A practical device designed to concentrate cells for cytomorphological studies of biological fluids.

In the present work, a device designed for concentrating cells from biological fluids is described. The instrument consists of a tube in which the inner cavity has a conical shape at one of its ends and a small orifice is found at the bottom, while the tube's exterior maintains its cylindrical shape. The tube is placed inside a second tube that ends on a flat surface on which a glass cover slide is placed. The sample to be studied is placed in the inner tube of the assembled device and spun in a regular clinical centrifuge. Cells are collected on the glass slide, fixed and stained for microscopical studies. The device was tested using 23 samples of cerebrospinal fluid (CSF) from patients with lymphoproliferative diseases. An adequate number of intact cells was recovered for observation, and a precise diagnosis was possible. Cells from three aliquots of each CSF sample were concentrated by this method, and by the more expensive standard commercial cytocentrifuge, with similar results. The device described here provides an easy, efficient and inexpensive method, for the concentration of cells from organic fluids.

Tumor-like lesion due to Chagas' disease in a patient with lymphocytic leukemia

Paciente masculino, 73 anos, do interior de Mato Grosso do Sul, com diagnostico inicial de sinusite, evoluiu em 3 dias para quadro de hipertensao intracraniana severa. Transferido para o servico de neurologia, os exames evidenciaram leucemia linfocitica e indicaram ainda processo inflamatorio expansivo como abscesso ou tumor (exame do liquido e tomografia). Instituiu-se Ceftriaxone e Decadron. Foi feita nova puncao lombar, injetou-se metotrexate considerando possivel infiltracao leucemica. No liquido observou-se formas flagelares de T. cruzi. Iniciou-se benzonidazol. Apos 4 dias o liquor apresentou formas fracionadas de tripomastigotas. O nivel de proteinas se estabilizou em 27 por cento. Cessou a sintomatologia da hipertensao. Tomografia e ressonancia magnetica posterior mostraram importante reducao da formacao tumoral observada anteriormente. Houve melhora das condicoes clinicas do paciente

Proteínas totais no LCR antes e após radioterapia / Csf protein contents before and after radiotherapy

Com o uso da radioterapia e quimioterapia intratecal associados, passou-se a observar alteraçöes do sistema nervoso central que denotaram lesöes neuronais e vasculares. A atribuiçäo da causalidade dessas alteraçöes a um dos dois fatores terapêuticos passou a ser estudada. Quanto ao papel da radioterapia, há trabalhos experimentais e clínicos que apontam para a possibilidade de ser um fator lesivo. E cogitada a possibilidade da radioterapia lesar a barreira hematencefálica, permitindo com isso a entrada de amethopterin no sistema nervoso. Como as dosagens de irradiaçäo usadas experimentalmente säo bem amiores que as terapêuticas, a analogia feita com o tratamento profilático de pacientes leucêmicos näo parece bastante clara. O propósito desse trabalho é estudar a taxa de proteínas totais no líquido cefalorraquino de pacientes com leucemia aguda, sem comprometimento do sistema nervoso, submetidos a radioterapia, como parametro de avaliaçäo de pertubaçäo da barreira hematencefálica. Para esse estudo foram selecionados 18 pacientes com leucemia aguda, sendo 16 com leucemia linfóide aguda, 2 com leucemia mielóide aguda e que näo tinham sinais ou sintomas clínicos de comprometimento neurológico, nem células blásticas no LCR. As amostras de LCR para este estduo foram colhidas até 8 dias antes do início da radioterapia e de 1 semana até 6 meses após o término dessa mesma sequência radioterápica. A dosagem da taxa de proteínas totais foi feita pelo método de Denis & Ayer. Em conclusäo, pelo método de avaliaçäo empregado, näo se pode evidenciar alteraçäo de permeabilidade da barreira hematencefálica, decorrente de irradiaçäo do sistema nervoso central para profilaxia da neuroleucemia.

Significance and limits of cerebrospinal fluid beta-2-microglobulin measurement in course of acute lymphoblastic leukemia.

Cerebrospinal fluid beta-2-microglobulin (CSF-beta 2m) was measured longitudinally in 48 patients affected by acute lymphoblastic leukemia (ALL). Thirteen developed a central nervous system (CNS) involvement during the course of the disease; although moderately higher mean CSF-beta 2m levels were found in these subjects, no significant statistical differences were observed in comparison with patients without this complication and compared with the control group. No correlations were found between beta 2m and other biochemical parameters in CSF. Furthermore, CSF-beta 2m levels appeared to be influenced by previous combined chemoradiotherapeutic treatment for CNS prophylaxis, presence of meningeal non-neoplastic infiltrates, patients' ages, amount of CSF blasts, and their immunological phenotype. In particular, only clearly B-committed leukemic cells, when tested, showed a strong surface expression of beta 2m, as demonstrated by immunocytochemical detection of this protein on cell membrane. However, in specific cases, CSF beta 2m measurement and CSF/serum beta 2m ratio were helpful in diagnosing and monitoring isolated CNS disease. Such findings suggest that CSF-beta 2m assay may be a useful tool in the management of CNS involvement in the course of ALL in only selected patients, as several factors can modify the outcome.

Elevated cerebrospinal fluid leukocyte count and protein concentration at diagnosis: independent risk factors in children with acute lymphoblastic leukemia.

The aim of this study was to investigate whether determination of the initial cerebrospinal fluid (CSF) protein concentration and leukocyte count in children with acute lymphoblastic leukemia (ALL) could yield useful information about the patient's central nervous system status and prognosis. The population-based unselected series comprised 160 children. The mean follow-up time was 72 months (range 25-143 months). Both the CSF protein concentration and the leukocyte count, if elevated, were significantly, although not independently, associated with diminished probability of event-free survival. The patients were divided into three groups for the final analyses: those without any abnormalities in the CSF (n = 133), those with elevated protein concentration and/or elevated leukocyte count, but with no malignant lymphoblasts in the CSF (n = 21), and those with malignant lymphoblasts in the CSF (n = 6). The probabilities of 5-year event-free survival for the first and second group were 65% and 15%; the probability of 2-year event-free survival for the third group was 17%. These differences were statistically significant (p less than 0.001). In multivariate analysis the relative risks of death or relapse for these groups were 1, 2.8 (95% confidence limits 1.5-4.9), and 7.6 (2.4-24.3), respectively (p less than 0.001). The inclusion of an elevated CSF protein concentration or leukocyte count in the risk group criteria of further trials should be considered.

Diagnosis of meningeal involvement in childhood acute lymphoblastic leukemia: cytomorphology and TdT.

Between December, 1984, and May, 1986, 98 CSF samples were sent to a central laboratory by postal express. The samples could be kept in a medium for up to 24 hours after the lumbar puncture. The quality of the cells proved to be good. Excluded were 5 samples delayed in delivery and 13 samples contaminated with blood, defined as the macroscopical presence of blood. The microscopical presence of erythrocytes in the cytocentrifuge preparation can make interpretation of the results difficult. Especially when leukemic blasts are present in the blood, extreme caution is necessary. A total of 71 samples could be studied by cytomorphology as well as by TdT-IF. When cytomorphological leukemic blasts were present, this was confirmed by TdT-IF positivity in all cases. But in 6 of 71 samples, TdT-IF was positive without the presence of cytomorphological leukemic blasts. Follow-up of these patients will show whether the therapeutic regimen has to be changed.

6-Mercaptopurine in cerebrospinal fluid during oral maintenance therapy of children with acute lymphoblastic leukemia.

In three children receiving oral remission maintenance therapy for acute lymphoblastic leukemia, the concentrations of 6-mercaptopurine (6-MP) in cerebrospinal fluid (CSF), plasma and red blood cells were compared. CSF samples were obtained from an Ommaya reservoir previously inserted for treatment of CNS relapse. At the time of the study, the children were all in remission and had been on oral 6-MP (42-63 mg m-2) once daily for at least 24 weeks. Immediately before dose intake on the day of study (about 24 h after last dose), the concentrations of 6-MP in CSF, plasma and red blood cells were rather similar and below 20 ng ml-1 in all patients. After dose intake, the concentrations in plasma and in red blood cells increased to 40-200 ng nl-1 within 0.5-4 h. In contrast, the concentration of 6-MP in the CSF remained fairly constant around 4-10 ng ml-1 throughout the time period studied (up to 4 h). It is concluded that 6-MP can be detected in CSF during oral maintenance therapy and that the drug has different pharmacokinetic profile in CSF to that in plasma and red blood cells. Further studies are necessary to evaluate the significance of the 6-MP concentrations obtained in CSF for the prevention of CNS relapse.

Shedding of leukemia-associated P24 antigen by lymphoblastoid cell lines.

We report the development of a unique enzyme-linked immunosorbent assay (ELISA) which makes possible the detection of leukemia-associated P24 antigen, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4 simultaneously. Using the RCA1/SJ-9A4-ELISA, P24 antigen, as few as 50 X 10(3) cells from a common acute lymphoblastic leukemia (C-ALL) cell line could be detected. The presence of D-galactose gave complete and specific inhibition of P24 antigen binding to RCA1. Matched concentrations of D-glucose and D-sucrose had no effect on binding. The release of the P24 antigen into the culture medium by a C-ALL cell line maintained at 37 degrees C could be detected; however, no P24 antigen was present in the culture medium when the cells were maintained at 4 degrees C. Sequential analysis of the culture medium for soluble P24 antigen revealed that release of the P24 antigen associated with cell growth. Molecular sieve chromatography of concentrated culture medium indicated that shed P24 antigen was eluted in the macromolecule fraction. P24 antigen was detected in the cerebrospinal fluid (CSF) of four patients with P24 positive ALL at the time of relapse of the central nervous system (CNS) and was undetectable while in complete remission. The CSF from three patients with P24 negative ALL and three patients with aseptic meningitis had no detectable activity.

Cytodiagnosis and monitoring of acute lymphocytic leukemia and eosinophilia in cerebrospinal fluid.

A case of adult aleukemic leukemia with an isolated CNS relapse diagnosed by cytologic examination of the CSF is reported. CSF hypereosinophilia of uncertain significance was documented. Immunologic marker studies (CALLA, HTA, Tdt) were performed on the CSF and showed a null cell acute lymphocytic leukemia. Sequential CSF specimens were obtained to determine the continued presence of lymphoblasts. Cytologic monitoring of the CSF in acute leukemia is a useful technique to determine disease status and efficacy of therapy. We advocate the use of cell morphology for monitoring, reserving the use of cell markers for initial identification of malignant cells and for use when the cell morphology is altered.

Immunocytochemistry of cerebrospinal fluid.

In order to determine how best to study cells in cerebrospinal fluid (CSF) by immunocytochemical techniques, several crucial technical variables and five immunocytochemical methods were examined. Immunocytochemical studies could be performed on either cell suspensions or smears. The method using cell suspensions was more sensitive, producing less background staining, but requiring more cells than that using smears. Among the five methods examined, indirect immunoperoxidase (IP) and indirect immunoalkaline phosphatase (IAP) were comparable in sensitivity. The peroxidase-antiperoxidase (PAP), alkaline phosphatase-antialkaline phosphatase (APAAP) and avidin-biotin complex-immunoalkaline phosphatase (ABC-AP) methods were comparable in sensitivity and were more sensitive than either the IP or IAP technique. The peroxidase methods were plagued with problems related to endogenous enzyme activity and the ABC-AP method may exhibit undesirable background staining. Therefore, the IAP method should be used for cell suspensions and the APAAP for cells on smears. In CSF specimens with a small number of cells, immunocytochemical studies should be done on smears by the APAAP method. These conclusions are supported by our experience with CSF specimens from patients with reactive and neoplastic lymphocytoses.

Effect of dose and repeat intravenous 24 hr infusions of methotrexate on cerebrospinal fluid availability in children with hematological malignancies.

This pharmacokinetic study examined the relationship between methotrexate (MTX) dose and drug concentrations in blood and cerebrospinal fluid (CSF) during repeated 24 hr infusions. Two regimens were used: an intermediate dose (ID) of 0.5 g/m2 (7 patients, 23 cycles) and a high dose (HD) of 2.5 g/m2 (8 patients, 39 cycles). Inter-patient variability in the drug concentration was apparent in serum and CSF for both doses. The dispersion was particularly wide in CSF for HD MTX. Considering median values, serum and CSF MTX were linked to dose escalation. Individual CSF/serum drug ratios were not modified by the dose (1.1% for ID MTX versus 1.4% for HD MTX). A potentially cytotoxic drug level in CSF (10(-6) M) was never obtained for ID MTX cycles, but was achieved in 44% of HD MTX cycles: for HD MTX, this corresponded to 88% of patients (7/8). Total body clearance did not modify the degree of CSF MTX passage. A positive, significant correlation (r = 0.62, P less than 0.05) was observed for ID MTX between individual serum and CSF MTX; no such relationship was seen with HD MTX. Individual cycle-to-cycle variations in the MTX concentration were particularly marked in CSF and for HD MTX, without strict concordance with blood levels.

CSF myelin basic protein levels in leptomeningeal metastases. Relationship to disease activity.

Myelin basic protein (MBP) was serially measured in 177 CSF samples of 33 patients with leptomeningeal metastases and in 34 cancer controls. The mean level in cancer controls (free of neural involvement) was 5.7 +/- 0.33 ng/ml (normal less than 5 ng/ml) with abnormal elevation of MBP detected in 17%. The activity of the leptomeningeal disease was classified as either acute-progressive, stable or in remission on the basis of clinical and CSF cytological findings. CSF MBP levels were analysed in each stage. Abnormal elevation of MBP was detected in 74% of the 68 samples obtained in the acute-progressive stage (mean +/- SEM: 18.25 +/- 1.4 ng/ml, P less than 0.0001), in 24% of the 79 samples in the stable phase (mean: 7.99 +/- 0.8 ng/ml, NS) and in 20% of the 30 samples in remission (mean 5.7 +/- 0.3 ng/ml, NS). Similar changes in levels of CSF MBP were also observed in longitudinal studies of patients responding to treatment or relapsing to the acute stage. Eight patients developed treatment induced necrotizing leukoencephalopathy with typical CT-scan findings; elevated levels of CSF MBP were detected in 7 of them (mean: 21 +/- 3 ng/ml) when measured within 2 weeks of diagnosis but not when measured 2 months earlier. Our study suggests that in leptomeningeal metastases, CSF MBP levels are indicators of the disease activity, particularly if longitudinal determinations are used.

Carcinoembryonic antigen (CEA), alphafetoprotein (AFP) alpha and beta subunits of human chorionic gonadotropin (hCG) in cerebrospinal fluid of children with acute lymphoblastic leukaemia.

The cerebrospinal fluid (CSF) and plasma levels of CEA, AFP, alpha and beta hCG were determined by radioimmunoassay in 19 children with acute lymphoblastic leukaemia (ALL). CSF in 15 patients at the onset of ALL was examined in the first week after diagnosis and subsequently every two months. In 4 other children in second complete remission of ALL, CSF was examined every two months as well. Elevated values of CEACSF were present in 1/15 patients at the onset of ALL, of AFPCSF 0/15, alpha hCGCSF in 2/15, beta hCGCSF in 2/15 cases. The elevated levels of these markers in CSF became normal in successive lumbar punctures and none of these children developed central nervous system (CNS) relapse in further follow-up. Isolated CNS relapses were diagnosed 13 times in 7 children. Elevated CEACSF levels were found in 6/13 cases (maximum, 25.0 ng/ml) and in one patient CEACSF levels correlated well with pleocytosis. Elevated AFPCSF values were present in 0/13 cases, alpha hCGCSF in 0/13 and beta hCGCSF in 3/13 patients and became normal by the next CSF examination. The determination of CEA, AFP, alpha and beta hCG in plasma did not play a role in monitoring CNS relapse in ALL patients.

Pregnancy-specific beta 1 glycoprotein (SP1) in the cerebrospinal fluid.

Pregnancy-specific beta 1 glycoprotein (SP1) was assayed by Particle Counting Assay in the cerebrospinal fluid (CSF) from 26 non-neurological patients, from 190 patients with various neurological disorders and from 84 patients with malignant hemopathies. With a sensitivity limit of 0.5 microgram/l, SP1 was undetectable in normal CSF. High levels were observed in CSF from one pregnant woman with herpetic encephalitis and from another woman with post-puerperal thrombophlebitis as a result of high serum concentrations and leakage of the blood-brain barrier. SP1 was detected at low levels in the CSF from 1 patient out of 5 with Creutzfeldt-Jakob disease and from a patient with Behçet's disease. Seven patients out of 84 with malignant hemopathies presented cerebral involvement; 3 of them had detectable SP1. However, SP1 was also detected in the CSF of 2 patients in apparently complete remission. The determination of SP1 in CSF appears to be of limited value in the diagnosis of neurological disorders and in the early detection of a cerebral localization of malignant hemopathies.

[Selenium concentration in the cerebrospinal fluid of children]. / Die Selenkonzentration im Liquor cerebrospinalis von Kindern.

Following a wet digestion of 0.5-2.0 ml cerebrospinal fluid in an open system using 2.0 ml nitric acid and 1.0 ml perchloric acid (240 degrees C) and a reduction step with 1.0 ml hydrochloric acid, Selenium can be determined polarographically after adding 100 micrograms Copper(II)-ions to the analyte (15 ml; water/perchloric acid). Selenium concentrations in cerebrospinal fluid of children younger than one year (2.49 +/- 1.67 ng/ml) are significantly higher (p = 0.0074) than those of older children (1.28 +/- 0.97 ng/ml). Independent of the childrens age and diseases the Selenium concentrations correlate distinctly with cell numbers and protein contents. A correlation between Selenium content and cell numbers alone could not be proved. The non-significant differences between the Selenium concentrations in cerebrospinal fluids of children with hydrocephalus, leukemia (with or without involvement of the central nervous system), and other diseases, respectively, may be interpreted by considering the protein content of the cerebrospinal fluid and the age of the children.

Elevated titers of cell-free interleukin-2 receptor in serum and cerebrospinal fluid specimens of patients with acquired immunodeficiency syndrome.

A sensitive monoclonal antibody based ELISA was used to detect cell-free interleukin-2 receptor (IL-2R) in the body fluids of patients with acquired immune deficiency syndrome (AIDS), a variety of other disease conditions and a control group of apparently healthy (heterosexual and homosexual) males. Two of the 25 control donors showed low titers (1:8) of IL-2 receptor in the serum samples; the cerebrospinal fluid (CSF) specimens from these individuals proved negative. However, serum and CSF specimens from all the 9 patients with AIDS showed significantly elevated titers (range 1:128 to 1:4096) of IL-2 receptor. The presence of moderate titers (range 1:128 to 1:512) of circulating IL-2 receptor could also be detected in all of the 4 patients with acute lymphocytic leukemia. IL-2 receptor was detectable in the CSF and/or serum specimens from 3 of 3 patients with lung cancer, 3 of 4 patients with acute hepatitis B infection, and 2 of 3 patients with multiple sclerosis. IL-2 receptor could not be detected in the serum or CSF specimens originating from patients with legionellosis (3/3), asthma (3/3), or those with non-pulmonary febrile bacterial infections (4/4). It is concluded that soluble IL-2 receptor may be found in serum or CSF specimens from patients with certain (but not all) disease conditions including AIDS. The conspicuously elevated titers of cell-free IL-2R in the body fluids of patients with AIDS may contribute to the drastic impairment of the immune system regulation observed in such patients.