Strand displacement amplification and fluorescence polarization.
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Fluorescence Polarization↗
Biomedical subjects
D Seckinger
Publications and source records attributed to D Seckinger.
Pleiotropic drug resistance and survival advantage in leukemic cells with diminished apoptotic response.
Cell line R9 generated by continuous exposure of MOLT-4 cells to adriamycin was cross-resistant to a variety of unrelated drugs. The following data indicate that diminished apoptotic response was the mechanism of acquired pleiotropic drug resistance: (i) apoptosis was a common mechanism of cell death for agents expressing cross-resistance; (ii) induction of apoptosis by drugs, medium depletion and serum deprivation was decreased in R9 cells; (iii) DNA degradation in apoptotic cells was lower in resistant lines, probably reflecting a modification of apoptotic pathway in resistant cells; (iv) inhibition of cell division and DNA synthesis by drugs was similar in sensitive and resistant cells. These data indicated a similar level of initial damage, as typical for resistance based on modified apoptotic response. There was no difference in bcl-2 protein level between sensitive and resistant cells. Thus acquired pleiotropic resistance and diminished apoptotic response in R9 cells were induced by a bcl-2-independent mechanism. Surface T-cell antigen CD4 was expressed in MOLT-4 and lost in R9 cells. The role of CD4 down-regulation in apoptosis-related drug resistance remains to be explored. The association between acquired pleiotropic drug resistance and increased survival capacity in unfavorable growth conditions indicated that drug-induced selection of cells with diminished apoptotic response may stimulate neoplastic progression. Alkylating agents induced similar cytotoxicity and only slightly lower apoptosis in R9 cells in comparison with MOLT-4 cells. Our data show that some drugs may overcome acquired pleiotropic drug resistance based on the modified apoptotic pathway.
Antineoplastic Agents↗
Apoptosis (programmed cell death) and the evaluation of chemosensitivity in chronic lymphocytic leukemia and lymphoma.
Chronic lymphocytic leukemia and lymphoma cells were treated with antitumor drugs in vitro and analyzed by flow cytometry to measure the number of apoptotic (AP) cells and DNA damage in the cells that escaped apoptotic death. AP cells were identified by a high sensitivity of DNA to thermal denaturation, which induced binding of antibody to single-stranded DNA, and by decreased stainability of cells with the intercalating DNA dye propidium iodide. The appearance of AP cells was prevented by Zn++ and inhibited by phorbol ester. AP cells were induced by alkylating agents, antimetabolites, and anthracyclines. A linear relationship between L-phenylalanine mustard dose and the number of AP cells was observed. A synergistic interaction between drugs was detected by an increased number of AP cells and by the intensity of DNA damage in non-apoptotic cells. A most interesting example of synergism was the combination of alkylating agents with fludarabine. Linearity of dose-response curves, and the capability to detect drug synergism and to evaluate variable response of cells from different patients to single agents and combinations suggest that flow cytometry of apoptosis will provide a basis for chemosensitivity tests in leukemia and lymphoma.
Antineoplastic Agents↗
Intercellular transfer of drug resistance.
The effect of L-phenylalanine mustard (L-PAM) on heterogeneous cell populations containing sensitive and resistant cells was evaluated by flow cytometric analysis of DNA damage. Cell cultures were treated with L-PAM for 1 h, fixed, and stained with anti-DNA monoclonal antibody which detects DNA damage induced by alkylating agents. DNA damage was significantly lower in sensitive A2780 cells cocultured with resistant A549 or A2780/PAM cells than in A2780 cells grown separately. Decrease of DNA damage in sensitive cells did not occur when sensitive and resistant cells were grown in common medium without direct contact. Transfer of drug resistance in cocultures was prevented by phorbol ester which is known to inhibit metabolic cooperation via cell junctions. Treatment of cocultures with buthionine sulfoximine increased DNA damage in resistant cells and prevented decrease of DNA damage in sensitive cells. Glutathione (GSH) content in A2780 cells cocultured with A549 cells was significantly higher than GSH content in A2780 cells grown separately. We conclude that decreased response of sensitive cells in cocultures was induced by contact transfer of GSH from GSH-rich resistant cells to sensitive cells. Intercellular transfer of drug resistance demonstrated by analysis of DNA damage was confirmed by colony formation assay. Treatment with L-PAM and Adriamycin killed all cells in A2780/MDR and A549 cultures. Coculture of these lines survived combination treatment because transfer of GSH to multidrug-resistant cells from GSH-rich A549 cells induced resistance to L-PAM and Adriamycin in a single cell. The presence of 2% A549 cells increased resistance of A2780/MDR cells to L-PAM. Phorbol ester eliminated resistance of coculture to combination treatment. Metabolic cooperation between cell subsets with different mechanisms of drug resistance induced resistance to treatment with drugs of different classes (multiclass drug resistance). Inhibition of cell cooperation may improve the response of tumors to combination chemotherapy.
Antimetabolites↗
Flow cytometric analysis of DNA damage and repair in the cells resistant to alkylating agents.
DNA damage in the cells sensitive and resistant to alkylating agents was determined by flow cytometry analysis of cells stained with anti-DNA monoclonal antibody (MOAB) F7-26. MOAB F7-26 interacted with single-stranded regions in alkylated DNA, and the binding of antibody to the cells increased in proportion to the decrease in cell viability. Development of resistance to L-phenylalanine mustard (L-PAM) in A2780 cells was associated with decreased immunoreactivity of DNA with MOAB F7-26. Fluorescence was significantly lower in resistant cells than in sensitive cells, and the difference in the binding of MOAB between two cell types increased with the dose of L-PAM. The enhancement of L-PAM cytotoxicity to resistant cells by buthionine sulfoximine and hyperthermia was accompanied by a proportional increase of MOAB F7-26 binding to DNA. The same relative potential of sensitization regimens was established by cell survival and MOAB staining. The time course of DNA repair established by decrease of MOAB binding after L-PAM removal was similar in sensitive and resistant cells. Resistance of A2780 cells to L-PAM was associated with low initial level of DNA damage and with decreased cytotoxicity per unit of damage. We conclude that resistant cells could be distinguished from sensitive cells by staining with MOAB F7-26 and that the sensitization of resistant cells could be quantitatively predicted by flow cytometry analysis of MOAB binding.
Alkylating Agents↗
Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage.
DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.
Alkylating Agents↗
DNA content in human cancer. Application in pathology and clinical medicine.
The relationship between flow cytometry measurements (DNA ploidy, S-phase index) of solid tumors and survival is reviewed. Breast, ovarian, colorectal, pulmonary, urinary bladder, renal, thyroid, and endometrial cervical carcinoma and melanoma are discussed. Correlations between tumor stage or grade and flow cytometry-derived data are considered. Tetraploidy, S-phase indexes, and data derived from paraffin-embedded material have been the basis for seemingly controversial interpretations. Related methods are covered in detail and comparative aspects of flow cytometry and cytophotometry are reviewed.
Cytophotometry↗
Differential photoabsorption using argon laser radiation on atherosclerotic plaque in non-hemolyzed and hemolyzed blood.
Since argon laser radiation (488 to 514 nm) can vaporize human atherosclerotic plaque, we determined whether different blood media--hemolyzed versus non-hemolyzed blood--can alter photoabsorption of atherosclerotic plaque. Forty cadaveric aortic fibrous plaque samples were fitted onto small vials containing 0.02 ml of either non-hemolyzed blood or hemolyzed blood over the surface of the plaque. The distal end of a 400-mu core diameter quartz fiber was directed onto the surface of the plaque and the proximal end of the fiber was connected to an argon laser. The vaporized area and depth of plaque penetration were measured and the estimated volume of crater formation was derived. Following 2.5, 5, 10 and 20 J of laser energies, vaporized volumes were 0.12, 0.72, 0.97 and 4.09 mm3, respectively, for hemolyzed blood and were 0 (p less than 0.01), 0 (p less than 0.01), 0.92 (NS) and 4.39 mm3 (NS), respectively, for non-hemolyzed blood. Laser radiation destroys red blood cells; the higher the energies, the greater the hemolysis. Thus, different blood media such as hemolyzed and non-hemolyzed blood can alter photoabsorption of atherosclerotic plaque. Low level argon laser absorption upon plaque can occur under hemolyzed blood but not under non-hemolyzed blood. Since higher levels of argon laser energies cause greater lysis of red blood cells, comparable degrees of plaque ablation are observed under either blood medium.
Arteriosclerosis↗
Characteristics of monoclonal antibody measurements in human peripheral blood.
Three areas of monoclonal antibody measurements using flow cytometry have been presented. These include a description of a dual immunofluorescent method for measuring two antibodies simultaneously, the effects of blood storage on enumeration of helper (H) and suppressor (S) cells, and the relationship between absolute lymphocyte count and H/S ratio in both control and AIDS patients. These studies reveal that a dual immunofluorescent labeling method is useful for enumerating lymphocytes from peripheral blood which bear the helper, suppressor and/or thymus-derived (T) cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate helper T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for enumerating suppressor T-lymphocytes. Dual immunofluorescently stained lymphocytes, prepared from whole blood, were analyzed by flow cytometry. Two light scatter parameters, (forward and 90 degree scatter) were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper and suppressor distributions from 167 control patients were as follows: The average percentage +/- SD of the helper and suppressor cells were 42.8 +/- 7.5 and 21.6 +/- 6.4, respectively. The H/S ratio was 2.17 +/- .75. These studies show that the H/S ratio can be determined in a single preparative sample and analyzed by dual immunofluorescence in a single flow cytometric analysis even though the H/S ratio may vary from normal during a disease condition. The dual immunofluorescent assay enables one to correlate the activities of two antibodies against cell surface receptors and allows the measurement of a large number of samples in a minimal time. This study also compared the effects of anticoagulant, storage time, and temperature on the phenotypic determination of the percentages of helper and suppressor T-lymphocytes in human peripheral blood. Blood was drawn in ACD, heparin, and EDTA and stored for up to 4 days at room temperature or 4 degrees C. Phenotypic determination of helper/suppressor lymphocytes was most stable for ACD or heparinized blood at room temperature. Marked changes were observed in the percentages of helper cells at 4 degrees C, whereas the percentages of suppressor cells did not change appreciably regardless of the anticoagulant storage time or temperature. Finally, the relationship between ALC and the H/S ratio in control and AIDS patients was determined. The ALC varied considerably in both control and patient populations as a function of time. Conversely, the H/S ratio remained constant.(ABSTRACT TRUNCATED AT 400 WORDS)
Acquired Immunodeficiency Syndrome↗
Joint venturing from the pathologist's perspective: an in-hospital model.
The prospect of forming a joint venture need not be frightening. Three years into the operation of one such venture, a pathologist explains why and how the formation of a marketing organization enhanced this laboratory's ability to provide broad-based community laboratory services while working within the hospital environment.
Hospital Administration↗
Potential complications of coronary laser angioplasty.
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Angioplasty, Balloon↗
Dual immunofluorescent analysis of human peripheral blood lymphocytes.
These studies reveal that a dual immunofluorescent labeling method is useful for enumerating cells from human peripheral blood that bear the helper, suppressor, and/or T-cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate Helper (H) T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for quantitating suppressor (S) T-lymphocytes. FL-conjugated Leu-4 antibodies were used to measure the T-lymphocyte activity. Dual immunofluorescent stained lymphocytes, prepared either from whole blood or by Ficoll-Hypaque, gradient cell separation, were analyzed by flow cytometry. Two light scatter parameters, forward and 90 degree, were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper, suppressor, and T-lymphocyte distributions from 100 controls were as follows: The average percentages +/- SD of the helper and suppressor cells were 41.2 +/- 7.2 and 23.0 +/- 7.2, respectively. The H/S ratio was 1.99 +/- 0.77, while the T-cell distribution on 28 patients was 71.4 +/- 7.7. The Ficoll-Hypaque purified lymphocytes and lysed whole blood lymphocytes compared favorably in their H/S ratios. A comparison was made between the percentages of helper and suppressor cells enumerated by fluorescent microscopy and flow cytometry in which correlation coefficients of 0.80 and 0.86 were determined, respectively. These studies show that helper and suppressor T-lymphocytes can be quantitated simultaneously by flow cytometry, which enables one to correlate the phenotypic activities of two antibodies against cell surface receptors and permits the measurement of a large number of samples in a minimal time.
Adult↗
The effects of anticoagulant and temperature on the measurements of helper and suppressor cells.
This study compared the effects of anticoagulant, storage time, and temperature on the phenotypic determination of the percentages of helper and suppressor Thymus-derived (T) lymphocytes in human peripheral blood. Blood was drawn in ACD, heparin, and EDTA and stored for up to 4 days at room temperature or 4 degrees C. A dual immunofluorescent labeling method, using fluoresceinated-helper (LEU 3a + b) and B-phycoerythrinated-suppressor (LEU 2a) antibodies, was used to simultaneously determine the percentages of the lymphocyte types in whole blood preparations by flow cytometry. Light scatter distributions were stable for ACD or heparinized blood at room temperature, whereas EDTA or 4 degrees C caused changes in the granulocyte distributions. Phenotypic determination of helper/suppressor lymphocytes was most stable for ACD or heparinized blood at room temperature. However, heparinized and EDTA blood showed marked decreases in the percentages of helper cells at 4 degrees C, and EDTA blood stored at room temperature showed an increase in helper cells. The percentages of suppressor cells did not change appreciably regardless of the anticoagulant, storage time, or temperature.
Anticoagulants↗
Subcutaneous phaeohyphomycosis caused by Cladosporium devriesii, sp. nov.
A 26-year-old woman from Grand Cayman Island with a 3-month history of a lump in the left breast was presumptively diagnosed as having a carcinoma. A segmental resection was attempted. Histologic examination of the biopsied tissue showed extensive granulomatous inflammation, necrosis, and septate, branched, dematiaceous hyphae in the intralobular ducts as well as in the surrounding tissues. When cultured on Sabouraud dextrose and brain heart infusion agars, the tissue yielded a slow-growing, downy, dematiaceous mould. Microscopically, it produced short, branched and unbranched chains of holoblastic conidia in acropetal manner from erect conidiophores that were characteristic of the genus Cladosporium. The isolate grew well at 36-37 degrees C, but failed to grow above 37 degrees C. It did not liquefy gelatin nor decompose casein, xanthine, hypoxanthine, or tyrosine, but did hydrolyze urea. Comparative studies with other pathogenic and saprophytic species of Cladosporium revealed that it represented an undescribed species. It is named Cladosporium devriesii, sp. nov.
Adult↗
Prognostic indicators including DNA histogram type, receptor content, and staging related to human breast cancer patient survival.
Primary tumors from breast cancer patients were evaluated for the biochemical presence of three steroid cytosolic receptors and by DNA histogram analysis using flow cytometry. These parameters were compared with the histological and staging diagnoses and the patients' survival over a 36-month period. A total of 74 patients with primary breast tumors were evaluated. The breast samples invariably demonstrated a peak population of diploid G0/1 cells which contained 2C amounts of DNA, as determined by mixing experiments using normal human breast tissues or trout erythrocytes as fixed standards. The tumors were classified into five DNA histogram types based on their DNA index distributions established by flow cytometry. These results showed that 21% of the tumors were diploid and indistinguishable from the diploid population of normal breast cells, 8% were hypodiploid, 11% were hypertetraploid, 8% were multiploid, and the remaining 52% were hyperdiploid. The DNA index values varied from 0.78 (hypodiploid) to 2.60 (hypertetraploid). The percentages of S-phase cells were lowest in the diploid and hypertetraploid tumors and highest in the hypodiploid tumors. Among the 24 patients who died during the 36-month follow-up, 92% (22 of 24) were classified in one of the aneuploid groups. Three high-risk groups identified on the basis of survival after 36 months were distinguished: hypodiploid (50% survival); multiploid (43% survival); and hyperdiploid (50% survival). Rates of survival in the diploid and hyperdiploid groups were 87 and 71%, respectively. The hypodiploid group was distinguished by having the lowest mean estrogen cytosolic receptor value [26 +/- 13 (S.D.) fmol/mg], progesterone cytosolic receptor value (13 +/- 15 fmol/ mg), and androgen cytosolic receptor value (less than 1 +/- 1 fmol/mg). In contrast, the diploid tumors had some of the highest receptor values, with mean estrogen cytosolic receptor value equal to 102 +/- 114 fmol/mg, progesterone cytosolic receptor value equal to 74 +/- 110 fmol/mg, and androgen cytosolic receptor value equal to 65 +/- 80 fmol/mg. The lowest survival rates (17% after 36 months) occurred in patients over 67 years of age who had aneuploid tumors, compared to 100% survival in patients over 67 years of age with diploid tumors. Our results demonstrate the value of using flow cytometry and steroid receptor values as supplements to histopathology for the characterization of subgroups of mammary cancer patients. The ability to identify patients with a good prognosis compared to those at high risk of recurrence and death will be valuable in the design of future prospective treatment studies.
Aneuploidy↗
Multiple gastric lipomas.
A patient with gastric ulceration and hemorrhage due to multiple antral gastric submucosal lipomas is presented. The number and close apposition of the lipomas resulted in loss of the usual gastroscopic and radiologic findings associated with these benign tumors. Complications arising from both singly occurring and multiple gastric lipomas are discussed.
Gastrectomy↗
Effects of dilute acetic acid on cancer cytology. I. Effects on normal tissue.
The simultaneous use of exfoliative cytology and colposcopy is occasionally indicated in the detection of cervical cancer. When there is a fragile epithelium in the perimenopausal or postmenopausal woman, the taking of a cytology preparation may cause sufficient vascular injury so that subsequent colposcopy is not possible. A series of patients was subjected to primary colposcopy followed by cytologic testing in order to evaluate the effect of dilute acetic acid on the Papanicolaou preparation. The minimal alterations caused by dilute acetic acid are easily recognized and do not impair the validity of the cytologic technique for cancer detection.
Acetates↗
Estrogen and progesterone receptors in human breast cancer.
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Breast Neoplasms↗