CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED

COLLEGE OF MEDICAL TECHNOLOGY

LABORATORY
WORKSHEET IN
HISTOPATHOLOGY

HARLEY ROSE B. BAUTISTA, RMT, MPh

CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY
A.Y 2023-2024

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

RISK MANAGEMENT AND SAFETY PRECAUTIONS

Risk management is a process of ensuring and maintaining personal and environmental health
and safety in the laboratory.
How?
• By identifying all potential hazards.
• By following detailed standard operating procedures (SOP)
• Personal hygiene practices
• Procedures for handling hazardous substances and materials

Types of Hazards
• Chemical hazard
• Physical hazard
• Biological hazard
• Electrical hazard
• Sharps hazard
• Fire hazard
• Radioactive hazard

CHEMICAL HAZARDS
• Potential harm or injury comes from misuse and mishandling of chemicals.
• May cause fire, irritations, allergic reactions
Examples:
 Cleaning agents/disinfectants
 Reagents
 Drugs
 Gases
PRECAUTIONS:
1. Use of proper PPE
2. Proper hand hygiene (immediately after exposure)
3. Always CHECK THE LABELS.
• Name of chemical
• Expiration date
• Hazard warnings and safety procedures
4. Proper storage and usage
• Chemicals must be placed below countertop height.
• Never mix chemicals.
• Use of fume hood

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

PHYSICAL HAZARDS
• Mostly are self-accidents.
• Includes ergonomic hazards of lifting, pulling, pushing, and repetitive tasks.
• Improper postures for a long period
• Improper way of lifting heavy objects
• Other unnoticed are electrical, mechanical, thermal, and acoustic that ignoring these may
soon cause serious consequences.

Other examples:
• Slippery floor
• Running
• Unstable objects
• Heavy objects
• Broken glassware, slides, sharps
• Dangling jewelries
• Long hair

BIOLOGICAL HAZARDS
• Anything that cause disease to humans
• Includes infectious agents, toxins, specimens, contaminated solutions or objects
• Always treat human samples as potentially biohazardous
Examples:
• Blood/tubes
• HIV
• HbsAg
• Urine and other body fluids
• Aerosols, droplets
• Sputum
• M.tuberculosis
• Stool
• Parasites and ova
• Fresh tissues

Other biohazards (biomaterials)

Once exposed to human/human samples:
• Needles
• Slides
• Pipette tips
• Masks
• Gloves
Precautions
• Use of proper PPE
• Proper hand hygiene
• Proper disposal of samples and of materials
• Slides and needles must be disposed of in a puncture-proof container.
• Careful handling of human samples

LABORATORY WORKSHEET HISTOPATHOLOGY

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• Disposed in the yellow plastic bags.

• Respiratory hygiene/cough etiquette
• Use of masks and/or facing away from other people

ELECTRICAL HAZARD
• Can result from faulty electrical equipment or wiring, or improper use of cords or
extensions.
• May cause fire, shock, explosions, electrocution.
• Life-threatening but can easily be avoided.
Precautions
• Do not touch or operate electrical equipment, or wiring if your hands are wet.
• Wet equipment should be unplugged and should be allowed to dry completely before
reusing
• Equipment must also be unplugged before cleaning.
• Overloaded circuits must be reported
When electrical shock occurs
• Do not touch the person with bare hands
• Turn off the circuit breaker, unplugging the equipment or cord
• Moving the equipment or the person must be done using nonconductive materials such as
glass or wood
• Victim should receive immediate medical attention
• Cardiopulmonary resuscitation (CPR) if necessary

Other Hazards:

FIRE HAZARDS
• Flammable liquids or gasses
• Electrical appliances, equipment, exposed wirings
• Combustible Chemicals
Must remember:
When a fire is discovered, all employees are expected to take the actions in the acronym RACE:
Rescue—rescue anyone in immediate danger
Alarm—activate the institutional fire alarm system
Contain—close all doors to potentially affected areas
Extinguish/Evacuate—Attempt to extinguish the fire, if possible or evacuate, closing the door
• Familiarize yourselves with the fire exits and fire extinguisher locations

LABORATORY WORKSHEET HISTOPATHOLOGY

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• When using a fire extinguisher remember, PASS:

Pull the pin
Aim at the base of the fire
Squeeze handles
Sweep nozzle side to side

NOTE:
NOT ALL TYPES OF FIRES CAN BE EXTINGUISHED BY WATER!

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

GENERAL LABORATORY SAFETY RULES

1. Make sure you know where your lab's safety equipment—including first aid kit(s), fire
extinguishers, eye wash stations, and safety showers—is located and how to properly use it.
2. Open flames should never be used in the laboratory unless you have permission from a
qualified supervisor.
3. Make sure you are aware of where your lab's exits and fire alarms are located.
4. Always work in properly ventilated areas.
5. Do not chew gum, drink, or eat while working in the lab.
6. Laboratory glassware should never be utilized as food or beverage containers.
7. Each time you use glassware, be sure to check it for chips and cracks. Notify your lab
instructor of any damaged glassware so it can be properly disposed of.
8. Never use lab equipment that you are not approved by your instructor to operate.
9. If an instrument or piece of equipment fails during use, or isn't operating properly, report
the issue to the instructor right away. Never try to repair an equipment problem on your own.
10. Do not work alone in the lab. Always ask for the supervision of your instruction when
doing an experiment.
11. Never leave an ongoing experiment unattended.
12. Do not pipette by mouth.
13. Make sure you always follow the proper procedures for disposing of lab waste.
14. Report all injuries, accidents, and broken equipment or glass right away, even if the
incident seems small or unimportant.
15. In the event of a chemical splashing into your eye(s) or on your skin, immediately flush
the affected area(s) with running water for at least 20 minutes.
16. If you notice any unsafe conditions in the lab, let your instructor know as soon as possible.

PERSONAL PROTECTION SAFETY RULES

1. When working with equipment, hazardous materials, glassware, heat, and/or chemicals,
always wear face shields or safety glasses.
2. When handling any toxic or hazardous agent, always wear the appropriate gloves.
3. When performing laboratory experiments, you should always wear a smock or lab coat.
4. Before leaving the lab or eating, always wash your hands.
5. After performing an experiment, you should always wash your hands with soap and water.
6. When using lab equipment and chemicals, be sure to keep your hands away from your body,
mouth, eyes, and face

LABORATORY WORKSHEET HISTOPATHOLOGY

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WHEN USING MICROSCOPE

Wear Protective Clothing
Before you begin using a microscope, put on protective clothing. This includes a lab coat,
a pair of safety glasses and a pair of disposable gloves. The slides you are examining under the
microscope could contain dangerous chemicals or biological material so, it is important to protect
all parts of your body.
Carry with Two Hands
To prevent damage to the microscope and to protect yourself from injury, always carry the
microscope with two hands. Place one hand on the arm of the microscope and place the other hand
underneath the base of the microscope. This method will give the microscope the most support. If
you are walking with it, always hold it up high to avoid hitting tables or chairs. If you are not
careful with the microscope and hit something, you could cause small pieces of the microscope to
break off and create a tripping hazard.
Do Not Touch the Lens
Never touch the lens of the microscope with your bare hands. This could damage the
functioning of the microscope. Instead, use a special lens paper to clean it. You may also use a soft
cloth dipped in a small amount of isopropyl alcohol to clean the lens, according to the Utah State
Office of Education.
Do Not Look into the Light
If you are using a microscope with a mirror, never use direct sunlight as a light source. This
could cause eye damage when looking into the microscope. If you are using a microscope with a
light, do not look directly into the light. This could cause eye damage as well. Also, remember to
turn off the light of the microscope when it is not in use.
Be Cautious Handling Slides
Always be careful when handling glass slides and cover slips. If the slide or cover slip
breaks, use protective gloves to clean up the broken glasses. This will help prevent cuts and
contamination from slide contents. Dispose of the glass in a designated sharps container in the
laboratory.
Storing
After you have finished using the microscope, always clean the slides off and wipe down
the microscope with a damp cloth. Microscopes should be stored on the lowest objective with the
nosepiece turned down to its lowest position. If you are using a microscope with a light, remember
to turn off the light before unplugging it from the outlet. Then, cover the microscope with a dust
cover and return it to the designated storage area.

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

CELL INJURIES, DEATH, AND INFLAMMATION

Basic terminologies:
Histo- tissue
Pathos- pain
Histopathology- study of tissue diseases, deals with functions and structures.
Other term: Pathobiology

CONCEPTS OF PATHOLOGY
 Etiology- origin
 Disease process
 Morphological changes
 Clinical significance

DIVISIONS OF PATHOLOGY
 Gross and Microscopic Pathology
 Anatomical Pathology (Autopsy)
 Clinical Pathology (CM, CC, Hematology, Microbiology, Blood Bank)

CELL ADAPTATION- the ability of cells to respond to various types of stimuli

• Atrophy- shrinkage in the size of the cells
• Physiologic Condition: decreased work load
• Pathologic Condition: degeneration of muscles
• Hypertrophy- increased size of cells; no new cells
• Physiologic condition: exercise
• Pathologic condition: obstruction of heart valves (increased work load)
• Hyperplasia- increased cell number; with cell division
• Physiologic condition: hormonal (breast and uterus)
• Pathologic condition: viral infection (HPV: warts)

Abnormal Cell Growths

• Aplasia- defective or incomplete cell growth
• Seen commonly in kidneys, gonads, adrenals
• Agenesia- non-appearance of cells
• Atresia- failure of an organ to form an opening
• Biliary atresia
• Hypoplasia- failure of an organ to fully mature

Other Abnormal Cell Growths

• Metaplasia
- one cell type changes to another cell type
- Adaptation to abnormal localized environmental changes or to new demands
• Smokers: air passage is from columnar to squamous
• Barrette's Esophagus: squamous to columnar due to acid reflux
• Dysplasia- pleomorphism
- also known as Atypical Hyperplasia
- Abnormal growth and differentiation
- Variation in size, shape and orientation
• Seen in Preneoplastic lesion (a stage in the cellular evolution to cancer)
• Anaplasia
- also known as Undifferentiated cells
- From more to less differentiated forms of cell or tissue

LABORATORY WORKSHEET HISTOPATHOLOGY

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CELL INJURY/ CELL DAMAGE

Causes:
 Genetic defects
 Oxygen deprivation
 Physical agents
 Chemical agents
 Immunologic reactions
 Infectious agents
 Nutritional agents

Types of cell injury

• Reversible cell injury
• Cellular swelling and vacuoles formation
• Hydropic changes: edematous
• Fatty changes: fatty liver
• Irreversible cell injury/ Necrosis
• Leads to cell death
• Cell nucleus
• Pyknosis: shrinkage
• Karyolysis: fading
• Karyorrhexis: fragmentation

CELL DEATH
• Necrosis- changes produced by enzymatic digestion
- More on environmental factors that trigger the release of enzymes
• Apoptosis- programmed cell death
- Method of the body that blocks potential cancer cells.

Patterns of Necrosis in Tissues or Organs

1. Coagulative necrosis- cell death caused by ischemia
• Heart attack
2. Liquefactive necrosis- complete destruction of cells; commonly seen in brains. Due to high
levels of lysosomes, brain tissues are liquefied later on turning into pus.
• Cerebral infarction
3. Caseous necrosis- forms cheesy and white appearance
• Tuberculosis (MTB)
4. Fat necrosis- fat destruction due to the release of pancreatic lipases; Chalk white appearance
• Breast fat necrosis due to injury, surgery, trauma
• Pancreatic trauma, pancreatic carcinoma
5. Gangrenous necrosis- due to lack of blood flow or a serious bacterial infection.
Types of Gangrene:
• Dry gangrene- caused by arterial occlusion
-tissue shrinks and turns black
-poor blood circulation

LABORATORY WORKSHEET HISTOPATHOLOGY

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• Wet gangrene- result of venous occlusion

-certain bacteria invades the tissue results to swelling
-there is presence of pus

SOMATIC DEATH- death of an organism as a whole

A. Primary changes
• Respiratory system
• Circulatory system
• Central Nervous system
B. Secondary changes- seen in post-mortem examination
• Algor mortis
• Rigor mortis
• Livor mortis
• Putrefaction
• Autolysis
• Post-mortem clotting
• Desiccation

1. Algor mortis- cooling of the body

• cooling of the body to equalize the environment
• Faster in cold weather in lean malnourished individual
• Delayed in infectious disease following increased temperature
2. Rigor mortis- stiffening of the body
• Stiffness
• Occurs first in the muscles of neck and head
• Starts at 2-3hrs after death; completes at 6-8hrs
• Remains stiff for 12-36hrs; lasts for 3-4 days
• Faster in warm environment; infants
• Delayed in cold temperature; obese individuals
3. Livor mortis- purplish discoloration of the body
• Caused by the sinking of the blood into the capillaries of the dependent part of the body
• Due to stasis
• Discoloration disappears upon application of pressure
• Blood oozes if incised
Difference with Ecchymosis
• Ecchymosis is due to trauma
• Does not disappear after application of pressure
• Blood does not ooze if incised
4. Putrefaction- decomposition of the body due to bacterial action
- Color changes to greenish blue due to iron sulfide
- Muscles softening
- Corneal retraction
- Loss of rigor mortis
- Skin peeling, swelling of face
- Clostridium cadaveris- described as putrefying flora

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5. Autolysis- liberation of hydrolytic enzymes

- Self digestion of the cells
- Enzymes are released from the cells due to the lack of normal processes in the body.
- Putrefactive bacteria enhances cellular destruction
6. Post-mortem Clotting- occurs slowly or immediately after death.
- Setting and separation of red blood cells from the fluid phase
- Must be differentiated from Ante-mortem clot (before death)
PMC:
Definite setting of RBCs from fluid phase
Currant jelly: sedimentation of RBC; assume shape of blood vessel.
Consistency: Rubbery
AMC:
Fibrin clot is tangled
Seldom resembles the blood vessels
Friable
7. Desiccation- drying or wrinkling of the cornea and anterior chamber
- Moisture is gradually fades.

INFLAMMATION- “-itis"
Consists of :
1. Vascular response
2. Migration of cells
3. Activation of leukocytes
4. Systemic reactions
It is a protective response of the body to remove the initial cause of injury and its consequences
or products
Terminated when the inciting agent is eliminated, and the mediators are degenerated.

Cardinal signs of Inflammation

1. Rubor – redness
2. Tumor – swelling: increased vascular permeability; fluid leaks to tissues
3. Calor – heat
4. Dolor – pain: release of prostaglandins
5. Functio Laesa – loss of function

Types of Inflammation (Acute, Chronic)

1. Acute inflammation- Rapid response to injurious agents
- Hallmark sign: increased vascular permeability
- Infiltration by Polymorphonuclear cells
- Chemotaxis: migration of cells to the site of injury
Examples of causes:
 Allergic reactions- rhinitis
 Infection- folliculitis
 Chemical irritants- dermatitis

Acute inflammation results to edema and exudation

• Edema- excess fluid to the interstitial tissue or to the serous cavities
• Exudation- escape of fluid proteins and blood cells from the vascular system into the
interstitial tissue and serous cavities

2 types of fluids:
i. Exudate- such as pus. Fluid with cells, proteins, solid materials that leaks into the tissue.
ii. Transudate- fluid the leaks into the tissue due to systemic conditions.

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2. Chronic Inflammation- an inflammation of prolonged duration

- infiltration of mononuclear leukocytes
- Tissue destruction
- Attempts at healing by connective tissues replacements
- Angiogenesis- formation of new blood vessels
Example:
 Autoimmune diseases- rheumatoid arthritis

Granulomatous inflammation
-a pattern of chronic inflammation
-characterized by formation of granuloma which forms lumps
Seen in:
Tuberculosis
Leprosy
Syphilis
Schistosomiasis

HEALING- resolution of inflammation

• Simple resolution:
• No destructed normal tissue
• Agent neutralized
• Vessels return to normal state
• Excess fluid is reabsorbed
• Regeneration- replacement of necrotic tissue with a new tissue

NEOPLASIA- process of “new growth”

• Neoplasm/tumor
Oncology- study of tumor/neoplasm
Cancer- common term for all malignant tumor
Neoplasia can either be
• benign
• malignant “cancerous”

Benign- usually localized and doesn’t metastasize

- Amendable to surgery
- Uses the suffix “-oma”

Benign tumors of:

 Mesenchymal cells- connective tissue
• Fibroblast- fibroma
• Chondroblast- chondroma
• Osteoblast- osteoma
• Lipoblast- lipoma
 Epithelial cells:
• Glandular pattern- adenoma
• Finger-like or warty projections- papilloma
• Large cystic mass- cystadenoma

Malignant- invasive and destroys adjacent areas.

Uses the suffix “-sarcoma” or “-carcinoma”
Malignant tumors of:
 Mesenchymal cells:
• Fibrosarcoma
• Chondrosarcoma
• Osteosarcoma

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• Leukemia/lymphoma
 Epithelial cells:
• Adenocarcinoma
• Squamous cell carcinoma

Pathways of spreading malignancy:

-carcinoma: through lymphatic spread
-sarcoma: hematogenous spread
-common in the liver and lungs

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TYPES OF BIOPSIES

Histology is the microscopic study of normal tissues.

Histopathology is the microscopic study of tissues affected by a disease.
The procedures used to prepare these tissues are termed as histologic or histopathologic
techniques and it starts with the collection of samples.
How do we acquire these samples?
Through surgery, biopsy, or autopsy.
From large sizes of samples or whole organs to tiny fragments of tissues.

What is a Biopsy?
Bio- means life
Psy- to see, appearance
A process of excision or examination of tissue samples coming from a living person.
The gold standard in diagnosing malignant disease.

Types of biopsies
• Fine needle aspiration biopsy (FNAB)- least invasive, only uses the smallest needle to
aspirate cells from the area of abnormality.

• Core needle biopsy- removes not only cells but also a small amount of surrounding tissue.

• Incisional biopsy- takes only a small part of an organ, tissue, or tumor.

If the organ, tissue, or tumor is found to be cancerous, additional surgery is needed to remove the
whole tumor.
• Excisional biopsy- generally removes the whole tumor.

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• Punch biopsy- used for obtaining diagnostic full-thickness of skin specimens. The
procedure uses a circular blade that is rotated down to epidermis, dermis, and into the
subcutaneous fats forming a 3-4mm cylindrical core of skin tissue sample.

• Cutaneous biopsy- also used for skin biopsy. For diagnosing melanoma
• Shave biopsy- usually done in the skin. Fragments are “shaved” from the surface.

• Wedge biopsy- an incision from a solid organ “wedge” shaped.

• Curettings- uses scoops or spoon-like apparatus to remove tissue growths from the body
cavity such as endometrium or cervical canal.
• Exfoliative cytology- a microscopic study of desquamated cells from epithelial surfaces.
Used to diagnose cervical cancer.
An example is a Pap smear.

LABORATORY WORKSHEET HISTOPATHOLOGY

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Tissue samples usually come in the laboratory in fixative such as formalin or sometimes they
arrive fresh and must be immediately fixed.
They arrive in the lab together with the patient’s basic information, clinical history, sample
description, and site of origin.

Upon receiving, they are then accessioned or assigned by a number as an identifier- an

important step in the pre-analytical phase to avoid the risk of mislabeling misidentification.
Some of the steps in accessioning a sample.
Surgery
S23- 0001
Autopsy
A23- 0001
Cytology
C23- 0001

Where “23” is for the year, the procedure was done, and 0001 is for the specimen number.

Histologic or Histopathologic techniques are done in either fresh or preserved tissues.

The difference is that fresh tissues are temporary and are only observed for a shorter period,
prone to lysis and rapid changes in cell structure.
Only done when there’s a need for immediate evaluation.
On the other hand, preserved tissues are permanent, better, and more effective means of
studying tissues. They are also best for long-time keeping, due to having stained, and mounted in
glass slides with coverslips.

Methods for Fresh Tissue Examination

1. Teasing or Dissociation
- The selected tissue is immersed in NSS or Ringer’s solution in a watch glass or petri dish
and dissected with a needle. Pieces of tissues are viewed in a microscope in a wet
preparation underneath a coverslip.
- Cells are examined in a living state allowing movement and mitotic division to be observed.

2. Squash preparation (Crushing)

- A process where a small tissue is crushed or forcibly compressed between two slides or a
coverslip.
- If necessary, a Supravital stain may be used by allowing it to be absorbed by the cells or
tissue.

3. Smear preparation
- The general rule is smears are made by spreading a portion of the specimen over the surface
of the slide using a platinum loop.
- Alternatively making a smear using a second slide to obtain a uniform distribution of
secretion or sample.
Three ways of Smear preparation:
 Streaking- Using an applicator stick or platinum loop, the sample (body fluid) is
rapidly and gently applied in a direct or zigzag line throughout the slide.
 Spreading- a selected portion of a sample is transferred to a clean slide teasing the
mucous strands apart with an applicator stick. More tedious than streaking.
Commonly done with sputum, mucoid secretions, and bronchial aspirates.
 Pull-apart- done by dropping a sample into a clean slide and facing it to another slide
allowing to first to disperse evenly in both slides. Pulling both slides in opposite
directions.

4. Touch preparation

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- Smear preparation whereby the surface of a freshly cut tissue is brought into contact and
pressed on the surface of a clean glass slide allowing cells to be transferred into the slide.

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ALL ABOUT YOURSELF

NAME: ______________________________________________________________________________
YEAR AND SECTION: _________________________________
GROUP NUMBER: ____________________________________
CONTACT NUMBER: __________________________________

REASONS WHY YOU TOOK BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY

HOW DO YOU SEE YOURSELF AFTER COLLEGE?

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GROUP PHOTO

NAMES OF GROUPMATES:

SELF PHOTO

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Date: ______________ Score: ____________

Laboratory Activity No. 1
GROSS EXAMINATION / MACROSCOPIC EXAMINATION OF SPECIMEN AND
TISSUE SAMPLING
Gross examination or “grossing” is the process by which the pathology specimens are inspected
with the bare eye to obtain diagnostic information while being processed for further microscopic
examination.
Gross examination of surgical specimens is typically performed by a pathologist, or by a
pathologist's assistant working within a pathology practice. Individuals trained in these fields are
often able to gather diagnostically critical information in this stage of processing, including the
stage and margin status of surgically removed tumors.
The initial step in any examination of a clinical specimen is confirmation of the identity of the
[patient]and the anatomical site from which the specimen was obtained. Sufficient clinical data
should be communicated by the clinical team to the pathology team in order to guide the
appropriate diagnostic examination and interpretation of the specimen - if such information is not
provided, it must be obtained by the examiner prior to processing the specimen.
There are usually two end products of the gross examination of a surgical specimen. The first is
the gross description, a document that serves as the written record of the examiner's findings and
is included in the final pathology report. The second product is a set of tissue blocks, typically
postage stamp-sized portions of tissue sealed in plastic cassettes, which will be processed into
slides for microscopic examination. Since only a minority of the tissue from a large specimen can
reasonably be subject to microscopic examination, the success of the final histological diagnosis
is highly dependent on the skill of the professional performing the gross examination. The gross
examiner may sample portions of the specimen for other types of ancillary tests as diagnostically
indicated; these include microbiological culture, flow cytometry, cytogenetics, or electron
microscopy.
OBJECTIVE
• At the end of this activity, the students are able to properly demonstrate and perform
macroscopic examination and sectioning of histopathologic samples.
MATERIALS
• Liver as specimen
• Pencil
• Ruler or tape measure
• Scalpel or cutter
• Cassettes
• Forceps
• Personal protective equipment
• Chopping board
• Newspaper
PROCEDURE
1. Wear your personal protective equipment.
2. Identify the type of specimen.
3. Record the size in centimeters (length, width, thickness) the consistency, shape, and color
of the specimen.
4. Using a scalpel or a cutter, carefully cut a small piece of sample with a length and width of
not more than 5mm and a thickness of not more that 4mm.
5. Using forceps, put the tissue section in the cassette, secure and properly label it with pencil.

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GROSS EXAMINATION
Type of specimen: __________________
Size of specimen: (in centimeter)
o Length: ______________
o Width: _______________
o Thickness: ___________
Consistency: _______________________
Shape: ____________________________
Color: ____________________________

PICTURES/DOCUMENTATION
Picture of Specimen: Gross appearance

Tissue sample in the cassette

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PERFORMANCE EVALUATION CHECKLIST

Laboratory No.: ____
Title: ________________________________________________________________________
Name: _________________________________________
Year and section: ________________________________

Rate the student’s performance by putting a number in the appropriate box using the following
criteria:

5- Excellent = performs task competently and mastery is greatly evident.

4- Very satisfactory = performs task competently and mastery is moderately evident.
3- Satisfactory = performs task competently and mastery is minimally evident.
2- Fair = performs task competently and mastery is less evident.
1- Poor = performance needs further improvement

CRITERIA RATING
Set up and equipment care
Following instructions
Proper examination of specimen
Safety
Clean up

Final rating: ___________

Comment/s:____________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

_____________________ _____________________ ___________

Student’s signature Instructor Date

LABORATORY WORKSHEET HISTOPATHOLOGY

RMT317/0922-002
 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

Date: ______________ Score: ____________

Laboratory Activity No. 2
EXAMINATION OF FRESH TISSUES
OBJECTIVES
• By the end of this activity, the students are able to properly demonstrate and perform
fresh tissue examination.
• And to be able to determine the importance of performing fresh tissue examination.
MATERIALS
• Liver as specimen
• Scalpel or cutter
• Needle
• Applicator sticks
• Forceps
• Slides
• Coverslips
• Inoculating loop
• Normal Saline Solution
• Personal protective equipment
• Chopping board
• Newspaper
Histologic or Histopathologic techniques are done in either fresh or preserved tissues. The
difference is that preserved tissues are permanent, better, and more effective means of studying
tissues. They are also best for long-time keeping, due to having stained, and mounted in glass
slides with coverslips.
On the other hand, fresh tissues are temporary and are only observed for a shorter period, prone
to lysis and rapid changes in cell structure, and examinations are only done when there’s a need
for immediate evaluation and diagnosis. Another advantage of examining fresh tissues is the
observation of protoplasmic activities such as movements, mitosis, and phagocytosis.

Methods for Fresh Tissue Examination

1. Teasing or Dissociation
- The selected tissue is immersed in NSS or Ringer’s solution in a watch glass or petri dish
and dissected with a needle. Selected pieces of tissues are transferred carefully to a slide
and mounted as wet preparation underneath a cover glass. It is either stained with
Supravital stain or unstained using phase contrast or bright field microscopy. Cells are
examined in a living state allowing movement and mitotic division to be observed.

2. Squash preparation (Crushing)

- A process where a small tissue (not more than 1 mm in diameter) is crushed or forcibly
compressed between two slides or a coverslip. If necessary, a Supravital stain may be used
by allowing it to be absorbed by the cells or tissue.

3. Smear preparation
- The general rule is smears are made by spreading a portion of the specimen over the surface
of the slide using a platinum loop. Alternatively, make a smear using a second slide to
obtain a uniform distribution of secretion or sample. Too-thin or too-thick smears have to
be avoided. This is useful for preparing smears of thick secretions such as serous fluids,
sputum, enzymatic lavage, from the gastrointestinal tract, and blood smears. This is
particularly useful in cytological examinations, particularly cancer diagnosis.
Three ways of Smear preparation:

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

 Streaking- Using an applicator stick or platinum loop, the sample (body fluid) is
rapidly and gently applied in a direct or zigzag line throughout the slide, attempting
to obtain a uniform distribution of secretions. Avoid too thick or too thin smears.
 Spreading- a selected portion of a sample is transferred to a clean slide teasing the
mucous strands apart with an applicator stick. More tedious than streaking.
Commonly done with sputum, mucoid secretions, and bronchial aspirates.
 Pull-apart- done by dropping a sample into a clean slide and facing it to another slide
allowing to first to disperse evenly in both slides. Pulling both slides in opposite
directions.

4. Touch preparation
- Smear preparation whereby the surface of a freshly cut tissue is brought into contact and
pressed on the surface of a clean glass slide allowing cells to be transferred into the slide
and examined using Phase Contrast microscopy or stained using light microscopy.

PICTURES/DOCUMENTATION

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

PERFORMANCE EVALUATION CHECKLIST

Laboratory No.: ____
Title: ________________________________________________________________________
Name: _________________________________________
Year and section: ________________________________

Rate the student’s performance by putting a number in the appropriate box using the following
criteria:

5- Excellent = performs task competently and mastery is greatly evident.

4- Very satisfactory = performs task competently and mastery is moderately evident.
3- Satisfactory = performs task competently and mastery is minimally evident.
2- Fair = performs task competently and mastery is less evident.
1- Poor = performance needs further improvement

CRITERIA RATING
Set up and equipment care
Following instructions
Proper examination of specimen
Safety
Clean up

Final rating: ___________

Comment/s:____________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

_____________________ _____________________ ___________

Student’s signature Instructor Date

LABORATORY WORKSHEET HISTOPATHOLOGY

RMT317/0922-002
 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

Date: ______________ Score: ____________

Laboratory Activity No. 3
HISTOPATHOLOGIC TECHNIQUES (PREPARATION OF TISSUE BLOCKS)
OBJECTIVES
• At the end of the activity, the students are able to demonstrate the proper histopathologic
techniques from fixative to embedding,
• To be able to identify the importance to each step of histopathologic techniques.

MATERIALS
• Fresh tissue specimen
• Tissue cassette
• Formalin
• Beaker
• 70% alcohol
• 90% alcohol
• 100% alcohol
• Xylene
• Paraffin wax
• Paper box as mold
• Personal protective equipment

PROCEDURE
Fixation
The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin)
in a 1:20 ratio. This will slowly penetrate the tissue causing chemical and physical changes that
will harden and preserve the tissue and protect it against subsequent processing steps. There are a
limited number of reagents that can be used for fixation as they must possess particular properties
that make them suitable for this purpose. For example, tissue components must retain some
chemical reactivity so that specific staining techniques can be applied subsequently. Formalin,
usually as a phosphate-buffered solution, is the most popular fixative for preserving tissues that
will be processed to prepare paraffin sections. Ideally, specimens should remain in fixative for
long enough for the fixative to penetrate into every part of the tissue and then for an additional
period to allow the chemical reactions of fixation to reach equilibrium (fixation time). Generally,
this will mean that the specimen should be fixed for between 6 and 24 hours. Most laboratories
will use a fixative step as the first station on their processor. Following fixation, the specimens
may require further dissection to select appropriate areas for examination. Specimens that are to
be processed will be placed in suitably labeled cassettes (small, perforated baskets) to segregate
them from other specimens. The duration of the processing schedule used to process the specimens
will depend on the type and dimensions of the largest and smallest specimens, the particular
processor employed, the solvents chosen, the solvent temperatures, and other factors. The
following example is based on a six-hour schedule suitable for use on a Leica Peloris™ rapid tissue
processor.

Dehydration
Because melted paraffin wax is hydrophobic (immiscible with water), most of the water in a
specimen must be removed before it can be infiltrated with wax. This process is commonly carried
out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration
until pure, water-free alcohol is reached. Ethanol is miscible with water in all proportions so that

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

the water in the specimen is progressively replaced by the alcohol. A series of increasing
concentrations is used to avoid excessive distortion of the tissue.

A typical dehydration sequence for specimens not more than 4mm thick would be:
1. 70% ethanol 15 min
2. 90% ethanol 15 min
3. 100% ethanol 15 min
4. 100% ethanol 15 min
5. 100% ethanol 30 min
6. 100% ethanol 45 min

At this point all but a tiny residue of tightly bound (molecular) water should have been removed
from the specimen.

Clearing
Unfortunately, although the tissue is now essentially water-free, we still cannot infiltrate it with
wax because wax and ethanol are largely immiscible. We therefore have to use an intermediate
solvent that is fully miscible with both ethanol and paraffin wax. This solvent will displace the
ethanol in the tissue, then this in turn will be displaced by molten paraffin wax. This stage in the
process is called “clearing” and the reagent used is called a “clearing agent”. The term “clearing”
was chosen because many (but not all) clearing agents impart an optical clarity or transparency to
the tissue due to their relatively high refractive index. Another important role of the clearing agent
is to remove a substantial amount of fat from the tissue which otherwise presents a barrier to wax
infiltration.
A popular clearing agent is xylene and multiple changes are required to completely displace
ethanol.
A typical clearing sequence for specimens not more than 4mm thick would be:
1. xylene 20 min
2. xylene 20 min
3. xylene 45 min

Wax infiltration/Impregnation
The tissue can now be infiltrated with a suitable histological wax. Although many different
reagents have been evaluated and used for this purpose over many years, the paraffin wax-based
histological waxes are the most popular. A typical wax is liquid at 60°C and can be infiltrated into
tissue at this temperature then allowed to cool to 20°C where it solidifies to a consistency that
allows sections to be consistently cut. These waxes are mixtures of purified paraffin wax and
various additives that may include resins such as styrene or polyethylene. It should be appreciated
that these wax formulations have very particular physical properties which allow tissues infiltrated
with the wax to be sectioned at a thickness down to at least 2 µm, to form ribbons as the sections
are cut on the microtome, and to retain sufficient elasticity to flatten fully during flotation on a
warm water bath.

A typical infiltration sequence for specimens not more than 4mm thick would be:
1. wax 30 min
2. wax 30 min
3. wax 45 min

Embedding or Blocking
Now that the specimen is thoroughly infiltrated with wax, it must now be formed into a “block”
which can be clamped into a microtome for section cutting. This step is carried out using an
“embedding centre” where a mould is filled with molten wax and the specimen placed into it. The

LABORATORY WORKSHEET HISTOPATHOLOGY

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

specimen is very carefully orientated in the mould because its placement will determine the “plane
of section”, an important consideration in both diagnostic and research histology. A cassette is
placed on top of the mould, topped up with more wax and the whole thing is placed on a cold plate

to solidify. When this is completed, the block with its attached cassette can be removed from the
mould and is ready for microtomy. It should be noted that, if tissue processing is properly carried
out, the wax blocks containing the tissue specimens are very stable and represent an important
source of archival material.

PICTURES/DOCUMENTATION

LABORATORY WORKSHEET HISTOPATHOLOGY

RMT317/0922-002
 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

PERFORMANCE EVALUATION CHECKLIST

Laboratory No.: ____
Title: ________________________________________________________________________
Name: _________________________________________
Year and section: ________________________________

Rate the student’s performance by putting a number in the appropriate box using the following
criteria:

5- Excellent = performs task competently and mastery is greatly evident.

4- Very satisfactory = performs task competently and mastery is moderately evident.
3- Satisfactory = performs task competently and mastery is minimally evident.
2- Fair = performs task competently and mastery is less evident.
1- Poor = performance needs further improvement

CRITERIA RATING
Set up and equipment care
Following instructions
Preparation of sample
Safety
Clean up

Final rating: ___________

Comment/s:____________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

_____________________ _____________________ ___________

Student’s signature Instructor Date

LABORATORY WORKSHEET HISTOPATHOLOGY

RMT317/0922-002
 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

Date: ______________ Score: ____________

Laboratory Activity No. 4
TISSUE SLIDE PREPARATION

OBJECTIVES
• At the end of this activity, students are expected to be able to demonstrate how to use the
microtome with safety and proper precaution.
• To be able to know the parts and functions of a microtome.
• To be able to know and demonstrate the histopathologic steps in preparation of tissue slide
from microtomy, staining, to mounting.

MATERIALS and EQUIPMENTS

• Tissue block
• Microtome
• Brush
• Applicator sticks
• Warm water bath
• Slides
• Oven
• Hematoxylin and Eosin Staining
• Xylene
• 100% alcohol
• 95% alcohol
• Water
• Mounting medium
• Cover slip
• Personal protective equipment

PROCEDURE

Trimming
Trimming is a process by which the technologist removes the excess wax of the tissue block.
After the tissues are embedded and the wax has solidified, the block is removed from the mold,
accession number is noted, the excess wax is cut off from the block to expose the tissue surface in
preparation for actual cutting. Trim the sides, top and bottom of the tissue block until perfect level
is achieved and all sides are parallel, almost to the edge of the tissue. Care should be taken to avoid
removing too much tissue.

Section-cutting (Microtomy)
Once the tissues have been embedded, they must be cut into sections thin enough to be placed on
a slide. This is done with a microtome. Good microtomy techniques are the one that minimizes the
artifacts on a tissue block for better staining and later on, for better tissue diagnosis.
After removing excess wax surrounding the tissue blocks, proceed with section-cutting with the
following procedure:
1. Clamp the tissue block onto the block holder.
2. Set the angle of the knife to 0-15°
3. Advance the block into the knife with continued cutting until complete sections (ribbons)
come out of the block while a regular cutting rhythm is maintained.

Continue the procedure by floating of the section onto the slide.

Floatation should expand the section to its original dimensions and ensure that the section is
completely flat.
4. Float the section on a warm water bath set at 45-50°C
5. Select a section by immersing the slide in the water bath in a near vertical position as close
as possible to the section.
6. When the slide touches the section, lift it vertically out of the water and drain.
7. Place the slide in the oven with 70°C to remove excess water, if water is no longer visible,
8. Place the slide in the oven with 2-5°C above the melting point of the paraffin.

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 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

9. Label the slide with the accession number using diamond pencil.

Staining
Staining is the process whereby the tissue components are made visible in microscopic sections by
direct interaction with a dye or stating solution. Cells’ and/or tissues’ different affinities to dyes
produce contrast so morphological changes are more easily identified as well as the characteristics
and structural relationships of tissues for the evaluation and the establishment of the presence or
absence of a disease.

Routine Hematoxylin and Eosin staining procedure:

1. Clear paraffin embedded sections in first xylene bath for 3 minutes
2. Transfer to second xylene bath for 2-3 minutes
3. Ethanol for 2 minutes
4. 95% ethanol for 1-2 minutes
5. Rinse with running water for 1 minute
6. Stain with Harris alum hematoxylin for 5 minutes (Ehrlich’s hematoxylin for 15-30
minutes)
7. Rinse excess stain with running water
8. Dip into 1% acid alcohol (1ml concentrated HCl to 99ml of 80% ethanol) for 10-30 seconds
9. Rinse with tap water
10. Blue in ammonia water for 5 minutes or in 1% aqueous lithium carbonate until sections
appear blue for about 30 seconds
11. Wash in running water for 5 minutes
12. Counter stain with 5% aqueous eosin for 5 minutes. If alcoholic eosin is used, dip only for
30 seconds to 1 minute
13. If aqueous eosin is used, wash with water. For alcoholic eosin, use 70% alcohol
14. Dehydrate, clear, and mount.

Mounting
Mounting is the last step in tissue processing that results in a permanent histological preparation
suitable for microscopy, after adhesion to the slide of the sections and appropriate staining of the
tissue. Mounting improves the light refraction between glass slides and the tissue so that little,
microscopic details can be appreciated therefore sections are impregnated with a transparent
medium that has an index of refraction close to that of the glass and the tissue. The mounting
medium bonds the specimen, slide and coverslip together with a clear durable film.

Mounting procedure:
1. Dip the slide one last time with xylene and wipe the excess off from the back of the slide
and from around the section.
2. Drop a mounting medium at the center of the slide.
3. Place a clean, dry coverslip on the edge of the slide, gradually inclined downward until it
touches the mounting medium.
4. Gently press it on to the slide while the mounting medium quickly spreads though the
whole area of the section.
Excessive mounting medium should be wiped out using a cloth with xylene.

LABORATORY WORKSHEET HISTOPATHOLOGY

RMT317/0922-002
 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

PICTURES/DOCUMENTATION

LABORATORY WORKSHEET HISTOPATHOLOGY

RMT317/0922-002
 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

PERFORMANCE EVALUATION CHECKLIST

Laboratory No.: ____
Title: ________________________________________________________________________
Name: _________________________________________
Year and section: ________________________________

Rate the student’s performance by putting a number in the appropriate box using the following
criteria:

5- Excellent = performs task competently and mastery is greatly evident.

4- Very satisfactory = performs task competently and mastery is moderately evident.
3- Satisfactory = performs task competently and mastery is minimally evident.
2- Fair = performs task competently and mastery is less evident.
1- Poor = performance needs further improvement

CRITERIA RATING
Set up and equipment care
Following instructions
Proper use of microtome
Floatation
Staining
Mounting
Safety
Clean up

Final rating: ___________

Comment/s:____________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

_____________________ _____________________ ___________

Student’s signature Instructor Date

LABORATORY WORKSHEET HISTOPATHOLOGY

RMT317/0922-002
 CALAYAN EDUCATIONAL FOUNDATION, INCORPORATED
COLLEGE OF MEDICAL TECHNOLOGY

LABORATORY WORKSHEET HISTOPATHOLOGY

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