EP0394404A1 - Procede automatise d'essais "in vivo" des micronoyaux et dispositif pour son execution - Google Patents

Procede automatise d'essais "in vivo" des micronoyaux et dispositif pour son execution

Info

Publication number
EP0394404A1
EP0394404A1 EP19890911362 EP89911362A EP0394404A1 EP 0394404 A1 EP0394404 A1 EP 0394404A1 EP 19890911362 EP19890911362 EP 19890911362 EP 89911362 A EP89911362 A EP 89911362A EP 0394404 A1 EP0394404 A1 EP 0394404A1
Authority
EP
European Patent Office
Prior art keywords
cells
cellulose
mixture
filter
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP19890911362
Other languages
German (de)
English (en)
Inventor
Felix Romagna
Gerhard Johannsen
Wilfried Frieauff
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leica Microsystems Holdings GmbH
Original Assignee
Wild Leitz GmbH
Leica Industrieverwaltung GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wild Leitz GmbH, Leica Industrieverwaltung GmbH filed Critical Wild Leitz GmbH
Publication of EP0394404A1 publication Critical patent/EP0394404A1/fr
Ceased legal-status Critical Current

Links

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
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • 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
    • G01N2001/2846—Cytocentrifuge method

Definitions

  • the invention relates to an automated "in vivo" microkernel test method in mammals, in particular in humans, and in rats and mice, and a corresponding preparation device for carrying out the same.
  • the micronucleus test is an "in vivo" test for the detection of agent-induced chromosome damage and damage to the spindle apparatus. For this purpose, erythrocyte-like cells of the bone marrow or peripheral blood of mammals are examined for cells containing micronuclei. The micronucleus test in the bone marrow of the mouse is most frequently used according to W. Schmid's modified method, cf. Mutation Res., 31 (1975), 9-15.
  • the main advantages of the present invention are that an increase in the sensitivity of the classic micronucleus test in the mouse bone marrow could be achieved and that above all the adaptability of the new method for examining the bone marrow is also given to the rat.
  • 1 shows, in a purely schematic representation in cross section, a first device according to the invention for the separation of cells to be examined from the bone marrow or peripheral blood of mammals (human; mouse, rat, etc.).
  • a vertical column made of glass or plastic can be fastened to a stand 12, for example a disposable syringe 7 with a centrally positioned cannula 9 pointing downwards.
  • the filling of the syringe 7 consists of a cellulose mixture 2 with a defined column height. Above this mixture there is a free space 1 for receiving the sample suspension 13 to be separated, which contains a carrier liquid, for example an isotonic solution or a fetal calf serum.
  • a lid 11 closes the free space 1 towards the top.
  • the syringe 7 has a filter reservoir 4a in which one or more filters 4 with a mesh size between 5 and 20 ⁇ m, preferably between 10 and 12 ⁇ m , are provided.
  • the filter reservoir 4a is preferably formed as an individual component and can accordingly be pushed onto the cannula 9 of the syringe 7 with a precise fit. However, it is also possible to form the syringe 7 in one piece together with the filter reservoir.
  • a collecting container 5 is provided for the eluate ⁇ , which consists of a suspension of mature and immature erythrocytes.
  • the collecting container 5 can be dimensioned such that it can be detachably connected to the disposable syringe 7, for example by being pushed on. It can have air passage openings 10. This ensures that with the container 5 attached, after filling a sample suspension 13 into the free space 1, a slow leakage of this substance through the individual filter regions 2 - 8 - 4 with normal pressure compensation is possible.
  • the procedure is as follows: after filling in the sample suspension 13 - in the present case rat bone marrow is entered - the sample penetrates into the cellulose mixture 2.
  • the cellulose mixture consists of microcrystalline cellulose and ⁇ -cellulose in a weight ratio of preferably 1: 1. For certain applications, this weight ratio can also vary between 1.5: 1 and 1: 1.5.
  • a similar method has already been developed by Beutler et al; however, this was limited only to peripheral blood and, moreover, was never used in connection with a micronucleus test, cf. J. Lab. Clin. Med. 88 (2), 1976, 328-333.
  • Standard cell type 50 from SIGMA, St. Louis, USA, for example, can be used as the microcrystalline cellulose; a product of the company mentioned can also be used as ⁇ -cellulose in fiber form.
  • Bei.de cellulose modifications are shaken vigorously for several minutes before being placed in the vertical column. The "column height" of the cellulose mixture 2 and its packing density in the syringe 7 is of great importance for the success of the preparation process.
  • the eluate 6 collected in the container 5 contains only the two desired cell types, namely the immature (polychromatophilic) and the mature erythrocytes. A total elimination of the nucleated hematopoietic cells was thus achieved.
  • rat bone marrow the cells are now cleaned using a Percoll step gradient method. This process step is essential for the micronucleus test in the rat bone marrow (removal of the remaining leukocytic granules).
  • the solutions described by Rennie et al in Klin. Chem. Acta, 98 (1979), pages 119-125 are used for the cleaning process mentioned.
  • a centrifuge tube for example with a volume of 5 ml, 1.5 ml of an 80% Percoll solution are first introduced. 1.5 ml of a 30% Percoll solution are layered on top.
  • the cells to be examined accumulate in the boundary layer area between the 80% and the 30% Percoll solution.
  • the cells to be examined must be arranged very flat on the slide. The cells are therefore placed on slides treated with polylysine by cytocentrifugation. The flattened and homogeneously distributed cells in this way can then be colored using the known May-Grünwald / Giemsa standard staining. However, coloring with fluorescent dyes, preferably with acridine orange, is also possible.
  • the cells which have been separated, prepared and labeled in this way are then available for subsequent automatic quantitative microscopic image analysis.
  • an enrichment process of the polychromatophilic erythrocytes occurs in samples from peripheral blood by means of a modified Percoll step gradient method.
  • the device for carrying out this enrichment method differs from that for cleaning rat bone marrow samples only in that instead of the 80% Percoll solution to be entered first, 1.5 ml of a 55-65% Percoll solution. Solution can be entered.
  • the MIAMED image analysis system developed by WILD-LEITZ The most important prerequisite for this is the previous elimination of the nucleated cells of hematopoiesis described above with the help of the mentioned preparation or separation method in the manner of a "column chromatography".
  • the MIAMED consists on the one hand of the automated WILD-LEITZ microscope MEDILUX and on the other hand of the image processing system MIAC (Modular Image Analysis Computer).
  • the microscope has a scanning table that can hold up to sixteen slides, a focus drive, lamp control and a nosepiece.
  • the table, focus and objective revolver are motor-driven and are controlled by software via the MIAC.
  • a video camera adapted to the microscope and MIAC enables the microscope images to be recorded electronically and transferred to the image processing system.
  • the MIAC is a modular bus-oriented system of great flexibility.
  • a new type of bus system enables fast and flexible communication between the various MIAC modules.
  • the modules include image memories and special image processors for fast processing of gray and binary images.
  • the architecture of the system is designed for so-called pipeline processing, that is to say for continuous and parallel processing of the images delivered by the camera.
  • the modularity also enables the system to be configured to suit the problem in order to achieve an optimal price / performance ratio.
  • the program package for the automatic detection of micronuclei is embedded in a software architecture that is specially designed for so-called "rare evenf” strategies and is adapted to the fast MIAMED pipeline structure. According to this strategy, the user with the three Programmte len data entry, automatic detection and do resulting ⁇ nis analyses. There may also be a so-called "superuser” program, which is not accessible to the routine user. It is used to set special system parameters.
  • the interactive actions of the user are limited to the setting of general search parameters to the entry of some special problem-dependent parameters that control the automatic search process of the system.
  • Essential input data are, for example, default values for the search areas on the slides, the setting of the focus level of the start field and the specification of the number of cells to be evaluated.
  • Automatic detection is a program that, without supervision, uses the input data to automatically and fully scan the slides spirally field by field for cells containing micronuclei. It is composed of a problem-independent "detection frame", which is responsible for the exact control and adjustment of the image fields, and a problem-specific "detection core”, which guarantees the extraction of the desired image information by appropriate algorithms. All data obtained in the automatic search run, in particular the positions of the detected micronucleus-containing cells, are stored in a special database, as was the input data previously. The search can be interrupted at any time by the user to output current interim results. In addition, the user has the option of Interruption turn on the display for the graphical representation of the single ⁇ NEN detection steps, so as to verify the selection of parameters and to modify, if necessary.
  • a program part for the evaluation and analysis of the automatically detected micronucleus-containing cells completes the program package.
  • the analysis program the general part of which enables the detected cells to be quickly demonstrated again, also contains a special part for problem-specific analysis of the generated data. This makes it possible to prepare and format all statistically relevant data in such a way that they can also be processed further by commercially available PC programs.
  • the present method it is possible for the first time to reliably carry out the micronucleus test in the bone marrow of the rat. This is possible because the new column method and the subsequent cleaning of the cells using the Percoll step gradient method can remove all artifact-producing leukocytic granules, so that a high-quality cell preparation results. This can then be examined - either manually or fully automatically with the aid of a special image analysis method - for erythrocytes containing micronuclei.
  • the polychro atophilic erythrocytes can be enriched using a Percoll step gradient method; in the "rat" species, for example, from originally 2 to 5% to over 80%.
  • Another advantage of the method according to the invention is that good adaptability to other mammalian species, including humans. The genotoxicity test in vivo has thus become considerably more flexible.
  • the column arrangement shown schematically in FIG. 1 is designed here as a laboratory device. It is of course also possible to provide this arrangement in one piece as a "disposable set" 11-7-4-6. This gives greater flexibility in handling at any location.
  • the lid of the disposable set device has a different interior shape than that of the laboratory arrangement shown in FIG. 1, since it is designed as a cylindrical inner plug which takes up the entire free space 1 including the partial volume 13. This ensures that the preparation set has a constant, reproducible, defined adjustable column filling height of the cellulose mixture 2 before it is used.
  • the amount of the cellulose mixture 2 to be used depends on the species. It is within the scope of the present invention to optimize the loading of the disposable syringe 7 with cellulose mixture fillings and / or with filter paper (s) 8 and / or with filter material (4) for the species to be examined in each case.
  • FIG. 2 shows a simplified cross-sectional view of a simplified variant, which is also designed as a disposable set and represents a compact cell separation device 22.
  • the preferably rotationally symmetrical device has an inlet opening 14, into which the cannula, for example of a disposable syringe, can be inserted.
  • the cellulose mixture 2 which has already been described in more detail in FIG. 1, is located in its central region. This mixture is by means of a upper seal 15 and a lower seal 16 held in the central region of the device 22.
  • the upper seal 15 prevents the cellulose mixture 2 from escaping and at the same time allows the sample suspension to be separated to pass through. Two embodiments are possible.
  • the seal 15 can be made of plastic and can be designed as a removable cover or as a cell-permeable grid.
  • the lower seal 16 likewise prevents the cellulose mixture 2 from passing through and allows the cell suspension to pass through and is advantageously designed as a cell-permeable plastic grid.
  • the special filter or filters 18 serve, in analogy to the function of the filter 4 in FIG. 1, for post-filtration. They can be designed as a cellulose or polycarbonate membrane with mesh sizes between 3 and 8 ⁇ m, preferably 5 ⁇ m, or as a cellulose ester mixture, MF from Nuclepore, USA.
  • the filter pad 20 is also expediently a cell-permeable plastic grid.
  • the reference number 19 denotes a conically tapered sealing wall which deliberately narrows the filter space downwards and ensures a firm fit and a solid peripheral seal of the special filter (s) 18.
  • the exit opening 21 adjoining at the bottom is the exit point for the fraction of the pure mature and immature erythrocyte suspension. It is preferably closable.
  • the preferred size standard of the Connection is: Luer or Record.
  • the cell separation device 22 is made of plastic or glass. In a preferred embodiment, the outer diameter ⁇ is between 10 and 12 mm; the height b (without taking into account the inlet opening 14 designed as a socket) between 13 and 15 mm. In this simple variant of a cell separation device, shown in FIG.
  • a syringe containing the sample suspension for example a disposable syringe with a volume of 2 or 5 ml
  • a syringe containing the sample suspension for example a disposable syringe with a volume of 2 or 5 ml
  • the cell suspension is slowly and carefully pressed through the device 22 and the separated cells are collected in a collecting vessel (not shown in FIG. 2).
  • the cell separation device 22 shown in FIG. 2 represents an optimized design variant as a spatial form. Only structural changes to what is shown are within the scope of the present invention, insofar as the sequence in the process sequence is maintained while maintaining the essential features: cellulose mixture cell separation and post-filtering using a special filter (n) is observed.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé automatisé d'essais ''in vivo'' sur des micronoyaux pour l'analyse de la moelle osseuse et du sang périphérique chez toutes les espèces concernées. On sépare les cellules hématopoïétiques contenant des noyaux par filtration en colonne, la suspension passant comme dans une chromatographie en colonne. Les érythrocytes jeunes ou mûrs ainsi séparés sont soumis à un processus du type "percoll" pour purifier ou enrichir les érythrocytes jeunes (réticulocytes), ensuite ils sont filtrés, puis applatis et positionnés de manière homogène sur des supports traités à la polylysine par cytocentrifugation, puis teints et enfin soumis à un processus entièrement automatisé d'analyse d'image microscopique.
EP19890911362 1988-10-19 1989-10-18 Procede automatise d'essais "in vivo" des micronoyaux et dispositif pour son execution Ceased EP0394404A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE3835654 1988-10-19
DE3835654 1988-10-19
DE3934625 1989-10-17
DE19893934625 DE3934625A1 (de) 1988-10-19 1989-10-17 Automatisiertes "in vivo"-mikrokerntest-verfahren und vorrichtung zur durchfuehrung desselben

Publications (1)

Publication Number Publication Date
EP0394404A1 true EP0394404A1 (fr) 1990-10-31

Family

ID=25873411

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19890911362 Ceased EP0394404A1 (fr) 1988-10-19 1989-10-18 Procede automatise d'essais "in vivo" des micronoyaux et dispositif pour son execution

Country Status (4)

Country Link
EP (1) EP0394404A1 (fr)
JP (1) JPH03502736A (fr)
DE (1) DE3934625A1 (fr)
WO (1) WO1990004784A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5240856A (en) * 1991-10-23 1993-08-31 Cellpro Incorporated Apparatus for cell separation
US5672481A (en) * 1991-10-23 1997-09-30 Cellpro, Incorporated Apparatus and method for particle separation in a closed field
CN104198262B (zh) * 2014-09-10 2017-02-15 国家海洋局第三海洋研究所 多功能水体放射性核素现场快速富集处理器

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2903855A1 (de) * 1979-02-01 1980-08-14 Bloss Werner H Prof Dr Ing Verfahren zum automatischen markieren von zellen und bestimmung der merkmale von zellen aus zytologischen abstrichpraeparaten

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9004784A1 *

Also Published As

Publication number Publication date
JPH03502736A (ja) 1991-06-20
DE3934625A1 (de) 1990-04-26
WO1990004784A1 (fr) 1990-05-03

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