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Common compound fixatives are:
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Formaldehyde-glutaraldehyde(4CF-1G) consists of:
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Compound fixatives
| Common compound fixatives are: | B-5, BOUIN, DAVIDSON, GENDRE, HOLLANDE, FORMALDEHYDE-GLUTARALDEHYDE, ZENKER & HELLY, ORTH, ZAMBONI, AND ZINC FORMALIN |
| Formaldehyde-glutaraldehyde(4CF-1G) consists of: | dissolve 50mL of water, monobasic sodium phosphate(NaH2PO4) 1.16g, dissolve in 38 mL of water, sodium hydroxide(NaOH) 0.27g, combine the 2 solutions, then add: formaldehyde,40% 10 mL, Glutaraldehyde, 50% 2 mL mix well and store at 4 C |
| Dual-purpose fixative, preserves tissues for routine histopathology while keeping the specimen suitable for electron microscopy after prolonged storage, stable for at least 3 months if stored at 4 C. | Formaldehyde-glutaraldehyde(4CF-1G) |
| What is another name of Davidson fixative | Hartmann |
| What are the fixation times for the Davidson versions? | version 1= 24-72 hrs version 2= 24 hrs |
| What are both versions of Davidson solutions stored in? | Both are stored in either 70% alcohol or NBF. |
| Davidson frequently substitutes what other solution and why? | Bouin, because Davidson lacks picric acid, which requires the removal before staining and is a safety hazard. |
| Davidson version 1 are popular for what organ and why? | eyes, because of the lack of hardness and the retina of the eye also does not tend to detach as it does with formalin fixation. |
| Both versions are used for ______,____________,___________________, ___________ and other _________(marine animals, fish, rodent eyes). | eyes, testis, lymph nodes, various soft tissues, and other species |
| What happens to retinas in prolonged fixation? | may cause excessive swelling, shattering of the lens, cataract type artifacts, defects in some eye components, indistinct rods and cones, retinal detachment, and inconsistent staining. |
| ____________________________ is easier after total fixation and firming action by 70% alcohol storage for > or equal to 2 days. | Slicing of eyes |
| After initial NBF fixation of colorectal resections, the ___________ can be refixed in what fixative to reveal white lymph nodes at gross dissection. | mesentary, modified Davidson |
| Both Davidson formulas are not useful in what type of microscopy? | electron, however IHC staining is successful |
| Potassium dichromate 2.5g, Sodium sulfate 1g, Distilled water 100 mL and just before use add Formaldehyde,37-40% 10 mL | Orth Solution |
| Characteristics of Orth solution | not a good general purpose fixative but is preferred for the subsequent demonstration of chromaffin(chromate-loving) granules in the cytoplasm of cells of the adrenal medulla. These granules are colored orange to brown with chromate. |
| Why are chromaffin granules in orth solution important? | The demonstration of these granules may be important in the diagnosis of a pheochromocytoma. |
| Why should mercury-based solutions should no longer be used in the laboratory and why are some pathologists reluctant to discontinue it? | Because of extreme toxicity, mercury fixative formulations should no longer be used in the lab. However, some pathologist are reluctant to discontinue its use on trephine biopsies, because of the excellent nuclear detail provided. |
| True or false: B-5 Stock solution contains mercuric chloride, sodium acetate(anhydrous), and distilled water | True |
| B-5 stock solution | Mercuric chloride 12g, sodium acetate(anhydrous) 2.5g, and distilled water 200 ml |
| B-5 working solution | B-5 stock solution 20 mL and formaldehyde 2 mL |
| The fixative has found wide acceptance for hematopoietic and lymphoreticular tissue, because of its ability to demonstrate beautiful nuclear detail | B-5 working Solution |
| What is the protocol for tissue fixed in b-5? | Tissue cannot remain in this solution indefinitely, after fixation, wet tissue must be placed in a storage solution, most frequently 70% alcohol. |
| This fixative gives excellent results with many special stains and is an excellent fixative for may tissue antigens to be demonstrated on paraffin embedded tissue. | B-5 solution |
| What reagent substitutes mercury? | Zinc |
| Zenker and helly stock solution | mercuric chloride 50 g, potassium dichromate 25 g, sodium sulfate(optional) 10g, and distilled water 1,000 mL |
| Zenker-helly stock solution 95 mL and acetic acid, glacial 5 mL | Zenker working solution |
| Is Zenker solution stable or unstable? | stable therefore it can be made up into large quantities if desired |
| Helly working solution | Zenker-helly stock solution 95 mL and formaldehyde 37-40% 5 mL |
| Is helly working solution stable or unstable? | unstable, because formaldehyde is a reducing agent |
| True or false: In both Zenker and Helly working solutions the tissue does not have to be treated for mercury pigment. | False, it does have to be treated for mercury pigment |
| What happens if excess fixative is not removed by washing with water in Zenker and Helly solutions? | The reduction of potassium dichromate by the dehydrating alcohol may cause the formation of chrome pigment. |
| What other pigment can be formed with Helly solution? | Formalin pigment |
| What is the difference in Zenker and Helly with RBCs? | Zenker solution will lyse erythrocytes because of its acetic acid content and Helly solution will preserve the erythrocytes. |
| Is Zenker or Helly solution the better nuclear fixative and why? | Zenker because the presence of acetic acid |
| Zenker solution uses | used to fix and decalcify needle biopsy specimens of bone marrow, but it can dissolve iron and If the Mallory phosphotungstic acid hematoxylin stain is to be applied |
| _____________________ techniques are unsatisfactory after fixation in either Helly or Zenker solution. | Silver |
| Max. fixation time for Zenker and Helly solutions and why | 24 hrs, the tissue becomes overhardened and nuclear basophilia is decreased |
| Zenker and Helly solutions safety hazards | Very toxic, fatal if inhaled, ingested, or absorbed through the skin. Target organs are the kidneys, central nervous system, and liver. Carcinogen in humans and should be used under a fume hood |
| Picric acid based solutions include: | Bouin, Gendre, Hollande, Zamboni(PAF) |
| Picric acid, saturated aqueous solution(1.2%) 750 mL, formaldehyde 37-40% 250 mL, and acetic acid,glacial 50 mL | Bouin Solution |
| This solution is excellent for tissue that is to be trichrome stained and for preserving structure with soft and delicate textures. | Bouin |
| True or false: Bouin preserves RBCs because of its formaldehyde content. | False: Bouin solution lyses RBCs because of its acetic acid content. |
| How is the swelling effect in acetic acid balanced in bouin solution? | The swelling effect of acetic acid is balanced by the shrinking effect of picric acid |
| How must the yellow color caused by picric acid in Bouin solution be removed? | The yellow color must be removed by washing, traditionally done with 50-70% alcohol, or 70% alcohol saturated with lithium carbonate. |
| What happens to embedded tissue if excess picric acid is left? | If excess picric acid is left in embedded tissue, the staining will deteriorate over time. |
| How should remaining wet tissue be stored after Bouin fixation? | Remaining wet tissue should be stored in 70-80% alcohol because the tissue cannot be stored in bouin fixative indefinitely |
| Max. fixation time in Bouin | <24 hrs |
| According to Kiernan, tissue stored in Bouin solution for several months is sometimes still usable, but the acid solution extracts RNA and partly hydrolyzes DNA. What are the results? | This results in the nuclei giving a positive reaction with Schiff reagent. |
| Bouin uses | excellent for use on biopsy specimens of the gastrointestinal tract because the nuclei are much crisper and better stained than with 10% NBF |
| What type of tissue fix well in bouin | tissue of the endocrine system |
| Bouin solution may be used as a routine fixative but cannot be used for ...... | the preservation of tissue that must be examined ultrastructurally(with electron microscopy) or in which nucleic acids must be demonstrated. |
| Gendre solution | Alcohol, 95% saturated with picric acid 800 mL, formaldehyde, 37-40% 150 mL, and glacial acetic acid 50 mL |
| Gendre solution is great for ______________________________. | this alcoholic bouin solution is excellent for the preservation of some carbohydrates, especially glycogen. |
| Hollande Solution | Copper acetate 25g, picric acid 40 g, formaldehyde 37-40% 100 mL, Acetic acid 15 mL, Distilled water 1,000 mL |
| Is Hollande solution stable and what is its action? | This modification of bouin solution is stable and will decalcify small specimens of bone |
| Hollande solution uses | widely used as a fixative for biopsy specimens of the gastrointestinal tract, can be stained successfully with most special stains |
| What does the cupric acetate in hollande solution do? | The cupric acetate present in the solution stabilizes RBC membranes and the granules of eosinophils and endocrine cells, so that lysis that occurs is less than that seen with Bouin solution |
| Why must Hollande solution be washed out and when? | The fixative must be washed out before the specimen is placed in a phosphate-buffered formalin solution on the tissue processor because salts present in the solution will form an insoluble phosphate precipitate. |
| Health hazard of Hollande solution | may cause dermatitis and toxic if ingested |
| Zamboni(buffered picric acid-formaldehyde or PAF) | Paraformaldehyde 20g, picric cacid, saturated aqueous(double filtered) 150 mL, NaH2PO4,h20 3.31g, NaHPO4(anhydrous) 17.88 g, distilled water 1,000 mL |
| What should the final pH of Zamboni solution be | pH of 7.3 |
| Characteristics of Zamboni solution | stable, good general purpose solution, allows secondary fixation with osmium and because it is easy to use and preserves the morphologic characteristics accurately some lab prefer it as the primary fixative for electron microscopy |
| Zinc formalin solutions | Aqueous zinc formalin(original formula), unbuffered aqueous zinc formalin, alcoholic zinc chloride formalin |
| _______________________is not lost with the long-term storage of wet tissue in zinc sulfate formalin solutions. | Antigenicity |
| What is prevent by zinc? | crosslinking |
| Zinc ions hold macromolecules in their native conformation via corrdinate bonds. The prevents the damaging cross-linkages that formaldehyde creates. What results from this? | The result is enhanced preservation of antigenicity, rare need for antigen retrieval and the possibility of greater dilution of antibodies |
| Which fixes faster: formaldehyde or zinc formalin | zinc formalin fixes more rapidly than formaldehyde alone, specimens fixed in zinc formalin for a few hours are comparable with those fixed in formaldehyde for 30 hours. |
| How can some antigen activity be recovered? | Some antigen activity can be recovered by backing up tissue that has been formalin fixed and embedded in paraffin, refixing with zinc formalin, and reprocessing |
| Zinc sulfate, heptahydrate 10g, formaldehyde, 37-40% 100 mL, and distilled water 900 mL | Aqueous zinc formalin(original formula) |
| Is zinc soluble in 70% alcohol used in the first processor dehydrating station in aqueous zinc formalin? What happens? | no, zinc is not very soluble therefore, this solution tends to precipitate in processors and inside the tissue specimen causing difficult microtomy |
| Unbuffered aqueous zinc formalin | zinc sulfate 20g, distilled water 900 mL, formaldehyde 37-40% 100 mL |
| fixation times for unbuffered aqueous zinc formalin | a min. of 4-6 hrs should be allowed for fixation of biopsy tissues and 6-8 hrs for most other tissues |
| Alcoholic zinc chloride formalin | Zinc chloride 4.5g, distilled or deioized water 1000 mL, isopropyl alcohol,99% 2,000 mL and formaldehyde 37-40% 400 ML |
| Which solution was recommended as a postfixative solution follwoing fixation with NBF? | Alcoholic zinc formalin |
| Is zinc chloride or zinc sulfate corrosive | Zinc chloride, but when used in alcholic zinc chloride formalin it is a very dilute concentration and should not harm the processor |
| How much faster does alcoholic zinc formalin solutions fix compared to auqeous solutions? | 1.5X |
| Alcoholic solutions are recommended if | 6-8 hrs cannot be allotted for fixation and these solutions are also better for fatty tissues |
| Bonds et al's study was to find a safe mercury free alternative to b-5, what where his findings? | He found that acetic zinc formalin fixed tissue gave results equal to that seen with tissue fixed with B-5; this included immunohistochemical results. |
| Nonaqeuous, nonadditive, and coagulating fixatives | acetone and alcohol(methyl and ethyl) |
| Acetone uses | used when the demonstration enzymes, especially acid and alkaline phosphatase, were indicated on tissue to be processed for paraffin embedding, used as a fixative for brain tissue when subsequent staining techniques for rabies diagnosis are needed. |
| Fixation in acetone | done rapidly at refrigerator temperatures and most of the dehydration is accomplished at the same time |
| frequently used on frozen sections of tissue to be stained for cell surface antigens by immunohistochemical techniques | Acetone |
| OSHA TWA and NIOSH TWA of acetone | 1,000 ppm(OSHA) and 250 ppm(NIOSH) |
| Health hazards of acetone | It is a narcotic in high concentrations and skin contact can lead to defatting and dermatitis, moderately toxic on ingestion |
| What is the flash point of acetone? | 4 C |
| What are methyl and ethyl alcohol use for in fixation? | Methyl alcohol is used frequently as a fixative for touch preparations and blood smears and ethyl alcohol is used to preserve water soluble tissue components are glycogen and the urate crystals that are deposited in gout. |
| Preserves most pigments, dissolves fat, and overhardens and shrinks the tissue. | Ethyl alcohol |
| Alcoholic formalin uses | fixes tissue, begins dehydration, preserves glycogen very well and penetrates qucikly. |
| TWA of ethyl and methyl alcohol | TWA ethyl- 1,000 ppm TWA methyl- 200 ppm |
| Which is more toxic methyl or ethyl alcohol | Methyl ingestion of methanol may result in blindness and death |
| Absolute ethyl alcohol 60 mL, chloroform 30 mL, and acetic acid, glacial 10 mL | Carnoy Solution |
| ________________are lysed by carnoy solution | Erythrocytes(RBC) |
| Characteristics of carnoy solution | rapid acting, preserves glycogen, and exhibits good nuclear preservation, but causes excessive shrinkage and hardening |
| How long should fixation be prolonged for in carnoy solution | Fixation should not be prolonged >4 hrs. |
| When should carnoy solution be used? | This fixative should be used only as indicated for the preservation of special tissue components lost through routine fixation |
| What happens if there is repeated or prolonged fixation in carnoy solution? | Repeated or prolonged exposure can produce damage to the central nervous system, liver, kidneys, and eyes |
| What solution subtitutes methyl alchol for ethyl alcohol in carnoy solution | methacarn |
| Absolute alcohol 300 mL and glacial acetic acid 100 mL | Clarke fluid |
| Clarke fluid is used for | one of the oldest fixatives and is excellent for subsequent paraffin embedding |
| What is the protocol for a unfixed tissue to be held for a brief period or transported a short distance? | It is best to place the specimen on saline dampened(excess saline squeezed out) gauze, enclose it in a tightly closed plastic container, and then place it on ice. |
| If a unfixed tissue is to be held for several days or transported over a long distance what transport medium is recommended | Michel transport medium |
| Michel medium is recommended for ______________, but not for ____________ biopsies. | for kidney biopsies, but not for muscle biopsies |
| Michel transport medium | Anhydrous citric acid 4.803g, ammonium sulfate 412.3 g, N-ethylmaleimide 625 mg(0.625g), magnesium sulfate 1.23 g, distilled water to 1 L |
| What does N-ethylmaleimide do in Michel medium | N-ethylmaleimide minimizes proteolytic activity and the ammonium sulfate fixes tissue bound immunoglobulins |
| 2 PBS buffer stock solution | potassium phosphate, dibasic(K2HPO4) 188g, potassium phosphate, monobasic(NaH2PO4) 33g, and sodium chloride 180 g |
| PBS 10% sucrose solution | stock PBS solution 4mL, distilled water 96 mL and sucrose 10 g |
| According to Elias, tissue for immune complex deposit studies can be stored in the _______________________ for 2 weeks without affecting susequent immunofluorescence or immunohistochemical studies. | 10% PBS-sucrose solution |
| Primary fixatives for ultrastructural strudies(electron microscopy) are: | osmium tetroxide, aldehydes(formaldehydes and glutaraldehyde), and buffered PAF(Zamboni) solutions |
| With the exception of _____________________, the oslutions can also be used for light microscopy. | osmium tetroxide |
| Criteria for good ultrastructural preservation | undilated space between the inner and outer nuclear membranes, no swelling or disruption of mitochondria, regular width and arrangement of channels of endoplasmic reticulin |
| WHat are the most sensitive indicators for poor fixation? | Mitochondria |
| Advantages of primary osmium tetroxide fixation | provides excellent preservation of cyctologic detail, renders lipids insoluble, giving excellent membrane preservation |
| Disadvantages of primary osmium tetroxide fixation | specimens cannot be left in fixative >2-4 hrs, penetration is poor; specimens must be minced to 1mm cubes, histochemical studies cannot be performed |
| Advantages of primary aldehyde fixation | allows better penetration of fixative, histochemical studies can be performed, electron microscopy can be performed if formaldehyde is used, formaldehyde and f-g mixtures can be used as a dual purpose, and can be used easily for perfusion of tissues |
| Disadvantages of primary aldehyde fixation | lipids are not preserved unless 2nd osmium tetroxide fixation is used, membrane bound cavities have a tendency to be slightly enlarged, membranes are electron lucent unless 2nd fixation of osmium occurs |
| advantages of primary buffered PAF fixation | specimens can remain in fixative at room temp indefinitely, penetrates rapidly and stabilizes cellular proteins, can be used to fix tissues for both light and electron microscopy |
| disadvantages of primary buffered PAF fixation | lipids are not well preserved unless secondary osmium tetroxide is used, some cytoplasmic granules and lysosomes may not be preserved, and some background substances may not be well preserved |
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