EP0220269A1 - Method of impregnating a tissue specimen with paraffin - Google Patents
Method of impregnating a tissue specimen with paraffinInfo
- Publication number
- EP0220269A1 EP0220269A1 EP19860902936 EP86902936A EP0220269A1 EP 0220269 A1 EP0220269 A1 EP 0220269A1 EP 19860902936 EP19860902936 EP 19860902936 EP 86902936 A EP86902936 A EP 86902936A EP 0220269 A1 EP0220269 A1 EP 0220269A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- bath
- pressure
- paraffin
- tissue specimen
- tissue sample
- 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.)
- Withdrawn
Links
- 239000012188 paraffin wax Substances 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 13
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229940057995 liquid paraffin Drugs 0.000 claims abstract description 5
- 239000012024 dehydrating agents Substances 0.000 abstract description 10
- 239000000470 constituent Substances 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 30
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000001744 histochemical effect Effects 0.000 description 2
- 238000005470 impregnation Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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
-
- 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
- G01N2001/305—Fixative compositions
Definitions
- the invention refers to a method of impregnating a tissue specimen with paraffin.
- tissue specimens are trea with a dehydrating agent, alcohol and acetone, respective before the tissue specimens are immersed in a bath of liquid paraffin.
- the pressure of the bath is lowered so muc that the respective dehydrating agent is vaporized and thereby evaporates from the tissue specimens as well-as from the paraffin bath, the pressure of the bath then being increased again to atmospheric pressure.
- An object of the invention is to provide a method of impregnation that eliminates the disadvantages of the methods known hitherto.
- Another object of the invention is to provide a method of impregnation in which the treatment with a dehydrating agent can be eliminated.
- the tissue specimen is immersed, either directly after an optional treatment of the tissue specimen with a fixing agent or after the tissue specimen optionally has been treated with a fixing agent and/or a dehydrating agent, in a bath of liquid paraffin which is kept below a predetermined temperature at which thermolabile component and properties of the tissue specimen undergo changes, and that thereafter, the pressure of the bath is lowered from atmospheric pressure to a predetermined pressure, at which water and/or, when appropriate, the dehydrating agent vaporizes. Then the pressure of the bath is increased again to atmospheric pressure.
- a tissue specimen When carrying out a histochemical diagnosis, normally a tissue specimen is first treated with a fixing agent, usually formalin, and thereafter with a dehydrating agent, e.g. methanol, ethanol or acetone, before the tissue specimen is immersed in a bath of liquid paraffin. In this bath the tissue specimen is impregnated with paraffin. The tissue specimen impregnated in this way with paraffin is then embedded in a paraffin block which can be cut by means of a microtome knife to obtain thin cuts (1-5 urn thic which can thereafter be examined in a microscope after the paraffin has been removed and after dyeing.
- a fixing agent usually formalin
- a dehydrating agent e.g. methanol, ethanol or acetone
- the tissue specimen is directly immersed into the paraffin bath.
- a paraffin having a melting point of about 37-44 C has been chosen and the bath is kept at a temperature of about 47 C at the most, i.e. below the temperature at which it has been observed that thermolabile components and properties of the tissue specimen begin to change. It should be pointed out that this temperature can vary somewhat depending on which type of tissue specimen one wants to examine.
- the pressure of the paraffin bath is thereafter lowered from atmospheric pressure to at least about 76 mm Hg, i.e. the pressure at which water boils at a temperature of about 46 C.
- the pressure of the bath is increased again to atomospheric pressure.
- the lowering of the pressure as well as the pressure increase must not take place too abrupt.
- the tissue material and cells present therein can burst through sudden pressure changes during the pressure lowering phase and can be compressed during the pressure increasing phase.
- the tissue specimen can also be treated with a dehydrating agent, for example methanol, ethanol or acetone, before the sample is immersed into the paraffin bath which also in this case is kept below the temperature at which thermolabile components and properties of the tissue specimen are changed.
- a dehydrating agent for example methanol, ethanol or acetone
- the pressure of the paraffin bath has to be lowered to at least about 510 mm Hg, i.e» the pressure at which acetone vaporizes at a temperature of about 45 C.
- the acetone will vaporize and evaporate from the tissue specimen as well as from the paraffin bath.
- the pressure of the bath must also in this case be lowered to at least 76 mm Hg.
Abstract
Dans un procédé d'imprégnation d'un échantillon de tissu avec de la paraffine soit directement après un traitement éventuel de l'échantillon de tissu avec un agent de fixation soit après que l'échantillon de tissu ait été traité éventuellement avec un agent de fixation et/ou un agent de déshydratation, l'échantillon de tissu est immergé dans un bain de paraffine liquide. Le bain est maintenu au-dessous d'une température prédéterminée à laquelle les propriétes et les constituants thermolabiles de l'échantillon de tissu subissent des modifications. La pression du bain est alors abaissée de la pression atmosphérique à une pression prédéterminée à laquelle l'eau présente dans l'échantillon de tissu et/ou le cas échéant, l'agent de déshydratation, se vaporize, la pression du bain étant alors à nouveau portée à la pression atmosphérique.In a method of impregnating a tissue sample with paraffin either directly after optional treatment of the tissue sample with a fixing agent or after the tissue sample has optionally been treated with a fixing agent and / or a dehydrating agent, the tissue sample is immersed in a bath of liquid paraffin. The bath is maintained below a predetermined temperature at which the properties and thermolabile constituents of the tissue sample undergo changes. The pressure of the bath is then lowered from atmospheric pressure to a predetermined pressure at which the water present in the tissue sample and / or, where appropriate, the dehydrating agent, vaporizes, the pressure of the bath then being at again brought to atmospheric pressure.
Description
Method of impregnating a tissue specimen with paraffin.
TECHNICAL FIELD
The invention refers to a method of impregnating a tissue specimen with paraffin.
BACKGROUND ART
Such methods are known from GB 1 536 422 and GB-A- 2 065 9
In both these known methods the tissue specimens are trea with a dehydrating agent, alcohol and acetone, respective before the tissue specimens are immersed in a bath of liquid paraffin.
After the tissue specimens have been immersed in the paraffin bath, the pressure of the bath is lowered so muc that the respective dehydrating agent is vaporized and thereby evaporates from the tissue specimens as well-as from the paraffin bath, the pressure of the bath then being increased again to atmospheric pressure.
From GB 1 536 422 it is apparent that the operating temperature is 65 C, while no temperature information can be found in GB-A-2 065 912. However, it could be assumed that the operating temperature also in the last mentioned case is about 60-65 C.
However, it has been found that a temperature higher'.than about 45-47 C destroys the structure of the tissue specimens and also has a shrivelling effect on the tissue i has also been found that proteins and other thermolabi tissue components as well as thermolabile properties are changed at higher temperature in such a way that antigen structures cannot be recognized to a sufficient extent in a subsequent histochemical tissue diagnosis. Moreover, membranes and organelles become shrivelled and are destroyed.
DISCLOSURE OF INVENTION
An object of the invention is to provide a method of impregnation that eliminates the disadvantages of the methods known hitherto.
Another object of the invention is to provide a method of impregnation in which the treatment with a dehydrating agent can be eliminated.
This is attained by the method according to the invention in that the tissue specimen is immersed, either directly after an optional treatment of the tissue specimen with a fixing agent or after the tissue specimen optionally has been treated with a fixing agent and/or a dehydrating agent, in a bath of liquid paraffin which is kept below a predetermined temperature at which thermolabile component and properties of the tissue specimen undergo changes, and that thereafter, the pressure of the bath is lowered from atmospheric pressure to a predetermined pressure, at which water and/or, when appropriate, the dehydrating agent vaporizes. Then the pressure of the bath is increased again to atmospheric pressure.
PREFERRED EMBODIMENTS
When carrying out a histochemical diagnosis, normally a tissue specimen is first treated with a fixing agent, usually formalin, and thereafter with a dehydrating agent, e.g. methanol, ethanol or acetone, before the tissue specimen is immersed in a bath of liquid paraffin. In this bath the tissue specimen is impregnated with paraffin. The tissue specimen impregnated in this way with paraffin is then embedded in a paraffin block which can be cut by means of a microtome knife to obtain thin cuts (1-5 urn thic which can thereafter be examined in a microscope after the paraffin has been removed and after dyeing.
According to the invention it is suggested that after an optional treatment of the tissue specimen with a fixing
agent, the tissue specimen is directly immersed into the paraffin bath. For this bath a paraffin having a melting point of about 37-44 C has been chosen and the bath is kept at a temperature of about 47 C at the most, i.e. below the temperature at which it has been observed that thermolabile components and properties of the tissue specimen begin to change. It should be pointed out that this temperature can vary somewhat depending on which type of tissue specimen one wants to examine.
According to the invention the pressure of the paraffin bath is thereafter lowered from atmospheric pressure to at least about 76 mm Hg, i.e. the pressure at which water boils at a temperature of about 46 C.
In this way, water present in the tissue specimen will vaporize and evaporate from the tissue specimen as well as from the paraffin bath.
Then, the pressure of the bath is increased again to atomospheric pressure. The lowering of the pressure as well as the pressure increase must not take place too abrupt. The tissue material and cells present therein can burst through sudden pressure changes during the pressure lowering phase and can be compressed during the pressure increasing phase.
According to another embodiment of the invention the tissue specimen can also be treated with a dehydrating agent, for example methanol, ethanol or acetone, before the sample is immersed into the paraffin bath which also in this case is kept below the temperature at which thermolabile components and properties of the tissue specimen are changed.
When acetone is used as a dehydrating agent, the pressure of the paraffin bath has to be lowered to at least about 510 mm Hg, i.e» the pressure at which acetone vaporizes at a temperature of about 45 C.
Thus, the acetone will vaporize and evaporate from the tissue specimen as well as from the paraffin bath.
To ensure that any remaining water also will evaporate from the tissue specimen, the pressure of the bath must also in this case be lowered to at least 76 mm Hg.
By the paraffin impregnating method according to the invention it has been found that the quality of the tissue specimens is improved compared to paraffin impregnating methods known hitherto.
Claims
[received by the International Bureau on 12 September 1986 (12.09.86); original claims 1—4 replaced by new claim 1 (1 page)]
Method of .impregnating a tissue specimen with paraffin by immersing the tissue specimen in a bath of liquid paraffin which is kept at a temperature of about 47 C at the most, characterized in that the tissue specimen is immersed into the bath directly after an optional treatment of the tissue specimen with a fixing agent, that the pressure of the bath in then lowered from atmospheric pressure to at least about 75 mm Hg to vaporize water present in the tissue specimen, whereupon the pressure of the bath is increased to atmospheric pressure.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE8501959 | 1985-04-23 | ||
| SE8501959A SE8501959L (en) | 1985-04-23 | 1985-04-23 | PREPARATION OF A TISSUE PREPARATION |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0220269A1 true EP0220269A1 (en) | 1987-05-06 |
Family
ID=20359937
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19860902936 Withdrawn EP0220269A1 (en) | 1985-04-23 | 1986-04-22 | Method of impregnating a tissue specimen with paraffin |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0220269A1 (en) |
| SE (1) | SE8501959L (en) |
| WO (1) | WO1986006479A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5089288A (en) * | 1989-06-24 | 1992-02-18 | Berger Hermann J | Method for impregnating tissue samples in paraffin |
| ATE169112T1 (en) * | 1996-08-02 | 1998-08-15 | Milestone Srl | METHOD FOR PROCESSING ORGANIC SAMPLES |
| US6793890B2 (en) | 1997-08-20 | 2004-09-21 | The University Of Miami | Rapid tissue processor |
| ATE407353T1 (en) * | 1997-08-20 | 2008-09-15 | Univ Miami | HIGH-QUALITY CONTINUOUS PROCESS FOR FIXATION, DEHYDRATION, DEGREASING AND IMPREGNATION OF TISSUES |
| WO2003029845A2 (en) * | 2001-10-01 | 2003-04-10 | Vision Biosystems Limited | Histological tissue specimen treatment |
| EP1673608A4 (en) | 2003-09-29 | 2008-05-07 | Vision Biosystems Ltd | System and method for histological tissue specimen processing |
| US20090298172A1 (en) | 2008-05-28 | 2009-12-03 | Steven Paul Wheeler | Histological specimen treatment apparatus and method |
| US10365189B2 (en) | 2015-05-07 | 2019-07-30 | Steven Wheeler | Histological specimen treatment |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2932112A1 (en) * | 1979-08-08 | 1981-02-26 | Carlos Prof Dr Thomas | METHOD AND DEVICE FOR EMBEDDING DRAINED TISSUE SAMPLES IN PARAFFIN |
-
1985
- 1985-04-23 SE SE8501959A patent/SE8501959L/en not_active Application Discontinuation
-
1986
- 1986-04-22 WO PCT/SE1986/000185 patent/WO1986006479A1/en unknown
- 1986-04-22 EP EP19860902936 patent/EP0220269A1/en not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO8606479A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| SE8501959L (en) | 1986-10-24 |
| SE8501959D0 (en) | 1985-04-23 |
| WO1986006479A1 (en) | 1986-11-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE FR GB IT LI LU NL SE |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 19870125 |