HISTOPATHOLOGY – LECTURE

Instructor: MR. CHARLES ANDRE VILLACERAN

Transcribed by: PERRIE M. DE VILLA

LECTURE 1: RISK MANAGEMENT, SAFETY AND INSTRUMENTATION

 Saw microtome: for hard materials (for teeth and
RISK MANAGEMENT bones)
 identifying, assessing and controlling threats to every  Laser microtome: for contact-free slizing; used for
individual in a facility (FIRST STEP). modern generations
 First step in risk management is to identify all
hazards in and emanating from the laboratory.
Note: on histotech, hazards are emphasized because we deal
with a lot of equipments and chemicals, hence, we are prone to CRYOSTAT
chemical and ergonomic hazards. Once we are inside the  CRYO- = cold ; -STAT = stable
HistoTech Department, our number one hazard would be the  used to preserve tissue samples
reagents and and followed by the equipment proper  can slice tissue samples thin enough for microscopic
handling. examinations
 Standard Operating Procedures (SOP) = must be  can also be used for
detailed to include control of hazardous substances, enzyme histochemistry
risk assessments and other health and safety  used when doctors
information relevant to handling specimens. demand STAT tissue
 HistoTech = equipment malfunction due to poor cutting.
maintenance and poor quality reagents can result  for rapid relying
in poor processing of tissues or inaccurate information of results
staining results.  enable rapid and fast
Note: Poor processing of tissues, inaccurate staining results, result generation.
and specimen loss leads to misdiagnosis or it affects px’s  entire process in using
prognosis. cryostat is performed
 MOST COMMON ACCIDENTS (in HistoTech): under cryogenic temperatures (-150 - -273 deg
Cutting of one’s finger or hand on microtome knives. celsius)
Note: Be very cautious at all times when inside the HistoTech.  4 DIFFERENT TYPES OF CRYOSTAT
o SINGLE COMPRESSOR CRYOSTAT
INSTRUMENTATION IN HISTOTECHNOLOGY o DOUBLE COMPRESSOR CRYOSTAT
Care, appropriate use, and equipment maintenance are o ROTARY CRYOSTAT
essential to ensure quality control in HistoTech. o MOTORIZED CRYOSTAT

MAJOR EQUIPMENTS AUTOTECHNICON

 automatically prepares tissues for the tissue
MICROSCOPE processing.
 Bread and butter of MTs.

MICROTOME
 cutting tool used to produce extremely thin slices of
AUTOMATED COVERSLIPPER
material called sections (small cuts). Can provide up
 automatically provides coverslip after smear
to 1-10 mm of thickness.
preparation.
 Manual rotary: more
 Covers slide to avoid any errors that involve during
preferred because we
coverslipping.
can control the hand
wheel. MOST
COMMON IN
PHILIPPINES.
 Automated Rotary
Microtome: hard to
control; provides inaccurate and unprecise tissue
cutting in some cases.
 Cryomicrotome/Cryostat: for frozen samples; Cryo =
cold and –stat = stable.
 Ultramicrotome: for extremely thin samples
 Vibrating microtome: for biological samples

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 AUTOMATED H&E STAINER HAZARDS AND HANDLING OF COMMON
 fill the trays with H&E stains and the machine will dip HISTOLOGICAL CHEMICALS
the slides into the containers that contains the stains. ACETIC ACID
 direct contact with concentrated acid can irritate the
ff:
o Skin
o Eyes
o Respiratory System
 1-10% dilute solution is relatively safe.
 concentrated (Glacial) Hac should not be mixed with
the ff:
o Chromic Acid
o Nitric Acid
o Sodium/Potassium hydroxide
DESIGN AND CUT TYPES
 mixing causes explosion.
 planar concave, wedge-shaped, or chisel-shaped 
designs.
AMMONIUM HYDROXIDE
 Planar concave microtome Knives:
o extremely sharp  should be stored away from acids and should not be
o very delicate mixed with formaldehyde
o only used for very soft samples e.g  o this generates heat and toxic vapors that
fascia, adipose and connective tissues. can be irritating to the respiratory system.

 Wedge Profile Knives: ANILINE

o more stable
o used in moderately hard marterials
 Epoxy or Cryogenic Sample
Cuttings.
o For frozen samples.
 Chisel Profile
o blunt edge: raises knife stability while
requiring significantly more force to cut.
TAKE NOTE:
 For an optimal result, this angle must be chosen
appropriately. (Adjust block holder and knife holder to
achieve the desired thickness).
 The optimal angel depends upon the knife geometry,
cut and many other parameters.
 If the angle is too large, the sample can crumple and
the knife can induce periodic thickness variations in
the cut. By further increasing the angle such that is too
large, one can damage the knife blade itself.
 It is also imperative that the laboratory maintain a
current file for every piece of equipment in the
laboratory. File should contain the following
 information:
o Name, manufacturer, model number and
Serial Number
o Preventive maintenance performed record—
as prescribed by the manufacturer
o Service call records and repairs performed
 proves that technicians have done a
service of proper maintenance.
o Operating manual copy
 The first and most important step in the operation of
any piece of equipment is to read the manual that
accompanies the equipment.
o reading the manual is applied to every
sections in the laboratory.
 always learn the basics of any machine’s operation
before using.
 have a checklist (the more precise the checklist, the
better the operator).
o checklist : insures the safe and effective use
of the laboratory equipment.
 toxic when absorbed by the skin
 can cause severe irritation of the eyes
 a potential carcinogen.
 Excessive exposure may cause the ff:
o drowsiness
o headache
o nausea
o cyanosis
 Routine use of this dangerous reagent should be
avoided.
CHLOROFORM
 a very dangerous chemical that is toxic when inhaled
or ingested.
 carcinogenic and can affect the ff:
o Liver
o Reproductive Organs
o CNS
o Blood
o G.I tract
 Excessive Vapor Exposure causes the ff:
o Disorientation
o Consciousness Loss
o Death
CHROMIC ACID
 can cause cancer and is toxic to the kidneys.
 corrosive to skin and mucus membranes
 All forms of skin contact must be AVOIDED.
 Any solution containing chromium, including post-
fixation solutions or rinses after staining with solutions
containing chromates should not be subjected to
drain disposal because it is an environment toxin.
ETHANOL
 mild to moderate irritation of skin and eyes.
Dangerously flammable and extremely volatile.
o Flammable = easily sets on fire
o Volatile = easily and quickly evaporates
o Combustible = needs heat before ignition/

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Note: Flammable and combustible materials differ based on
temperatures they must be expose before they set on things on MERCURIC CHLORIDE/ MERCURIC OXIDE
fire.  can cause severe irritation to the eyes and skin.
 corrosive to metal due to mercury content.
ETHYLENE GLYCOL  most processing solutions will be contaminated with
 toxic to the ff when inhaled: mercury if specimen is fixed in the ff fixatives:
o Reproductive System o B-5
o Urinary System o Helly’s
o Blood systems o zenker’s fixative
 Propylene-based Glycol ethers should be used as a  reagents used to de-zenkerize the sections will
substitute for ethylene-based glycol and the reagent release the mercury and must not go through drain
must be handled under a fume hood with butyl disposal.
gloves.  To avoid expensive disposal, mercuric fixative may be
replaced with zinc formalin or glyoxal solutions.
FORMALDEHYDE Note: De-zenkerize simply means mercury removal.
 all workers exposed to formaldehyde should be
periodically monitored for exposure levels METHANOL
 formalin waste can be recycled by distillation or by  moderate skin and eye irritant; toxic by ingestion and
drain disposal inhalation.
 can be detoxified by a commercial product or can be  may cause blindness and death in taken in
disposed of by a licensed waste hauler. excessive amounts.
Note: Formaldehyde is the simplest aldehyde and type of gas
dissolves in water to form Formalin. Formalin saturated NITRIC ACID
formaldehyde solution mixed with water and is composed of
 corrosive to the ff:
estimated amount of 37-40% water.
o skin
o mucous membranes
FORMIC ACID o most metals
 can irritate the skin and eyes, and can corrode metal.  toxic by inhalation
 Should be handled under a chemical fume hood.
NITROGEN (LIQUID)
GLUTARALDEHYDE  can cause frostbites or thermal (cold) burns
 can cause severe irritation of the eyes and skin; toxic
by ingestion. OSMIUM TETROXIDE
 corrosive to eyes and mucous membranes
HYDROCHLORIC ACID  vials must be scored, broken, and opened under a
 concentrated acid is particularly dangerous because of hood, not in open air.
its fumes; and should be handled under a fume hood
using the ff: OXALIC ACID
o Goggles  relatively safe when used in dilutions prescribed for
o Apron histologic use
o Gloves
 when concentrated, it is corrosive and causes severe
burns in the ff:
HYDROGEN PEROXIDE o eyes
 harmless if used in concentrations less than 5% o skin
o mucous membranes
HYDROXIDE (SODIUM AND POTASSIUM)  repeated skin contact can cause dermatitis and slow
healing ulcers
 very corrosive to eyes and skin
PERIODIC ACID
ISOPENTANE
 relatively safe when used in quantities prescribed for
 extremely flammable and highly volatile
histology
 should be stored only in a refrigerator or freezer that
is especially suited for explosive atmosphere.
 chilled isopentane: causes frostbite
PHENOL
 excessive vapor exposure causes the ff:  readily absorbed through skin
o Respiratory tract irritation  may cause the ff:
o cough o increased heart rate
o irregular breathing o convulsions
o death
ISOPROPANOL  may burn eyes and skin
 combustible and should be used with extreme
 can cause mild to moderate irritation to the eyes and
caution under a hood especially when mixing with
is toxic by ingestion
concentrated formaldehyde

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PICRIC ACID SULFURIC ACID
 explosive when dry or when combine with metal and  strong irritant to the ff:
metallic salts o skin
 should not be disposed by disposal since they may o eyes
form explosive picrates with metal pipes. o respiratory system
Note: Very yellow reagent.
TOLUENE (FOR CLEARING)
POTASSIUM FERRICYANIDE/ POTASSIUM  repeated exposure can cause the ff:
FERROCYANIDE o Impaired memory
o Poor Coordination
 relatively safe when handled in concentrations
o Mood swings
prescribed by histologic use
o Permanent nerve damage
 should be restricted or avoided if possible, except as a
POTASSIUM PERMANGANATE diluent in mounting media or for coverslip removals
 can cause irritation of skin and eyes
o because it is a strong oxidant. XYLENE (FOR CLEARING)
 should not be mixed with the ff:
 same risk as toluene
o Acetic Acid
o Ammonium Hydroxide
o Ethnol ZINC CHLORIDE
o Ethylene Glycol  corrosive to most metals, including stainless steel
o Formaldehyde  should not be used in tissue processors
o Glycerol
o Hydrochloric Acid
o Hydrogen Peroxide
o Sulfuric Acid

PROPYLENE GLYCOL
 less toxic substitute for ethylene-based ethers

SILVER SALTS
 relatively safe when used as a fresh solution
 can be explosive when solution becomes old.
 a serious environmental hazard and should not be
discarded by drain disposal.

SODIUM AZIDE
 very toxic and may be fatal when ingested or
absorbed thru the skin or when mixed with acids.
 can explode when placed in contact with metals and
should not be discarded by drain disposal.

SODIUM BISULFATE
 relatively safe when diluted.
 should be kept away from OXIDANTS.

SODIUM HYPOCHLORITE (LIQUID CHLORINE

BLEACH)
 strong oxidant ; eye irritant and corrosive to most
metals
 do NOT mix with formaldehyde or diaminobenzene

SODIUM IODATE
 may be used to replace mercuric oxide when
reconstituting Harris Hematoxylin

SODIUM THIOSULFATE
 carries minimal health risk when used in histology
under normal conditions

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