In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56–66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30 h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1 Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.     

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.     

In vitro assay of cytogenetic damage induced in bone marrow cells in vivo by chemical carcinogens

Bone marrow cells explanted after the host animal has been exposed to chemicals can be assayed in vitro for cytogenetic damage. Cells were grown in medium for two cell cycles in the presence of 5-bromodeoxyuridine and then analyzed for the frequency of sister chromatid exchanges and cell replication kinetics. The patterns for induction of these endpoints as assayed in vitro were very similar to those observed if the assay was performed totally in vivo. Several chemicals were tested in this system and shown to be positive; hycanthone, ethidium bromide, 4-fluoro-3-nitro-phenyl azide, mitomycin-C, 2-aminoanthracene, benzo(a)pyrene, and cyclophosphamide. Dehydroemetine, known as a nonmutagenic drug, was negative in this assay. Since this assay incorporates host metabolism, is rapid, and has a wide dynamic range, it may be useful to perform to determine the potential of chemicals to induce genetic damage.     

A novel assay for assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.

BACKGROUND: Assessment of CD8(+) T-cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV-specific cytotoxicity by multiparameter flow cytometry. METHODS: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE(hi)PI(-)) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The (51)Cr-release assay and IFN-gamma ELISpot were performed by standard procedures. RESULTS: A comparison with the Cr-release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01). CONCLUSION: This new assay provides both specific and sensitive results when employed for the detection of HIV-specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels.     

In vivo and in vitro cytogenetic damage induced by sodium nitrite

The mutagenicity of sodium nitrite was assayed by in vivo and in vitro experiments. The in vivo experiments were carried out in male rats and mice intragastrically treated twice, with an interval of 24 h, with nitrite in doses of 1.72, 5.18, 15.55 and 46.66 mg/kg body weight and in male rabbits treated with the same doses of nitrite administered daily in drinking water for 3 months. Chromosomal aberration analysis was conducted in all 3 species of animals and micronucleus induction was only evaluated in mice. Nitrite induced increases in aberrant metaphases in all 3 species of animals. Likewise, in mice it induced increases of the numbers of micronucleated polychromatic erythrocytes and a light bone marrow depression. Neither in the increases of the numbers of chromosomal aberrations nor in that of micronuclei, were dose-related responses observed. The in vitro experiments were carried out on BSC-1 and HeLa cells grown in cultures with nitrite in doses of 0.265 and 0.530 mg/ml for 24 h. Both doses produced significant increases of the percentage of chromosomal aberrations but also without demonstration of positive dose-effect relationships.     

In vitro protein kinase activity measurement by flow cytometry

Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.     

Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry

BACKGROUND: The evaluation of the safety of drugs and other chemicals is an important aspect of toxicology work. The mouse micronucleus assay is a standard in vivo genotoxicity assay. Chromosomal damage is an indicator of genotoxicity, which manifests in the formation of micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. The assay is laborious to perform by manual counting. The laser scanning cytometer allows automated and rapid quantitation of cellular and subcellular fluorescence in monodisperse cell samples on a microscope slide. The object of this study was to evaluate the application of this new technology in the mouse micronucleus genotoxicity assay. Materials and Methods One hundred forty-four mice of various strains were dosed with combinations of carcinogens and antioxidants. Duplicate blood films were prepared 3 days later. One set of slides was stained with acridine orange, and the proportion of micronucleated erythrocytes was counted in 5,000 cells per slide. The duplicates were stained with propidium iodide (40 microg/ml). Five thousand cells per sample were examined using a laser scanning cytometer. The proportion of micronucleated erythrocytes was measured. RESULTS: A coefficient of correlation of 0.96 was found between the data from the two assays. The automation of the assay on the LSC produced a considerable time saving and efficiency gain. CONCLUSIONS: We conclude that with further development, laser scanning cytometry is likely to become the preferred modality for the performance of standard genotoxicity assays.     

The in vitro micronucleus assay for detection of cytogenetic effects induced by mutagen-carcinogens: comparison with the in vitro sister-chromatid exchange assay

The sensitivity of a cytogenetic assay, as expressed by the in vitro induction of micronuclei (MN), was compared to the in vitro induction of sister-chromatid exchanges (SCEs). Chinese hamster lung (V79) cells were exposed to 3 known alkylating agents: methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and to 5 newly synthesized naphthofurans: 2-nitro-7-methoxynaphtho[2,1-b]furan (A), 2-nitro-8-methoxynaphtho[2,1-b]furan (B), 2-nitronaphtho[2,1-b]furan (C), 2-nitro-7-bromonaphtho[2,1-b]furan (D) and 7-methoxynaphtho[2,1-b]furan (E). The induction of MN only was also analysed after exposure of the cells to 4 alcohols: ethanol, methanol, butanol and propanol. The lowest dose at which a significant effect could be observed was determined. In both assays, MNNG, MMS and EMS were equally active with the following order of potency: MNNG greater than MMS greater than EMS, the latter being a very weak inducer of MN and SCE. Compounds A and B were also very effective in both assays. Compound C was a more active inducer of SCE than MN. Compounds D and E were not active in either assay. None of the 4 alcohols induced MN. Our results are compared with the previously published data on in vitro and in vivo induction of SCE and MN. We conclude that the MN in vitro assay which detects clastogens as well as agents affecting the spindle apparatus, is a good indicator of genotoxicity, though slightly less sensitive than the in vitro SCE test. It could provide a rapid, simple and inexpensive complementary short-term test for the evaluation of potentially mutagenic chemicals.     

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream^X is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream^X. Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream^X, and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.     

In vitro evaluation of crystalline silicon nanoparticles cytotoxicity

The effects of silicon-based nanoparticles on viability and cellular organelle state were evaluated in human lymphocytes in vitro. We did not find any changes in cell viability in experimental groups compared to control. Cell death occurred mainly due to apoptosis and late apoptosis, and necrosis/apoptosis ratio in the control and after exposure to nanoparticles remained unchanged. All silicon-based nanoparticles (Si, SiB, SiPd) caused an increase of reactive oxygen species in the cells. Evaluation of mitochondria and lysosomes state after interaction with modified nanoparticles demonstrated slight decrease in its function. Thus, modification of silicon nanoparticles did not significantly reduce their biocompatibility.     

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56–66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei—the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.     

Sensitivity of the erythrocyte micronucleus assay: Dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., ≤0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals

The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.     

Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

In vitro cytotoxicity evaluation of some substituted isatin derivatives

A range of substituted 1H-indole-2,3-diones (isatins) were synthesized using standard procedures and their cytotoxicity evaluated against the human monocyte-like histiocytic lymphoma (U937) cell line in vitro. SAR studies identified C(5), C(6), and C(7) substitution greatly enhanced activity with some di- and tri-halogenated isatins giving IC(50) values <10 microM. Of the 23 compounds tested, four were selected for further screening against a panel of five human cancer cell lines. These compounds, in general, showed greater selectivity toward leukemia and lymphoma cells over breast, prostate, and colorectal carcinoma cell lines. The most active compound, 5,6,7-tribromoisatin (2p), was found to be antiproliferative at low micromolar concentrations and also activated the effector caspases 3 and 7 in a dose-dependent manner. These results indicate that di- and tri-substituted isatins may be useful leads for anticancer drug development in the future.     

FDA/PI flow cytometry assay of complement-mediated cytotoxicity of antibodies generated during xenotransplantation.

BACKGROUND: A flow cytometry complement-mediated cytotoxicity assay (FCCA) using fluorescein diacetate (FDA) and propidium iodide (PI) to measure antibody-dependent toxicity is useful to determine the success of xenotransplant organs. We evaluated the validity of different mathematical models as a measure of cytotoxicity in FCCA. METHODS: Sera from untreated baboons (n = 7) and from immunosuppressed animals (n = 5) undergoing different xenotransplantation protocols with pig organs were tested by endogenous FCCA and a similar assay also using exogenous complement, and the results were compared with those of a complement-dependent hemolytic assay to detect anti-pig antibodies (APHA). The influence of PI/FDA staining and the use of several mathematical models were analyzed. RESULTS: For both groups of animals, we observed high correlations between the endogenous and exogenous FCCA pathways and between calculations based on PI and FDA staining. Of the four mathematical models tested--the Von Krogh equation, two exponential models, and area under the curve--the Von Krogh equation was the most appropriate in terms of goodness of fit and concordance with APHA. CONCLUSIONS: FDA/PI FCCA is useful to measure endogenous and exogenous complement-mediated cytotoxicities, and it has advantages related to identification of potential new xenoantibodies. Although all four mathematical models produced acceptable solutions, the Von Krogh equation was the best option.     

Genotoxicity of analgesic compounds assessed by an in vitro micronucleus assay

Several analgesic compounds and mixtures of analgesics were examined for both cytotoxicity and ability to induce chromosomal damage in the normal rat-kidney cell line NRK-49F. Chromosomal damage was assessed using an in vitro micronucleus assay. Of all the compounds tested, only N-hydroxyparacetamol caused a high degree of cell death at the concentrations used. 4 analgesic compounds were found to be inducers of micronuclei in NRK cells; in order of decreasing potency these were: N-hydroxyparacetamol, N-hydroxyphenacetin, caffeine and paracetamol. An aspirin, phenacetin, caffeine mixture (APC) failed to induce micronuclei above the background level, and a paracetamol-codeine combination did not increase the level of micronuclei induction above that induced by paracetamol alone. This report suggests paracetamol and some related compounds are capable of inducing chromosomal damage in mammalian cells in vitro, which is consistent with recent reports of a possible paracetamol-DNA interaction.     

Rapid in vitro biocompatibility assay of endovascular stents by flow cytometry using platelet activation and platelet-leukocyte aggregation

Clinical studies suggest that stent design and surface texture are responsible for differences in biocompatibility of metallic endovascular stents. A simple in vitro experimental setup was established to test stent-induced degree of platelet and leukocyte activation and platelet-leukocyte aggregation by flow cytometry. Heparin-coated tantalum stents and gold-coated and uncoated stainless steel stents were tested. Stents were implanted into silicone tubes and exposed to blood from healthy volunteers. Platelet and leukocyte activation and percentage of leukocyte-platelet aggregates were determined in a whole-blood assay by subsequent staining for activation-associated antigens (CD41a, CD42b, CD62p, and fibrinogen binding) and leukocyte antigens (CD14 and CD45) and flow cytometric analysis. Blood taken directly after venous puncture or exposed to the silicone tube alone was used as negative controls. Positive control was in vitro stimulation with thrombin receptor activating peptide (TRAP-6). Low degree of platelet activation and significant increase in monocyte- and neutrophil-platelet aggregation were observed in blood exposed to stents (P < 0.05). In addition, leukocyte activation was induced as measured by increased CD45 and CD14 expression. Heparin coated stents continuously induced less platelet activation and leukocyte-platelet aggregation than uncoated stainless steel stents of the same length and shorter stents of the same structure. Stent surface coating and texture plays a role in platelet and leukocyte activation and leukocyte-platelet aggregation. Using this simple in vitro assay and whole blood and flow cytometry, it seems possible to differentiate stents by their potency to activate platelets and/or leukocytes. This assay could be applied for improving the biocompatibility of coronary stents.     

Combined boyden-flow cytometry assay improves quantification and provides phenotypification of leukocyte chemotaxis

Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response.