HISTO RA Flashcards by Armani Yulong

It is a step in tissue processing that involves preservation of tissues as close to original as possible

FIXATION

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Primary Goal of Fixation

to preserve cells and tissues as close as to the original as possible

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Secondary Goals of Fixation (2)

to HARDEN tissues to facilitate EASY cutting
to PROTECT the tissue from the TRAUMA of further HANDLING

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FACTORS THAT INFLUENCE FIXATION

Thickness/ Size
Hydrogen Ion Concentration
Osmolality
Concentration of Fixative
Volume
Time and Duration
Temperature

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THICKNESS of tissue in FIXATION

3-5 mm

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SIZE of tissue in FIXATION and also in LUNG specimen

3x2 cm; 1-2 cm

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Hydrogen Ion Concentration of tissue in FIXATION

Ideal pH between 6-8

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IDEAL Osmolality of tissue in FIXATION

SLIGHTLY HYPERTONIC

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In OSMOLALITY, HYPERTONIC solution causes

CELL SHRINKAGE

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In OSMOLALITY, HYPOTONIC solution causes

CELL SWELLING

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BUT in PRACTICE osmolality can be

ISOTONIC SOLUTION

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T/F
In Concentration of Fixative, We use concentrated solution as it can be damaging to tissues

FALSE; WE NEVER USE

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In Concentration of Fixative, this is diluted to produce 10% formalin

FORMALDEHYDE

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In Concentration of Fixative, GLUTARALDEHYDE is diluted to produce and is used for

a. 3% Glutaraldehyde – Electron microscopy
b. 0.25% – Immunoelectron microscopy

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The maximum effective or VOLUME of tissue

20 TIMES the volume of specimen (routine)

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Fixative to Tissue Ratio:

15-20:1

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Penetration Rate of Formalin:

1 mm/ hour

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Volume of Specimen for Osmium Tetroxide (Electron Microscopy) and reason for this

5-10 times the volume of specimen as it is quite expensive

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For Museum Preparations:

should not be less than 50-100 times the volume of the specimen

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Time and Duration of tissues

Usually subjected to fixative within 24-48 hours

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Temperature of tissue for MANUAL FIXATION

ROOM TEMPERATURE

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FIXATION Temperature of tissue for EM and Histochemistry

0-4 degC

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FIXATION Temperature of tissue for AUTOTECHNICON and why?

40 degC (hastens fixation)

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T/F
Does application of heat shortens fixation time

TRUE

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Temperature range that will ACCELERATE fixation:

37-56 degC

FACTORS TO CONSIDER WHEN CHOOSING THE RIGHT FIXATIVE

1. Urgency of the case/ need for immediate examination 2. Type of specimen/ tissue to be processed 3. Tissue structure to be studied 4. Type of section to be made 5. Staining technique to be applied

In Urgency of the case/ need for immediate examination, you must use a fixative that is ____

FAST-ACTING

In Urgency of the case/ need for immediate examination, this is used for rapid in action, thus it’s the most commonly used fixative

FORMALIN

Type of specimen/ tissue to be processed that is for preservation of brain tissue for rabies

Carnoy’s & Acetone

Type of specimen/ tissue to be processed that is not used for kidneys

Bouin’s

In Tissue structure to be studied, it is for glycogen

Picric acid fixatives (Brasil’s fluid)

In Tissue structure to be studied, for preservation of fats it is not used as it dissolve fats

ACETONE

In Tissue structure to be studied, it is for enzymes, lipases, phosphatase

ACETONE

Types of section to be made

1. Serial 2. Individual

Staining technique to be applied that is a ROUTINE STAIN

H&E

This can inhibit HEMATOXYLIN

OSMIUM TETROXIDE

FACTORS THAT WILL RETARD OR PROLONG FIXATION PROCESS

1. Cold Temperature 2. Presence of blood and mucus 3. Presence of Fat/ Fatty Tissues 4. Size and thickness

FILL IN THE BLANKS (RETARD OR PROLONG FIXATION) 1. Cold Temperature: it can _____ enzymes 2. Presence of blood and mucus: Remedy ___ 3. Presence of Fat/ Fatty Tissues: Remedy ___ 4. Size if it is ___ and thickness if it is ___

can inactivate enzymes can be removed by washing the tissue with NSS cut the specimen thinly to ensure rapid entry of fixative larger; thicker

FACTORS THAT WILL ACCELERATE OR SHORTENS FIXATION PROCESS

1. Heat Application 2. Agitation 3. Size & Thickness

FILL IN THE BLANKS (ACCELERATE OR SHORTENS FIXATION) 1. Heat Application ____; beyond this temp, it can lead to _____ 2. Agitation 3. Size if it is _____ & Thickness if it is ___

37-56°C ONLY; damage continuous mixing (autotechnicon) smaller; thinner

Problem in FIXATION: Loss of substances soluble in fixing agent/ Loss or inactivation of enzyme; Cause

May result from wrong choice of fixative

Problem in FIXATION: Soft tissues & feather like consistency; Cause

Incomplete washing of fixative

Problem in FIXATION: Brittle & hard tissues; cell shrinkage & swelling; Cause

Overfixation/ prolonged fixation

Problem in FIXATION: Presence of artifacts on tissues; Cause

Inadequate or incomplete washing

Problem in FIXATION: Failure to arrest early autolysis; Cause

Insufficient fixative; failure to fix abruptly

FIXATION METHODS

1. Physical Method 2. Chemical Method

TYPES of PHYSICAL METHOD

HEAT FIXATION MICROWAVE TECHNIQUE

Type of Physical Method that involves thermal coagulation of proteins

HEAT FIXATION

FIXATION METHOD that is commonly done and uses chemical fixatives

CHEMICAL METHOD

TYPE OF FIXATIVES

I. According to Mechanism of Action (as to manner of preservation) II. According to Action (as to purpose) II. According to Active Component Substance (as the main ingredient)

FILL IN THE BLANKS I. According to Mechanism of Action: as to ____ II. According to Action: as to ____ II. According to Active Component Substance: as the ____

manner of preservation purpose main ingredient

Types of FIXATIVES According to Mechanism of Action (as to manner of preservation)

ADDITIVE FIXATIVES NON-ADDITIVE FIXATIVES

Type of FIXATIVES According to Mechanism of Action (as to manner of preservation) that when used are combined or absorbed by the tissues

ADDITIVE FIXATIVE

In ADDITIVE FIXATIVES when used are combined or absorbed by the tissues it becomes ____

part of the tissue

ALL the fixatives available are additive except:

Acetone & Alcohol

Type of FIXATIVES According to Mechanism of Action (as to manner of preservation) that do not bind with the tissue

NON-ADDITIVE FIXATIVES

In NON-ADDITVE FIXATIVES it does not bind with the tissue for it is ____

not absorbed or taken in by the tissue

Examples of NON-ADDITIVE FIXATIVES

Acetone and Alcohol

Types of FIXATIVES according to Action (as to purpose)

1. Microanatomical 2. Cytological 3. Histochemical

Type of FIXATIVES according to Action (as to purpose) that allows general microscopic study of tissue structure

MICROANATOMICAL

Type of FIXATIVES according to Action (as to purpose) that preserve parts of the cell (either nucleus or cytoplasm)

CYTOLOGICAL

Types of FIXATIVES in Cytological

1. Nuclear 2. Cytoplasmic

Fixative that is for nuclear chromatin, chromosomes, etc.

NUCLEAR FIXATIVE

NUCLEAR FIXATIVE ___ glacial acetic acid pH with ___ 4.6

WITH LESS THAN

Nuclear Fixative EXAMPLES

Bouin’s Carnoy’s Heidenhein’s Susa Newcomer’s Flemming’s with HAc

Fixative that is for golgi body, mitochondria, etc.

CYTOPLASMIC FIXATIVES

CYTOPLASMIC FIXATIVE ___ glacial acetic acid pH with ___ 4.6

WITHOUT MORE THAN

Cytoplasmic Fixatives EXAMPLES

Regaud’s/ Moller’s Orth’s Formalin with post chroming Flemming’s without HAc

Type of FIXATIVES according to Action (as to purpose) that preserve the chemical components of tissues like enzyme

HISTOCHEMICAL

EXAMPLES of Histochemical

Acetone Absolute ethyl alcohol Newcomer’s 10% Formol Saline

Types of Fixatives According to Active Component Substance (as the main ingredient)

1. Aldehyde Fixatives 2. Metallic Fixatives 3. Picric Acid Fixatives 4. Alcoholic Fixatives 5. Glacial Acetic Acid 6. Acetone 7. Osmium Tetroxide 8. Trichloacetic Acid

Types of Aldehyde Fixatives

A. FORMALDEHYDE/ FORMALIN FIXATIVES B: GLUTARALDEHYDE FIXATIVES C: GLYOXAL

Types of FORMALDEHYDE/ FORMALIN FIXATIVES

a. 10% Formalin b. 10% Formol Saline c. 10% Neutral Buffered Formalin d. Formol Corrosive/ Formol Sublimate e. Alcoholic Formalin/ Gendres

ROUTINELY USED Formaldehyde/ Formalin Fixative

10% Formalin

10% Formalin preparation:

1 L or 1000 mL Formalin: Combine 100 ml of 37-40% formalin + 900 ml distilled water

Formaldehyde/ Formalin Fixative that is to preserve CNS and post mortem tissues

10% Formol Saline

10% Formol Saline uses ____ as diluent

NSS

Formaldehyde/ Formalin Fixative that is for tissues with iron pigments

10% Neutral Buffered Formalin

10% Neutral Buffered Formalin preparation

Same with 10% formalin, but sodium phosphate was added as buffer

Formaldehyde/ Formalin Fixative that is best for lipids & neutral phospholipids

Formol Corrosive/ Formol Sublimate

Formol Corrosive/ Formol Sublimate preparation

Formaldehyde + Mercuric Chloride

Formaldehyde/ Formalin Fixative that is for sputum and microincineration techniques

Alcoholic Formalin/ Gendres

Alcoholic Formalin/ Gendres preparation

Combination of 95% Ethanol + Glacial Acetic Acid (HAc) + Formaldehyde

Disadvantage of Formalin:

It may precipitate upon longer storage

To prevent precipitation of formaldehyde to white paraformaldehyde:

Add 10% Methanol

Aldehyde Fixative that is for Electron Microscopy

GLUTARALDEHYDE FIXATIVE

Types of GLUTARALDEHYDE FIXATIVES

a. 2-5% Solution b. 4% Solution

Glutaraldehyde fixative that is for small tissue fragments/ needle biopsies

2-5% Solution

Glutaraldehyde fixative that is for larger tissues less than 4 mm thick

4% Solution

Aldehyde Fixative that is SMALLEST ALDEHYDE

GLYOXAL

Types of Metallic fixatives

A. MERCURIC CHLORIDE B. LEAD FIXATIVES C. CHROMATE FIXATIVES

It is the most common metallic fixative

MERCURIC CHLORIDE

Metallic Fixative that is excellent for trichrome staining & tissue photography

MERCURIC CHLORIDE

Types of MERCURIC CHLORIDE

a. Zenker’s Fluid b. B5 c. Heidenhein’s Susa d. Zenker Formol/ Helly’s Fluid

MERCURIC CHLORIDE fixative that is for Liver, spleen, connective tissue fibers & nuclei

Zenker’s Fluid

MERCURIC CHLORIDE fixative that is for bone marrow

MERCURIC CHLORIDE fixative that is for Tumor skin biopsies and an excellent cytologic fixative

Heidenhein’s Susa

MERCURIC CHLORIDE fixative that is for Pituitary gland, bone marrow, spleen, and liver

Zenker Formol/ Helly’s Fluid

DISADVANTAGE of MERCURIC CHLORIDE:

They produce black mercury deposits

REMEDY for MERCURIC CHLORIDE:

Alcohol Iodine is used to wash out excess mercuric fixatives

It is proposed as mercuric chloride substitute as it is toxic to man

ZINC SULFATE

METALLIC FIXATIVE that is for acid mucopolysaccharide

LEAD FIXATIVE

METALLIC FIXATIVE that have potassium dichromate

CHROMATE FIXATIVES

Types of CHROMATE FIXATIVES

a. 1-2% Chromic Acid b. 3% Potassium Dichromate c. Regaud’s/ Moller’s d. Orth’s Fluid

CHROMATE FIXATIVE that is for carbohydrates

1-2% Chromic Acid

CHROMATE FIXATIVE that is for lipids & mitochondria

3% Potassium Dichromate

CHROMATE FIXATIVE that is for mitochondria, RBC & colloid containing tissues

Regaud’s/ Moller’s

CHROMATE FIXATIVE that is for Rickettsiae & other bacteria, tissue necrosis

Orth's Fluid

Type of Fixative According to Active Component Substance (as the main ingredient) that is an excellent for glycogen demonstration

PICRIC ACID FIXATIVES

This is used as fixative, decalcifying agent, and stain

PICRIC ACID FIXATIVES

Types of PICRIC ACID FIXATIVES

a. Bouin’s Solution b. Brasil’s Alcoholic Picformol c. Hollande’s Solution

Type of Picric Acid Fixative that is for embryos, pituitary biopsies & endometrial curetting

Bouin’s Solution

T/F Bouin’s Solution is for KIDNEY STRUCTURES

FALSE; IT IS NOT

T/F Bouin’s Solution is compatible with feulgen's stain

FALSE; IT IS NOT

This is used to wash out excess amount of picric acid fixatives

50-70% Alcohol

This acts as both fixative and dehydrating agent

ALCOHOL

Types of ALCOHOL FIXATIVES

a. 95% Isopropyl Alcohol/ IPA b. Methyl Alcohol (100%) c. Ethyl Alcohol (70-100%) d. Carnoy’s e. Newcomer’s

Type of ALCOHOL FIXATIVE that is for touch preparations

95% Isopropyl Alcohol/ IPA

Type of ALCOHOL FIXATIVE that is the MOST RAPID tissue fixative

CARNOY'S

Type of ALCOHOL FIXATIVE that is for fixing chromosomes & lymph glands

CARNOY'S

Type of ALCOHOL FIXATIVE that is for mucopolysaccharides (histochemical) & nuclear protein

NEWCOMER'S

Type of Fixative According to Active Component Substance (as the main ingredient) that solidifies at 17 degC

GLACIAL ACETIC ACID

It is Contraindicated for cytoplasmic fixation as it is a fixative for nucleus

GLACIAL ACETIC ACID

This is used in combination with other fixatives to produce a compound fixative (but can be used alone)

GLACIAL ACETIC ACID

Type of Fixative According to Active Component Substance (as the main ingredient) that is used both fixative & dehydrating agent

ACETONE

This must be used at ICE COLD TEMPERATURE between -5 to 4 degC

ACETONE

This is for phosphatases & lipases enzymes

ACETONE

DISADVANTAGE of ACETONE

Evaporates easily & dissolves fats

Type of Fixative According to Active Component Substance (as the main ingredient) that can be used for myelin & peripheral nerves

OSMIUM TETROXIDE

Type of Fixative According to Active Component Substance (as the main ingredient) that is a fixative & a weak and slow decalcifying agent

TRICHLOROACETIC ACID

Fixatives for brain tissues for the diagnosis of rabies

1. Carnoy’s Fluid – MOST RAPID FIXATIVE 2. Acetone

Fixatives for Enzyme Histochemistry

1. 4% Formaldehyde 2. Formol Saline

Fixatives for Electron Microscopy

1. Osmium Tetroxide 2. Glutaraldehyde 3. Paraformaldehyde

Fixatives for Enzyme Histochemistry & Electron Immunocytochemistry

1. Karnovsky’s 2. Glutaraldehyde 3. Paraformaldehyde

Fixatives often used in conjunction with other fixatives to produce a compound fixative

1. Glacial acetic acid 2. Trichloroacetic acid/ TCA

HISTO RA Flashcards

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