Histotechnology Midterm 1

What chemical commonly used in histology is a known sensitizer ?

Formalin

Fixative should be __ times the volume of the tissue

15-20

What should be done when a fresh specimen has to wait for processing?

Place saline dampened gauze on it and refrigerate 4C

What do we include in gross descriptions?

measurements (l w d)

how many pieces there is

weight

colour

consistency

shape/texture

A dusky colour in tissue indicates

necrosis

How thick should tissue pieces be for when they are placed in a cassette?

3 mm

How do we prevent fresh tissues from decomposing until they get processed?

store in 4 degrees celcius

When placing a small specimen in a cassette, what is used to prevent pieces from falling through the gaps?

sponge

What is autolysis and putrefaction?

Autolysis- breakdown from proteolytic enzymes released by lysozymes

- Causes loss of nuclear detail, weak or no staining, epithelium detached from bassement membrane

Putrefaction- Bacterial overgrowth

- causes air bubbles, fixation artifacts and smells bad

What is the most noticeable microscopic sign that a sample is putrefied?

air bubbles caused by microbial metabolism

What is the difference between a coagulant and a non-coagulant fixative?

Non coagulant fixatives cross link proteins

transform cytoplasm into gel

-formaldehyde, formalin, gluteraldehyde, osmium tetroxide, acetic acid

coagulant fixatives precipitates/denatures/coagulates protein

removes free water in cells

creates protein meshwork

-Ethanol, picric acid, mercuric chloride

Potassium dichromate is non coagulant at pH >3.4-3.8 and coagulant at pH <3.4-3.8

What is the difference between an additive and a non-additive fixative?

additives crosslink with proteins in the cell

non-additives dissociate bound water molecules(alcohols)

What is the difference between tolerant and a non-tolerant fixative?

tolerant fixatives can be used to store tissues

-formaldehyde, formalin, glutaraldehyde, potassium dichromate

non-tolerant fixatives make tissues hard and brittle and may destroy tissues if they are stored in it for too long

-osmium tetroxide, ethanol, picric acid, mercuric chloride, acetic acid

Describe formaldehyde

Additive/Non-coagulant

cross links proteins and indirectly fixes glycogen

leaves behind formalin pigment or acid formaldehyde haematin

sensitizer, carcinogenic

describe 10% neutral buffered formalin

Additive/noncoagulant/tolerant

composed of 4% formaldehyde, sodium phosphate monobasic and sodium phosphate dibasic as buffers

Describe formol

10% alcoholic formalin

fixates faster, preserves glycogen better, dissolves fats and lipids, does not preserve hemosiderin

What are the two buffers used in formalin? Why do we need a buffer?

sodium phosphate monobasic

sodium phosphate dibasic

buffers prevent the formation of formalin hematin pigment that forms at pH under 6

describe gluteraldehyde

additive/non-coagulant/tolerant

cross links proteins, penetrates slowly

used for electron microscopy, often followed by osmium tetroxide.

stored at 4 degrees C

Describe osmium tetroxide

additive/non-coagulant/intolerant

secondary fixative for electron microscopy usually follows glutaraldehyde

can be used to stain lipids in frozen sections

Describe acetic acid

non coagulant/non-additive/intolerant

fixes nucleoproteins and swells tissues

lyses RBCs

Describe mercuric chloride

coagulant/additive/intolerant

powerful protein coagulant

precipitates all proteins and reacts with amino acids

remove mercuric pigment with iodine-sodium thiosulfate

do not dispose into sewage

ingredient of Zenker's, Helly's, B5

Describe picric acid

coagulant/additive/intolerant

does not fix carbs or lipids. preserves glycogen

good nucleoprotein coagulant

can be used as a fixative and a stain

picrates must be washed out with alcohol

explosive when crystallized

makes tissue soft and easier to penetrate

Describe potassium dichromate

additive

pH dependent: non coagulative and tolerant until pH is <3.4 then it is a coagulant

Preserves mitocondria and amines

extremely toxic vapours

must be thoroughly washed after fixation to remove chromate pigment

Describe acetone

coagulant/non-additive/intolerant

often used as a dehydrating agent

fixes rapidly but causes brittleness

Describe ethanol

coagulant/non-additive/intolerant

denatures proteins by replacing water and disrupting bonds

preserves glycogen and urate

dissolves fat

flammable

controlled by federal government so often is replaced by isopropyl alcohol which is not controlled

What is used to fix bone marrow smears?

methanol

Describe Zenker's

mercuric chloride, potassium dichromate, acetic acid

fixative used for bloody specimens and bone marrow biopsies good for trichrome stain

fixed tissue should be washed overnight to remove chromate pigment

lyses rbcs

Describe Helly's

Mercuric chloride, potassium dichromate, formalin

bone marrow fixative

wash to remove chromate

Describe B5

Mercuric chloride, formaldehyde

good fixative for bone marrow,lymph, spleen

preserves nuclear detail, stains well,

fixates in 4-8 hours

Describe Carnoy's

Chloroform, acetic acid, ethanol

no water

fixes in 1-3 hours - very rapid fixation

preserves nuclear detail and glycogen - used as cytological fixative

use only for small tissues

fixed tissue should be processed immediately or moved to 80% alcohol

Describe Bouin's

Picric acid, formaldehyde, acetic acid

good for GI biopsies and connective tissue.

fixed tissue should be transferred to 50-70% alcohol to prevent picrates

lyses RBCS, dissolves calcium and iron

should not be used for electron microcopy

What does a hypertonic fixative solution do? What does a hypotonic fixative solution do?

Hypertonic - causes shrinkage

hypotonic - causes swelling

What fixative has the worst fixation rate?

Glutaraldehyde

How are formalin pigments prevented?

by using sodium phosphate monobasic and dibasic buffers

How are mercuric pigments prevented?

Treat with iodine-sodium thiosulphate

How are chrome pigments removed?

Wash off well with water

Unfixed tissue that is to be held for several days or transported over a long distance should be placed in

Michel's Transport Medium

Why do we use ascending grades of alcohol for dehydration?

Minimizes shrinkage

Why do we need a clearing agent? What clearing agent do we use?

to remove alcohol from dehydration and replace it with a solvent for paraffin

xylene is the clearing agent we use

it is miscible with both alcohol and paraffin

Why shouldn't tissues be over exposed to xylene? What can be used instead of xylene?

can cause over-hardening

Toluene can be used instead without this effect

When embedding with wax, what temperature do we use it at?

2-3 degrees Celsius above the melting point of wax

we have ours set at 60 degrees

What is the melting point of paraffin?

55-58 degrees Celsius

Paraffin wax solidifies by _

crystallization

What benefit does toluene have over xylene as a clearing agent?

toluene does not overharden tissue

The addition of __ during processing can increase the rate of infiltration

vacuum

T/F: The carousel tissue processor is a closed system

False it is an open system

Fluid exchange processors are closed

Rapid processing is not recommended for what tissues?

fatty tissue and dense tissue

What does microwave processing do?

speeds up tissue processing from hours to minutes

What is the purge cycle for the automated processor?

Two step xylene and alcohol cycles to clear any wax build up

Paraffin temperatures must be recorded _

daily

What does overdehydration do?

shrinks and hardens

causes microchatter

What does underdehydration do?

Causes mushy, soft tissue

T/F: Potassium Dichromate is an effective fixative that is often used alone

False

potassium dichromate is never used alone, but it is used in many compound fixatives as an ingredient

What is the best fixative to preserve glycogen?

Ethanol

How are melanin pigments removed?

By placing in a solution of potassium permanganate for 20 minutes

What are the two fixatives used for electron microscopy?

Glutaraldehyde and osmium tetroxide

What are the 3 steps to tissue processing?

dehydration, clearing, wax infiltration

What can be used to substitute ethanol as a dehydrating agent?

Isopropyl alcohol

Cloudy toluene is indicative of

contamination with water and must be changed

What tissue processor is usually used by big hospital laboratories?

fluid exchange processor

What is used to embed hard or fragile tissues instead of paraffin?

Celloidin

When placing multiple pieces of tissues inside a single cassette, they should be oriented _

horizontally

How do we embed tissues that have a lumen?

Standing up on end

How should needle biopsies be arranged during embedding?

parallel on a diagonal

How should bone be embedded?

diagonally

A white, mushy tissue in a tissue block indicates

underdehydration

What is coarse trimming?

done to expose tissue surface at 10-30 um

What causes moth holes?

Aggressive trimming

What is fine trimming?

trimming done at 4-5um to obtain tissue sections that will be mounted onto a slide

What can be used prior to a water bath to help reduce folds in tissue sections?

20% alcohol

What is the temperature of the water bath used in microtomy?

8-10 degrees below the melting point of wax

What is the angle used in the cryotome?

45 degrees

What do we use to section fresh, unfixed tissues for rapid diagnosis?

Cryotome

What is the name of the part that holds the tissue in cryotomy?

A chuck

What is the embedding medium used for the cryotome?

OCT compound (optimal cutting temperature)

glycerol and resin compound

What is the anti roll plate?

Prevents individual cryotome sections from rolling and directs section onto the knife

T/F: The cryotome does not cut a ribbon, it cuts individual pieces

True

Tissues that contain more water will section best at _ temperatures

warmer

Hard tissue will section best at _ temperatures

lower

Chattering or shattering of sections indicates

temperature is too low

What is the Peltier effect?

heat is emitted or absorbed according to the direction of the current between two dissimilar metals

this is used by the heat extractor in the cryotome

What temperature is the cryotome kept at?

-23C

When does formalin pigment form?

When pH is below 6

What is used in compound fixatives to correct the shrinkage effect of other fixatives?

glacial acetic acid

What is a key property of a clearing agent?

Must be miscible with both paraffin and alcohol

we use xylene

toluene can be used to prevent over hardening of tissues

What are microtome blades made of?

Steel

When is decalcification done

following fixation

How does calcium appear on an H&E stained slide?

amorphous basophilic blue deposits

What are the 4 methods of decalcification?

acid, ion-exchange, electrolytic, chelating agents

Why shouldn't decalcification be done on tissues fixed in mercury-based fixatives?

will render calcium radio opaque - false positives in xrays

What do we use to cut bone specimens?

band saw

diamond blades help prevent bone dust

Describe acid decalcification

acid releases calcium from bone through ionic exchange. Calcium migrates to acid solution

done with formic acid, nitric acid, HCl,

fast but damaging must be closely monitored

Why should formalin be washed before being decalcified in HCL?

it would form bis-chloromethyl ether, a carcinogen

What acid decalcifiers are used for rapid diagnosis of small biopsies?

HCL and HNO3

What is the best all-around decalcifier?

10% formic acid

Describe the chelation method for decalcifying

organic compound like edta binds to calcium

pH dependent - 8 and above is best

extremely slow but gentle

Describe the ion-exchange method for decalcifying

Uses a mixture of formic acid and commercial ion exchange resin

calcium is removed by formic acid into the resin

Describe the electrolytic method for decalcifying

formic acid or HCl is used as electrolytic medium. calcium ions move to the cathode

heat generated by this process can damage tissues

How do we perform chemical end point of decalcification testing?

add amoniom hydroxide to decal fluid.

if it becomes cloudy it is grossly under decalcified

if clear, add ammonium oxalate and set aside for 30 minutes

precipitation = under decalcified

clear = good to go

HINT: h comes before o in the alphabet Hydroxide first, Oxalate after

Precipitation of the decal fluid after ammonium hydroxide indicates

grossly underdecalcified