Comparison of bromodeoxyuridine labeling indices obtained from tissue sections and flow cytometry of brain tumors

1988 ◽  
Vol 68 (3) ◽  
pp. 388-392 ◽  
Author(s):  
Tadashi Nagashima ◽  
Takao Hoshino ◽  
Kyung G. Cho ◽  
Morris Senegor ◽  
Frederick Waldman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Brain Tumors ◽  
Deoxyribonucleic Acid ◽  
Single Cells ◽  
Fluorescein Isothiocyanate ◽  
Content Type ◽  
Treatment Regimens ◽  
Tissue Sections ◽  
The Difference

Sixteen patients with brain tumors were given a 30- to 60-minute intravenous infusion of bromodeoxyuridine (BUdR), 200 mg/sq m. Grossly viable fragments were taken from the biopsied tumor specimens and divided into two portions. One portion was dissociated into single cells, stained both with fluorescein isothiocyanate (FITC) using anti-BUdR monoclonal antibody as the first antibody and with propidium iodide (for deoxyribonucleic acid), and analyzed by flow cytometry (FCM). The labeling index (LI) was calculated as the number of FITC-labeled cells expressed as a percentage of the total number of cells analyzed. The other portion was fixed in 70% ethanol, embedded in paraffin, sectioned, and stained with immunoperoxidase using anti-BUdR monoclonal antibody as the first antibody. The LI of these tissue sections was calculated in two ways: from selected areas in which the labeled cells were evenly distributed and from the entire tissue section. The LI's obtained by FCM correlated closely with those from the entire tissue sections (r = 0.99, p < 0.000001) and were usually lower than LI's from selected areas of tissue sections. The LI's determined by FCM also correlated well with the LI's from selected areas of tissue sections (r = 0.82, p < 0.00012), despite the difference in values between them. Thus, the FCM-derived LI and the tissue LI can both provide useful information for predicting the biological malignancy of individual tumors and for designing treatment regimens for individual patients with brain tumors; however, different standards should be used to interpret the LI's obtained by these two methods.

Pressure in the sagittal sinus during intracranial hypertension in man

1974 ◽  
Vol 40 (5) ◽  
pp. 603-608 ◽  
Author(s):  
Albert N. Martins ◽  
Arthur I. Kobrine ◽  
Douglas F. Larsen
Keyword(s):  
Brain Tumors ◽  
Intracranial Hypertension ◽  
Cerebral Blood Volume ◽  
Content Type ◽  
Sagittal Sinus ◽  
Sagittal Sinus Pressure ◽  
The Mean ◽  
The Difference ◽  
Spatial Compensation ◽  
Sinus Pressure

✓ Intracranial pressure (ICP) and sagittal sinus pressure (SSP) were measured simultaneously in 12 patients with brain tumors and secondary intracranial hypertension (ICH). In nine, the mean SSP was largely unaffected by changes in ICP. In three, SSP changed with the ICP. In all but one patient, the ICP remained higher than SSP and, as the ICP increased, the difference between the two also increased. Sinograms performed during ICH demonstrated partial collapse of the sinuses in some patients and not in others. The mean SSP in adults with brain tumors appears to respond unpredictably to changes in ICP. Since the rate of cerebrospinal fluid drainage depends upon the gradient between ICP and SSP, intracranial spatial compensation is probably influenced by the response of SSP to ICP. Individuals with gradients that rapidly increase because their sinuses do not collapse probably compensate more rapidly than those whose sinuses do collapse. This assumed difference in the rate of spatial compensation may account for some of the variability of the ICP response to an enlarging intracranial mass or a change in cerebral blood volume.

Dexamethasone-induced abolition of the inflammatory response in an experimental glioma model: a flow cytometry study

2000 ◽  
Vol 93 (4) ◽  
pp. 634-639 ◽  
Author(s):  
Behnam Badie ◽  
Jill M. Schartner ◽  
Jasmeet Paul ◽  
Becky A. Bartley ◽  
Jessica Vorpahl ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Brain Tumors ◽  
Ex Vivo ◽  
Cell Infiltration ◽  
Cytotoxic T Cell ◽  
Content Type ◽  
Rat Glioma ◽  
Infiltrating Cells ◽  
Glioma Model ◽  
Fine Print

Object. Commonly used for management of cerebral edema in patients with brain tumors, steroid medications also have immunosuppressive functions. To characterize the effects of steroids on the central nervous system's response to tumors more clearly, flow cytometry was used to quantify the extent of inflammatory cell infiltration in an immunogenic rat glioma model.Methods. Freshly prepared 11-day-old intracranial C6 tumors that had been excised from dexamethasone-treated and untreated rats were labeled ex vivo with monoclonal antibodies against CD11b/c, CD45, and CD8a antigens. The extent of microglia (CD11b/c—highly positive, CD45—slightly positive cell), macrophage (CD11b/c—highly positive, CD45—highly positive cell), lymphocyte (CD11b/c-negative, CD45—highly positive cell), and cytotoxic T-cell (CD8a-positive cell) infiltration into each rat's tumor, tumor periphery, and contralateral tumor-free hemisphere was analyzed using flow cytometry.Microglia and lymphocytes constituted a significant component of infiltrating cells in this model, comprising 23 ± 3% and 33 ± 5% of viable cells, respectively. Macrophages, on the other hand, accounted for only 9 ± 1% of infiltrating cells. Treatment of rats with a 7-day course of low-dose dexamethasone (0.1 mg/kg/day) resulted in a greater than 50% inhibition of microglia (p = 0.03) and lymphocyte (p = 0.001) infiltration into tumors. Increasing the dexamethasone dose to 1 mg/kg/day further abolished lymphocyte infiltration (89% inhibition, p = 0.001) but had no additional inhibitory effect on microglia invasion. Macrophage infiltration of tumors was not inhibited at the dexamethasone doses used in this study (p = 0.42).Conclusions. Flow cytometry is a valuable technique for characterizing tumor-associated inflammatory cells in gliomas. Even at low doses, dexamethasone was found to inhibit significantly the infiltration of brain tumors by lymphocytes and microglia. These findings should be considered when experimental immunotherapeutic strategies are evaluated for clinical application.

scholarly journals Development of Protective Immunity in New Zealand White Rabbits Challenged with Bacillus anthracis Spores and Treated with Antibiotics and Obiltoxaximab, a Monoclonal Antibody against Protective Antigen

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Lisa N. Henning ◽  
Sarah Carpenter ◽  
Gregory V. Stark ◽  
Natalya V. Serbina
Keyword(s):  
Monoclonal Antibody ◽  
New Zealand ◽  
Bacillus Anthracis ◽  
Protective Immunity ◽  
Protective Antigen ◽  
Lethal Dose ◽  
Survival Rates ◽  
Anamnestic Response ◽  
Content Type ◽  
Treatment Regimens

ABSTRACT The recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response.

Characterization of neuroectodermal antigen by a monoclonal antibody and its application in CSF diagnosis of human glioma

1988 ◽  
Vol 68 (3) ◽  
pp. 449-455 ◽  
Author(s):  
Toshihiko Wakabayashi ◽  
Jun Yoshida ◽  
Hisao Seo ◽  
Kyoto Kazo ◽  
Yoshiharu Murata ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Brain Tumors ◽  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cell Line ◽  
Human Glioma ◽  
Content Type ◽  
Sds Page ◽  
Fine Print

✓ Monoclonal antibodies were produced by immunization of the human glioma cell line SK-MG-4. One of the antibodies, designated G-22, reacted with 18 of 20 glioma cell lines, two melanoma cell lines, and three lung cancer cell lines, but not with 39 cell lines derived from sarcoma, carcinoma, or hematopoietic tumors. The antigen was expressed in the brain of human fetuses in early gestation (9 weeks) but not in late gestation (8 months) or in normal adult brain, suggesting that the antibody recognizes neural differentiation antigens expressed by neuroectodermal origin. A high incidence of positive antigens has been observed in gliomas but not in the other neural tumors, such as ependymomas, meningiomas, and neuroblastomas. Thus, the antigen defined by the G-22 monoclonal antibody could be defined as glioma-associated antigen. Pulse-labeling with tritiated leucine and subsequent immunoprecipitation of the solubilized cell membrane revealed that the antigen recognized by this antibody had a molecular weight of 67 kD on sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). It was shown by dot-blot enzyme-linked immunospecific assay (ELISA) that the antigen could be detected in the cerebrospinal fluid (CSF) from patients with gliomas. From analysis of affinity chromatography and SDS-PAGE, the antigen present in the CSF had a molecular weight similar to that of a 1% Nonidet P-40 (NP-40) extract from a glioma cell line. When the antigen in CSF was quantitatively assayed by ELISA, the mean antigen level (expressed as optical density at 450 nm) in the CSF of seven patients was 0.8 ± 0.28 (mean ± standard deviation), which was significantly higher than the 0.38 ± 0.14 level observed in the CSF of 15 patients with nonglioma brain tumors and the 0.23 ± 0.09 level in the CSF of four patients without brain tumors. These results indicate that the monoclonal antibody G-22 is useful for the diagnosis of glioma.

scholarly journals Multi-set Pre-processing of Multicolor Flow Cytometry Data

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Rita Folcarelli ◽  
Gerjen H. Tinnevelt ◽  
Bart Hilvering ◽  
Kristiaan Wouters ◽  
Selma van Staveren ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Classification Performance ◽  
Multivariate Methods ◽  
Measurement Variability ◽  
Flow Cytometry Data ◽  
Cell Sample ◽  
The Difference ◽  
Analytical Technology ◽  
Number Of Cells

Abstract Flow Cytometry is an analytical technology to simultaneously measure multiple markers per single cell. Ten thousands to millions of single cells can be measured per sample and each sample may contain a different number of cells. All samples may be bundled together, leading to a ‘multi-set’ structure. Many multivariate methods have been developed for Flow Cytometry data but none of them considers this structure in their quantitative handling of the data. The standard pre-processing used by existing multivariate methods provides models mainly influenced by the samples with more cells, while such a model should provide a balanced view of the biomedical information within all measurements. We propose an alternative ‘multi-set’ preprocessing that corrects for the difference in number of cells measured, balancing the relative importance of each multi-cell sample in the data while using all data collected from these expensive analyses. Moreover, one case example shows how multi-set pre-processing may benefit removal of undesired measurement-to-measurement variability and another where class-based multi-set pre-processing enhances the studied response upon comparison to the control reference samples. Our results show that adjusting data analysis algorithms to consider this multi-set structure may greatly benefit immunological insight and classification performance of Flow Cytometry data.

Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets

1991 ◽  
Vol 260 (2) ◽  
pp. C242-C248 ◽  
Author(s):  
A. Oda ◽  
J. F. Daley ◽  
J. Kang ◽  
M. Smith ◽  
J. A. Ware ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Fluorescein Isothiocyanate ◽  
Cytosolic Calcium ◽  
Human Platelets ◽  
Calcium Mobilization ◽  
The Other ◽  
Calcium Response ◽  
External Calcium ◽  
Granule Release

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.

Quantitative analysis of glutathione in human brain tumors

1990 ◽  
Vol 72 (4) ◽  
pp. 610-615 ◽  
Author(s):  
Hiroshi Kudo ◽  
Takaya Mio ◽  
Takashi Kokunai ◽  
Norihiko Tamaki ◽  
Kimiaki Sumino ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Free Radicals ◽  
Brain Tumors ◽  
Cellular Response ◽  
Chemotherapeutic Agents ◽  
Normal Brain ◽  
Cytochemical Study ◽  
Content Type ◽  
The Difference ◽  
Brain Tissues

✓ Reduced glutathione (γ-glutamylcysteinylglycine, GSH) plays an important role in the protection of cells against damage from free radicals and other electrophils and also influences cellular radiosensitivity, cellular response to hyperthermia, and cytotoxicity to some kinds of chemotherapeutic agents. The concentrations of GSH in 40 primary and metastatic brain tumors were quantitatively analyzed, and GSH was localized in these tumors by a novel o-phthalaldehyde histofluorescence method. The level of GSH was 195.2 ± 57.1 µg/gm (mean ± standard deviation) in glioblastomas multiforme, 444.1 ± 105.1 µg/gm in normal brain tissues, and 614.4 ± 237.4 µg/gm in meningiomas. The differences in GSH levels between glioblastomas and normal brain tissues and between glioblastomas and meningiomas were statistically significant (p < 0.01). The mean GSH level in astrocytoma grades II and III was 321.9 ± 11.8 µg/gm. The difference in the GSH level between glioblastomas and astrocytomas was statistically significant (p < 0.05). Radiosensitive tumors, such as multiple myeloma, germinoma, and small-cell carcinoma, showed low GSH levels. These data suggest the possibility that the GSH may be a predictor for the efficacy of radiation therapy. The cytochemical study showed GSH localized in the cytoplasm; although it stained well in meningioma tissue, GSH was not well stained in sections of multiple myeloma. The endothelial proliferation did not stain well in glioblastoma, which seems to imply that this area is vulnerable to attack by free radicals from irradiation and/or chemotherapy.

DNA index and S-phase in primary brain tumors

1994 ◽  
Vol 80 (1) ◽  
pp. 85-89 ◽  
Author(s):  
Ludek Vavruch ◽  
Sverker Eneström ◽  
John Carstensen ◽  
Bo Nordenskjöld ◽  
Sten Wingren
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Deoxyribonucleic Acid ◽  
Close Correlation ◽  
S Phase ◽  
Intracranial Tumors ◽  
Dna Index ◽  
Content Type ◽  
Tumor Specimen ◽  
Fine Print

✓ Forty-nine primary intracranial tumors (33 astrocytomas, eight acoustic schwannomas, five oligodendrogliomas, and three miscellaneous tumors) were studied by flow cytometry. Each tumor specimen was divided into two portions: one was studied as an unfixed suspension stained with propidium iodide and the other after formalin fixation and paraffin embedding. The 50-µm sections from the paraffin blocks were deparaffinized, rehydrated, and enzymatically disintegrated, and the cells in suspension were stained with propidium iodide. Flow cytometry of the two portions showed a significant correlation between fresh and fixed specimens regarding the S-phase (r = 0.87) and a very close correlation of the deoxyribonucleic acid (DNA) index (r = 0.95). When the 33 astrocytomas were analyzed separately, similar results were obtained (r = 0.86 for S-phase and r = 0.93 for DNA index, respectively). This study demonstrated a high correlation between fresh and fixed specimens for DNA ploidy and S-phase both in primary intracranial tumors in general and also in the selected subgroup of astrocytomas.

scholarly journals Flow Cytometric Analysis of Microsporidia Belonging to the Genus Encephalitozoon

1999 ◽  
Vol 37 (2) ◽  
pp. 371-375 ◽  
Author(s):  
Delynn M. Moss ◽  
Gian P. Croppo ◽  
Sara Wallace ◽  
Govinda S. Visvesvara
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Polyclonal Antibodies ◽  
Flow Cytometric Analysis ◽  
Rabbit Antiserum ◽  
Fluorescein Isothiocyanate ◽  
Epidemiologic Studies ◽  
Cross Reactivity ◽  
Light Scatter ◽  
Flow Cytometric

Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-E. hellem serum was reactive to E. hellem spores but also had cross-reactivity to spores of E. cuniculi andE. intestinalis. On the other hand, fluorescence histograms showed that rabbit anti-E. cuniculi and rabbit anti-E. intestinalis sera were reactive with homologous spores only. Monoclonal antibody prepared against E. hellemreacted only with spores of E. hellem. Neither the polyclonal antibodies nor the monoclonal antibodies reacted withCryptosporidium parvum oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of Encephalitozoonwere identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of Encephalitozoon spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.

Flow cytometric determination of modal DNA population in relation to proliferative potential of human intracranial neoplasms

1988 ◽  
Vol 69 (4) ◽  
pp. 588-592 ◽  
Author(s):  
Kyung G. Cho ◽  
Tadashi Nagashima ◽  
Stanley Barnwell ◽  
Takao Hoshino
Keyword(s):  
Flow Cytometry ◽  
Brain Tumors ◽  
Dna Analysis ◽  
Pituitary Tumors ◽  
Biological Behavior ◽  
Proliferative Potential ◽  
Content Type ◽  
Flow Cytometric ◽  
Slow Growing

✓ Paraffin-embedded specimens of brain tumors from 256 patients who had received an intravenous infusion of bromodeoxyuridine (BUdR) at the time of craniotomy were analyzed retrospectively by flow cytometry to determine the modal deoxyribonucleic acid (DNA) population. A single G1 peak was considered to represent a unimodal DNA population; two or more G1 peaks indicated a multimodal population. Most of the pituitary tumors and moderately anaplastic astrocytomas had unimodal DNA populations, whereas a higher percentage of other slow-growing tumors, such as meningiomas, ependymomas, and neurilemomas, had multimodal populations (46.2%, 50.0%, and 60.0%, respectively). A relatively high percentage of the rapidly growing or highly malignant brain tumors, including highly anaplastic astrocytomas, glioblastomas multiforme, metastatic tumors, and medulloblastomas, also had multimodal populations (52.9%, 48.7%, 57.1%, and 66.7%, respectively). In most tumor groups, however, the percentage of tumors with a multimodal DNA population did not correlate with the BUdR labeling index or with the percentage of BUdR-labeled S-phase cells. Thus, modal DNA analysis by flow cytometry may provide information about the degree of heterogeneity and the biological behavior of individual brain tumors, but the results do not necessarily correlate with the rate of tumor growth or the prognosis in individual patients.

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