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  • 优先权
    • 专利摘要:
    Process for obtaining a product comprising a gelatinous support of great transparency and strength containing a part of Klotz, wherein said support is suitable for keeping biological samples inside for long periods of time, and where that support allows visual access and physical to the samples of its interior as well as the passage of the ultrasounds of the echographs without distortions so it is optimal for learning. The support comprises gelatin and Klotz (which in turn comprises Chloral hydrate, Sodium sulfate anhydrous, Potassium Sulfate, Sodium Chloride, Sodium Bicarbonate, Ascorbic acid, water (most of the solution) and a small proportion of formaldehyde). The product obtained is also subject to the patent. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2673443A2
    申请号:ES201631640
    申请日:2016-12-21
    公开日:2018-06-21
    发明作者:Agustín BARRAGÁN HERNÁNDEZ；Vicente ESTEVE BERNET
    申请人:CARDENAL HERRERA CEU, University of；UNIVERSIDAD CARDENAL HERRERA CEU；
    IPC主号:
    专利说明:

    PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ AND PRODUCT OBTAINED WITH SUCH PROCEDURE

    As the name implies, the present invention relates to a method for the conservation of biological samples and the product obtained and used for such preservation.
    The patent therefore comprises the procedure for the conservation of biological samples and the product obtained with said procedure and which is used for the conservation of such samples.
    What is pursued is the obtaining of a semi-solid block of great resistance and transparency that allows biological samples to be stored inside and that these are preserved for long periods of time all without prejudice to allowing access to such samples, both visual access , as a physicist (through punctures or other means and surgical systems) or through ultrasound, being of great value for use in learning. fifteen

    BACKGROUND OF THE INVENTION
    There are different types of fixation in which they are used to process biological samples. They are grouped into physical methods (such as freezing in liquid nitrogen) or chemical methods. twenty
    Within the chemical methods the most used fixative is formalin (40% formaldehyde). This compound is highly carcinogenic and its handling has to be carried out with many precautions, so substitutes are being sought to eliminate its use.
    There are already some formaldehyde substitutes such as Histofix® on the market that are not so dangerous and also their smell is not strong. 25
    There are also solutions that carry a small proportion of formaldehyde, such as the Bouin and Klotz solution.
    The Bouin solution is useful especially for performing immunohistochemical techniques because it produces a large nuclear fixation although it has the disadvantage that destroys some cytoplasmic components. 30
    On the other hand, Klotz is a solution composed of Chloral Hydrate, Sodium Sulfate Anhydrous, Potassium Sulfate, Sodium Chloride, Sodium Bicarbonate, Ascorbic Acid, Water (most of the solution) and a small proportion of formaldehyde (5% of the total ).
    For the manufacture of 5 liters of Klotz the applicant uses the following quantities of the following elements.
    For 5 L
    - Chloral Hydrate 49.94g
    - Sodium sulfate anhydrous 79.93g
    - Potassium Sulfate 1.26g
    - Sodium Chloride 1.47g
    - Sodium bicarbonate 3g
    - Ascorbic acid 2.64g
    - Water 4900gr
    - Formol 24.97 ml

    The Klotz has the benefit that it only has a small proportion of formalin and allows the biological samples (organs, tissues, etc ...) to have a macroscopic appearance similar to what they would have just taken out of the corpse and their texture would also be similar for what it is Very useful for storing viscera for anatomical and pathological reviews.
    To fix the samples with Klotz, you have to immerse them in the solution for a minimum of 5 days, and once set they can already be kept submerged in the solution, provided they do not dry, although the solution must be changed from time to time.
    To avoid having to change the solution, whatever it was, and avoid drying out the biological samples, one possibility was to gel the fixative so that both the biological sample and the fixative were "trapped" in a semi-solid block and visually accessible, allowing the practice of punctures and other tests as well as useful for the use of ultrasound and ultrasound systems.
    The use of gelatin, due to its chemical and physical characteristics, was considered suitable.
    Powdered gelatin is used in the food industry to make meals and desserts, but it has also been used in ultrasound, with water, to perform symptoms, which are 20 models for the practice of ultrasound study. The different symptoms used so far use materials that pretend to simulate the appearance of organs (olives, macaroni) or slaughterhouse viscera (chicken breasts, lamb livers) that, if not fixed, cannot be maintained over time.
    The use of jellies is already anticipated in some documents of both patents 25 and non-patents.
    As an example, we can mention the article by JRV Pulvertaft, from the Westmister School Hospital of Medicine in London, “Museum Techniques, a review” (1950), which he describes on page. 16 in the preparation of transparent specimens, the use of compositions that may include 5% gelatin and 0.4% formalin, being that in that case the specimens must be placed in the refrigerator for the gelatin to solidify. 5
    WO0025580A1 refers to a method that uses a biological material preservation solution that is in gel form at temperatures below 10 degrees and pressures greater than 79 atm, wherein the solution comprises gelatin, preferably in amounts less than 5 %, in addition to sodium chloride and sugars. 10
    GB1253340A describes a means for the preservation of tissues and organs comprising among other compounds hydrated gelatin, honey and glycerin. It becomes liquid at temperatures above 40 ° C.
    None of the documents cited solve how to obtain a semi-solid and highly transparent composition that remains stable at room temperature (20-40 ° C) and is suitable for the conservation of biological samples.
    Some of the gelation tests carried out - and which are described below - also did not result in a semi-solid block, highly transparent and stable at room temperature.
    Thus, for example, in gelation with formalin, gelatin precipitated upon contact with it, making it impossible to form a compact block.
    On the other hand, in gelation with Histofix®, the resulting gelatinous compound was also very resistant but more yellowish in color and, in addition, at temperatures of 40⁰ Celsius it is liquefied so it does not offer the desired stability.
    The proposed invention overcomes the problems presented by obtaining a semi-solid block 25 of great strength and transparency that allows the biological samples stored for long periods of time to be stored inside and allows access to such samples both visually and physically (a through punctures or other means and surgical systems) or through ultrasound, being of great value for use for learning. 30

    DESCRIPTION OF THE INVENTION
    To facilitate the use of fixed viscera and for use in other disciplines such as diagnostic imaging, the use of a gelatinous support with preservative properties is proposed. 35
    For this purpose, a procedure has been developed to obtain a product that comprises a gelatinous support of great transparency and resistance that contains a part of Klotz, where that support is suitable for keeping biological samples inside it for long periods of time, and where This support allows visual and physical access to the samples inside, as well as the passage of ultrasound 5 of the ultrasound scanners without distortion, making it optimal for learning.
    The support comprises gelatin and Klotz (which in turn comprises Chloral Hydrate, Sodium Anhydrous Sulfate, Potassium Sulfate, Sodium Chloride, Sodium Bicarbonate, Ascorbic Acid, water (most of the solution) and a small proportion of formaldehyde).
    The tests have been done with powdered gelatin so the explanations and 10 correspondences are made based on this type of gelatin, however it is not ruled out that it can be performed with other types of gelatin, such as sheet gelatin or instant gelatin , having to look for the equivalences between a form of presentation of the product and another. As an example we cite that 10gr of gelatin powder equals 6 sheets of gelatin in sheets, according to the information in the portal www.gelatine.org of the Gelatine Manufacturers of Europe (GME).
    The procedure for the conservation of biological samples begins with the preparation of the compound that will contain them, which we will call "product" and which is also the subject of this patent.
    Previously it is necessary both to prepare the Klotz, preferably according to the formula that has been exposed and which, since it is already disclosed, is not claimed, and how to process the biological samples to be preserved by fixing them and washing them to eliminate the remains of blood.
    The washing of the biological samples and especially the elimination of the remains of blood, can be done with water but preferably it must be done with alcohol since it clears the tissue more and eliminates the blood better.
    Once this has been done, the procedure covered by this patent is followed and includes:
    1.- A first heating phase of approximately 1/3 of the Klotz to be used.
    The heating is carried out under hot plate stirring, the temperature rising slowly and slowly (approximately 30 degrees every minute) until the Klotz reaches a temperature between 80⁰ and 99⁰, preferably 90⁰ centigrade. Above 100⁰ centigrade the reagents would evaporate. The Klotz is maintained at that temperature for a period of 5 to 10 minutes, preferably 7 minutes. 35

    2.- A second phase, independent of the first phase, in which the jelly is dissolved in the remaining 2/3 of the Klotz to be used. The amount of gelatin that dissolves is between 6% and 8%, preferably between 6.5% and 7.5% of the total final amount of product to be prepared. If the amount of gelatin is reduced, the consistency would not be as desired, and if it increases, there would be virtually no change, only the medium would become more turbid and the cost would increase.
    3.- A third phase in which, before the three minutes following the solution exposed in the second phase, the hot Klotz of the first phase is mixed with the Klotz compound and gelatin of the second phase.
    If this third phase is done after 3 minutes from phase 2, the gelatin may precipitate and the gelidified support may not form correctly.
    With these first three phases, the product claimed in this patent is obtained.
    4.- A fourth phase where the product is poured into a mold with the organs that you want to keep inside, there are alternatives to the procedure depending on the use that you want to give the samples.
    5.- In a fifth phase, the product, with the organs inside, becomes refrigerated.
    As a result of this gelation, a practically transparent gelatin is obtained, more resistant than normal since the gelatin proteins have been fixed. In addition, this jelly allows the ultrasound of ultrasound machines to pass without practically the appearance of any type of device.
    Some of the advantages of this conservation product is that:
    - It allows to conserve organs with very little amount of formalin for long periods of time.
    - Allows the conservation and review of less frequent lesions at both macroscopic and ultrasound levels.
    - Allows the practice of ultrasound guided punctures, both at the teaching level and at the level of professional clinicians.
    As stated, depending on the type of use that is intended to be given to biological samples, there may be alternatives in the execution of the fourth phase of the procedure, 30 for example:
    To create suitable means for ultrasound, it is best to create a product bed in the mold to prevent the organs from being in contact with the bottom of the mold. Once the base is made, the organs are placed on top, fixed with needles or with sutures and filled with product before solidifying. 35
    For the preparation of models for cystocentesis, specific models can be made using urinary bladders with urethral catheters inside and a route applied to this catheter, so that the urinary bladder can be filled as many times as necessary.
    For this, the following steps must be followed: 5
    - Fix the bladder in Klotz
    - Insert a urinary catheter through the bladder neck, closing the neck around the catheter with a non-absorbable suture.
    - Create a product bed in the mold and fix the bladder to that base with sutures or needles. 10
    - Fill the bladder with fluid (water or other colored liquid).
    - Fill the mold with liquid gelled Klotz product before solidifying and refrigerate.
    - Remove the needles.
     fifteen
    DESCRIPTION OF A MODE OF CARRYING OUT THE INVENTION

    As it has been exposed, the procedure for the conservation of biological samples has a previous phase of preparation of one of the components, the Klotz, which is prepared according to the following quantities and weights. twenty
    For the manufacture of 5 liters of Klotz the applicant uses the following quantities of the following elements.
    For 5 L
    - Chloral Hydrate 49.94g
    - Sodium sulfate anhydrous 79.93g
    - Potassium Sulfate 1.26g
    - Sodium Chloride 1.47g
    - Sodium bicarbonate 3g
    - Ascorbic acid 2.64g
    - Water 4900gr
    - Formol 24.97 ml


     25




     30



    Likewise, the biological samples, prior to undergoing the conservation process, must be processed which includes:
    1. Fix them by using a type of fixation, preferably the Klotz although other means may be used.
    2. Wash them to remove the remains of blood, an operation that is normally carried out with 96⁰ alcohol and the remains of blood must be removed (it can be removed with water but the alcohol clears the tissue more and removes the blood better).
    The process of preservation of biological samples claimed 10 starts with the preparation of the product, and thus, for 150ml of product the following procedure is followed:
    1.- A first phase of heating of 50ml of Klotz:
    The heating is carried out under hot plate agitation, raising the temperature at a rate of 30⁰ degrees per minute until the Klotz reaches a temperature of 90⁰ 15 centigrade, temperature at which it must be maintained for 7 minutes.
    2.- A second phase, independent of the previous one, in which 10gr of standard powdered gelatin is dissolved in 100ml of Klotz.
    3.- Less than three minutes after the previous phase, a third phase takes place in which the Klotz is mixed at 90⁰ Celsius of the first phase with the compound of Klotz and gelatin of the second phase.
    With these first three phases the product that is also claimed in the present patent is obtained.
    This product is characterized in that it comprises Klotz (chloral hydrate, sodium anhydrous sulfate, potassium sulfate, sodium chloride, sodium bicarbonate, ascorbic acid, water, 25 formalin) and gelatin, the content of gelatin being between 6% and 8%, preferably between 6.5% and 7.5% of the total weight of the product, and because it also remains stable at a temperature between 20⁰ and 40⁰.
    4.- For the preservation of the biological samples, the procedure comprises a fourth phase in which the product is poured into a mold and the organs to be stored are deposited in said mold being coated with the product.
    Depending on the intended use of biological samples, the organs will be deposited in different ways that are alternatives to the preservation procedure.
    4.a. To preserve the organs so that they are suitable for ultrasound, the procedure includes, in phase 4, the following subphases:
     Creation of a product bed in the mold to prevent the organs from being in contact with the bottom of the container.
     Once the base is made, the organs are placed on it and fixed with 5 needles or with sutures.
     The mold is filled with the product until at least the sample is covered to subsequently remove the needles.
    4.b. For the preservation of organs that serve as a model for cystocentesis, so that the bladder can be filled as many times as necessary, the procedure 10 comprises, within its phase 4 the following subphases:
    - Insert a urinary catheter through the bladder neck, closing the neck around the catheter with a non-absorbable suture.
    - Create a product bed at the base of the mold and fix the bladder to the bed with sutures or needles. fifteen
    - Fill the bladder with fluid (water or other colored liquid).
    - Fill the mold with product until at least cover the sample.
    - Later the needles will be removed.

    5.- Finally, a fifth phase of the procedure for the conservation of biological samples 20, includes the refrigeration (between 7 and 14 degrees) of the product with the organs inside.
    As a result of this gelation, a practically transparent gelatin is obtained, more resistant than normal since the gelatin proteins have been fixed. In addition, this jelly allows the ultrasound of ultrasound machines to pass without practically the appearance of any type of device.
    The organs that get inside molds can be practically all (tested with kidneys, intestines, urinary bladder liver, brains, lungs and hearts). These organs must be pre-fixed, preferably in Klotz as described above and in addition they should preferably be washed with 96⁰ alcohol to remove 30 traces of blood.
    权利要求:
    Claims (13)
    [1]
    1.- PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ where the samples are previously fixed and washed characterized by comprising:
    A first phase of heating of 1/3 of the Klotz to be used and where that Klotz is heated, for a time of between 2 and 4 minutes, until reaching a temperature of between 80⁰ and 99⁰, keeping at that temperature for a period of time between 5 and nine minutes.
    A second phase, independent of the first phase, in which it proceeds to dissolve gelatin in the remaining 2/3 of the Klotz to be used. The amount of gelatin that dissolves is between 10 6% and 8%, of the total final amount of product to be prepared.
    A third phase in which, before the three minutes following the solution exposed in the second phase, the hot Klotz of the first phase is mixed with the Klotz compound and gelatin of the second phase.
    [2]
    2. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USING 15 KLOTZ according to claim 1 characterized in that it further comprises:
    A fourth phase in which the product obtained with the method set forth in claim 1 and the biological samples to be preserved are introduced into a mold.
    A fifth phase in which the result of the fourth phase stops to be refrigerated at a temperature between 7⁰ and 14⁰. twenty
    [3]
    3. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ according to claim 2, characterized in that the introduction of the product and the samples to be stored in the mold is carried out according to the following sub-phases:
     Creation of a product bed in the mold.
     Placing the samples to keep on the bed holding them. 25
     Fill the mold with the product at least until the samples to be preserved are covered.
     Removal of the means for fixing the samples to the bed.
    [4]
    4. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ according to claim 2 characterized in that the samples comprise at least one bladder and that the introduction of the product and the samples to be preserved in the mold is carried out according to the following subphases:
    - Introduction of a catheter through the bladder neck, closing the neck around the catheter with a non-absorbable suture.
    - Creation of a product bed in the mold. 35
    - Fastening the bladder to the bed.
    - Filling the bladder with fluid.
    - Fill the mold with the product at least until the samples to be preserved are covered.
    - Removal of the means for securing the samples to the bed.
    [5]
    5. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USING 5 KLOTZ according to claim 1 characterized in that the klotz is heated under stirring and gradually.
    [6]
    6. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ according to claim 1 characterized in that the heating stage of the klotz until reaching its proper temperature is 3 minutes. 10
    [7]
    7. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ according to claim 1, characterized in that the appropriate temperature that the klotz must reach is 90⁰.
    [8]
    8. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ according to claim 1 characterized in that the time that the klotz must remain at the appropriate temperature is 7 minutes.
    [9]
    9. PROCEDURE FOR THE CONSERVATION OF BIOLOGICAL SAMPLES USED BY KLOTZ according to claim 1 characterized in that the amount of gelatin that dissolves is between 6.5% and 7.5% of the total final amount of product to be prepared. . twenty
    [10]
    10. PRODUCT OBTAINED by the method described in claim 1 characterized in that it comprises Klotz (chloral hydrate, sodium anhydrous sulfate, potassium sulfate, sodium chloride, sodium bicarbonate, ascorbic acid, water, formalin) and gelatin being the amount of gelatin between 6% and 8%.
    [11]
    11.- PRODUCT OBTAINED according to the preceding claim characterized in that 5 25 liters of klotz comprise
    - Chloral Hydrate 49.94g
    - Sodium sulfate anhydrous 79.93g
    - Potassium Sulfate 1.26g
    - Sodium Chloride 1.47g 30
    - Sodium bicarbonate 3g
    - Ascorbic acid 2.64g
    - Water 4900gr
    Formol 24.97 ml
    [12]
    12.- PRODUCT OBTAINED according to claim 10 characterized in that the amount of gelatin is between 6.5% and 7.5% of the total weight of the product obtained.

    [13]
    13.- PRODUCT OBTAINED by the method described in claim 1 characterized in that it is semi-solid at a temperature between 20º and 40º.
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    同族专利:
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    ES2673443B1|2019-05-03|
    ES2673443R1|2018-07-04|
    引用文献:
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    GB352001A|1929-12-28|1931-06-29|Jose Oriol Fenes|Process for the preservation of bodies and anatomical specimens|
    US3249502A|1963-06-27|1966-05-03|Michel & Pelton Co|Embalming material and method|
    CN102246742B|2011-05-25|2013-03-13|中国人民解放军第三军医大学第三附属医院|Biological tissue fixing agent and preparation method thereof|
    CN103053510B|2013-01-31|2014-10-15|南京医科大学|Simple Manufacturing Method for Long-Term Preserving Softened Human Anatomy Viscous Specimen|
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