DE2207787A1 - Transporting- and storage medium for tissue biopsies - using aq chlorohexidene and buffer - Google Patents

Abstract

The medium contains 0.02-2% of chlorohexidine (1,6-di-(N,N'-p-chlorophenyl-guanidino-N5,N'5)-hexane and additionally a known buffering agent to maintain the pH at 7.0-7.8, pref. 7.2-7.5, such as kakodylate buffer. The medium should be hypertonic and contains therefore known agents such as polyvinylpyrrolidone, polyvinylalcohol, glucose solns. etc. Fungicides, antibiotics etc. may be added. The medium may be used for the transport or storage of tissue biopsies such as in the transport of tissue from operating theatre to cryogenic laboratories. The medium prevents bacterial growth, enzyme-histochemical methods may be used on the tissue after storage of up to 48 hrs. The medium appears to sterilise stabilised enzyme protein and allows reactive gps. to maintain a certain activity in vitro. It prevents visible shrinking, swelling, deformation, autolysis and bacterial invasion in tissue.

Description

Transport or storage medium for tissue biopsies.

The present invention relates to a transport or storage medium for tissue biopsies. Allow tissues stored in the medium according to the invention reliable determinations of the enzyme activity within a few days after the Ezcision of the tissues can be performed. The morphology of the cells remains well preserved after storage in the medium. The medium comprises an aqueous solution, the chlorhexidine (1,6-DI- (TJ N '-p-chlorophenylguanidino-N5N51) -hexane) and a buffering agent to maintain the pH of the solution from 7.0 to 7.8.

In histopathology, diagnoses are usually based on of morphological-histological and histochemical determinations of tissue sections performed under the light microscope.

The biopsy specimens intended for diagnosis are often placed in fixation fluids for routine drainage and embedding in paraffin into the Laboratories transported. It is known that the commonly used fixatives, e.g. physiological saline solution, formalin or glutaraldehyde solutions very strong have enzyme-inhibiting and protein-extracting effects. In addition, that requires pretty much complicated embedding technique with paraffin wax takes a lot of time and delays the pathological diagnosis.

By using freezing methods, diagnosis can be made with the Light microscope can usually be performed shortly after the sample arrives. Freeze-smith microtome techniques enable the detection of chemical components as well as enzymes to facilitate diagnosis. The frozen tissue can then fixed, watered, embedded in paraffin and stored in the usual way, for further histological studies.

The advantages of freshly frozen sections for the histopathological Diagnosis can be summarized as follows: relatively quick handling and the possibility of using enzyme activity determinations without the usual morphological ones Limit studies.

A major disadvantage of freezing methods is that they are instantaneous Freezing the tissue samples is not always feasible in the operating room. In many Cases, the excision of tissues is subjected to the histopathological diagnosis are to be carried out in locations that are far away from a laboratory, that is equipped for the cryo-method.

It would therefore be desirable to have a transport medium for tissue biopsies that prevents the growth of bacteria, that prevents the enzyme proteins and their activities well enough so that they can be included in histochemical techniques, when the samples reach the laboratory with the equipment for freezing technology. Such transport solutions may affect the tissue morphology do not destroy.

The transport medium according to the invention, which is a buffered chlorhexidine solution can be used for specimens for pathological-anatomical diagnosis can be used with simultaneous inhibition of bacterial growth, always nor the frozen section method, without subsequent washing, to remove the Medium is possible.

Enzyme histochemical demonstrations to distinguish e.g.

Diaphorase activities are even 48 hours after the immersion Tissue samples possible. The coloring reactions become noticeable after 72 hours of treatment reinforced. Tissues immersed in the same solution for a week were exposed of oxidative enzyme activities, but still showed acidic phosphatase and APase activities.

Enzyme histochemicals can be performed within 48 hours of surgery Methods to be applied to the tissues. Determinations of enzyme activity can be carried out are using reference data, the possible'nonenzymatic reactions concern and when the normal occurrence of enzyme activity in the current regions is known.

The period before the significant damage to the enzyme activity occurs up to 48 hours, is sufficient to transport the tissue biopsies in the transport medium to enable over long distances, e.g. from one continent to another or even for mailing the tissues if they are packed in thermal containers.

The exercise medium of the present invention appears to establish enzyme proteins and allow reactive groups to retain some activity in vitro. It also protects tissue from visible shrinkage, swelling, deformation and autolysis and bacterial invasion.

The chlorhexidine component of the transport or storage medium according to the invention is in the form of a water-soluble salt, e.g. the dihydrochloride or diacetate, used. If higher concentrations of chlorhexidine are desired, it can It may be appropriate to use a salt of a polyhydroxy acid, e.g. gluconic acid. The amount of qilahexidine, calculated as the free base, should be in the range of 0.02 to 2% by weight, preferably 0.07-1, based on the transport medium.

The medium should contain a buffer capable of maintaining the pH of the medium of 7.0-7.8, preferably 7.2-7.5, so as not to maintain Destroying the pH of the tissue biopsies, resulting in the histopathological determinations can make impossible. The selection of the buffering agent is not critical, any known one Buffer giving a pH in the above range can be used. as Cacodylate buffer can be named as an example of a suitable buffering agent. It is important that the buffering agent does not have any insoluble salts with the chlorhexidine component forms. However, this does not pose a problem for the person skilled in the art, through simple tests suitable buffering agents can be determined.

Since the medium according to the invention is used as a transport or storage medium to be used for tissue it must be hypertonic with respect to the tissues. Hypertension is created by introducing known compounds with this property obtained, e.g. from polyvinylpyrrolidone, polyvinyl alcohol, glucose solutions, etc. The The amounts and molecular weights of these polymers are known and can be known to the person skilled in the art can be found in the literature. As an example of a suitable addition to Achieving hypertension can polyvinylpyrrolidone with a molecular weight of 24,000, introduced in an amount of 7.5 wt. Based on the transport medium, be cited.

Further suitable additives can be used in conjunction with the inventive Transport- or storage medium, e.g. fungicides, antibiotics, etc.

The best results in the preservation of those in the invention Medium transported or stored tissue biopsies are obtained when the medium and the tissue is kept cool, e.g. at temperatures between + O - 80C, the temperature from 2 to 600 is preferred. The transport or storage can be carried out in Isolation vessels are carried out.

The following example shows the effect of the transport according to the invention or storage medium. It explains the invention without describing it.

Example: biopsies were taken from the gingiva of 10 patients, the.

Receive prophylactic periodontal treatment 2 weeks beforehand had to reduce the clinical signs of acute inflammation and around to obtain a comparable test sample. The biopsy samples were. into little pieces divided (about 5 sm3 each) for comparative studies ("between subject" and "within subject") after storage in different solutions. By histological Examination and histochemical examination for chemical components or on The effects of chlorhexidine solutions on the samples were compared with those of enzymes 10% neutral buffered formalin, 3% glutaraldehyde in 0.675 mol cacodylate buffer (pH 7.2) and physiological saline solution. The tissues of all 10 patients passed all tests. Freshly frozen tissues were used as controls in the studies included.

Estperioden: The tissue samples were taken immediately after the excision and Subdivision described above in different solutions (+ 4 ° C) introduced. They were stored in a refrigerator for 24, 48 and 42 hours before moving in cryostatic injectors were placed in and in liquid nitrogen were frozen. Long-term effects were seen after 7 days of immersion determined.

Chlorohexidine solutions: The test solutions (final pH 7.2) passed from 0.22 itol, 2.2 mmol and 22 mmol chlorhexidine digluconate in 0.5 mol cacodylate buffer (pH 7.5) and 7.5% polyvinylpyrrolidone (PVP, MW 24,000, Sigma R, USA). The amounts of chlorhexidine, calculated as the free base, were 0.012, 0.12 and 1.2% by weight, based on the solution. The effects of the buffer alone and the chlorhexidine solutions without buffer and PVP Were tested.

Light and electron microscopic examination: tissue sections (8 microns) were in a cryostat (System Dittes-Duspiva, Heidelberg, Germany) -200C cut. They were made with hematoxylin and eosin or according to the von Gieson technique colored. The morphological determinations were made as double blind tests of the various Treatments performed under the light microscope.

The mean values of 10 different determinations were determined and expressed in signs (+) listed in Table 1. Thick cuts (40 microns) of the same frozen samples were used as electron microscopic controls used without the usual fixation in glutaraldehyde. The frozen sections were treated directly with 1% O. ^ O4 and 0.14 M veronal buffer (pH 7.2) for one hour, embedded in Epon 812 (Ladd Research Industries, Inc., USA) and in an LKB Ultrotome IIIR (Sweden) cut (500 Å). The thin sections were made with uranyl acetate and lead citrate stained. Control samples (frozen) were in 3% glutaraldehyde and 0.075 moles of sodium cacodylate buffer, pH 7.2, for 3 hours prior to the above Treatment fixed.

Investigation of histochemical components: frozen sections with the Microtome (8 microns) were used to determine for sulphydryl groups by the DDD method stained (Barrnett, R.J. + Seligman, A.M .: Histochemical demonstration of sulphydryl and disulfide group of protein. J. Nat. Canc. Inst. 14: 769, 1952), for determination of acidic mucosubstances with 0.1% toluidine blue in 30% ethanol (pH 5.7) and for the determination of unsaturated hydrophobic lipids with the OTAN technique (Adams, C.W.M .: A histochemical method for the simultaneous demonstration of normal and degenerating myelin. J. Path. Bact. 77: 649, 1959) stained.

Load cells were stained using the Bloom + Kelly astrablau technique (Bloom G. and Kelly, J.W .: The copper phthalocyanine dye "astrablau" and its staining properties, especially the staining of mast cells Histochemie 2: 48, 1960). the The results are shown in Table 1.

Histochemical enzyme studies: frozen sections with the microtome (8 microns) were incubated for 15 minutes at 37 ° O for NADH2 and NADPH2 diaphorase activities to demonstrate Chayen J., Bitensky, L., Butcher, R. + Poulter, L .: A Guide to Pracitcal Histochemistry. Oliver + Boyd, Edinburgh, 1969) and the ATPase activity at pH 7.2 (Jacobsen, iT.0. + Leth Jorgensen, P .: A quantitative biochemical and histochemical study of the lead method for localization of adenosine triphosphate hydrolyzing enzymes. J. Histochem. Cytochem. 17: 443, 9), the ATPase activity at pH 9.4 (Padykula, H.A. + Hermann, E .: The specificity of the histochemical method for adenosine triphosphatase. J. Histochem. Cytochem. 3: 170, 155) and the acid phosphatase activity (azo dye method, modified by Burstone; Barka, T.

+ Anderson, P.J .: Histochemistry: Theory, Practice and Bibliography. Hoeber Med. Div., Harper + Row, New York 1963). Control cuts were made without the respective substrates and after enzyme-inhibiting treatments (10% formals on the tissue sections) to indicate possible non-enzymatic staining.

The determinations under the light microscope were 3 different from each other conducted by independent researchers. The mean values of the 10 different examinations were determined and expressed in signs (+), which can be seen from Table 2.

In the tables, the results obtained with freshly frozen tissues are used as controls. If a value is better than the control, this is indicated with +++. If no determinations could be carried out, eg if the samples were destroyed during storage, this is noted with an O. As can be seen from the tables, the samples stored in a chlorhexidine solution according to the invention are in most cases the same or even better than the control samples, whereas the values obtained with the samples stored in known fixation solutions show that the tissue has been destroyed. Table 1 Determinations of the histological and histochemical staining results under the light microscope, after various treatments of the tissue samples before the frozen section with the microtome Tests: morphology of acid mast cells lipids sulfhydryl groups Mucosub punch Staining Techniques Haematoxylin Toluidine Astra Blue Otan DDD and eosin blue Fresh frozen ++ ++ ++ ++ ++ Saline solution 24 * + + ++ ++ ++ (physiological) 48 "+ + ++ ++ ++ 72 "0 + ++ ++ ++ Glutaraldehyde 24H 0 + + + 0 (3% buffered) 48 "0 + + 0 0 72 "0 + + 0 0 Formalin 24H 0 + + + + (10% neutral 48 "0 + + + + buffered) 72 "0 + + + + Chlorohexidine 24H ++ ++ ++ ++ +++ (0.2% 2.2 mM, 48 "++ ++ ++ +++ +++ with buffer and 72 "++ ++ ++ ++ ++ Polyvinylpyrro lidon) * Hours at + 4 ° C. Table 2 Histochemical determinations of retained enzyme activities after various treatments of the tissue samples Enzyme activities NADH2 diaphorase NADH2 diaphorase ATP-ase ATP-ase, acidic phosphorus pH 9.4 pH 7.2 phase freshly frozen ++ ++ ++ ++ ++ Saline solution 24H * ++ ++ ++ ++ ++ (physiological) 48 "+ ++ + + + 72 "0 + + + + Glutaraldehyde 24H + 0 0 + 0 (3% buffered) 48 "0 0 0 0 0 72 "0 0 0 0 0 Formalin 24H + + 0 0 0 (10% neutral) 48 "0 0 0 0 0 buffered) 72 "0 0 0 0 0 Chlorhexidine 24H ++ ++ +++ ++ ++ (0.2% 2.2mH, 48 "++ ++ ++ + ++ with buffer and 72 "++ + ++ + ++ Polyvinylpyrrolidone) * Hours at + 4 ° C

Claims (3)

Claims 1. Transport or storage medium for tissue bopes, characterized in that it contains 0.02-2% by weight of chlorhexidine. 2. Medium according to claim 1, characterized in that it is additionally contains a well-known buffering agent to maintain the pH from 7.0 to 7.8. 3. Medium according to claim 2, characterized in that the buffering agent Is cacodylate buffer.