Cerebrospinal Fluid Flow Cytometry Research Articles

Severe CSF immune cell alterations in cryptococcal meningitis gradually resolve during antifungal therapy

Cryptococcal meningitis (CM) is a severe fungal disease in immunocompromised patients affecting the central nervous system (CNS). Host response and immunological alterations in the cerebrospinal fluid (CSF) after invasion of Cryptococcus neoformans to the central nervous system have been investigated before but rigorous and comprehensive studies examining cellular changes in the CSF of patients with cryptococccal meningitis are still rare. We retrospectively collected CSF analysis and flow cytometry data of CSF and blood in patients with CM (n = 7) and compared them to HIV positive patients without meningitis (n = 13) and HIV negative healthy controls (n = 7). Within the group of patients with CM we compared those with HIV infection (n = 3) or other immunocompromised conditions (n = 4). Flow cytometry analysis revealed an elevation of natural killer cells and natural killer T cells in the CSF and blood of HIV negative patients with CM, pointing to innate immune activation in early stages after fungal invasion. HIV positive patients with CM exhibited stronger blood-CSF-barrier disruption. Follow-up CSF analysis over up to 150 days showed heterogeneous cellular courses in CM patients with slow normalization of CSF after induction of antifungal therapy.

Diagnostic significance of cerebrospinal fluid flow cytometry in Chinese children with B lineage acute lymphoblastic leukemia

BackgroundCentral nervous system leukemia (CNSL) is one of the major causes of the poor prognosis of childhood leukemia. We aimed to compare the sensitivity of cytomorphology (CM) and flow cytometry (FCM) in diagnosing CNSL, emphasizing the importance of FCM in the diagnosis process.MethodsOne-hundred-sixty-five children with newly diagnosed B-cell Acute Lymphoblastic Leukemia (B-cell ALL) were included in this study. Cerebrospinal fluid (CSF) samples were taken for routine CSF analysis, CM analysis, and FCM examination. Computed tomography scans and/or magnetic resonance imaging were performed at diagnosis. Patients with CNS2, CNS3, and traumatic lumbar puncture (TLP) at diagnosis received two additional courses of triple intrathecal injections during induction treatment. We compared the sensitivity of FCM and CM in the diagnosis of children with CNSL.ResultsOne hundred and twenty-eight (77.58%) CSF samples were negative by either CM or FCM (CM−/FCM−), four (2.42%) were positive by both CM and FCM (CM+/FCM+), and thirty-three (20%) displayed a single positive finding by FCM (CM−/FCM+) (p = 0.044). By adding two intrathecal injections in the induction treatment, ten children with TLP+ had no CNS relapse, like those with TLP−. However, compared to CNS1 and TLP, the event-free survival (EFS) did not significantly improve in patients with CNS2 and CNS3. Moreover, CNSL status was associated with worse 3-year EFS (p < 0.05).ConclusionsWe have validated that FCM is more accurate in stratifying the status of the CNS compared to CM analysis. However, to improve the EFS rate of childhood leukemia, it is necessary to combine CM examination, FCM, and cranial imaging for the early diagnosis of CNSL.

Incidence, risk factors, and outcome of asymptomatic central nervous system involvement in adult patients with acute myeloid leukemia.

Examination of central nervous system (CNS) involvement is not routine diagnostic practice in adult patients with acute myeloid leukemia (AML). Therefore, many asymptomatic patients with CNS involvement might go undetected. The effect of CNS involvement on the AML disease course is not well defined, with conflicting results regarding clinical outcome. This study aimed to determine the incidence of asymptomatic CNS involvement in AML estimated by multiparametric flow cytometry of cerebrospinal fluid (MFC-CSF) at diagnosis, the related potential risk factors, and prognosis. In total, 645 patients with de novo AML were screened; 183 (28.4%) of them fulfilled institutional practice for MFC-CSF analysis based on presence of CNS symptoms and/or clinical features. CNS symptoms and signs were observed in 8/183 (4.4%) patients, but most patients (175/183, 95.6%) were asymptomatic. In the asymptomatic group, 73/175 (41.7%) patients had positive or suspicious cerebrospinal fluid (CSF) findings categorized as CNS positive (CNSpos) and 102/175 (58.3%) had normal CNS findings categorized as CNS negative (CNSneg). The presence of leukemic blasts was confirmed in 81/183 (44.3%) patients; the total incidence of CNS involvement in the whole AML group was 12.6% (81/645). Compared with asymptomatic patients with CNSneg, those with CNSpos had a significantly higher frequency of lymphadenopathy, white blood cell count ≥30×109/L, presence of the monocytic phenotype, and a high percentage of bone marrow (BM) blasts. The multivariate logistic regression model identified monocytic phenotype (p=0.047) and high percentage of BM blasts (p=0.042) as predictors for CNSpos. CNSpos did not affect overall survival in patients with AML. There was a higher incidence of CNS involvement in asymptomatic adult patients with de novo AML, emphasizing possible undervalued rates of CNS disease at diagnosis. Prospective studies should determine whether diagnostic lumbar puncture for MFC-CSF analysis and CNS prophylaxis could contribute to better selection and prognosis in this patient population.

Intravascular Lymphoma-Associated Stroke: A Systematic Review of Case Studies.

Intravascular lymphoma (IVL) is an aggressive systemic large B-cell lymphoma that is a rare cause of stroke. The clinical characteristics of stroke associated with IVL remain underexplored, contributing to diagnostic complexities and a high mortality rate. This study endeavors to elucidate the salient clinical and investigative features of stroke linked to this condition. A systematic review was performed using the PubMed database from the incident to August 2023 including search categories for IVL and stroke. All studies, excluding review articles, were included in this study. There were 58 cases with a confirmed diagnosis of IVL associated with stroke, with a mean age of 62.9 ± 9.6 years (female 50%). Classical lateralizing stroke symptoms were noted in only 69% of cases. Other clinical syndromes included altered sensorium (31%), rapidly progressive cognitive impairment (23%), seizures (22%), and gait disturbances (19%). Common hematological abnormalities included elevated lactate dehydrogenase (LDH, 97%), erythrocyte sedimentation rate (ESR, 79%), C-reactive protein (CRP, 61%), interleukin-2, microglobulins, and cerebrospinal fluid (CSF) protein. CSF flow cytometry was not diagnostic, and cytology was mostly negative. The dynamic pattern for DWI/T2 lesions was predominant and primarily located in the subcortical regions. Diffuse background slowing (64%) was a major finding in the electroencephalogram. Seventy-one percent of cases died (n=45) mostly due to delayed diagnosis. Only 31% were treated with first-line R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine, prednisone) chemotherapy, among whom 25% died. This study suggests that IVL-associated strokes carry a high mortality rate, largely due to challenges in timely diagnosis and therapy. Unlike classical stroke syndrome, key indicators to aid in early diagnosis include a clinical syndrome of multiple non-lateralizing neurological symptoms, dynamic MRI DWI/T2-lesions primarily located in subcortical regions, elevated serum LDH, ESR, CRP, interleukins, microglobulin, CSF protein, and CSF polymerase chain reaction analysis, apart from tissue examination. Larger studies should be performed to establish diagnostic and predictive scores.

Cerebrospinal fluid cytology-assisted diagnosis of T-lymphoblastic lymphoma: A case report

Background: Lymphoblastic lymphoma is a rare form of highly aggressive non-Hodgkin lymphoma. The most common clinical manifestations are superficial lymphadenopathy and mediastinal mass. In a few cases, invasion of the central nervous system is the first manifestation. It is also difficult to diagnose patients using the central nervous system as the first manifestation. Here, we report the case of a 26-year-old man with central nervous system disease as the primary manifestation; we used cerebrospinal fluid cytology (CSF-C) for early diagnosis and shared the importance of CSF-C for early diagnosis of T-cell lymphoblastic lymphoma. Case presentation: The patient was admitted to the hospital because of “right eyelid closure weakness with headache for 1 month and exacerbation with sluggish response for 1 week.” Physical examination revealed a bilateral Kernig sign (+) and Lasgue sign (+). The Mini-Mental State Examination and Montreal Cognitive Assessment scores were 20 (out of 30). When there was no abnormality in the imaging examination, the patient was misdiagnosed with meningoencephalitis and received anti-inflammatory treatment because the initial symptom was a clinical manifestation of the central nervous system, and the imaging and blood tests showed no definite abnormality. Cerebrospinal fluid has been studied and second-generation sequencing detection, such as after CSF-C tip to abnormal lymphocytes, to open the breakthrough of the diagnosis of lymphoma. Conclusions: In the cases with central nervous system injury as the first manifestation, CSF-C was combined with immunohistochemistry and cerebrospinal fluid flow cytometry to provide a clear and effective method and evidence for the early diagnosis of T-cell lymphoblastic lymphoma.

Paired Circulating Tumor DNA in Cerebrospinal Fluid and Peripheral Blood: Reliable Technology for Diagnosing and Monitoring Primary and Secondary Central Nervous Lymphoma

Objective: This research delineates the temporal alterations of cell-free circulating tumor DNA (ctDNA) in the plasma and cerebrospinal fluid (CSF) of patients harboring primary and secondary central nervous system (CNS) lymphoma. The aim is to establish that CSF serves as a superior reservoir of ctDNA compared to plasma, thereby aiding in diagnostic processes and monitoring therapeutic efficacy. Concurrently, this study underscores the enhanced response and survival rates in patients administered Bruton's tyrosine kinase (BTK) inhibitors over an extended period exceeding three months. Methods: Data including cranial-enhanced magnetic resonance imaging (MRI) scans, leukocyte and protein concentrations in the CSF, exfoliated CSF cytology, flow cytometry, interleukin-10 to interleukin-6 ratios, ctDNA in plasma and CSF, were gathered from patients presenting primary and secondary CNS lymphoma（7 PCNSL,9 SCNSL）. The temporal ctDNA modifications in plasma and CSF during the patient's disease progression-initial onset, remission, and relapse-were scrutinized. Additionally, the implications of BTK inhibitor usage on patient response rates and survival rates were assessed. Results: Within the cohort of primary CNS lymphoma patients, ctDNA was detected in the CSF and plasma of 2 and 5 patients, respectively. Conversely, in patients with secondary CNS lymphoma, CSF and plasma ctDNA were discovered in 8 and 4 cases, respectively. The detection rate was contingent upon the central lesion's magnitude and position and the patient's surgical history related to the brain. Four patients, including two from each group, underwent cranial-enhanced MRI to appraise tumor burden at various disease stages, simultaneously with the monitoring of ctDNA in plasma and CSF. It was revealed that ctDNA levels in the CSF were in synchrony with the tumor burden, whereas plasma ctDNA levels remained relatively static. This indicates that CSF ctDNA may serve as a viable tool to assess therapeutic responses in CNS lymphoma patients. In two patients where surgical biopsy was unfeasible and both CSF exfoliation cytology and flow cytometry returned negative results, CSF ctDNA was positive, thereby underscoring its auxiliary role in diagnosing CNS lymphoma. Furthermore, the study indicated improved response and survival rates in patients subjected to BTK inhibitors for over three months. Conclusion: CSF ctDNA demonstrates potential in detecting CNS lymphoma and in the dynamic monitoring of therapeutic responses. Compared to cranial-enhanced MRI, exfoliated CSF cytology, and flow cytometry, CSF ctDNA testing offers superior sensitivity. For patients for whom brain surgical biopsy is not an option, CSF ctDNA testing emerges as a valuable supplementary diagnostic tool. CSF ctDNA allows for dynamic monitoring of therapeutic responses in CNS lymphoma patients, enabling the detection of residual lesions, and thereby guiding the formulation and duration of the treatment regimen. Patients administered oral BTK inhibitors for more than three months demonstrate higher treatment response and survival rates.

Clinical Features and Therapeutic Evaluation of Central Nervous System Involvement in Pediatric Anaplastic Large Cell Lymphoma

Objective：To analyze the clinical features of central nervous system involvement (CNS3) in pediatric anaplastic large cell lymphoma (ALCL) and evaluate the efficacy of CNCL-ALCL-2017 protocol for the treatment of CNS3. Methods：The clinical data of 215 pediatric ALCL patients ≤18 years old enrolled in the Chinese Children's Cancer Group (CNCL) between April 2017 and March 2023 were collected sequentially. All enrolled patients staging and CNS layering were according to the IPNHLSS staging system and treated with CNCL-ALCL-2017 protocol (modified BFM-ALCL99 protocol). The clinical features at diagnosis and treatment outcomes of CNS3 patients were analyzed. All patients were followed up until June 30, 2023. Data were collected using a unified information platform. Associations between patient characteristics were analyzed with Chi-squared or Fisher's exact tests. Eventfree survival (EFS) and overall survival (OS) curves were estimated according to the KaplanMeier method and compared by Logrank test. Furthermore, statistical analysis was performed using SPSS 22.0 software. P&amp;lt;0.05 was considered statistically significant. Results: Among the 215 ALCL pediatric patients, 17 (7.9%) had CNS3, including 13 males and 4 females with median age of 8.0 (range 1.0-14.0) years. In this patients, brain parenchyma and spinal cord involvement were found in 58.8% (10/17) patients on imaging, with space-occupying lesions in 7 cases and abnormal signals on MRI in 4 cases. Concurrent space-occupying lesions and abnormal signals on MRI was detected in 1 case. Cerebrospinal fluid (CSF) positivity was detected in 35.3% (6/17) patients, including 2 cases with positive ALK gene and 5 cases with positive flow cytometry, with 1 case positive for both. Cranial nerve deficits such as facial nerve palsy, visual or hearing impairment were observed in 23.5% (4/17) patients. Concurrent CSF, imaging and cranial nerve abnormalities were present in 5.9% (1/17), while two of this were present in 29.4% (5/17). Isolated CSF positivity without other abnormalities was detected in 23.5% (4/17). Pathological subtypes included common type (76.5%, 13/17), histiocyte-rich variant (5.9%, 1/17), small cell variant (5.9%, 1/17), others (11.8%, 2/17). Immunohistochemistry showed ALK + in 94.1% (16/17) and CD3 + in 76.5% (13/17). Concurrent ALK gene positivity in both peripheral blood and bone marrow was detected in 64.7% (11/17), with 58.8% (10/17) positive in both samples. The median follow-up of all patients was 35.4 months (range 0.5-74.9). All CNS3 patients had stage IV disease and were treated with regimen D (with 5 g/m 2 methotrexate). The 3-year OS was 94.3%, 98.2% and 93.3% for CNS1 (144, 67.0%), CNS2 (54, 25.1%) and CNS3 (17, 7.9%) patients, respectively; the 3-year EFS was 85.6%, 90.3% and 86.7%, respectively. No significant differences were found among the three groups. The 3-year OS and EFS for the whole cohort were 95.1% and 84.7%, respectively, also with no significant differences from the CNS3 group. Of the CNS3 patients, 1 was lost to follow-up after the first course, 1 died during treatment, and the interim CR rate was 75.0% (12/16). CSF conversion was achieved in 3/6 (50%) CSF-positive patients. 6.25% (1/16) patient had CSF ALK gene re-positivity during maintenance and persistent positivity despite additional ALK inhibitor alectinib and intrathecal therapy. In the CNS1 group, 2 cases were lost to follow-up and 4 cases died. The relapse rate was 11.4% (16/140), and the rate of central nervous system relapse was 2.1% (3/140). Additionally, the rate of CNS1 relapse was slightly higher but not statistically significantly different for CNS2. Conclusions: CNS involvement is relatively rare in pediatric ALCL. Detection rate of CNS3 can be improved by CSF flow cytometry and gene screening. The prognosis of CNS3 was significantly improved with CNCL-ALCL-2017 protocol, which may be related to the increased dose of methotrexate in the protocol. The short follow-up time and the small number of cases in this group may affect the results. Furthermore, CNS relapse rate of CNS2 was not markedly higher compared to CNS1. Keywords: Anaplastic large cell lymphoma; Pediatric; Central nervous system; Clinical features; Prognosis

Diagnostic and Prognostic Value of Circulating Cell-Free DNA in Cerebrospinal Fluid and Plasma in Newly Diagnosed Diffuse Large B Cell Lymphoma

Background:Diffuse large B cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous non-Hodgkin lymphoma. The clinical risk assessment for central nervous system (CNS) recurrence is still challenging in DLBCL due to the limited conventional biomarkers and techniques available. Circulating cell-free DNA (cfDNA), which reflects comprehensive tumor genetic profiles, has emerged as a potential novel strategy in the era of precision medicine. However, the clinical value of cfDNA detected in different samples in newly diagnosed DLBCL in China remains elusive. This study aims to explore the diagnostic and prognostic value of cfDNA detection in cerebrospinal fluid (CSF) and plasma in DLBCL. Methods:Next-generation sequencing (NGS) was performed on cfDNA derived from CSF, plasma, encompassing single nucleotide variants (SNV), insertion/deletions, copy number variants (CNV), IG related fusions, and clonotypic immunoglobulin gene rearrangements，alongside systemic tumor tissues in newly diagnosed DLBCL patients. Results:We recruited 30 patients, among whom CSF cfDNA was detected in 19 patients, with 6 (31.6%) being positive for SNV only. The positive rate of CSF cfDNA increased to 36.8% when considering both SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement. Notably, cfDNA was detected in 100% of CSF samples from 3 patients with known CNS involvement, and in 4 patients without known CNS involvement based on absent evidence of abnormal CSF cytology, flow cytometry, or radiography. The positive rates of SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement in CSF were 80%, 40%, and 40%, respectively, relatively confirmed by available paired tumor tissues or bone marrow samples. CfDNA was detected in 100% of plasma samples. When accounting for both SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement, the positive rate increased to 76.7% from 73.3%, which was much higher than paired peripheral blood genomic DNA (20%). The cfDNA detected in plasma exhibited high concordance rates with matched systemic tumor tissues or bone marrow samples, with concordance rates of 100%, 76.9%, and 76.5% for SNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement, respectively. Interestingly, cfDNA was detected in plasma samples of 2 patients while tumor tissues' genomic DNA was negative. In total, 24 gene alterations were identified in CSF, 172 in plasma, and 160 in tumor tissue or bone marrow. Commonly mutated genes in plasma and CSF cfDNA included PIM1 (36.7% vs. 14.2%), MYD88 (33.3% vs. 28.6%), TET2 (30% vs. 28.6%), and CD79B (26.7% vs. 28.6%). The most common types of immunoglobulin gene rearrangement were IGK in different samples, and the common IG related fusions were IGH:: BCL6, IGH:: MYC, and IGH:: BCL2. Conclusion:Our study suggests that plasma cfDNA exhibits a high concordance rate with tumor tissues and can aid in the precise diagnosis and measurable residual disease monitoring. CSF cfDNA is more sensitive than conventional methods for diagnosing CNS invasion and could serve as a more accurate biomarker for predicting the risk of CNS recurrence among patients with newly diagnosed DLBCL, helping to select patients who may require enhanced CNS prophylaxis. Moreover, synchronous detection of SNV, CNV, IG related fusion, and clonotypic immunoglobulin gene rearrangement could be helpful in improving the positive rate of cfDNA.

P06.19.A BEYOND T CELL TOXICITY - INTRATHECAL CXCL13 ELEVATION INDICATES B-CELL INVOLVEMENT IN NEUROLOGICAL IMMUNE-RELATED ADVERSE EVENTS FOLLOWING IMMUNE CHECKPOINT INHIBITION: 2 CASE OBSERVATIONS AND COMPREHENSIVE REVIEW

Abstract BACKGROUND Neurological immune-related adverse events (nirAE) can occur following cancer treatment with immune checkpoint inhibitors (ICI). Mainly attributed to autoreactive T-cells, nirAE usually respond to first line therapy with high-dose steroids (GC). Severe or fatal outcomes are rare &amp; most frequently reported in clinically overlapping syndromes with accumulating evidence of B cell autoimmunity. MATERIAL AND METHODS We present 2 patients who developed grade 3-4 nirAE with evidence suggestive for B cell involvement. RESULTS Case 1: A 75y female with metastatic melanoma developed double vision &amp; general weakness 2 weeks after the 1st cycle of ipilimumab &amp; nivolumab. Diagnostic workup showed pleocytosis (27 cells/µl) with elevated CXCL13 (316 pg/ml) in cerebrospinal fluid (CSF). Increases of creatine kinase, troponin T, elevated levels of anti-acetylcholine receptor antibodies &amp; positive anti-titin antibodies led to the diagnosis of myocarditis &amp; myositis/myasthenia gravis overlap syndrome. The patient received pulsed high-dose GC with tapering. CSF flow cytometry revealed intrathecal recruitment of CXCL13-responsive B cell helper T cells of a predominant inflammatory phenotype &amp; a highly increased pathological CD4/CD8 T cell ratio in peripheral blood. Clinically non-responding to GC, the patient received additional therapy with immunoglobulins (IVIG). 2 weeks later she was readmitted due to generalized myopathy with dropped head &amp; received a second cycle of high-dose GC since she refused plasmapheresis. Further escalation with rituximab could not be realized because the patient rapidly deteriorated &amp; deceased. Case 2: A 75y female with metastatic invasive enteric adenocarcinoma of the lung developed gait ataxia &amp; ataxia of the lower limbs 5 months after pembrolizumab therapy. CSF analysis showed a pleocytosis (248 cells/µl) with plasma cells, pronounced CXCL13 elevation (&amp;gt;488 pg/ml) &amp; positive screening for anti-neuronal cerebellar antibodies, suggesting ICI-mediated cerebellitis. The patient received high-dose GC &amp; IVIG for 5 days each, was discharged but readmitted 5 months later due to subacute onset of spastic paraparesis. Magnetic resonance imaging of the spine revealed thoracic myelopathy &amp; CSF showed mild pleocytosis (5 cells/µl), intrathecal immunoglobulin synthesis &amp; mild CXCL13 elevation (33 pg/ml). The patient completed a second cycle of steroids followed by rituximab targeting B-cell involvement &amp; currently 4 months later is clinically stable. CONCLUSION Intrathecal CXCL13 may serve as diagnostic tool of B cell involvement, here supported by intrathecal immunoglobulin synthesis, presence of plasma cells and/or severely altered immune cell compositions. Early treatment escalation with B cell depleting agents such as rituximab may help prevent fatal clinical courses.

Case 317: Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia.

A 44-year-old previously healthy man with a 9-month history of progressive cognitive decline, depression, urinary incontinence, and inability to perform tasks of daily living presented to the emergency department with worsening cognitive and neuropsychiatric symptoms. He had become more distressed, and his family noticed him departing the house without closing doors, leaving water faucets running, and sending his children to school on Sundays. History taken from the patient's wife revealed that his brother had passed away in his late 30s after a slowly progressing functional and cognitive decline over the course of 5 years. No further detailed family history could be obtained. The review of systems was negative; he had no prior medical, psychiatric, or surgical history; and he denied any history of recent travel, camping, hiking, or vaccination. The patient was not taking any dietary supplements, nor was he taking any over-the-counter or prescription medication. Examination revealed vital signs were within normal limits. Neurocognitive assessment revealed a conscious, coherent, and alert patient with impaired memory and concentration. He showed poor attention, depressed mood, and restricted affect. He was unable to spell the word world forward, nor was he able to understand a request to spell it backward. The rest of the physical and neurologic examination revealed no abnormalities. Extensive laboratory work-up was conducted and included the following: toxicology screening; screening for HIV-1, HIV-2, and syphilis treponemal antibodies; COVID-19 polymerase chain reaction; and measurement of B1 and B12 levels. The results of screening were negative. Cerebrospinal fluid (CSF) assays, including CSF oligoclonal bands and CSF flow cytometry, revealed values within normal limits. CT of the brain without intravenous contrast material was performed in the emergency department to rule out acute intracranial abnormality. Multiplanar multisequence MRI of the brain without and with intravenous contrast material was ordered for further assessment. CT images of chest, abdomen, and pelvis were unremarkable (images not shown).

Case 317

A 44-year-old previously healthy man with a 9-month history of progressive cognitive decline, depression, urinary incontinence, and inability to perform tasks of daily living presented to the emergency department with worsening cognitive and neuropsychiatric symptoms. He had become more distressed, and his family noticed him departing the house without closing doors, leaving water faucets running, and sending his children to school on Sundays. History taken from the patient's wife revealed that his brother had passed away in his late 30s after a slowly progressing functional and cognitive decline over the course of 5 years. No further detailed family history could be obtained. The review of systems was negative; he had no prior medical, psychiatric, or surgical history; and he denied any history of recent travel, camping, hiking, or vaccination. The patient was not taking any dietary supplements, nor was he taking any over-the-counter or prescription medication. Examination revealed vital signs were within normal limits. Neurocognitive assessment revealed a conscious, coherent, and alert patient with impaired memory and concentration. He showed poor attention, depressed mood, and restricted affect. He was unable to spell the word world forward, nor was he able to understand a request to spell it backward. The rest of the physical and neurologic examination revealed no abnormalities. Extensive laboratory work-up was conducted and included the following: toxicology screening; screening for HIV-1, HIV-2, and syphilis treponemal antibodies; COVID-19 polymerase chain reaction; and measurement of B1 and B12 levels. The results of screening were negative. Cerebrospinal fluid (CSF) assays, including CSF oligoclonal bands and CSF flow cytometry, revealed values within normal limits. CT of the brain without intravenous contrast material was performed in the emergency department to rule out acute intracranial abnormality (Fig 1). Multiplanar multisequence MRI of the brain without and with intravenous contrast material was ordered for further assessment (Figs 2-4). CT images of chest, abdomen, and pelvis were unremarkable (images not shown).

Multiple Cranial Neuropathies with Orbital Myositis as an Initial Presentation of Lyme Disease (P9-10.002)

<h3>Objective:</h3> To report a case of Lyme disease presenting with subacute multiple cranial neuropathies and orbital myositis. <h3>Background:</h3> Lyme disease can present with multiple cranial neuropathies. Orbital myositis is not well described as one of the rare complications of Lyme disease. Here, we describe a patient with multiple cranial neuropathies and orbital myositis as an initial manifestation of Lyme disease. <h3>Design/Methods:</h3> A 69-year-old male with a history of hyperthyroidism status post thyroidectomy, presented with a new onset headache, double vision, numbness of the face, and dysphagia. On examination, he had left lower motor neuron facial weakness, left eye proptosis, and findings suggestive of III and VI nerve palsies. Brain MRI with contrast revealed abnormal enhancement along the left V2 and V3 nerves and enhancement of the bilateral III cranial nerve. Abnormal enhancement was also seen at the left orbital apex in addition to enhancement along the left retro-orbital optic nerve. MRI of the orbit showed asymmetric enlargement and subtle enhancement of the left extra orbital muscles with mild orbital fat infiltration, compatible with orbital myositis. Lyme IgG/IgM antibody screen was positive which was confirmed by Lyme immunoblot assay which was also positive for Lyme IgG (5 bands) and IgM (3 bands) antibodies. B2 Glycoprotein IgM was elevated. Cerebrospinal fluid (CSF) analysis revealed lymphocytic pleocytosis and elevated protein. CSF cytology and flow cytometry were negative for malignant cells. TSH, thyroxin, and anti-thyroid peroxidase antibodies were unremarkable. Sarcoidosis, IgG4-related disease, autoimmune, and infectious workups were unrevealing. The patient received IV ceftriaxone with significant improvement and with near resolution of the symptoms and clinical findings after 1 month. <h3>Results:</h3> NA <h3>Conclusions:</h3> Orbital myositis is a rare complication of Lyme disease. <b>Disclosure:</b> Dr. Biso has nothing to disclose. Dr. Philip has nothing to disclose. Dr. Chevula has nothing to disclose. Dr. El-Dokla has nothing to disclose.

African-Americans With Multiple Sclerosis Have a Higher Proportion of B-Cell Lineage Cells in Their Cerebrospinal Fluid (CSF) Than White Patients (P9-3.010)

<h3>Objective:</h3> To compare proportions of B-cell lineage CD19+ and CD20+ cells in CSF of African-Americans (AA) and White (W) patients with MS. <h3>Background:</h3> Compared to W patients, AA patients with MS are more likely to have oligoclonal bands in CSF; have higher IgG index in CSF; and have higher circulating plasmablasts in blood. It is unknown whether the proportion of B-cells in CSF differs between AA and white patients in MS. <h3>Design/Methods:</h3> Demographics, disease-related information, and treatment history were retrospectively collected on patients with neurologist-diagnosed MS who self-identified as AA or W, received care at NYU MS Care Center, and underwent flow cytometry of CSF during diagnostic work-up. Lymphocyte subsets (B-lymphocytes, T-lymphocytes, NK cells), monocytes and plasma cells were analyzed with flow cytometry (BD FACSCanto™ and FACSCanto™ II Cell Analyzers). <h3>Results:</h3> 20 AA and 57 W MS patients fulfilled inclusion criteria. The groups had similar age at lumbar puncture, sex ratio, CSF cell counts, protein and glucose CSF concentrations, and oligoclonal band number. IgG index was higher in AA compared to W (<i>1.08 v 0.87, p=0.044</i>). With respect to mononuclear cell counts in CSF, AA had higher proportions of CD19+ (<i>5.45% AA v. 2.27% W, p=0.0061</i>) and CD20+ (<i>5.00% AA v. 1.95% W, p=0.0046</i>) cells, but did not significantly differ in proportion of CD4+, CD8+, CD38+ bright B-cells, NK cells and monocytes. Sensitivity analyses that excluded all patients on disease-modifying therapy at the time of lumbar puncture (n=8) yielded similar conclusions. <h3>Conclusions:</h3> B-cells are overrepresented in the CSF of African American patients with MS relative to Whites. Whether this finding bears on the increased severity of MS disease course in AA patients warrants investigation. <b>Disclosure:</b> Mr. Xue has nothing to disclose. Dr. Arbini has nothing to disclose. Mr. Melton has received personal compensation for serving as an employee of Janssen Pharmaceuticals. Dr. Kister has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Genentech-Roche. The institution of Dr. Kister has received research support from Genentech. Dr. Kister has received publishing royalties from a publication relating to health care.

Supporting the differential diagnosis of connective tissue diseases with neurological involvement by blood and cerebrospinal fluid flow cytometry

ObjectiveNeurological manifestations of autoimmune connective tissue diseases (CTD) are poorly understood and difficult to diagnose. We here aimed to address this shortcoming by studying immune cell compositions in CTD patients with and without neurological manifestation.MethodsUsing flow cytometry, we retrospectively investigated paired cerebrospinal fluid (CSF) and blood samples of 28 CTD patients without neurological manifestation, 38 CTD patients with neurological manifestation (N-CTD), 38 non-inflammatory controls, and 38 multiple sclerosis (MS) patients, a paradigmatic primary neuroinflammatory disease.ResultsWe detected an expansion of plasma cells in the blood of both N-CTD and CTD compared to non-inflammatory controls and MS. Blood plasma cells alone distinguished the clinically similar entities N-CTD and MS with high discriminatory performance (AUC: 0.81). Classical blood monocytes indicated higher disease activity in systemic lupus erythematosus (SLE) patients. Surprisingly, immune cells in the CSF did not differ significantly between N-CTD and CTD, while CD4+ T cells and the CD4+/CD8+ ratio were elevated in the blood of N-CTD compared to CTD. Several B cell-associated parameters partially overlapped in the CSF in MS and N-CTD. We built a machine learning model that distinguished N-CTD from MS with high discriminatory power using either blood or CSF.ConclusionWe here find that blood flow cytometry alone surprisingly suffices to distinguish CTD with neurological manifestations from clinically similar entities, suggesting that a rapid blood test could support clinicians in the differential diagnosis of N-CTD.

Flow Cytometric Detection of Malignant Blasts in Cerebrospinal Fluid: A Biomarker of Central Nervous System Involvement in Childhood Acute Lymphoblastic Leukemia.

Despite the excellent prognosis for children and adolescents with acute lymphoblastic lymphoma (ALL), the involvement of the central nervous system (CNS) represents a major therapeutic challenge. Patients who develop CNS relapse have a very poor prognosis, and since current methods cannot reliably identify patients with CNS involvement or patients at high risk of CNS relapse, all children with ALL receive CNS-directed treatment. The current golden standard for detecting CNS involvement is the assessment of cytomorphology on cytospin slides of cerebrospinal fluid (CSF). This technique is inadequate due to low sensitivity and reproducibility. Flow cytometric analysis of CSF represent a novel, highly specific and sensitive technique for the detection of leukemic cells in the CNS. In prospective studies, CSF flow cytometry demonstrated two to three times higher rates of CNS involvement at diagnosis of childhood ALL than conventional cytospin, and especially demonstrated superior sensitivity in detecting low-level CNS disease. CNS involvement determined via flow cytometry has been linked to a higher risk of CNS relapse and poor outcomes in several studies. In this review, we discuss the central analytical concepts of CSF flow cytometry and summarize the current evidence supporting the use of flow cytometric detection of malignant blasts as a biomarker of CNS involvement in childhood ALL.

Case 302: Supratentorial Lymphocytic Inflammation with Parenchymal Perivascular Enhancement Responsive to Steroids.

Part one of this case appeared 4 months previously and may contain larger images. A 21-year-old immunocompetent man who was a long-term resident of Qatar presented to the emergency department with recurrent episodes of unprovoked generalized tonic-clonic seizures lasting 2-3 minutes that spontaneously resolved and were associated with postictal confusion. The patient also had progressive mild diplopia, intermittent dizziness, and numbness in the left arm over the course of 3 months. The patient did not have any other systemic symptoms or chronic medical diseases. He did not have any history of intake of illicit drugs, supplements, or regular medications; he had not received any recent vaccinations; and he had not undergone any surgical procedures. He had no history of travel. At presentation, vital signs were normal. Neurologic examination showed mild left homonymous hemianopia, normal gait with no cerebellar signs, and preserved sensations, power, tone, and reflexes in all four limbs. An electroencephalogram showed no epileptiform discharges. Chest CT and extensive laboratory work-up, including viral, fungal, bacterial, and parasite work-up, thyroid function tests, and immunologic blood tests yielded normal results. Those included normal complete and differential blood counts and normal serum chemistry. Serum analysis was negative for antinuclear antibody, Sjögren syndrome antigens A and B, cytoplasmic antineutrophil cvtoplasmic antibody, and paraneoplastic profile. Serum evaluation was also negative for human immunodeficiency virus type 1 and type 2 RNA, and Brucella, Schistosoma, and toxoplasma antibodies. Venereal Disease Research Laboratory (VDRL) and rapid plasma regain (RPR) test results were negative. Cerebrospinal fluid (CSF) analysis revealed clear fluid and normal pressure and biochemistry, except for elevated protein concentration (0.48 g/L) (normal range, 0.15-0.45 g/L). There were 43 leukocytes/µL (99% lymphocytes) (normal range, 0-5 leukocytes/µL; lymphocytes range, 40%-80%), with no atypical or malignant cells. CSF Gram staining, acid-fast staining, cryptococcal antigen, varicella-zoster virus polymerase chain reaction (PCR), herpes simplex virus PCR, VDRL, and RPR test results were negative. CSF cultures did not show any evidence of growth of bacteria, fungi, or acid-fast bacillus. CSF flow cytometry did not show a monoclonal lymphoid population. No CSF oligoclonal bands were detected. Conventional brain MRI with intravenous administration of contrast material and perfusion study were performed and included different sequences (Figs 1-3).

Case 302.

History A 21-year-old immunocompetent man who was a long-term resident of Qatar presented to the emergency department with recurrent episodes of unprovoked generalized tonic-clonic seizures lasting 2-3 minutes that spontaneously resolved and were associated with postictal confusion. The patient also had progressive mild diplopia, intermittent dizziness, and numbness in the left arm over the course of 3 months. The patient did not have any other systemic symptoms or chronic medical diseases. He did not have any history of intake of illicit drugs, supplements, or regular medications; he had not received any recent vaccinations; and he had not undergone any surgical procedures. He had no history of travel. At presentation, vital signs were normal. Neurologic examination showed mild left homonymous hemianopia, normal gait with no cerebellar signs, and preserved sensations, power, tone, and reflexes in all four limbs. An electroencephalogram showed no epileptiform discharges. Chest CT and extensive laboratory work-up, including viral, fungal, bacterial, and parasite work-up, thyroid function tests, and immunologic blood tests yielded normal results. Those included normal complete and differential blood counts and normal serum chemistry. Serum analysis was negative for antinuclear antibody, Sjögren syndrome antigens A and B, cytoplasmic antineutrophil cvtoplasmic antibody, and paraneoplastic profile. Serum evaluation was also negative for human immunodeficiency virus type 1 and type 2 RNA, and Brucella, Schistosoma, and toxoplasma antibodies. Venereal Disease Research Laboratory (VDRL) and rapid plasma regain (RPR) test results were negative. Cerebrospinal fluid (CSF) analysis revealed clear fluid and normal pressure and biochemistry, except for elevated protein concentration (0.48 g/L) (normal range, 0.15-0.45 g/L). There were 43 leukocytes/µL (99% lymphocytes) (normal range, 0-5 leukocytes/µL; lymphocytes range, 40%-80%), with no atypical or malignant cells. CSF Gram staining, acid-fast staining, cryptococcal antigen, varicella-zoster virus polymerase chain reaction (PCR), herpes simplex virus PCR, VDRL, and RPR test results were negative. CSF cultures did not show any evidence of growth of bacteria, fungi, or acid-fast bacillus. CSF flow cytometry did not show a monoclonal lymphoid population. No CSF oligoclonal bands were detected. Conventional brain MRI with intravenous administration of contrast material and a perfusion study were performed and included different sequences (Figs 1-3).

Cerebrospinal fluid flow cytometry and risk of central nervous system relapse after hyperCVAD in adults with acute lymphoblastic leukemia.

Potential involvement of the central nervous system (CNS) by acute lymphoblastic leukemia is typically evaluated by a conventional cytospin (CC) of cerebrospinal fluid (CSF). Multiparameter flow cytometry (MFC) is generally more sensitive and specific than morphology, but data to guide its use versus CC are limited. This study identified 92 patients who had MFC performed on their initial CSF specimen and received at least 4 cycles of hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with methotrexate and cytarabine (hyperCVAD) as their initial treatment. Eighteen (20%) were CSF+ by MFC at the baseline, and only 6 of these patients were positive by CC. In contrast, 0 of 51 patients who were negative by MFC and had CC available were positive by CC. Despite the receipt of significantly more intra-CSF chemotherapy (P < .001), the cumulative incidence of CNS relapse by MFC was 22% among CSF+ patients versus 5% among those who were CSF- (P = .044). No such association was observed between CNS relapse and CC results (P = .42). None of the 74 CSF- patients became CSF+ during their initial treatment despite being tested a median of 5 times (range, 2-10). CSF positivity by MFC was the factor most strongly associated with CNS relapse in a series of univariate Cox models (hazard ratio, 3.7; P = .067). The initial CSF status by MFC had no significant impact on overall or event-free survival. MFC of CSF is superior to CC of CSF in identifying adults at high risk for CNS relapse after treatment with hyperCVAD. Surveillance of CSF by MFC has limited utility.

Detection of Neoplasms by Metagenomic Next-Generation Sequencing of Cerebrospinal Fluid

Cerebrospinal fluid (CSF) cytologic testing and flow cytometry are insensitive for diagnosing neoplasms of the central nervous system (CNS). Such clinical phenotypes can mimic infectious and autoimmune causes of meningoencephalitis. To ascertain whether CSF metagenomic next-generation sequencing (mNGS) can identify aneuploidy, a hallmark of malignant neoplasms, in difficult-to-diagnose cases of CNS malignant neoplasm. Two case-control studies were performed at the University of California, San Francisco (UCSF). The first study used CSF specimens collected at the UCSF Clinical Laboratories between July 1, 2017, and December 31, 2019, and evaluated test performance in specimens from patients with a CNS malignant neoplasm (positive controls) or without (negative controls). The results were compared with those from CSF cytologic testing and/or flow cytometry. The second study evaluated patients who were enrolled in an ongoing prospective study between April 1, 2014, and July 31, 2019, with presentations that were suggestive of neuroinflammatory disease but who were ultimately diagnosed with a CNS malignant neoplasm. Cases of individuals whose tumors could have been detected earlier without additional invasive testing are discussed. The primary outcome measures were the sensitivity and specificity of aneuploidy detection by CSF mNGS. Secondary subset analyses included a comparison of CSF and tumor tissue chromosomal abnormalities and the identification of neuroimaging characteristics that were associated with test performance. Across both studies, 130 participants were included (median [interquartile range] age, 57.5 [43.3-68.0] years; 72 men [55.4%]). The test performance study used 125 residual laboratory CSF specimens from 47 patients with a CNS malignant neoplasm and 56 patients with other neurological diseases. The neuroinflammatory disease study enrolled 12 patients and 17 matched control participants. The sensitivity of the CSF mNGS assay was 75% (95% CI, 63%-85%), and the specificity was 100% (95% CI, 96%-100%). Aneuploidy was detected in 64% (95% CI, 41%-83%) of the patients in the test performance study with nondiagnostic cytologic testing and/or flow cytometry, and in 55% (95% CI, 23%-83%) of patients in the neuroinflammatory disease study who were ultimately diagnosed with a CNS malignant neoplasm. Of the patients in whom aneuploidy was detected, 38 (90.5%) had multiple copy number variations with tumor fractions ranging from 31% to 49%. This case-control study showed that CSF mNGS, which has low specimen volume requirements, does not require the preservation of cell integrity, and was orginally developed to diagnose neurologic infections, can also detect genetic evidence of a CNS malignant neoplasm in patients in whom CSF cytologic testing and/or flow cytometry yielded negative results with a low risk of false-positive results.

Flow cytometric analysis of cerebrospinal fluid improves detection of leukaemic blasts in infants with acute lymphoblastic leukaemia.

Infants with acute lymphoblastic leukaemia (ALL) have a high frequency of central nervous system (CNS) involvement. Flow cytometric analysis of cerebrospinal fluid (CSF) was recently demonstrated to be a sensitive method for detecting CNS involvement in childhood ALL. In the present study, CSF from 14 infants was collected at routine lumbar punctures and analysed by multicolour flow cytometry. At initial diagnosis, leukaemic blasts were detected in CSF by flow cytometry in 11 patients (78·6%) compared to seven patients (50%) by cytospin. Larger studies are needed to determine if CSF flow cytometry has prognostic value in infant ALL.