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At least 109 records · Page 6Linked to original sources

PubMed1986

Assay of protein C in human plasma: comparison of amidolytic, coagulation, and immunochemical assays.

We studied functional protein C activity, both anticoagulant and amidolytic, as well as protein C antigen in 30 normal subjects, several members of a family with congenital protein C deficiency, 18 patients with severe preeclampsia, 27 patients with coronary heart disease, including 15 patients with myocardial infarction and 12 with angina pectoris, 20 patients on stable oral anticoagulant therapy (thrombotest values: 3-12%) and three patients with disseminated intravascular coagulation. Protein C values measured by the coagulant assay were compared to those obtained with amidolytic and immunochemical assays. In all the groups studied, the activity assays (amidolytic and coagulant) correlated significantly with each other as well as with the immunochemical assay. In patients on oral anticoagulant therapy the coagulant assay gave lower protein C values than amidolytic and immunochemical assays. A good correlation was found between immunological and amidolytic protein C assays (r=0.90, p less than 0.001), immunological and coagulant protein C assays (r=0.93, p less than 0.001), and amidolytic and coagulant protein C assays (r=0.95, p less than 0.001) in all the samples studied without including the protein C values of patients on oral anticoagulant therapy. These results allow us to recommend the functional protein C coagulant assay in patients on stable oral anticoagulant therapy because only this assay evaluates the "in vivo" protein C function in these patients.

Angina Pectoris↗

PubMed2006

Significant correlation between the CD63 assay and the histamine release assay in chronic urticaria.

BACKGROUND: Antibodies directed to the alpha subunit of the high affinity IgE receptor and the IgE molecule are proposed to be of pathogenetic relevance in a group of patients with chronic urticaria (CU). The diagnosis of autoimmune chronic urticaria (ACU) is difficult; the autologous serum skin test (ASST) seems to be a useful screening test, but reliable, additional confirmatory methods are needed. OBJECTIVES: To assess the diagnostic value of a modified serum-induced basophil activation test, the CD63 expression assay, in the diagnosis of ACU by comparing the results of the CD63 assay with the results of the histamine release (HR) test, the ASST and serum levels of soluble CD40 ligand (sCD40L). METHODS: Using basophils from an atopic (DA) and a nonatopic (DNA) donor the activity of sera of 72 patients with CU were measured in HR assay by enzyme-linked immunosorbent assay and in CD63 expression assay by flow cytometry. An ASST was carried out in all patients; in 30 of the 72 patients sCD40L was detected and correlations were derived between the different assays. Sera of 20 normal controls and 26 patients with systemic autoimmune diseases were also tested in the HR assay and in the CD63 expression assay. RESULTS: Histamine-releasing activity was detected in the sera of 51% (DA) and 32% (DNA) of CU patients and 57% (DA) and 28% (DNA) of sera upregulated CD63 expression on the surface of basophils from the different donors. There was a significant correlation between the HR and the CD63 assays carried out on both donors, but the ASST showed a strong correlation with the HR assay only for basophils from the DA. The serum level of sCD40L was significantly higher in patients with CU compared with controls, but the difference between the autoimmune and the nonautoimmune groups was not significant. CONCLUSIONS: The CD63 expression assay seems to be a reliable functional test in the diagnosis of ACU, particularly if highly sensitive donor basophils are used, but the determination of the sCD40L serum level was not sufficient to differentiate between the autoimmune and the nonautoimmune patient groups.

Adolescent↗

PubMed2003

An ELISA assay for the detection of factor VIII antibodies - comparison with the conventional Bethesda assay in a large cohort of haemophilia samples.

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure factor VIII antibodies in haemophilia patients. The assay utilizes binding of the antibodies in the plasma to solid phase antigen, i.e. recombinant factor VIII which was subsequently detected by a human polyclonal IgG labelled with the alkaline phosphatase-p-nitrophenyl phosphate substrate system. Comparisons were made with the Bethesda assay for the quantitation of factor VIIII inhibitors. Dose response curves for the reference standards were consistently linear and reproducible. The assay was specific for factor VIII antibodies, showing a negative reaction for antibodies to other coagulation factors, antinuclear factors and antiphospholipid antibodies. Using this method, 312 samples from haemophilia A patients and 31 samples from healthy controls were screened for the presence of inhibitors and compared with the conventional Bethesda assay. Twenty-four cases were found to be positive for inhibitors in both the ELISA and Bethesda assay. Five additional cases were also found to be positive in the ELISA assay, which, however, were negative in Bethesda assay. One patient who was initially positive for factor VIII inhibitors both by the Bethesda assay and ELISA eventually became negative for the Bethesda assay (<0.5 BU/ml) but was still positive for the ELISA assay. The ELISA thus described had a specificity of 97.8% and a sensitivity of 100% when tested against a large cohort of haemophilia A samples.

Case-Control Studies↗

PubMedDate not supplied

Evaluation of MTT assay in agarose for chemosensitivity testing of human cancers: comparison with MTT assay.

The MTT assay in agarose, a simple colorimetric test performed in double-layer agarose, has been evaluated for chemosensitivity testing of fresh tumor samples from human cancers in comparison with the MTT assay. The absorbance of cells from fibroblast cell lines or normal tissues was markedly reduced in agarose. The chemosensitivity of cells from a carcinoma cell line or fresh tumor tissues was not apparently affected by the presence of almost 50% of fibroblast or nonmalignant cells in the MTT assay in agarose, whereas it did so in the MTT assay. The frequency of the differences between chemosensitivity of fresh tumor samples in both assays was increased, when the tumor tissue cells contained a higher proportion of nonmalignant cells, i.e. vimentin-positive cells. In 173 patients with various carcinomas, in vitro sensitivity to 7 drugs in the MTT assay in agarose was significantly greater than that in the MTT assay. Further, the MTT assay in agarose had a higher accuracy for prediction of either sensitivity or resistance than the MTT assay in a total of 38 in vitro-in vivo correlations. These results indicated that the MTT assay in agarose was a more suitable technique for the application to chemosensitivity of fresh tumor samples from patients with various carcinomas as compared to the MTT assay.

Adult↗

PubMed1997

Measurement of antibodies against glutamic acid decarboxylase 65 (GADA): two new 125I assays compared with [35S]GAD 65-ligand binding assay.

Recently, 65-kDa glutamic acid decarboxylase (GAD 65) antibodies (GADA) have been introduced as autoimmune markers in blood to confirm the diagnosis of insulin-dependent diabetes mellitus (IDDM). In this study, to evaluate two new assays that use 125I-labeled GAD 65, we assayed samples from 100 children with recent onset of diabetes and 100 control children, the results were compared with those of a [35S]GADA assay and with results for islet cell antibodies (ICA), the conventional autoimmune marker. Receiver operating characteristic (ROC) curve analysis showed one of the new assays (from RSR) to be more sensitive (P = 0.01) than the comparison ([35S]GADA) assay, whereas the second new assay (from Elias) was less sensitive (P < 0.001). The GADA frequency at 97.5% specificity was greatest in the comparison assay: 63 of 100 vs 41 of 100 (P < 0.01) and 53 of 100 (P = 0.16) in the RSR and Elias assays, respectively. Almost all GADA-positive patients had ICA, but one-third of the ICA-positive patients was GADA-negative. Accordingly, adding GADA analysis results to ICA testing increased the frequency of detection of autoimmune markers only slightly (from 81% to 85%). In conclusion, at 97.5% specificity the [35S]GADA assay seemed to be more efficient than the 125I assays, although the difference was significant only for the Elias 125I assay. Antigen-specific antibodies other than GADA may explain the difference in GADA and ICA frequencies.

Adolescent↗

PubMed1997

MTT-hybrid assay incorporates the advantages of both clonogenic and MTT assay radiosensitivity testing for fresh tumor samples.

After devising a 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-Hybrid assay that incorporates the advantages of both the clonogenic and standard MTT assays, we evaluated the validity of this system using a malignant fibrous histiocytoma (MFH) cell and four xenografted tumors. In the MFH cells the surviving fractions were correlated with radiation dose at three assays. In xenografted tumor cells, the surviving fractions determined by the MTT assay did not show a similar dose response, possibly because of contamination by normal cells. Changes in the surviving fraction of the xenografted cells were correlated to the radiation dose as determined by the clonogenic assays, but this assay took longer performance times and was not useful for evaluating tumors with a low planting efficiency. With the MTT-hybrid assay, changes in fractions of the four xenografted tumor cells were correlated to radiation dose. The MTT-hybrid assay required a smaller optimal number seeding cells and shorter assay time than those of the clonogenic assay. Therefore, the MTT-Hybrid assay can be used successfully to assess the radiosensitivity of both established tumor cell lines and fresh tumor samples.

Animals↗

PubMed1986

A comparison of the macrophage migration inhibition (MMI) assay and the semi-automated leucocyte adherence inhibition (SALAI) assay.

The present study was undertaken in order to compare simultaneously the semi-automated leucocyte adherence inhibition (SALAI) assay with the two-stage macrophage migration inhibition (MMI) assay in patients with colorectal disease. Eighty-six patients were assayed simultaneously in the MMI assay and the SALAI assays using patients' leucocytes and 83 patients were assayed simultaneously in the MMI assay and the SALAI assay using patients' serum. The SALAI assay using patients' leucocytes was more sensitive (70% vs 40%) and more specific (87% vs 73.3%) than the MMI assay. The SALAI assay was simple to perform, allowed many replicate samples and was more specific and sensitive in relation to the detection of colorectal cancer than other more commonly advocated tests. It may be useful as a screening test for colorectal cancer and it is suggested that further studies should be undertaken in this area.

Colonic Neoplasms↗

PubMed2006

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.

Animals↗

PubMed1987

Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture.

We compared an antigen capture assay (Abbott Laboratories, North Chicago, Ill.) with a reverse transcriptase assay to identify and quantify human immunodeficiency virus (HIV) in culture. In direct comparisons of serial dilutions of lymphadenopathy-associated virus type 1, the antigen assay was 100-fold more sensitive than the reverse transcriptase assay in detecting the virus. The antigen assay reacted strongly with 60 different HIV isolates but did not cross-react with human T-cell lymphotropic virus type I, human T-cell lymphotropic virus type II, cytomegalovirus, varicella-zoster virus, herpes simplex virus type 1, Epstein-Barr virus, adenovirus type 5, or poliovirus type 1 or with extracts from four different control human cell lines and eight different phytohemagglutinin-stimulated normal human lymphocytes. Peripheral blood lymphocyte samples from 50 individuals were evaluated by both the antigen assay and the reverse transcriptase assay. The cells from the 34 seropositive individuals were all positive by the antigen assay (range, 3 to 9 days; average time, 5.9 days) and the reverse transcriptase assay (range, 7 to 16 days; average time, 9.6 days). Cells from the 16 seronegative individuals were negative by both assays. These results indicate that the antigen assay is an important addition to the monitoring of HIV production in the lymphocytes of infected patients.

Cell Line↗

PubMed1999

Comparison of the PACE 2 assay, two amplification assays, and Clearview EIA for detection of Chlamydia trachomatis in female endocervical and urine specimens.

Screening for sexually transmitted diseases (STDs) in a greater proportion of sexually active patients has become an accepted protocol by most health care providers. The purpose of this study was to compare the current test methods for detection of Chlamydia trachomatis used at the University of South Alabama, the PACE 2 assay (Gen-Probe) and the Clearview EIA (Wampole Laboratories), with two amplification technologies, the AMP CT (Gen-Probe) and LCx (Abbott) assays. In addition, a number of demographic parameters were ascertained by asking questions at the time of examination as well as for health care provider concerns and preferences. One urine and four endocervical swab specimens were collected in random order from 787 female patients attending one of four obstetrics-gynecology clinics. Eighty-seven percent of patients had no STD-related symptoms. Patients were considered positive for C. trachomatis if three or more assays (swab and/or urine) were positive. Abbott and Gen-Probe confirmed discrepant results by alternate amplified assays. A total of 66 true-positive specimens were detected by use of the combination of endocervical swabs and urine specimens. After discrepant analysis, sensitivities for endocervical swab specimens for the EIA and the PACE 2, LCx, and AMP CT assays were 50, 81, 97, and 100%, respectively. Sensitivities for the LCx and AMP CT assays with urine specimens were 98 and 81%, respectively. The prevalence of C. trachomatis was 8.4%, as determined by amplification technology. Overall, the amplification technologies were the most sensitive methods with either swab (AMP CT assay) or urine (LCx assay) specimens. The PACE 2 assay offered the advantage of a simpler and less expensive assay with acceptable sensitivity. The clearview CT EIA, while yielding a rapid in-office result, had unacceptably low sensitivity. The wide variation in performance with amplification assays with urine specimens as reported in both this study and the literature obviates the need to clarify optimal parameters for this specimen type.

Alabama↗

PubMed2000

Comparative evaluation of three human immunodeficiency virus genotyping systems: the HIV-GenotypR method, the HIV PRT GeneChip assay, and the HIV-1 RT line probe assay.

Evaluation of drug resistance by human immunodeficiency virus (HIV) genotyping has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. We compared three genotyping methods for identification of mutations known to confer drug resistance in the reverse transcriptase (RT) and protease genes of HIV type 1 (HIV-1). The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.) with the ABI 377 DNA sequencer (Applied Biosystems Inc.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) were used to genotype plasma samples from HIV-infected patients attending the University of Wisconsin Hospitals and Clinics and the Mayo Clinic. At the time of analysis, patients were failing combination therapy (n = 18) or were treatment naive (n = 6). Forty codons of the RT and protease genes were analyzed by GenotypR and GeneChip for resistance-associated mutations. LiPA analyzed seven RT codons for mutations. Each sample was genotyped by all three assays, and each assay was subjected to pairwise comparisons. At least 92% of the codons tested (by the three assays) in paired comparisons were concordant. GenotypR and GeneChip demonstrated 96.6% concordance over the 40 codons tested. GenotypR identified slightly more mutations than GeneChip and LiPA; GeneChip identified all primary mutations that corresponded to failing treatment regimens. Each assay identified at least 84% of the mutations identified by the other assays. Mutations that were discordant between the assays mainly comprised secondary mutations and natural polymorphisms. The assays had better concordance for mutations that corresponded to current failing regimens, present in the more predominant viral quasispecies. In the treatment-naive patients, GenotypR, GeneChip, and LiPA mainly identified wild-type virus. Only the LiPA identified K70R, a possible transmitted zidovudine resistance mutation, in the RT gene of a treatment-naive patient. We conclude that although discrepancies in results exist between assays, each assay showed a similar capacity to identify potentially clinically relevant mutations related to patient treatment regimens.

Anti-HIV Agents↗

PubMed2000

Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays.

AIM: To evaluate the clinical usefulness of three commercially available assays for Her-2/neu oncogene and protein measurements. The Her-2/neu protein is overexpressed, mostly as a result of gene amplification, in 20-30% of human breast cancers, and has been shown to have prognostic and predictive value for treatment with chemotherapy or the new monoclonal antibody, Herceptin. METHODS: An immunohistochemistry (IHC) assay using the Dako polyclonal antibody A0485, which measures the Her-2/neu protein, was compared with two new Food and Drug Administration (FDA) approved fluorescence in situ hybridisation (FISH) assays--INFORM and PathVysion, in a cohort of 52 formalin fixed, paraffin wax embedded breast tissues. These tissues were selected randomly from 84 consecutive infiltrating breast cancer specimens, which were first stratified according to the Her-2/neu protein levels as measured by IHC. RESULTS: The two FISH assays achieved a 98% concordance rate: 14 specimens (27%) showed Her-2/neu gene amplification and 37 specimens (71%) showed no Her-2/neu gene amplification. The PathVysion assay had certain advantages over the INFORM assay. In contrast, the IHC assay detected Her-2/neu overexpression in a high percentage of cases, including 13 high positive specimens (25%) and 13 medium positive specimens (25%). Although 10 of these 13 IHC high positive specimens showed gene amplification by FISH, nine of 13 IHC medium positive specimens showed no gene amplification. Statistical analyses showed that the differences between IHC and FISH assays were primarily in the specimens with medium positive IHC, but negative FISH results. CONCLUSIONS: Because of the increasing importance of the Her-2/neu oncogene and oncoprotein in the clinical management of patients with breast cancer, the accurate and consistent evaluation of Her-2/neu status is crucial. This study suggests that the best approach is to combine both IHC and FISH assays; that is, to use the IHC assay as a triage step, followed by the PathVysion FISH assay to analyse the IHC medium and high positive cases.

Biomarkers, Tumor↗

PubMed1990

Monitoring therapy with LMW heparin: a comparison of three chromogenic substrate assays and the Heptest clotting assay.

Three LMW heparins (LMWH), one unfractionated heparin (UH), and international standards of LMWH and UH were compared in three chromogenic substrate (CS) assays and the 'Heptest' clotting assay. With a two-stage CS assay, linear standard curves were obtained in the 0.1-1.0 U/ml range, nearly coinciding for all preparations. With the one-stage CS assays, standard curves were curvilinear and similar for UH and the LMWH groups. In the Heptest assay, standard curves were linear for UH but not for LMWH. Mean recovery of LMWH, added to patients' plasma samples was 70-98% for the four assays. Variation between individual recoveries was much greater with Heptest (coefficient of variation (CV) 35-44%) than with one-stage CS assays (CV 14-21%) or two-stage CS assays (CV 7-8%). For monitoring LMW heparin therapy, CS assays seem preferable to Heptest. The two-stage CS assay had superior accuracy, but the one-stage CS assays were easier to perform.

Automation↗

PubMed2003

The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity.

BACKGROUND: The interferon-gamma (IFN-gamma) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-gamma secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-gamma secretion. METHODS: We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-gamma ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (alphaEN-EBV and alphaJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. RESULTS: When the GrB ELISPOT was directly compared to the IFN-gamma ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-gamma secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. CONCLUSION: Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses.

Journal Article↗

PubMed2000

Monitoring unfractionated heparin therapy: relationship between eight anti-Xa assays and a protamine titration assay.

Several studies have demonstrated that heparin assays, such as anti-activated factor X (anti-Xa) assays, can be successfully substituted for activated partial thromboplastin time for heparin dosage monitoring. A number of different assays are available and the relationship between results with different techniques is largely unknown. The aim of the present study was to assess the relationship between heparin assays by protamine titration and anti-Xa assays. Samples were collected from 43 patients receiving unfractionated heparin (UFH). In each sample, the heparin level was determined using a protamine titration assay and eight commercially available anti-Xa assays. The mean heparin level by protamine titration was 0.31 U/ml. Mean anti-Xa activity results ranged from 0.40 to 0.42 IU/ml for the three clotting-based assays, and from 0.32 to 0.40 IU/ml for five chromogenic assays. Thus mean results of different anti-Xa assays varied by up to 30%. The range of anti-Xa activity equivalent, on average, to 0.2-0.4 U/ml by protamine titration, considered to be the therapeutic range, was approximately 0.25-0.5 IU/ml, depending on the assay. The relationship between results of clotting and chromogenic methods was similar irrespective of whether or not warfarin-induced prolongation of international normalized ratios was present.

Antibodies↗

PubMed1993

Enhanced chemosensitivity in acute myeloid leukemia by hematopoietic growth factors: a comparison of the MTT assay with a clonogenic assay.

The MTT assay, a colorimetric assay, is found to be suitable for chemosensitivity testing. Recently, it has been suggested that hematopoietic growth factors (HGF) may enhance the effects of cytostatic drugs in acute myeloid leukemia (AML). We therefore studied the effects of granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with cytosine arabinoside (Ara-C), daunorubicin (DNR), mitoxantrone (MXT), or etoposide (VP-16) by using the MTT assay. The results were compared with in vitro clonogenic assays. Briefly, AML cells of nine patients were incubated in the presence or absence of G-CSF, IL-3, or GM-CSF under serum-free conditions for 24 hours. Next, for the MTT assay, Ara-C (final dilution range: 0.0024-240 micrograms/ml), DNR (final dilution range: 0.05-3.2 micrograms/ml), MXT (final dilution range: 0.05-3.2 micrograms/ml), or VP-16 (final dilution range: 0.1-100 micrograms/ml) were added and incubated for 48 hours. Cell survival was determined and IC75 values (75% reduction as compared to control cultures) were calculated. For clonogenic assays, the three lowest drug concentrations were used. After 48 hours, the clonogenic response was determined in serum-free, semi-solid cultures with G-CSF, IL-3, or GM-CSF. The results obtained by the MTT assay showed no significant enhancement of cytotoxicity by HGF on cytostatic drug preincubated cells compared to cytostatic drugs alone. The results obtained by the clonogenic assays showed increased cytotoxicity of Ara-C combined with G-CSF, IL-3, or GM-CSF. The median IC75 values of Ara-C decreased from 0.056 to 0.0168 microgram/ml with G-CSF (p = 0.01), from 0.108 to 0.0168 microgram/ml with IL-3 (p = 0.004) and from 0.12 to 0.0204 microgram/ml for GM-CSF (p = 0.02). Only moderate enhanced cytotoxicity was observed when VP-16 was combined with IL-3 (p = 0.036) or GM-CSF (p = 0.036), but not with G-CSF. No enhanced cytotoxicity of DNR and MXT to clonogenic AML cells was found when these agents were combined with HGF stimulation. The results indicate that the MTT assay underestimates HGF enhanced cytotoxicity of Ara-C or VP-16 to clonogenic cells. Therefore, the assay is not useful for accurately detecting differences of clonogenic response due to the proliferative status of cells. In this paper, the potential explanations for the failure of the MTT assay are discussed.

Adult↗

PubMed1989

Predictability of in vivo chemosensitivity by in vitro MTT assay with reference to the clonogenic assay.

The MTT assay reported by Mosmann is a rapid and convenient colorimetric assay for cellular growth and survival in vitro. In this paper, the MTT assay was modified as a chemosensitivity test, and its potential was investigated. Using 10 human tumor xenografts in athymic nude mice, the predictability of in vitro antitumor effects of drugs using the MTT assay was compared with that using the clonogenic assay. The MTT assay showed excellent reproducibility, and the predictable rate in this assay was 86.7%, with 100% true-positive and 77.8% true-negative rates, almost equivalent to the 90.0% predictable rate of the clonogenic assay. This method also has several advantages with respect to rapidity, quantitation, management of many samples, and cell number required for the assay. Application of this assay to chemosensitivity testing seems to be valuable and useful.

Animals↗

PubMed2003

Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop.

The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.

Animals↗