Fixation Flashcards

Question

# BOC Study Guide Question A pigment caused by chromate-containing fixatives can be prevented by treating the tissue prior to processing with: A. running water B.iodine C. piciric acid D. Potasium permanganate ## Footnote HT level

Answer 1

A. Running water Chromate-containing fixatives shoud be washed (with water) before processing becuase the dehydrating alcohol can couase an insoluble pigment to form ## Footnote See Potassium chromate flash card

Answer 2

**Picric acid-based** >modified Bouin solution * Protein coagulant additive + * Nucleoprotein ppts, DNA soluble/partially hydrolyzed+/- * Nill lipid and carbohydrate reaction - * Very slow penetration - * Inhibits enzyme activity + * Poor EM/ultrastructural preservation - * Stablizes (cupric acetate) RBC membranes, granuels of eosinophils & endocrine cells + * Excellent for GI, testies, endocrine, & connective | Piric acid = exploseive dry ## Footnote See Part 2

Answer 3

**Picric acid-based** >modified Bouin solution * Will decal small bone spec. + * *Should not be used with Feulgen reaction** becasue of false pasitve with Schiffs due to DNA hydrolysis- * * Fixative must be washed out before phosphate buff formalin (insoluble phosphate ppt) **Ingredients: **copper acetate, *piric acid*, formaldehyde, acetic acid (blances shrinkage), DI water | Piric acid = exploseive dry ## Footnote See Part 1

Answer 4

**Picric acid-based** * Protein coagulant additive + * Nucleoprotein ppts, DNA soluble/partially hydrolyzed+/- * Nill lipid and carbohydrate reaction - * Very slow penetration - * Inhibits enzyme activity + * Good EM/ultrastructural preservation if postfixed with osmium +/- * Good general fixative+ | Piric acid = exploseive dry ## Footnote See Part 2

Answer 5

**Picric acid-based** * pH 7.3 so somewhat stable + * Should not be used with Feulgen reaction* - * The paraformaldehyde needs to be slightly alkaline in order to dissociate it into formaldehyde **Ingredients: **paraformaldehyde, saturated aqueous picric acid, sodium hydroxide, phospate buffer, & DI | Piric acid = exploseive dry ## Footnote See Part 1

Answer 6

**Not usually used alone** **Nonadditive fixative** * Nucleoprotein/DNA coagulant nonadditive + * Nill protein, lipid, &carbohydrate reactions - * Rapid penetration + * Counter acts shrinking + (protein swelling more than anyother fixative, collagen swells because the acid breaks protein links exposing hydrophilic which pulls water in) * Unknown enzyme activity +/- * Poor EM/ultrastructural preservation - * Lysis RBC - ## Footnote See Acetic Acid chemial structure

Answer 7

**Not Used Alone** **Additive fixative** **Orth Solution (only real example)** * Basic compared to chromic acid * pH > 3.5 specific use for chromaffin granuels (orange/brown) in cytoplasm of adreanl medulla; diag phenochromocytoma+ * Tissue fixed in solution with potassium dichromate will be very receptive to eosin staining + * Tissue must be washed (with water) after fixation- * Can produce artifactual pigment, easily preventable but only removed with 1% HCl in 70% alcohol for 30min -/+ * Attaches to some lipids & makes insoulble +/- * Nil carbohydrate reaction * Rapid penetration + ## Footnote See also chromic acid flash card

Answer 8

**Not Used Alone** **Additive fixative** **Orth Solution (only real example)** * Chromium is hightly toxic- * Inhibits enzyme activity+ * Poor/distorted EM/ultrastrucutral preservation * Protein noncoagulant additive+ * Dissolves DNA/precipitates nucleoprotein +/- * Attaches to some lipids & makes insoulble +/- **Orth Ingredients:** potassium dichromate, sodium sulfate, DI, dilute with formaldehyde ## Footnote See also chromic acid flash card

Answer 9

**Not Used Alone** **Additive fixative** * Potassium dichromate in an acidic solution * Tissue fixed in solution with potassium dichromate will be very receptive to eosin staining + * Tissue must be washed after fixation- * Can produce artifactual pigment, easily preventable but only removed with 1% HCl in 70% alcohol for 30min -/+ * Chromium is hightly toxic- * Inhibits enzyme activity+ * Poor/distorted EM/ultrastrucutral preservation * Protein coagulant additive+ * Nucleic acids coagulant+ * Oxidizes unsat. fatty acids- * Oxidizes carbohydrates to aldehydes- * Slow penetration- ## Footnote See potassium dichromate chemial structure

Answer 10

**Dialdehyde** **Additive fixative** ** Noncogulant fixative** * Much less toxic than formaldehyde+ * Protein additive (form n-hydroxymethl adduct with 2 carbon atoms)+ * Nucleic acid additive + * Nil lipid & carbohydrate reactions - * Rapid penetration ( with little crosslinking) + * Enzyme activity inferior to formaldehyde- * Unkown EM/ultrastructural preservation - * Chromatin & membranes perserved with excellent clarity+ * Stains for H. pylori (silver) unsatisfactory- * Antigen retrieval not needed except if arginine is th epitope +/- * Despite one free aldehyde group, no PAS reaction problems + * Lysis RBCs - * Crosslinks specifically +/- * Slight reduction is staning especially after longer periods of storage ## Footnote See [Glyoxal chemial structure](https://upload.wikimedia.org/wikipedia/commons/thumb/8/8d/Structural_formula_of_glyoxal.svg/1200px-Structural_formula_of_glyoxal.svg.png)

Answer 11

Nonaqueous fixative **Nonadditive fixative** **Noncogulant fixative** * Rapid acting + (should not go longer than 4hrs) * Preserves glycogen + * Good nuclear preservation + * Excessive shrinkage - * Excessive hardening - * Lysis RBCs - * Helps strip RBCs for cyology spec + * Prolonged exposure can damage CNS, liver, kidneys, & eyes * Carcinogenic due to chloroform **Carnoy Ingredients:** absolute alcohol, *chloroform*, acetic acid

Answer 12

Aqueous fixative **Additive fixative** **Noncogulant fixative** ! The extra alddehyde not involved in crosslinking reacts tot PAS stain causing false positives! * Penetrates slowly & poorly (thin gross sections) * Crosslinking rapidly * Good EM/ultrastructure preservation + -->usualy post fixed with osmium * Excessive hardening if prolonged - !Unstable= breaks down when oxygen exposed * Noncoagulant of nucelic acids+ * Perserves lipids with gradual loss-/+ * Carbohydrates nill- * Inhibits enzyme activity >formaldehyde+ **Glutaraldehyde Ingredients:** sodium phosphate/monobasic, sodium hydroxide, DI, & 25% gluaraldehyde

Answer 13

**Additive fixative** **Coagulant fixative** **B-5 Solution (only real example)** * Protein coagulant + * Nucleoprotein/DNA coagulant + * Nill carbohydrate reactions - * "Unmaskes" some lipids +/- * Rapid penetration + * Inhibits enzyme activity - * Organelles preserved so EM/ultrastructural preservation + -Not used alone- **B-5 Ingredients:** mercuric chloride, sodium acetate, DI, dilute with formaldehyde ## Footnote See B-5 solution flash card

Answer 14

**Additive fixative** **Noncoagulant fixative** Primarly postfixative for EM to ensure preservation of lipids * Protein additve noncoagulant + * Nucleoprotein/DNA noncoagulant + * Nill carbohydrate reactions - * Reacts with and adds to doulbe bone in lipids makes them insoluble + * Very slow penetration (1mm cubes) - * Inhibits enzyme activity + * Excellent organelle & membrane preservation so excellent EM/ultrastructural + -Used alone- !Vaporizes easily, so expsensive & hazardous --> TWA 0.002ppm

Answer 15

A reagent used to link the stain or dye molecules to the tissue. Many in histo are metals espeically those linking hematein to tissue Example: Hematoxylin is an acid, but as it is almost always used in conjunction with alum or iron (the mordant) it becomes a basic stain

Answer 16

A chemical or substance that adds on to/links/combines with, another substance altering it usally improving/strengthening. The electrical charge at attachment site changes. Many add on to tissue proteins. Examples: **1. Mercuric chloride**-proteins **2. Picric acid**-proteins **3. Potassium chromate**-proteins **4. Formaldehyde**-proteins **5. Glutaraldehyde**-proteins **6. Glyoxal**-proteins & nucleic acids **7. Osmium tetroxide**-proteins **8. Zinc sulfate/chloride**-proteins ## Footnote See the mercuric chloride, picric acid, potassium chromate, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, and zinc sulfate/choride flash cards

Answer 17

Waster based fixatives. Lipid preservation is possible with only a few. Examples: **1. Acetic acid** **2. Formaldehyde**-some gradual lipid loss **3. Glutaraldehyde**-some gradual lipid loss **4. Glyoxal**- **5. Mercuric chloride**-Unmasks some lipids **6. Osmium tetroxide**-reacts & adds to double bond in lippids making them insoluble **7. Picric acid** **8. Potassium dichromate low pH**-oxidizes unsaturated fatty acids (so no preservation) **9. Potassum dichromate hi pH**- attacehds to some & makes them insoluble **10. Zinc sulfate/chloride**

Answer 18

A chemical or substance that act on tissue without chemically combining with it. Mostly organic compouds that primarly dissociate bound water molecules from tissue protein groups, protein solubility is then lost and it precipitates (PPT). Excessive water removal shrinks & harden tissue. Examples: **1. Alcohols** (nonaqueous, can be used alone) **2. Acetone** (nonaqueous, can be used alone) **3. Acetic acid** (noted the only sweller) ## Footnote Compare to Additive fixatives

Answer 19

"Metal Mesh" Creates a network in tisue that allows solutions to gain entry into the interior of the tissue easily (easy penetration). Examples: 1. Zinc salts 2. Mercuric chloride 3. Cupric sulfate 4. Eythl alcohol 5. Acetone 6. Picric Acid 7. Acetic acid- Nucleic acids | Hold mesh ball when learning above examples ## Footnote Compare to Noncoagulant fixatives

Answer 20

"Jello" Fixatives that create gel that make penetration by subsequent solutions difficult. Examples: 1. Formaldehye 2. Gulteraldehyde 3. Glyoxal 4. Osmium tetroxide 5. Postassium dichromate | Draw jello cup with all six fixatives above ## Footnote Compare to Coagulant fixatives

Answer 21

The irreversible condensation of Chromatins in nucleii. It can happen when the solution temperature in microwave exceeds 68C.

Answer 22

Cause: Unbuffered formaldehyde solutions (acidic pH4-5, includes unbuff-zinc formaldehye solutions too) react to atmospheric oxygen which forms formic acid. The solutions forms black acid hematin/ formalin pigment when added to blood rich tissue. Prevention: Maintain solution pH closer to 7 (buffering) & appropriate volume ratio of fixative solution. Correction: Treat tissue sections with alcoholic picric aid or alkaline alcohol. ## Footnote See [microscopic image example](http://)

Answer 23

Correction: It can partially be removed by the addition of acid alcohol.

Answer 24

Correction: It can be removed by the addition of Iodine and sodium thiosulfate. | Think "MIS" aka Mercury = Iodine & soium thiosulfate

Answer 25

Correction: It can be removed by the addition of alcoholic picric acid and alkaline alcohol.

Answer 26

1. Temperature 2. Size 3. Volume Ratio 4. Time 5. Choice of Fixative 6. Penetration 7. pH 8. Osmolality 9. Storage ## Footnote See respective flash cards for each factor

Answer 27

Can affect tissue morphology Increasing it also increases rate of fixation but also rate of autolysis 45C max ## Footnote See respective flash cards for each factor

Answer 28

Also: Black acid hematin Cause: Formic acid added to blood rich tissue from unbuffered formaldehyde solutions (acidic pH4-5, includes unbuff-zinc formaldehye solutions too) that react to atmospheric oxygen. Prevention: Stop formic acid from forming. So maintain solution pH closer to 7 (buffering) & appropriate volume ratio of fixative solution. Correction: Treat tissue sections with alcoholic picric acid or alkaline alcohol. ## Footnote See [microscopic image example](http://)

Answer 29

**Additive fixative** **!Contraindicated for tissues destine for Feulgen reaction as fixation hydrolyzes the nuclei excessively so stain will be unsatisfactory!** -Bouin -Gendre -Hollande -Zamboni (PAF or picric acid formalin) See [Picric Acid chemical structure](https://upload.wikimedia.org/wikipedia/commons/thumb/e/e4/Pikrins%C3%A4ure.svg/800px-Pikrins%C3%A4ure.svg.png) ## Footnote See Bouin, Gendre, Hollande, & Zamboni flash cards respectively

Answer 30

Give poor ultrastructural preservation because Zinc is a coagulant. ## Footnote See coagulant & formalin flash cards

Answer 31

**Not Used Alone** **Orth Solution (only real example)** * (pH > 3.5) Specific use for chromaffin granuels (orange/brown) in cytoplasm of adreanl medulla; diag phenochromocytoma+ * Tissue fixed in solution with potassium dichromate will be very receptive to eosin staining + * Tissue must be washed after fixation- * Can produce artifactual pigment, easily preventable but only removed with 1% HCl in 70% alcohol for 30min -/+ * Chromium is hightly toxic- * Inhibits enzyme activity+ * poor/distorted EM/ultrastrucutral preservation * pH effects alot of characteristics * <3.5 pH: protein coagulant additive+, nucleic acids coagulant+, oxidizes unsat. fatty acids-, oxidizes carbohydrates to aldehydes-, & slow penetration- * >3.5 pH: protein noncoagulant additive+, dissolves DNA/precipitates nucleoprotein +/-, attaches to some lipids & makes insoulble +/-, nil carbohydrate reaction, rapid penetration + **Orth Ingredients:** potassium dichromate, sodium sulfate, DI, dilute with formaldehyde ## Footnote See Potassium dichromate chemial structure

Answer 32

"thickness" Effects reagent penetration, as the more distanceto cross the longer it will take but autolysis of deeper/inner tissue will occur first ## Footnote See respective flash cards for each factor

Answer 33

Volume of fixative must be at least 15x-20x > tissue volume If not,the fixative composition will be compromised as water & serum proteins are displaced ## Footnote See respective flash cards for each factor

Answer 34

Ischemic (blood supply cut off) & duration Inadequate duration will not preserve morph Avoid autolysis, putrefication, & desquamation of epithelium ## Footnote See respective flash cards for each factor

Answer 35

Appropriate choice, as often no correction possible I.F. , enzyme study, or fat = frozen Staining reaction might require post fixation (ex trichrome) Water soluble element (urate cyrstals) = 100% ROH ## Footnote See respective flash cards for each factor

Answer 36

Deepth =k sq(t) k= diffusibility coefficient (unique to each fixative) Type of tissue influences it (muscle fiber, capillaries, & high surface area Heat Coagulant vs noncoagulant ## Footnote See respective flash cards for each factor

Answer 37

Additional studies wet tissue reserve NBF=indefinet storage but IHC ideal is to move to 70% ROH to stop crosslinking ## Footnote See respective flash cards for each factor

Answer 38

the # of particles in solution Critical/Important in ultrastructural/EM studies Most body fluids 0.3 Osm 1 Osm = 1 formula weight of nondissociating compound per 1000g solution (ex sucrose) Isotonic [sol particles] = cytosol Hypo [sol particles] < cytosol Hyper [sol particles] > cytosol ## Footnote See respective flash cards for each factor

Answer 39

Effects reactivity of fixative and EM/ultrastructural preservation | Remember, pickling in high acidity does not bode well for EM ## Footnote See respective flash cards for each factor

Answer 40

A. Zenker fluid The sections must have been fixed in Zenker fluid, which**contains chromium trioxide**, and **not washed carefully before placing the tissue in alcohol**. The alcohol will reduce the chromium and** forms an insoluble pigment in the tissue.** Zenker Ingredients: DI, Mercuric chloride, **Potassium dichromate**, Glacial acetic acid, Fixation time: 4 – 24 hours

Fixation Flashcards

(64 cards)