SCHOOL OF MEDICAL

LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

OUTLINE A. RECEIVING: DIAMOND PEN

I. Routine Procedure Instrumentations
A. Diamond Pen, Pencils, Pen
B. Gross Table
C. Automated Tissue Processor
D. Paraffin Dispenser
E. Embedding Center
F. Embedding Molds
G. Refrigerator
H. Microtome
I. Flotation Bath
J. Slide Dryers
K. Stainers (Coplin Jars/Automated Stainers
L. Microscope
II. Storge, Retention, and Disposal
III. Recommended Minimum Retention Time of Records, Reports,
Specimens
Figure 1. Diamond Pen

I. ROUTINE PROCEDURE INSTRUMENTATIONS ● Used on a non-frosted slides

● The slide that has a rough portion is called a frosted slide.
Receiving Diamond pen, Pencils, Pen → Lead pencil is used to label on the rough part of the slide
→ It does not have an ink but it is pointed at the tip. The
Gross Examination Gross Table purpose is to write a label permanent and directly in the
● Forceps glass slide by etching it
● Scalpel
● Chopping Board B. GROSS EXAMINATION: GROSS TABLE
● Weighing Balance
● Ruler
● Orientation Markers
● Watch Glass/Petri Dish
● Tissue Cassettes

Fixation
Decalcification
Automated Tissue Processor/Elliot
Dehydration
Bench Type Processor
Clearing
Infiltration

Embedding/Casting/Molding/Bl Paraffin Dispenser

ocking Embedding Center
Embedding Molds
Refrigerator

Trimming Microtome
Sectioning/Microtomy Flotation Bath
Slide Dryers

Staining Stainers (Coplin Jars/ Automated

Mounting Stainers) Figure 2. (Left) Materials found in the Gross Table; (Right) Gross Table
Labelling Microscope
● Gross Examination - create a gross examination:
→ Type of Specimen
Molder - Disposable type: Paper → Color: either brown, tan, or white
Boat
▪ The sample is not expected to be red because it is placed
in formalin. It is tan in color. Blood accumulation will be
removed because of the application of formalin
▪ Make sure that the sample must be fully soaked in
formalin. It is considered to be a differentiating factor. This
Slide Dryer
is to preserve the sample.
→ Size
▪ We also check the size of the sample wherein the unit is
in centimeters. Length x width x height is the computation
→ Weight
→ Consistency

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

▪ We also identify the texture and composition of the ▪ Two different types of tissue processor:
specimen as well − Tissue- transfer
→ Texture
→ NOTE: For complicated tissues, there are other description
from doctors
● It is the task of the pathologist to perform a gross examination.
The role of the medtech on the other hand is to be only the
assistant. We write down the doctor's description during the
gross examination of a specimen. However, there are some
instances that we are allowed to perform gross examination
given that the sample is simple wherein basic description are
the only things that we need to input
● Gross table Figure 4. Tissue-Transfer
→ this where we perform gross examination
→ There is also an advanced type in which we can be able to o Principle/mechanism: “dip and dunk”
record and etc. o The tissue will move from one container to another.
o Most laboratories use this one
− Fluid-transfer
C. FIXATION TO INFILTRATION: AUTOMATED TISSUE
PROCESSOR

Figure 5. Fluid-Transfer

o Principle/mechanism: “enclosed”.
o Fixed
o Tissue sample does not move but is the fluid that
will move in and out to the sample
o The fluid is transferred, not the tissue.
o Using this fluid-transfer, there are instances that
some dehydrating or clearing agent will not be
removed and will be mixed with the next processes.
o That is why this is not mostly used in the histopath
lab.

D. EMBEDDING/CASTING/MOLDING: EMBEDDING
CENTER, FREEZER, REFRIGERATORS
Figure 3. Fixation to Infiltration

● MANUAL METHOD
→ From fixation down to infiltration
▪ We are using beakers in performing this manual method
in Histopath
▪ We transfer the sample from one beaker to another. We
perform manual method
→ Fixation – 10% formalin is used
→ Decalcification – not used for soft tissues but for calcified
ones such as bones, teeth, cartilages. Purpose is to soften
tissues
● AUTOMATED MACHINES
→ Automated tissue processors - the machine itself will
transfer the sample from one container into another. It will
take 16 hours to process Figure 6. Embedding Center
▪ Cannot perform Decalcification
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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

● EMBEDDING CENTER
→ where we perform, embedding or casting. We are arranging
the tissue for us to form a tissue block. We have four
different components.
→ Has 4 parts:
▪ Paraffin dispenser
− This is where we place the paraffin wax in which it is
solid in form.
− Temperature is 2-4oC higher than the melting point of
paraffin wax in order for it to melt so that we can form
the tissue block
− Why should the temperature of the paraffin dispenser
higher than the paraffin wax?
o Our goal is to have a liquid form of paraffin wax
because upon purchasing, paraffin wax is like
pellets or in solid form. So, we put the wax in this Figure 8. Refrigerators
container in order for it to melt.
▪ Orientation stage ● Freezer (Thermal Requirement - 20oC
− Other term for orientation is “arranging” ● Refrigerators (Thermal Requirement: 4oC
− It is where we arrange the tissue. We place a sample in
the right orientation so that the area that needs to be Note: Not all Histopathology Laboratories have refrigerators
appreciated will be examined by the pathologist. because most of our reagents have a storage temperature of room
− For example in the case of the fallopian tube, the temp. Some reagents that should be refrigerated will be put on the
lumen must be seen as much as possible. refrigerators of other areas in the laboratory.
− Embedding can be used one twice or thrice
▪ Cold/Chilling Plate – to solidify the paraffin wax
▪ Warm storage D. TRIMMING & SECTIONING/MICROTOMY:
● Before we start with the orientation, place the tissue cassettes MICROTOME
to melt the paraffin wax that was built up in our tissue. Then, we
can now start with the orientation and after that, place the
molder onto our cold plate in order to harden the paraffin wax.

Figure 9. Microtome

Figure 7. Parts of the Embedding Center ● MICROTOME

→ Best friend is microtome.
● EMBEDDING MOLDER → Used for cutting tissue sections
→ Three main parts:
▪ Block holder – this where we place the tissue block
▪ Knife holder – holds the knife
▪ Pawl and Feedwheel Mechanism – what is being rotate
in order for the tissue holder to come in contact with blade

Figure 8. Embedding Molder

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

D.1. KINDS OF MICROTOME SLIDING MICROTOME

ROTARY MICROTOME

Figure 10. Rotary Microtome Figure 13. Sliding Microtome

● Most commonly used. ● much dangerous because the blade is exposed.

● Designed for tissue sections that are embedded using paraffin ● Used if the embedding medium is cellulose nitrate.
wax ● Used for cutting large types of samples/tissue sections
● Inventor: Minot with hard blocks
● Tissue Cut: 3 to 5 micrometers
FREEZING MICROTOME
CRYOSTAT

Figure 11. Rotary Microtome

Figure 14. Freezing Microtome
● Used for STAT diagnosis.
● A rotary microtome is kept inside a cold chamber maintained at ● Freezing - same mechanism with cryostat
-5°C to - 30°C
ULTRATHIN
ROCKING MICROTOME ● The embedding medium is epoxy resin.
● Tissue Cut: 60-100 nm

D.2. FLOATATION BATH

Figure 12. Rocking Microtome

● Mechanism is just like a rocking chair.
● The oldest, simplest, and first microtome invented.
● Invented by Trefall
Figure 15. Tissue Sections after using a microtome
● “Tissue sections is the term used for the thin cut products from
the microtome”. After cutting, they are placed in the floatation
bath.

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

● Temperature must 5-10oC lower the melting point of paraffin AUTOMATED STAINER
wax so that the paraffin wax will not melt

Figure 19. Automated Stainer

COVERSLIPS

Figure 16. Flotation Bath

● Must be against a black background for us to see our tissue

sections
● Method is fish/fishing out technique
→ used in order to arrange our tissue sections in the slide.
→ must be centered

D.3. SLIDE DRYER

Figure 20. Coverslips

● Cover the tissue and allow longer storage

F. STAINING, MOUNTING, & LABELING

BRIGHTFIELD MICROSCOPE

Figure 17. Slide Dryer

● 5-10oC HIGHER higher than the melting point of paraffin

● Goal: to remove the excess paraffin wax that was accumulated
during fishing out.
● Only the tissue should remain on the slide. If there is paraffin, it
will be a hindrance during the staining method.
● Note: Overheating causes uneven staining, artifacts formation,
and tissue destruction
→ We should only place our slides in the slide dryer within 3-5 Figure 21. Brightfield Microscope
minutes only.
● Brightfield Microscope was commonly used during the Histology
Laboratory.
E. STAINING, MOUNTING, & LABELING ● In this type of microscope, upon viewing, the background is very
light because there is direct passing through of light source.
COUPLIN JARS, STAINING RACK
DARKFIELD MICROSCOPE

Figure 18. Couplin Jars

Figure 22. Darkfield Microscope
● Manual Staining
● There is a cover in between the light source and the sample.

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

● Upon looking at the microscope, the background is dark and ● The usual appearance of the tissues under this type of
the sample looks illuminating. microscope is green.
→ Unstained Sample - it must be unstained and freshly
prepared because we cannot properly view the slide when it FLUORESCENCE MICROSCOPE
is stained.
▪ Ex. Purple-stained sample over a dark background

PHASE CONTRAST

Figure 26. Fluorescence Microscope

● This only allows observing a specific part of the tissue. We

need to add a stain for us to have a fluorescence appearance
under the microscope.
● This is a specialized type of microscope.

FLUORESCENCE MICROSCOPE

Figure 23. Phase Contrast

● In this type, we also utilize unstained samples.

● The sample must be colorless/transparent for us to observe
the features of the tissues.

POLARIZING MICROSCOPE

Figure 27. Electron Microscope

● This is used for small/tiny tissues

● Its illuminating power is increased compared to the other
mentioned microscopes.
● This is the most advanced type of microscope.

Figure 24. Polarizing Microscope

Figure 25. Usual Appearance of a Polarizing Microscope (Green)

● This is designed to examine the birefringent properties of our

tissues with anisotropism.
→ Ex. Amyloid
▪ This type of tissue is different from other tissues which
needs a direct light or one angle only
▪ It can also divide one single light that is focused on the
tissue.

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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

II. STORAGE, RETENTION, AND DISPOSAL Table 2. Paraffin Blocks and Slides
SURGICAL SPECIMENS
Table 1. Surgical Specimens STORAGE ● Must be in a cool, dry place
SURGICAL SPECIMENS ○ To avoid melting of the paraffin blocks
STORAGE ● Return to their original (leak-proof and ● Free from vermin and insects
airtight) containers ● Arranged according to year and surgical
○ After gross examination, we do not use pathology accession number for easy
the entire tissue during fixation, only a retrieval
portion. DISPOSAL ● Slides: sharp container
○ Upon receiving, we must check and ● Paraffin blocks: pathologic waste bags
ensure that the container would last for a ● Slides may be used by resident
longer period of time. pathologists and med students; may be
○ Leak-proof since formalin has the used for further researches
tendency to spill this sample will not be ● Patient confidentiality
preserved. ○ Labels must be removed
● Group chronologically for ease of finding REQUESTS ● To be viewed by another institution
○ Storage of tissues must be 10 years so FOR ○ When patient is not convinced, patients
our storage room in histopath is big. BLOCKS may borrow the blocks and slides
○ Ex. A minimum of 15 samples per day AND SLIDES ● Logbook must be available
resulting to high amount of samples in a ● Slides are transported with damage and
year. breakage prevention
○ So it should be big enough to accomodate ● Deposit and charging for new slides are
the number of tissues in histopath. customary practices
○ The purpose of this is for easy retrieval ○ Deposit charges will be returned to the
when needed as evidence or additional patients once the samples are returned
tests ● Preserve the original state of the material
● Must be free from human and animal
interference III. RECOMMENDED MINIMUM RETENTION TIME OF
DISPOSAL ● Placed in biohazard bags for incineration or RECORDS, REPORTS, SPECIMENS
burial
○ We must first remove the tissues from the ● This is suggested by the National Pathology Accreditation
container and only them tissue will be Advisory Council (NPAAC)
buried for it to be decomposed. → This includes the following number of years that records,
RETURNING ● Guidelines must be according to the samples and other documents must be stored.
OF hospital
SPECIMEN ○ Before, patients can bring their samples Table 3. Records
TO with them at home given that the sample RECORDS STORAGE
PATIENT is benign (if malignant, it is needed for
Request, Accession logs, Maintenance 2 years
further reading)
and Quality Control logs
○ Now, hospitals are not allowing the
patients anymore because of formalin. So, BB Quality Control 5 years
laboratories are tasked to properly BB employee signatures, patient 10 years
dispose the samples. records, donor and recipient records
● Ensure proper documentation Records of indefinitely/permanently Indefinitely
● Limbs and fetuses deferred donors, forensic accession logs
○ religious purposes
○ Miscarriage: the samples are called the Table 4. Report
“products of conception” REPORTS (RESULTS) STORAGE
■ Taken during the process of raspa. Clinical Pathology (e.g. CC, Hema) 2 years
■ Mothers are allowed to bring those Anatomical/Surgical Pathology 10 years
tissues at home Cytogenetics (final resorts and 20 years
● Bullets, breast implants, and foreign photographs)
bodies Forensic Autopsy Indefinitely
○ evidences for crime; released to police
authorities and with properly documented
Table 5. Specimen
Chain of Custody (COC) form
○ Separate logbooks SPECIMEN) STORAGE
Urine 24 hours
Serum and other body fluids 48 hours
Microbiology and blood smears (routine) 7 days
BB donor/recipient (blood) 7 days
post-transfusion
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 SCHOOL OF MEDICAL
LABORATORY SCIENCE
HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC.)
SAN PEDRO COLLEGE – MAIN
CAMPUS
Instructor’s Name: Ma’am. Doren Venus Otod, RMT
AY 2022 – 2023 - 2ND SEMESTER WEEK 05 - ROUTINE PROCEDURE INSTRUMENTATIONS

Surgical Tissues 10 years

Cytogenetic slides 3 years
Cytology slides (e.g. pap) 5 years
Tissue, BM, FNA Slides 10 years
Paraffin blocks 10 years
Forensic: Blocks, slides Indefinitely

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