L2.2 HISTOPATH & CYTO LAB - Histopathologic Techniques

Lead Fixatives

↳ Are used in 4% aqueous solution of basic lead acetate.

.

↳ Recommended for acid mucopolysaccharides.

.

↳ Disadvantage = takes up carbon dioxide forming insoluble lead carbonate (blackish deposit) which can be removed via filtration or acetic acid.

4

acetate

polysaccharides

carbon

filtration

LEAD FIXATIVES:

.

↳ Are used in ___% aqueous solution of basic lead __________.

.

↳ Recommended for acid muco___________________.

.

↳ Disadvantage = takes up __________ dioxide forming insoluble lead carbonate (blackish deposit) which can be removed via _____________ or acetic acid.

Picric Acid Fixatives

Glacial Acetic Acid Fixatives

Alcoholic Fixative

Osmium Tetroxide (Osmic Acid)

4 Terms under Lead Fixatives

(PAF, GAAF, AF, OT)

Picric Acid Fixatives

TERM UNDER LEAD FIXATIVE:

.

↳ 1% aqueous solution.

.

↳ Produces yellow discoloration of tissue. Can be removed by using acid dye, lithium carbonate or 70% ethanol with 5% sodium thiosulfate.

.

↳ Excellent for glycogen demonstration.

1

yellow

lithium

thio

glycogen

PICRIC ACID FIXATIVES:

.

↳ __% aqueous solution.

.

↳ Produces __________ discoloration of tissue. Can be removed by using acid dye, ___________ carbonate or 70% ethanol with 5% sodium _____sulfate.

.

↳ Excellent for _____________ demonstration.

Bouin's Solution

Brasil's Alcoholic Picroformal Fixative

2 Types of Picric Acid Fixatives

(BS, BAPF)

Bouin's Solution

TYPES OF PICRIC ACID FIXATIVES:

.

➙ Recommended for embryos and pituitary biopsies.

.

➙ Preferred fixative for Masson's trichrome.

pituitary

Masson

BOUIN'S SOLUTION:

.

➙ Recommended for embryos and _____________ biopsies.

.

➙ Preferred fixative for _________’s trichrome.

Brasil's Alcoholic Picroformal Fixative

TYPES OF PICRIC ACID FIXATIVES:

.

➙ Contains Formaldehyde 37%, picric acid, ethanol or isopropyl and TCA (Trichloroacetic acid).

.

➙ Better and less messy than Bouins.

37

isopropyl

messy

BRASIL'S ALCOHOLIC PICROFORMAL FIXATIVE:

.

➙ Contains Formaldehyde ____%, picric acid, ethanol or _____________ and TCA (Trichloroacetic acid).

.

➙ Better and less __________ than Bouins.

Glacial Acetic Acid Fixatives

TERM UNDER LEAD FIXATIVE:

.

↳ Is a compound fixative use in conjunction with other fixative.

.

↳ Precipitates chromosomes, chromatic material.

.

↳ Constituent of most common compound nuclear fixative.

conjunction

chromosomes

nuclear

GLACIAL ACETIC ACID FIXATIVES:

.

↳ Is a compound fixative use in ________________ with other fixative.

.

↳ Precipitates __________________, chromatic material.

.

↳ Constituent of most common compound ___________ fixative.

Alcoholic Fixative

TERM UNDER LEAD FIXATIVE:

.

↳ Rapidly denatures and precipitates proteins.

.

↳ Used in concentration 70-100%.

.

↳ Excellent for glycogen preservation.

.

↳ Used doing dehydration. Can both fix and dehydrate tissue.

proteins

100

preservation

dehydration

ALCOHOLIC FIXATIVE:

.

↳ Rapidly denatures and precipitates __________.

.

↳ Used in concentration 70-______%.

.

↳ Excellent for glycogen _________________.

.

↳ Used doing _______________. Can both fix and dehydrate tissue.

Methyl Alcohol 100%

Isopropyl Alcohol 95%

Ethyl Alcohol

3 Types of Alcohol Fixative

(MA, IA, EA)

Methyl Alcohol 100%

TYPES OF ALCOHOL FIXATIVE:

.

↳ Excellent for dry and wet smears, bone marrow tissues.

.

↳ Toxic agent.

bone

Toxic

METHYL ALCOHOL 100%:

.

↳ Excellent for dry and wet smears, ________ marrow tissues.

.

↳ _________ agent.

Isopropyl Alcohol 95%

TYPES OF ALCOHOL FIXATIVE:

.

↳ For touch preparations.

touch

ISOPROPYL ALCOHOL 95%:

.

↳ For _________ preparations.

Ethyl Alcohol

TYPES OF ALCOHOL FIXATIVE:

.

↳ Used in conc. 70-100%.

.

↳ Fixes blood, tissue film, smears.

.

↳ Does not fix glycogen.

100

tissue

glycogen

ETHYL ALCOHOL:

.

↳ Used in conc. 70-______%.

.

↳ Fixes blood, _________ film, smears.

.

↳ Does not fix _____________.

Osmium Tetroxide (Osmic Acid)

TERM UNDER LEAD FIXATIVE:

.

↳ Dissolves in water (up to 6% at 20 degrees celsius).

.

↳ Is a pale-yellow powder that preserves cytoplasmic and golgi bodies.

.

↳ Fixes myelin and peripheral nerves.

water

20

yellow

golgi

peripheral

OSMIUM TETROXIDE (OSMIC ACID):

.

↳ Dissolves in ________ (up to 6% at ___ degrees celsius).

.

↳ Is a pale-___________ powder that preserves cytoplasmic and _________ bodies.

.

↳ Fixes myelin and _______________ nerves.

Fleming's Solution

Fleming's Solution without Acetic Acid

2 Types of Osmium Tetroxide (Osmic Acid)

(FS, FSWAA)

Fleming's Solution

TYPES OF OSMIUM TETROXIDE (OSMIC ACID):

.

↳ Most common chrome-osmium acetic acid fixative.

.

↳ Recommended for nuclear preparation.

.

↳ Detriments cytoplasm.

common

nuclear

cytoplasm

FLEMING'S SOLUTION:

.

↳ Most __________ chrome-osmium acetic acid fixative.

.

↳ Recommended for ___________ preparation.

.

↳ Detriments _______________.

Fleming's Solution without Acetic Acid

TYPES OF OSMIUM TETROXIDE (OSMIC ACID):

.

↳ Contains only chromic and osmic acid.

.

↳ Recommended for cytoplasmic structure.

chromic

cytoplasmic

FLEMING'S SOLUTION WITHOUT ACETIC ACID:

.

↳ Contains only __________ and osmic acid.

.

↳ Recommended for _________________ structure.

Carnoy's Fluid

↳ Rapidly fix biopsies.

.

↳ It can both fix and dehydrate.

.

↳ Preserves Nissi granules and cytoplasmic granules.

.

↳ Used to fix brains for rabies diagnosis.

biopsies

fix

Nissi

rabies

CARNOY'S FLUID:

.

↳ Rapidly fix ___________.

.

↳ It can both ____ and dehydrate.

.

↳ Preserves ________ granules and cytoplasmic granules.

.

↳ Used to fix brains for __________ diagnosis.

Newcomer's Fluid

↳ Recommended for fixing mucopolysaccharides and nuclear proteins.

.

↳ Acts as a nuclear and histochemical fixative.

muco

nuclear

histochemical

NEWCOMER'S FLUID:

.

↳ Recommended for fixing ______polysaccharides and ____________ proteins.

.

↳ Acts as a nuclear and ________________ fixative.

Trichloro Acetic Acid

↳ Sometimes incorporated into compound fixatives.

.

↳ May be used as weak decalcifying agent.

compound

weak

TRICHLORO ACETIC ACID:

.

↳ Sometimes incorporated into ____________ fixatives.

.

↳ May be used as ________ decalcifying agent.

Acetone

↳ Used at ice cold temperature ranging -5 to 4 degrees Celsius.

.

↳ Recommended for water diffusible enzymes (phosphatase and lipase).

.

↳ Used to fix brains for rabies diagnosis.

cold

-5

water

lipase

brains

ACETONE:

.

↳ Used at ice _______ temperature ranging ____ to 4 degrees Celsius.

.

↳ Recommended for ________ diffusible enzymes (phosphatase and __________).

.

↳ Used to fix _________ for rabies diagnosis.

Heat Fixation

↳ Involves thermal coagulation of tissue proteins.

.

↳ Preserves nuclear and cytoplasmic detail.

.

↳ It dissolves starch and glycogen.

.

↳ Employed for frozen section and bacteriologic smears.

coagulation

nuclear

starch

bacteriologic

HEAT FIXATION:

.

↳ Involves thermal _______________ of tissue proteins.

.

↳ Preserves __________ and cytoplasmic detail.

.

↳ It dissolves _________ and glycogen.

.

↳ Employed for frozen section and ____________________ smears.

Secondary Fixation

↳ Process of placing an already fixed tissue in a second fixative.

.

↳ Purpose:

.

…..➙ To improve demonstration of particular substances.

.

…..➙ For further hardening and preservation of tissue.

.

…..➙ For special staining techniques.

.

……..- Mordant = substance that enhances the affinity between the stain and tissue.

second

substances

preservation

staining

affinity

SECONDARY FIXATION:

.

↳ Process of placing an already fixed tissue in a ___________ fixative.

.

↳ Purpose:

.

…..➙ To improve demonstration of particular _____________.

.

…..➙ For further hardening and ________________ of tissue.

.

…..➙ For special ____________ techniques.

.

……..- Mordant = substance that enhances the ___________ between the stain and tissue.

Washing Out

↳ Removal of excess fixative.

.

…..➙ Tap water = remove excess chromates.

.

…..➙ 50-70% alcohol = removes excess picric acid.

.

…..➙ Alcoholic iodine = remove excess mercuric fixative.

.

↳ Preparation for the next step, dehydration. Not all fixative should undergo this right after use, some go straight to dehydrations.

fixative

chromates

70

picric

iodine

mercuric

dehydration

WASHING OUT:

.

↳ Removal of excess __________.

.

…..➙ Tap water = remove excess _____________.

.

…..➙ 50-____% alcohol = removes excess _________ acid.

.

…..➙ Alcoholic ___________ = remove excess ______________ fixative.

.

↳ Preparation for the next step, _________________. Not all fixative should undergo this right after use, some go straight to dehydrations.

Microwave Technique

↳ Works as a physical agent.

.

↳ Frequency = 2,450 MHz.

.

↳ Accelerates:

.

…..➙ Fixation

.

…..➙ Staining

.

…..➙ Decalcification

.

…..➙ Immunohistochemistry

.

…..➙ Electron microscopy

physical

2,450

Staining

Immuno

MICROWAVE TECHNIQUE:

.

↳ Works as a ____________ agent.

.

↳ Frequency = __________ MHz.

.

↳ Accelerates:

.

…..➙ Fixation

.

…..➙ ____________

.

…..➙ Decalcification

.

…..➙ ___________histochemistry

.

…..➙ Electron microscopy

Retarded by:

FACTORS THAT AFFECT FIXATION:

.

↳ Size and thickness of the tissue specimen. (The thicker the specimen, the longer the fixation time).

.

↳ Presence of mucus, fat and blood affects penetration of fixative to the tissue which can interfere the fixation process. (Mucus and blood can be removed by Normal Saline Solution (NSS) and fat can be just cut thinner or fixed longer).

.

↳ Cold temperature - slow down decomposition of tissues by inactivating enzyme activities.

thickness

longer

penetration

Saline

Cold

enzyme

RETARDED BY:

.

↳ Size and _________________ of the tissue specimen. (The thicker the specimen, the _________ the fixation time).

.

↳ Presence of mucus, fat and blood affects ________________ of fixative to the tissue which can interfere the fixation process. (Mucus and blood can be removed by Normal _________ Solution (NSS) and fat can be just cut thinner or fixed longer).

.

↳ ________ temperature - slow down decomposition of tissues by inactivating _________ activities.

Enhanced by:

FACTORS THAT AFFECT FIXATION:

.

↳ Size and thickness of tissues.

.

↳ Agitation

.

↳ Moderate heat at 37-56 degree Celsius but hasten autolytic changes and enzyme destruction.

Size

Agitation

56

autolytic

destruction

ENHANCED BY:

.

↳ _______ and thickness of tissues.

.

↳ ______________

.

↳ Moderate heat at 37-____ degree Celsius but hasten __________ changes and enzyme ____________.

Decalcification

UNDER TISSUE PROCESSING:

.

↳ Procedure whereby calcium or lime salts are removed from tissues.

.

↳ Done after fixation.

.

↳ Not part of routine tissue processing since not all specimens are needed to be decalcified.

.

↳ Some specimens:

.

…..➙ Bone

.

…..➙ Teeth

.

…..➙ Tuberculous lungs

.

…..➙ Arteriosclerosis vessels

.

↳ Agents:

.

…..➙ Acids

.

…..➙ Chelating agents

.

…..➙ Ion exchange resin

.

…..➙ Electrical ionization

lime

fixation

routine

lungs

Arteriosclerosis

ionization

DECALCIFICATION:

.

↳ Procedure whereby calcium or ________ salts are removed from tissues.

.

↳ Done after _____________.

.

↳ Not part of ___________ tissue processing since not all specimens are needed to be decalcified.

.

↳ Some specimens:

.

…..➙ Bone

.

…..➙ Teeth

.

…..➙ Tuberculous _________

.

…..➙ __________________ vessels

.

↳ Agents:

.

…..➙ Acids

.

…..➙ Chelating agents

.

…..➙ Ion exchange resin

.

…..➙ Electrical ________________

Acid Decalcifying Agent

↳ Most widely used agents for routine decalcification of large amounts of bony tissue.

.

↳ Most common = acid.

routine

bony

acid

ACID DECALCIFYING AGENT:

.

↳ Most widely used agents for ___________ decalcification of large amounts of _________ tissue.

.

↳ Most common = ________.

Nitric Acid

Hydrochloric Acid

Formic Acid

Formic Acid-Sodium Citrate Solution

Trichloricacetic Acid

Sulfurous Acid

Chromic Acid (Flemming's Fluid)

6 Acid Decalcifying Agents

(NA, HA, FA, FASCS, TA, SA, CA)

Nitric Acid

TYPE OF ACID DECALCIFYING AGENTS:

.

↳ Most common and fastest decalcifying agent.

.

↳ Recommended concentration 5-10%.

fastest

10

NITRIC ACID:

.

↳ Most common and ___________ decalcifying agent.

.

↳ Recommended concentration 5-___%.

Aqueous Nitric Acid Solution

Formol-Nitric Acid

Perenyi's Fluid

Phloroglucin-Nitric Acid

4 Types of Nitric Acid

(ANAS, FNA, PF, PNA)

Aqueous Nitric Acid Solution

TYPES OF NITRIC ACID:

.

↳ Decalcification time: 12-24 hours.

24

AQUEOUS NITRIC ACID SOLUTION:

.

↳ Decalcification time: 12-___ hours.

Formol-Nitric Acid

TYPES OF NITRIC ACID:

.

↳ Decalcification time: 1-3 days.

.

↳ Produces yellow discoloration. Can be removed by 0.1% or pure concentrated nitric acid.

3

yellow

0.1

nitric

FORMOL-NITRIC ACID:

.

↳ Decalcification time: 1-__ days.

.

↳ Produces _________ discoloration. Can be removed by ____% or pure concentrated _________ acid.

Perenyi's Fluid

TYPES OF NITRIC ACID:

.

↳ Decalcification time: 2-7 days.

.

↳ Both a decalcifying agent and a tissue softener.

7

softener

PERENYI'S FLUID:

.

↳ Decalcification time: 2-__ days.

.

↳ Both a decalcifying agent and a tissue _____________.

Phloroglucin-Nitric Acid

TYPES OF NITRIC ACID:

.

↳ Decalcification time: 12-24 hours.

.

↳ Fastest decalcifying agent.

12

Fastest

PHLOROGLUCIN-NITRIC ACID:

.

↳ Decalcification time: ___-24 hours.

.

↳ ___________ decalcifying agent.

Hydrochloric Acid

TYPE OF ACID DECALCIFYING AGENTS:

.

↳ Inferior compare to nitric acid in its role as decalcifying agent because of slower action and greater distortion of tissue.

.

↳ Good for nuclear stain (1% HCL with 70% alcohol).

.

↳ 1% = good for surface decalcification of tissue block.

slower

distortion

nuclear

70

surface

HYDROCHLORIC ACID:

.

↳ Inferior compare to nitric acid in its role as decalcifying agent because of __________ action and greater ______________ of tissue.

.

↳ Good for __________ stain (1% HCL with ___% alcohol).

.

↳ 1% = good for __________ decalcification of tissue block.

Formic Acid

TYPE OF ACID DECALCIFYING AGENTS:

.

↳ Moderate-acting decalcifying agent which procedures better nuclear staining.

.

↳ Recommended for routine decalcification of post-mortem research tissues.

.

↳ Formula:

.

…..➙ Acid: 10 mL

.

…..➙ Saline: 90 mL

.

↳ Decalcification time: 2-7 days.

Moderate

staining

research

90

2

FORMIC ACID:

.

↳ _____________-acting decalcifying agent which procedures better nuclear ____________.

.

↳ Recommended for routine decalcification of post-mortem ____________ tissues.

.

↳ Formula:

.

…..➙ Acid: 10 mL

.

…..➙ Saline: __ mL

.

↳ Decalcification time: __-7 days.

Formic Acid-Sodium Citrate Solution

UNDER FORMIC ACID:

.

↳ Formula:

.

…..➙ Aqueous sodium citrate 20% = 50 mL.

.

…..➙ Formic acid 45% = 50 mL

.

…..➙ Decalcification time = 3-14 days.

citrate

50

45

14

FORMIC ACID-SODIUM CITRATE SOLUTION:

.

↳ Formula:

.

…..➙ Aqueous sodium ________ 20%: ____ mL.

.

…..➙ Formic acid ____%: 50 mL

.

…..➙ Decalcification time: 3-___ days.

Trichloroacetic Acid

TYPE OF ACID DECALCIFYING AGENTS:

.

↳ Formula:

.

…..➙ 5 gm

.

…..➙ Formal saline 10% = 95 mL

.

↳ Decalcification time: 4-8 days.

5

10

95

8

TRICHLORICACETIC ACID:

.

↳ Formula:

.

…..➙ Trichloroacetic acid: ___ gm

.

…..➙ Formal saline __%: __ mL

.

↳ Decalcification time: 4-___ days.

Sulfurous Acid

TYPE OF ACID DECALCIFYING AGENTS:

.

↳ A very weak decalcifying solution suitable only for minute pieces of bone.

minute

SULFUROUS ACID:

.

↳ A very weak decalcifying solution suitable only for __________ pieces of bone.

Chromic Acid (Flemming's Fluid)

TYPE OF ACID DECALCIFYING AGENTS:

.

↳ May be used as a fixative and decalcifying agent.

.

↳ Carcinogenic and highly corrosive.

fixative

Carcinogenic

CHROMIC ACID (FLEMMING'S FLUID):

.

↳ May be used as a ___________ and decalcifying agent.

.

↳ __________________ and highly corrosive.

EDTA

CHELATING AGENT:

.

↳ Most common chelating agent.

.

↳ It is a very slow decalcifying agent.

.

↳ 1-3 weeks for small specimens.

.

↳ 6-8 weeks or longer for dense bones.

common

slow

3

6

dense

EDTA:

.

↳ Most _____________ chelating agent.

.

↳ It is a very ________ decalcifying agent.

.

↳ 1-__ weeks for small specimens.

.

↳ __-8 weeks or longer for _________ bones.

Ion Exchange Resin

↳ Hastens decalcification by removing calcium ions from formic acid-conditioning decalcifying solutions.

.

↳ Not recommended for fluids containing mineral acids (nitric acid and HCL).

calcium

formic

mineral

ION EXCHANGE RESIN:

.

↳ Hastens decalcification by removing _________ ions from ________ acid-conditioning decalcifying solutions.

.

↳ Not recommended for fluids containing __________ acids (nitric acid and HCL).

Electrophoresis (Electrical Ionization)

↳ Is a process whereby positively charged calcium are attracted to a negative electrode and subsequently removed from the decalcifying agent.

.

↳ Example = a small bone that has calcium is positively charged. With this process, the bone will move toward the negative electrode, removing its calcium, resulting in the bone's softening.

negative

calcium

softening

ELECTROPHORESIS (ELECTRICAL IONIZATION):

.

↳ Is a process whereby positively charged calcium are attracted to a _____________ electrode and subsequently removed from the decalcifying agent.

.

↳ Example = a small bone that has ____________ is positively charged. With this process, the bone will move toward the negative electrode, removing its calcium, resulting in the bone's ________________.

Factors Influencing Rate of Decalcification

↳ Ratio of fluid tissue to is 20:1 (recommended).

.

↳ Heat will hasten the decalcification.

.

↳ Agitation.

20:1

Heat

Agitation

FACTORS INFLUENCING RATE OF DECALCIFICATION:

.

↳ Ratio of fluid tissue to is _____ (recommended).

.

↳ _______ will hasten the decalcification.

.

↳ ______________.

Physical or Mechanical

X-ray or Radiological Method

Chemical Method (Calcium Oxalate Test)

3 Measuring Extent of Decalcification

(P or M, X or RM, CM)

Physical or Mechanical

MEASURING EXTENT OF DECALCIFICATION:

.

↳ Done by touching or bending the tissue with the fingers. Can be subjective.

.

↳ Alternative method is pricking the tissue with needle. Too much puncture though, can be mistaken as a microfracture by the pathologist.

bending

subjective

needle

microfracture

PHYSICAL OR MECHANICAL:

.

↳ Done by touching or ____________ the tissue with the fingers. Can be ______________.

.

↳ Alternative method is pricking the tissue with ___________. Too much puncture though, can be mistaken as a _________________ by the pathologist.

X-ray or Radiological Method

MEASURING EXTENT OF DECALCIFICATION:

.

↳ Very expensive although most ideal, most sensitive and most reliable method.

.

↳ Tissue is placed in a waterproof polyethylene sheet then is placed on top of an X-ray field.

.

↳ Exposes tissue to radiation for 1 min under 30 kilovolts.

.

↳ Can show degree of decalcification:

.

…..➙ Opaque/gray = calcified.

.

…..➙ Clear/black = decalcified.

sensitive

polyethylene

30

Opaque

black

X-RAY OR RADIOLOGICAL METHOD:

.

↳ Very expensive although most ideal, most _____________ and most reliable method.

.

↳ Tissue is placed in a waterproof __________________ sheet then is placed on top of an xray field.

.

↳ Exposes tissue to radiation for 1 min under ___ kilovolts.

.

↳ Can show degree of decalcification:

.

…..➙ __________/gray = calcified.

.

…..➙ Clear/_________ = decalcified.

Chemical Method (Calcium Oxalate Test)

MEASURING EXTENT OF DECALCIFICATION:

.

↳ Recommended for routine purposes, to detect the presence of calcium in the decalcifying agent.

.

↳ Solutions:

.

…..➙ Concentrated ammonia (added drop by drop).

.

…..➙ 0.5 mL saturated aqueous ammonium oxalate (allow to stand for 30 minutes).

routine

calcium

ammonia

0.5

oxalate

30

CHEMICAL METHOD (CALCIUM OXALATE TEST):

.

↳ Recommended for ___________ purposes, to detect the presence of __________ in the decalcifying agent.

.

↳ Solutions:

.

…..➙ Concentrated ____________ (added drop by drop).

.

…..➙ ____ mL saturated aqueous ammonium __________ (allow to stand for ____ minutes).

Post Decalcification

↳ Saturated lithium carbonate or 5-10% aqueous sodium.

.

↳ Common = running water (30 minutes for small sample), (1-4 hours large samples) for several hours.

.

↳ Is similar to washing out. Removes decalcifying agent to specimen.

lithium

sodium

30

4

decalcifying

POST DECALCIFICATION:

.

↳ Saturated __________ carbonate or 5-10% aqueous _________.

.

↳ Common = running water (_____ minutes for small sample), (1-__ hours large samples) for several hours.

.

↳ Is similar to washing out. Removes ________________ agent to specimen.