L2.1 HISTOPATH & CYTO LAB - Histopathologic Techniques

Preparing Specimen for Examination

↳ Tissue or liquid specimen preparations for microscopic analysis.

liquid

microscopic

PREPARING SPECIMEN FOR EXAMINATION:

.

↳ Tissue or __________ specimen preparations for ___________________ analysis.

Fresh Tissue Examination

↳ The method of tissue examination may vary according to:

.

…..➙ Structural and chemical components of the cell.

.

…..➙ Nature and amount of tissue.

.

…..➙ The need for an immediate examination.

chemical

Nature

immediate

FRESH TISSUE EXAMINATION:

.

↳ The method of tissue examination may vary according to:

.

…..➙ Structural and ___________ components of the cell.

.

…..➙ _________ and amount of tissue.

.

…..➙ The need for an _____________ examination.

General Methods of Preparing Specimen for Examination

1. Fresh tissue specimen preparation.

.

…..➙ Teasing or Dissociation

.

…..➙ Squash or Crushing

.

…..➙ Smear Preparations

.

…..➙ Frozen section

.

2. Sectioning of wax embedded specimen.

.

3. Microincineration

.

4. Autoradiography

.

5. Ground sections

Dissociation

Squash

Smear

Frozen

embedded

Micro

radiography

Ground

GENERAL METHODS OF PREPARING SPECIMEN FOR EXAMINATION:

.

1. Fresh tissue specimen preparation.

.

…..➙ Teasing or _______________

.

…..➙ ___________ or Crushing

.

…..➙ ________ Preparations

.

…..➙ ___________ Section

.

2. Sectioning of wax _______________ specimen.

.

3. _________incineration

.

4. Auto_________________

.

5. ________ Section

Teasing or Dissociation

Squash Preparation

Smear Preparation

Frozen Section

4 Methods of Fresh Tissue Examination

(T or D, SP, SP, FS)

Teasing or Dissociation

METHODS OF FRESH TISSUE EXAMINATION:

.

➙ A tissue specimen is immersed in an isotonic salt solution and carefully pulled apart and dissected with needles.

isotonic

needles

TEASING OR DISSOCIATION:

.

➙ A tissue specimen is immersed in an ___________ salt solution and carefully pulled apart and dissected with __________.

Squash Preparation

METHODS OF FRESH TISSUE EXAMINATION:

.

➙ A tiny piece of fresh tissue is compressed between two glass slides to spread cells thin.

two

thin

SQUASH PREPARATION:

.

➙ A tiny piece of fresh tissue is compressed between _____ glass slides to spread cells _______.

Smear Preparation

METHODS OF FRESH TISSUE EXAMINATION:

.

➙ Is the process of examining sections or sediments.

sediments

SMEAR PREPARATION:

.

➙ Is the process of examining sections or _____________.

Frozen Section

METHODS OF FRESH TISSUE EXAMINATION:

.

➙ Use Cryostat (built in rotary microtome machine).

.

➙ 5-10 micra in thickness (fresh tissue).

.

➙ Optimum temp -18 to -20.

.

➙ Are transferred to a slide.

Cryostat

10

-18

slide

FROZEN SECTION:

.

➙ Use ___________ (built in rotary microtome machine).

.

➙ 5-___ micra in thickness (fresh tissue).

.

➙ Optimum temp ____ to -20.

.

➙ Frozen sections are transferred to a _______.

Liquid nitrogen

Isopentane cooled by liquid nitrogen.

Carbon dioxide gas

Aerosol sprays

4 Commonly Used Methods of Freezing

(LN, ICLN, CDG, AS)

Liquid nitrogen

COMMONLY USED METHODS OF FREEZING:

.

➙ Vapor phase - uneven cooling.

Vapor

cooling

LIQUID NITROGEN:

.

➙ _______ phase - uneven ____________.

Isopentane cooled by liquid nitrogen

COMMONLY USED METHODS OF FREEZING:

.

➙ Liquid nitrogen does not directly cool the specimen.

directly

ISOPENTANE COOLED BY LIQUID NITROGEN:

.

➙ Liquid nitrogen does not __________ cool the specimen.

Aerosol sprays

COMMONLY USED METHODS OF FREEZING:

.

➙ Uses fluorinated hydrocarbons.

fluorinated

AEROSOL SPRAYS:

.

➙ Uses ______________ hydrocarbons.

Fixation

TISSUE PROCESSING:

.

↳ Most critical step.

.

↳ Prevents:

.

…..➙ Degeneration

.

…..➙ Decomposition

.

…..➙ Putrefaction

.

…..➙ Distortion of tissues

critical

Degeneration

Putrefaction

tissues

FIXATION:

.

↳ Most _________ step.

.

↳ Fixation prevents:

.

…..➙ ________________

.

…..➙ Decomposition

.

…..➙ _______________

.

…..➙ Distortion of __________

Additive Fixation

Non-Additive Fixation

2 Basic Mechanisms in Fixation

Additive Fixation

BASIC MECHANISMS IN FIXATION:

.

➙ Chemical constituent of fixative becomes part of the tissue by forming cross-links.

Chemical

cross

ADDITIVE FIXATION:

.

➙ ___________ constituent of fixative becomes part of the tissue by forming _______-links.

Non-Additive Fixation

BASIC MECHANISMS IN FIXATION:

.

➙ Not incorporated but alters the composition of tissues by removing the bound water attached to H-bonds.

composition

bound

H

NON-ADDITIVE FIXATION:

.

➙ Not incorporated but alters the _________________ of tissues by removing the ________ water attached to ___-bonds.

Hydrogen Ion Concentration

Temperature

Thickness of Section

Osmolality

Concentration

Duration of Fixation

6 Main Factors Involved in Fixation

(HIC, T, T of S, O, C, D of F)

Hydrogen Ion Concentration

MAIN FACTORS INVOLVED IN FIXATION:

.

↳ pH 6 - 8

8

HYDROGEN ION CONCENTRATION:

.

↳ pH 6 - ___

Temperature

MAIN FACTORS INVOLVED IN FIXATION:

.

↳ Most room temperature for routine surgical specimens.

.

↳ 0-4 degrees Celsius (electron microscopy and histochemistry).

.

↳ 60 ℃ (rapid fixation).

.

↳ 100 ℃ (tissue w/ tuberculosis).

surgical

4

histochemistry

60

rapid

100

tuberculosis

TEMPERATURE:

.

↳ Most room temperature for routine ____________ specimens.

.

↳ 0-__ degrees Celsius (electron microscopy and _____________________).

.

↳ ____ ℃ (_______ fixation).

.

↳ ______ ℃ (tissue w/ ________________).

Thickness of Section

MAIN FACTORS INVOLVED IN FIXATION:

.

↳ 1 to 2 mm² (electron microscopy).

.

↳ 2 cm² and not more than 4mm thick (light microscopy).

2

electron

4

light

THICKNESS OF SECTION:

.

↳ 1 to __ mm² (__________ microscopy).

.

↳ 2 cm² and not more than __mm thick (________ microscopy).

Osmolality

MAIN FACTORS INVOLVED IN FIXATION:

.

↳ 400-450 mOsm.

450

OSMOLALITY:

.

↳ 400-_____ mOsm.

Concentration

MAIN FACTORS INVOLVED IN FIXATION:

.

↳ Formaldehyde (10%) = prevents precipitation of acids.

.

↳ Glutaraldehyde (3%).

.

↳ Glutaraldehyde (0.25%) = ideal for immunoelectron microscopy.

Formaldehyde

0.25

immunoelectron

CONCENTRATION:

.

↳ __________________ (10%) = prevents precipitation of acids.

.

↳ Glutaraldehyde (3%).

.

↳ Glutaraldehyde (_____%) = ideal for _________________ microscopy.

Duration of Fixation

MAIN FACTORS INVOLVED IN FIXATION:

.

↳ Primary fixation (2-6 hours) but can remain in fixative over the weekend.

.

↳ 3 hours for electron microscopy then placed in a holding buffer.

.

↳ Representative sample = 2 cm² not more than 4 mm thick.

.

↳ For fixation of tissue not exceeding 5mm, there is complete fixation after 6-12 hours. Usual fixation time is 24 hours.

.

↳ Over fixed specimen = cause hardening and shrinkage of tissues. Results will be affected as it inhibits immunological activity that can be restored by washing tissue with running water.

.

↳ Whole brain = should be fixed in a 10% neutral buffered formalin for 2 to 3 weeks.

6

weekend

3

holding

2

DURATION OF FIXATION:

.

↳ Primary fixation (2-__ hours) but can remain in fixative over the ____________.

.

↳ __ hours for electron microscopy then placed in a _____________ buffer.

.

↳ Representative sample = __ cm² not more than 4 mm thick

5

12

shrinkage

immunological

formalin

weeks

DURATION OF FIXATION:

.

↳ For fixation of tissue not exceeding __mm, there is complete fixation after 6-___ hours. Usual fixation time is 24 hours.

.

↳ Over fixed specimen = cause hardening and _____________ of tissues. Results will be affected as it inhibits ____________________ activity that can be restored by washing tissue with running water.

.

↳ Whole brain = should be fixed in a 10% neutral buffered ____________ for 2 to 3 _______.

Speed

Penetration

Volume

Duration of Fixation

4 Practical Considerations of Fixation

(S, P, V, D of F)

Speed

PRACTICAL CONSIDERATIONS OF FIXATION:

.

↳ Specimen should be fixed immediately. Unless the specimen is for frozen section.

immediately

frozen

SPEED:

.

↳ Specimen should be fixed ________________. Unless the specimen is for __________ section.

Penetration

PRACTICAL CONSIDERATIONS OF FIXATION:

.

↳ Formalin diffuses into the tissue at the rate of 1 mm per hour.

Formalin

1

hour

PENETRATION:

.

↳ __________ diffuses into the tissue at the rate of __ mm per ______.

Volume

PRACTICAL CONSIDERATIONS OF FIXATION:

.

↳ 10-25 times the tissue volume.

.

↳ Ratio = 20 fixative : 1 tissue.

25

20

VOLUME:

.

↳ 10-____ times the tissue volume.

.

↳ Ratio = ___ fixative : 1 tissue.

Duration of Fixation.

PRACTICAL CONSIDERATIONS OF FIXATION:

.

↳ Depends on their structure (fibrous organs such as uterus, intestinal tract takes longer to fix).

.

↳ Ways to cut down fixation time:

.

…..➙ Heat

.

…..➙ Vacuum

.

…..➙ Agitation

.

…..➙ Microwave technique

.

…..➙ Refrigeration technique (slow down decomposition of tissue especially those that will undergo photography).

Fibrous

Vacuum

Microwave

photography

DURATION OF FIXATION:

.

↳ Depends on their structure (__________ organs such as uterus, intestinal tract takes longer to fix).

.

↳ Ways to cut down fixation time:

.

…..➙ Heat

.

…..➙ ___________

.

…..➙ Agitation

.

…..➙ _____________ technique

.

…..➙ Refrigeration technique (Slow down decomposition of tissue especially those that will undergo ________________).

Simple Fixatives

Compound Fixatives

2 Types of Fixative (According to Composition)

(SF, CF)

Simple Fixatives

TYPES OF FIXATIVE (ACCORDING TO COMPOSITION):

.

↳ Composed of only one type of fixative.

one

SIMPLE FIXATIVE:

.

↳ Composed of only _____ type of fixative.

Compound Fixatives

TYPES OF FIXATIVE (ACCORDING TO COMPOSITION):

.

↳ Composed of two or more types of fixative.

.

…..➙ Some fixatives have a negative effect to tissues when used as is, but if it is mixed with another fixative, the negative effect is diminished.

more

negative

COMPOUND FIXATIVE:

.

↳ Composed of two or ______ types of fixative.

.

…..➙ Some fixatives have a _____________ effect to tissues when used as is, but if it is mixed with another fixative, the negative effect is diminished.

Microanatomical Fixative

Cytological Fixative

2 Types of Fixative (According to Action)

(MF, CF)

Microanatomical Fixative

TYPES OF FIXATIVE (ACCORDING TO ACTION):

.

↳ It permits microscopic study of tissue without altering the structural pattern and intracellular relationship of tissue.

structural

intracellular

MICROANATOMICAL FIXATIVE:

.

↳ It permits microscopic study of tissue without altering the ____________ pattern and ________________ relationship of tissue.

Cytological Fixative

TYPES OF FIXATIVE (ACCORDING TO ACTION):

.

↳ Preserves specific parts and elements of the cell itself. Specific, not whole.

.

…..➙ Nuclear fixative

.

…..➙ Cytoplasmic fixative

.

…..➙ Histochemical fixative

specific

Histochemical

CYTOLOGICAL FIXATIVE:

.

↳ Preserves ___________ parts and elements of the cell itself. Specific, not whole.

.

…..➙ Nuclear fixative

.

…..➙ Cytoplasmic fixative

.

…..➙ __________________ fixative

Aldehyde Fixatives

Term under Simple Fixative

(AF)

Formalin

10% Formol-Saline

10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH7)

Formal-Corrosive (Formal-Sublimate)

Gendre's Fixative

Glutaraldehyde

6 Types of Aldehyde Fixatives

(F, FS, NBF, FC, GF, G)

Formalin

TYPES OF ALDEHYDE FIXATIVES:

.

↳ A gas produced by the oxidation of methyl alcohol.

.

↳ 37 - 40% weight in volume.

.

↳ Commonly used as a 4% solution, giving 10% formalin for tissue fixation.

.

↳ Working solution for formaldehyde = 10% (routine tissue processing).

.

↳ Stock solution of formaldehyde = 37-40%. Tissue will harden if submerged with stock solution.

methyl

40

4

Working

Stock

harden

FORMALIN:

.

↳ A gas produced by the oxidation of _________ alcohol.

.

↳ 37 - ____% weight in volume.

.

↳ Commonly used as a __% solution, giving 10% formalin for tissue fixation.

.

↳ _____________ solution for formaldehyde = 10% (routine tissue processing).

.

↳ _________ solution of formaldehyde = 37-40%. Tissue will ___________ if submerged with stock solution.

10% Formol-Saline

TYPES OF ALDEHYDE FIXATIVES:

.

↳ Saturated formaldehyde (40% by weight volume diluted to 10% sodium chloride).

.

↳ For central nervous tissue and general post-mortem tissues.

Saturated

40

chloride

central

mortem

10% FORMOL-SALINE:

.

↳ ___________ formaldehyde (____% by weight volume diluted to 10% sodium _________).

.

↳ For _________ nervous tissue and general post-___________ tissues.

10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH7)

TYPES OF ALDEHYDE FIXATIVES:

.

↳ Prevents precipitation of acid formalin pigments.

.

↳ Preparation:

.

…..➙ Sodium dihydrogen phosphate (anhydrous) 3.5 gm

.

…..➙ Disodium hydrogen phosphate (anhydrous) 6.5 gm

.

…..➙ Formaldehyde 40% = 100 mL

.

…..➙ Distilled water = 900 mL

.

↳ Preparation (not buffered):

.

…..➙ Formaldehyde 40% = 100 mL

.

…..➙ Distilled water = 900 mL

formalin

dihydrogen

Disodium

6.5

10% NEUTRAL BUFFERED FORMALIN OR PHOSPHATE-BUFFERED FORMALIN (pH7):

.

↳ Prevents precipitation of acid ____________ pigments.

.

↳ Preparation:

.

…..➙ Sodium _______________ phosphate (anhydrous) 3.5 gm

.

…..➙ ____________ hydrogen phosphate (anhydrous) ____ gm

.

…..➙ Formaldehyde 40% = 100 mL

.

…..➙ Distilled water = 900 mL

40

900

10% NEUTRAL BUFFERED FORMALIN OR PHOSPHATE-BUFFERED FORMALIN (pH7):

.

↳ Preparation (not buffered):

.

…..➙ Formaldehyde ____% = 100 mL

.

…..➙ Distilled water = _______ mL

Formal-Corrosive (Formal-Sublimate)

TYPES OF ALDEHYDE FIXATIVES:

.

↳ Formal-Mercuric chloride solution for routine post-mortem.

.

↳ No need for washing out.

.

↳ Fixes lipids, neutral fats and phospholipids.

Mercuric

mortem

washing

neutral

FORMAL-CORROSIVE (FORMAL-SUBLIMATE):

.

↳ Formal-____________ chloride solution for routine post-_________.

.

↳ No need for _____________ out.

.

↳ Fixes lipids, __________ fats and phospholipids.

Gendre's Fixative

TYPES OF ALDEHYDE FIXATIVES:

.

↳ Alcohol formalin.

.

↳ Post fixation with phenol-formalin for 6 hours.

.

↳ Enhances immunoperoxidase studies.

.

↳ Advantages:

.

…..➙ Faster fixation time (reduces to one half).

.

…..➙ For rapid diagnosis (fix and dehydrates at the same time).

.

…..➙ Use to fix sputum.

Alcohol

phenol

6

peroxidase

half

dehydrates

sputum

GENDRE'S FIXATIVE:

.

↳ ___________ formalin.

.

↳ Post fixation with _________-formalin for __ hours.

.

↳ Enhances immuno_____________ studies.

.

↳ Advantages:

.

…..➙ Faster fixation time (reduces to one _______).

.

…..➙ For rapid diagnosis (fix and ______________ at the same time).

.

…..➙ Use to fix __________.

Glutaraldehyde

TYPES OF ALDEHYDE FIXATIVES:

.

↳ 2 formaldehyde residues linked by 3 carbon chain.

.

↳ 2.5% for small tissue fragments and needle biopsies fixed in 2-4 hours at room temperature.

.

↳ 4% for large tissues less than 4mm thick in 6-8 hours up to 24 hours.

.

↳ Especially used for central nervous system tissues.

carbon

2.5

needle

room

4

8

central

GLUTARALDEHYDE:

.

↳ 2 formaldehyde residues linked by 3 _________ chain.

.

↳ ___% for small tissue fragments and __________ biopsies fixed in 2-4 hours at _______ temperature.

.

↳ ___% for large tissues less than 4mm thick in 6-__ hours up to 24 hours.

.

↳ Especially used for __________ nervous system tissues.

Mercury Chloride

Chromate Fixative

2 Term under Metallic Fixative

(MC, CF)

Mercury Chloride

TERM UNDER METALLIC FIXATIVE:

.

↳ Most common metallic fixative.

.

↳ Solution of 5-7% included in compound fixatives.

.

↳ Produces black precipitates of mercury.

.

…..➙ Removed by 0.5% iodine in 70% ethanol for 5-10 mins.

.

↳ Trichrome staining is excellent.

.

…..➙ Use to stain collagen, elastic fiber and connected tissue.

.

↳ Fixative of choice for cell detail in tissue photography.

common

7

compound

black

ethanol

10

Trichrome

elastic

photography

MERCURY CHLORIDE:

.

↳ Most ____________ metallic fixative.

.

↳ Solution of 5-___% included in ______________ fixatives.

.

↳ Produces _________ precipitates of mercury.

.

…..➙ Removed by 0.5% iodine in 70% ___________ for 5-___ mins.

.

↳ _____________ staining is excellent.

.

…..➙ Use to stain collagen, __________ fiber and connected tissue.

.

↳ Fixative of choice for cell detail in tissue ________________.

Zenker's Fluid (Mercuric Chloride with Glacial Acetic Acid)

Zenker-Formal (Helly's Solution)

Heidenhain's Susa Solution

B-5 Fixative

4 Types of Mercury Chloride

(ZF, ZF, HSS, B5F)

Zenker's Fluid

TYPES OF MERCURY CHLORIDE:

.

↳ De-zenkerization = removes mercuric or black deposits.

.

↳ Use to fix liver, spleen, connective tissue fibers and nuclei.

.

↳ AKA = Mercuric Chloride with Glacial Acetic Acid

zenkerization

black

spleen

nuclei

Glacial

ZENKER'S FLUID (MERCURIC CHLORIDE WITH GLACIAL ACETIC ACID):

.

↳ De-_________________ = removes mercuric or ________ deposits.

.

↳ Use to fix liver, _________, connective tissue fibers and _________.

.

↳ AKA = Mercuric Chloride with ________ Acetic Acid

Zenker-Formal (Helly's Solution)

TYPES OF MERCURY CHLORIDE:

.

↳ Contains mercuric chloride, potassium dichromate and 40% formalin.

.

↳ Produces brown pigment (bloody organs). Can be removed by picric acid or sodium hydroxide.

potassium

40

brown

picric

hydroxide

ZENKER-FORMAL (HELLY'S SOLUTION):

.

↳ Contains mercuric chloride, _____________ dichromate and ____% formalin.

.

↳ Produces _________ pigment (bloody organs). Can be removed by ________ acid or sodium ____________.

Heidenhain's Susa Solution

TYPES OF MERCURY CHLORIDE:

.

↳ Recommended for tumor biopsies (skin).

.

↳ Tissues can be directly transferred to 95% or absolute alcohol.

.

↳ Fixes tissues rapidly. 3 - 12 hours fixation.

.

↳ Does not produce black deposit.

tumor

95

alcohol

12

produce

HEIDENHAIN'S SUSA SOLUTION:

.

↳ Recommended for _________ biopsies (skin).

.

↳ Tissues can be directly transferred to ___% or absolute ___________.

.

↳ Fixes tissues rapidly. 3 - ____ hours fixation.

.

↳ Does not ____________ black deposit.

B-5 Fixative

TYPES OF MERCURY CHLORIDE:

.

↳ Commonly used for bone marrow smears.

.

↳ Contains distilled water, mercuric chloride, sodium acetate.

.

↳ 30 minutes to 2 hours fixation.

.

↳ Good for cytology and bone marrow biopsy.

smears

acetate

30

cytology

B-5 FIXATIVE:

.

↳ Commonly used for bone marrow ___________.

.

↳ Contains distilled water, mercuric chloride, sodium _________.

.

↳ ____ minutes to 2 hours fixation.

.

↳ Good for ____________ and bone marrow biopsy.

Chromic Acid

Potassium Dichromate

Regaud's (Muller's) Fluid

Orth's Fluid

4 Types of Chromate Fixative

(CA, PD, RMF, OF)

Chromic Acid

TYPES OF CHROMATE FIXATIVE:

.

↳ 1-2% aqueous solution included in compound fixatives.

.

↳ Precipitates all proteins.

.

↳ Preserves all carbohydrates.

2

compound

proteins

Preserves

CHROMIC ACID:

.

↳ 1-__% aqueous solution included in _____________ fixatives.

.

↳ Precipitates all _________.

.

↳ _____________ all carbohydrates.

Potassium Dichromate

TYPES OF CHROMATE FIXATIVE:

.

↳ 3% aqueous solution.

.

↳ Preserves lipids and mitochondria given that the pH is 4.5 to 5.2.

3

mitochondria

5.2

POTASSIUM DICHROMATE:

.

↳ ___% aqueous solution.

.

↳ Preserves lipids and _______________ given that the pH is 4.5 to _____.

Regaud's (Muller's) Fluid

TYPES OF CHROMATE FIXATIVE:

.

↳ Contains 3% potassium dichromate and 40% formaldehyde.

.

↳ Recommended for chromatin, mitochondria.

.

↳ Hardens tissue better and more rapidly than Orth's.

.

↳ Contraindicated for carbohydrates since it is not good in penetrating glycogen.

3

formaldehyde

chromatin

rapidly

carbohydrates

REGAUD'S (MULLER'S) FLUID:

.

↳ Contains ___% potassium dichromate and 40% ______________.

.

↳ Recommended for ______________, mitochondria.

.

↳ Hardens tissue better and more __________ than Orth's.

.

↳ Contraindicated for ___________________ since it is not good in penetrating glycogen.

Orth's Fluid

TYPES OF CHROMATE FIXATIVE:

.

↳ Recommended for early degenerative processes and tissue necrosis.

.

↳ Demonstrates rickettsiae.

degenerative

necrosis

rickettsiae

ORTH'S FLUID:

.

↳ Recommended for early ________________ processes and tissue ___________.

.

↳ Demonstrates _______________.