Abstract:
Tissue fixatives, such as diazolidinyl urea, which are free of aldehydes and toxic chemicals are described. When used, either in aqueous or alcoholic solutions, good tissue preservation is attained. In addition, tissue antigens are retained which makes the fixative useful for immunostaining procedures.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to compositions for the fixation of cells and tissues and to methods for the fixation of cells and tissues using as the fixing agents certain compounds. 
     The objective of tissue fixation is to provide as much detail of the cell as possible. To do this, it is necessary to maintain the cells in their original unaltered morphology so that maximum cellular detail is observed under the microscope. With the development of immunostaining there is also the requirement that the antigens of the cells are not altered by the method of fixation or stabilization. Although the microscope is the usual means for examining cells that are fixed and stained, they may also be examined by the laser or the flow cytometer. The flow cytometer is an important method for examining a large number of cells in a brief time. 
     The usual formulations for stabilization of cells contain one or more agents which react vigorously with the proteins of the cells to denature, coagulate and insolubilize the components of the cell. Typical of this type of agent is picric acid, mercuric ions, formaldehyde and glutaraldehyde. In addition, some less toxic compounds which can also denature and stabilize the proteins are acetic and formic acid. Unfortunately, the toxicity associated with these compounds renders their use less than satisfactory. 
     OBJECTS OF THE INVENTION 
     Thus, it is an object of the invention to provide a fixative solution for tissues and cells which has an extremely low toxicity yet meets all of the requirements of a model fixative. 
     Another object of the invention is to provide a fixative solution for tissues and cells that preserves tissues and cells and their cellular detail. 
     Yet another object of the invention is to provide a fixative solution that provides an unaltered antigenic surface for reaction with specific antibodies. 
     SUMMARY OF THE INVENTION 
     These and other objects of the invention are obtained by a fixative solution for tissues and cells comprising histological fixing amounts of at least one active agent selected from the group consisting of: 
     i) diazolidinyl urea 
     ii) imidazolidinyl urea 
     iii) dimethylol-5,5-dimethylhydantoin 
     iv) dimethylol urea 
     v) 2-bromo-2-nitropropane-1,3-diol; and 
     vi) quaternary adamantane (e.g. 1-(3-chloroallyl)-3,5,7-tri-aza-1-azoiadamantiane-chloride, N-(3-chloroallyl)-hexammonium chloride such as Dowicil 200; Dowicide Q; Preventol D1) 
     in a solvent selected from water and an alcohol and mixtures thereof. 
     In another aspect, the invention comprises an improvement in a method of fixing tissues and cells with a histological fixative wherein the histological fixative is an active agent selected from at least one of the group consisting of: 
     i) diazolindinyl urea 
     ii) imidazolidinyl urea 
     iii) dimethylol-5,5-dimethylhydantoin 
     iv) dimethylol urea 
     v) 2-bromo-2-nitropropane-1,3-diol; and 
     vi) quaternary adamantane. 
     Unlike the typical histological fixing agents, the active agents of the invention have extremely low toxicity. For example, toxicity studies comparing diazolidinyl urea of the invention with formaldehyde of the prior art show the following: 
     
         ______________________________________     Inhalation             Dermal     Toxicity             Toxicity    LD 50______________________________________Formaldehyde       500 mg/Kg  270 mg/Kg   800 mg/KgDiazolidinyl urea       None      2000 mg/Kg  2570 mg/Kg______________________________________ 
    
     This reduced toxicity makes disposal and handling less of a problem. In addition, since there is no inhalation toxicity, there are no badge detection devices required as there are for formaldehyde. 
     Another advantage offered by the active agents of the invention is the fact that they are not flammable and therefore do not present a fire hazard as do many of the prior art fixatives. 
     The mechanism by which the active agents of the invention provide the desired tissue and cell membrane is not known for certain. It is believed that the active agent binds in some fashion to the cell membrane or tissue to stabilize. This hypothesis is drawn because many of the active agents of the invention are known disinfectants which kill bacteria by binding to cell structures. This is not a full explanation of the mechanism responsible for the results of the invention since many other disinfectants such as Kathon and Omadine fail to provide tissue and cell stabilizing effects. 
     The ability of the active agents of the invention to preserve antigens is also not understood but it is probably due to a difference in the reaction between the active agents of the invention and prior art fixatives such as formaldehyde with proteins. Formaldehyde crosslinks with itself and proteins to obscure the antigen. To determine if this is true, diazolidinyl urea was added to the protein albumin to stabilize it. After incubation of diazolidinyl urea and protein mixture for 24 hours, discgel electrophoresis indicated no change in the rate of migration of the protein. When this experiment is conducted with formaldehyde, a large number of multimers and insoluble protein results. 
     In another aspect of the invention, it has been found that the addition of alkali metal salts of ascorbic acid increases the activity of the active agents of the invention in fixing the tissue or cell membrane. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The fixative solutions of the invention are comprised of the active agents in solvent selected from water, an alcohol and mixtures thereof. 
     The alcohol solvent comprises one or more alkanols such as methanol, ethanol, propanol and butanol; polyols, e.g. diols or triols such as ethylene glycol, glycerol, propylene glycol and trimethylene glycol and mixtures of alkanols and polyols. 
     Whether the solvent employed is water, alcohol solvent or a mixture thereof depends principally upon the tissue or membrane being fixed. For example, where large pieces of tissue are being fixed, it is preferred to use an alcohol solvent or aqueous alcohol solvent since the alcohol solvents increase penetration. Also, in fixing cells such as Pap smears, the alcoholic preparations are preferred because they cause the cells to stick to the slides. When aqueous alcoholic solutions are employed as the solvent for the active agents of the invention, the ratio of alcohol to water will fall in the range of 4:1 to 2:1. 
     The amount of the active agents in the formulation of the invention is that effective to fix or stabilize the tissue or cell membrane. Generally, this amount falls in the range of abut 20 to 100 grams per liter, preferably 50 to 75 grams per liter. 
     When alkali metal ascorbic acid salts such as sodium ascorbate are included to increase the activity of the active agents to fix the tissue or cells, they are added in an amount of about 0.25 to 1 grams per liter. 
     The solute in the preparations of the invention may also include any of the other addendum conventionally added to histological fixative preparations. These addendum include mordants, buffers, penetration increasers, osmotically active substances and nuclear detail improvers and nuclear size increasers. 
     Examples of suitable mordants are salts with a metal ion having an oxidation state of two or more. Illustrative are zinc, strontium, calcium, barium and chromium salts. The preferred salt is zinc sulfate. 
     Suitable buffers include alkali metal phosphate salts such as sodium phosphate and potassium phosphate. 
     Osmotically active substances that may be included in the formulation of the invention are alkali metal salts such as sodium chloride. In addition, sugars such as the polysaccharides, sucrose, glucose and the like may be employed. 
     Nuclear detail improvers and nuclear size increasers include acetic acid and lithium salts such as lithium chloride. Zinc salts such as zinc sulfate not only improve nuclear definition but also improves staining. 
     Illustrative of substances which increase the rate of penetration of the fixing agent are dimethylsulfoxide and ethanol. 
     The following examples are illustrative of formulations of the invention. 
     EXAMPLE I 
     
         ______________________________________Diazolidinyl urea      50     g/LNa.sub.2 HPO.sub.4     0.73   g/LKHPO.sub.4             0.02   g/LNaCl                   8.50   g/LDistilled H.sub.2 O to one liter______________________________________ 
    
     EXAMPLE II 
     
         ______________________________________Diazolidinyl urea      50     g/LEthanol                500    mlAcetic acid, conc.     10     mlDistilled H.sub.2 O to one liter______________________________________ 
    
     EXAMPLE III 
     
         ______________________________________Diazolidinyl urea      50     g/LLithium chloride       6.35   g/LDistilled H.sub.2 O to one liter______________________________________ 
    
     EXAMPLE IV 
     
         ______________________________________Diazolidinyl urea      50     g/LDimethylsulfoxide      100    mlDistilled H.sub.2 O to one liter______________________________________ 
    
     EXAMPLE V 
     
         ______________________________________Diazolidinyl urea      50     g/LDimethylsulfoxide      100    mlZinc chloride          5.8    g/LDistilled H.sub.2 O to one liter______________________________________ 
    
     EXAMPLE VI 
     
         ______________________________________Diazolidinyl urea      50     g/LAscorbic acid, sodium  .25    g/LDistilled H.sub.2 O to one liter______________________________________ 
    
     The following is an example of the use of fixatives of the invention. 
     EXAMPLE VII 
     Tissue is immersed in the fixative of Example I for four hours. The treated tissue is then dehydrated through a series of graded alcohols, cleared in xylene and impregnated with molten paraffin. This procedure is performed under heat and vacuum/pressure in a 12-hour cycle using a Fisher Histomatic (Model 166 MP) tissue processor. The tissue is then blocked, paraffin embedded, rehydrated in ice water for a minimum of three hours to enhance sectioning, and sectioned at 4-5 microns. The tissue is mounted on a glass slide, deparaffinized, stained, coverslipped and evaluated microscopically. 
     The following example demonstrates the satisfactory results obtained with the fixative of the invention using various staining methods. 
     EXAMPLE VIII 
     Example VII is repeated using the staining method identified. The results in each case are as follows: 
     
         ______________________________________Staining Method      Results______________________________________Mayer&#39;s mucicarmine  Demonstrable;                well-definedElastin              Satisfactory detailMovat&#39;s reticulin stain                Satisfactory detail;                minimal shrinkageGomori&#39;s trichrome stain                Fibrous tissue well-                definedPeriodic Acid-Schiff (PAS)                Non-specific staining                not evidenced as in                formalin-fixed prep.Geimsa               Satisfactory detailHematoxylineosin     Satisfactory detail______________________________________ 
    
     The following example demonstrates the ability of the fixative of the invention in retaining tissue antigens in immunostaining procedures. 
     EXAMPLE IX 
     The tissues identified below having the antigenic sites identified below are fixed with the fixative formulation of Example I and immunohistochemically stained using avidin-biotin stainings. 
     
         ______________________________________Tissue         Markers Detected______________________________________Lymph node     LN-1          LN-2          LN-3          UCA          L-26          LCHL-1Brain          Neurofilament          Glial Fibrillary Acidic ProteinHodgkins node  Ber H.sub.2          Leu M.sub.1Colon          Cytokeratin MAK-6          Cytokeratin AE1/AE3Muscle         DesminPituitary      S-100Thyroid        ThyroglobulinBreast         α-lactalbumin______________________________________ 
    
     None of the antigenic sites are affected by the immunostaining.