FIXATION Flashcards by Janus Palermo

Steps (in order) in tissue processing

Fixation
Decalcification
Dehydration
Clearing
Infiltration or impregnation
Embedding
Trimming
Sectioning or Cutting
Staining
Mounting
Labeling

MNEMONIC:
Fuck DDis CIET Sige Share Mo Lang

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Process of identifying the specimen WITHOUT WRITING THE PATIENT’S NAME on the specimen tag

NUMBERING

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In numbering:

S is for ________
A is for ________
C is for _______

S is for SURGERY
A is for AUTOPSY
C is for CYTOLOGY

NOTE:

YEAR is indicated usually in 2 digits
ACCESSION NUMBER
PENCIL is utilized to label

Example: S23-143
S - Surgery
23 - Year sample received
143 - Accession number

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FIRST and MOST CRITICAL STEP in routine tissue processing; The process of stopping all cellular activities so that cells can be viewed under the microscope

FIXATION

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Most important reaction in maintaining the morphology of the tissue in fixation

Stabilization of proteins

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PRIMARY AIM OF FIXATION:

Preserve the MORPHOLOGICAL AND CHEMICAL INTEGRITY of the cell in as life-like manner as possible

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SECONDARY AIM OF FIXATION:

HARDEN AND PROTECT the tissue from trauma of further handling, so that it is easier to cut during gross examination

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Main factors involved in fixation:

Speed
Rate of penetration
pH
Temperature
Thickness of section
Osmolality
Concentration
Amount of fixative
Duration of fixation

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Speed for fixation:

IMMEDIATELY fix after removal

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Rate of penetration of fixation:

1 mm/hr for formalin from periphery to interior

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pH of fixatives should be:

NEUTRAL (between 6 and 8)

BEST pH: 7 (formalin pH)

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Temperature for fixation

Routine tissue processing:
For urgent biopsies/rapid fixation:
Electron microscope (EM):
Automatic tissue processor:

Routine tissue processing: ROOM TEMPERATURE
For urgent biopsies/rapid fixation: 60C
Electron microscope (EM): 0 to 4C
Automatic tissue processor: 40 to 42C

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Thickness of section

Recommended size of the tissue:
Electron microscope (EM):
Light microscopy:

Thickness of section

Recommended size of the tissue:
- 2cm^2 and no more than 4mm thick

Electron microscope (EM): 1-2mm thick

Light microscopy: 4mm thick

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Osmolality in fixation:

SLIGHTLY HYPERTONIC SOLUTIONS usually at 400-450 mOsm

<400 = SWELL
>450 = SHRINK

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Concentration of fixatives

Formalin:
Glutaraldehyde:

Concentration of fixatives

Formalin: 10% working solution
Glutaraldehyde: 3% normal solution; for EM: 0.25% solution

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Amount of fixative

Routine tissue processing:
Osmium tetroxide:
Electron microscope (EM):
Museum preparation:

Amount of fixative

Routine tissue processing: 20 TIMES the volume of the tissue

Osmium tetroxide: 5 to 10 TIMES the volume of the tissue

Electron microscope (EM): 20 TIMES the volume of the tissue

Museum preparation: <50 TIMES the volume of the tissue

NOTE:
RATIO OF TISSUE TO FIXATIVE = 1:20
RATIO OF FIXATIVE TO TISSUE = 20:1
**Traditionally 10-20 times the tissue

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Duration of fixation:

Adequate fixation is 4 to 6 hours

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Prolonged fixation can lead to:

SHRINKAGE
TOO MUCH HARDENING
INHIBITS CHEMICAL REACTIONS

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Factors that HASTENS/ENHANCE fixation:

Thin tissue sections
Agitation
Moderated heat (room temperature)

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Factors that RETARDS/SLOWS fixation:

Thick tissue sections
Presence of FATS, MUCUS, and BLOOD
Cold temperature

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Remedies of there are presence of fats mucus and blood in tissue sections:

FATS - cut the tissue into smaller pieces
MUCUS and BLOOD - wash with NSS

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Characteristics of GOOD FIXATIVE

Cheap, stable and safe to handle
Kills cells quickly; must inhibit bacterial decomposition and autolysis
Make tissue resistant to damage and distortion
Produce minimum shrinkage of tissue
Rapid and even penetration of tissues
Harden the tissue
Must make cellular components INsoluble to hypotonic solutions and render them INSENSITIVE to subsequent processing (RECALL)

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TWO (2) mechanisms of fixation:

Additive fixation
Non-additive fixation

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Chemical constituent of the fixative is TAKEN IN and becomes a part of tissue

ADDITIVE FIXATION

Examples of fixatives in additive fixation:

1. Formaldehyde fixatives 2. Mercuric chloride fixatives 3. Osmium tetroxide fixatives

Fixing agent is NOT TAKEN into the tissue but it alters the tissue composition thus, stabilizing the tissue which makes them unstainable for bacterial decomposition

NON-ADDITIVE FIXATION

Example of a Non-additive fixative:

Alcohol fixatives

FIXATIVES according to composition:

1. Simple fixatives 2. Compound fixatives

Fixative that is made up of ONLY ONE component of substance

SIMPLE FIXATIVES

Examples of SIMPLE FIXATIVES:

1. Aldehyde fixatives 2. Metallic fixatives 3. Picric acid fixatives 4. Alcohol fixatives 5. Osmium tetroxide (Osmic acid) mnemonic: AMPAO

Fixatives under Aldehyde fixatives:

1. FORMALDEHYDE - 10% formol saline - 10% Neutral buffered formalin or phosphate buffered formalin (pH7) - Formol-corrosive (formol sublimate) - Alcohol-formalin (Gendre's fixative) 2. GLUTARALDEHYDE mnemonic: Feeling Good si Aldehyde

Fixatives under Metallic fixatives:

1. MERCURIC CHLORIDE 2. CHROMATE FIXATIVES 3. LEAD FIXATIVES mnemonic = madaming METALS sa Malaysia, China, at Laos 1. MERCURIC CHLORIDE - B-5 fixative - Heidenhain's Susa solution - Zenker's fluid - Zenker-formol (Hely's fluid) mnemonic = ang mga tao sa Malysia ay laging BHZZe (busy) 2. CHROMATE FIXATIVES - Chromic acid - Regaud's fluid - Orth's fluid - Potassium dichromate mnemonic = ang mga tao sa China ay laging naka-CROP top 3. LEAD FIXATIVES mnemonic = masarap mag-isa sa Laos

Fixatives under Picric acid:

1. Bouin's solution 2. Brasil's alcoholic picroformol mnemonic: BaBy ni Picric acid

Fixatives under alcohol fixatives:

1. Isopropyl alcohol 95% 2. Carnoy's fluid 3. Ethyl alcohol 70-100% 4. Methyl alcohol 100% 5. Newcomer's fluid mnemonic: ICE MuNa bago ALCOHOL

Fixatives under Osmium tetroxide (Osmium acid)

1. Flemming's solution 2. Flemming's solution without acetic acid Feel na Feel ni Osmium tetroxide REMEMBER: Glacial acetic acid - goods na goods sa nucleus, bad na bad sa cytoplasm **Nuclear fixatives - WITH Glacial acetic acid **Cytoplasm fixatives - WITHOUT Glacial acetic acid

REMEMBER: Glacial acetic acid - goods na goods sa nucleus, bad na bad sa cytoplasm **Nuclear fixatives - WITH Glacial acetic acid **Cytoplasm fixatives - WITHOUT Glacial acetic acid

Made up of TWO or MORE fixatives which have been added together

COMPOUND FIXATIVES

Bouin's solution is a compound fixative that contains:

- Formaldehyde - Picric acid - Glacial acetic acid

Carnoy's fluid is a compound fixative that contains:

- Glacial acetic acid - Absolute alcohol - Chloroform

FIXATIVES according to Action

1. Microanatomical fixatives 2. Histochemical fixatives 3. Cytological fixatives

Permit the GENERAL microscopic study of tissue structures

MICROANATOMICAL FIXATIVES

Microanatomical fixatives should never contain _____________ because it inhibits hematoxylin

Microanatomical fixatives should NEVER CONTAIN OSMIUM TETROXIDE (OSMIC ACID) because it inhibits hematoxylin

Examples of Microanatomical fixatives:

10% Formol Saline 10% Neutral Buffered Formalin Heidenhain's Susa Formol sublimate (Formol corrosive) Zenker's solution Zenker-Formol (Helly's) Bouin's solution Brasil's solution Mnemonic: 1010HF ZZBB

Preserve the CHEMICAL constituents of cells and tissues:

HISTOCHEMICAL FIXATIVES

Examples of Histochemical fixatives:

10% Formol Saline Absolute ethyl Alcohol Acetone Newcomer's mnemonic: FAAN

Preserves SPECIFIC parts and particular microscopic element of the cell

CYTOLOGICAL FIXATIVES

Examples of Cytological fixatives

A. NUCLEAR FIXATIVES B. CYTOPLASMIC FIXATIVES

Nuclear fixatives usually contains __________________ due to its affinity for nuclear chromatin

CONTAINS GLACIAL ACETIC ACID REMEMBER: Glacial acetic acid - goods na goods sa nucleus, bad na bad sa cytoplasm **Nuclear fixatives - WITH Glacial acetic acid **Cytoplasm fixatives - WITHOUT Glacial acetic acid

pH of Nuclear fixatives should have:

pH of 4.6 or less (<4.6)

Examples of Nuclear fixatives:

Bouin's solution Flemming's with glacial acetic acid Carnoy's Newcomer's Heidenhain's Susa mnemonic: BE(F)NCH "BENCH"

Cytoplasmic fixatives should ________________ which destroys mitochondria and Golgi bodies.

Cytoplasmic fixatives should NEVER CONTAIN GLACIAL ACETIC ACID which destroys mitochondria and Golgi bodies. REMEMBER: Glacial acetic acid - goods na goods sa nucleus, bad na bad sa cytoplasm **Nuclear fixatives - WITH Glacial acetic acid **Cytoplasm fixatives - WITHOUT Glacial acetic acid

pH of Cytoplasmic fixatives should have:

pH more than 4.6 (>4.6)

Examples of Cytoplasmic fixatives:

Flemming's fluid without acetic acid Formalin with post chroming Orth's fluid Helly's Regaud's mnemonic = FFOHR da Cytoplasm

Chemical fixatives that act by creating covalent chemical bonds between protein tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue/Enhance stabilization of protein

CROSS-LINKING FIXATIVES

TWO MAJOR GROUPS OF CHEMICAL FIXATIVES

1. CROSS-LINKING FIXATIVES (e.g., Aldehydes) 2. PRECIPITATING (OR DENATURING FIXATIVES) (e.g., Alcoholic fixatives)

Chemical fixatives that act by reducing the solubility of protein molecules and (often) by disrupting the hydrophobic interactions that give many proteins their tertiary structure. the precipitation and aggregation of proteins is a very different process from the crosslinking that occurs with the aldehyde fixatives/Destroys/further slow down stabilization

PRECIPITATING (OR DENATURING FIXATIVES)

TWO (2) METHODS OF FIXATION

1. Physical methods 2. Chemical method

_____ methods include HEATING, MICROWAVING, and CRYO-PRESERVATION (FREEZE-CRYING)

Physical methods

_____ fixation is usually achieved by immersing the specimen in the fixative solution (IMMERSION FIXATION) or, in the case of small animals or some whole organs such as a lung, by perfusing or injecting the vascular system with fixative (PERFUSION FIXATION)

Chemical fixation/method