Pre and Post Analytical Procedures: Subtopic: Receiving, Accessioning, & Releasing of Results
Pre and Post Analytical Procedures: Subtopic: Receiving, Accessioning, & Releasing of Results
JLLD 1
Histopathology and Laboratory Management
para sa due date (5 days TAT; some There are referral letters from
hospital has 1 month TAT) other hospitals directed to the
doctors, so the letters must be
collected so that we can know
- Specimen Rejection Criteria kung unsa na na work up ang
o Dili pasabot na dili na gyud dawaton buhaton ani na patient
ever 2. SPECIMEN CHARGING
Ex. Appendix must be accepted - In this process, this is the time that you can
but we have to call the double check the specimens received. Please
watcher/doctor or anyone take note also the date and time and the person
related to the patient so that we who received the specimen. You may also fill
can explain or clarify ang mg out a claim stub to be given to the
ana wrong at wala napasok sa watcher/nurse/patient.
ating criteria - Different hospitals have different charging:
Mahirap magpa-recollect so received three specimen
tanggapin na lang but if we will o 1 – uterus
be based it to the book it should o 1 – left and right fallopian tube
be rejected – still depends on o Sizes of specimen:
lab protocol.
Fallopian tubes = small biopsy
o Unlabeled or wrong label on the
Uterus = large biopsy
specimen o Charging of specimens: pila mabayaran
Can be accepted, but in the
sa patient
result form you must write that
o Ma-incorporate pila ka specimen ang na
the specimen is unlabeled.
receive in case na need mag back track
o Incomplete patient information/clinical
- Transaction Slip
history
o Billing information
There are times when
o HIS: nurses can input some remarks
pathologists will ask for
additional information because additional remark is what type of
there are cases that is hard to specimens
diagnose. One big container + many
o Left unfixed or unrefrigerated for an specimen = extra large tissue
biopsy charging
extended period.
- Claim Stub
Esp if biopsy fixed sample is a
o Name
must.
o Putrefied or autolyzed specimens o Age/Sex
o Damaged specimen or broken slides o Room no.
o Insufficient sample for processing o Examination
In real life situation, no matter o Additional Test
how small the amount the o Date Requested & Due Date
specimen is, we must process o Prepared by (sino nag receive siya nag
it. prepare)
And be careful in processing it This can be noted in a surgical
because it can be a specimen pathology worksheet
loss.
o Spilled or contaminated specimens 3. ACCESSIONING
Containers must be full of - Assign the laboratory number/accession
formalin to measure that the number unique to that patient.
specimen is fixed properly
o Failure of the requesting physician to HOW TO ACCESSION?
enter the request in the computer. - Choose the appropriate logbook.
o Empty containers without the specimen o Surgical Pathology (e.g. biopsy)
or form o IHC: Immunohistochemical (further
o Referral Pathology consultation material evaluation right after the surgical
without Histopathology report or pathology if the doctor request for it)
referring hospital. o Cytology/Cell Block (e.g. body fluids)
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Histopathology and Laboratory Management
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Histopathology and Laboratory Management
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Histopathology and Laboratory Management
o Instructions for embedding and block o 0.5% acetic acid solution is used as
making fixative
Pathologists will also instruct Fixative for the ink
the histotech paano and ano o E.g. breast is submitted in lab to
ang gusto niya i-pa expose examine if to what extent nag abot ang
Orientation of tissue defines the tumor cells
plane of section in embedding - Surgical margins
- Crucial in clinical management and provide o Lateral
important prognostic data o Medial
o Superior
GROSSING AREA
o Inferior
- Grossing stations allow gross examination of o Basal
surgical specimens which are typically
o Border where the surgeon cut
performed by a pathologist, or by a pathologists’
Tested for the positive or
assistant working within a pathology practice.
negative as a tumor cell
o Maraming gamit
Ideally dapat negative kasi it
o Gripo: formalin
means high chance na nakuha
o Inks: inking (for margin) na lahat ang tumor cells
o Scissors - Ink is for label of what margin
o Guide
o They sometimes self-record and say GENERAL PRINCIPLE OF GROSS EXAMINATION
what they see and the medtech will later 1. Specimen Identification
transcribe it a. Doon sa sample result makita ang
- Instruments received in one-part neutral buffered
o Gloves formalin and labeled as…
o Formalin 2. Identify all the anatomical structures present
o Blade – mas prefer sa doctors mag use a. E.g. appendix: fibro fatty tissue
ng blade when they are going to set the 3. Orientation markers should be identified
size of the sample to be put in the a. Possible lang ito siya sa malalaking
cassette (2x2 cm) and the specimen specimens for marking and identification
must fit into the cassette 4. Measurements: Length width height (3D)
o Ruler – the pathologist will measure the 5. Weight: especially parenchymatous organ
mass and the specimen (Prostate chips)
o Forceps 6. Examine the external surface
o Cassette a. Smooth, rough, serosa, what you see is
o Cutting board / Chopping Board what you describe
7. Cut all the organs at intervals of 1 cm thickness
o Large Scissor – used in tubular
a. Bread loafing
specimens like intestines
8. Describe cut surface, identity pathologic process
o Scalpel – alternative for blades
9. If suspected lesion is present, measure,
o Dissecting scissors – for small areas or describe with the color and consistency
specimens (e.g., lymph node) a. Separate measurement for the lesion
o Saw – for hard specimens (e.g., identified
amputated leg) 10. Surgical Margins
11. Histological section
INKING
- When working in histopathology as a surgical SMALL BIOPSIES
crossing tech you have to learn a new language When will we work on this? How much time do we need
and this language must be clear and concise so to execute this?
there are a few terms that you may see
(e.g., endometrial curettage and skin punch biopsy,
o Flat pigmented lesion = macule
“specimen consists of tan to black fragments of tissue
- Madaming new terms from the doctors
aggregately measuring 0.5 cm -one dimension)
- Histopathological evaluation of surgical margins
of a resected tumor specimen can give an 1. Number of fragments, aggregate dimension
insight about the extent of tumor spread 2. Greatest dimension of largest fragment
3. Shape of fragment, Color and Consistency
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Histopathology and Laboratory Management
a. Tan to brown soft irregular piece of - The broad objective of tissue fixation is to
tissue (piece of tissue = one) preserve cells and tissue components in a “life-
4. Supported within the gauze or filter paper to like state” and to do this in such a way as to
prevent tissue loss during processing allow for the preparation of thin, stained
5. All fragments are submitted sections.
6. “Submitted in one block” “block 2” (depends on o From the time na nakuha ng surgeon
the cassette; 2 cassette = 2 block) ang specimen nalagay na agad sa
APPENDIX fixative to stop autolysis
Autolysis: results in tissue
Procedure digestion by intracellular
1. Measure organ (length and greatest diameter) enzymes released when
2. Divide specimen in two by cutting a cross- organelle membrane rupture
section 2 cm from tip Bacterial decomposition or
3. Cut cross-sections of proximal fragments at 5 putrefaction brought about by
mm intervals the microorganism
4. Divide distal fragments in two by a longitudinal - For practical purposes fixation aims to prevent
cut or arrest the degenerative processes which
5. Description commence as soon as a tissue is deprived of its
a. L*W*H blood supply
b. External surface o Loss & diffusion of soluble substances
c. Perforation must be avoided as far as possible
d. Wall Thickness - Tissue processor:
e. Mucosa
f. Lumen
g. Content
“Specimen consists of tan to red appendix measuring
4x3x1 cm, the external surface is smooth, cut section
shows a dilated lumen with a fecaloid material, wall
thickness is 0.1 cm. block 2”
- Section for histology
- Proximal 1/3 close to surgical margin; one cross-
section
- Mid 1/3: one cross section
- Distal 1/3: one longitudinal section
- If tumor is present, perform inking and take an
additional section from it
- Tissue Basket: kung saan ilalagay ang cassettes
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Histopathology and Laboratory Management
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Histopathology and Laboratory Management
o Ideally 3 mm thick should provide - Non coagulant fixatives which are additive in
excellent fixation and processing nature formed extensive crosslinks producing a
o Penetration of formalin to the tissue is 1 less permeable gel
mm per hour o Still encountered in a modern
o So the bigger the specimen is mas hindi histological literature
na makapenetrate ang formalin
o Bigger specimen = longer fixation DENATURATION
process - Most commonly this effect is induced by
4. Volume of the fixative dehydrants such as the alcohols or acetone.
o 20:1 Fixative to tissue ratio These reagents remove and replace free water
o It is important to have an excessive in cells and tissues and cause a change in the
volume of fixative in relation to the tertiary structure of proteins by destabilizing
volume or to the total volume of tissue hydrophobic bonding.
o Because with additive fixatives the o Hydrophobic areas frequently found on
effective concentration of reagent is the inside of protein molecules and are
depleted as fixation proceeds released from the repulsion of water and
o In a small total volume this could have become free to occupy a greater area
o Hydrophilic areas well mixed with water
an effect on fixation quality
5. Temperature are loosely bound by hydrogen bonds
o Higher temperature = higher rate of and removal of water destabilizes these
bonds
diffusion = higher rate of chemical
o So the changes produce in the
reaction between the fixative and tissue
elements conformation of the protein molecules
o It can also increase the rate of tissue cause a change in the solubility of the
protein
degeneration in unfixed areas of tissue
o So rendering water soluble proteins in
o Microwave fixation may involve higher
soluble
temperature up to 65 degrees Celsius
o So a change that is largely irreversible
but for relatively short periods only
6. Time is the protein is returned to an aqueous
o Time varies depending on the fixatives environment
o Fixative has to penetrate by diffusion to **a paraffin section from the mucosa of small intestine
the center of the specimen and that has been fixed in 95% ethanol, a denaturing
sufficient time has to be allowed for the fixative. While nuclear presentation is fair there is a
reactions of the fixation to occur substantial shrinkage of cytoplasmic and extracellular
o FORMALIN = 24 hours elements.
o Pre-fixed tissue that is received is
processed the next day so that it is well
fixed for processing (minsan sobra pa
24 hours)
7. Penetration rate
o 1 mm per hour
o Depends on diffusion characteristics
o Varies from one reagent to another
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Histopathology and Laboratory Management
- Tissue fixed with formaldehyde stains poorly o Ethanol: most widely used dehydrant, is
with eosin because formaldehyde reacts a drinking alcohol and hence strictly
extensively with amino groups and forms controlled
methylene bridges and thus these groups are no o Methanol
longer available to bind negatively charged dye o Isopropanol
molecules such as those of eosin o Butanol
- The extent to which additive fixatives form - Reagent: Denatured Alcohol Ethanol
cross-links vary considerably - 70%-95%-100% gradually increasing
o E.g., glutaraldehyde is more effective at concentration
forming crosslinks than formaldehyde
DEHYDRATING AGENT IDEAL
o Explains why glutaraldehyde effectively CHARACTERISTICS
preserves the ultrastructure of cells and 1. Dehydrates rapidly without producing
is the fixative of choice for electron considerable tissue damage
microscopy 2. Not evaporate too fast
3. Not harden tissue excessively
**a paraffin section of the mucosa of small intestine that 4. Not remove stains
has been fixed in neutral buffered formalin, a cross- 5. Not a fire hazard
linking fixative. Nuclear and cytoplasmic preservation is 6. Not toxic to handler
satisfactory, but some cellular shrinkage is present. 7. Able to dehydrate a wide range of tissue
JLLD 9
Histopathology and Laboratory Management
- good ethanol substitute but rarely used for 1. Dissolution of calcium by a diluted mineral ACID
routine processing 2. Using of Chelating Agents
- volatile, flammable, costly - Most common chelating agent: EDTA (pH:
- poor lipid solvent 7)
- does not dissolve nitrocellulose (unless mixed - Function: Chelates calcium to form a soluble
with acetone) salt.
ISOPROPANOL - Forms: VERSENE (SOLID) and
SEQUESTRENE (LIQUID)
- universal solvent 3. Removal of calcium by using a diluted mineral
- slightly slower in action and not as hygroscopic and along with ION EXCHANGE RESIN to keep
as ethanol the decalcifying fluid free of calcium.
- superior lipid solvent 4. Electrolytic removal of calcium ions from tissue
- since specimens submitted are not usually fatty by the use of electric current:
tissues ethanol is still most preferred ELECTROPHORESIS
5. Microwave oven decalcification – faster than the
routine procedure (inc tissue damage)
STRONG ACIDS
A. NITRIC ACID
- COMPOSITION
o Concentrated Nitric Acid: 10 mL
o Distilled Water: 100 mL
- ADVANTAGES
o Rapid in action
o Produces minimum shrinkage
o Produces good nuclear staining
DECALCIFICATION o Recommended for urgent biopsy,
- REMOVAL OF CALCIUM ions from a bone or needle and small biopsy
calcified tissue through a histological process - DISADVANTAGES
that makes them flexible and easier to cut. o Prolonged decalcification may lead to
tissue distortion
PATHOLOGIC CALCIFICATION o Damage tissue stainability
- Abnormal tissue deposition of Calcium salts o Imparts a yellow color with nitrous acid
- Smaller amounts of Fe, Mg, & other mineral o Strong acids tend to be more damaging
salts to tissue antigens (histochemical
- DYSTROPHIC – deposition occurs locally in staining)
dying tissues
- METASTATIC – deposition in normal tissues
B. PERENYI’S FLUID
DECALCIFYING IS AFFECTED BY THE - COMPOSITION
FOLLOWING: o 10% Nitric Acid: 40 mL
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Histopathology and Laboratory Management
C. PHLOROGLUCIN-NITRIC ACID
- COMPOSITION
o Concentrated Nitric Acid: 10 mL
A. FORMIC ACID
o Phloroglucin: 1 gm - COMPOSITION
o 10% Nitric Acid: 100 mL o Formic acid: 10 mL
- ADVANTAGES o 10% Normal saline: 90 mL
o Most rapid decalcifying agent - ADVANTAGES
o Recommended for urgent cases o Both a fixative and decalcifying agent
- DISADVANTAGES o Permits excellent nuclear and
o Nuclear staining is poor cytoplasmic staining
o Prolonged decalcification produces o Recommended for small pieces of
extreme tissue distortion bones and teeth
o Yellow color must be neutralized with o Suitable for most routine surgical
5% sodium sulphate and thoroughly specimens
washed with running tap - DISADVANTAGES
o water (24 hours) o Relatively slow, not suitable for urgent
o Complete decalcification cannot be specimens
determined by chemical means o Requires neutralization with 5% sodium
sulfate
D. VON EBNER’S FLUID
- COMPOSITION B. TRICHLOROACETIC ACID
o Saturated aqueous solution of NaCl: 50 - COMPOSITION
mL o Trichloroacetic acid: 5 gm
o 36% Concentrated HCl: 8 mL o 10% Formal saline: 95 mL
o Distilled Water: 50 mL - ADVANTAGES
- ADVANTAGES o Permits good nuclear and cytoplasmic
o Permits good cytologic staining staining
o Moderately rapid decalcifying agent o Does not require washing out
o Does not require washing - DISADVANTAGES
o Recommended for teeth and small o Weak decalcifying agent, not used for
pieces of bone dense tissues
- DISADVANTAGES o Very slow-acting, not recommended for
o Extent of decalcification cannot be urgent examinations
measured by chemical test
WEAK ACID C. CHROMIC ACID
- COMPOSITION
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Histopathology and Laboratory Management
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Histopathology and Laboratory Management
Removal of excess acid is done by immersing the o (Miscible-is the ability of a substance to be
decalcified tissue in: mixed well Solubility-is the ability of the
- Saturated Lithium carbonate substance to be dissolved)
- 5- 10% Aqeous Sodium bicarbonate o (Clearing is the bridge between Dehydration
and infiltration that why it is one of the
COMMON PRACTICE:
crucial steps)
- Rinse the decalcified specimens with running o (Note: In H.E STAINING the first 3 clearing
tap water, agents are for the final deparaffinization.
o (In PAPSMEAR when used after tissue has
been stained, the clearing agent will make
microscopic tissue preparation transparent
due to their high index of refraction)
Since most alcohols and paraffin are NOT miscible,
another step, known as clearing, is introduced.
After the dehydrating agent is removed, the tissue will
have a translucent appearance = thus the procedure
is called “clearing”
o The clearing of the tissue is due to the
increase in the refractive index of the tissue
caused by the clearing agents
THE PURPOSE OF THE CLEARING IS TO
FACILITATE THE PENETRATION OF THE
TISSUE BY THE EMBEDDING MEDIUM WHICH
IS THE WAX
XYLENE
CLEARING “DEALCOHOLIZATION” 30 mins to 1 hour
Most commonly used clearing agent in routine
Process whereby alcohol or a dehydrating agent is biopsies
removed by the tissue and replaced it with a Used for clearing, both embedding and mounting
substance that will dissolve with the wax that will be procedures
used in impregnation
Colorless chemical and inexpensive
Process of replacing the dehydrating fluid with a fluid
that is miscible with both the dehydrating fluid and ADVANTAGES
impregnation or embedding medium
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Histopathology and Laboratory Management
CLEARING TIME: 15-30 MINS (MOST RAPID At very high levels of exposure, xylene can injure
CLEARING AGENT) the liver and kidneys.
Miscible with alcohol and paraffin
Makes tissue transparent BENZENE
Does not affect staining RAPID ACTING: CLEARING TIME: 15-60MINS
Evaporates rapidly in the oven thus can easily be Also recommended for urgent biopsies
replaced by the melted paraffin Same as xylene, highly volatile inside the oven
DISADAVATANGES thus is easily replaced by paraffin wax
Miscible in alcohol and makes tissue transparent
Highly flammable Unlike xylene, it is preferred by some because it
Tissue immersed in xylene for more than 3hrs doesn’t cause tissue to become hard and brittle
become hard and brittle If tissue is immersed in benzene for a longer
Can cause tissue shrinkage thus not period of time, there is considerable tissue
recommended for biopsies of lymph nodes and shrinkage
nerves Chronic exposure to Benzene can cause bone
Becomes milky if there is still water in the tissue marrow suppression resulting to aplastic anemia
NOTE: Benzene is also a carcinogenic – acute myeloid
Make sure dehydration process is done leukemia
completely o Aplastic anemia
Repeat dehydration using absolute alcohol o Acute myeloid leukemia
Exposure to high levels of xylene can irritate the Exposure to benzene may be harmful to the
lungs, causing chest pain and shortness of breath. reproductive organs.
Extreme overexposure (e.g., in a confined space) Some women workers who breathed high levels
can result in pulmonary edema, a potentially life- of benzene for many months had irregular
threatening condition in which the lungs fill with menstrual periods. When examined, these
fluid. women showed a decrease in the size of their
Its vapor is heavier than air and may accumulate ovaries.
in low-lying areas.
Children exposed to the same levels of xylene TOLUENE
vapor as adults may receive a larger dose CLEARING TIME: 1-2HRS
because they have greater lung surface area: Used as substitute for xylene and benzene
body weight ratios. Recommended for routine purposes
In addition, they may be exposed to higher levels ADVANTAGES:
than adults in the same location because of their
short stature and the higher levels of xylene vapor Even if tissue is left for 24hrs in toluene, there is
found nearer to the ground. no shrinkage, brittleness and hardening
Inhalation of xylene vapors in small amounts can NOT carcinogenic unlike Benzene
cause headache, dizziness, drowsiness, and DISADVANTAGE
nausea. Slower than xylene and is EXPENSIVE
Effect of xylene on the central nervous system is Emits fume that can be toxic to humans
attributed to the liposolubility of xylene in the CNS effects (headache, dizziness, drowsiness,
neuronal membrane.
euphoria, hallucinations, tremor, and seizures)
It has been suggested that xylene disturbs the o Liver and kidney damage
action of proteins essential to normal neuronal
o Cardiac arrythmia
function either by disruption of the lipid
environment in which the membrane protein’s Breathing high levels of toluene during
function or by direct interaction with the proteins in pregnancy has been shown to result in children
the membranes. with birth defects and to retard mental abilities
and growth.
Xylene vapor is only mildly irritating to mucous
membranes; however, xylene splashed in the There is evidence that exposure to toluene at
eyes can result in corneal injury. work is associated with spontaneous abortion.
Repeated or prolonged skin contact with liquid
xylene can defat the skin, causing it to crack and
peel (it will dissolve the natural oils in the skin) CHLOROFORM
CLEARING TIME: 6-24HRS
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Histopathology and Laboratory Management
JLLD 15
Histopathology and Laboratory Management
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Histopathology and Laboratory Management
To keep the medium in liquid state during At least 4 changes of wax are required at 15
infiltration, the medium should be in melted. minutes intervals in order to insure complete
Higher melting point means the wax is difficult to removal of clearing agent from the tissue.
melt and therefore, it is a hard type of wax! The specimen is then immersed in another
Conclusion: The higher melting point, the harder fresh solution of melted paraffin for
the wax…. approximately 3 hours to insure
The harder the wax, the easier it is to make thin completeness of embedding or casting
sections...
AUTOMATIC PROCESSING
Hard wax requires heavy duty type of microtome
during sectioning!!! Makes use of an automatic tissue processing
PARAFFIN WAX
• commonly available paraffin waxes have the ff
melting point in:
o 45 degree Celsius
o 52 degree Celsius
o 56 degree Celsius – most commonly
used
o 58 degree Celsius
The recommended temperature for infiltration is
usually 2-5 degree Celsius above the melting machine which fixes, dehydrates, clears and
point of the wax to ensure it is completely infiltrates tissues thereby decreasing the time
melted. and labor needed during the processing of
56-degree Celsius wax = Temp 58-61 tissues resulting to rapid diagnosis with less
58-degree Celsius wax = Temp: 60-63 technical errors
o Higher temperatures can tissue to Oscillating tissue basket
become brittle and shrunken and these • The tissue basket oscillates up and down in
changes will start at temperatures above each station at three-second intervals to ensure
60 degree Celsius thorough and even mixing of the reagents and
optimum tissue infiltration
In a room temperature (20-24 degree Celsius), • Beaker III – fixative. 30- 45 minutes
paraffin wax with a melting point of 54-58 degree • Beaker IV – 70% alcohol. 30 minutes
Celsius is recommended • Beaker V – 90% alcohol. 30 minutes
If the laboratory temperature is between 15-18 • Transparent beakers
degree Celsius, the melting point of the wax to
• Beaker VI – Absolute alcohol. 1 hour
be used should be between 50 - 54 degree
Celsius • Beaker VII – Absolute alcohol. 1 hour
• Beaker VIII – Absolute alcohol. 1 hour
THREE WAYS OF PARAFFIN WAX
• Beaker IX – Xylene. 1-2 hours
IMPREGNATION AND EMBEDDING
1 By manual processing • Beaker X – Xylene 45 minutes – 1 hour
2 By automatic processing • Thermostatically controlled beakers
3 By vacuum embedding • Wax bath I (done at 45°c) 2 hours
MANUAL PROCESSING • Wax bath II. 2 hours
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Histopathology and Laboratory Management
When it’s time for tissue to be transferred to the next Involves the wax impregnation under negative
beaker or jar, the cover of the machine is raised up, and atmospheric pressure inside an impregnation
the lifting mechanism carefully removes the tissue and embedding oven to hasten the removal of
basket and gently transfers it to the next beaker. the air bubbles
The machine is mounted on rollers to permit and clearing agent from the tissue block thereby
turning of platforms and easy access to beakers promoting a more rapid tissue impregnation
and wax bath
It removes residual bubbles such in the lungs
Makes use of 12 individual processing steps
with 10 one Liter capacity glass beakers and 2 Particularly recommended for urgent biopsies
thermostatically controlled wax baths with a and for delicate tissues such as lungs, brain,
safety device cut-out switch to protect wax from decalcified bones, eyes, spleen and lymph
over heating nodes
There is also 2-3 changes of wax needed to o The tissue is then not over-exposed to
remove the clearing agent and to impregnate the heat, brittleness, shrinkage, and
specimen hardening
o This is made possible due to constant
Time required is reduced from 25% to 75% of
tissue agitation which accelerates and
the normal time required. (Fastest)
improves tissue penetration giving rise
to more consistent results Composed of flat-bottomed heavy brass
chamber with heavy glass lid with thick rubber
ADVANTAGES OF AUTOMATED PROCESSING valves which produces an airtight seal when the
It’s very efficient chamber is being used.
Saves time and energy to operate
The vacuum chamber is enclosed in a
Cost effective and user friendly thermostatically controlled water jacket which is
Can process different tissues same time usually maintained at a temperature 2-4 degree
The machine does the transfer of tissue from Celsius above the melting point of the wax
one bath to another.
PROCESS
PRECAUTIONS 1. After clearing with xylene
Presence of any odor of the clearing agent
2. Place tissue in paraffin wax, in vacuum chamber
during final paraffin wax bath indicated that the
and make the oven airtight
paraffin should be changed
Dehydrating agents should be frequently 3. Exhaust the air slowly by means of vacuum
changed since dehydration is the most critical pump until there is negative pressure of 400 to
stage of tissue processing and inadequate 500nmHg
dehydration is difficult to correct once tissue is in 4. Leave for 15 minutes then slowly readmit air
paraffin until normal atmospheric pressure is reached
After two complete processing runs of load, the
5. Place the tissue in a fresh wax.
first 100% alcohol bath should be always
discarded and the other moved down so that the 6. Repeat steps 3 and 4
final batch has fresh absolute 100% alcohol o Place the tissue in a fresh wax
The clearing agent and the other diluted
7. Repeat step 3 and leave for 30-45 minutes
alcohols can be changed at least once a week
To avoid spillage, fluid and wax containers must 8. Bring to normal atmospheric pressure and
be filled to the appropriate level proceed to embedding
Wax accumulating on the surface of the beaker
should be removed FACTORS AFFECTING PARAFFIN
Any spillage should be wiped away IMPREGNATION
Wax bath thermostat should be maintained at o bone, teeth, brain and eye are difficult to
least 3 degrees above the melting point of the infiltrate (BeeBee) using paraffin (its hard to
wax and should be checked when loading the infiltrate the heart of my Beebee crush)
machine o Require prolonged period of immersion with
VACUUM EMBEDDING paraffin
o REMEMBER: prolonged immersion with paraffin
causes tissue shrinkage
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Histopathology and Laboratory Management
o THUS there are other recommended embedding More elastic and resilient because of the plastic
medium for these tissues component
o Benzene and Xylene are easily removed from o permitting large dense tissue blocks
the tissues while chloroform and cedarwood oil such as bones, teeth and brain to be cut
requires frequent wax changes. easily with the same result as in double
embedding.
o Adding benzene to cedarwood oil would hasten Serial sections can be cut with ease without
the process cooling the block, thereby preventing formation
of ice crystals
PRECAUTIONS OBSERVED IN PARAFFIN WAX It is soluble in common clearing agents and
IMPREGNATION IN GENERAL follows the same time schedule for paraffin
Prolonged treatment with paraffin causes tissue impregnation and does not tend to crack like
shrinkage and hardening of the tissue, making other paraffin wax substitutes.
cutting difficult
EMBEDDOL
Overheated paraffin (above 60 degree Celsius)
can cause tissue shrinkage and hardening Similar to Paraplast
Paraffin wax must be pure (less brittle and less compressible than
Free from dust, water droplets and foreign paraplast)
matter Can be use as substitute
Fresh wax should be filtered at 2 degree Celsius Melting point of 56-58 degree Celsius
higher than its melting point BIOLOID
Wax that has been trimmed away from the
Semisynthetic wax recommended for
impregnated tissue may be melted and filtered
embedding eyes
for future use with a coarse filter paper
For organs with thin wall and circular shaped
ALWAYS FILTER FRESH WAX sections
• If wax is suspected to be contaminated with TISSUE MAT
water, it can be subjected to 100-105 degree
Celsius to remove the water. Paraffin with rubber
With same property as Paraplast
• Paraffin wax can only be reused once.
ESTER WAX
PARAFFIN EMBEDDING Has lower melting point than paraffin (46-48
Tissue will now be embedded in paraffin wax degree Celsius) but is harder than paraffin
melted at 5-10 degree Celsius above the melting Main component: Diethylene glycol distearate
point o a very hard and brittle
Cooling is done: Added with:
o Refrigeration at -5 degree Celsius o Castor oil
o Immersion in cold water o Ethyl cellulose
o Tissue tek with cold plate o Stearic acid
Good for sections as thin 2-4 microns
SUBSTITUTES FOR PARAFFIN WAX
DIETHYLENE GLYCOL
1 Paraplast
Not soluble in water but soluble in 95% ethyl
2 Ester Wax
alcohol
3 Water Soluble Waxes o Tissue can be impregnated without prior
PARAPLAST clearing
o Cellosolve and xylene may be used as
Paraplast is suitable for tissue infiltration.
clearing agents (depending on the
o (Mixture of highly purified paraffin +
protocol of the hospital or preferences of
synthetic plastic polymers)
the pathologist)
Melting point: 56-57 degree Celsius
Clearing agent must be gradually removed
It is a refined combination of highly purified o Tissue must be placed in a solution
paraffin with plastic polymers.
containing equal amount of clearing
Paraplast – Paraffin plus Plastic!!! agent and ester wax for 3-6 hrs before
transferring the tissue to pure ester wax
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Histopathology and Laboratory Management
o 3-4 changes of wax should be done to Tissue block must not have contact with
ensure complete tissue impregnation water or ice
Sectioning of Ester wax-impregnated tissue Tissue sections are difficult to float out and
should be done on a heavy duty microtome mount out due to its extreme solubility in
(sledge or sliding type) due to the relative water
hardness of the wax o Add soap in water to reduce
distortion and promote flattening
WATER-SOLUBLE WAXES and floating out of sections
Composed of polyethylene glycol with It will not infiltrate tissue however when
melting points of 38-42 or 45-56 degree there is large amounts of fat in the tissue.
Celsius Tissue such as central nervous tissue needs
Carbowax – polyethylene glycol composed long periods of impregnation.
of 18 carbons or more which appears solid
at room temperature
EMBEDDING
CARBOWAX
Also known as Casting or Blocking
Carbowax is soluble in water hence does Process by which the impregnated tissue is placed
not require dehydration and clearing of into a precisely arranged position in a mold
tissue containing a medium which is then allowed to solidify
o After the tissue is fixed, it is washed out The medium used to infiltrate the tissue is usually
to remove excess fixative and the same medium utilized for embedding
precipitates and transferred directly to
melted carbowax After impregnation, the tissue is placed into a
o Processing time is reduced mold containing the embedding medium and this
Harmful effects of dehydrating agents and medium is allowed to solidify.
clearing agents are avoided Paraffin embedded tissues are
Fats which are usually removed by arranged at the bottom of the mold and
dehydrating and clearing agents are not immersed in melted paraffin at a temperature
removed between 5 – 10oC above its melting point and;
Eating too much CARBs can make you FAT! then cooled rapidly
Fat is not removed from the body!!! o Refrigeration at -5oC
o Immersion in cold water
Tissues are not exposed to too much heat
This allows hardening of tissues, giving them a
o There is no excessive hardening, firmer consistency and better support, thereby
shrinkage and brittleness of tissue facilitating the cutting of sections.
o Cytologic details are preserved Orientation - process by which a tissue is
arranged in precise positions in the mold during
Eating too much CARBs can make you FAT! embedding, on the microtome before cutting,
No gym workout = No HEAT = No sweat! and on the slide before staining
No heat = No shrinkage and hardening of Generally speaking, the surface of the section to
muscle! be cut should be placed parallel to the bottom of
Suitable for enzyme histochemical studies the mold in which it is oriented.
because enzymes are denatured greatly by
heat SEVERAL TYPES OF BLOCKING OUT MOLDS
Four changes of carbowax are needed (56 MAY BE USED:
degree Celsius) 1. Leuckhart’s Embedding Mold’
o Once in 70% for 30 mins
2. Compound Embedding Unit
o Once in 95% for 45 mins
o Twice in 100% Carbowax for 1 hr 3. Plastic Embedding Rings and Base mold
each with agitation 4. Disposable Embedding Molds
o Specimen is then embedded in 1. LEUCKHART’S EMBEDDING MOLD’
carbowax and rapidly cooled in
refrigerator consists of two L–shaped strips of heavy
Hygroscopic property = easily dissolve in brass or metal arranged in a flat metal plate
water
JLLD 20
Histopathology and Laboratory Management
and which can be moved to adjust the size o Blocks can be filed up after
of the mold to the size of the specimen. sectioning
The L- mould are kept on a metal plate. 4. DISPOSABLE EMBEDDING MOLDS
Tissue is placed the surface of the glass and
is oriented Peel–away disposable thin plastic
The plate is then filled with molten wax. The embedding molds
wax is then cooled. L-moulds are then o available in 3 different sizes, are
removed simply needed off one at a time, as
Easy to procure soon as the wax has solidified,
Reusable giving perfect even block without
Blocks produced are even, with parallel trimming.
sides, and with a fairly shaped initial setting Plastic Ice Trays
of the wax. o used in ordinary refrigerators may
Adjustable to make different size of blocks be recommended for busy routine
It is recommended for routine use, although laboratories.
too slow and cumbersome for use in a busy o Each compartment may be utilized
laboratory. for embedding one tissue block,
which may then be removed by
2. COMPOUND EMBEDDING UNIT bending the plastic tray once the
made up of series of interlocking plates wax has solidified: or by smearing
resting on a flat metal base, forming several the inner mold with glycerin or
compartments. before embedding.
It has the advantage of embedding more
specimens at a time, thereby reducing the
time needed for blocking Paper boats
o are normally utilized for embedding
3. PLASTIC EMBEDDING RINGS AND BASE celloidin blocks but are equally
MOLDS useful for paraffin wax blocks.
consist of a special stainless steel base o They have the advantage of being
mold fitted with a plastic embedding ring cheap and easy to make. They
which later serves as the block holder during provide easy and accurate
cutting. identification of specimen, thereby
One model, the so called Tissue Tek, is a avoiding confusion and interchange
machine equipped with a warm plate to of tissue blocks.
manage the impregnated specimen, and a o Rapid embedding of small or large
cold plate at –5oC for rapid solidification of volume of individual specimens is
the block. possible, since paper molds can be
The base mold is filled up with meltin made to suit any size of tissue.
paraffin.
The sample is then oriented in the mold DOUBLE EMBEDDING METHOD
The plastic embedding ring is placed in Peterfyi's celloidin-paraffin wax technique
position and filled up with wax. the process in which tissues are first infiltrated
The tissue is then allowed to cool down on with celloidin and subsequently embedded in
the cold plate of the Tissue tek equipment paraffin mass.
Upon hardening (5minutes), This is used to facilitate cutting of large blocks of
o the tissue is taken out together with dense firm tissues like the brain, bone, and teeth
the embedding ring and also recommended for making small sections of
o is immediately ready for cutting celloidin blocks.
without having to undergo trimming Gelatin impregnation is rarely used except
thereby saving time and effort. o when dehydration and clearing is to be
Advantage of Tissue Tek avoided because gelatin is water soluble
o Tissue is firmly attached to the o when tissues are to be subjected to
holder with permanent identification histochemical and enzyme/lipids
o Resectioning is easily done studies.
JLLD 21
Histopathology and Laboratory Management
Fixatives (such as 10% formalin) should still be The next day, an equal volume (50 mL) of ether
washed out by running water before tissue is is added and intermittently mixed until an evenly
infiltrated by gelatin consistent solution is obtained
It is used as an embedding medium for delicate Dense tissues which are hard to infiltrate (e.g.
specimens and frozen tissue sections bones and brains) and specimen which tend to
o because it prevents fragmentation of collapse easily due to air spaces (e.g. eyes and
tough and friable tissues when frozen lungs) are supported better, thereby avoiding the
sections are cut. crumbling of tissues during sectioning.
It has a lower melting point and does not cause
overhardening of tissue by heating. When eye sections are embedded by the
paraffin method, the retina may be detached
Used along with 1% phenol to prevent growth of
from the harder tissues (e.g. sclera and choroid)
molds
that encircle it.
GELATIN IMPREGNATION Its rubbery consistency allows tissue blocks that
After the fixative has been completely washed are either very hard or of varying consistency, to
out, the tissue is be cut without undue distortion.
o placed in 10% Gelatin with 1% Phenol It permits cutting of tissue sections which are
for 24 hours thicker than paraffin wax, and is therefore
o transferred to 20% Gelatin with 1% recommended for processing of neurological
Phenol for the next 12 hours, and tissues.
o finally to another fresh solution of 20% o Thicker sections contain higher number
Gelatin with 1% Phenol which is then of neurons
allowed to cool in a refrigerator until
impregnation and embedding are
completed.
CELLOIDIN BLOCKS
CELLOIDIN
There are two methods for celloidin impregnation of
Purified form of nitrocellulose soluble in many tissue
solvents, suitable for the following specimens 1 Wet Celloidin method
o Organs with large hollow cavities (eye, 2 Dry Celloidin method
lungs)
o For hard and dense tissues (bone and WET CELLOIDIN METHOD
teeth) Recommended for bones, brain sections and
o Delicate tissues (embryos) whole organs.
It is supplied in After the usual fixation and dehydration of the
tissue, it is place in equal parts of ether and
o thin (2%), medium alcohol for 12 – 24 hours.
o medium (4%) and o Clearing can be by passed
The tissue is then placed in thin celloidin (2–4%)
o thick (8%) solutions of cellulose
for 5–7 days,
o dissolved in equal parts of ether and transferred to medium celloidin (4–6 %) for
alcohol (solvent). another 5–7 days,
For example, to prepare 8% celloidin drained off and poured with thick celloidin (8–
12%) until the specimen has become
o Get 8ml celloidin. impregnated, usually between 3–5 days.
o Mix it with 92ml solvent (50 ml ether + o + 7 + 5 = 19 days!!!!! (Slow process)
50 ml alcohol) The specimen is removed from the celloidin and
Preparation transferred to an embedding medium containing
freshly poured thick celloidin and kept in a tightly
The fastest way to dissolve celloidin is to soak it covered jar or dessicator in order to evaporate
first in half the final volume of anhydrous ethanol the alcohol – ether solvent.
to soften it (50 mL for each 8 grams celloidin), The dessicator top is removed for a few second,
with intermittent mixing in a tightly stoppered time and again, to admit fresh air and harden
container. tissue block.
JLLD 22
Histopathology and Laboratory Management
Evaporation must be gradual to achieve a Comparing the thin preparations of celloidin and
consistent, uniform degree of hardness LVN, the latter has better tissue penetration or
throughout the block and prevent the formation infiltration
of air bubbles. LVN is also cheaper than celloidin
When the ball of the finger leaves no mark on Celloidin is available as thin (2%) , medium (4%)
the surface of the tissue leaves no mark on the and thick (8%) preparations (lowest viscosity to
surface of the tissue block, evaporation and highest viscosity)
consequently, embedding, is considered to be The equivalent concentrations for LVN are 5%,
complete. 10% and 20%.
o Use the thumb, not your fingernails!!! Note: at same the viscosity, LVN has a higher
The tissue block is then stored in 70 – 80% concentration. It means it has better tissue
alcohol until ready for cutting. This is done to penetration
avoid dehydration and shrinkage of tissues. It forms a harder tissue block and makes cutting
Prolonged contact with air dries out and hardens of thinner sections possible.
celloidin. Sections tend to crack during staining
DRY CELLOIDIN o The tendency of tissues to crack may be
prevented by adding plasticizers (e.g.
Preferred for processing of whole eye sections
Oleum Ricini or Castor oil)
The principle and procedure of this method is More explosive than celloidin and should
similar to wet celloidin method, except that therefore be handled with care.
70% alcohol is not used for storage before
When dry, striking or dropping the container will
cutting.
cause the substance to explode.
Instead, Gilson’s mixture, made up of equal
o It is usually marketed while wet with
parts of chloroform and cedarwood oil is added
alcohol.
to the celloidin block before hardening.
The container must be kept tightly covered and
The cedarwood oil used in the Dry Celloidin
protected from sunlight to avoid evaporation of
Technique helps to soften the layers of eye
alcohol.
preventing the retinal detachment.
o Cedarwood oil is added twice daily for When no longer needed for future use, the
nitrocellulose should be carefully disposed,
10 days until the mixture is composed of
since the material becomes increasingly
90% percent cedarwood oil.
dangerous as the alcohol continues to
o The purpose of this method is to make
evaporate.
the tissue transparent
o Alcohol is not used in Dry method
because it will dissolve the cedarwood
oil. PLASTIC RESIN EMBEDDING
Provided superior results for light microscopic
LOW VISCOSITY NITROCELLULOSE
studies
Another form of celloidin soluble in equal Thinner sections – high resolution microscopy
concentration of ether and alcohol with a lower such as electron microscope
viscosity, allowing it to be used in higher o Renal biopsies
concentrations and still penetrate tissues rapidly.
EPOXY PLASTICS
Composed of Epoxy plastics, catalysts and
Take note: accelerators
Higher viscosity means poor tissue penetration o Araldite (bisphenol A)
or infiltration slowest, large molecule with
Higher concentration means good tissue high viscosity
penetration o Glycerol-based (epon)
Celloidin = higher concentration means higher faster, smaller size than araldite,
viscosity lower viscosity but cannot be
Conclusion: prepared in pure form
o Cyclohexene dioxide (spurr)
At lower preparations, both celloidin and LVN low viscosity and can obstained
have the same viscosity. However, at the same in pure form
level viscosity, LVN has a higher concentration
JLLD 23
Histopathology and Laboratory Management
Efficiency is based on size of molecules, • Use forceps or brush instead of your fingers to
viscosity and purity pick up sections or wax fragments from blade or
Hydrophobic – not compatible with most water block face.
soluble stains • The handwheel brake will lock the microtome
Irritation on skin and airways when the handle is in any position and is used
Take note: Gloves must be worn while when realigning a block face or adjusting the
processing and processing should be done coarse feed.
inside fume hoods to eliminate toxic vapor and • The knife or blade should be removed from the
avoid irritation to the airways microtome when the instrument is left
Contains vinylcyclohexene dioxide – unattended or when cleaning the instrument.
carcinogenic - skin cancer • Never place a knife or blade on the bench or in a
Reduces antigenicity – not recommended for box with the cutting edge facing up.
immunohistochemical studies
MICROTOME KNIVES
Example: Cytokeratin 18 is overexpressed on
Microtome knives may be generally grouped into:
the surface of cells in colon cancer
1. standard thick metal
ACRYLIC PLASTICS
2. thin disposable blades
Composed of acrylic or methacrylic acid for light 3. glass knives
microscopy 4. diamond knives
Less viscous than plastic resins = less time
needed for embedding SET BLADE CLEARANCE ANGLE OPTIMALLY
Polyglycol methacrylate (GMA) Blade clearance angle is adjustable and must be set for
o Hydrophilic – compatible with water optimum performance.
soluble stains • The clearance angle prevents contact between
Methyl methacrylate (GMA) the knife facet and the face of the block.
o Preferred for its relative hardness = • The facet angle is the angle between the two
better sectioning facets that form the cutting edge. For routine use
Prematurely polymerize in the presence of light, knives and disposable blades are made with a
that’s why acrylics should be stored in facet angle of approximately 35°, but this angle
amber(dark) bottle can vary with the blade type and from
manufacturer to manufacturer
• Follow the microtome manufacturer’s guidelines
SECTIONING for the recommended angle setting. For Leica
• Aka microtomy or cutting knife and blade holders a setting of between 1°
• process where tissues are cut into uniformly thin and 5° is recommended.
slices to facilitate microscopic studies
• Instrument: microtome TAKE CARE WHEN “TRIMMING”, “FACING”, OR
“ROUGHING”
LOCATE MICROTOME APPROPRIATELY This stage in microtomy requires great care as tissue of
The location of the microtome in the laboratory is diagnostic importance can easily be lost or the block
important. surface damaged.
• Position the microtome on a stable bench, away • Before you commence trimming always make
from air drafts, doorways and passing staff. certain that all of the clamping mechanisms are
• A height-adjustable bench and ergonomic chair tightened securely
are preferred. • The goal of properly trimming a block is to
conservatively expose the tissue down to a level
UTILIZE SAFETY FEATURES PROPERLY where a representative section can be obtained.
You must be familiar with the safety features of the • Trimming is usually done at thicknesses
microtome you are using and observe some basic rules between 10 and 30 μm.
when cutting sections.
CONSIDER FACTORS AFFECTING SECTION
• Microtome knives and disposable blades are THICKNESS
extremely sharp and can inflict serious injuries Set the microtome at the desired setting but note that
unless appropriate care is taken when working there are a number of factors that determine the actual
with them. section thickness.
JLLD 24
Histopathology and Laboratory Management
• A cohesive section of 4 μm may provide more Proper drying ensures that sections are completely
information than a severely disrupted section of dehydrated, free of heat damage, flat and unlikely to lift
2 μm. during staining.
• The actual thickness of the first couple of • Drain excess water from beneath the section
sections in a ribbon may be thicker than before drying. This is vital if slides are dried flat
indicated because of thermal expansion, when on a hot plate .
cutting a cold block (as seen in sections 1, 2 & 3 • Slides can be stored in racks in an upright
below). - Next picture position, then dried in an oven.
• Other factors such as speed of rotation, • Generally drying temperatures should not
clearance angle setting and the condition of the exceed 65 ˚C.
cutting edge can influence the actual thickness • Excessive heat can cause droplets of water
achieved. underneath a section to boil and this will cause
damage. • Dry sections for between 10 and 30
ENSURE BLOCKS ARE COLD
minutes.
Sectioning is generally improved when the specimen • Some delicate specimens will produce best
and the wax are well matched in hardness. It is for this results when dried at 37 ˚C for a longer time
reason that most paraffin blocks must be cold when (several hours to overnight).
sections are cut. The actual method used to chill the
block is important. CLEAN AND MAINTAIN THE MICROTOME
• Cold wax provides better support for the harder THOROUGHLY
elements in a specimen allowing thinner It is important to remove accumulated tissue debris and
sections to be obtained. wax after use. Regular preventative maintenance is
• Place the blocks on a cold plate or a cold wet important.
surface for a few minutes (such as the surface of 1 Clean the instrument daily.
melting ice). 2 Always remove the knife or blade before
cleaning.
LEARN THE TECHNIQUES FOR CUTTING
3 The knife holder can easily be removed to
CONSISTENT, HIGH-QUALITY, THIN SECTIONS
facilitate access for cleaning.
There is no substitute for experience but there are. some 4 Section waste is best removed with a dry
fundamental steps that will make the task easier. paintbrush.
• Use a section of blade that has not been used 5 Do not clean the outer surfaces with alcohol or
for rough trimming. xylene as they are not resistant to these
• Re-chilling of the block may be required if the solvents and exposure to xylene should be
block face becomes warm or if deeper levels are avoided. Paraffin remover, mild commercial
required. household cleaners or soap and water are
• Generally a slow, uniform cutting stroke recommended.
produces the best results and the least 6 No fluid must enter the inside of the instrument
compression during cleaning.
7 Have the instrument inspected at least once a
FLOAT OUT SECTIONS CAREFULLY year by a qualified service technician.
Floatation should expand the section to its original 8 Follow the lubrication instructions provided in
dimensions and ensure it is completely flat your instruction manual using recommended
• Monitor the temperature carefully. The lubricants.
temperature will need to be 5 - 9 ˚C below the
melting point of the wax.
• Make sure the water is clean and free of bubbles
and section waste (to avoid cross- LEARN TO RECOGNIZE AND CORRECT
contamination). COMMON FAULTS
• Examine each section as it floats on the water Some of the most common faults seen in paraffin
surface as imperfections can be readily seen. sections are:
• Leave the section on the water surface just long
enough for it to flatten. Overexpansion can spoil 1 Wrong micrometer setting
the morphology in susceptible sections 2 First section in ribbon chosen
3 Sectioning at too great a speed
DRY SLIDES ADEQUATELY 4 Poor processing
JLLD 25
Histopathology and Laboratory Management
1. PROGRESSIVE STAINING
STAINING
• Stained in a definite sequence, until desired
• Process of applying dyes on the section to see intensity of color is attained.
and study the architectural pattern of the tissue • Once the dye is taken up by the tissue, it is
and physical characteristics of the cells. NOT washed/decolorized.
• Basic dyes – has affinity to acidic part (e. g. • It is somewhat less favored than regressive
NUCLEUS stained with hematoxylin) due to: producing insufficiently intense
• Acidic dyes – has affinity to basic part of the cell staining producing diffused/obscured details.
(e. g. CYTOPLASM stained with eosin) 2. REGRESSIVE STAINING
JLLD 26
Histopathology and Laboratory Management
• The tissue is first OVERSTAINED then • Reagent should not be exposed to sunlight
DECOLORIZED to prevent explosion
3. DIFFERENTIATION (DECOLORIZATION) • Inactivated by NaCI/HCI before discarding
• Selective removal of excess stain, during 7. VITAL STAINING
regressive staining. • The selective staining of living cell
• In general: if the primary stain used is Basic, constituents demonstrating cytoplasmic
the differentiation is caried by an acid structures by phagocytosis of the dye
4. METACHOROMATIC STAINING particle
• Most dyes stain tissue • Nucleus is resistant to vital staining, the
ORTHOCHROMOATICALLY – stain tissues permeability of the dye in nucleus signifies
with same color of the dye CELL DEATH
• Some is METACHROMATIC – stain tissues 8. INTRAVITAL STAINING
different from the color of the dye • Done by injecting the dye into any part of the
• This stain belongs to the Thizine & animal body (intraperitoneal, intravenous)
Triphenylmathane group • Lithium, Carmine, India ink
• All Metachromatic dyes are Cations or 9. SUPRAVITAL STAINING
Basic • Stain living cells immediately after removal from
o Ex. the living body
1. Tolouidine Blue • Thin slices of tissue are placed in staining
2. Cresyl Blue dishes
3. Safranin • Common dyes used are:
4. Basic Fuschin O Neutral red
5. Azure A, B, C O Janus green
6. Methyl Violet/Crystal Violet O Trypan blue
7. Bismarck Brown O Nile Blue
a. Methylene Blue O Thionine
5. COUNTERSTAINING O Tolouidine Blue
• Used to differentiate, to provide contrast and
background
O Cytoplasmic Stains STAINS & STAINING SOLUTIONS
Eosin Y 1. NATURAL DYES
Eosin B • Cochineal dyes, logwood dyes, and
Phloxine B vegetable extracts.
Picric acid
Orange B HEMATOXYLIN
Rose Bengal • Extracted from the core or heartwood of
Light Green Mexican tree “Hematocylin
Lissamine Green Campechianum”
O Nuclear Stains • HEMATIN – the active coloring agent
Neutral Red formed by the oxidation also known as
Safranin O “Ripening”
Carmine Hematoxylin • Ripening/Oxidation
Celestine Blue o May be done by exposing the
Methylene Blue substance to air and sunlight
Tolouidine Blue (SLOW)
o May be done by adding oxidizing
agents such as:
6. METALLIC IMPREGNATION Hydrogen peroxide
• Tissues are demonstrated not by stain but Mercuric oxide – ripening
by colorless solution of Metallic salts agent of HARRIS
• Producing BLACK DEPOSITS through HEMATOXYLIN
reduction Potassium permanganate
• Different from stains it doesn’t incorporate Potassium perborate
with the tissue but it forms ppt. within the Sodium perborate
tissue (e. g. Gold Chloride, Silver Nitrate)
JLLD 27
Histopathology and Laboratory Management
JLLD 28
Histopathology and Laboratory Management
RINGING
Sealing of Margin of the coverslip
Use to prevent escape of fluid or semi-fluid
samples
Evaporation of mountant
To immobilize the coverslip.
To prevent sticking of the slides upon storage
1. Kronig’s Cement
2. Durofix
JLLD 29