WO1993005650A1

WO1993005650A1 - Methods and compositions with aldehyde stabilizing solution

Info

Publication number: WO1993005650A1
Application number: PCT/US1992/007925
Authority: WO
Inventors: Gerald W. Camiener
Original assignee: Camiener Gerald W
Priority date: 1991-09-20
Filing date: 1992-09-18
Publication date: 1993-04-01
Also published as: CA2119554C; AU669673B2; JPH07507266A; EP0627879A1; US5439667A; AU2683592A; EP0627879A4; CA2119554A1

Abstract

Methods are provided to prepare a variety of tissues in a pathology-stable form keyed to the use of an aqueous solution of glyoxal and/or its addition products, including glyoxal in an amount sufficient to prevent autolysis and other degradative changes in various tissues. The said solution is capable of treating a tissue so as to maintain the tissue in a condition suitable for pathology and/or other experimental observation, and/or diagnosis. The stabilizing solution is also useful to prepare parts or whole animals, parasites, and plants in an anatomically preserved state for a prolonged period of time, and compositions useful in that method.

Description

METHODS AND COMPOSITIONS WITH ALDEHYDE STABILIZING SOLUTION

BACKGROUND OF THE INVENTION

The prior art is replete with formaldehyde treatments for various purposes. Chang et al.,. U.S. 4,654,312, teaches a composition for destroying erythrocytes while maintaining leukocytes that comprises formaldehyde. In a representative composition, Example 1, discloses 1 % formaldehyde, 3 % ethylene glycol and 0.25 % sodium citrate at a pH 7.7 ± 0.5. Chang et al. describe this as a "lysing solution" (col. 5, line 33). It is also stated by Chang et al. in a generic disclosure that in lieu of formaldehyde they contemplate about 0.5 to about 4 percent of any short chain aliphatic aldehyde, but preferably formaldehyde (col. 3, line 65 - col. 4, line 2). It is stated that this aldehyde acts as a fixing agent to stabilize the white cells while the distilled water that is in the mixture acts as a lysing agent to rupture the erythrocytes. While Chang et al. disclose that their composition destroys erythrocytes, Falkowski et al., U.S. Patent 4,136,161, disclose that glyoxal with sodium citrate serves to stabilize erythrocytes. There is no suggestion of the use of an alkanol with the glyoxal. It is also to be noted that contrary to the teachings of Falkowski et al., while monovalent ions such as potassium are indicated in the prior art as useful for stabilization of glutaraldehyde, research that has been conducted subsequently has shown that the aldehyde is destroyed and not saved by the potassium ion treatment. Buchalter, U.S. 3,983,252, discloses at col. 7, Example 1, a 50 % aqueous solution, 40 cc. glutaraldehyde with 7 gm sodium citrate and 50 cc propylene glycol, and 960 cc water. The use of this composition is as a disinfectant, with no suggestion of providing histological stabilization. In a generic recitation of various other ingredients, glyoxal is included as a recitation of one of the aldehydes that might be used. Jones, U.S. Patent 2,333,182, at

Example 1, discloses a mixture of 37 % water, 30 % glyoxal at 40 % strength and 20 % methanol, with a use as an embalming fluid. Myron Yanoff et al., "Glutaraldehyde Fixation of Whole Eyes", American J. Clinical Pathology, Vol. 44, pp. 167-171 (1965), discloses the glutaraldehyde fixation of whole eyes. Rendon, U.S. Patent 3,057,775, in Examples 1 and 3 discloses an aqueous mixture of an alkanol, glutaraldehyde, sodium acetate as well as phenol, glycerin, and a wetting agent for use as an embalming fluid. Specific use of glyoxal is not suggested. Ryan, U.S. Patent 4,198,206, discloses stabilization of platelet with the use of glutaraldehyde, and suggests that ethylene glycol is used as an "antifreeze", i.e., to protect against freezing of the stabilizing solution, col. 1, lines 42-47.

DETAILED DESCRIPTION

In accordance with a first aspect of the invention there is provided a method of providing pathology-stable tissue which comprises treating said tissue with an aqueous solution (I) of glyoxal in an amount sufficient to prevent major degenerative changes in said tissue, whereby said tissue remains in a state suitable for micro-, or macroscopic examination sufficient for pathological or experimental examination and diagnosis.

The glyoxal is used alone or in admixture with other components. The glyoxal is used in combination with a balanced mixture of other components. In the absence of such a proper mixture, good microscopic appearances are not obtained. In one embodiment, the glyoxal is an aldehyde addition product in the form of a bisulfite, hydrate or alcohol addition product. When the glyoxal is an aldehyde addition product in the form of a bisulfite, hydrate, alcohol, or glycol addition product, the addition product may be used as a liquid form, or in the solid form to which water and/or an alkanol can be added to provide a liquid mixture, or in combination with other materials. When the bisulfite is used, it is preferably in the form of an metal salt, preferably the sodium or potassium salt. When in the form of an alcohol addition product, the addition product used is preferably a glycol such as ethylene glycol, the product of which is known as dioxanediol. The use of a solid form of glyoxal is particularly advantageous for kits in that leakage, shipping, and stability problems are eliminated. Water and/or an alkanol is added by the user shortly before a sample is added to the kit.

Tissues suitable for glyoxal treatment include tissues of animal, or plant origin, but particularly those of mammalian and human origin.

In one embodiment, glyoxal is present in an amount of from about 0.08 % to about 36 % of the overall solution. More preferably, glyoxal is present in an amount of from about 0.1 % to about 20 % percent of the overall solution. Also included within the solution may be from about 0.15 % to about 36 % of a C_χ_₄ alkane-mono- di- or triol. Alkanols include monools like ethanol, diols like ethylene glycol and triols like glycerol. Glycerol is particularly suited for a stabilizing solution (I) that has as one of its objectives the maintenance of a tissue sample in a more pliable condition. Mono- and diol-alkanes are particularly useful in permitting faster tissue penetration of the stabilizing solution which is very important for preserving of larger tissue samples and organs. Mono- and diol-alkanes also are particularly effective in stabilizing dialdehydes in solution, thereby providing active reactants for longer times.

In another preferred embodiment, the stabilizing .solution is prepared by mixing an appropriate amount of glyoxal bisulfite addition product in water, together with sodium or potassium phosphates, to give a pH of 6.5. The resulting pH buffered stabilizing solution maintains physiologic conditions while it is stabilizing the tissue, is preventing autolysis, and is maintaining the tissue in a condition suitable for pathology observation. In a preferred embodiment, said solution includes at least one C_χ_₄ alkane mono-, di- or triol at a concentration of from about 0.4 % to about 32 % of said solution, whereby the penetration of said solution into said tissue is facilitated. In a further embodiment, said solution includes an ionic or nonionic chemical in an amount of from about 0.1 % to about 14 % of said solution, whereby the osmotic pressure effects of said solution is altered, thereby modifying and/or stabilizing said tissue for subsequent examination. In one such embodiment, sea lamprey eels are stabilized using a glyoxal solution containing 3 % sodium chloride.

In other embodiments, additional and specific micro-anatomical stabilization effects are obtained by the inclusion of various specific reactants including mercuric salts, trichloroacetic acid, acetic acid, picric acid and potassium dichromate, in amounts of from abut 0.07 % to about 40.0 %. In one preferred embodiment, said glyoxal solution contains 37 % picric acid, and 5 % glacial acetic acid. Another preferred embodiment, said glyoxal solution contains 4.5 % mercuric chloride, 0.5 % sodium chloride and 2 % trichloracetic acid. Other reactants can include chromium trioxide, copper salts, and various other heavy-metal and transition-metal salts.

In a further aspect of the invention, there is provided a method for simultaneously providing a pathology-stable tissue while decalcifying calcified regions within said tissue wherein said solution includes an acid and or acid-salt such as formic, hydrochloric, citric and nitric acids and/or their alkali-metal or ammonium salt forms in amount of from about 0.3 % to about 75 %. In this embodiment, for the simultaneous preservation of a pathology-stable tissue and decalcification of any calciferous regions within said tissue, the solution may additionally, or alternatively, include a chelating agent, in an amount from about 0.1 % to about 18 %, such as ethylene-diamine-tetra-acetic acid, or an alkali-metal-salt or ammonium-salt thereof.

By a "pathology-stable tissue," there is intended to mean that a tissue sample has been stabilized in a state suitable for pathology and/or experimental examination, and/or diagnosis. In such a stabilized condition, the microscopic and macroscopic cell, tissue and organ constituents will have been maintained in as lifelike a condition as possible, substantially free from the autolytic and other changes that occur when normal tissue is removed from a living being or organism (as in the case of a biopsy sample) , or when the entire body or organism dies and a portion of the body is to be preserved for subsequent examination. As the term is used herein, a "tissue" refers to any form of tissue, whether a thin slice taken, for example, from a possibly cancerous tissue for purpose of a biopsy, or any larger portion that includes such tissue, for example, a liver or a kidney. By applying the solution as soon as possible after removal or death, the application of the solution (I) serves to stabilize the tissue in as normal as possible a state, and immediately stops metabolic reactions, autolytic changes and microbial degradative changes. Autolysis is a process whereby the normal cellular enzymes and components degrade and destroy the cell structure itself.

In a further aspect of the invention there is provided a kit for maintaining a pathology-stable preparation which comprises a female receptacle means and a male closure means, and contained within said female receptacle means an aqueous solution containing a glyoxal addition product in an amount sufficient to prevent major degenerative changes in the sample reserved for pathological examination. Such a kit also can contain a solid form of a glyoxal addition product to which water and/or alkanol is added prior to the sample being introduced. The stabilizing solution also can contain various other pH buffering and osmotic-pressure affecting chemicals, as well as various special-effect reactants as described earlier.

In one embodiment, the kit is for maintaining a stool sample in a stabilized form so that parasites, worms and/or protozoans contained in the stool sample are maintained in a condition suitable for analysis and evaluation at a later time by an appropriate laboratory. Said kit includes a female receptacle means and a male closure means and a stabilizing solution containing glyoxal addition product. In an embodiment, said tissue is in the form of an organ or an entire organism which is .to be preserved for a prolonged period of time, whereby said solution prevents substantial degenerative changes within this period. Other embodiments employ said kit for the collection, stabilization, and maintenance of other bodily fluids, solids, exudates and/or secretions such as phlegm, mucous, semen, pus and urine. In a further aspect of the invention, there is provided a composition capable of providing pathology-stable tissue when a tissue is immersed in said composition, said composition being an aqueous solution of a glyoxal addition product in an amount sufficient to prevent major degenerative changes in said tissue, glyoxal being present in an amount of from about 0.08 % to about 36 % percent of the overall solution, and said solution including from about 0.15 % to about 36 % of a C₂_₄ alkane- mono-, di-, or triol to stabilize said solution. In this aspect, a further embodiment provides a solution that includes at least one C₂_₄ mono-, di-, or triol alkane at a concentration of from about 0.4 % to about 32 % of said solution, whereby the penetration of said solution into said tissue is facilitated. In this embodiment, preferably said solution includes an ionic or nonionic chemical in an amount of from about 0.1 % to about 14 % of said solution, whereby the osmotic pressure effects of said solution is altered, thereby stabilizing said tissue for subsequent examination. A further embodiment, the preferably said solution contains an acid and/or an acid-salt and/or a chelating agent to effect decalcification of calcified portions of tissues.

In one embodiment of this first aspect of the invention there is provided a small sample of tissue from either a biopsy or a fresh cadaver that is treated with the solution (I) in an amount sufficient to prevent autolysis of the tissue by native enzymes that upon cessation of living state of the tissue operate to digest and to disintegrate the tissue, and to prevent other degenerative changes in the tissue.

In a further embodiment of this first aspect of the invention, there is provided a test-stable stool sample kit comprising means for maintaining a stool sample in a condition for evaluation at a later time by an appropriate laboratory including a female receptacle means and a male closure means and the stabilizing solution (I) . As a typical female receptacle means may be mentioned wide mouth jar containers of plastic or glass or the like with a screw pattern to accommodates a male screw cap closure. Another female receptacle means could be a bag or pouch, and the "male" closure could be a tie or clamp. It is to be understood that the female receptacle means may be of any shape, size or form provided it is such as to accommodate a sample of tissue, organ or bodily product that is to be inserted into the female receptacle means.

While various fixatives are known in the art, the stabilizing solution (I) reacts quickly with a variety of groups in the cell to stop enzyme actions, to fix structural features, to stop microbial actions and generally to maintain the sample in a manner as close to the form and appearance of the living tissue as possible, whereby the pathology examination is facilitated. While formaldehyde and other usually-used fixatives, when used alone, will provide acceptable microscopic appearing preparations, glyoxal used alone will not. The additional combination of components is required to provide good microscopic appearances.

In a second aspect of the invention, the stabilizing solution (I) is used to prepare parts or whole animals or plants in an anatomically preserved state for a prolonged period of time, and compositions useful in that method. The stabilizing solution (I) of the invention may be used to preserve frogs or other small animals for school laboratory dissection, or entire cadavers or individual organs for medical school instructional and other uses. The stabilizing solution (I) also facilitates the preservation of the specimen by killing and/or inhibiting any microorganisms that otherwise might be present or develop and thereby degrade the specimen.

Whereas in the first aspect of the invention, stabilization of a tissue sample was desired for a period of days, in this second aspect of the invention, stabilization for a prolonged period preferably measured in years, is the object. It is intended to be used with specimens such as frogs, fetal pigs, cadavers, cats, worms, insects or large organs that are to be used for study or dissection whether in a high school, college medical school, or autopsy setting, or other preservation uses.

It should be noted that the tissue may be of animal or plant origin. While human tissue, organs, or the entire organism are contemplated, it should be noted that the invention is contemplated

as applicable to any mammal, amphibian, reptile, bird, bony fish, cartilaginous fish, insect, arthropod, cephlapod, insect, multicellular microorganism, round worm, flat worm, segmented worm or any other animal or plant group. Depending on the specific application, it may be desirable to include at least one additional chemical such as mercuric salts, trichloroacetic acid, acetic acid, picric acid, potassium dichromate, phenol, biphenol, phenols, cresols, quaternary ammonium compounds, surfactants, antibiotics copper salts, sulfosalicylic acid, osmic acid, platinum salts, cadmium salts, cobalt salts and uranyl salts. Generally, the amount of any of these additional chemicals would be from about 0.07 % to about 16.5 %.

The following examples illustrate the invention:

EXAMPLE 1

To a glass container, there is added 100 ml of a 40 % solution of glyoxal and 900 ml of deionized water. With mixing, 5.0 g of sodium acid phosphate monohydrate and 6.5 g of anhydrous disodium phosphate are added and dissolved, the pH is slightly acid. Small pieces of dog tissue are immersed in the solution for several hours, and then are processed for histological examination. Good cellular detail is observed, with no evidence of autolysis or other degradative changes.

EXAMPLE 2

To a glass container, there is added, in order with mixing, 700 ml of deionized water, 200 ml of denatured ethyl alcohol and 150 ml of a 40 % glyoxal solution. Kidneys are cut in half so.that one- end serves as a control for the other end. Kidney halves are immersed in at least 10 times their volume of the said prepared solution and allowed to stand for varying periods of time. They then are prepared for pathological and histological examination. After 6 hours of exposure to the solution, the center-most portions of the kidney show good penetration of the solution whilst the controls that were immersed in the same solution without ethyl alcohol show very much poorer penetration in the center-most portions.

EXAMPLE 3

Kidneys prepared and treated in accordance with Example 2 are examined after 7 and 21 days of immersion in said solution. The kidneys remain in good stabilized condition suitable for pathologic and histologic examination. Good cellular detail is observed, without observable degradative changes.

EXAMPLE 4

To a glass container, there is added, in order with mixing, 700 ml of deionized water, 200 ml of ethylene glycol and 100 ml of a 40 % glyoxal solution. The solution is adjusted to pH 6.5 by the drop- wise addition of sodium hydroxide solution under conditions of continuous mixing. The solution is stored for a few days and then is used to stabilize pieces of human tissue prior to histologic processing and examination. Tissues processed with said solution show good preservation and cellular detail. Control tissues treated with the same solution absent the ethylene glycol show poorer preservation. Independent chemical analysis shows that the ethylene-glycol-containing solution has a higher glyoxal concentration than the solution absent the ethylene glycol.

EXAMPLE 5

To a glass container, there is added, in order with mixing, 500 ml of deionized water, 200 ml of denatured ethyl alcohol, 150 ml of glycerol, 60 ml of a 40 % glyoxal solution, and 50 ml of a 40 % solution of glyoxal. Intact rat livers are separated into individual lobes and the lobes are used as controls for different experimental conditions. In this example, lobes were immersed for 14 days in 10-fold excesses of said solution. The lobes are then processed and examined. Lobes treated with said solution show good preservation and good tissue consistency with no - evidence of "crumbliness." Control lobes immersed in the same solution absent the glycerol did not show the same tissue "pliability" and "softness." Microscopic examination of the test lobes treated with the solution in this example showed good cellular and microcellular detail. Control lobes treated with same solution except that the combination of dialdehydes was not used also show good cellular detail, but the uptake of stain was not as uniform in some sub- cellular details as was seen when the combination of dialdehydes was used.

EXAMPLE 6

To a glass container, there is added, in order with mixing, 850 ml of denatured ethyl alcohol, 50 ml of glacial acetic acid and 100 ml of a 40 % glyoxal solution. Various arachnids, insects and plant parts were immersed in said solution and were examined after one week, three weeks and nine months of immersion. All tissues, cells and organisms showed good preservation and good cellular detail.

EXAMPLE 7

To a glass container, there .is added, in order with mixing, 800 ml of deionized water, 45 g mercuric chloride, 5 g of sodium chloride, 20 g of trichloroacetic acid, 40 ml of glacial acetic acid, and 200 ml of 40 % glyoxal solution. Pieces of mammalian tissue are immersed in the solution for 3 to 24 hours. Slices less than 2 mm in thickness need only 3 hours of treatment. Subsequent processing and examination showed good preservation and good cellular detail with negligible shrinkage of connective tissue.

EXAMPLE 8 To a glass container, there is added, in order with mixing, 850 ml of deionized water, 10 g of formic acid, 100 g of trisodium citrate, and 100 ml of a 40 % glyoxal solution. Pieces of mammalian bone are immersed in at least a 10-fold excess of said solution and left for 5-7 days. Subsequent processing and examination show good preservation and good cellular detail. Calcified areas are completely decalcified and shrinkage is not observed.

EXAMPLE 9

The formic acid and sodium citrate in Example 8 were replaced with 100 g of disodium ethylene-diamine tetraacetate. Mammalian teeth were immersed in said modified solution and left for 7-10 days. Subsequent processing and examination showed good preservation and good structure Decalcification appears to complete.

EXAMPLE 10

A test-stable stool sample kit is provided which includes means for maintaining a stool sample in a condition suitable for analysis at a later time by an appropriate laboratory include a female receptacle means and a male closure means and 30 ml. of an aqueous solution which contains 40 % glyoxal. As a typical female receptacle means may be mentioned a 2 oz. wide mouth plastic container with a screw pattern to accommodates a male screw cap closure. It is to be understood that the female receptacle means may be of any shape, size or form provided it is such to accommodate a small scraping of bowel sample that is to be inserted into the female receptacle means.

Parasites which may be in the stool sample and which may be an object of laboratory examination are preserved in the stool samples to permit the stool sample to be properly used to identify the presence of such parasites.

EXAMPLE 11 In the test-stable stool sample kit of Example 10, the mentioned aqueous solution is replaced by 800 mg of dioxanediol (solid) , which is dissolved in 20 ml of an aqueous solution prior to inserting the stool sample. Parasites which may be in the stool sample and which may be an object of laboratory examination are preserved in the stool samples to permit the stool sample to be properly used to identify the presence of such parasites.

EXAMPLE 12

A freshly excised primate heart is perfused with a solution of glyoxal as prepared in Example 4 except that the pH was not adjusted. Following perfusion, the heart was left immersed in a closed container for 10 months, and then examined macroscopically and microscopically. The heart is preserved in good condition with no apparent degradative changes, and is suitable for student observation.

EXAMPLE 13

An adult bull frog is extinguished under ether, and is perfused with the solution described in Example 12 to which sodium chloride (0.6 %) and cetyltrimethylammonium bromide (0.15 %) have been added. The frog is kept moistened with same solution (under wetted cotton) in a closed container. The frog remains suitable for student dissection for a period of at least one year.

Claims

WHAT I CLAIM IS :

1. A method of providing pathology-stable tissue which comprises treating said tissue with an aqueous solution of a glyoxal in an amount sufficient to prevent major degenerative changes in said tissue, whereby said tissue remains in a state suitable for examination for micro- or macroscopic examination sufficient for a pathological or experimental examination.

2. A method of claim 1 wherein said glyoxal is in the form of an addition product.

3. A method of claim 1 wherein said tissue is of animal origin.

4. A method of claim 1 wherein said tissue is of mammalian origin.

5. A method of claim 1 wherein said tissue is of plant origin.

6. A method of claim 2 wherein said glyoxal is a glyoxal addition product comprised of glyoxal and a bisulfite, water, alcohol, or glycol addition product.

7. A method of claim 1, wherein said glyoxal is present in an amount of from about 0.08 % to about 36 % percent of the overall solution.

8. A method of claim 1, wherein said glyoxal is present in an amount of from about 0.2 % to about 20 % percent of the overall solution.

9. A method of claim 1, wherein said solution contains from about 0.15 % to about 36 % of a C_1-4 alkane mono-, di-, or triol, whereby said solution is stabilized.

10. A method of claim 1 wherein said solution includes at least one C₁_₄ mono-, di-, or triol alkane at a concentration of from about 0.4 % to about 32 % of said solution, whereby the penetration of said solution into said tissue is facilitated.

11. A method of claim 1 wherein said solution includes an ionic or nonionic chemical in an amount of from about 0.1 % to about 14 % of said solution, whereby the osmotic pressure effects of said solution is altered, thereby stabilizing said tissue for subsequent examination.

12. A method of claim 1 wherein said solution includes from about 0.01 moles/liter to about 4.2 moles/liter of a pH buffering agent.

13. A method of claim 1 for simultaneously providing a pathology- stable tissue while decalcifying calcified regions within said tissue, wherein said solution contains an acid such as formic, hydrochloric, citric and/or nitric acids, and/or their alkali- metal or ammonium salts in amount of from about 0.3 % to about 75 %.

14. A method of claim 1 for simultaneously providing a pathology- stable tissue decalcifying calcified regions within said tissue, wherein said solution includes a chelating agent in an amount from about 0.1 % to about 18 %.

15. A method of claim 14 wherein said chelating agent is ethylene- dia ine-tetra-acetic acid, or an alkali-metal salt or ammonium salt thereof.

16. A kit for maintaining a pathology-stable preparation which comprises a female receptacle means and a male closure means and contained within said female receptacle means an aqueous solution of a glyoxal of claim 1 in an amount sufficient to prevent major degenerative changes in the sample reserved for pathological or experimental examination.

17. A kit for maintaining a stool sample in a form suitable for subsequent examination and recognition of parasites, worms and protozoans, which comprises a female receptacle means and a male closure means and the stabilizing solution of claim 1.

18. A method of claim 1 wherein said tissue is in the form of an organ or an entire organism which is to be preserved for a prolonged period of time, whereby said solution blocks the substantial degenerative changes within this period.

19. A composition capable of providing pathology-stable tissue when a tissue is immersed in said composition, said composition being an aqueous solution of a glyoxal addition product in an amount sufficient to prevent, major degenerative changes in said tissue, said glyoxal product being present in an amount of from about 0.08 % to about 35 % percent of the overall solution, and said solution including from about 0.15 % to about 36 % of a C₂_₄ mono, di-, or triol alkane as a stabilizing agent.

20. A composition of claim 19 wherein said solution includes at least one C₂_₄ mono, di- or triol alkane at a concentration of from about 0.4 % to about 32 % of said solution, whereby the penetration of said solution into said tissue is facilitated.

21. A composition of claim 19 wherein said solution includes an ionic or nonionic chemical in an amount of from about 0.1 % to about 14 % of said solution, whereby the osmotic pressure effects of said solution is altered, thereby stabilizing said tissue for subsequent examination.

22. A composition of claim 19 wherein said solution includes from about 0.01 moles/liter to about 4.2 moles/liter of a pH buffering agent.

23. A composition of claim 19 for simultaneously providing a pathology-stable tissue while decalcifying calcified regions within said tissue, wherein said solution contains an acid or an acid- salt which is formic, hydrochloric, citric or nitric acid in amount of from about 0.3 % to about 75 %.

24. A composition of claim 19 for simultaneously providing a pathology-stable tissuewhile decalcifying calcified regions within said tissue, wherein said solution includes a chelating agent in an amount from about 0.1 % to about 18 %.

25. A composition of claim 24 wherein said chelating agent is ethylene-diamine-tetra-acetic acid, or an alkali-metal salt or ammonium salt thereof.