HISTOPATHOLOGY UNIT

PRETEST
Automatic Tissue Processor A machine that prepares tissues for sectioning thru series of
(Leica TP1020) alcohol, clearing agent and embedding medium.

Embedding Center After 16 hours in the automatic tissue processor, tissues are
(Histostar) then brought to the embedding center for embedding, it
composts of a hot plate, paraffin dispenser and cold plate.

Tissue Molds Where tissues are placed properly so that it can be easily
sectioned using a rotary microtome.

Rotary Microtome Used to produce thin sections ranging from 4 to 6 um

(Microtec)

Freezing Microtome (Cryostat) Used when Rush Frozen Section is requested, fresh unfixed
tissues are placed under extreme cold temperature, using
cryomatrix instead of paraffin to hold the tissue when its being
cut. Diagnosis should be done around 15 minutes or less.

Dry heat Oven After placing the tissue sections on the slide its placed inside
the dry heat oven to facilitate initial deparaffinization and final
fixation.

Automatic Tissue Where the staining takes place whether routine hematoxylin
Stainer (Varistain) eosin staining or papaniculau staining procedure.

Centrifuge Equipment needed to produce cell block and pull apart smears
from fluid specimens for fluid cytology.

Cytospin Usually used for clear pathologic fluid, an example of

cerebrospinal fluid so than the cells are concentrated in an
area readily seen by the pathologists.

Immunohistochemical Stains One of the advancements in histopathology, aids physicians

on how they treat cancer patients by knowing the primary
cause of malignancies and how they can block it thru proper
medications.
 INTRODUCTION

Ang histopathology section ay nakakatulong sa Service Offered:

pagsiyasat ukol sa cancer. Dito, ipinadadala ang • Pap Smear
biopsy na mula sa iba’t ibang bahagi ng katawan. • Histopathology
Ang specimens na ito ay dadaan sa tissue • Routine
processing, stainig atsaka ilalagay sa slides upang • Rush Frozen Section (RFS)
masuri ng isang pathologist.Sa histopathology unit • Cytopathology
din isinasagawa ang otopsiya. •Immunohistochemistry
• ER
Rooms: • CD-20
• Histopathology Unit • PR
• Staining Room • CD-7
• Gross Room • HER 2 NEU

Automated Tissue Processing For cutting ribbons of tissue sample embedded in

paraffin

Machine used to maintain low cryogenic temperature of

tissue for rush frozen section.

LECTURE
Anatomic Pathology
Step 1. Upon receiveing the specimen, see to it that the Step 2. Assign an accession number and also
data is complete and the specimen is properly labeled. remember that the official request form should be used.
Label the accession number on the top of the body in
the specimen container. Put the appropriate fixative for
the tissue types. Respectively, used 10% buffered
formalin for tissue biopsy and for cytopathology, 95%
ethanol.
Step 3. After brushing the specimen by the pathologist, Step 4. After brushing the specimen by the pathologist,
we load the specimen on a tissue cassette and put them we load the specimen in the tissue cassettes and put
in our automatic tissue processor. For cytopathology, them in our automatic tissue processor
note that we use 2 tube method so that we can obtain a
cell block and a pull apart smear from the fluid
specimen.

Step 5. Embedding the tissue on tissue molds is one Step 6. For routine tissue biopsies, we use rotary
way of making a tissue block from the samples. microtome for tissue sectioning. For rush sections, we
are using freezing methods

Step 7. Placing the tissue

section in the floatation bath
and putting them on a glass
slide by teasing
 Automated Tissue Processor (Leica TP1020)
Reagents in each of its stations
Fixation Formalin, buffered or unbuffered
Picric acid
Dehydration Ethanol (Ethyl Alcohol)
-ol Isopropyl alcohol
Methanol (Methyl Alcohol)
Butyl alcohol
Industrial alcohol
Clearing Xylene and xylene substitutes
CAT BTX Toluene
Benzene
Chloroform
Trichlorethane
Acetone
Paraffin Paraffin
Preparations for tissue processing
Filling the reagent stations • Lift the carousel cover.
• Fill all stations with the corresponding reagents. Make sure to
observe the minimum and maximum level indication marks.
• Spilled reagents have to be wiped away immediately.
In case of long-term exposure, the instrument surfaces
are only conditionally resistant to solvents.
• Mount every container onto the station holder at the
corresponding station.
• The container rims and sealing rings of the lids always
have to be clean. The lids have to close tightly -
otherwise larger amounts of solvent fumes will escape
and, in instruments with vacuum function, vacuum will
not be generated.
Filling the paraffin stations The heated wax baths may only be used with paraffin.
Under no circumstances may they be filled with solvents.
When solvents heat, a highly explosive mixture builds up!
Caution! The interior containers of the paraffin stations
become very hot when the heating function is activated! Do
not touch the gray upper rim of the containers with your
hands! Risk of injury! Caution when handling hot paraffin!
Risk of injury!
• Altering the standard working temperature
• Factory-set standard working temperature is 65 °C (70
°C on the special paraffin station model that is resistant
to chloroform). When working with paraffin that has a
melting point below 58 °C, the instrument working
temperature can be readjusted with the corresponding
setting screw.
• Do not overfill the paraffin stations! Make sure the
paraffin level is not below the minimum or above the
maximum level indicator.
• Use a screw driver to turn the setting screw (1) to the
desired value.
• If you find that the paraffin does not melt completely
after lowering the working temperature, slightly readjust
again.
• To fill the paraffin stations, use wax pellets or paraffin which
has already been liquefied.
• When filling the station, make sure the paraffin level is
not below the minimum level in which case there is a
 risk that not all specimens will be entirely immersed in
paraffin and thus will not be infiltrated completely.
• It may take several hours to liquefy solid paraffin.
Make
sure to calculate the waiting time! When refilling wax
pellets, again make sure to observe the waiting time for
complete liquefaction.
• Place the paraffin station onto the corresponding station
holder and push the cable into the notch at the edge of the
platform.

Check for each paraffin station whether it is actually installed

at same station number it is connected to at the rear of the
instrument.
• When filling the station, make sure the paraffin level is
not below the minimum level in which case there is a
risk that not all specimens will be entirely immersed in
paraffin and thus will not be infiltrated completely.
Reagents in 12 stations
Perocesses involved Fixation
Dehydration
Clearing
Infiltration/ Impregnation
Fixation 10% Formalin
10% Formalin
Dehydration Increasing concentration
70%, 75%, 80%, 85%, 90%, 95%
Clearing Xylene (3 times)
Xylene I
Xylene II
Xylene III
Infiltration/ Impregnation Wax Bath (3 times)
Paraffin Wax I
Paraffin Wax II
Paraffin Wax III

ACTIVITY 1
A urine cytology together with urine culture and sensitivity The specimen should be processed first by the
was submitted to the laboratory how would you process it? bacteriology unit; after that it must be forwarded
a) Reject the specimen because its only one to histopathology unit for two tube method way
b) Demand a separate specimen from the ward of processing
c) The specimen should be processed first by the
bacteriology unit; after that it must be forwarded to Bacteriology unit needs sterile specimen so they
histopathology unit for two tube method way of should go first.
processing
d) Get a separate sample from the specimen brought
An amputated foot was brought to the laboratory for Reject the specimen because of absence of
disposal, problem is there is an absence of request form. proper request form
What will you do?
a) Accept the specimen then just make a request by Reject the amputated foot because lacks proper
yourself request form
b) Burn the amputated foot before it decomposes
c) Dispose the amputated foot because it will stink
d) Reject the specimen because of absence of proper
request form
An outpatient was trying to get a copy of his/her biopsy Follow up on the pathologist about the matter
result on the date you indicated on the claim stub but upon and relay to the patient what the pathologist said
checking in the logbook there was no result yet, what will
you do? Post analytical for anatomical pathology are under
a) Tell the patient to come back tomorrow the Pathologists
b) Follow up on the pathologist about the matter and
relay to the patient what the pathologist said
c) Give the patient other tissue biopsy result that is
already available
d) Give your number to the patient and tell them to
contact you for the result
A rush frozen section tissue was brought to the laboratory Enter the main organ with the same specimen
at 9:00 am after it was diagnosed as malignant by the number you gave on the frozen tissue specimen
pathologist you entered it in the logbook for routine tissue
biopsy, after three hours the main organ was brought in the Receiving tissue specimens on the same day use
laboratory. What will you do? only the same accession number
a) Discard the second specimen because the
pathologist already diagnosed it as malignant
b) Reject the specimen because you had already
received one
c) Enter the main organ with the same specimen
number you gave on the frozen tissue specimen
d) Enter the main organ with a new specimen number
Sputum specimen for cytology was brought to the lab in Cut the segment of the ETA tube then spin in the
endotracheal aspirate tube how would you process it? centrifuge and prepare for 2 direct smear slides
a) Request for a new sputum specimen in a specimen and cell block
bottle
b) Aspirate using syringes It’s advisable to centrifuge ETA tubes when
c) Blow the ETA tube using rubber aspirator preparing slides and cell block
d) Cut the segment of the ETA tube then spin in the
centrifuge and prepare for 2 direct smear slides and
cell block

ACTIVITY 2
This is an automated tissue processor used in Veterans
Memorial Medical Center, Histopathology Unit.
Determine the reagents in each of the 12 stations by
matching it with the answers in thee other column.

1st station: 70% ethanol

2nd station:75% ethanol
3rd station: 80% ethanol
4th station: 85% ethanol
5th station: 90% ethanol
6th station: 95% ethanol
7th station: Xylene
8th station: 95% ethanol
9th station: 90% ethanol
10th station: 85% ethanol
11th station: 80% ethanol
12th station: Wax Bath

SHIFTING EXAM
Determine whether the word refers to as Malignant or 1. Adenoma - Benign
Benign. 2. Nevus- Benign
 1. Adenoma 3. Fibroma- Benign
2. Nevus 4. Renal Cell Carcinoma- Malignant
3. Fibroma 5. Hepato cellular carcinoma- Malignant
4. Renal Cell Carcinoma 6. Leukemia- Malignant
5. Hepato cellular carcinoma 7. Fibroma- Benign
6. Leukemia 8. Seminoma- Malignant
7. Fibroma 9. Lipoma- Benign
8. Seminoma 10. Hepatoma- Malignant
9. Lipoma
10. Hepatoma

Temperature of paraffin during embedding procedure

a) All of the choices
b) 5 to 10 degrees Celsius below melting point of the
paraffin
c) 5 to 10 degrees Celsius above melting point of the
paraffin
d) 5 to 10 degrees Celsius above melting point of the
paraffin
The color of the nuclei after staining of Hematoxylin and True
Eosin is Blue.
a) True
b) False
4-6 microns in thickness of section is used in Rush Frozen False
Section.
a) True
b) False
Oxidizing Agent of Harris Hematoxylin is Mercuric Oxide. True
a) True
b) False
Cause of chatter or microscopic vibration on the sections Dull blade
a) All of the above
b) Over dehydration or lack of moisture in tissue
c) Dull blade
d) Cutting too rapidly
Suitable temperature for floatation baths for paraffin
sections
a) Both A and C are correct
b) 6 to 12 degrees Celsius above the melting point of
the paraffin
c) 5 to 10 degrees Celsius above the melting point of
the paraffin
d) 5 to 10 degrees Celsius below the melting point of
the paraffin
Hematoxylin is derived from the heartwood of the Mexican True
tree known as Hematoxylon Campechianum.
a) True
b) False
Regressive Staining is used in Rush Frozen Section False
procedure.
a) True
b) False
Most widely used counter stain routine staining of sections Eosin
a) OG-6
b) Wright’s giemsa
c) Eosin
d) Hematoxylin
Refractive Index is the ratio of speed of light in air and True
speed of light in a specific medium.
a) True
b) False
An intern received a FNAB of Thyroid which consists of four False
slides only s/he instructed the patient that the result would
be after three working days.
a) True
b) False
Cause of white artifact on tissue sections. Can be due to incomplete removal of paraffin
a) Presence of water in the stain
b) Can be due to incomplete removal of paraffin
c) Contaminated stain
d) None of the above
Temperature of the melted paraffin need for embedding is True
5-10 degree Celsius above the melting point.
a) True
b) False
Cause of mounted stained sections are not as crisp as
usual when viewed microscopically
a) Mounting medium on top of the cover glass
b) Mounting medium is too thick
c) Mounting medium is too thin
d) All of the above
All uses formalin (10%) is disposed properly in the sink with
a continuous flow of running tap water for how many
minutes?
a) At least 2 minutes
b) At least 5 minutes
c) At least 4 minutes
d) At least a minute
OG-6 stains the cytoplasm of the mature superficial cells. True
a) True
b) False
Canada Balsam is the mountant used in VMMC. False
True
False
Rotary microtome is most commonly used in specimen Paraffin-embedded
embedded with which medium?
a) Paraffin-embedded
b) Neither
c) Glycerol methracylate
d) Both
What are the commonly used adhesive for slides. Mayer’s egg albumin
a) Poly-L-lysine
b) All of the above
c) Mayer’s egg albumin
d) Columbus
Cause of irregular, skipped or excessively thick or thin
sections
a) Paraffin sticking in the back site of the holder
b) Cutting too rapidly
c) Too warm room
d) Result of either too little or too much blade tilt
Normally used when rapid diagnosis of tissue is required. Rush Frozen Section

First and most critical stage in Histotechnology Fixation

Fixative used in Bone Marrow Core Preparation Bouin’s Solution
Also known as Dealchoholization Clearing
Its aim is to remove the fixative and water from the tissue Dehydration
Temperature of paraffin for infiltration 2-5 degrees Celsius above melting point
Temperature of paraffin used in embedding 5-10 degrees Celsius above melting point
Temperature of the flotation bath (water bath) 6-10 degrees Celsius below melting point
Process of removing excess paraffin Trimming
Process by which the tissue is cut in uniformly thin slices Sectioning
Type of microtome used in VMMC Rotary
What is the clearance angle 0-15 degrees
What is the bevel angle 27-32 degrees
A natural dye derived from the heartwood of the Mexican Hematoxylin
tree
Routinely used as a counterstain in histopathology after Eosin
hematoxylin
What type of mounting media is Entallan Resinous
An example of aqueous mounting media Paramount
Fixative to Tissue Ratio 20:1
Concentration of ethanol used for urine cytopathology 95%
Concentration of clearing agent used in our automatic 100%
tissue processor