Cerebrospinal-fluid-derived immunoglobulin G of different multiple sclerosis patients shares mutated sequences in complementarity determining regions.

B lymphocytes play a pivotal role in multiple sclerosis pathology, possibly via both antibody-dependent and -independent pathways. Intrathecal immunoglobulin G in multiple sclerosis is produced by clonally expanded B-cell populations. Recent studies indicate that the complementarity determining regions of immunoglobulins specific for certain antigens are frequently shared between different individuals. In this study, our main objective was to identify specific proteomic profiles of mutated complementarity determining regions of immunoglobulin G present in multiple sclerosis patients but absent in healthy controls. To achieve this objective, we purified immunoglobulin G from the cerebrospinal fluid of 29 multiple sclerosis patients and 30 healthy controls and separated the corresponding heavy and light chains via SDS-PAGE. Subsequently, bands were excised, trypsinized, and measured with high-resolution mass spectrometry. We sequenced 841 heavy and 771 light chain variable region peptides. We observed 24 heavy and 26 light chain complementarity determining regions that were solely present in a number of multiple sclerosis patients. Using stringent criteria for the identification of common peptides, we found five complementarity determining regions shared in three or more patients and not in controls. Interestingly, one complementarity determining region with a single mutation was found in six patients. Additionally, one other patient carrying a similar complementarity determining region with another mutation was observed. In addition, we found a skew in the κ-to-λ ratio and in the usage of certain variable heavy regions that was previously observed at the transcriptome level. At the protein level, cerebrospinal fluid immunoglobulin G shares common characteristics in the antigen binding region among different multiple sclerosis patients. The indication of a shared fingerprint may indicate common antigens for B-cell activation.

Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples.

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.

Evaluation of the new body fluid mode on the Sysmex XE-5000 for counting leukocytes and erythrocytes in cerebrospinal fluid and other body fluids.

BACKGROUND: We evaluated the body fluid (BF) mode on the new Sysmex XE-5000 analyzer. METHODS: Red (RBC) and white blood cell (WBC) (differential) counts of BFs (139 patient samples and 87 normal samples) were measured and compared to the Fuchs-Rosenthal chamber and stained cytospin slides. RESULTS: Extended cell counting using the BF mode was noted to have an improved WBC detection limit (CV(20)%) of 10 x 10(6)/L. Excellent agreement with the manual method was observed for most BFs [mean bias +2 to 6 x 10(6)/L for cerebrospinal fluid (CSF) and -1 to 12 x 10(6)/L for other fluids]. In CSF, the BF-mode counted more WBC (polymorphic nuclear cells) compared with the manual method (mean bias +5 to 6 x 10(6)/L), especially in samples with low cell counts (<20 x 10(6)/L). Carry over was negligible (mostly <0.17%) and linearity was excellent (mean bias <5%). The reference ranges for CSF (n=87) were RBC 0 x 10(6)/L, WBC and mononuclear <7 x 10(6)/L, and polymorph nucleated cells <3 x 10(6)/L. CONCLUSIONS: The BF mode on the Sysmex XE-5000 offers rapid and accurate RBC and WBC (differential) counts in clinically relevant concentration ranges in CSF and other fluids. In addition, the exclusion of high fluorescent cells, such as mesothelial cells and macrophages from WBC counting may reduce the number of manual analyses in pleural fluids and ascites.

Central memory CD4+ T cells dominate the normal cerebrospinal fluid.

BACKGROUND: To use cerebrospinal fluid (CSF) immune phenotyping as a diagnostic and research tool, we have set out to establish reference values of white blood cell (WBC) subsets in CSF. METHODS: We assessed the absolute numbers and percentages of WBC subsets by 6-color flow cytometry in paired CSF and blood samples of 84 individuals without neurological disease who underwent spinal anaesthesia for surgery. Leukocyte (i.e., lymphocytes, granulocytes, and monocytes), lymphocyte (i.e., T [CD4(+) and CD8(+) ], NK, NKT and B cells), T cell (i.e., naïve, central memory, effector memory, and regulatory) and dendritic cell subsets (i.e., myeloid and plasmacytoid) were studied. RESULTS: CSF showed a predominance of T cells, while granulocytes, B and NK cells were relatively rare compared to blood. The majority of T cells in CSF consisted of CD4(+) T cells (∼70%), most of them (∼90%) with a central memory phenotype, while B cells were almost absent (<1%). Among the small population of dendritic cells in CSF, those of the myeloid subtype were more frequent than plasmacytoid dendritic cells (medians: 1.7% and 0.4% of leukocytes, respectively), whilst both subsets made up 0.2% of leukocytes in blood. CONCLUSIONS: This study reports reference values of absolute numbers and percentages of WBC subsets in CSF, which are essential for further investigation of the immunopathogenesis of neuro-inflammatory diseases. Furthermore, the relative abundance of CD4(+) T cells, mainly with a central memory phenotype, and the presence of dendritic cells in CSF suggests an active adaptive immune response under normal conditions in the central nervous system (CNS).

Addition of serum-containing medium to cerebrospinal fluid prevents cellular loss over time.

Immediately after sampling, leukocyte counts in native cerebrospinal fluid (CSF) start to decrease rapidly. As the time lapse between CSF collection to analysis is not routinely registered, the clinical significance of decreasing cell counts in native CSF is not known. Earlier data suggest that addition of serum-containing medium to CSF directly after sampling prevents this rapid decrease in leukocyte counts and, thus, may improve the accuracy of CSF cell counting and cell characterization. Here, we prospectively examined the effect of storage time after lumbar puncture on counts of leukocytes and their major subsets in both native CSF and after immediate addition of serum-containing medium, measured by flow cytometry and microscopy. We collected CSF samples of 69 patients in tubes with and tubes without serum-containing medium and determined counts of leukocytes and subsets at 30 minutes, 1 hour, and 5 hours after sampling. Compared to cell counts at 30 minutes, no significant decrease in cell number was observed in CSF with serum-containing medium 1 and 5 hours after sampling, except for the granulocytes at 1 hour. In native CSF, approximately 50% of leukocytes and all their subsets were lost after 1 hour, both in flow cytometric and microscopic counting. In 6/7 (86%) samples with mild pleocytosis (5-15 × 10(6) leukocytes/l), native CSF at 1 hour was incorrectly diagnosed as normocellular. In conclusion, addition of serum-containing medium to CSF directly after sampling prevents cell loss and allows longer preservation of CSF cells prior to analysis, both for microscopic and flow cytometric enumeration. We suggest that this protocol results in more accurate CSF cell counts and may prevent incorrect conclusions based on underestimated CSF cell counts.

[Antidote against local anaesthetic intoxication: new use of lipid emulsion for intravenous administration]. / Tegengif voor intoxicatie door lokale anesthetica: nieuwe toepassing van vetemulsie voor intraveneuze toediening.

Local anaesthetics are routinely used for several indications, but despite local administration their use may lead to systemic toxicity. The symptoms include numbness of the tongue, dizziness, tinnitus, visual disturbances, muscle spasms, convulsions, coma, and respiratory and cardiac arrest. Recently, an intravenous lipid emulsion was reported to act as a novel potential antidote for systemic toxicity due to local anaesthetics. We describe the application of this lipid emulsion in a 27-year-old patient with generalized seizures and coma due to local anaesthetic toxicity. She recovered quickly and was responsive again 10 minutes after the intravenous administration of the lipid emulsion.