Molecular analysis of the CDR3 encoding region of the immunoglobulin heavy chain locus in cerebrospinal fluid cells as a diagnostic tool in lymphomatous meningitis

The diagnosis of leptomeningeal B-cell lymphoma is based on the identification of malignant B cells in the cerebrospinal fluid (CSF). Frequently, cytology does not allow clear distinction between neoplastic lymphoid cells and reactively transformed mononuclear cells. Individual B-cell clones can be identified on the basis of DNA sequences that encode the highly diverse complementarity-determining region (CDR3) of the immunoglobulin heavy chain locus (IgH). We studied CSF samples from 5 patients with B-cell malignancies and cytological evidence of leptomeningeal involvement, using polymerase chain reaction (PCR)-based high-resolution capillary electrophoresis and automated fluorescence analysis to detect PCR fragments. As controls, we assessed CSF specimens from 7 patients with inflammatory neurological diseases and three samples without pathological findings. In all patients with B-cell malignancies, a single PCR product was generated, indicating that CDR3-specific fragments were derived from monoclonal cell populations. CSF samples from patients with inflammatory diseases yielded multiple CDR3 amplicons, suggesting the presence of a polyclonal B-cell activation. No PCR product could be amplified in normal CSF samples. Automated fluorescence detection of CDR3 fragments is a highly sensitive and rapid method to distinguish neoplastic monoclonal and reactive polyclonal B-cell populations in the CSF.     

A case of neoplastic angioendotheliosis--serial study of magnetic resonance imaging

The case of a 59-year-old man who was diagnosed as having neoplastic angioendotheliosis by biopsy of a small hemangioma on the skin is reported. The clinical features were characterized by hypersomnia, memory disturbance, disorientation to time and mild left hemiplegia including the face. Laboratory findings showed an elevated erythrocyte sedimentation rate, increased serum LDH, increased CSF protein and pleocytosis in the CSF. The CSF level of IgG was also elevated and was associated with the appearance of oligoclonal IgG bands. The biopsy specimen of the hemangioma on the skin revealed that some small vessels were packed with atypical mononuclear cells which were positive for anti-B cell antibody. Magnetic resonance imaging (MRI) of the brain detected multiple lesions located in the cerebellum, thalamus and caudate nucleus. The left paramedian thalamic lesion might be responsible for his characteristic mood and behavioral changes. The serial MRI study disclosed that some lesions progressively enlarged and duplicated in number. These findings might be typical for neoplastic angioendotheliosis, in which the rapidly proliferating cells occluded small vessels one after another in the central nervous system. The serial study of MRI may serve an important diagnostic purpose in this disease, although most patients with this disease, so far, have been diagnosed by autopsy.     

Monocytes and histiocytes in cell cultures of cerebrospinal fluid

Summary A method of CSF cell culturing, based on observations of cultured cells isolated from 700 CSF specimens obtained for routine diagnostic procedures by lumbar puncture from patients who had no proven or suspected neoplastic disease, is described which enables the demonstration of proliferating mononuclear elements even when they are present in specimens with low cell count. Spread on surfaces of plastic and glass material, monocytes and histiocytes in CSF cell cultures can appear as polygonal or crescent shaped epitheloid cells, may assume spindle shapes, or transform into multinucleated giant cells. Some cells give rise to clones with different rates of proliferation, up to the formation of a monolayer. After short term culturing the cytochemical characteristics of the cells are comparable to those of the native cells. Phagocytosis in culture is possible. Cells with a high rate of proliferation can be isolated from CSF specimens in subacute non-bacterial inflammatory processes, in chronic meningitis, in the state of repair of bacterial meningitis and subarachnoid hemorrhage, after repeated lumbar punctures and other unspecific irritations such as myelography and pneumencephalography, and in the course of intrathecal cytostatic therapy.Supported by the Deutsche Forschungsgemeinschaft.     

Supplementary cytodiagnostic analyses of mononuclear cells of the cerebrospinal fluid using cytological markers

Summary Various methods of CSF cell differentiation are discussed. These methods provide additional information regarding the origin and, therefore, the types of pathological alterations occurring in the leptomeninges. They include the application of immunological and cytochemical markers to differentiate and identify B and T lymphocytes as well as cells from the monocyte-macrophage series and precursor cells of polymorphnuclear leukocytes. Application of these methods to CSF cells and the differentiation of CSF cells from 16 patients with inflammatory or neoplastic alterations were discussed. B cell or T cell type lymphocytes predominate with multiple sclerosis, T lymphocytes with tubercular meningitis. Varying quantities of B and T lymphocytes are found with viral meningitis. In one case the tumor cells of a reticulum cell sarcoma were identified in the CSF as T cells; in one case of plasmacytoma, tumor cells in the CSF were identified as B lymphocytes. In selected cases of leukemic or carcinomatous infiltration of the meninges and of medulloblastoma, CSF cells did not react to treatment with immunological markers.This study was supported by the Deutsche Forschungsgemeinschaft     

Cerebrospinal fluid immunoglobulins and beta 2-microglobulin in lymphoproliferative and other neoplastic diseases of the central nervous system

Humoral immune aberrations may occur in the cerebrospinal fluid (CSF) of patients with lymphoproliferative and other neoplastic diseases infiltrating the central nervous system (CNS). Such aberrations may be of diagnostic importance. We therefore studied CSF and serum from 47 patients with lymphoproliferative diseases and from 16 patients with various nonlymphoid neoplasias; 17 patients and 12 patients, respectively, had neoplastic CNS involvement. Elevated CSF IgM index and oligoclonal IgG bands in CSF and serum were commonly found, especially in patients with CNS involvement. Cerebrospinal fluid IgG and IgA indexes were usually normal. Increased CSF to serum albumin ratio, reflecting blood-brain barrier dysfunction, and increased CSF beta 2-microglobulin concentration were most common in patients with CNS involvement. The results indicate that neoplastic CNS disease should be borne in mind when CSF humoral immune aberrations are found.     

Influence of the cerebrospinal fluid laboratory parameters in the ELISA test for neurocysticercosis using a total cysticerci antigen

To evaluate if the cerebrospinal fluid (CSF) parameters may influence the cysticercosis immunoreactivity response in the CSF. CSF samples of 109 patients were analyzed and classified in three groups, according to the neurological manifestations and the reactivity in antibody-enzyme linked immunosorbent assay (Ab-ELISA) testing in CSF for neurocysticercosis (NC): group A, 18 patients with neurological disorders compatible with NC and reactive Ab-ELISA in CSF for NC; group B, 50 patients with neurological disorders non-compatible with NC and reactive Ab-ELISA for NC; group C, 41 patients with neurological disorders non-compatible with NC and non-reactive Ab-ELISA in CSF for NC. The CSF analysis in group A was compatible with NC. The group B in comparison to the groups A and C presents higher frequency and intensity of hypercytosis, presence of red blood cells in CSF, protein concentration and immunological reactive test for other etiological agents (p<0.05). Based on the present data, we suggest that the inflammatory process and high protein concentration may determine false positive reactions in the Ab-ELISA test for NC in the CSF.     

Single-cell PCR analysis of the immunoglobulin heavy-chain CDR3 region for the diagnosis of leptomeningeal involvement of B-cell malignancies using standard cerebrospinal fluid cytospins

The diagnosis of leptomeningeal B-cell malignancies is based on the identification of malignant B cells in the cerebrospinal fluid (CSF). We have established a polymerase chain reaction (PCR) approach to characterize the clonally diverse gene encoding the immunoglobulin heavy-chain (IgH) third complementarity determining region (CDR3) of single B cells. We demonstrate that single-cell PCR is readily applicable to individual cells derived from routine CSF cytospins and is a powerful method to discriminate monoclonal neoplastic from polyclonal reactive B-cell responses. Single-cell PCR analysis, as a new tool for the diagnosis and monitoring of neoplastic meningitis associated with B-cell malignancies, is particularly important if cytology, immunocytochemistry, flow cytometry and automated gene scanning of CSF samples are unable to detect malignant monoclonal proliferation.     

The effects of natalizumab on inflammatory mediators in multiple sclerosis: prospects for treatment‐sensitive biomarkers

Background: Natalizumab affects systemic cytokine expressions and clinical course in relapsing–remitting multiple sclerosis (RRMS). We analyzed levels of inflammatory cytokines in cerebrospinal fluid (CSF) cells and peripheral blood mononuclear cells (PBMCs), levels of matrix metalloproteinase (MMP)‐9 and osteopontin (OPN) in CSF, and clinical outcome measures in 22 natalizumab‐treated RRMS patients. Methods: mRNA levels of cytokines in cells were detected with real‐time RT‐PCR. Protein levels of OPN and MMP‐9 were measured by ELISA. Results: Natalizumab reduced CSF cell counts (P < 0.0001). Tumor necrosis factor (TNF) and interferon‐γ (IFN‐γ) mRNAs were significantly increased in PBMCs. In contrast, expressions of IFN‐γ and interleukin (IL)‐23 were decreased but IL‐10 increased in the CSF cells. OPN and MMP‐9 were reduced in the CSF. Patients being in remission at baseline showed the same deviations of mediators as those in relapse after natalizumab treatment. The open label clinical outcome measures were either stable or improved during therapy. Conclusions: Natalizumab attenuates pro‐inflammatory mediators intrathecally and the reduced pro‐inflammatory milieu may allow increased production of the anti‐inflammatory mediator IL‐10. The increased systemic cytokines may impede the improvement of certain clinical measures like fatigue. The affected mediators seem to be sensitive to an immune‐modifying treatment which could be used as biomarkers for this therapy.     

脑膜淋巴瘤的临床脑脊液细胞学研究

目的探讨脑脊液细胞学、免疫细胞化学和流式细胞分析在脑膜淋巴瘤诊断中的灵敏性和特异性。方法13例诊断为脑膜淋巴瘤的患者，其中原发性中枢神经系统淋巴瘤5例，均为非霍奇金淋巴瘤B细胞型（NHL-B）；继发性8例，包括NHL-B6例，非霍奇金淋巴瘤T细胞型（NHL-T）1例，mantle型1例。神经系统表现为头痛、视乳头水肿、脑脊膜刺激征、脊髓腰骶神经根受累和多脑神经麻痹等。腰穿脑脊液压力升高，细胞计数、蛋白均升高，糖减低。所有患者均采用沉淀池法，免疫细胞化学单克隆抗体CD19、CD20、CD79a、CD34和免疫球蛋白轻链等；其中3例脑脊液进行淋巴细胞亚群流式细胞分析。以4例病毒性脑炎和5例炎性脱髓鞘病患者的脑脊液为对照。结果脑脊液细胞学发现每例患者均有淋巴瘤细胞或异型淋巴细胞。免疫细胞化学发现5例患者多数细胞B细胞标记物阳性；例6多数细胞CD34阳性；例8CD20阳性细胞比例升高；例7CD19、CD20阴性。除例8外，其余脑脊液中细胞呈CD4和CD8阴性或极少数阳性。对照组CD4阳性细胞为主，伴有少量CD8阳性细胞，CD19和CD20阴性或极少数阳性。脑脊液流式细胞分析发现例9和例11的B淋巴细胞占85．9％～97．4％，提示异常的B细胞克隆。对照组以CD4、CD8阳性细胞为主，CD19阳性细胞低于4．0％。结论脑脊液细胞学和免疫细胞化学是诊断脑膜淋巴瘤的重要方法，脑脊液淋巴细胞亚群的流式分析能够对诊断脑膜淋巴瘤有所帮助。     

Analysis of B-cell activation of cerebrospinal fluid lymphocytes in multiple sclerosis

We have developed a microculture system to study pokeweed mitogen (PWM)-induced B-cell differentiation responses of CSF lymphocytes from patients with multiple sclerosis (MS) and other neurologic diseases (OND). B-cell differentiation was assessed by (1) enumeration of immunoglobulin-secreting cells (IgSC) by a protein A reverse hemolytic plaque assay; and (2) quantitation of supernatant IgG by ELISA. Cultures of MS CSF cells and OND CSF cells responded to PWM with a similar frequency, with responses in CSF cell cultures exceeding responses in corresponding blood cell cultures in several instances in both groups of patients. Numbers of IgSC in unstimulated cultures of MS CSF cells exceeded numbers in cultures of autologous peripheral blood mononuclear cells (PBM). Results suggest that CSF cells may be a particularly reactive population compared with PBM.     

Cell surface markers for diagnosis of central nervous system involvement in lymphoproliferative diseases

To diagnose lymphoproliferative central nervous system (CNS) involvement we have used monoclonal antibodies in an immunocytochemical method for differentiation of cells in cerebrospinal fluid (CSF) and peripheral blood. The cell distribution in 9 patients with B-cell lymphoma and 7 patients with chronic lymphatic leukemia was compared to that in a group of patients with aseptic meningitis. Most patients with neoplastic CNS involvement showed a high proportion of CSF B cells (OKB2+ and/or OKB7+) and a concurrently low proportion of CSF T cells (anti-Leu 1+). Proliferating cells expressing transferrin receptor (OKT9 labeled) were increased in the CSF of 2 patients with neoplastic CNS involvement. In 2 patients with infectious CNS complications, the cell distribution in CSF did not differ from that in patients with aseptic meningitis. Patients with leukemia who had no CNS symptoms, and also 1 patient with meningitis and blood-brain barrier damage, showed a normal cell distribution in CSF despite high B-cell numbers in the peripheral blood. This indicates a selective passage of leukocytes into the CNS and/or local proliferation.     

The proliferation rate of T and B lymphocytes in cerebrospinal fluid

Summary An immunocytochemical method is presented by which individual proliferating T and B lymphocytes can be detected in the cerebrospinal fluid (CSF) of patients with inflammatory and neoplastic diseases of the central nervous system. The absolute numbers of proliferating T and B lymphocytes were approximately equal, so that only a minority of the strongly prevailing T-cell population proliferates in the CSF during the immune process. The highest proliferation (25.7%) was found within the small subset of activated, i.e. cytoplasmic immunoglobulin-containing, B lymphocytes. This provides further evidence that these cells are not the terminally differentiated plasma cells of classical cytology but progenitor cells capable of further proliferation. B-cell lymphomas could be easily identified. The detection of abnormally proliferating cell subsets could help in the differentiation of opportunistic infection from leukaemic infiltration     

Neoplastic meningitis

Neoplastic meningitis is a common problem in neuro-oncology, occurring in approximately 5% of all patients with cancer. Notwithstanding frequent focal signs and symptoms, neoplastic meningitis is a disease affecting the entire neuraxis; therefore, staging and treatment must encompass all cerebrospinal fluid (CSF) compartments. Central nervous system staging of neoplastic meningitis includes contrast-enhanced cranial computerized tomography or magnetic resonance imaging, contrast-enhanced spine magnetic resonance imaging, or computerized tomographic myelography and radionuclide CSF flow study (FS). Treatment of neoplastic meningitis includes involved-field radiotherapy of bulky or symptomatic disease sites and intra-CSF drug therapy. The inclusion of concomitant systemic therapy may benefit patients with neoplastic meningitis and may obviate the need for intra-CSF chemotherapy. At present, intra-CSF drug therapy is confined to three chemotherapeutic agents (i.e., methotrexate, cytosine arabinoside, and thio-triethylenephosphoramide) administered by a variety of schedules either by intralumbar or intraventricular drug delivery. Although treatment of neoplastic meningitis is palliative with an expected median patient survival of 2 to 6 months, it often affords stabilization and protection from further neurological deterioration.     

CNS T-cell lymphoma: an under-recognized entity?

The incidence of CNS lymphoma has increased significantly in the past 30 years, primarily in the elderly and immunocompromised. While T-cell lymphomas comprise 15–20% of systemic lymphomas, they comprise less than 4% of primary CNS lymphomas, suggesting that they may be under-recognized compared to their systemic counterparts. To investigate this, we studied brain biopsies from three patients who were diagnosed with T-cell lymphoma confined to the brain. They had enhancing lesions by MRI, arising in the cerebellum and brainstem in one and temporal lobe in two. We compared these to biopsies from three patients who had reactive lymphoid infiltrates and who had clinical signs/symptoms and radiographic findings that were indistinguishable from the lymphoma group. Biopsies from both the lymphoma group and reactive group showed considerable cytomorphologic heterogeneity. Although one lymphoma case contained large atypical cells, the other two contained small, mature lymphocytes within a heterogeneous infiltrate of neoplastic and reactive inflammatory cells. Surface marker aberrancies were present in two lymphoma cases, but this alone could not reliably diagnose T-cell lymphoma. The proliferation index was not useful for differentiating lymphoma from reactive infiltrates. In five of the six cases the diagnosis was most influenced by clonality studies for T-cell receptor-gamma gene rearrangements. We conclude that because of the high degree of overlap in cytomorphologic and immunophenotypic features between T-cell lymphoma and reactive infiltrates, T-cell lymphoma may not be recognized unless studies for T-cell receptor gene rearrangements are performed for CNS lesions composed of a polymorphous but predominantly T-cell infiltrate. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microglial cerebrospinal fluid antibodies

Hallmark lesions of Alzheimer disease (AD) are filled with reactive immunocompetent microglia, suggesting that immunological aderrations may participate in the pathophysiology of this disorder. If immune-mediated processes are closely linked to neuronal break-down, it would be of importance to have a reliable means to detect these processes. Cerebrospinal fluid (CSF) antibodies are discussed as such potential sources. The seredipitous use of the developing rat central nervous system (CNS) unexpectedly demonstrated that some AD CSF recognize amoeboid microglial cells. Similarly, AD CSF specifically stains activated microglia and neural macrophages in experimentally induced lesions. A cell-culture technique is described that allows rapid screening of CSF antibodies. Examination of CSF from a diversified dementia population revealed that AD CSF, in contrast to other dementia CSF, displayed remarkable selectivity toward microglial cells. Cortical biopsies from patients suspected to have AD were incubated with the patient's own CSF and that of confirmed AD patients. Both CSF samples recognized microglial cells in the cortical biopsy. AD CSF microglial antibodies appear to be significant in view of the increasing association between microglia and neuro degenerative processes in AD. These findings add further support to the concept that inflammation and similar immune mechanisms may contribute to to AD pathogenesis.     

Neurologic evaluation of non-Hodgkin's lymphoma in adult patients: a prospective study

This prospective study included 67 adult patients with low, intermediate or high malignancy degrees of non-Hodgkin's lymphomas according to the Working Formulation. Patients with or without anti-HIV antibodies in the serum were considered. All patients were submitted to neurologic evaluation, and 63 of them to examination of the cerebrospinal fluid (CSF). Patients presenting neurologic signs and symptoms were 42 (62.7%). Neurologic findings and CSF changes were correlated. The association of localized thoraco-lumbar pain and CSF changes (presence of neoplastic cells, increased protein concentration and/or increased gamma globulin content) was statistically significant, as the association of abnormal muscle strength in the lower limbs and CSF changes in patients without HIV antibodies in the serum. Cranial nerve dysfunction (III, IV and VI cranial nerves) correlated with the finding of neoplastic cells in the cerebrospinal fluid.     

Internalization of plasma proteins by cerebellar Purkinje cells

Animal studies suggest that Purkinje cells internalize proteins from the blood and CSF. This process may relate to the pathogenesis of paraneoplastic cerebellar degeneration in patients with anti-Purkinje cell antibodies. To determine if human Purkinje cells may also internalize plasma proteins, cerebellar tissue was taken from routine autopsies of eight patients without neurologic or neoplastic disease. Several plasma proteins including IgG, IgA, IgM, transferrin, albumin and alpha-2-macroglobulin were detected by immunohisto-chemistry within the cytoplasm of Purkinje cells. Internalized proteins frequently filled the entire soma and major dendrites, sparing the nucleus. Vascular structures were also immunolabeled, while glia internalized plasma proteins differentially, with oligodendrocytes selectively internalizing transferrin. Purkinje cells were the most numerous and heavily labeled neuronal cell type in spite of their small numerical representation in the cerebellar neuronal population. Our results are compatible with previous animal studies, and suggest that internalization of specific antibodies could contribute to the pathogenesis of Purkinje cell loss in paraneoplastic cerebellar degeneration.     

Lower initial red blood cell count in cerebrospinal fluid predicts good functional outcome in patients with spontaneous subarachnoid haemorrhage

Background and purpose

Red blood cell (RBC) degradation after subarachnoid haemorrhage (SAH) negatively affects functional outcome. Although the detection of RBCs in the cerebrospinal fluid (CSF) is a widely available part of neurological routine diagnostics, the prognostic value as a biomarker remains unclear. This study was undertaken to investigate whether CSF RBC count correlates with established radiological markers of SAH volume and whether the CSF RBC count can predict functional outcome in SAH patients.

Methods

A total of 121 consecutive spontaneous SAH patients were retrospectively analyzed. CSF was collected from external ventricular drain as part of routine diagnostic procedures. We used multivariable binary logistic regression to investigate associations between CSF RBC counts and functional outcome 3 months after SAH or hospital survival. Good functional outcome was defined as modified Rankin Scale ≤ 2.

Results

Patients' age was 60 ± 14 years, and the median admission Hunt & Hess grade (H&H) was 4. CSF samples were collected 2 days after intensive care unit admission. High CSF RBC counts positively correlated with radiological measurements for SAH volume, for example, modified Fisher score (p = 0.002) and Hijdra ventricle score (p = 0.016). Multivariable regression analysis adjusted for age, H&H grade, modified Fisher and Hijdra scores showed that low CSF RBC counts predicted hospital survival (per 100,000 CSF RBCs: adjusted odds ratio [adjOR] = 0.74, 95% confidence interval [CI] = 0.61–0.89, p = 0.001) and good functional outcome after 3 months (per 100,000 CSF RBC: adjOR = 0.76, 95% CI = 0.60–0.96, p = 0.020).

Conclusions

CSF RBC counts correlate with radiographic scores quantifying SAH volume and may serve as an early independent biomarker for hospital survival and good functional 3-month outcome in patients requiring ventriculostomy after SAH.     

IgG intrathecal synthesis and specific antibody index in patients with neurocysticercosis

We analyzed cerebrospinal fluid (CSF) and blood serum from 55 patients with neurocysticercosis (NC) at different clinical stages. According to inflammatory activity in the CSF, three stages were identified: (1) reactive, when there was at least an increase in the number of cells; (2) weakly reactive, when significant alterations were found in the CSF, including an increase in gamma globulins, albeit without hypercytosis; (3) non-reactive, when there was neither hypercytosis nor increase in gamma globulins. Nineteen patients had the reactive form; 18 had the weakly reactive form; 18 displayed the non-reactive form. Local immunoproduction was intense in the reactive group, moderate in the weakly reactive group, and absent in the non-reactive group. The specific antibody index was raised in approximately 2/3 of patients with the reactive form, 2/3 in those with the weakly reactive form, and 1/3 in those with the non-reactive form. In conclusion: (1) the classical CSF syndrome in NC can present both in complete and partial modes; (2) local immunoproduction can occur in weakly reactive forms; (3) a raised specific antibody index can occur in the absence of an inflammatory reaction in the CSF.     

Myelin basic protein in the cerebrospinal fluid of patients infected with HIV

Summary The major pathological abnormalities of HIV encephalopathy are infiltrates of macrophages, multinucleated giant cells, microglial nodules and demyelination. Elevated myelin basic protein (MBP) levels in the cerebrospinal fluid (CSF) provide a marker for central nervous system demyelination. The purpose of this study was to investigate the possible role of CSF MBP as a useful and early marker for HIV encephalopathy. The CSF of 40 consecutive patients with HIV infection of various clinical stages was investigated, including 13 patients with clinical signs of HIV encephalopathy. CSF MBP was elevated in 2 patients (5.0 and 5.3 ng/ml), both of whom had moderate to severe HIV encephalopathy. The course of the disease was rapid in both patients. In the remaining 38 patients, CSF MBP levels were marginally elevated (n=12) or normal (n=26). Our results suggest that CSF MBP is not a sensitive marker for the diagnosis and evaluation of HIV encephalopathy, but may be an indicator of prognosis for the course of the disease. There were only few findings of elevated CSF MBP levels in patients with HIV encephalopathy in the current study, and this may be because the disorder progressed slowly in most patients. It is possible that CSF MBP levels in HIV encephalopathy may only be elevated with acute clinical deterioration but are normal in slowly progressive forms of demyelination, as seen in multiple sclerosis.