WO1989006162A1 - Procedes et appareil d'evaluation de la resistance des tumeurs - Google Patents

Procedes et appareil d'evaluation de la resistance des tumeurs Download PDF

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Publication number
WO1989006162A1
WO1989006162A1 PCT/US1988/004574 US8804574W WO8906162A1 WO 1989006162 A1 WO1989006162 A1 WO 1989006162A1 US 8804574 W US8804574 W US 8804574W WO 8906162 A1 WO8906162 A1 WO 8906162A1
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WIPO (PCT)
Prior art keywords
cells
tumor cells
tube holder
slide
chambers
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PCT/US1988/004574
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English (en)
Inventor
Larry M. Weisenthal
Thomas Edward Duarte
Robert A. Nagourney
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Oncotech Incorporated
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Publication of WO1989006162A1 publication Critical patent/WO1989006162A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5021—Test tubes specially adapted for centrifugation purposes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B5/00—Other centrifuges
    • B04B5/04—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers
    • B04B5/0407—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles
    • B04B5/0414—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles comprising test tubes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an improved in vitro method and apparatus to detect the resistance of tumor cells to chemotherapy.
  • it relates to using .iri vitro tests to monitor the acquisition of resistance to chemotherapy in human tumors and methods to assess alternative therapeutic regimes.
  • the assay provides a means for individually counting the number of tumor cells surviving in the presence or absence of drug when the biopsied tumor cells are subjected to the assay, and has been used to predict the chemosensitivity of human tumors to various drugs (Weisenthal, L.M., et al., Cancer 51:1490-1495 (1983)).
  • Weisenthal, L.M., et al., Cancer 51:1490-1495 (1983) A review of the development and relevance of this assay is set forth in Shoesnthal, L.M., et al., in Recent Results in Cancer Research 94:161-173, Springer Verlag, Berlin-Heidelberg (1984).
  • a typical assay using 60 to 96 individual culture tubes requires 2 to 3 hours for slide preparation and up to 8 hours for slide reading.
  • a rapid method for estimating the lowest effective concentration of chemotherapeutic agent at which most of the tumor cells in a sample are killed would improve the usefulness and practicability of the DiSC assay.
  • the invention provides a method and apparatus for performing a differential staining cytoxicity assay to determine the sensitivity of tumor cells .in vitro to a therapeutic agent administered .in vitro by detecting the ability of cells to exclude a dye after incubation with the agent.
  • the method includes assaying multiple samples of tumor cells from an individual subject using a range of concentrations of the therapeutic agent to provide an estimate of the lowest effective concentra ⁇ tion capable of killing most of the tumor cells. This method obviates the necessity for tedious cell counting, while retaining the advantages of the assay in identifying tumor cells in the presence of normal cells included in the sample.
  • the ' apparatus facilitates processing of multiple samples of tumor cells covering the range of concentrations of the therapeutic agents selected, and includes a centrifuge tube holder containing a plurality of centrifuge tube chambers, a slide to receive the centrifuged cells from each of the inserted tubes, separated from the tubes by a filter punctured with circular openings matching the top circumferences of the tubes to absorb liquid during centrifugation, and to permit the passage of cellular material from the chambers to the slide that is placed over the filter on top of the centrifuge tube holder.
  • a clamp is used to fasten the filter and slide on top of the tube holder after the tubes are inserted.
  • Figure 1 is an assembled isometric view of the apparatus and clamp for securing the apparatus together
  • Figure 2 is an exploded isometric view of the apparatus at the point of assembly showing the centrifuge tube holder, centrifuge tubes, filter and glass slide.
  • Figure 3 is a staggered, assembled vertical section of the apparatus having the centrifuge tubes inserted.
  • Figure 4 is an enlarged view of a portion of Figure 3 showing the centrifuge tubes in and extending above the chambers that receive the centrifuge tubes.
  • Figure 5 is a photograph of a slide containing tumor cells treated with five dilutions of a chemothera ⁇ Commissionic drug using the apparatus of the invention.
  • Figure 6A-F are photographs of disks from a slide prepared using the apparatus of the invention, each disk containing tumor cells treated with a different concen ⁇ tration of the drug nitrogen mustard.
  • Figure 7 is a graph of the comparison of the percent cell survival method and the EMT 90 method.
  • Figure 8 is a graph of results of testing different chemotherapeutic agents on tumor cells using the percent cell survival method.
  • Figure 9 shows results of different chemotherapeutic agents on tumor cells determined by the EMT 90 method of the invention.
  • Figure 10 compares the response of tumor cells to the drug CDDP alone and with V/L obtained using the percent cell survival criterion and EMT/90 method.
  • the present invention employs the .in vitro DiSC assay procedure described in detail by Weisenthal et al., in Cancer Treatment Reports, 70(11) :1283-1295 (1986), incorporated by reference herein, modified as described below.
  • the invention also comprises an apparatus for carrying out the centrifugation of multiple samples containing tumor cells constituting a range of concentrations of a therapeutic agent for use in the method of the invention.
  • the method relies on the use of serial dilutions of the therapeutic compound to estimate the minimum effective concentration of drug at which 90% of the tumor cells in a population from a tumor sample are killed (i.e. 10% tumor cell survival). This endpoint concentration may be used to predict the concentration of therapeutic agent below which the tumor cells will be increasingly resistant to the agent.
  • the relative drug sensitivity or resistance of the specimens may be determined.
  • the method and apparatus may be used in conjunction with strategies to evaluate the continued effectiveness of the particular therapeutic agent, and/or to modify a therapeutic protocol in a human subject, as described by Weisenthal et al., Cancer Research, supra. General Methods
  • bone marrow specimens, peripheral blood buffy coats (approximately 1 cm 3 ), or other appropriate tumor cell specimens are diluted with 6 to 10 volumes of complete RPMI-1640 medium containing 10 units/ml of heparin. This is underlayered with Ficoll-diatrizoate (Lymphocyte Separation Medium, Organon Teknika Corporation, Durham, NC) and centrifuged. The interface layer is washed and counted. Solid tumors are manually teased apart after incubation with collagenase/DNAse.
  • Cells (3 to 8 X 10 4 ) are aliquoted into 0.8 ml capacity conical polystyrene or polypropylene tubes and brought to a volume of 0.1 ml with complete RPMI-1640 medium plus 10% heat-inactivated fetal calf serum.
  • concentrations, preferably five serial dilutions, of a drug solution are added to the cell suspensions and incubated for time periods according to the drug to be assayed. For example cells may be incubated either short term (e.g. 1 hour), longer term or continuously.
  • DRBC acetaldehyde-fixed duck red blood cells
  • DRBC acetaldehyde-fixed duck red blood cells
  • 0.2 ml Fast Green (solid tumors) or Fast Green Nigrosin (hematologic neoplasms) (1% Fast Green and 0.5% Nigrosin) in 0.15M NaCl is added to stain cells killed by the drug and the tubes vortexed.
  • the cell suspensions are again vortexed, and then transferred into a centrifuge apparatus, such as the apparatus described herein, to apply the cells onto a microscope slide in order of lowest to highest drug concentration over the dilution range.
  • the cell disks located on the slide after centri- fugation are counterstained with hematoxylin and eosin (H & E) (solid tumors) or are fixed with methanol for 20 seconds and counterstained with Wright-Giemsa (hema- tologic neoplasms) or with another stain such as PAS or immunoperoxidase to indicate living cells.
  • H & E hematoxylin and eosin
  • methanol methanol for 20 seconds
  • Wright-Giemsa hema- tologic neoplasms
  • PAS immunoperoxidase
  • EMT 90 a multiple dilution concentration range (preferably 5 dilutions) is tested.
  • the assay endpoint is the lowest drug concentration (dilution) which results in essentially total tumor cell kill (cell kill estimated to exceed 90%).
  • drugs in the selected range of concentrations e.g. serial dilutions in the ranges of 1:2, 1:3, 1:5 or 1:10 depending on the therapeutic agent being tested
  • the lowest con ⁇ centration (X) of therapeutic agent is determined individually for each drug for example using the clinical pharmacokinetic data available in the literature and from experiments to obtain preliminary test data. Times of exposure in the assay may be determined by performing preliminary experiments to determine which assay conditions most closely predict clinical outcomes.
  • the assay should be calibrated for different drugs with either a short-term, longer term or a continuous exposure. Continuous exposures are simpler and the assay can be calibrated on the basis of a continuous exposure for most drugs.
  • specimen preparation depends on the nature of the tumor sample being taken, and follows standard guidelines for obtaining specimens for human studies.
  • specimens may include EDTA anticoagulated blood or marrow specimens and solid tumor biopsy specimens. Solid tumors are collected in complete tissue culture medium with antibiotics.
  • Stained slides are then first read on a computerized Image Analyzer (Image Technology, Model 3000, Deer Park, New York).
  • the Image Analyzer is initially set to count only the DRBC, to determine that all of the 5 disks on the slide contain approximately the same number of DRBC. If a particular disk has less than one-half the amount of DRBC as the- other disks, that particular assay dilution is deemed to be unreliable. If the disk has one-half or more the "standard" number of DRBC, then the surviving cell estimate for that particular disk is divided by the ratio of the actual number of DRBC for that disk to the actual number of DRBC in the "standard" control disks. (This is done for quality control purposes and to normalize cell numbers in cases where there may be some loss of fluid during the cytocentrifuge process.)
  • the slides are then screened by experienced technicians who are capable of recognizing tumor cells from non-tumor cells by detecting the living cells that have counterstained.
  • the technician begins at the highest drug concentration, and proceeds to view the other slide cell disks in succession.
  • the technician determines the first disk in which there appear to be greater than 10% surviving tumor cells relative to the control cultures.
  • each disk is evaluated as a "yes” or “no" endpoint; "yes”, if there are more than 10% surviving cells relative to the control cultures, and "no", if there are fewer than 10% surviving cells relative to the control cultures.
  • the EMT 90 The lowest concentration capable of killing 90% of the tumor cells is designated "the EMT 90.”
  • the first disk corresponding to the highest drug concentration (e.g. 16X) will contain predominantly dead tumor cells.
  • the final disk containing the lowest drug concentration (e.g. IX) will contain predominantly living tumor cells.
  • the first disk with tumor cell survival estimated to be less than 10% of the control culture will present the lowest drug concentration capable of 90% cell kill.
  • the following disk corresponding to the next lowest drug dilution will contain numerous living tumor cells, obviously more than 10% of the control cultures.
  • tumor cell kill rather than 100% tumor cell kill is that there are occasionally situations in which one or two tumor cells are difficult to distinquish from normal cells; that is, either the tumor cells look relatively normal (e.g. resembling macrophages) or the normal cells may look abnormal so as to resemble tumor cells. In most assays, the vast majority of the cells can be accurately distinguished by an experienced reader as either being tumor cells or normal cells, but there may occasionally be a few cells which are difficult to distinguish.
  • Slides are preferably independently scored by two different readers. If the EMT 90 value agrees within one serial dilution, i.e. one unit of EMT 90 dilution X to 2X, between two observers, the results are averaged (e.g. to 1.5X). If the results disagree by more than one EMT 90 unit, then slides are reevaluated by the same observers and by additional observers until acceptable agreement is obtained within one EMT 90 unit and the corresponding results are then averaged and standard deviations are presented. Based on numerous tests, observations from experienced observers agree within one EMT 90 unit more than 90% of the time on first reading. Because the criterion of 90% tumor kill is so stringent, even substantial variation in the number of tumor cells/DRBC in each disk does not cause major differences in the EMT 90 value.
  • Centrifugation of the samples containing tumor cells treated with a range of dilutions of chemotherapeutic drug are preferably performed in the centrifugation apparatus shown in Figures 1-4 and described herein.
  • This apparatus allows multiple cell disks to be placed on a single slide, permitting a technician or automated reader device to rapidly scan an entire therapeutic agent concentration range from lowest to highest.
  • Centrifuge tube holder 10 is shown in Figure 2 having a base 12 and top 14 mounted in spaced generally parallel arrangement with the base 12.
  • a ledge 16 is formed by an extension of the top 14 of the assembly over the body 18.
  • the combination of the apertures 20 and chambers 22 is designed to receive a plurality of centrifuge tubes 24.
  • the base 12 contains a plurality of apertures 26 connected to the chambers 22 for facilitating removal of the centrifuge tubes 24.
  • The. top 14 also may contain raised projections 28 for holding a filter 30 as described below.
  • the tube holder 10 is preferably made of a hard plastic such as acrylic (e.g. Lucite) or other material such as aluminum.
  • the centrifuge tubes 24 preferably taper to a diameter that is smaller at the bottom of the tube than at the top as shown in Figure 2 and include a cuff 32 at the top of the tube that extends above the aperture 20 and the top 14 of the assembly.
  • the tubes may be constructed of a plastic such as polypropylene or polystyrene or similar material.
  • the tube holder 10 is designed to be used with a planar substrate such as a glass slide 34 for receiving centrifuged cellular material.
  • the slide 34 may be manufactured so as to contain visible discrete regions, (e.g. circular disks, to receive cellular material from samples contained in the centrifuge tubes 24).
  • the filter 30 is inserted between the tube holder 10 and the slide 34 to absorb fluids from the samples in the centrifuge tubes 24 during operation.
  • the filter 30 has apertures 38 that correspond to the apertures 20 in the top 14 of the tube holder 10, and openings 40 that receive the pegs 28 on the top 14 of the tube holder 10 to secure the filter onto the tube holder and to maintain alignment with the apertures 38 on the filter, and the cuffs 32 of the tubes.
  • the typical order of assembly is the insertion of centrifuge tubes 24 containing a liquid sample into the chambers 22 in the tube holder 10, placement of the filter 30 onto the top 14 of the tube holder aligning the apertures 38 over the apertures 20 in the top 14 of the tube holder 10, followed by placing the slide 34 on top of the filter 30.
  • the volume of fluid sample used in the centrifuge tubes of the apparatus is preferably in a range of from 0.08 to 0.12 ml to prevent saturation of the filter paper with liquid.
  • the slide 34 and filter 30 are preferably secured onto the tube holder 10 by a clamp 42.
  • the position of the centrifuge tubes 24 within the chambers 22 is shown in Figure 3, which presents two tubes 24 in section and a tube 44 in elevation.
  • Figure 4 when assembled, the tops 32 of the centrifuge tubes 24 are pressed against the filter and slide so as to reduce the deposit of cellular material from each centrifuge tube 24 outside of the region on the slide corresponding to the aperture 20 in the tube holder 10.
  • a particular clamp 42 is shown in the Figures for the assembly of the present invention, it is envisioned that other types of clamps could be used embodying the same principles of attachment.
  • a generally rectangular arrangement with five (5) chambers 22 has been shown for the assembly, it is contemplated that other configurations and numbers of chambers could be used without departing from the scope of the invention.
  • centrifuge tubes 24 Once the desired number of centrifuge tubes 24 have been placed in the tube holder 10 and the filter 30 and slide 34 have been clamped in place, the entire assembly is designed to be placed in a centrifuge such as a Shandon cytocentrifuge (Shandon Southern Instruments, Inc., Sewickley, PA) which is subjected to centrifugal forces during a centrifugation run.
  • a centrifuge such as a Shandon cytocentrifuge (Shandon Southern Instruments, Inc., Sewickley, PA) which is subjected to centrifugal forces during a centrifugation run.
  • a typical slide having five disks containing cells treated with five concentrations of a drug is depicted in Figure 5.
  • the EMT 90 values obtained can be used as an index of the efficacy of possible treatments for tumor cells biopsied on an individual basis.
  • the EMT 90 values if obtained at various stages of drug therapy for the tumor can serve as an index for monitoring the acquisition of resistance to the treatment tested.
  • the prospective efficacy of proposed changes in treatment protocol can also be evaluated.
  • the invention method can be used to compare sensitivity to various therapies, for example, those used in chemotherapy.
  • Commonly used drugs include, for example, mechlorethamine, melphalan, carmustine, cytarabine, dexamethasone, doxorubicin, cisplatin, etoposide, vincristine and related antineoplastic agents as well as immune effectors such as monoclonal antibodies, lymphokines, lymphokine- activated effector cells and interferons.
  • the sensitivity of tumors to these materials can also be evaluated.
  • chemotherapeutic agents which have complementary functions, such as membrane-altering drugs.
  • the potency of a chemotherapeutic agent is thus altered by coad inistration of, for example, cofactors and drugs used for other purposes. Whether such supplementation will potentiate a drug to which resistance has been acquired can be determined by comparing the EMT 90 for the drug with and without the supplement.
  • tumor cells were obtained by bone marrow aspiration on a patient having acute lymphoblastic leukemia (ALL) (Memorial Hospital Medical Center, Long Beach, CA) .
  • ALL acute lymphoblastic leukemia
  • the cells were isolated according to the methods described in Cancer Treat. Rep. 70:1283-95 (1986), incorporated by reference herein.
  • the cells were placed in polypropylene microtubes (0.8 ml) and were exposed to fivefold dilutions (0.25 mg/ml or IX; 0.5 mg/ml or 2X; , 1.0 mg/ml or 4X; 2.0 mg/ml or 8X and 4.0 mg/ml or 16X) of the drug nitrogen mustard (mechlorethamine hydrochloride, Merck, Sharp and Dohme, West Point, PA) for a continuous 4 day period of culture.
  • Acetaldehyde-fixed DRBC (50,000) were then added to each tube along with 20 ml Fast Green (Sigma Chemical Co., St. Louis, MO.) and Nigrosin (Sigma Chemical Co., St. Louis, MO.).
  • Culture tubes were then transferred to the 5 chamber acrylic centrifuge tube holder apparatus and the filter paper, planar slide and clamp were assembled as depicted in Figures 1 and 2.
  • cytocentrifugation at 500 rpm on a Shandon cytocentrifuge (Shandon Southern Instruments, Inc., Sewickley, PA) the slides were disassembled from the apparatus and were counterstained with Wright-Giemsa Stain (Sigma Chemical Co., St. Louis, MO).
  • FIG. 6A-F Photomicrographs (original magnification 400X) of cells treated with the five concentrations of nitrogen mustard are shown in Figure 6A-F.
  • Figure 6A control; Figure 6B: 0.25 mg/ml; Figure 6C: 0.50 ml/ml; Figure 6D: 1.0 mg/ml; Figure 6E: 2.0 mg/ml and Figure 6F: 4.0 mg/ml.
  • Figure 6A control; Figure 6B: 0.25 mg/ml; Figure 6C: 0.50 ml/ml; Figure 6D: 1.0 mg/ml; Figure 6E: 2.0 mg/ml and Figure 6F: 4.0 mg/ml.
  • Each photograph depicts a representative portion of a disk.
  • This figure demonstrates the ease with which an assay may be performed and a single slide visually scanned by a technician to determine the drug cutoff concentration for 90% tumor cell kill.
  • almost all of the tumor cells in the first disk IX concentration or 0.25 mg/ml; Figure 6B) are viable.
  • the method and apparatus of the invention were used to assay resistance of tumor cells from two patients (Memorial Hospital Medical Center, Long Beach, CA) with acute lymphoblastic leukemia to the drugs vincristine (Eli Lilly Co., Indianapolis, IN) and doxorubicin (Adria Laboratories, Columbus, OH).
  • Cells were obtained from two different patients by means of bone marrow aspiration. Cells were isolated according to the method described in Cancer Treat. Rep. 70:1283-95, (1986), incorporated by reference herein. Cells were exposed to fivefold drug dilutions (as shown in the legend to the X axis in Figure 7), for a continuous 4 day period of culture.
  • the method and apparatus of the invention were used to determine the ability of additional drugs to 5 circumvent acquired resistance of tumor cells and the results compared to those obtained in the same assay by calculating percent cell survival.
  • LPAM mephalan
  • VCR vincristine
  • BCNU carmustine
  • HN2 nitrogen mustard
  • VP16 etoposide °
  • V/L verapamil/lidocaine
  • verapamil hydrochloride Searle, Chicago, IL
  • lidocaine Astra Pharmaceutical Products, Inc., Westboro, MA.
  • concentrations of drugs were used (concentrations were determined by training set data including results from correlation of .in vitro and .in 5 vivo applications of the drug): LPAM, 3.12(4X); VCR,
  • Cells were obtained from a single patient having acute lymphoblastic leukemia and were assayed as described above using a series of 5 dilutions of each drug and plated on slides in groups of five using the apparatus of Figures 1-4. The results were determined using the percent cell survival method and the EMT 90 assay as described in Example I.
  • Figure 9 summarizes the results for the response of tumor cells to five concentrations of five different drugs using the EMT 90 method.
  • V/L V/L. (A one unit variation or greater was determined to be significant since the difference between 16X and 8X is within the range of human observer variability, whereas the difference between 32X and 8X exceeds the range of observer variability.)
  • ovarian cancer The patient had failed three previous therapeutic regimes using (1) cyclophosphamide, doxo ⁇ rubicin, cisplatin; (2) high dose intraperitoneal cisplatin; and (3) cisplatin plus cytarabine.
  • the drug concentrations selected for the other four drugs provided less than 90% tumor cell kill.
  • Tumor cells were obtained from a paracentesis of the patient's ovarian tumor ascites fluid and were incubated for four days with the following concentrations of cisplatin (Bristol-Myers Laboratories, Syracuse, NY), (CDDP) 0.75 mg/ml (IX); 1.5 mg/ml (2X); 3.0 mg/ml (4X) ; 6.0 mg/ml (8X) and 12 mg/ml (16X) alone and in combination with V/L (0.5 mg/ml verapamil and 6 mg/ml lidocaine) , and processed as described above in Example II.
  • the EMT 90 method provides an assay comparable to or more sensitive than the percent cell survival DiSC assay, and is also capable of indicating dramatic shifts in cell sensitivity with alternative therapeutic regimes.
  • the method and apparatus of the present invention relying on an effective drug concentration rather than percent cell survival, provide greater efficiency and reproducibility for assays to determine chemosensitivity using differential staining of tumor cells.
  • the problems inherent in individual cell counting including tumor cell clumping are eliminated, and the speed of determining results are dramatically increased.
  • a trained technician can review 12 different assay slides (60 individual disks) and quantitate results at a rate of approximately 1 minute per slide or 12 minutes total with the EMT 90 method, compared to an average time of up to six (6) hours using the percent cell survival method previously described.

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Abstract

Le procédé amélioré permettant d'évaluer les chances de succès de thérapie pour le traitement de tumeurs est fondé sur l'estimation de la plus basse concentration effective d'un agent thérapeutique pouvant tuer une majorité de cellules tumorales in vitro. Le procédé comprend un test et un appareil pour analyser simultanément une série de dilutions d'un agent thérapeutique en utilisant un indice de destruction de cellules de 90 % comme concentration cytotoxique effective de l'agent.
PCT/US1988/004574 1988-01-04 1988-12-21 Procedes et appareil d'evaluation de la resistance des tumeurs WO1989006162A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994009352A2 (fr) * 1992-10-14 1994-04-28 Andrew George Bosanquet Procede et appareil permettant de realiser des tests, notamment des tests comparatifs
FR2762911A1 (fr) * 1997-04-30 1998-11-06 Cytotech Distribution Sa Dispositif et methode d'analyse de preparations cytologiques
WO2010135468A1 (fr) * 2009-05-19 2010-11-25 Vivia Biotech S.L. Procédés permettant de fournir des essais de médicaments personnalisés ex vivo pour des tumeurs hématologiques
EP2335825A1 (fr) * 2009-12-21 2011-06-22 F. Hoffmann-La Roche AG Unité et dispositif pour la préparation de cellules et/ou de particules dans un liquide et procédé d'analyse microscopique
EP2372367A1 (fr) * 2010-04-01 2011-10-05 F. Hoffmann-La Roche AG Procédé informatisé pour utiliser une cellule d'échantillon automatique

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WO1994009352A2 (fr) * 1992-10-14 1994-04-28 Andrew George Bosanquet Procede et appareil permettant de realiser des tests, notamment des tests comparatifs
WO1994009352A3 (fr) * 1992-10-14 1994-06-23 Andrew George Bosanquet Procede et appareil permettant de realiser des tests, notamment des tests comparatifs
EP0732575A2 (fr) * 1992-10-14 1996-09-18 Andrew George Bosanquet Procédé permettant de réaliser des tests, notamment des tests comparatifs
EP0732575A3 (fr) * 1992-10-14 1996-12-04 Andrew George Bosanquet Procédé permettant de réaliser des tests, notamment des tests comparatifs
US5650125A (en) * 1992-10-14 1997-07-22 Bosanquet; Andrew George Method and apparatus for conducting tests
FR2762911A1 (fr) * 1997-04-30 1998-11-06 Cytotech Distribution Sa Dispositif et methode d'analyse de preparations cytologiques
WO2010135468A1 (fr) * 2009-05-19 2010-11-25 Vivia Biotech S.L. Procédés permettant de fournir des essais de médicaments personnalisés ex vivo pour des tumeurs hématologiques
EP2335825A1 (fr) * 2009-12-21 2011-06-22 F. Hoffmann-La Roche AG Unité et dispositif pour la préparation de cellules et/ou de particules dans un liquide et procédé d'analyse microscopique
WO2011076705A1 (fr) * 2009-12-21 2011-06-30 F. Hoffmann-La Roche Ag Unité et dispositif de préparation de cellules et/ou de particules dans un liquide et procédé d'analyse microscopique
JP2013515235A (ja) * 2009-12-21 2013-05-02 エフ．ホフマン−ラ ロッシュ アーゲー 液体中の細胞および／または粒子を調製するためのユニットおよび装置ならびに顕微分析法
EP2372367A1 (fr) * 2010-04-01 2011-10-05 F. Hoffmann-La Roche AG Procédé informatisé pour utiliser une cellule d'échantillon automatique
US8617041B2 (en) 2010-04-01 2013-12-31 Roche Diagnostics Operations, Inc. Automated sample workcell and method of operation
US9505012B2 (en) 2010-04-01 2016-11-29 Roche Diagnostics Operations, Inc. Automated sample workcell and method of operation

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