to be differentiated from NMOSD, multiple sclerosis, VKH syndrome, and other diseases. Early diagnosis and molecular targeted therapy can improve the quality of life and prolong life.

MYD88 p.(L265P) detection on cell-free DNA in liquid biopsies of patients with primary central nervous system lymphoma.

MYD88 p.(L265P) detection on cell-free DNA in liquid biopsies of patients with primary central nervous system lymphoma.

The use of dried cerebrospinal fluid filter paper spots as a substrate for PCR diagnosis of the aetiology of bacterial meningitis in the Lao PDR

AbstractWe investigated whether dried cerebrospinal fluid (CSF) conserved on filter paper can be used as a substrate for accurate PCR diagnosis of important causes of bacterial meningitis in the Lao PDR. Using mock CSF, we investigated and optimized filter paper varieties, paper punch sizes, elution volumes and quantities of DNA template to achieve sensitive and reliable detection of bacterial DNA from filter paper specimens. FTA Elute Micro CardTM (Whatman, Maidstone, UK) was the most sensitive, consistent and practical variety of filter paper. Following optimization, the lower limit of detection for Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/μL (interquartile range 5.5 GE/μL) or 230 (IQR 65) colony forming units/mL. A prospective clinical evaluation for S. pneumoniae, S. suis and Neisseria meningitidis was performed. Culture and PCR performed on fresh liquid CSF from patients admitted with a clinical diagnosis of meningitis (n = 73) were compared with results derived from dried CSF spots. Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one patient under the limit of detection. In a retrospective study of S. pneumoniae samples (n = 20), the median (IQR; range) CSF S. pneumoniae bacterial load was 1.1 × 104 GE/μL (1.2 × 105; 1 to ×.1 × 106 DNA GE/μL). Utilizing the optimized methodology, we estimate an extrapolated sensitivity of 90%, based on the range of CSF genome counts found in Laos. Dried CSF filter paper spots could potentially help us to better understand the epidemiology of bacterial meningitis in resource-poor settings and guide empirical treatments and vaccination policies.

The cerebrospinal fluid cytological features of tuberculous meningitis in children

Background Tuberculous meningitis (TBM) is a common central nervous system (CNS) infectious disease,and is a serious threat to people's health. Due to its diverse clinical manifestations, especially atypical in infants and young children, it is easy to be misdiagnosed and can lead to high disablility rate and mortality rate. At present, cerebrospinal fluid (CSF) cytology still plays an important role in the etiological diagnosis of CNS infectious diseases. Based on the observation of CSF cytological changes of children with TBM, this study is to further understand the performance and evolution characteristics of this disease, so as to improve the ability of early diagnosis and judgment on the treatment effect. Methods Fresh CSF was collected to make smears by using cytological slide centrifugation, and apply May-Grunwald-Giemsa (MGG) staining for morphological classification. Results CSF cytology of all patients were abnormal. The average leukocyte count of children with TBM was (310.19 ± 156.26) × 106/L, and was significantly different from that of children with purulent meningitis ( P = 0.000). Lymphocyte proportion of TBM group was (66.56 ± 14.08)% , and there were significant differences among different groups ( P = 0.037, 0.000). Compared to children with purulent meningitis, higher proportion of monocytes ( P = 0.000, 0.020) and plasma cells ( P = 0.001, 0.004) were found in children with TBM and viral meningitis. The positive detection rate of plasma cells in children with TBM was higher than that in children with purulent meningitis ( P = 0.001). Conclusion The CSF cytology changing characteristics of children with TBM are revealed. In the beginning the lymphocyte proportion is dominant in CSF cytology, and then different extents of mixed cellular response appear as disease develops. For children with effective treatment, the proportion of neutrophils decreases rapidly and turns to lymphocyte reaction gradually, with monocytes and plasma cells, lasting for a long time. Long-term mixed cellular response will happen in children with ineffective treatment. All in all, the CSF cytology changing characteristics are significant in clinical diagnosis and differential diagnosis of TBM in children.