Beruflich Dokumente
Kultur Dokumente
QUALITY CONTROL
Laboratory Staining: check before issuing to the
pathologist
Special stains: accompanied by a control stain
e.g. Giemsa stain for Helicobacter pylori
Standard Operating Procedure (SOP): mandated by
accrediting/ regulatory agency (ASCP)
MSDS
-
HEALTH HAZARDS
1.
2.
3.
4.
5.
6.
Biohazards
o Cause diseases in humans
o Infectious agents, contaminated solution,
specimen or object
Irritants
o Cause reversible inflammatory effects at site of
contact with living tissue
Corrosive chemicals
o Cause destruction/ irreversible alterations
when exposed to living tissue/ destroy
inanimate surfaces (generally metals)
Sensitizers
o Cause allergic reaction in substantial portion of
exposed subjects
o May occur at work (exposure levels)
Carcinogens
o May induce tumors
o Chloroform, chromic acid, formaldehyde
Toxic materials
o Capable of causing death by ingestion, direct
contact/ inhalation at certain specific
concentration
PHYSICAL HAZARDS
1.
2.
3.
4.
Combustible
o Ignite at or above certain temperature (flash
point) at which vapors ignite in presence of
ignition source
Flammables
o Flash points below 1000F/ 37.80C
o Requires specially designed cabinets
Explosives
o Chemicals that cause sudden instantaneous
release of pressure, gas and heat when
subjected to sudden shocks, pressure or
increase in temperature
Oxidizers
o Harmless alone but irritates combustion in
other materials
o Can cause fire (through release of oxygen or
other gas)
LABELLING
-
Conventional cabinet
Dangerous liquids must be stored below countertop
for minimum risk of bodily exposure
Reagents: stored in plastic containers/ plastic
coated glass bottle
Do not store flammable liquids at ref temperature
HANDLING SPILLS
-
Gloves
Disposable plastic aprons (for possible chemical
spill)
Disposable gowns (for biohazard)
Dustpan and brush (for powders)
Sponge, towel, mop (for liquid)
Absorbent material (commercial absorbent)
Bleach (NaOCl for biohazard)
Baking soda (for acids)
Vinegar (for alkali)
Commercial formalin neutralizing product
Sealable plastic bucket
2.
Skin contact
o Wash 15-30 mins
o Emergency shower
o Soap with water (if not water soluble)
o Remove contaminated clothing
Splashing on eyes
o Eyewash solution
o Water temp: 15-350C
o Rinsing: 15-30 mins
Ethylene Glycol
HAZARDS AND HANDLING COMMON
HISTOLOGICAL CHEMICALS
-
Acetic Acid
-
Toxic by inhalation/ingestion
Toxic to reproductive, urinary & blood systems &
with additional exposure toxic to skin
Use propylene-based glycol ethers as substitute
Handle under fume hood with butyl gloves
Ammonium Hydroxide
Formic Acid
Aniline
Glutaraldehyde
Chloroform
-
Hydrochloric Acid
-
Hydrogen Peroxide
-
Hydroxide (Na/K)
Isopentane
Toxic to kidneys
Corrosive to skin, mucous membranes
Carcinogenic
Enviromental toxin
Do not subject to drain disposal
Ethanol
-
Ether
-
Extremely flammable
Highly volatile
Store only in refrigerator/freezer
Chilled isopentane can cause frostbite
Excessive exposure causes irritation of RT, cough,
irregular breathing
Accidental ingestion can cause vomiting,
headache, depression & abdominal swelling
Isopropanol
-
Propylene Glycol
Methanol
Silver Salts
Nitric Acid
-
Nitrogen (Liquid)
-
Osmium Tetroxide
-
Oxalic Acid
-
Periodic Acid
-
Sodium Azide
-
Toxic
Fatal when swallowed/ absorbed through skin/
mixed with acids
Can explode when in contact with metals
Do not subject to drain disposal
Sodium Bisulfate
-
Sodium Hypoclorite
-
Sodium Iodate
-
Phenol
Sodium Thiosulfate
Picric Acid
-
Potassium Permanganate
Sulfuric Acid
-
Toluene
-
Virus
Fungi
Microsporidian parasite
Xylene
MICROTOME
1.
Zinc Chloride
-
Parts
o
TYPES OF MICROSCOPE
1.
2.
3.
4.
5.
6.
7.
Bacteria
Sperm trails
Crystals in urine
Biochemistry
Biomedical research
Fluorescence Microscope
o Use fluorochrome to stain microorganism
o Thru excitation of fluorescent dye by light
o Light: Xenon arc lamp/ Mercury-vapor lamp
Rotary Microtome
o Highest performance
o For manual/motorized sectioning of biological
specimen
o
o
o
o
o
o
o
o
o
2.
3.
4.
5.
Safety lock
Fine advance micrometer dial/ thickness scale
Specimen holder
Object orientation adapter
Freezing Microtome
o For cutting thin to semi-thin fresh frozen tissue
o Freeze using liquid CO2 or temperature
recirculating coolant
Rocking Microtome
o 2-24 microns in steps of 2 microns each
Sledge Microtome
o For cutting large locks of paraffin and resin
embedded
o For large and hard blocks
Sliding Microtome
o Optimal stability
o Consistent high quality sections
6.
7.
4.
MICROTOME KNIVES
-
1.
2.
3.
A.
-
Precautions
B. Stropping
burr formed during honing is removed & cutting
edge of knife is polished
Knife is stropped before every object is sectioned
Paddle strop made up of horse leather (attached to
slid back)
Toe to heel direction
40-120 strokes
Disposable blades
-
Common
Cheaper conventional steel knives
Can cut up to 2-4 micron thick sections
Types of Hone
Glass knives
1.
2.
3.
Belgium yellow
o For manual sharpening when cutting edge has
been rendered blunt/nicked
Arkansas
o More polishing effect than Belgium yellow
Fine carborundum
o Much more coarser than the two
o Used only for badly nicked knives
Diamond knives
-
2.
3.
Procedure
1.
2.
Paraffin oven
o wax oven
o Maintain temperature of 2-50C above melting
point of wax (routinely 560C)
o Store paraffin in its liquid form
o Allow section to dry
Hot plate
o May be used instead of paraffin oven
c.
3.
4.
5.
6.
a.
b.
c.
d.
2.
3.
4.
Celloidin/ Nitrocellulose
o Use to be recommended for embedding hard
tissues such as bones, teeth and for large
sections
Double embedding method
o Process in which tissues are first infiltrated with
celloidin and then embedded in paraffin mass
Resin embedding
o For electron microscopy
o For undecalcified bone and for high resolution
light microscopy of tissue sections thinner than
usual 2-6um such as renal biopsy
Plastics: Epoxy, polyester, acrylic
a. Epoxy
Hydrophobic
Carcinogenic
Glycerol (Epon)
For EM
Acrylic plastic
Used for LM
Leica EG1160
Compact bench top TEC which enables the user to
produce paraffin embedded tissues that can later
be successfully sectioned with ease
Features digital program interface for individual
temperature setting for the paraffin reservoir,
cassette bath, mold warmer and work surface
Essential Parts
1.
2.
3.
4.
5.
6.
Refrigerating system
Paraffin melting chamber
Microscreen (filters particle/sediments)
Hot and cold orientation platforms
Waste drawer
Hot well (for preheating forceps)
3.
4.
5.
6.
7.
8.
Paraffin reservoir
o Holds 3L paraffin
o 45-700C (paraffin liquid temp)
Paraffin dispenser with illumination
o Dispenser is separately heated and always has
the same temp as paraffin reservoir
o Dispenser handle is used for manually
operating the paraffin flow with a dispenser
handle and extension clamp
Mold warmer
o 33-700C
Cassette bath
o 45-700C
o Can hold more than 100 cassettes
Cold plate
o -50C
o Optimal consistency of the blocks minimizes
the risk of brittleness as a result of rapid
cooling and high level of productivity
Refrigeration spot
o Integrated in the cold plate ensuring consistent
low temp
o Mold containing the sample filled with liquid
paraffin (1/3) are placed in refrigeration spot to
allow partial solidifying
Paraffin collecting tray
o Located under the heated work area to collect
excess paraffin derived from surface
Work area
o 45-700C
o Embedding area, forceps holder, recessed area
for cassettes and space to reove the lids
o Forceps holder is separately heated
Demonstration of soluble
substances such as lipids and
carbohydrates
Immunofluorescent and
immunohistochemical staining
Formation of vapor
phase (uneven pulling
of tissue
PROCESSING OF TISSUES
Fixation
Dehydration
Clearing
Infiltration (Impregnation)
Embedding
Trimming
Section-Cutting
Staining
Mounting
Labeling
FIXATION AND FIXATIVES
Fixation the most and critical step in
histotechnology involves fixing or preserving fresh
tissue for examination.
Aim:
o To preserve the morphologic and
chemical integrity of the cell in as life
like a manner as possible.
o To harden and protect the tissue from
the trauma of further handling.
Fixatives have the property of forming cross
links between proteins.
Soluble proteins are fixed to structural proteins
and thus rendered insoluble.
Fixation Prevents:
Degeneration
Decomposition
Putrefaction
Distortion of tissues
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
o
o
5. Concentration
o Formaldehyde: 10%
o Glutaraldehyde :3%
o Glutaraldehyde: (0.25) ideal for
immune-electron microscopy
6. Duration of fixation
o Primary fixation in buffered formalin is
usually carried out for 2-6 hours but
can remain in fixative over the
weekend
o Electron microscopy: 3 hours then
placed in a holding buffer
Practical consideration of Fixation:
1. Speed specimen should be fixed
immediately
2. Penetration formalin diffuses into the
tissues at the rate of 1mm per hour
3. Volume 10-25 times the volume
4. Duration of fixation fibrous organs take
longer fixation
*fixation time can be cut down by using heat, vacuum,
agitation or microwave
Types of fixative ACCORDING TO COMPOSITION
1. Simple Fixatives are made up of only one
component substance
a. Aldehydes
b. Metallic Fixatives
i. Mercuric chloride
ii. Chromate fixatives
iii. Lead fixatives
iv. Heat
Formalin A gas produced by the oxidation of
methyl alcohol, and is soluble in water to the
extent of 37-40% weight in volume
o Commonly used as a 4% solution,
giving 10% formalin for tissue fixation
o Buffered to pH 7 with phosphate buffer.
-
Distilled h2o
-
900ml
2.
DECALCIFICATION
Is it a process whereby calcium or lime salts
are removed from tissues (most especially
bone and teeth: tuberculous organs and
arteriosclerotic vessels).
It should be done after fixation
Calcium may be removed by the following:
o Acid
o Chelating agents
o Ion exchange resins
o Electrical ionization (electrophoresis)
ACID DECALCIFYING AGENTS
They are the most widely used agents for
routine decalcification of large amounts of
bony tissues because they are stable, easily
available and relatively inexpensive.
1. NITRIC ACID
Recommended concentration:
5-10%
Disadvantage: inhibiting
nuclear stains and destroying
tissues especially in
concentrated solutions.
A. Aqueous Nitric Add solution
10%
Decalcification time: 12 24
hours
B. Formol-Nitric Acid
C. Perenyis fluid
Decalcification time: 12 24
hours
2. HYDROCHLORIC ACID
Is a moderate acting
decalcifying agent which
produces better nuclear
staining
Formula:
Decalcification time: 3 14
days
4.
TRICHLOROACETIC ACID
Formula:
Trichloroacetic acid 5
g
Formol saline 10 % - 95
ml
CHELATING AGENTS
Substances which combine with calcium ions
and other salts
The most common chelating agents: EDTA
Tissue is placed in EDTA from 1 3 weeks for
small specimens, but it may take 6 8 weeks
or longer to totally decalcify dense cortical
bone.
pH : 7 7.4
Formula:
o EDTA disodium salt 5.5 gm
o Distilled water 90 ml
o Formaldehyde 10 ml
DEHYDRATION
Process or removing intercellular and
extracellular water from the tissue following
fixation and prior to wax impregnation.
A. Alcohol
o Ethyl alcohol is the alcohol
recommended for routine dehydration
of tissues.
o It is a clear, colorless and flammable
fluid
o Fast acting
o Methyl Alcohol is a toxic dehydrating
agent, primarily employed for blood
and tissue films and for smear
preparations
o Butyl alcohol is utilized in plant and
animal micro-techniques, Slow
dehydrating agent
o Temperature: 37C will hasten
dehydration
B. Acetone
o It is a cheap, rapid acting dehydrating
agent utilized for most urgent biopsies
which it dehydrates in to 2 hours.
o Limited only to small pieces of tissues
due to its extreme volatility and
inflammability
C. Dioxane
o Is an excellent dehydrating and
clearing agent readily miscible in
water, melted paraffin, alcohol and
xylol.
o Ribbon poorly
D. Cellosolve
o It dehydrates rapidly
o Ethylene glycol ethers are combustible
ar 110 120F
o Toxic in inhalation, skin contact, and
ingestion
E. Triethyl phosphate
o It is soluble in alcohol, water, ether,
benzene, chloroform, acetone and
xylene
F. Tetrahydrofuran
o It both dehydrates and clears tissue
since it is miscible in both water and
paraffin.
o May cause conjunctival irritation
*when 4% phenol is added to each 95% ethanol baths
part of dehydration process, it acts as a softener for
hard tissues such as tendon, nail, dense fibrous tissue.
-
CLEARING
Is the process whereby alcohol or a
dehydrating agent is removes from the tissue
A.
B.
C.
D.
E.
o By automatic processing
o By vacuum embedding
MANUAL PROCESSING
o At least four changes of wax are
required at 15 minutes intervals in
order to insure complete removal of the
clearing agent from the tissue.
o Immersed the specimen for another 3
hours in the paraffin wax to insure
complete embedding or casting of
tissue.
o FIXATION
Xylene/Toluene- 1 hour
o IMPREGNATION
c.
3 types:
Bisphenol A (araldite)
Glycerol (Epon)
Cyclohexene dioxide
(spurr)
Disadvantages:
Hydrophobic
Reduce antigenicity
Vinylcyclohexane dioxide
carcinogenic
o POLYESTER: were originally introduced
for electron microscopy.
o ACRYLIC PLASTICS: are made up of
esters of acrylic of methacrylic acid.