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WO2010068129A1 - Procédé de traitement et d'imprégnation d'échantillons histologiques ou biologiques - Google Patents

Procédé de traitement et d'imprégnation d'échantillons histologiques ou biologiques Download PDF

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Publication number
WO2010068129A1
WO2010068129A1 PCT/RU2008/000769 RU2008000769W WO2010068129A1 WO 2010068129 A1 WO2010068129 A1 WO 2010068129A1 RU 2008000769 W RU2008000769 W RU 2008000769W WO 2010068129 A1 WO2010068129 A1 WO 2010068129A1
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WO
WIPO (PCT)
Prior art keywords
samples
processing
paraffin
impregnation
liquid
Prior art date
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Ceased
Application number
PCT/RU2008/000769
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English (en)
Russian (ru)
Inventor
Илья Борисович ИЗВОЗЧИКОВ
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Individual
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Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to PCT/RU2008/000769 priority Critical patent/WO2010068129A1/fr
Publication of WO2010068129A1 publication Critical patent/WO2010068129A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • the technical field The invention relates to research methods, and in particular to methods for preparing histological and biological samples for microscopic examination.
  • the samples taken for research before they can be microtomized must be fixed, for example, with an aqueous or aqueous-alcoholic solution of formaldehyde, then dehydrated with a liquid miscible with water, then clarified with an organic liquid that mixes well with dehydrating liquid and paraffin (or other wax) then waxed.
  • a waxed sample is suitable for microtomation.
  • Fixation is a necessary procedure in histological studies, since tissues isolated from the body undergo changes very soon. Fixation allows you to fix (fix) cells and formed elements of tissues in a form that corresponds to intravital. Not fully fixed tissue continues to change [B.Romeis. Microscopic technique. M. IL, 1953, Ch. IY].
  • the duration of each stage depends on many parameters, the most important of which are the volume of the sample, the diffusion rate of the replacement fluid deep into the sample and replaced from its surface, processing temperature, etc.
  • the most common is the method of processing and impregnating histological and biological samples, in which the samples are sequentially incubated in at least two portions of a fixing aqueous or water-alcohol solution of formaldehyde, in three portions of a dehydrating liquid containing water, in three portions of a completely anhydrous dehydrating liquid, in two servings antireflection liquid and in two portions of paraffin melt [R. Lilly. Pathological technique and practical histochemistry. M., Mir, 1969, ch. IY].
  • the specified method allows you to replace the water contained in the cells and tissues of histological and biological samples, with paraffin without disturbing the tissue and cell structure. This method is widely used in laboratories, but it is very long-lasting and requires a large number of processing fluids.
  • a known method of processing and impregnation of histological and biological samples by successively immersing a container (basket) with samples in a fixing aqueous solution of formaldehyde, then in a dehydrating liquid (ethyl alcohol), then in a bleaching liquid (xylene), then in two portions of paraffin, only 12 liquids in which the placed sample basket remains stationary [EP 0269316, M.cl. 4 GOlN 1/28, 1988].
  • the disadvantage of this method is its duration.
  • the residence time in each fluid is from 15 minutes (endoscopic biopsies) to 2 hours (large pieces, for example, surgical material) and may be longer if samples of large volume are processed.
  • the liquids are mixed when a container with samples is lowered into them by rotating the container; in addition, by rotating the container outside the liquid, the remaining liquid in the container is removed before dipping it in the next liquid.
  • Periodic dipping and mixing of the processing fluids accelerates the processing, and the removal of residues of the previous fluid before immersion in the next helps to reduce the contamination of the processing fluids of the previous ones.
  • this method also requires a large number of shifts of processing reagents.
  • the disadvantage of the described method is that it does not provide for the removal of vapors from the tissue processing zone. As a result of this, with intense evaporation, the vapor pressure in the confined space increases, the steam becomes saturated and further evaporation, and hence the removal of the polluting liquid, ceases.
  • a known method of processing and impregnation of histological and biological samples including fixing the samples in a fixing liquid containing formaldehyde, dehydrating the samples with ethanol, xylene clarification and paraffin impregnation, all stages are carried out with stirring, excess treatment fluid is removed before impregnation, and the paraffin impregnation stage is carried out under a vacuum.
  • all processing steps are carried out under microwave irradiation and mixing.
  • the specified method can be used for processing samples with a thickness of 5 mm or more and at the same time a large number of them (210 samples).
  • the basis of the invention is the task of reducing the consumption of paraffin used for the impregnation of histological and biological samples.
  • the basis of the invention is also the task of reducing processing time by reducing the residence time in the melt of paraffin.
  • the claimed invention is also directed to reducing energy consumption for impregnation with molten paraffin at elevated temperatures.
  • paraffin impregnation in a method for processing and impregnating histological and biological samples, including fixing the samples in a fixing liquid, dehydrating them, bleaching and paraffin impregnation with stirring, paraffin impregnation is carried out under conditions of forced condensation of vapors of volatile treatment fluids with the removal of condensate from the treatment zone.
  • Vapors are condensed at a temperature 10 to 75 ° C below the treatment temperature
  • liquids miscible with water and with a paraffin melt can be used as a dehydrating liquid.
  • liquids miscible with paraffin melt and water include isopropanol, n-butanol, 2-butanol, isobutanol (methylpropanol-1), l-methoxy-2-propanol, isopropylacetone.
  • the boiling point (at atmospheric pressure) of isopropanol is 78 ° C, 2-butanol 99.5 ° C, isobutanol 108 ° C, n-butanol 117 ° C, isopropyl acetone 118 ° C, l-methoxy-2-propanol 120 0 C.
  • Mixing in the inventive method is carried out by constant periodic immersion of samples in dehydrating and impregnating liquids and removing from them with a frequency of 6-10 times per minute; when extracting samples from a dehydrating or impregnating liquid, these liquids flow down, which helps to accelerate the diffusion of the medium being replaced from the surface of the sample.
  • Such mixing may be carried out, for example, in a drum-type device, such as that described in RU 2 150 197, or in any other device where continuous immersion of samples in a medium and their extraction into the gas phase can be performed. In this case, the evaporation of volatile liquids occurs not only from the mirror of the liquid surface, but also from the surface of the samples. Subsequent immersion does not allow samples to dry.
  • a refrigerator on the surface of which water vapor and dehydrating liquids condense, can be placed inside the chamber in which the treatment takes place; In this case, there should be a tray under the refrigerator - a condensate collector. Condensation is discharged from the camera. However, the refrigerator can be placed in an additional chamber, in which free or forced access of vapors is made.
  • the temperature of the paraffin melt is maintained at a level exceeding the melting temperature of paraffin by at least 2 0 C.
  • the dehydration fluid remaining in the samples evaporates and condenses, and the paraffin remains unpolluted with the processing fluids. Since the contamination of paraffin with processing fluids in the inventive method is minimized, it becomes possible to reduce the number of stations with paraffin from four to two, to reduce the total time of impregnation of the samples and, consequently, the time of exposure to high temperature fabrics, and to reduce energy consumption.
  • liquids used in the claimed method miscible with both water and paraffin melt, make it possible to exclude the use of antireflection liquids and a separate stage of enlightenment.
  • Example 1 Example 1
  • the processing fluid battery includes 10 containers of 3 l each, respectively containing:
  • the residence time of the fixing solutions in the device is 10 hours (2 hours the first solution and 8 hours the second).
  • the residence time in the device of each portion of isopropanol is 30 minutes
  • a pressure of 500 mmHg is set in the device.
  • the cooling liquid entering the refrigerator in the form of a coil located under the lid of the chamber was cooled to room temperature (20 ° C). Under these conditions, the samples are incubated for 45 min in two portions of paraffin melt.
  • 65 ml of isopropanol containing less than 1% water is condensed; condensate is discharged from the device.
  • the experiment is carried out as in example 3, but at the stage of impregnation, the pressure h was maintained at 350 mmHg, the temperature in the chamber was 65 ° C (the boiling point of isopropanol at 350 mmHg was 60 ° C), and the coolant received in the refrigerator, cooled to - 10 ° C.
  • the processing time in each change of paraffin melt is 25 minutes.
  • paraffin returned after impregnation to the 9th container contains a trace amount of isopropanol, and the paraffin returned to the 10th container does not contain isopropanol.
  • the processing fluid battery includes 10 containers of 3 l each, respectively containing:
  • the processing time in each portion of isobutanol is 30 minutes, the temperature in the working chamber is 50 ° C.
  • coolant is supplied to the refrigerator at room temperature (25 ° C) and the vacuum pump is turned on to create a pressure of 500 mmHg in the device.
  • the processing time in each portion of paraffin is 45 minutes at a temperature of 65 ° C and a pressure of 500 mm Hg, coolant is supplied to the refrigerator at room temperature (25 ° C).
  • the paraffin returned to the 6th container contains a trace amount of isobutanol, and the paraffin returned to the 7th container does not contain isobutanol.
  • Example 4 The experiment was carried out as in example 3, but at the stage of impregnation the pressure was maintained at the level of 100 mmHg, the temperature in the chamber was 65 ° C (the boiling point of isobutanol at 100 mmHg was 60 ° C), and the coolant entering the refrigerator was cooled to -10 ° C.
  • the processing time in each change of paraffin melt is 25 minutes.
  • Isobutanol returned to the 5th container does not contain water.
  • the paraffin returned to the 6th container contains a trace amount of isobutanol, and the paraffin returned to the 7th container does not contain isobutanol.
  • the inventive method of processing and impregnation of histological and biological samples can be used in laboratories specializing in histological (pathological and anatomical) and cytological studies, as well as in any other biological and medical institutions involved in microscopic studies of tissues and cells.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

Le procédé de l'invention comprend la fixation d'échantillons dans un liquide de fixation, leur déshydratation, clarification et imprégnation avec de la paraffine pendant le mélangeage. Le stade d'imprégnation avec de la paraffine est mis en oeuvre dans des conditions de condensation forcée de vapeurs et d'évacuation du condensat de la zone de traitement.
PCT/RU2008/000769 2008-12-10 2008-12-10 Procédé de traitement et d'imprégnation d'échantillons histologiques ou biologiques Ceased WO2010068129A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/RU2008/000769 WO2010068129A1 (fr) 2008-12-10 2008-12-10 Procédé de traitement et d'imprégnation d'échantillons histologiques ou biologiques

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/RU2008/000769 WO2010068129A1 (fr) 2008-12-10 2008-12-10 Procédé de traitement et d'imprégnation d'échantillons histologiques ou biologiques

Publications (1)

Publication Number Publication Date
WO2010068129A1 true WO2010068129A1 (fr) 2010-06-17

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PCT/RU2008/000769 Ceased WO2010068129A1 (fr) 2008-12-10 2008-12-10 Procédé de traitement et d'imprégnation d'échantillons histologiques ou biologiques

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WO (1) WO2010068129A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110031279A (zh) * 2019-03-30 2019-07-19 吴淑华 标本固定台
CN110823665A (zh) * 2019-11-27 2020-02-21 李雄 正丁醇在制备用于活检组织标本脱水和透明制片的低毒脱水剂中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1317307A1 (ru) * 1982-11-03 1987-06-15 В.Я. Супоницкий Способ подготовки образцов животной ткани дл микроскопировани
DE4404544A1 (de) * 1994-02-12 1995-08-17 Schubert Werner Verfahren und Vorrichtung für die Stabilisation frischer Gewebs-Probeexzisionen
RU2089871C1 (ru) * 1994-09-26 1997-09-10 Извозчиков Илья Борисович Способ подготовки биологического образца к гистологическому исследованию
EP1533604A1 (fr) * 2003-11-18 2005-05-25 Bio Optica-Milano S.p.A. Procédé de préparation d'un échantillon histologique pour la coloration

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1317307A1 (ru) * 1982-11-03 1987-06-15 В.Я. Супоницкий Способ подготовки образцов животной ткани дл микроскопировани
DE4404544A1 (de) * 1994-02-12 1995-08-17 Schubert Werner Verfahren und Vorrichtung für die Stabilisation frischer Gewebs-Probeexzisionen
RU2089871C1 (ru) * 1994-09-26 1997-09-10 Извозчиков Илья Борисович Способ подготовки биологического образца к гистологическому исследованию
EP1533604A1 (fr) * 2003-11-18 2005-05-25 Bio Optica-Milano S.p.A. Procédé de préparation d'un échantillon histologique pour la coloration

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110031279A (zh) * 2019-03-30 2019-07-19 吴淑华 标本固定台
CN110823665A (zh) * 2019-11-27 2020-02-21 李雄 正丁醇在制备用于活检组织标本脱水和透明制片的低毒脱水剂中的应用

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