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compound fixatives
characteristics and uses
| Name compound fixatives | B-5, BOUIN, DAVIDSON, GENDRE, HOLLANDE, FORMALDEHYDE-GLUTARALDEHYDE, ZENKER & HELLY, ORTH, ZAMBONI, AND ZINC FORMALIN |
| What compound fixatives are aldehyde based solutions? | Formaldehyde-glutaraldehyde and Davidson(version 1 & 2) |
| Monobasic sodium phosphate(NaH2PO4) 1.16g, Sodium hydroxide(NaOH) 0.27g, formaldehyde 10 mL and glutaraldehyde 2mL | Formaldehyde-glutaraldehyde |
| Dual purpose fixative, preserves tissue for routine histopathology while keeping the specimen suitable for electron microscopy, stable for at least 3 months if stored at 4 C , PAS reaction is not substantially affected | Characteristics of Formaldehyde-glutaraldehyde |
| uses of Formaldehyde-glutaraldehyde | routine and light microscopy and electron microscopy |
| 95% ethanol 30 mL, 10% NBF 20 mL, Glacial acetic acid 10 mL, Distilled water 30 mL | Davidson version 1 |
| 37% stock formaldehyde 50 mL, absolute ethanol 75 mL, 1.2 glacial acetic acid 2.5 mL, distilled water 75 mL | Modified Davidson version 2 |
| Another name for Davidson fixative | Hartmann |
| Fixation time for Davidson version 1 | 24 to 72 hours |
| Fixation time for Davidson version 2 | 24 hours |
| Either version of Davidson can be stored in | 70 % alcohol or NBF |
| Uses of Davidson | recommended for eyes, testis, lymph nodes, various soft tissues, and other species(marine animals, fish, rodent eyes), bone marrow |
| What fixative has similar formulations to Davidson solution? | Bouin |
| What is the difference between Davidson and Bouin solution? | Davidson lacks picric acid which requires removal before staining and alcohol balances the swelling effect of acetic acid so tissues are not overly hard in microtomy |
| What is noted about the retina of the eye if fixed in Davidson ? | does not tend to detach as it does with formalin fixation |
| Why should overexposure in modified Davidson (version 2) be avoided? | damaging effects of acetic acid on nuclear detail |
| Retinas are well preserved but prolonged fixation may cause excessive swelling, shattering of lens, cataract type artifacts, defects in some eye components, indistinct rods and cones, retinal detachment, and inconsistent staining | Effects of eyes in Davidson solution |
| How many days is recommended for eyes? | > or equal to two days |
| What can be refixed in modified Davidson to reveal white lymph nodes at gross dissection after initial NBF fixation? | mesentary |
| Davdison is not useful for | electron microscopy but immunohistochemical staining has been successful |
| What fixative is a chromate based solution? | Orth Solution |
| Potassium dichromate 2.5 g , sodium sulfate 1 g , distilled water 100 mL , formaldehyde 10 mL | Orth |
| Uses of Orth solution | preferred for the subsequent demonstration of chromaffin granules in the cytoplasm of cells of the adrenal medulla , important in the diagnosis of pheochromocytoma |
| Safety regulations for Orth solution | The tissue must be washed after fixation, the time of fixation must be controlled, and the remaining wet tissue stored in 70-80% alcohol |
| Characteristic of Orth | not a good general purpose fixative, chromaffin granules are colored orange to brown with chromate, reaction most intense at pH 5-6 |
| Mercury based solutions | B-5, Zenker and Helly |
| Mercury fixative formulations should no longer be used in the lab. Why are some pathologists reluctant to discontinue? | trephine biopsies and the excellent nuclear detail |
| Mercuric chloride 12 g , sodium acetate(anhydrous) 2.5 g , distilled water 200 mL | B-5 stock solution |
| B-5 stock solution(mercuric chloride, sodium acetate,distilled water) 20 mL and formaldehyde 2 mL | B-5 working solution |
| B-5 uses | hematopoietic and lymphoreticular tissue because of its ability to demonstrate beautiful nuclear detail, special stains, paraffin embedded tissue |
| B-5 characteristics | sodium acetate raises pH to 5.8-6.0, formalin pigment may or may not be obtained, track mercury cradle to grave , must be washed for mercury pigment |
| removal of mercury pigment | iodine followed by sodium thiosulfate |
| Zenker and Helly stock solutions | Mercuric chloride 50 g , potassium dichromate 25 g , sodium sulfate 10 g , distilled water 1,000 mL |
| Zenker-helly stock solution(mercuric chloride, potassium dichromate, sodium sulfate, distilled water) 95 mL and acetic acid ,glacial 5 mL | Zenker working solution |
| Characteristics of Zenker working solution | stable, large quantities can be prepared , treat for mercury pigment, chrome pigment can be formed , lyse erythrocytes, better nuclear fixative because of acetic acid , best of all fixatives but needs to be avoided because of mercury , silver stain=no |
| Zenker-Helly stock solution 95 mL and formaldehyde 5 mL | Helly working solution |
| Characteristics of Helly working solution | not stable, must be treating for mercury pigment , chrome and formaldehyde pigment can form , preserve the erythrocytes, silver staining unsatisfactory |
| Uses of Zenker working solution | fix and decalcify needle biopsy specimens of bone marrow , dissolves iron, recommended for Mallory phosphotungstic acid hematoxylin applied |
| fixation time for zenker and helly solution | should not exceed twenty four hours or the tissue becomes overhardened and nuclear basophilia is decreased |
| Zenker and helly solution fixed tissue should be washed with? | running water and stored in 70-80% alcohol |
| Safety Zenker and Helly | can be fatal if inhaled, ingested, or absorbed through the skin. Target organs are the kidneys, central nervous sytem, and liver. Carcinogen |
| Picric-acid based solutions | bouin, Gendre, hollande, and zamboni(PAF) |
| Picric acid, saturated aqueous 750 mL formaldehyde 250 mL, acetic acid glacial 50 mL | Bouin solution |
| Characteristics of Bouin solution | lyses RBCs, iron and small calcium deposits dissolve and formalin pigment may be obtained, extracts RNA and hyrdolyzes DNA resulting in positive Schiff reagent, brilliant nuclear cytoplasmic staining |
| Uses for Bouin | Trichrome stain and preserving structure with soft and delicate textures , biopsy specimens for the GI tract, tissue of endocrine system, routine fixative |
| How is picric acid removed? | 50-70% alcohol or 70% alcohol saturated with lithium carbonate |
| Can tissue be held in Bouin fixative indefinitely? | no should be sored in 70-80% alcohol |
| Max fix time in bouin | < twenty four hours |
| Bouin solution can not be used for? | the preservation of tissue that must be examined ultrastructurally ( with electron microscopy) or in which nucleic acids must be demonstrated |
| Alcohol, 95% saturated with picric acid 800 mL , formaldehyde 150 mL , glacial acetic acid 50 mL | Gendre solution |
| Uses for Gendre | excellent for the preservation of some carbohydrates especially glycogen |
| Excess picric acid should be removed from Gendre with? | 80% alcohol |
| Copper acetate 25 g, picric acid 40 g , formaldehyde 100 mL, acetic acid 15 mL , distilled water 1,000 mL | Hollande solution |
| Characteristics of Hollande | stable , modification of Bouin solution, copper acetate stabilizes rbcs and granules of eosinophils and endocrine cells |
| Uses of Hollande | will decalcify small specimens of bone, biopsy specimens of GI tract |
| Why must Hollande solution be washed out before the specimen is placed in a phosphate buffered formalin solution on the tissue processor? | salts present in the solution will form an insoluble phosphate precipitate |
| Health hazards of Hollande | moderately toxic if ingested and may cause dermatitis |
| Paraformaldehyde 20 g , picric acid, saturated aqueous 150 mL, sodium phosphate monobasic 3.31 g, sodium phosphate dibasic 17.88 g, distilled water 1,000 mL | Zamboni ( buffered PAF) |
| Characteristics of Zamboni | Final pH of 7.3 , very stable, good general purpose , |
| Uses of Zamboni | provides secondary fixation with osmium and electron microscopy |
| What are the Zinc formalin solutions | Aqueous zinc formalin, unbuffered aqueous zinc formalin, and alcoholic zinc chloride formalin |
| Zinc sulfate, heptahydrate 10 g, formaldehyde 100 mL, and distilled water 900 mL | Aqueous zinc formalin |
| Chacateristics of aqueous zinc formalin | not very soluble in 70% alcohol used in the first processor dehydration station , precipitate in processors, precipitates inside the tissue causing microtomy issues |
| Zinc sulfate 20 g , distilled water 900 mL , formaldehyde 100 mL | Unbuffered aqueous zinc formalin |
| Characteristics of Unbuffered aqueous zinc formalin | formalin pigment can be produced, min of 4-6 hrs should be allowed for fixation of biopsy tissues and 6-8 hours for most other tissues |
| Zinc chloride 4.5 g , distilled or deionized water 1,000 mL, isopropyl alcohol 2,000 mL, formaldehyde 400 mL | Alcoholic Zinc Chloride formalin |
| uses of Alcoholic zinc chloride formalin | postfixative solution, following fixation with NBF, better for fatty tissues |
| Characteristics of Alcoholic Zinc chloride formalin | Antigenicity is enhanced, nuclear detail improved, corrosive compound, fixes 1.5 times faster than aqueous solutions , toxic effect recommends zinc sulfate solutions , |
| _________________ fixed tissue gaves results equal to that seen with tisue fixed in b_5; this included immunohistochemical results | acetic zinc formalin |
| Health hazards of Zinc sulfate | Inhalation can cause irritation to the respiratory tract and the salts may hydrolyze into acid if swallowed, ingestion of 10 g reported to cause a fatality, skin and eye irritant |
| Health hazards of Zinc chloride | severe health risk , corrosive, will cause burns, harmful if inhaled, ingested, or comes into contact with skin and eyes, OSHA PEL is 1 mg |
| Nonaqueous fixatives | Acetone, acetic acid, and alcohol |
| Acetone characteristics | nonadditive protein coagulant |
| acetone uses | fixative for brain tissue for rabies diagnosis, frozen sections of tissue to be stained for cell surface antigens by , immunohistochemical techniques., preservation of special tissue components |
| Characteristics of acetone | rapid acting, extreme shrinkage, distortion, overhardening, TWA of 1000 ppm (OSHA) and TWA of 250 ppm( NIOSH), narcotic in high concentrations, defatting and dermatitis , moderately toxic, highly flammable flashpoint 4 c |
| Methyl alcohol uses | touch preparations and blood smears |
| Ethyl alcohol uses | preserve water soluble tissue components glycogen and urate crystals deposited in gout , |
| Characteristics of alcohol | noadditive protein precipitant that acts by breaking hydrogen and ionic bonds |
| Ethyl alcohol characteristics | preserves most pigments, dissolves fat, and overhardens and shrinks the tissue, TWA is 1,000 ppm toxic by ingestion, flammable |
| Methyl alcohol characteristics | 200 ppm, toxic by ingestion, result in blindness and death, flammable |
| Absolute ethyl Alcohol 60 mL , chloroform 30 mL , acetic acid glacial 10 mL | Carnoy solution |
| Characteristics of Carnoy Solution | Erythrocytes are lysed, rapid acting, preserves gylcogen, exhibits good nuclear preservation, causes excessive hardening and shrinkage, fix time not longer than four hours |
| Uses of Carnoy solution | Preservation of special tissue components lost through routine fixation |
| Health hazards of carnoy solution | damage to central nervous system, liver, kidneys, and eyes, carcinogen, should be used in fume hood |
| Methyl alcohol 60 mL , chloroform 30 mL , acetic acid glacial 10 mL | Methacarn |
| Which fixative is recommended according to Vacca? | Methacarn |
| Characteristics of methacarn | it hardens and shrinks tissue less than carnoy fixative |
| Transport solutions | Michel transport medium, 2 PBS stock solution, and PBS- 10% sucrose solution |
| Anhydrous citric acid 4.803 g, ammonium sulfate 412..3 g, n-ethylmaleimide 625 mg, magnesium sulfate 1.23 g, and distilled water to 1 L | Michel transport medium |
| pH of Michel transport medium | 7.0-7.2 |
| potassium phosphate, dibasic 188 g , potassium phosphate, monobasic 33 g , and sodium chloride 180 g | 2 PBS buffer stock solution |
| Stock PBS solution 4 mL , distilled water 96 mL, and sucrose 10 g | PBS- 10% sucrose solution |
| Tissue for immune complex deposit studies can be stored in what transport solution? and for how long without affecting immunoflourescence or IHC studies? | PBS- 10% sucrose solution , two weeks |
| Primary fixatives for ultrastructural studies( electron microscopy) | osmium tetroxide, formaldehyde. glutaraldehyde, buffered PAF (Zamboni) solution |
| Used for light microscopy | formaldehyde, glutaraldehyde, Zamboni |
| the most sensitive indicators of poor fixation | Mitochondria |
| Advantages of primary osmium tetroxide fixation | provides excellent preservation of cytologic detail, renders lipids insoluble, giving excellent membrane preservation |
| Disadvantages of primary osmium tetroxide fixation | cannot be left in fixative > two to four hours, penetration is poor, must be minced to 1 mm cubes, histochemical studies cannot be performed |
| Advantages of primary aldehyde fixation | allows for better penetration, histochemical studies can be performed, electron microscopy can be performed( formaldehyde), can be used as dual purpose fixative, used easily for perfusion of tissues, cellular detail preserved |
| Disadvantages of primary aldehyde fixation | lipids are not preserved unless secondary osmium tetroxide is used, membrane bound cavities have a tendency to be slightly enlarged, membranes are electron lucent |
| Advantages of buffered PAF fixation | specimens can remain in the fixative solution at room temp indef., penetrates rapidly and stabilizes cellular proteins, can be used to fix both light and electron microscopy |
| Disadvantages of buffered PAF fixation | lipids are not well preserved, some cytoplasmic granules and lysosomes may not be preserved, some background substances may not be well preserved |
| Formalin pigment removal | absolute alcohol sat with picric acid for 10 minutes to 3 hours, wash with water or 70% alcohol, irnse in 1% acetic acid , wash again |
| Mercury pigment removal | Gram or lugol iodine for 10 min, wash sections briefly with running water, place the sections in 5% solution of sodium thiosulfate for 3 min, wash sections for 10 min and stain as desired |
| Lugol Iodine solution | iodine 1g Potassium Iodide 2g Distilled water 100 mL |
| Hallmarks of good fixation | nuclei with various crisp chromatin patterns and blue nuclear membrane , no cell shrinkage or artifactual spaces, cell cytoplasm well preserved and should stain well with eosin |
| Autolysis can not be prevented on what type of tissue? | autopsy |
| Autolysis can be prevent on surgical specimens by: | placing specimen in fixative asap(15-20X), opening uterus upon receipt , opening and pinning GI tract upon receipt, slicing organ resection into thin slices, bisecting lymph nodes |
| Incomplete fixation can show | smudgy nuclei with no chromatin pattern defined or nuclear bubbling |
| Corrective actions for incomplete fixation | increase time in fixative, change fixative, place formalin alcohol in first 3 stages of processing, gross sections are thin enough, volume is 15-20x, change solutions (no overuse), do not pack cassettes tightly, using agitation |
| Absolute alcohol 300 mL and glacial acetic acid 100 Ml | Clarke fluid |
| The oldest fixatives and is excellent for subsequent paraffin embedding | Clarke |
| Uses of clarke fluid and why | light microscopy because of microanatomical preservation |
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