Cytomorphologic and Flow Cytometric Analysis of Cerebrospinal Fluid with T-Cell Lymphoma Involvement: A Retrospective Study of Rare Cases.

OBJECTIVE: To compare the efficacy of conventional cytomorphologic analysis and flow cytometry (FC) in the diagnosis of T-cell lymphoma in the cerebrospinal fluid (CSF). STUDY DESIGN: We performed a retrospective review of CSF samples from 2002 to 2012 that showed involvement of a T-cell lymphoma, either by cytomorphologic analysis and/or FC. Patients' demographics, clinical history and follow-up were assessed. RESULTS: Thirty-nine CSF samples were identified from 9 patients. A definitive diagnosis of T-cell lymphoma involvement was made by cytomorphologic analysis and FC in 6 (15.4%) and 39 (100.0%) specimens, respectively. In specimens with definitive cytopathologic diagnoses, the cytomorphologic features included increased cellularity, a monotonous lymphoid population and large, atypical lymphoid cells. Considering cytomorphologic features only, 9 specimens demonstrated atypical lymphocytes not fulfilling the criteria for malignancy, and 24 specimens were negative for malignancy. CONCLUSIONS: CSF with T-cell lymphoma involvement may yield paucicellular or acellular specimens depending on the volume of the CSF, the time interval between specimen collection and specimen processing and the application of preservative to CSF. The rate of detection of T-cell lymphoma in the CSF by FC is unequivocally higher than by cytomorphologic analysis. Careful attention to clinical history is crucial, as FC testing may be tailored to evaluate for T-cell lymphoproliferative disorders in limited samples.

Utility of quantitative EBV DNA measurements in cerebrospinal fluid for diagnosis and monitoring of treatment of central nervous system EBV-associated post-transplant lymphoproliferative disorder after allogenic hematopoietic stem cell transplantation.

BACKGROUND: Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorder (PTLD) after hematopoietic stem cell transplantation (HSCT) is a rare but life- threatening condition. Despite routine EBV DNA-emia monitoring in blood, central nervous system involvement of the disease is still associated with diagnostic difficulties and poor outcome. CASE REPORT: We describe a 17-year-old female patient with severe aplastic anemia who developed central nervous system EBV-associated PTLD after matched unrelated allo-HSCT. EBV DNA concentration in cerebrospinal fluid was initially tested due to central nervous system-related symptoms. The sample was found to be highly EBV DNA-positive. Using quantitative real-time polymerase chain reaction (PCR) measurements, therapy efficiency was monitored until complete remission of the EBV-associated PTLD. CONCLUSIONS: The case report illustrates the high utility of quantitative real-time PCR EBV measurements in cerebrospinal fluid for rapid diagnosis and therapy monitoring of central nervous system EBV-associated PTLDs after allo-HSCT.

Use of flow cytometry in the diagnosis of lymphoproliferative disorders in fluid specimens.

The diagnostic evaluation of fluid specimens, including serous effusions and cerebrospinal fluids (CSFs), can be challenging for a number of reasons. The evaluation of lymphoid proliferations in these specimens can be particularly problematic, given the frequent presence of coexisting inflammatory conditions and the manner in which these specimens are processed. As a result, immunophenotypic analysis of hematopoietic cell populations by flow cytometry has emerged as a useful ancillary study in the diagnosis of these specimens, both in patients with and without a previous history of a lymphoproliferative disorder. In this study, we review our experience with flow cytometry in fluid specimens over a four-year period. Flow cytometry was performed in 184 of 6,925 total cases (2.7% of all fluids). Flow cytometry was performed in 4.8% of pleural fluids (positive findings in 38%, negative in 40%, and atypical in 18%), 1.1% of peritoneal fluids (positive in 40%, negative in 50%, and atypical in 10%), 1.9% of pericardial fluids (positive in 67%, negative in 33%), and 1.9% of CSFs (positive in 23%, negative in 55%, atypical in 3%). The specimen submitted was inadequate for analysis in 9.2% of cases, most commonly with CSF specimens, but was not related to the volume of fluid submitted. Atypical flow cytometry findings and atypical morphologic findings in the context of negative flow cytometry results led to the definitive diagnosis of a lymphoproliferative disorder in a significant number of cases when repeat procedures and ancillary studies were performed.

Epstein-Barr virus-positive post-transplant lymphoproliferative disorder of the central nervous system, after renal transplantation with a discrepancy in viral load between peripheral blood and cerebrospinal fluid.

A 43-year-old female developed an Epstein-Barr virus (EBV)-positive post-transplant lymphoproliferative disorder (PTLD) in the central nervous system (CNS), 14 years after renal transplantation. One year prior to presentation, the patients' treatment regimen was altered from cyclosporine, azathioprine, and prednisone to mycophenolate mofetil and prednisone. Magnetic resonance imaging of the brain revealed lesions suspect for malignant lymphoma. The EBV real-time polymerase chain reaction (PCR) on peripheral blood was negative, but highly positive on cerebrospinal fluid. EBV-positive PTLD was confirmed using histological analysis of cerebral biopsies. Despite tapering of immune suppressive medication and treatment with rituximab and chemotherapy, the patient deceased 50 days after presentation. This case illustrates that vigilance is required when presented with a negative EBV PCR result on peripheral blood when PTLD of the CNS is suspected. This late presentation suggests a relation to the switch in immunosuppressive regimen 1 year earlier.

Flow cytometric characterization of cerebrospinal fluid cells.

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.

CSF IgH gene rearrangement analysis in isolated post-transplant lymphoproliferative disorder of the central nervous system.

Isolated post-transplant lymphoproliferative disorder (PTLD) of the central nervous system is rare and its diagnosis can be challenging. A biopsy is usually required in order to distinguish the disease from opportunistic infections. We present a case in whom immunoglobulin heavy chain gene rearrangement analysis of cerebrospinal fluid (CSF) was instrumental in establishing the diagnosis.

Levels of serum and cerebrospinal fluid soluble CD27 in the diagnosis of leptomeningeal involvement of hematolymphoid malignancies.

Reportedly, soluble CD27 (sCD27) is a sensitive and specific marker for leptomeningeal involvement (LI) of CD27-expressing lymphoproliferations, such as B-cell non-Hodgkin's lymphoma and chronic B-lymphocytic leukemia. On morphologic analysis of cerebrospinal fluid (CSF), one third of patients suspected of LI have false negatives, so a diagnostic marker for LI in B-cell non-Hodgkin's lymphoma or B-lymphocytic leukemia would be extremely valuable. sCD27 was detected in the serum and CSF samples from 35 selected patients in whom 18 cases of acute lymphoblastic leukemia (ALL) (3 with LI), 7 of non-Hodgkin's lymphoma, and 5 of acute myelogenous leukemia (3 with LI) were submitted for (immuno)morphologic detection of malignant cells and intrathecal therapy, along with samples from 5 control patients (2 submitted for epidural hemorrhage, 3 for lumbar disc protrusion). Concentrations of CSF-sCD27 were determined by enzyme-linked immunosorbent assay (PeliKine Compact Human Soluble CD27 ELISA Kit, Cat. No. M1960; Research Diagnostics Inc., Concord, Mass). The cutoff value was 350 U/mL. Serum and CSF-sCD27 concentrations above the cutoff value were not detected. Although it is unlikely that LI would be present in patients with chronic lymphoproliferation who have normal sCD27 concentrations in CSF samples, the determination of CSF-sCD27 is not sufficiently specific to allow it to serve as a reliable tumor marker.

Immunoglobulin gene rearrangement analysis in cerebrospinal fluid of patients with lymphoproliferative processes.

OBJECTIVE: To determine the sensitivity and specificity of clonal immunoglobulin heavy chain gene rearrangement (IGHR) analysis in the distinction of benign and malignant lymphoproliferative diseases. METHODS: A retrospective analysis was conducted of patients in whom a malignant lymphoproliferative process was suspected. Cells of CSF samples were collected by centrifugation, resuspended in 100 microl of the supernatant and boiled. A 10 microl aliquot of this lysate served as template for semi-nested polymerase chain reaction using variable and joining region consensus primers. PCR products were analyzed by polyacrylamide gel electrophoresis. Cytopathological diagnosis and flow cytometry results were recorded. Sensitivity and specificity of IGHR analysis, cytopathology and flow cytometry were calculated. RESULTS: Eleven patients (12 specimens) had involvement of leptomeninges at the time of lumbar puncture. Another 25 cases (27 specimens) had normal CSF findings or were diagnosed with benign lymphoproliferative conditions. Sensitivity of CSF cytopathology, flow cytometry and IGHR analysis were 0.27 [95% confidence interval 0.06, 0.61], 0.1 [0.003, 0.45] and 0.58 [0.28, 0.85]. Specificity was 1 [0.86, 1], 0.95 [0.77, 1.0] and 0.85 [0.66, 0.96]. INTERPRETATION: IGHR analysis appears to be a useful addition to morphological and flow cytometry analysis of cerebrospinal fluid in the evaluation of CNS lymphoproliferative processes.

Diagnosing lymphoproliferative disorders involving the cerebrospinal fluid: increased sensitivity using flow cytometric analysis.

Flow cytometric immunophenotypic analysis (FCA) can be performed to evaluate lymphoid cells in cerebrospinal fluid (CSF). We compared this method with conventional cytologic diagnosis to determine its utility. A retrospective comparison of 35 consecutive CSF flow cytometry results with the corresponding cytologic diagnoses was undertaken. Twenty-five of 35 CSFs (71%) were successfully analyzed by flow cytometry. The 10 samples which could not be analyzed were either too old (greater than 3 days) or had an insufficient number of cells. A total of 9 lymphomas was detected: 4 by both flow cytometry and cytology; 2 by cytology alone; and 3 by flow cytometry alone. This represents a 50% increase in the detection of lymphoproliferative disorders in CSF by a combination of flow cytometry and cytology vs. cytology alone. Furthermore, in 3 cases with follow-up where the cytologic diagnosis was "atypical cells of undetermined significance" and the flow cytometric findings were negative for malignancy, the clinical course confirmed a benign pleocytosis in all three. We conclude that flow cytometric analysis markedly improves sensitivity when used in combination with cytology in the evaluation of lymphoid cells in CSF.

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.