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HISTOLOGICAL TECHNIQUES
LIVER CANCER

INTRODUCTION
Histological techniques deal with the preparation of tissue for microscopic
examination
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Histological techniques reveal normal tissue structure,
tissue abnormalities and cancerous conditions
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DNA
mutations cause cells begin to grow out of control and eventually form tumor
...

Cancer spreads to liver is more common than it begins in hepatocytes
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The most common type of liver cancer is
hepatocellular

carcinoma

while

intrahepatic

cholangiocarcinoma

and

hepatoblastoma are much less common
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Liver cancer cases rise from 65% in 2007~2011, to 74% in 2012~2016
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8%
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HBV is the common cause but it can actually prevented by taking vaccine and
maintaining a healthy lifestyle
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ORGAN AND SITE SELECTION

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OVERVIEW

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FIXATION
Objective:
1
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2
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3
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Procedure:
Fixation and Trimming
1
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2
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3
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4
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5
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6
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7
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Decalcification is required for
hard tissue samples such as bones and teeth
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Fit tissue pieces into the cassette
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Place a lid on the cassette
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10
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11
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Decalcification
1
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(20X tissue volume)
2
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Stirring agitation of fluid hasten decalcification
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Tissue samples may be mechanically or chemically tested
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Bend or pierce tissue samples with a sharp needle
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Decalcification requires generally 1~2 days
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Wash specimen for 24~48 hours in running water before tissue processing
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Duration of fixation depends on the size of sample and the fixative used
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Prolonged fixation will cause tissue harden and shrinkage
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Delayed fixation will cause autolysis
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Make sure the specimen is completely covered with fixative
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5
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6
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Troubleshooting:
Soft mushy tissue
Formation of acid formalin pigment
Incomplete fixation
Obtain better penetration
Enhance fixation

Adequate time for fixation
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Longer fixation
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Agitate specimen in fixative
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To embed tissues in solid medium
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To support the tissues
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To enable the knife to cut the section with little damage
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Prepare 80%, 95% and 100% alcohol solution and beakers or containers
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Dehydrate the tissue in a series of increasing alcohol concentrations:
i
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ii
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iii
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(twice)

3
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4
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5
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(three times) for rapid dehydrating process
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Clear the tissue with xylene (paraffin solvent) for 1 hour
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Clear the tissue with xylene (paraffin solvent) for 1
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Infiltration and impregnation
8
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Embedding
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10
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Transfer cassettes to the warm
compartment of the embedding station
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Transfer one cassette onto the hot plate
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Remove the cassette lid
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16
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(The specimen must not come into
contact with the edge of mold
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Transfer the mold onto the hot plate
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Pour melted paraffin wax from paraffin dispenser
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Transfer the mold carefully onto the cold plate
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When a thin film of semi-solid wax has formed on the base of mold, introduce the
tissue by using forceps
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21
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22
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24
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Make sure there are no air bubbles
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Transfer the mold and cassette onto the cold plate or to the refrigerator
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Automated tissue processing
1
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2
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Hook the carrier onto the arm of automated
tissue processor
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Lower the carrier into the first bar of 70% alcohol
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Set up the timer
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The automated tissue processor holds the beakers of alcohol of increasing
concentration (70% to 85% to 100%)
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Paraffin wax is not
soluble in alcohol
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Expose tissue to histolene (clearing agent) that is miscible with both alcohol and
paraffin wax
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The last 2 beakers in the
machine contain the clearing agent
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Immerse tissue for more than equal to 1 hour in pots of molten paraffin wax
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Transfer carrier to the embedding station
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9
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10
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11
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12
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Safety precaution:
1
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2
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3
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4
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5
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6
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7
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Troubleshooting:
Overdehydration
Tissues are not embedded at the same
level
Wax crystal
Different embedding media is required

Air bubbles trapped within tissue
Tissue dried out after processing

Shorten dehydration time
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Rapidly cool the wax
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 Affected by heat
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 Tissue is breaking away from wax
during sectioning
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Vacuum is required
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SECTIONING
Objective:
1
...

2
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3
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Procedure:
Microtomy
Preparation
1
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2
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Avoid prolonged cooling and very cold surfaces to prevent cracking in the block
surface
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Install the knife or disposable blade
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4
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5
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Trimming of tissue block
6
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Make sure the top and
bottom of tissue block is parallel to the edge of knife
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Keep 2~3 mm of paraffin wax around the tissue
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Fit the cassette paraffin tissue block onto the cassette holder of the microtome
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Orientate the tissue block so that its greatest axis is perpendicular to the edge of
knife
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Unlock the hand-wheel
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Advance or move the block forward until it is in contact with edge of knife
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If not, adjust the block orientation
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Set the section thickness around 15~20 µm
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Coarse cut the block until the whole surface of embedded tissue can be cut
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13
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Sectioning
14
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5~4 µm
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Remove wax debris from the knife with alcohol
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Install a new knife or move to an unused area of knife
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Install the cassette block onto the cassette holder again
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Cut the paraffin section with an optimal speed when rotating the hand-wheel of
microtome
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19
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20
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21
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Floating out and fishing
22
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Slightly drag when ribbon touches water
to produce tension and removes folds or wrinkles
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Gently separate the floating ribbon on the water bath with pressure from tips of
forceps
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Pick up the floating ribbon on a clean glass slide
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25
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Drying
26
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27
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For immunochemistry, slides
should be stored at 4ᵒC to minimize antigen loss
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Safety precaution:
1
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The cutting edge is
extremely sharp and can cause injuries
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Always remove the knife or blade before detaching the knife holder from the
instrument
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Always put the knife back into the knife case when not in use
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Never place a knife anywhere with the cutting edge facing upwards and never
try to catch a falling knife
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Avoid prolonged floating out of section on water bath to prevent tissue expand
and distort
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Reduce knife tilt or section thickness
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 Fix the knife angle
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 Trim away excess wax
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Section compression
 Cool the block in refrigerator
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Sections expand and disintegrate on  Return tissue to vacuum impregnation
water surface
container for a few hours
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 Clean the floatation bath
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To stain and identify certain features of specimen
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To enhance the tissue contrast
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To visualize metabolic processes
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To differentiate live cells and dead cells
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To examine the specimen under microscope
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Soak the section in 200 mL of xylene for 5 min and agitate
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Place section into another 200 mL of xylene for 5 min
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Transfer it to 200 mL of absolute alcohol for 1 min
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Transfer it to 200 mL of 90% alcohol for 1 min
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Transfer it to 200 mL of 70% alcohol for 1 min
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Wash it in running tap water
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7
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Wash it in running tap water
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Dip several times in 200 mL of 1% acid alcohol (2 mL hydrochloric acid in 198
mL of 70% alcohol) as a differentiator
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9
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Wash it in running tap water
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Counterstain in 200 mL of eosin for 1~2 min
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11
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12
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13
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(twice)
14
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(twice)
15
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Mount in DPX and label the slide
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Overstaining with hematoxylin gives illusion of understained eosin
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Overstaining with eosin cause hematoxylin to appear lighter than actual
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Add small amounts of acetic acid to hematoxylin periodically can aid in
maintaining appropriate pH value
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If the bluing step is missing, rehydrate the slides and then continue to bluing
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Dehydrate again slides before mounting
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Leave slide longer to differentiate
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Proper bluing required
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 Completely remove bluing agent
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 Dilute eosin
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MOUNTING
Objective:
1
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2
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3
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Procedure:
Slide method
1
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2
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3
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One end is placed on the blotting papper and the
other end slowly lowered until mounting media touches the coverslip
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Let the mounting media spread and invert the slide quickly
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Guide the coverslip into place with a dissecting needle
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Add mounting media on the coverslip in the center
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Invert the slide over the coverslip and let the surface tension pull the coverslip
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Safety precaution:
1
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2
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Too
much mounting media provide bad visualization
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Mounting media must be insoluble in water
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Choose mounting media with refractive index close to slide
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Excessive blotting will dry up the section, causing tissue shrinkage and cracking
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Make sure the label fits the specimen for proper identification
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Too much air bubbles
Remove the coverslip, clear it with
xylene and re-mount the section
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Obtain neutral pH for mounting media
Add potassium acetate
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Tissue distortion
Do not press down the coverslip
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Racheal
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https://rsscience
...
López-Terrada, Dolores & Alaggio, Rita & Garcia de Davila, Maria & Czauderna,
Piotr & Hiyama, Eiso & Katzenstein, Howard & Leuschner, Ivo & Malogolowkin,
Marcio & Meyers, Rebecka & Ranganathan, Sarangarajan & Tanaka, Yukichi &
Tomlinson, Gail & Fabre, Monique & Zimmermann, Arthur & Finegold, Milton
...
Towards an international pediatric liver tumor consensus
classification: proceedings of the Los Angeles COG liver tumors symposium
...
27
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1038/modpathol
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80
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researchgate
...
Trobaugh-Lotrario, Angela & O’Neill, Allison & Li Peng & Towbin, Alexander
& Weldon, Christopher & Lopéz-Terrada, Dolores & Malogolowkin, Marcio
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Advances in pediatric Liver Tumors
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16
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1007/s11901-017-00335-0
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researchgate
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Slaoui, Mohamed & Fiette, Laurence
...
Histopathology Procedures: From
Tissue Sampling to Histopathological Evaluation
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J
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69-82
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1007/978-1-60761-849-2_4
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researchgate
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Ravikumar S, Surekha R, Thavarajah R
...
J NTR
Univ Health Sci [serial online] 2014 (cited 2021, Nov 28); 3, Suppl S1:1-8
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jdrntruhs
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asp?2014/3/5/1/128479

VIDEO SOURCE (SELF-MADE)
https://drive
...
com/file/d/1vdzkcvfN0Urgy1Y2seLk-S7s2dzUfoBF/view?usp=sharing

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