WO1990007703A1 - Tissue preservation medium - Google Patents

Tissue preservation medium Download PDF

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Publication number
WO1990007703A1
WO1990007703A1 PCT/GB1989/001540 GB8901540W WO9007703A1 WO 1990007703 A1 WO1990007703 A1 WO 1990007703A1 GB 8901540 W GB8901540 W GB 8901540W WO 9007703 A1 WO9007703 A1 WO 9007703A1
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WIPO (PCT)
Prior art keywords
tissue
medium
preservation medium
tissue preservation
sections
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Application number
PCT/GB1989/001540
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French (fr)
Inventor
Ann Carmichael
George Reid Coghill
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The University Of Dundee
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
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Publication of WO1990007703A1 publication Critical patent/WO1990007703A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof

Definitions

  • This invention relates to a tissue preservation medium.
  • Frozen sections allow the demonstration of formalin- sensitive antigens, but the tissue must be frozen as soon as possible after it is obtained to preserve the antigens. This means that the patient must be brought to the hospital nearest to the laborator , necessitating admission or a further out-patient appointment.
  • the current alternative is for the tissue to be frozen at the outlying hospital or clinic and brought back to the main laboratory later in liquid nitrogen or carbon dioxide ice.
  • morphological detail tends to be poorer in frozen sections compared with that present in paraffin sections so that one often has the choice of immunocytochemistry or morphology, but not both.
  • reagents e.g. gl cerol, Histocon and Michel's Transport Medium
  • glycerol The properties of glycerol are thought to depend upon retaining adequate stabilisation of tissue with resulting immobilisation of the macromolecular systems of that tissue.
  • Histocon containing chlorhexidine and cacodylate buffer, is thought to have anti-bacterial and "preserving" properties and has been shown to allow combined histological, enzyme histochemical and electron microscope studies.
  • Michel et al produced a similar approach to a medium for transporting biopsy specimens with a further aim to using immunofluorescent techniques.
  • This liquid medium containing N-ethyl malemide buffer with ammonium sulphate, was used giving an excellent preservation of tissue fixed immunoglobulins. It has also been found to give satisfactory preservation of the antigenicity of surface markers on lymphocytes for immunological evaluation.
  • tissue preservation medium comprising an inhibitor of bacterial activity, a material which maintains the structural integrity of tissue and a material which maintains the isotonicity of tissue.
  • Gelatin, agar and polyethylene glycol can each be employed to maintain the structural integrity of the tissue, and the concentration of these materials is preferably selected to 'ensure that the stored tissue can be easily and effectively cut.
  • concentration of these materials is preferably selected to 'ensure that the stored tissue can be easily and effectively cut.
  • up to a 5% aqueous solution most preferably 1 to 3%, with the optimum being 1.5%, has been found to provide good cutting; too high a concentration of gelatin produces a tendency for the sections to shatter.
  • the structural integrity of the tissue can also be maintained using a sugar such as sucrose or glucose which prevent blood vessels splitting and assist in providing a good cutting facility for the tissue and medium. Again, the concentration of the sugar should be selected accordingly, and preferably should be between 3% and 9% with 7.5% being the optimum.
  • the bacterial activity inhibitor is preferably a cacodylate, for example sodium cacodylate which acts also as a buffer for maintaining the molarity of the medium.
  • Sodium cacodylate has the formula [(CH3)2 As02 Na.3H20] and it is suggested that the arsenic content assists in conferring tissue preservative properties on the medium.
  • the cacodylate also provides maintenance of the tissue's isotonicity. It is important to ensure sterility of the components used to make up the transport medium as far as possible.
  • tissue preservation medium comprising an aqueous solution of gelatin, a cacodylate, preferably sodium cacodylate, and a sugar, preferably sucrose.
  • the concentration of the medium is important. Too high a concentration makes it difficult for the medium to be frozen, and cutting of the tissue is then impaired. Too low a concentration reduces the preservative properties of the medium and adversely affects its ability to maintain tissue cohesion.
  • EXAMPLE 1 To 1 litre of distilled water were added 21.4g of sodium cacodylate and 75g of sucrose. The pH of the resulting solution was 7.0 - 7.2. lOg of gelatin was then added, and the solution was heated to 40 degrees C with stirring to dissolve the gelatin.
  • the medium thus produced was stored at 4 degrees C.
  • Lymphoid tissue in the form of tonsillectomies were used to test the effectiveness of the medium, pieces of freshly- removed tonsil being cut into 7 or 8mm cubes and placed in the medium for the stated number of days.
  • Tissue tonsil from 7 year old female Storage Time : 1 and 3 days
  • Macroscopic appearance the preserved tissue produced excellent results. It had similar consistency to that of freshly cut tissue and there was no evidence of swelling or colour loss within the tissue. Cutting: the preserved tissue cut exceptionally well giving flat sections completely free of wrinkles. The preserved tissue cut better than freshly-frozen tissue.
  • a series of skin biopsies from patients with contact dermatitis were used to compare the conventional fresh biopsy and immediate frozen section method with tissue kept in the transport medium for one week before frozen section.
  • Biopsies 4m skin punch biopsies were taken from 22 patients with contact dermatitis attending a dermatology out-patient department. Each biopsy was bisected immediately using a razor blade. One half was frozen immediately on metal foil using liquid nitrogen and sent to the laboratory. On receipt the biopsy was stored in liquid nitrogen for one week. The other half of the biopsy was placed in a transport medium and sent to the laboratory where it was kept in a refrigerator (0-4 C) for one week before it was mounted onto a chuck and frozen using liquid nitrogen. The medium is a gel at 0-4 C, but can be easily liquified in warm water so that the biopsy can be fully immersed in it. Both biopsies were then cut at 6 urn in a Slee crystat (Histological Equipment Ltd. Carshalton, Surrey, England) .
  • Immunoperoxidase staining Sections from both halves of each biopsy were stained simultaneously using an avidin- biotin complex (ABC) method (Vector Laboratories Ltd. , Peterborough, England) , with a batter of monoclonal ' antibodies against imflammatory cell antigens (table 1) . The sections were counterstained with Mayer's haemalum, dehydrated and mounted in DPX.
  • ABSC avidin- biotin complex
  • Table l Battery of monocional antibodies employed for immunoperoxidase staining.
  • Histological Assessmen The slides were assessed subjectively on a scale of 1 (excellent) to 5 (unreadable) for morphology and staining (see table 2) by a pathologist. Each slide was reviewed in turn with no attemp to randomise the frozen or transport medium slides. Table 2. Scale for assessment of staining and morphology.

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  • Health & Medical Sciences (AREA)
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  • Physics & Mathematics (AREA)
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Abstract

The requirement for unfixed tissue is a major complication in the use of immunocytochemistry for the diagnosis of inflammatory disease. A solution to this problem is provided by the use of a novel gel transport medium which is capable of preserving unfixed skin biopsies from contact dermatitis patients for one week prior to frozen section and immunocytochemistry for leukocyte antigens. The use of the medium provides a considerable subjective improvement in both morphology and staining quality. This is thought to be due to improved cutting of the sections and reduced background staining of collagen. This transport medium allows tissue to be taken at sites distant from a pathology laboratory for immunohistochemical examination.

Description

Tissue Preservation Medium
This invention relates to a tissue preservation medium.
The requirement for unfixed tissue is a major complication in the use of immunocytochemistry for the diagnosis of inflammatory disease. Tissue samples for diagnosis are usually transported to the laboratory for histological examination in a fixative, the most popular of which is buffered formalin (4% formaldehyde). This is a simple technique which allows thin paraffin-embedded tissue sections to be prepared for routine histology. However, if one wishes to demonstrate the presence of antigens in such tissue by immunocytochemistry, the cross-linking effects of the fixative on proteins often preclude this. Many laboratories therefore ask specimens requiring immunocytochemistry to be sent to them without fixation for subsequent frozen sectioning.
Frozen sections allow the demonstration of formalin- sensitive antigens, but the tissue must be frozen as soon as possible after it is obtained to preserve the antigens. This means that the patient must be brought to the hospital nearest to the laborator , necessitating admission or a further out-patient appointment. The current alternative is for the tissue to be frozen at the outlying hospital or clinic and brought back to the main laboratory later in liquid nitrogen or carbon dioxide ice.
In addition, morphological detail tends to be poorer in frozen sections compared with that present in paraffin sections so that one often has the choice of immunocytochemistry or morphology, but not both.
For some years, therefore there has been a requirement for a fixative-free method of transporting tissue to the laboratory from outlying clinics or hospitals.
Over the years, many reagents e.g. gl cerol, Histocon and Michel's Transport Medium, have been used for preserving tissue prior to, or just after, fixation. The properties of glycerol are thought to depend upon retaining adequate stabilisation of tissue with resulting immobilisation of the macromolecular systems of that tissue. Histocon, containing chlorhexidine and cacodylate buffer, is thought to have anti-bacterial and "preserving" properties and has been shown to allow combined histological, enzyme histochemical and electron microscope studies. In 1973, Michel et al produced a similar approach to a medium for transporting biopsy specimens with a further aim to using immunofluorescent techniques. This liquid medium, containing N-ethyl malemide buffer with ammonium sulphate, was used giving an excellent preservation of tissue fixed immunoglobulins. It has also been found to give satisfactory preservation of the antigenicity of surface markers on lymphocytes for immunological evaluation.
Although various forms of media have already been used, there has been little attempt to use such media for preserving tissues over a length of time. According to the present invention there is provided a tissue preservation medium comprising an inhibitor of bacterial activity, a material which maintains the structural integrity of tissue and a material which maintains the isotonicity of tissue.
Gelatin, agar and polyethylene glycol can each be employed to maintain the structural integrity of the tissue, and the concentration of these materials is preferably selected to 'ensure that the stored tissue can be easily and effectively cut. In the case of gelatin, for example, up to a 5% aqueous solution, most preferably 1 to 3%, with the optimum being 1.5%, has been found to provide good cutting; too high a concentration of gelatin produces a tendency for the sections to shatter. The structural integrity of the tissue can also be maintained using a sugar such as sucrose or glucose which prevent blood vessels splitting and assist in providing a good cutting facility for the tissue and medium. Again, the concentration of the sugar should be selected accordingly, and preferably should be between 3% and 9% with 7.5% being the optimum.
The bacterial activity inhibitor is preferably a cacodylate, for example sodium cacodylate which acts also as a buffer for maintaining the molarity of the medium. Sodium cacodylate has the formula [(CH3)2 As02 Na.3H20] and it is suggested that the arsenic content assists in conferring tissue preservative properties on the medium. The cacodylate also provides maintenance of the tissue's isotonicity. It is important to ensure sterility of the components used to make up the transport medium as far as possible.
Further according to the invention there is provided a tissue preservation medium comprising an aqueous solution of gelatin, a cacodylate, preferably sodium cacodylate, and a sugar, preferably sucrose.
The concentration of the medium is important. Too high a concentration makes it difficult for the medium to be frozen, and cutting of the tissue is then impaired. Too low a concentration reduces the preservative properties of the medium and adversely affects its ability to maintain tissue cohesion.
Embodiments of the present invention will now be described by way of illustration in the following Examples.
EXAMPLE 1 To 1 litre of distilled water were added 21.4g of sodium cacodylate and 75g of sucrose. The pH of the resulting solution was 7.0 - 7.2. lOg of gelatin was then added, and the solution was heated to 40 degrees C with stirring to dissolve the gelatin.
The medium thus produced was stored at 4 degrees C.
Lymphoid tissue in the form of tonsillectomies were used to test the effectiveness of the medium, pieces of freshly- removed tonsil being cut into 7 or 8mm cubes and placed in the medium for the stated number of days.
A. Tissue : tonsil from 7 year old female Storage Time : 1 and 3 days
Macroscopic appearance: the preserved tissue produced excellent results. It had similar consistency to that of freshly cut tissue and there was no evidence of swelling or colour loss within the tissue. Cutting: the preserved tissue cut exceptionally well giving flat sections completely free of wrinkles. The preserved tissue cut better than freshly-frozen tissue.
Microscopic appearance: the preserved tissue displayed good tissue preservation, and excellent staining with monoclonal antibodies.
B. Tissue which had been immersed in the medium for two weeks was smeared on Agar plates using a wire loop. the plates were stored overnight at 37oC and showed no sign of bacterial growth.
EXAMPLE 2
A series of skin biopsies from patients with contact dermatitis were used to compare the conventional fresh biopsy and immediate frozen section method with tissue kept in the transport medium for one week before frozen section.
Material and Methods
Biopsies: 4m skin punch biopsies were taken from 22 patients with contact dermatitis attending a dermatology out-patient department. Each biopsy was bisected immediately using a razor blade. One half was frozen immediately on metal foil using liquid nitrogen and sent to the laboratory. On receipt the biopsy was stored in liquid nitrogen for one week. The other half of the biopsy was placed in a transport medium and sent to the laboratory where it was kept in a refrigerator (0-4 C) for one week before it was mounted onto a chuck and frozen using liquid nitrogen. The medium is a gel at 0-4 C, but can be easily liquified in warm water so that the biopsy can be fully immersed in it. Both biopsies were then cut at 6 urn in a Slee crystat (Histological Equipment Ltd. Carshalton, Surrey, England) .
Immunoperoxidase staining: Sections from both halves of each biopsy were stained simultaneously using an avidin- biotin complex (ABC) method (Vector Laboratories Ltd. , Peterborough, England) , with a batter of monoclonal ' antibodies against imflammatory cell antigens (table 1) . The sections were counterstained with Mayer's haemalum, dehydrated and mounted in DPX.
Table l. Battery of monocional antibodies employed for immunoperoxidase staining.
Name Company Antigenic site
CD3 Dako Pan-T cell marker CD4 Dako Helper T cell subset CD8 Dako Suppressor/Cytotoxic T Cell subset CD1 Dako Langerhans cells CD19 Dako B cells Mo Dako Macrophages PC Dako Proliferating cell antigen
MC2 (CD15) Neutrophils, fucosyl galactosamine
Histological Assessmen: The slides were assessed subjectively on a scale of 1 (excellent) to 5 (unreadable) for morphology and staining (see table 2) by a pathologist. Each slide was reviewed in turn with no attemp to randomise the frozen or transport medium slides. Table 2. Scale for assessment of staining and morphology.
Grade Description
1 Excellent
2 Good
3 Average
4 Poor
5 Unreadable
RESULTS
Morphology and staining quality were consistently better in the transport medium sections than in conventional frozen sections. The average grades for each monoclonal antibody used are shown in table 3. There is some difference between monoclonal antibodies, but in most sections which had been in transport medium, the background collagen staining was remarkably low with normal or slightly reduced specific staining intensity. This was responsible for the better signal-to-noise reflected in the better staining grade given to sections from transport medium-treated biopsies.
The subjective improvement in morphology appears to have been due to reduced cutting artefact. On cutting the sections it was found that acceptable sections could be obtained from transport medium treated tissue with relative ease and there were fewer unusable or poor sections overall from transport medium treated tissue. Orientation of the biopsy on the chuck was also found to be easier after the tissue had been in the transport gel. A proportion of the biopsies were taken from sever contact dermatitis reactions with incipient blistering. This was clearly visible in transport medium treated biopsies, but not in the conventionally treated biopsies. Likewise, spongiotic vesicles were easily seen in transport medium treated biopsies.
Table 3. Differences in staining quality and morphological grade for each monoclonal antibody used (1 = excellent, 5 = unusable) .
Figure imgf000010_0001
These results illustrate the superiority of transport medium over conventional treatment of tissue for the preparation of frozen sections. Even after one week's storage at 0-4 C the tissue staining and morphology is better than that obtained in biopsies frozen immediately. Furthermore, this improvement in section quality can be obtained in biopsies taken at a considerable distance from the laboratory and transported to the main hospital before being processed. This avoids having patient travel long distances to special biopsy clinics in the main centr and means that biopsies can be done at the time of the first visi The improvement in quality is sufficient to permit image analysis of immunoperoxidase stained sections, something not previously possible.
Modifications and improvements may be made without departing from the scope of the invention.

Claims

1. A tissue preservation medium comprising an inhibitor of bacterial activity, a material which maintains the structural integrity of tissue and a material which maintains the isotonicity of tissue.
2. A tissue preservation medium as claimed in Claim 1, wherein the material employed to maintain the structural integrity of the tissue is gelatin, agar or polyethylene glycol.
3. A tissue preservation medium as claimed in Claim 1, wherein the material employed to maintain the structural integrity of the tissue is a less than 5% aqueous solution of gelatin.
4. A tissue preservation medium as claimed in Claim 3, wherein an aqueous solution in the range 1%. to 3% gelatin is employed.
5. A tissue preservation medium as claimed in Claim 4, wherein an aqueous solution of 1.5% gelatin is employed.
6. A tissue preservation medium as claimed in Claim 1, wherein the material employed to maintain the structural integrity of the tissue is a sugar solution.
7. A tissue preservation as claimed in Claim 6, wherein the sugar is a sucrose or glucose soluion of between 3% and 9% concentration.
8. A tissue preservation medium as claimed in Claim 7, wherein the sucrose or glucose solution has a 7.5% concentration.
9. A tissue preservation medium as claimed in any one of the preceding Claims, wherein the bacterial activity inhibitor is a cacodylate.
10. A tissue preservation medium as claimed in Claim 9, wherein the bacterial activity inhibitor is sodium cacodylate.
11. A tissue preservation medium comprising an aqueous solution of gelatin, a cacodylate and a sugar.
12. A tissue preservation medium as claimed in Claim 11, wherein the cacodylate is sodium cacodylate and the sugar is sucrose.
PCT/GB1989/001540 1988-12-23 1989-12-27 Tissue preservation medium WO1990007703A1 (en)

Applications Claiming Priority (2)

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GB8830197.3 1988-12-23
GB888830197A GB8830197D0 (en) 1988-12-23 1988-12-23 Tissue preservation medium

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WO1990007703A1 true WO1990007703A1 (en) 1990-07-12

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1253340A (en) * 1970-05-28 1971-11-10 Inst Pentru Controlul De Stat Preservation of tissues and organs
GB1416733A (en) * 1972-02-21 1975-12-03
GB2114291A (en) * 1982-02-04 1983-08-17 James H Harrison Preservative and fixative preparations for biological systems
US4506018A (en) * 1982-12-30 1985-03-19 Becton, Dickinson And Company Blood diluent
EP0262966A2 (en) * 1986-10-01 1988-04-06 Animal House, Inc. Sampling device
WO1988003028A1 (en) * 1984-11-29 1988-05-05 New England Medical Center Hospitals, Inc. Stabilization of leukocytes
JPS63168562A (en) * 1986-12-30 1988-07-12 Motohide Takahama Cell immobilizing/preserving liquid

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1253340A (en) * 1970-05-28 1971-11-10 Inst Pentru Controlul De Stat Preservation of tissues and organs
GB1416733A (en) * 1972-02-21 1975-12-03
GB2114291A (en) * 1982-02-04 1983-08-17 James H Harrison Preservative and fixative preparations for biological systems
US4506018A (en) * 1982-12-30 1985-03-19 Becton, Dickinson And Company Blood diluent
WO1988003028A1 (en) * 1984-11-29 1988-05-05 New England Medical Center Hospitals, Inc. Stabilization of leukocytes
EP0262966A2 (en) * 1986-10-01 1988-04-06 Animal House, Inc. Sampling device
JPS63168562A (en) * 1986-12-30 1988-07-12 Motohide Takahama Cell immobilizing/preserving liquid

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, volume 101, no. 25, 17 December 1984, (Columbus, Ohio, US), Albrecht Reith et al.: "The influence of mode of fixation, type of fixative and vehicles on the same rat liver: a morphometric/stereologicstudy by lig ht and electron microscopy", page 381, abstract 226048n, & Scanning Electron Microsc. 1984, 2(), 645-651. *
Chemical Abstracts, volume 111, no. 5, 31 July 1989, (Columbus, Ohio, US), see page 322, abstract 36257p, & JP,A,63 168 562 (12 July 1988) *
Chemical Abstracts, volume 90, no. 7, 12 February 1979, (Columbus, Ohio, US), Sugai Naonori et al : "Effects of pretreatment with sucrose-containing cacodylate buffer on alkaline phosphatase activity in rat tissue sections", page 211, abstract 50267w, & Fukushima J. Med. Sci. 1977, 24(30), 89-94. *

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EP0449925A1 (en) 1991-10-09
AU4832190A (en) 1990-08-01
GB8830197D0 (en) 1989-02-22

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