Processing

Question 1

incomplete dehydration

Answer 1

accounts for the vast majority of processing problems and results in mushy tissue

Question 2

alcohols

Answer 2

the most common dehydrating reagents

Question 3

clearing agents

Answer 3

must be miscible with both the dehydration agent and the infiltration medium
primary purpose is to remove the alcohol used to dehydrate the tissue

Question 4

prolonged clearing

Answer 4

results in hard, brittle tissues

Question 5

xylene

Answer 5

most commonly used clearing agent, turns cloudy in the presence of water

Question 6

toluene

Answer 6

does not over harden tissue as much as xylene

Question 7

overdehydration

Answer 7

seen as microchatter in H&E stained slides, commonly seen in biopsy specimens, can be corrected by processing biopsies separately from other tissues and decreasing the amount of time in the dehydrating solutions

Question 8

poor processing

Answer 8

evidenced by poor nuclear staining, often caused by water remaining in the tissue when it is placed in the clearing agent which results in poor clearing and infiltration, can also be caused by too much heat during processing

Question 9

melting point

Answer 9

high melting point results in hard blocks that section thinly but ribbon poorly, while low melting point ribbons easily but doesn’t section as thin. 55-58C is common melting point

Question 10

diagonal embedding

Answer 10

makes sectioning much easier

Question 11

forceps metastasis

Answer 11

when fragments of tissue are transferred between blocks if the forceps aren’t wiped down between specimens

Question 12

paraffin crystal size

Answer 12

smaller is better, allows for better support of the tissue

Question 13

mushy in the middle

Answer 13

tissue was cut too thick during grossing and needs to be reprocessed

Question 14

dehydrant

Answer 14

alcohol for removal of water

Question 15

clearing agent

Answer 15

xylene to remove the alcohol and make the tissue receptive to paraffin, high index of refraction renders tissue transparent

Question 16

infiltrating medium

Answer 16

paraffin infiltrates the tissue and gives it support

Question 17

open tissue processor

Answer 17

rarely used because of reagent evaporation

Question 18

closed tissue processor

Answer 18

preferred method

Question 19

Ethanol (ethyl alcohol)

Answer 19

clear, colorless, flammable
fast, best dehydrant
hydrophilic
70-95-100% steps help reduce tissue shrinkage

Question 20

Methanol (methyl alcohol)

Answer 20

clear, colorless, flammable, poisonous
rarely used
except for fixation of blood smears

Question 21

Isopropanol (Isopropyl alcohol)

Answer 21

flammable, toxic
excellent substitute for ethanol
eosin can’t stain with it
doesn’t harden or shrink tissue as much as ethanol

Question 22

Acetone

Answer 22

volatile, flammable 
dehydrates rapidly, cheap
excessive shrinkage
absorbs water when exposed to air
hard to maintain solution levels in open air processors

Question 23

xylene

Answer 23

most commonly used clearing agent
can overharden tissues
intolerant of water left in the tissue
flammable hazardous neurotoxin, do not pour down the sink

Question 24

toluene

Answer 24

flammable, more volatile than xylene

does not overharden tissue as much as xylene

Question 25

benzene

Answer 25

very volatile and toxic, carcinogen

fast acting, doesn’t overharden as much as xylene but not used in histology

Question 26

chloroform

Answer 26

volatile, carcinogen
makes tissue less brittle than xylene
clears tissue slowly
must use in tight containers because it easily absorbs water from the air
best for uterus muscle and tendon

Question 27

cedarwood oil

Answer 27

volatile, strong odor
hardens and damages tissue the least out of any reagent
clears quickly
used for special projects

Question 28

limonene

Answer 28

xylene substitute
harden tissues less than xylene but contaminates teh paraffin more often
can’t dispose down drain

Question 29

why is paraffin routinely used

Answer 29

large number of blocks can be processed in a short amount of time
easy to get serial sections as well as routine and special stains

Question 30

water present during dehydration

Answer 30

results in mushy tissue and incomplete clearing and infiltration

Question 31

how fixative pH affects the processing unit

Answer 31

if zinc formalin goes above pH 7 it can cause formation of precipitates

Question 32

precautions for handling tissue processing reagents

Answer 32

gloves, most of the reagents are dangerous or irritants

Question 33

importance of preventative maintenance on tissue processors

Answer 33

keep solutions clean so that there isn’t carryover of water causing underprocessing, maintain temperatures to prevent overprocessing and hardening of tissue

Question 34

why do we decalcify?

Answer 34

because calcium deposits damage the microtome blade and make sectioning very difficult

Question 35

why do we carefully monitor decalcification?

Answer 35

because it is essential to make sure the specimen is neither overdecacified nor underdecalicied since either state results in undesirable staining outcomes; no nuclear staining or left over calcium that stains very darkly and can damage the blade

Question 36

decalcification

Answer 36

occurs after fixation but before processing

Question 37

pros and cons of acid decalcifiers

Answer 37

fast is both a pro and con, need to carefully monitor the process so the sample isn’t over or underdecalcified

Question 38

function of acid decalcifiers

Answer 38

acidic solutions between pH 3 and 5, calcium salts dissolve and then ionize, migrating into the surrounding solution. This is the most common type of decalcification in histology

Question 39

determining the endpoint of decalcification

Answer 39

mechanical: test flexibility or scrape, no one does this method
chemical: the decalcifying solution is checked for the presence of calcium
radiography: x-rays are used to show that decalcification is complete

Question 40

why do we remove decalcifying agents prior to processing?

Answer 40

to prevent carryover of acids that would interfere with processing

Question 41

how to preform localized decalcification

Answer 41

once the block has been faced the surface can be placed in decal solution, then rinsed and sectioned to break down any calcium deposits that remain

Question 42

Max size

Answer 42

2cm square, 3-4mm thick