[Flow cytometry increases the proportion of valuable samples in cerebrospinal fluid with normal cell count in malignant blood diseases]. / La citometría de flujo aumenta la proporción de muestras valorables en líquido cefalorraquídeo con recuento celular normal en hemopatías malignas.

BACKGROUND: The alteration of cerebrospinal fluid (CSF) in hematologic neoplasms is a poor prognostic marker. The characteristics of CSF are usually analyzed by flow cytometry or cytology. However, paucicellular CSF samples (≤5 cells/dL) can sometimes be considered unsuitable for analysis due to the low number of events. OBJECTIVE: To evaluate the proportion of samples reported as suitable for analysis obtained by cytometry (FCM) and cytology in paucicellular CSF samples. MATERIAL AND METHODS: 169 samples ofpaucicellular CSF corresponding to 115 patients with hematologic neoplasms were selected. The samples were obtained by lumbar puncture in tubes conditioned with EDTA and Transfix®. We characterized the immunophenotype ofCSF samples with an 8-color panel, and 55 samples (32%) were in a small sample tube (SST). In all cases, monocytes were identified by CD14 labeling and T lymphocytes by CD3 labeling. The acquisition was carried out in a FACSCantoII® cytometer, and the analysis was performed using Infinicyt® software. RESULTS: The proportion of samples suitable for analysis was higher in FCM compared to cytology (98% vs 61%, p < 0.000). We identified the presence of T lymphocytes and/or monocytes in most samples (98% and 90%, respectively). In the SST samples, the number of events recorded in low-volume samples (< 1 mL) was lower than in samples with higher volume (140 vs 556, p < 0.001), with a median of identification of 3 cell populations. CONCLUSION: FCM allows the analysis of a higher proportion ofpaucicellular CSF samples than cytology in hematologic neoplasms study.

Cerebrospinal fluid findings in patients with hematologic neoplasms and meningeal infiltration.

Neoplastic cell infiltration into the central nervous system (CNS) is a serious complication of hematological neoplasms. Cytomorphology (CM) and flow cytometry (FC) have been used to detect meningeal infiltration. The association between CSF findings with the results of CM and FC is still poorly understood. We retrospectively evaluated CSF findings in 72 patients with hematological neoplasm and meningeal infiltration detected either by CM or FC. We compared CSF cell count, total protein concentration, and lactate concentration according to the type of hematological neoplasm. We also compared these CSF findings according to the FC and CM results (FC + CM + , FC + CM-, and FC-CM +). The proportion of patients with positive FC was higher than with CM (FC - 91.7%; CM - 63.9%). Thirty-five (48.6%) patients with meningeal infiltration had normal CSF cell count, normal total protein concentration, and normal lactate concentration. The proportion of cases in which these CSF parameters were normal did not differ according to the type of hematological neoplasm. The positivity of CM was significantly higher in patients with > 3 cell/mm3 (P = 0.015) but the positivity of FC was not significantly different between patients with > 3 cell/mm3 or ≤ 3 cells/mm3. Patients with positive CM had more CSF cells (P = 0.0005) and higher lactate concentration (P = 0.0165) than patients with negative CM. The absence of CSF changes in cell count and total protein and lactate concentrations does not exclude the presence of meningeal infiltration. Although CM is considered the gold standard, the probability of positive CM is low in patients without CSF abnormalities in these parameters. Patients with hematological neoplasm with suspected meningeal infiltration should be investigated with both methods.

Autotaxin and soluble IL-2 receptor concentrations in cerebrospinal fluids are useful for the diagnosis of central nervous system invasion caused by haematological malignancies.

BACKGROUND: Invasion of the central nervous system by haematological malignancies is diagnosed by cytological analyses of cerebrospinal fluid or diagnostic imaging, while quantitative biomarkers for central nervous system invasion are not available and needed to be developed. METHODS: In this study, we measured the concentrations of autotaxin and soluble IL-2 receptor in cerebrospinal fluid and evaluated their usefulness as biomarkers for central nervous system invasion. RESULTS: We observed that both the autotaxin and soluble IL-2 receptor concentrations in cerebrospinal fluid were higher in subjects with central nervous system invasion than in those without, and the cerebrospinal fluid concentrations were independent from the serum concentrations of these biomarkers. ROC analyses revealed that the soluble IL-2 receptor concentration in cerebrospinal fluid was a strong discriminator of central nervous system invasion in subjects with haematological malignancies, while the autotaxin concentration in cerebrospinal fluid also had a strong ability to discriminate central nervous system invasion when the subjects were limited to those with lymphoma. The combined measurement of autotaxin and soluble IL-2 receptor in cerebrospinal fluid improved the sensitivity without notably reducing the specificity for central nervous system invasion in subjects with lymphoma when central nervous system invasion was diagnosed in cases where either value was beyond the respective cut-off value. CONCLUSION: These results suggest the possible usefulness of soluble IL-2 receptor and autotaxin concentrations in cerebrospinal fluid for the diagnosis of central nervous system invasion.

Utility and proposed algorithm of CSF flow cytometry in hematologic malignancies.

In patients with hematologic malignancies, multiparameter flow cytometry (FCM) offers greater sensitivity than cytology in detecting malignant cells in the initial cerebrospinal fluid (CSF) specimen. However, the role of FCM in assessment of subsequent specimens is unclear. We developed an algorithm to reduce the number of low-yield FCM tests without significant impact on clinically meaningful results. Patients with hematologic malignancies were studied in a derivation cohort, and the following algorithm was developed: (1) cytology and FCM on all initial samples, (2) cytology on all subsequent samples, and (3) FCM on subsequent samples only if previous FCM was positive. A separate population served as the validation cohort. The derivation cohort included 197 patients representing 1157 cytology and 543 FCM samples. Common malignancies were B-Cell ALL (25.3%), diffuse large B cell lymphoma (29.4%), and Burkitt lymphoma (7.7%). In the derivation cohort, the algorithm yielded a sensitivity of 90.0% (95% CI, 81.2-95.6%) and a specificity of 100% (95% CI, 98.9-100.0%). The validation cohort included 132 patients with 563 cytology and 602 FCM samples. In the validation cohort, the testing algorithm yielded a sensitivity of 87.5% (95% CI, 75.9-94.8%) and a specificity of 100% (95% CI, 99.1-100.0%). Of the 15 samples that were missed by the algorithm, FCM findings did not impact patients' management because of known CNS disease (seven patients) or they were responding to treatment (eight patients). CSF testing in hematologic malignancies using the proposed algorithm presents an evidence-based approach to reduce the number of unnecessary FCM tests of CSF without compromising patient care.

Assessment of the Utility of Cytology and Flow Cytometry of Cerebrospinal Fluid Samples in Clinical Practice.

OBJECTIVES: We sought to assess the utility and limitations of both flow cytometry (FC) and cytology for the analysis of cerebrospinal fluid (CSF) in a practical clinical setting. METHODS: A total of 393 consecutive CSF samples from 171 patients submitted for both cytomorphologic and FC assessments were analyzed. RESULTS: Both FC and cytology findings were negative for malignancy in 315/393 samples (80%), and either positive (POS) or suspicious/atypical (SUSP/AT) in 7% of samples. This resulted in high agreement between FC and cytology (87%). Minor discrepancies were present in 4% of the cases. In 28 samples, an abnormal population was detected by FC but not by cytology. CONCLUSIONS: FC and cytology are important complementary methods for analyzing CSF samples. In cases where cytology is SUSP/AT and FC is inconclusive or negative, additional specimens should be submitted for immunostaining, cytogenetics, and/or molecular studies.

Serial cerebrospinal fluid examinations to diagnose hematological malignancy causing neurological disease.

To determine the diagnostic utility of serial cerebrospinal fluid (CSF) examinations for hematological malignancy causing neurological disease. All CSF cytology reports at Mayo Clinic Rochester from 2005 to 2014 (n = 20,018) were reviewed. Study inclusion criteria were: repeated CSF examinations within 1 year in patients without known hematological malignancy performed to determine if hematological malignancy was the cause of neurological disease. Exclusion criteria were: preexisting hematological malignancy; >1 year between CSF examinations, serial CSF examinations for infection, tumor surveillance or intrathecal therapies, and for assessment or treatment of CSF dynamics (e.g. idiopathic intracranial hypertension, CSF shunt or persistent CSF leak). The initial study population included patients undergoing three or more serial CSF examinations; subsequently those undergoing two serial CSF examinations were investigated. A total of 613 patients met the study criteria with 477 having two CSF examinations and 136 having three or greater CSF examinations. Of those with three or greater serial CSF examinations none were found to have hematological malignancy exclusively on the third or subsequent CSF examinations. Of those with two CSF examinations 0.4 % (2/477) were found to have hematological malignancy (large B cell lymphomas) exclusively on the second CSF. Ten patients (1.6 %) had suspicious hematological abnormalities on initial CSF examinations confirmed on subsequent CSF examinations. Serial CSF examinations are of low yield to diagnose hematological malignancy as a cause of neurological disease but may confirm atypical features observed in an initial CSF examination.

Cerebrospinal fluid chimerism analysis in patients with neurological symptoms after allogeneic cell transplantation.

Central nervous system (CNS) complications have been described in patients undergoing allogeneic hematopoietic cell transplantation (alloHCT). Cerebrospinal fluid (CSF) analysis is included in the diagnostic workup in patients with neurological symptoms after alloHCT. CSF donor-recipient chimerism analysis usually is not used to evaluate patients with neurological complications after alloHCT. To assess the potential contribution of CSF donor-recipient chimerism in patients with neurological complications, we analyzed 85 CSF samples from 50 patients with neurological complications after alloHCT. After alloHCT, 21 patients showed the presence of recipient-derived DNA. In 13 of these patients, recurrence of the underlying disease was detected in CSF. There was a moderate correlation between the recipient DNA percentage as detected by short tandem repeat (STR) amplification and the cell concentration in CSF (Spearmann r: 0.66 P=0.004). The percentage of cells with immunophenotypic abnormalities from patients relapsing in the CNS detected by flow cytometry showed a strong correlation with the percentage of recipient-derived DNA in CSF assessed by STR analysis (Spearmann r: 0.83 P=0.0008). Donor-recipient chimerism analysis in CSF in patients with neurological symptoms after alloHCT is a practical, feasible and useful complementary method to the already established methodologies included in the diagnostic workup.

Diagnostic utility of cerebrospinal fluid flow cytometry in patients with and without prior hematologic malignancy.

Flow cytometry (FCM) is an adjunct study to routine analysis of cerebrospinal fluid (CSF) to investigate for involvement by a hematologic malignancy. However, in our experience, FCM only infrequently detects abnormalities in CSF. To help optimize resources without forfeiting clinically important data, we sought to determine evidence-based indications and criteria for performing FCM on CSF. FCM results of 316 consecutive CSF specimens were retrospectively reviewed and correlated with clinical history, total nucleated cell (TNC) counts, and results of concurrent cytologic review. Of 255 samples adequate for analysis, 54% were from patients with a prior history of hematologic malignancy, of which 12% (17 cases) were abnormal by FCM. Corresponding TNC counts among samples with abnormal FCM ranged from 0-1050 cells/µL, and only 44% showed abnormal morphology on concurrent cytology. Of the remaining 46% of samples from patients with no known history of hematologic malignancy who had CSF sampling for neurological indications, only one (1%) was abnormal by FCM. This specimen had an elevated TNC count (39 cells/µL) but lacked clearly abnormal findings on concurrent cytology. These results support the use of CSF FCM only in patients with a history of hematologic malignancy or, in the absence of such a history, in samples showing pleocytosis. If these criteria were applied to the current cohort using a TNC count cut-off of >5 cells/µL, 23% of samples would have been deferred from testing, resulting in decreased cost, improved efficiency, and reduction in the need for unnecessary testing without a negative impact on clinical care.

Flow cytometric analysis of cerebrospinal fluid has low diagnostic yield in samples without atypical morphology or prior history of hematologic malignancy.

OBJECTIVES: To identify pretest characteristics of cerebrospinal fluid (CSF) specimens that will allow the rational use of flow cytometric analysis (FCA) in the diagnosis of hematologic malignancy. METHODS: Retrospective data were collected on 501 consecutive CSF samples submitted for FCA. RESULTS: A positive diagnosis of hematologic malignancy was made in 41 specimens (8.2%). Blasts or atypical lymphocytes were noted on Wright-stained slides in 98% of FCA-positive specimens (40/41), and a history of a hematologic malignancy was present in 89% of specimens (34/38). All FCA-positive specimens had atypical morphology or history of hematologic malignancy. Four hundred six specimens (81%) were FCA negative. Of FCA-negative specimens, 7% (30/406) had atypical morphology, and 3% (12/404) had future central nervous system involvement seen within 30 days. CONCLUSIONS: These data support a policy in which FCA of CSF is actively discouraged unless atypical lymphocytes or blasts are seen or a history of hematologic malignancy is present.

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

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

Diagnostic strategies to investigate cerebrospinal fluid involvement in haematological malignancies.

Central nervous system (CNS) involvement is a fatal complication of certain haematological malignancies with an incidence as high as 25% in specific leukaemia/lymphoma subtypes. It is often accompanied by 'occult' cerebrospinal fluid (CSF) involvement at diagnosis, which is frequently missed by conventional cytology examination. Unfortunately, a diagnostic gold standard is yet unavailable since CSF morphology may be negative for malignant cells in up to 45% of patients with suspected meningeal involvement. New technologies such as flow cytometry, molecular genetics and newer biomarkers may improve sensitivity and specificity facilitating the diagnosis of CNS involvement as well as effective prophylaxis and successful treatment.

Blastic plasmacytoid dendritic cell neoplasm: cytopathologic findings.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive hematopoietic neoplasm, which in the past was also known variously as blastic NK cell lymphoma, agranular CD4+ natural killer cell leukemia, and CD4+/CD56+ hematodermic neoplasm. BPDCN is now believed to arise from plasmacytoid dendritic cells, but its exact etiology is still unknown. We report here on the cerebrospinal fluid (CSF) cytology of a BPDCN, a hypercellular specimen comprised of malignant, singly dispersed cells with scant to moderate amounts of pale blue, agranular cytoplasm, and uniform round to oval nuclei, fine chromatin, prominent nucleoli, occasional cytoplasmic microvacuoles, and pseudopodia. Neither mitoses nor karyorrhexis were identified. Flow cytometry of the CSF demonstrated that the malignant cells expressed bright CD45, HLA-DR and CD33, dim CD4, heterogeneous CD56, and partial CD123. The importance of clinical, histopathological, and phenotypic correlation is emphasized. Clinical and histopathological correlation and a literature review are also presented. The poor clinical outcome makes it important to accurately report this rare tumor in a CSF specimen.

Flow cytometry and cytomorphology evaluation of hematologic malignancy in cerebrospinal fluids: comparison with retrospective clinical outcome.

An independent clinical assessment was compared with flow cytometry (FCM) and cytomorphology results obtained on 227 cerebrospinal fluids investigated for hematologic malignancy, in a retrospective longitudinal study with a median observation time of 11 months. A combined method assessment (CMA), defining "positive" a sample if at least one method gave "positive" results, was also tested. Eleven out of 55 screening samples and 53 out of 166 follow-up samples resulted positive at clinical evaluation. FCM and CM were concordant with positive clinical assessment in 68.5% and 51.5% of cases, respectively. According to CMA, 10.5% of samples (resulting false negative by either FCM or cytomorphology) were rescued as true positive. FCM retained significantly higher accuracy than cytomorphology (p=0.0065) and 100% sensitivity when at least 220 leukocytes were acquired. CMA accuracy was higher than FCM accuracy and significantly higher than cytomorphology accuracy in the analysis of all samples (p<0.0001), samples from mature B/T cell neoplasms (p=0.0021), and samples drawn after intrathecal treatment (p=0.0001). When acquiring ≤220 leukocytes, FCM accuracy was poor, and combining cytomorphology added statistically significant diagnostic advantage (p=0.0043). Although FCM is the best diagnostic tool for evaluating CSF, morphology seems helpful especially when clinically positive follow-up samples are nearly acellular.

Propofol-fentanyl versus propofol alone for lumbar puncture sedation in children with acute hematologic malignancies: propofol dosing and adverse events.

OBJECTIVE: We sought to determine whether the combination of propofol and fentanyl results in lower propofol doses and fewer adverse cardiopulmonary events than propofol and placebo for lumbar puncture in children with acute hematologic malignancies. DESIGN: Randomized, controlled, double blind, crossover study. SETTING: Pediatric Sedation Program. PATIENTS: Children with acute leukemia or lymphoma receiving sedation for lumbar puncture. INTERVENTIONS: Each patient received two sedations in random order, one with propofol/placebo and one with propofol/fentanyl. The study investigator and patient/parent were blinded to placebo or fentanyl. Data collected included patient age and diagnosis, propofol dose and adverse events. Adverse events included oxygen saturation <94%, airway obstruction, apnea, hypotension, and bradycardia (<5% mean for age). Logistic regression analysis was used to assess probability of adverse events and the Wilcoxon Signed Rank and McNemar's tests were used for paired comparisons. MEASUREMENTS AND MAIN RESULTS: Twenty-two patients were enrolled. Fourteen patients were male and eight were female. Each patient was studied twice for a total of 44 sedations. The median age was 5.0 yrs (range, 2.2-17.2 yrs). All procedures were successfully completed. The median total dose of propofol was 5.05 mg/kg (range, 2.4-10.2 mg/kg) for propofol/placebo vs. 3.00 mg/kg (range 1.4-10.5 mg/kg) for propofol/fentanyl (p < 0.001). Twelve adverse events occurred in 11 of 22 patients (50.0%) propofol/placebo compared with 6 of 22 (18.2%) propofol/fentanyl (p = 0.02). The most common adverse event was hypotension. CONCLUSIONS: The combination of propofol and fentanyl vs. propofol alone for lumbar puncture sedation in children with acute hematologic malignancies resulted in lower propofol doses and fewer adverse events.

CSF flow cytometry greatly improves diagnostic accuracy in CNS hematologic malignancies.

OBJECTIVE: To assess the diagnostic accuracy of flow cytometric immunophenotyping in comparison with classic cytomorphology for diagnosing CNS localizations of hematologic malignancies, and to evaluate the implications of CSF pleocytosis and protein content in this context. METHODS: We reviewed the results of diagnostic evaluations of all CSF samples analyzed for localization of a hematologic malignancy between 2001 and 2004 at our center. RESULTS: A total of 1,054 samples from 219 patients were available for analysis. Sixty patients had a CSF localization diagnosed by positive flow cytometry, cytomorphology, or both. The first sample was positive by flow cytometry in 44 (73%) patients, by cytomorphology in 19 (32%). Four first samples were positive by cytomorphology but negative by flow cytometry. Patients with positive cytomorphology had more frequent clinical symptomatology (95% vs 58%) and CSF pleocytosis (84% vs 25%), and tended to a poorer progression-free survival than patients with positive flow cytometry only. OR for CNS localization in case of CSF pleocytosis was 10.1 (95% CI 4.9 to 20.8); OR for CNS localization in case of elevated protein content was 2.9 (95% CI 1.5 to 5.4). Nevertheless, 26 of 137 (19%) patients with normal cell count and protein concentration had a CNS localization. CONCLUSIONS: The diagnostic value of flow cytometry is more than twice that of cytomorphology. However, cytomorphologic examination of the CSF has additional diagnostic and possibly prognostic value, and should still be performed in conjunction with flow cytometry.

Disease- and treatment-related elevation of the neurodegenerative marker tau in children with hematological malignancies.

Children acquire neuropsychologic dysfunctions after chemotherapy for hematologic malignancy. In this study, putative changes in levels of CSF-tau (a marker of neural dysintegrity) in leukemic children prior to and during chemotherapy were studied. Cerebrospinal fluid (CSF) samples were obtained before and during treatment from patients with B cell non-Hodgkin's lymphoma (NHL, n = 10), non-B cell acute lymphoblastic leukemia/NHL (non-B-ALL, n = 48), acute myeloid leukemia (AML, n = 9), other malignant diseases (n = 9), and six control children. A sandwich-type ELISA (INNOTEST hTAU-Ag) was used for measuring CSF-tau. Sixteen out of 50 patients with hematological malignancies, including the patients with proven leukemic CNS invasion, already showed high CSF-tau levels at baseline (>300 pg/ml). The pre-induction treatment for non-B-ALL, consisting of only corticosteroids and methotrexate (MTX), resulted in a significant increase of tau at day 8 (on average to 535 pg/ml). Larger increases as compared to baseline levels of CSF-tau were observed in patients treated for B-NHL with systemic vincristine, corticosteroids and cyclophosphamide, and intrathecal MTX (mean 776 pg/ml at day 8). In two AML patients with CNS invasion, CSF-tau increased during chemotherapy up to 1,500 and 948 pg/ml, respectively. In one non-B-ALL patient with MTX-induced clinical neurotoxicity, CSF-tau was above the detection limit of 2,000 pg/ml. Almost one-third of the patients with hematological malignancies had elevated CSF-tau levels at diagnosis. Transient high levels of CSF-tau, reaching levels observed in other neurodegenerative disorders, were observed during induction chemotherapy for non-B-ALL, B-NHL and CNS+ AML. The clinical implications of both observations will be the subject of further study.