1branch of biology which deals with STUDy of diseased tissue under microscope
Histopathology
2Removing of bit of Tissue from Living-being
Biopsy
3Removing of large Tisswe / Organ from Dead Body.
Autopsy
4Destruction of tissue due to its own enzyme.
Autolysis
5Destruction of tissue due to Environmental bacteria & microorganism
Putrefaction
6Why do we have to study Histopathology?
1. diagnose stage of cancer
2. diagnose pre-cancer stage
3. other inflammatory diseases
7Flow in Histopathology Laboratory
1. Sample collection
2. Sample receiving
3. Entering specimen details
4. Requested procedure changing
5. Processing of specimen
8Tissue are collected and fixed immediately in a _______________________
10% neutral buffered formalin
9- it causes chemical and physical changes
-it hardens and preserve the tissue
Formalin
10-it protect from degenaration, autolysis and putrefaction
Formalin
11___ times the volume of fixative in ratio to the size of the specimen
3 times (1:3 ratio)
12Fixative is __ to __ times greater than the size of the tissue
10 to 20 times
13______________ of the specimen is highly recommended
Tagging
14Process of cutting the specimen into 3 or more parts.
Loafing
15the surgical removal of the entire breast and all of the axillary lymph nodes under the adjacent arm
modified radical mastectomy (MRM)
16removal of the entire uterus, cervix, both ovaries, and both uterine tubes
total abdominal hysterectomy-bilateral salpingo-oophorectomy (TAH-BSO)
17-This specimen is wrapped in a gauze envelope
Small fragile specimen
18Example of Small fragile specimen
Punch biopsy and gastric tissues
19One of the biggest challenges Histopathology facing
Accurately label and tract specimens to avoid potential misidentification errors
20Specimen identification is maintained across steps like
1. Specimen labeling
2. Grossing
3. Block labeling
4. Slide labeling
21It involves precise and systematic description color , shape! size and consistency
Grossing
22are crucial parts of the pathologic examinations
Dissection and selection of sections for microscopic study
23- Done by PATHOLOGIST
- assisted by MEDICAL TECHNOLOGIST
- or HISTOTECHNICIAN
Dissection and selection of sections
24Cartilaginous, bony and hard tissues are placed in the decalcyfying solution for _____ days
I-7 days
25How to gross a specimen?
1. Describe: color, shape, consistency, and measurement
2. Cut the specimen on both ends
3. Place in tissue cassette
3. Cover the tissue cassette
4. Label with corresponding accession number
26In specimen grossing, cut the specimen at no mor than the thickness of _____mm
5mm
27A combined longitudinal and cross cut
tangential cut
28Once the tissue is properly fixed, it goes through a process which involves the following steps :
1. dehydration
2. clearing
3. infiltration/ impregnation
29A process of removal of water from aqueous-fixed tissue.
Dehydration
30Process of progressively immersed with ascending
concentration of alcohol
Dehydration
31Process of replacing the dehydrant with substance that will be miscible with the embedding medium.
Clearing
32- reagent that can miss both alcohol and war
- act as a bridge for alcohol and wax
Organic Solvent Xylene
33Process in which final xylene is replaced with molten wax which infiltrates the tissue.
Impregnation/Infiltration
34In Impregnation/Infiltration tissue is immersed in a molten wax at ____ degrees Celsius
60 degrees Celsius
35instrument where tissue processing is routinely done.
Automated Tissue Processor
36is a carousel type semi-enclosed tissue processor with 12 stations
Leica TP1020 tissue processor
37It is the perfect combination of proven technology and a modern, functionally enhanced design.
Leica TP1020 tissue processor
38Part of Leica TP1020 tissue processor that indicates the station parameters such as number of tissue baskets, vacuum function and remaining infiltration time, real time, start time (delayed start), overall duration and end of run time.
LCD
39Part of Leica TP1020 tissue processor that is moved up and down in the liquid at three-second intervals to ensure thorough and even mixing of the reagents and optimum infiltration of the tissue.
Tissue basket
40Part of Leica TP1020 tissue processor that reduces solution loss and therefore mission into the ambient atmosphere to a minimum
Sealing rings on the container lids
41Part of Leica TP1020 tissue processor that makes it easily accessible because it can be rotated nearly immediately and without difficulty
Integrated and adjustable rollers
42- can be applied to any of the stations both in manual and automatic operation.
-The advantage: substantially improved infiltration of tissue in a shorter time.
The Leica TP1020 tissue processor vacuum
43Answer the following reagent per station:
1st station =
2nd station -
3rd station -
4th station -
5th station -
6th station -
7th station -
8th station -
9th station -
10th station -
11th station -
12th station -
1st station = 10% neutral buffered formalin
2nd station - 95% alcohol
3rd station - 95% alcohol
4th station - 95% alcohol
5th station - 100% alcohol
6th station - 100% alcohol
7th station - 100% alcohol
8th station - Xylene
9th station - Xylene
10th station - Xylene
11th station - melted paraffin wax
12th station - melted paraffin wax
44Identify the Advantage of Automated Tissue Processing: Intuitive function means it's easy to learn to
how to use, which reduces training time.
Reduces training time
45Identify the Advantage of Automated Tissue Processing: 100 or 200 cassettes processing at a time supports
variable workloads.
Variable workloads
46Identify the Advantage of Automated Tissue Processing: Proven "workhorse" track record of high reliability.
High reliability
47Identify the Advantage of Automated Tissue Processing: Possible mechanical operation during power
outages keeps specimens from drying out.
Mechanical operation
48What are the steps in embedding
1. One cassette at a time to maintain specimen
integrity.
2. Choose the appropriate metal mold.
3. Orient the tissue correctly.
4. Place your cassette top over the mold and fill
with paraffin.
5. Oven for 1 hour at 70°C.
6. Set on to the cold plate neatly and in order for
30 minutes.
7. Remove the block from the mold
8. Clean the block of excess paraffin.
49instrument used to cut section from
tissue embedded in paraffin wax.
Microtome
50Part of microtome that is used to move the block downward across the blade
hand wheel
51Part of microtome that is used to move forward and backward
Front knob
52Part of microtome that is used for lateral adjustments
Mabhab
53Part of microtome that changes the angle of knife holder
Knife angle knob
54Steps in microtomy
1. Use the lever to secure the tissue block.
2. The hand wheel is used to move the block downward across the blade.
3. Safety knob must be turned downward and the handle pulled outward.
4. The blade should be replaced or adjusted before you cut. Use a new tissue block and make sure to be careful when handling the razor blades .
5. When it needs to be cleaned use only a brush on an upward motion.
6. Cutting works best when the knife holder is set to a fairly steep angle.
7. Remove the outer layer of wax in the block. (trimming)
8. Adjust the knife holder as close as possible to the tissue block.
9. Set the thickness indicator 5 microns.
10. Remove and place in an ice bath.
11. Begin to cut and make a ribbon of 6.
12. Place the ribbon on a warm water bath at 45 degrees Celsius.
13. Two of the ribbon strings will go to the slide.
14. Label with corresponding accession number and place in the staining rack.
55Steps in Deparaffinization
1. Incubate the slide for 30 minutes at 70 degrees celsius.
2. Place in two levels of xylene (5 minutes each level)
3. Five times dips of 95% ethyl alcohol.
4. 10 dips in running water.
56Steps in Staining (Hematoxylin and Eosin)
1. Hematoxylin stain for 20 minutes.
2. 10 time dip in running water.
3. One dip in 1% acid alcohol.
4. 10 dips in running water.
5. Two dips in ammonia water.
6. Five dips in running water.
7. 10 dips in a eosin.
8. 10 dips in 95% ethyl alcohol.
9. 10 dips in 95% ethyl alcohol.
10. Two levels of xylene for 2 minutes each level.
11. Blow dry the slides.
57It stains the extracellular matrix and
cytoplasm pink.
Eosin y
58Steps in Staining (Hematoxylin and Eosin)
1. Apply protective coverslip.
2. Send the slides to the pathologist for diagnostic examination.
59What are the 3 main process in Tissue Processing
dehydrating the specimen,
introducing a clearing agent, and embedding the
specimen in paraffin wax.
60- use to stain paps smear
Papanicolaou Stain or Pas Stain
61is used for cervical cancer screening
in gynecology.
paps smear
62What are the stains in Papanicolaou stain (pap stain)?
polychromatic pap stain contains 5 dyes in three
solutions:
- 1 nuclear stain (Haematoxylin)
- 2 cytoplasmic counter stain (OG-6 & EA-50)
63-stains cell nuclei in BLUE
-both progressive and regressive nuclear staining
can be used.
Nuclear stain (Haematoxylin)
64commonly used (regressive
technique)
Harris Haematoxylin
65What are the Two Cytoplasmic stain?
Orange G-6 and Eosin Azure-50
66- First acidic counter stain
- monochrome stain
- stains the protein keratin
(a normal component of the squamous
epithelial) in ORANGE
Orange G-6
67- second counter stain
- different formulation with a number, according
to the proportion of the constituent dye.
- commonly use
- polychromatic stain (mixiure of 3 stains)
Eosin Azure-50
68it stains the cytoplasmic of cells, in shades of
PINK or GREEN, depending on the pH.
Eosin Y
69What are the Principles and Procedures of Pap staining technique?
1. FIXATION
2. NUCLEAR STAINING (HAEMATOXYLIN)
3. DIFFERENTIATION (1% ACID ALCOHOL)
4. BLUING (AMMONIA)
5. DEHYDRATION BEFORE CYTOPLASMIC
STAINING
6. CYTOPLASMIC STAINING
7. CLEARING
8. BLOW DRY & COVER SLIPING (MOUNTING)
70- It uses fixed with 100% alcohol, 95% ethyl alcohol
or spray alcohol fixative
- stand for 20 minutes
FIXATION
71- uses Harris Haematoxylin for 5 minutes.
- nucleus is deliberately over stained
NUCLEAR STAINING (HAEMATOXYLIN)
72It involes the ff:
- take off additional haematoxylin stain
- as often results of hypochromasia
- contrast between counter stain and nuclear
stain will be lessened.
- rinsing in running water for 10 times
DIFFERENTIATION (1% ACID ALCOHOL)
73- serves as bluing agent
ammonia water
74how many dips in ammonia or bluing agent
2 dips
75-It is the use of ascending grades of alcohol
- it prepares the cell sample for uptake of the
counter stain.
DEHYDRATION BEFORE CYTOPLASMIC
STAINING
76What stains keratinized cell and how many dips?
Orange G-6: 5 dips
77What stains rest of the cell? For how many seconds?
Eosine Azure: 30 seconds
78- It helps to protect from dust
- It preserve the slide
BLOW DRY & COVER SLIPING (MOUNTING)
79an injury caused by freezing of the skin and underlying tissues
Frostbite
80advanced formulation that is designed to support tissue during cryotomy.
Tissue freezing medium
81Steps in Frozen Section (Cryostat)
1. Always wear gloves to avoid frostbite.
2. Tissue freezing medium advanced formulation that is designed to support tissue during cryotomy.
-Place around the tissue or in the mold when you're preparing to go on the block to be cut.
3. Pick them up with a microscope slide.
4. Consult your manual for the temperature suitable for the specimen.
82can be set to a very high or very low depending on the specimen to be cut
Thickness indicator
83it extra freeze samples
Pe button
84magnetic stainless steel disc for mounting
Specimen disc
85it secure the tissue block
Mounting area
86It is where the shaving off the block happens
Stage
87it flattens the sliced tissue
Anti-roll cover
88it locks and unlocks the blade
Blade level
89it manipulates the stage sideways, forward and backward
Stage lock
90it moves the mounting area microscopically
One arrow button
91it moves the mounting area at the greater distance
Two arrow button
92it most the mounting area on upward and downward movements
Hand wheel
93What are the steps in sample preparation?
1. Apply the media direct to the specimen.
2. Specimen should be on the center.
3. Use the heat extraction bar to immediately freeze the tissue sample.
94What are the steps in sectioning?
1. Put the specimen this in the mountain area.
2. Figure out how far away is the tissue to the blade.
3. May or may not initially see the tissue slices.4. You can always manipulate the thickness of the slice.
5. Using the brush technique.
6. Pick up with microscope slide.
95What are the steps in staining?
1. Immersed with Haematoxylin stain for 1 to 2 minutes.
2. Wash well in distilled water for 10 dips.
3. One dip in ammonia water.
4. Rinse in distilled or running water for 10 dips.
5. Counterstain in stain Eosin Y for 1-2 dips.
6. Dehydrate in two levels of 95% ethyl alcohol.
7. Blow dry the slides.
8. Apply protective coverslip. 9. Slide is now ready for microscopic examination.