Use of TransFix™ cerebrospinal fluid storage tubes prevents cellular loss and enhances flow cytometric detection of malignant hematological cells after 18 hours of storage.

Flow cytometry is a sensitive method for detection of leptomeningeal localizations of hematological malignancies (LHM) in cerebrospinal fluid (CSF). Rapid processing of CSF is needed, as leukocyte numbers appear to decline quickly after lumbar puncture. The cell-stabilizing agent TransFix™ may enhance the detection of LHM in CSF by preventing cellular loss. To study the effects of TransFix on leukocyte numbers and the detection of LHM, we prospectively collected 99 CSF samples from patients with suspected or proven LHM in tubes with (i) TransFix; (ii) serum-containing medium; and (iii) no cell-stabilizing agents (native CSF). Presence of LHM and absolute leukocyte numbers were determined by flow cytometry after 30 minutes and 18 hours of storage. Leukocyte numbers in TransFix-stabilized CSF were higher than in the corresponding native samples at both time points (1.4× and 2.3× respectively, P < 0.0001 on each occasion). After 18 hours of storage, TransFix enhanced the detection of LHM in CSF. In all discordant paired observations (13/99, P = 0.005), the level of suspicion (classified as positive, suspicious, or negative) in CSF with TransFix was higher than in native CSF. We conclude that the use of TransFix-containing CSF storage tubes prevents cellular loss and enhances flow cytometric detection of LHM after 18 hours of storage.

Elevated numbers of regulatory T cells, central memory T cells and class-switched B cells in cerebrospinal fluid of patients with anti-Hu antibody associated paraneoplastic neurological syndromes.

Multi-parametric flow cytometry was used to study lymphocyte subsets and dendritic cells in paired blood and CSF samples from 11 newly diagnosed patients with progressive anti-Hu antibody associated paraneoplastic neurological syndromes (Hu-PNS), 9 patients with other inflammatory neurologic disorders (IND), and 12 patients with other non-inflammatory neurologic disorders (OND). Hu-PNS patients had elevated numbers of regulatory T cells, central memory T cells, class-switched B cells and dendritic cells in their CSF. These findings support the hypothesis that the immune system is locally activated in Hu-PNS, and suggests common etiological pathways between Hu-PNS and other inflammatory central nervous system disorders.

Effector memory and late memory T cells accumulate in the blood of CMV-carrying individuals but not in their cerebrospinal fluid.

Cytomegalovirus (CMV)-carrying individuals have significantly higher levels of effector memory and late memory T lymphocytes in their blood than non-carriers. To date, it is well recognized that the central nervous system is subjected to active immunosurveillance, as evidenced by the presence of central memory T cells in cerebrospinal fluid (CSF) of healthy individuals. In order to investigate whether levels of effector memory and late memory T cells were also increased in the CSF of CMV-carrying individuals, we characterized CD4⁺ and CD8⁺ T-cell subsets in CSF and blood of both groups. Effector memory and late memory T cells were only rarely seen in CSF, which was similar in CMV carriers and non-carriers. In conclusion, there was no demonstrable difference in the numbers of CSF effector memory and late memory T cells between CMV seronegative and CMV seropositive individuals.

Three sensitive assays do not provide evidence for circulating HuD-specific T cells in the blood of patients with paraneoplastic neurological syndromes with anti-Hu antibodies.

Anti-Hu antibody-associated paraneoplastic neurological syndromes (Hu-PNSs) are severe and often precede the detection of a malignancy, usually small-cell lung cancer. In Hu-PNS, it is hypothesized that neuronal cells are destroyed by T cells targeted against HuD, a protein expressed by small-cell lung cancer cells and neurons. There is only limited evidence for the existence of HuD-specific T cells. To detect these T cells in the blood of Hu-PNS patients, we employed 3 highly sensitive assays that included T cell stimulation with dendritic cells (DCs) to specifically expand the number of any HuD-specific T cells. A total of 17 Hu-PNS patients were tested with 1 or more of the following 3 assays: (1) tetramer staining after stimulation of T cells with conventionally generated DCs (n = 9), (2) interleukin (IL)-13 enzyme-linked immunosorbent spot (ELISpot; n = 3), IL-4 and IL-5 and interferon (IFN)-γ multiplex cytokine bead array (n = 2) to assay cytokine production by T cells after stimulation with conventionally generated DCs, and (iii) IFN-γ ELISpot and tetramer staining after T cell stimulation with accelerated co-cultured DCs (n = 11). No circulating HuD-specific T cells were found. We suggest that either autoaggressive T cells in Hu-PNS are not targeted against HuD or that their numbers in the blood are too low for detection by highly sensitive techniques.

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.

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).