Jan Willem Gratama

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. © 2013 International Clinical Cytometry Society

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. © 2013 International Clinical Cytometry Society.

Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low‐cellular CSF samples and enable to define cell populations more accurately. © 2011 International Clinical Cytometry Society

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 (+ 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). © 2010 International Clinical Cytometry Society

To detect HuD-specific T cells in patients with Hu-antibody associated paraneoplastic neurological syndromes (Hu-PNS), we used short-term stimulation assays with HuD protein spanning peptide pools (PSPP) with purities of at least 70% and found reproducible false-positive CD8+ T-cell responses in three of 127 individuals (two healthy controls and one Hu-PNS patient), which all shared HLA-A*2402 and HLA-B*1801. After testing the 15-mer peptides of the HuD antigen separately, we discovered that the same three 15-mers yielded the CD8+ T cell response in those three individuals. This highly unusual result could not be reproduced when using new batches of peptides with a higher level of purity (>82% and >95%). Therefore, we assumed this response was not directed against the HuD peptides and analyzed the HuD 15-mers by Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS), which showed the presence of a cytomegalovirus (CMV)-encoded peptide (AIAEESDEEEAIVAY) as a contaminant. The three responding individuals all were CMV-seropositive and the contaminating peptide appeared to fit in the binding groove of HLA-B*18. Our data reveal that synthetic PSPP may contain immunogenic contaminations which may cause false positive results in T-cell stimulation assays. © 2008 International Society for Advancement of Cytometry