HISTOPATHOLOGY INTERVIEW
o Rocking – parrafinblocks or tough tissues
Interview topic I: RISK MANAGEMENT AND
SAFETY
1. Enumerate the PPE
Personal protective equipment- anything used or
worn by a person to minimize risk to person’s
health or safety and it includes a wide range of
clothing and safety equipment.
o Googles – protect eyes from fumes
o Mask – protect and avoid inhalation of
chemicals
o Gloves – protect hands from spillage
o Lab gown – protect user from spillage
o Head cap / hairnets
o Closed shoes
2. State the Universal precautions
o Treat all specimens as infectious
o All patients are considered to be
potentially carrier of blood born
pathogens
o The guideline recommends wearing gloves
when collecting or handling blood and
body fluids contaminated with blood and
wearing foreshields when there is danger
of blood splashing on mucous membranes
and when disposing needles and sharps.
3. Give 4 equipments used in histopathology
laboratory. Explain the mechanism and
purpose of each.
o Microscope – enlarges the minute details
of the object under the study; Allows
visualization of morphological cellular
details that are too small
o Automated processors – machine that
automatically fixes, dehydrates, clears,
and infiltrates the tissue; Advantage: saves
time for technician to process tissue. Used
in decalcification of bones and in staining
of tissue.
o Microtome – machine designed to cut very
thin section of tissue.
o Rotary - Parrafin, paraplast, carbowax
o Sliding – celloidin embedded
o Freezing – unembedded or gelatine
o Ultra-thin – thin sections from plastic or
epoxy
o Microtome knives – used to cut;
requirement for satisfactory cutting
o Incubator – used for infiltration of tissue in
paraffin and paraplast
o Oven
o Hone and strope
o Floatation bath – used to flatten or
smoothen and facilitate separation of
embedded tissue ribbon
o Tissue cassette – perforated plastic or
metal where the specimen is placed
4. What are the information needed in the
chemical label
o Chemical name and if its a mixture, all
ingredients must be written.
o Manufactrer’s name and address if
purchased commercially, or person
making the reagent.
o Date of purchase made.
o Expiration date must be written if
known.
o Hazard warnings and safety
precautions
o Date and name of person who
prepared (in lab)
o Name of recipient (if delivered to lab)
5. What is the biohazard symbol?
The image warns
people of possible
exposure to
biological
substance that
may contain virus,
toxins, or medical waste.
o Symbol that signifies the significant
health hazard of biological material
o Refers to biological subs. That pose a
threat to health
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 6. What is the NFPA hazard identification
system.
o Proves certain criteria for assessing the
health, flammability, instability and related
hazards presented by acute exposure to
material under conditions of fire, spill, or
similar emergencies.
o Symbol system used to inform individuals,
firefighters of the hazards they may
encounter with fires in a particular area.
o Diamond-shaped, color coded symbol
contains information relating to health,
flammability, reactivity, and personnal
protection/special precaution.
o Graded based on extent of concern
7. How are you going to handle spills in the
laboratory?
o Spill neutralizing and containment kits
should be available immediately outside the
hazardous area which includes PPE, such as
thick nitrile glves, disposable plastic aprons
for chem. Spills, and disposable gowns for
biohazards and absorbent materials, bleach
for biohazard,baking soda for acid, vinegar
for alkali.
o Limited spills are wiped off with towel or
sponge, hands with gloves
Interview topic II: FRESH TISSUE PREPARATION,
INFLAMMATION, CELLULAR DEATH, AND
AUTOPSY.
1. What is the purpose for fresh tissue
preparation?
o To examine tissue in living state,
allowing protoplasmic activities such
as motion, mitosis, phagocytosis, and
pinocytosis to be observed.
2. Enumerate the methods for fresh tissue
preparation. Explain the procedure for
each method.
o TEASING / DISSOCIATION
o Tissue Section immersed in isotonic
solution, dissected or separated,
avoiding maceration of muscle tissue is
used, teasing is done longitudinally &
tissue is mounted on the slide with
isotonic salt solution and examined.
o Vital stains used:Methylene blue or
methyl violet
o SQUASH PREPARATION (Crushing)
o Small pieces of tissue (not more than
1mm) are placed on a glass slide &
forcibly compressed with another slide
(coverglass)
o SMEAR PREPARATION
o Examination of sediments / Small
tissue sections by spreading materials
lightly on slide using applicator. This
technique is useful in cytological
examination.
o SP: STREAKING
o material directly applied on slide.direct
or zigzag line
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 o SP: SPREADING
o material (blood or viscid specimen) is
transferred on slide and gently spread
into a circular motion
o SP: PULL – APART
o material disperses evenly over surface
of two slides
o for thick secretions : serous fluid,
enzymatic lavage from GI tract,
concentrated sputum, blood smears.
o SP: TOUCH PREPARATION (
IMPRINT/IMPRESSION SMEAR)
o freshly cut tissue is brought in contact
& pressed on the surface of the slide,
stained & examined under phase
contrast or bright field microscope.
o Advantage: cells examined without
destroying their actual intracellular
relationship or without separating
them from their normal surroundings.
o FROZEN SECTION
o Rapid tissue diagnosis
o Recommended for sensitive tissue
components (for lipids & nervous
tissue elements, enzyme
histochemistry) that will undergo
denaturation
o Small pieces are frozen rapidly using
liquefied CO2, liquefied NO2. Fresh
tissue cut on freezing microtome or
cryostat(10-15um), transferred on a
dish containing a cold isotonic
solution, “fished” with a clean slide nd
stained with vital stain to facilitate
microscopic identification.
3. Enumerate 9 cellular degenerative
changes and explain.
a. Metaplasia – reversible changes
involving transformation in one type of
adult cell
b. Dysplasia – enlargement due to
proliferation of abnormal cells; when
cells may undergo a morphological
transformation in which increase cell
division is coupled with incomplete
maturation of the result; poor cellular
differentiation losing morphological
character of mature cells
c. Anaplasia – usually held as a criterion
toward malignancy form
d. Neoplasia - continuou abnormal
proliferation of cells without control
e. Agenesia – non appearance of an
organ; complete failure of growth of
an organ
f. Atropy – decrease in size of normally
mature tissue organ
g. Hypoplasia - failure of an organ to
reach its mature full size
h. Atresia – failure to form an opening in
an organ
i. Aplasia – incomplete/defective
development of tissue; usually seen in
one of the paired organ structure
(kidneys, adrenals, gonads)
4. Differentiate acute from chronic
inflammation.
ACUTE INFLAMMATION
o Sudden onset
o Vascular and exudates
o PMNs predominant
CHRONIC INFLAMMATION
o Long term
o Vascular and fibroblastic
o Mononuclear predominant
(marcrophage, lymphocytes,
plasma cell)
5. What are the cardinal signs of
inflammation?
o Rubor (REDNESS) – due to arteriolar and
capillary dilation with increase rate of
blood flow toards the site of injury
o Tumor (swelling) – due to transfer of
internal heat to site of injury bought about
by increase blood flow
o Calor (HEAT) – due to increase capillary
permeability causing extravasion of blood
flow
o Dolor (pain) – due to pressure upon the
sensory nerve; exudates/tumor
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o Functio laesa (diminished function) –
deteriorating functional units of the tissue
6. What is somatic death?
o The incomplete and irreversible
change of the circulation,
replication and brain function
o Death of entire body
7. What are the primary changes during
somatic death?
o Cessation of circulation; circulatory
failure
o Cessation of respiration;
respiratory failure
o Insensibility and loss of ECG
rhythm; CNS failure
8. What are the secondary changes after
somatic death?
a. Pallor and loss of elasticity of skin –
paleness hat occurs with those with
white skin
b. Algor mortis – cooling of the body (7 F
per hour) cause by the absence of
circulation
c. Rigor mortis – stiffening of the skeletal
muscles after death (formation of
insoluble muscle potency); 6-12 hrs
d. Liver mortis – post-mortem lividity
(purplish color of skin); 6 hrs
e. Post-mortem clotting – ante morton
clot due to platelet aggregation; clot
formed in heart/large BV after death
f. Drying – dryness and shrinking is seen
in skin and eyeballs
g. Putrefaction – decomposition of
protein after 48hrsof death and
eventual breakdown of cohesiveness
between tissues or organs
h. Autolysis begins to decompose
i. Skeletanization – final stage of death
in which the last vestiges of the soft
tissues of a corpse have decayed or
dried
9. What is autopsy?
o “autopsia” – to see with one’s own
eyes
o A thorough examination of a dead
body to determine the cause and
manner of death to evaluate if
there are underlying disease
o Interpret and correlate factors
leading death
o Identify the deceased
o Establish time of death
10. What are the two types of autopsy?
o Forensic autopsy – carried out
when the cause of the death is a
criminal matter
o Clinical or Academic autopsy –
performed to find the medical
cause of death and is used in cases
of unknown or certain death for
research purpose
11. What are the four autopsy techniques?
o Virchow technique – organs are
removed one by one and dissected
as removed organs (order of
removal); 1. Brain, 2. Spinal chord,
3. Abdominal, 4. Thorax
o Rokitansky – organs are removed
based on convenience; partial
insitu dissection with en bloc
removal
o En masse (Le tulle) technique – all
at one time, thoracic, cervical,
abdominal, and pelvic are removed
en masse and subsequently
dissected into organ
o Ghon or Zenker (En bloc) –
physically related organs (organ
bloc) are removed at the same
time (e.g thoracic block, celiac
block, urgenital block); deals with
large number of blocks at the same
time
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 - Formalin: 24hrs
Interview topic III: FIXED TISSUE PREPARATION
3. What is the purpose of secondary fixation?
1. Why is fixation important?
o Primary aim is to preserve morphological and o the process of placing an already fixed
chemical integrity of cell in its life-like matter tissue in second fixative in order to:
as possible. It prevents degeneration, o Facilitate and improve the demonstration
decomposition, putrefaction, and distortion of of a particular subs
tissues after removal from the body o ensure further and complete hardening
o Fixation improves staining, makes cells resist to and preservation of tissue
the effects of chemicals in processing o Make special staining techniques possible
(secondary fixative act as mordant)
2. What are the factors that affect fixation? 4. What is the purpose of washing out?
a. Hydrogen ion conc.
– satisfactory ph 6.8 o remove excess fixative from the tissue
- outside this changes may occur that are afyer fixation to improve staining and
detrimental to ultrastructural preservation of remobe artifacts from the tissue.
the tissue o may sue tap water, 50-70% alcohol,
b. Temperature alcoholic iodine
– usually at room temp, some lab at 40 C;
- EM and some histochem at 0-4 C; Post chromatization - fixation whereby a primarily
- nucleic acids at higher temp; fixed tissue placed in aqueous soultion of 2.5-3%
- rapid fixation of very urgent biopsy specimen K dichromate for 24hrs to act as a morsant foe
with formalin 60 C; bwtter staining effects and aid in cytological
- tissue with TB at 100 C preservation of tissue.
c. Thickness of section
- EM: 1-2mm2
- LM: 2cm2
- LM: <0.4cm
- Don’t compromise measurements in order to
obtain full penetration and satisfactory fixation
d. Osmolality
- Hypertonic – shrinkage
- Hypotonic/isotonic – swelling
- Slightly hypertonic – 400-450mOsm; sucrose is
positive to osmium tetroxide
e. Concentration
- 10% formaldehyde
- 3% glutaraldehyde
- 0.25% glutaraldehyde for immune EM
f. Duration
- Prolonged: shrinkage and hardening of
tissue an inhibit enzyme activity and
immunological reaction
- EM: 3hrs
- Buffered formalin: 2-6hrs, may remain

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5. Give one fixative, its composition, recommended tissue, two adv and disadv.
Fixative Composition Recommended tissue Advantages Disadvantages

Bovin's solution - saturated - embryos - minimal distortion of - penetrates large

(picrate/picric solution of picric microanatomical structures tissue poorly, use is
acid) acid - pituitary biopsy and can be used for general limited to small
and special stain fragments of tissue
- strong
formaldehyde - excellent preservative for - picrates soluble in
40% preserving soft and delicate water
structures
- glacial acetic - destroy cytoplasmic
acid structure

Formalin - 10% formalin - cheap, readily available, - irritating to nose,

from easy to prepare eyes, may cause rhinitis
formaldehyde
- preserve glycogen, fat - considerable
- gas produced mucin shrinkage of tissue
by oxidation of
methyl alcohol

6. What is the purpose of decalcification? B. Chelating agent

o calcium or lime salts are removed from
tissues ( esp. Bones and teeth) following
fixation by the use of chemical agents
(either with acids to form soluble Ca salts)
or with chelating agent that bind to Ca ions
o done after fixation and before
impregnation to ensure and facilitate
normal cutting of sections and prevent
obscuring the micro anatomical details of
sections by bone dust and other cellular
debris
o bones, teeth, calcified tissues (tuberculous
organs and arteriosclerotic vessel)
7. What are the 4 ways of calcium removal?
A. Acid decalcification
o Use for routine calcification of large
amount of bony tissue
o hydrolyze Ca salts and goes to solution
o Ca binds to acid - salt/ppt
o nitric acid, HCl, formic acid, TCA, sulfurous
acid, chromic acid, citric acid
Ca salt + acid -> Ca + acid -> Ca salt
o subs combine with Ca ions and other salts
to form weakly dissociated complex and
facilitate removal of Ca salt
o acid will alao hydrolyze Ca salt to form
weakly dissociated compound
o EDTA- versene
Ca + EDTA -> Ca EDTA chelate
C. Ion exchange resin
o hastens decalcification by removing Ca ions
from formic acid containing decalcifying
soltn, thereby increasing solubility from
the tidsue
o Ca ions is removed by binding with (-)
charge
o 1/2 inch thick IE Resin at bottom of
container specimen on top and agent
Ca + resin -> Ca bind to resin
D. Electrophoresis
o positively charged Ca ion are attracted to
negatively charged electrodes and
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 subsequently removed from the - To remove acids from tissues or to neutralize
decalcifying soltn them using either sat. Lithium carbonate soltn. Or
5-10% aqueous sodium bicarbonate soltn.

8. What are the methods of measuring the extent
of decalcification and explain each?
A. Physical or Mechanical
o done by touching of bending the tissue
with the fingers to detect the consistency
of tissue
o vague and inaccurate
B. Xray or Radiological method
o most reliable method due to its ability to
detect even the smallest focus of Ca which
appears on an xray plate
o expensive but most ideal
o bone placed on waterproof polyethylene
sheet on top of xray film, exposed to xray,
1 minute, 30 KV and examine
o X for mercuric chloride fixed tissue
because of its radio-opacity interfere
C. Chemical method (Ca oxalate)
o detects Ca in acid solutions by
precipitation of insoluble Ca hydroxide or
Ca oxalate
o Solutions: ammonium hydroxide (conc);
ammonium oxalate (sat. Aq)
9. What is the purpose of post decalcification?
10. What is the purpose of dehydration?
- removes water and fixative and its prior tow ax
impregnation.
- to remove intracellular and extra H2O from
tissues
Why is tissue immersed in increasing strengths of
alcohols?
- to remove aqueous tissue fluids with little
disruption to tissue caused by diffusion currents
- mostly: 70% ethyl alcohol in h2O; 95% & 100%
11. Give five characteristics of a good dehydrating
agent
A. should dehydrate rapidly without producing
considerable shrinkage or Distortion of tissue
B. should not evaporate very fast
C. Should be able to dehydrate even fatty tissue
D. Should not harden tissue excessively
E. Should not be toxic to the body
F. Should not remove stains

G. Should not be a fire hazard

12. Give one example of dehydrating agent.

DEHYDRATING AGENT ADVANTAGES DISADVANTAGES

DIOXANE (Diethylene dioxide) - less tissue shrinkage - tend to ribbon poorly

- dehydrating and clearing - expensive

- long period or prolonged dehydration cannot - dangerous

affect the consistency or staining property

TETRAHYDROFURAN (THF) - dehydrate and clears - toxic if ingested or inhaled

- less shrinkage - eye and skin irritant in prolonged

exposure= conjunctival irritation
- easy to cut sections

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CELLOSOLVE - dehydrate rapidly - ethylene glycol ethers are
combustible at 110-120F
- tissue may be stored in it for months without
hardening or distortion - toxic by inhalation, skin contact, and
ingestion

13. What is the purpose of clearing?

o Not produce excessive shrinkage,
- It removes the alcohol or dehydrating agent and hardening, or damagae of tissue
replaced with substance that will dissolve the wax o Not dissolve out aniline dye
with which the tissue is to be impregnated o Not evaporate quickly in water bath
14. Give 5 characteristics of a good clearing agent o Make tissue transparent

o Miscible with alcohol to promote rapid

removal of the dehydrating agent from
the tissue
o Miscible with and easily removed by
melted paraffin wax and by mounting
medium to facilitate impregnation and
mounting of section

15. Give example of clearing agent.

CLEARING AGENT ADVANTAGES DISADVANTAGES

XYLENE - most rapid clearing agent - higly inflammable and should

appropriately stored
- urgent biopsies ( 15-30min)
- prolonged=hard and brittle
- make tissue transparent
- milky when incompletely
- miscible with absolute alcohol and paraffin hdehydrated tissues is immersed in
it
- not extract out aniline dye

BENZENE - rapid acting=urgent biopsies (15-60min) - carcinogenic= damage to BM

resulting in aplastic anemia
- miscible with absolute alcohol
- highly inflammable
- used to make tissue hard and brittle

CHLOROFORM - miscible with absolute alcohol - toxic to liver=prolonged inhalation

- for tough tissues - does not make tissue transparent

- not flammable - complete clearing is difficult to

evaluate
- for routine work (16-24hrs)

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16. What is the purpose of impregnation?
- process whereby clearing agent is completely
removed from tissue and replaced by a medium
that will completely fill all the tissue cavities,
thereby giving firm consistency to specimen and
allow easier handling and cutting of suitably thin
sections without any damage or distortion to the
tissues and cellular components
How is impregnation done?
- label bottles with paraffin 1,2,3
- use properly melted paraffin
- remove tissue from last clearing bath, drain, or
blot
- Place in paraffin wax bottle1 and leave for 1-2
hrs
- Transfer to paraffin wax II for 1hr
17. What is the purpose of embedding?
- allow hardening of tissue, giving them a fitmer
consistency and better support, thereby
facilitating cutting of sections
18. What are the four types of embedding media?
A. Paraffin wax - simplest, most common and best
medium used
B. Celloidin - purified form of nitrocellulose
soluble in many solvents. Suitable for those eith
large, hollow cavities which tend to collapse, for
hard and dense tissues such as bones and teeth
and for large tissue sections (whole embryo)
C. Gelatin - histochem and enzyme studies; for
delicate specimen and frozen tissues
D. Plastic (Resin) - superior for light microscopic
studies (in hard tissues such as undecalcified
bone) and for tissue sections thinner than usual 4-
6um (renal and bone marrow biopsy)
Interview topic IV: TRIMMING,
HONING/STROPPING, FLOATING OUT/FISHING
AND ADHESIVENESS
1. What is the proper orientation of the tissue?
- select a proper sized mold and to allow
segments of the specimens to be embedded all
flat to the bottom at the container and still have a
margin of a few ml around all edges. Thr mold
must also be deep enough to allow the paraffin to
be added to about the thickness of the specimen.
2. What shape is the tissue block trimmed?
Truncate pyramid - to make more ribbon from
sections
- The excess paraffin wax is trimmed off from the
block to expose the tissue surface
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3. What are the five types of microtome?
A. Rotary (minot) 1885-1886 - 4-6um upward/downward

- for cutting of paraffin, paraplast, carbowax embedded blocks

- most common; rotation of flywheel causing reciprocal motion of knife over the block

B. Sliding (Adams) 1789 - 10-25um

- base sledge or standard sliding

- for cutting celloiding embedded block

- most dangerous because of movable exposed knife

C. Rocking (Cambridge) 1701 - 10-12um forward/backward

- for cutting of paraffin embedded blocks of tough tissues

- for cutting sections of large blocks or paraffin embedded tissues; cant form ribbons

- simplest; heavy base and 2 arms

D. Freezing ( Queckett) 1848 - 0-15um

- for cutting of unembedded or gelatin embedded blocks frozen in liquified CO2 and etc.

- rapid diagnosis, fat demostration, neurological structures

E. Ultra-thin - 0.5-1um

- for cutting of thin sections from a plastic or epoxy embedded block

4. What are the three types of conventional knives?

KNIVES LENGTH MICROTOME EMBEDDING MEDIA

BICONCAVE 120mm rotary paraffin

PLANE-CONCAVE 25mm sliding, rotary, rocking celloidin, paraffin

PLANE-WEDGE 100mm sliding or base sledge paraffin block

type

5. What is the purpose of honing? - remove irregularities from knife

- "edge first" "heel to toe"

- belgium yellow, arkansas, carborundum
- Lubricants: mineral and clover oil
- involves the removal of gross nicks on the knife
edge to remove blemishes and grinding the
cutting edge of the knife on a stone to acquire an
even edge.
6. What is the purpose of stropping?
- it removes "burr" created by honing and cutting
edge of the knife is polished.
- Its purpose is to polish and sharpen the cutting
edge

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7. What are the purpose and procedure for 8. What is the purpose and procedure for fishing
floating out? sections?

Purpose: done to flatten the sections and prepare Purpose: Thin coats of albumin are spread thinly
them for mounting into the slide. Also folds and so that the protein will not pick up the stain and
creates may be removed by stretching gently with distract the appearance of the slide; mount tissue
a pair of dissecting needles sections on a clean glass slide

Procedure: A section is selected for staining and
placed in a slide and floated out in a water bath
set approximately 6-10 C lower than the melting
point of the wax is used in embedding the tissue:
45-50 C in water bath
Procedure: apply a thin coat of albumin(adhesive)
on the slide and spread thinly to affix the section
to the slide.
9. What is the purpose of adhesive?

- alternative to drying is by the use of adhesive

which is spread thinly and evenly on the slide to
promote adhesion of sections and essential for
methods that require exposure of sections to
acids and alkalis during staining

10. Give an example of adhesive. Give its composition and two advantages.
ADHESIVE COMPOSITION ADVANTAGE

Mayer's egg albumin - egg white 50ml - easy to make

- glycerin 50ml - inexpensive

- thymol crystal 100mg - inconvinient

Dried albumin -5g dried egg albumin

- 5g NaCl

- 150ml of distilled water and crystal of thymol

Gelatin-formaldehyde - 5ml 1% gelatin

mixture
- 5ml 2% formaldehyde

GALLEGO.JG2017-2018
Interview topic V: STAINING AND MOUNTING
1. What is the principle of staining?
- It is the process of applying dyes on the sections
to see and study the architectural pattern of
tissues and physical characters of the cells. This is
made possible because diff. Tissues and cells
display varying affinities for most dyes and stains
used during the process, so thay they may
become more visible, morphologic changes are
more identified and the presence or absence of
disease process can be established.
o Histological staining - demonstrate tissue
constituents
o Histochemical - various constituents of
tissues are studied
o Immunohistochemical - phenotypic
markers are detected
Principle: On chemical basis, certain parts of cells
and tissues are acidic in character and have
greater affinity for basic dyes, while basic
constituents take more acid stains.
Acidin(nucleus) - take up basic dye (Hematoxylin)
Basic(cytoplasm) - take upnacidic dye (eosin)
2. What are the most commonly used stains in thr
lab?
o Hematoxylin - hematoxylin
campechianum; most valuable due to its
clear and chromatin staining; commonly
used for histological studies (stain nucleus)
o Eosin - most valuable stain used for
differentially staining CT and cytoplasm
o Crystal violet - nuclear or chromatin stain
used for staining amyloid in frozens
sections and platelets in blood
o Methylene blue - common basic nuclear
stain employed eith eosin to provide
marked differentiation of various
structures in the tissue.
3. Differentiate direct from indirect staining.
Direct staining
o dye + tissue due to opposite electric
charge
o process of giving color to sections by using
aqueous or alcoholic dye solutions
Indirect staining
o process whereby the action of the dye is
intensified by adding another agent or
mordant which serves as a link or bridge
between the tissue and the dye, to make
staining possible (K alum with hematoxylin
in Erlich's hematoxylin)
4. Differentiate progressive from regressive
staining.
Progressive staining
o tissue elements are stained in a definite
sequence.
o staining solutions is applied for specific
periods of time or until desired intensity of
coloring of diff. Tissue elements is attained
o wrights, romanowsky: eosin, mb
Regressive staining
o tissue is overstained
o excess stain is removed or decolorized
from unwanted parts of tissue
5. Differentiate metachromatic from
counterstaining.
Metachromatic staining
o entails the use of specific dyes which diff.
Particular subs. By staining them with
color that is diff. From that of the skin
itself.
o safranin, thionine
Counterstaining
o application of diff. Color or stain to
provide contrast abd background to the
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 staining of the structural components to
be demonstrated
o neutral red, safranin O, Carmine,
hematoxylin
6. Differentiate supravital from intravital staining.
Supravital staining
o stains living cells immediately after
removal from the body
o neutral red, janus green, trypan blue
Intravital staining
o done by injecting the dye into any part of
the animal body, producing specific
coloration of certain cells, particularly in
the reticuloendothelial system
o lithium, india ink
7. Explain the principle of metallic impregnation.
- Process where spec. Tissue elements are
demonstrated, not by stains, but by colourless
soltns. Of metallic salts which are thereby
reduced by the tissue, producing an opaque,
usually black deposit on the surface of the
tissue/bacteria
8. Give 5 special stains, the structure it
demonstrates and the positive color.
A. Periodic acid schiff - carbohydrates - red
magenta
B. Feulgen - DNA - red purple
C. Best carmine - glycogen - bright red
D. Congo red - amyloid - deep red
E. Weil's - myelin sheath - black
F. Sudan black - lipids - blue black
G. Van Greson - collagen - pink/deep red
9. What is the definition and purpose of
mounting?
o a synapsy fluid applied bet. The section and the
coverslip after staining, settling the section
firmly, preventing the movement of coverslip
o mounting prevents the stained section from
getting scratched, and from bleaching or
deterioration due to oxidation, thereby
preserving the slides for permanent keeping, to
facilitate easy handling and storage.
o to improve microscopic exam because the
medium used has a refractive index very close
to that of clear glass which is 1.065
o sealant: chroning cement, burofix?
10. Differentiate temporary from permanent
mountant
Temporary mountant
- chosen when urgent biopsy is needed and for
mounting of metachromatically stained sections
- water-miscible (glycerin gel)
Permanent mountant
- references
Natural/resinous - canada balsam
Synthetic - entellan
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Interview topic VI: DIAGNOSTIC CYTOLOGY
1. What is diagnostic cytology?
- Microscopic exam from diff body sites for
diagnostic purposes
2. What are the two branches of cytology?
Exfoliative cytology - deals with the microscopic
study of cells that have been desquamated from
epithelial surfaces. exfoliated cells may be found
in smears that had spontaneously been shed or
physically removed from epithelial and mucous
membranes such as Vagina, buccal cavity and
from body fluids( e.g sputum, urine,Etc)
Fine needle aspiration cytology - simple, safe and
Rapid such cytologic technique that had been
commonly used for diagnosis of cancer in the last
30 years; for palpable mass
3. Give the five applications of diagnostic
cytology.
o detection of malignant cells in body fluids;
mainly used for staging cancer
o Detection of precancerous cervical lesions
in women
o assessment of female hormonal status in
case of sterility and endocrine disorders
o for determination of genetic sex
o for detection of infectious agent
4. What are the ways of processing a cytological
specimen?
A. SMEARING (PAP SMEAR)
Viscous specimen -> smearing -> fixation ->
staining
Non viscous specimen ->fixation -> centrifuge -
> smearing -> staining
B. FILTER MEMBRANE TECHNOLOGY
Specimen(CSF, synovial) ->
ultracentrifugation(e.g nucleopor) -> tape
special filter on slide (cellulose acetate) ->
fixation -> staining
C. CELL BLOCK
Specimen (routine/effusion) -> fixation ->
centrifuge -> collect sediments, pack in special
paper -> fixation (Helly’s fluid) -> process as
biopsy sample
5. Explain the process of staining using modified
Pap's staining technique.
After fixation in 95% alcohol:
o Hydration - 80%, 70%, 50% alcohol (6 dips)
o Nuclear staining - Harris hematoxylin (6min)
o Differentiation - distilled h2o, 0.25/0.5% Hcl,
tap h2o running
o Dehydration - 50, 70, 80, 95% alcohol
o Counterstaining - OG - 6(1 & 1/2min)
o Differentiation - 95% ethanol; 3 changes
6. What are the cells found in the normal cervico-
vaginal smear? Diff. Each.
A. Mature superficial cells - polygonal squamous
cells measure 45 to 50 micrometers in diameter,
they're usually identified by the presence of pale,
pink staining cytoplasm and dark pyknotic nuclei;
<6um in diameter
True acidophilia - superficial vaginal cells under
estrogen influence
Pseudoacidophilia - drying of smear before
fixation
B. Intermediate cells - medium sized polyhedral or
elongated cells with basophilic vacuolated
C. Parabasal cells - round to oval cells with small
dents basophilic cytoplasm and the total cell
diameter of 15-30um; large vesicular nucleus; two
weeks of age to puberty, post child birth,
abortion, and post-menopause
D. Navicular cells - boat shaped intermediate cells
with strong tendency to fold or curl on edges;
latter half of menstrual cycle, pregnancy,
menopause
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E. Pregnancy cells - round, oval or boat shaped
cells with translucent basophilic cytoplasm
observed at the center of the cells due to:
glycogen accumulation, push nucleus to side or
toward CM
G. Endocervical glandular cells - occur in large
groups of a small sheets. The cytoplasm stained
will blue or gray and finely vacuolated cytoplasm

F. Endometrial cells - small cells, slightly

cylindrical with less basophilic cytoplasm
occurring in pact groups of three or more; 1-10
days after menstruation; shed in response to
ovarian hormone

7. What are the the grading systems for cytological specimens? Differentiate each.

Pap's grading

Class I: Absence of atypical cells

Class II: prsence of atyoical cytologic pic. But no evidence of malignancy

Class III: (+) severe dysplasia suggestive but not conclusive of malignancy

Class IVa: carcinoma insitu

Class IVb: invasive carcinoma

Bethesda system

Group I: Exclusively normal cells

Group II: Normal variant but not prone for malignancy

Group III: Moderate to severe dysplasia

Group IV: Carcinoma insitu

Group V: Invasive carcinoma

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