Sample Pre-Test

Histopathology Part 1
Matching type
COLUMN A COLUMN B

1. Zenker’s fluid A. 3 – 24 hours

2. 10% phosphate buffered formalin B. 12 – 24 hours

3. Formol – corrosive C. 12 – 48 hours

4. Heidenhain’s susa fixative D. 3 – 12 hours

5. Regaud’s fluid E. 4 – 24 hours

• A. Microanatomical fixative
• B. Nuclear fixative
• C. Cytoplasmic fixative
• D. Histochemical fixative
• E. NOTA

• 6. Acetone
• 7. Flemming’s fluid
• 8. 10% Neutral buffered formalin
• 9. Brasil’s solution
• 10. Flemming’s fluid without acetic acid
• A. Gendre’s fixative
• B. Helly’s solution
• C. Moller’s fluid
• D. Formol-sublimate
• E. Osmic acid
•
• 11. Alcoholic formalin fixative
• 12. Regaud’s fluid
• 13. Formol-corrosive
• 14. Osmium tetroxide
• 15. Zenker-formol
Matching Type
COLUMN A COLUMN B
16. Decalcification 35 A. 3rd step
17. De-alcoholization 34 B. 5th step
18. Fixation 33 C. 1st step
19. Infiltration 31 D. 2nd step
20. Dehydration 32 E. 4th step
21. What fixative is contra-indicated to radiological method in determining extent of
decalcification due to its characteristic radio-opacity?
A.Regaud’s fluid B. Orth’s fluid
22. Composition of Gilson’s mixture used in Dry celloidin method
A. 3ml chloroform + 2ml cedarwood oil
B. 2ml chloroform + 3ml cedarwood oil
23. Blueing agent recommended for thin paraffin sections
A. Scott’s T.W.S. B. Ammonia water
24. Fixative used for bacteriologic purposes
A. Carnoy’s fixative B. Acetone
25. Helly’s solution
A. Mercuric chloride + glacial acetic acid
B. Mercuric chloride + formalin
Rationale
CELLULAR ADAPTATIONS
ATROPHY (involution, diminution) refers to decrease in cell size
• ↓ Workload,
• loss of nerve supply
• ↓ Blood supply (ischemia),
• Inadequate nutrition (starvation)
• Loss of hormonal stimulation
• Physiologic atrophy occurs with aging in the parenchyma of organs
and allows for survival of cells with decrease function
• Physiologic atrophy begins in the thymus and intermediate lobe in
early adulthood and in the uterus after menopause
FIXATION
• Fixation is the foundation step of the histo-technology
• MUST be done IMMEDIATELY
• To preserve
• To harden
• It inhibits both putrefaction and autolysis
• The effectiveness is noted at 20x
• To date, formaldehyde as a 10% neutral buffered formalin is the most
widely used universal fixative because it preserves a wide range of
tissues and tissue components
• Methanol
FIXATION

• Glacial acetic acid is a component of compound fixative

• It solidifies at 17’C hence the name glacial
• Contraindicated for cytoplasmic fixation because it destroys mitochondria and Golgi
apparatus
• It causes the tissue to swell
• Zenker-formol (Helly’s solution) is a microanatomic fixative for pituitary gland, BM
and blood containing organs
CHARACTERISTICS OF FIXATIVES
 FIXATION

• Acetone is recommended for fixing brain tissues for diagnosis of rabies and water
diffusible enzymes such as lipases and phosphatases
• Carnoy’s fluid is the most rapid fixative. Recommended for urgent biopsises,
chromosomes and lymph nodes
• Bouin’s solution is recommended for fixation of embryos and pituitary biopsies
• WASHING-OUT is a process of removing excess fixative from the
tissue after fixation in order to improve staining and remove artefacts
• 50 to 70% alcohol for picric acid (Bouin’s)
• Alcoholic iodine for Mercuric chloride fixatives
• Water for all the rest like Flemmings, formalin, osmic acid, etc
• Formol-Corrosive (sublimate)
DECALCIFICATION
• DECALCIFICATION is a procedure whereby calcium or lime salts
are removed from calcified tissues
• The rate of decalcification may be influenced by several factors
like structure of the tissue, size of the tissue, concentration,
volume, mechanical agitation to facilitate fluid exchange,
accelerates the rate of diffusion, and temperature at which the
reaction takes place. The recommended ratio is 20:1
• It is done after fixation process and before impregnation to ensure
and facilitate normal cutting
• Non-removal of calcium salts may interfere with the accurate
evaluation and examination of histologic sections
• Mercuric chloride-fixed tissues are contraindicated to radiological
analysis due to its characteristic radio-opacity
EXOCRINE GLAND
• In merocrine glands, the secretory cells release their secretion by
exocytosis. Hence the discharge of the secretion does not result in the
loss of any part of the cell. Salivary glands & exocrine portion of
pancreas.
• In holocrine glands, release of secretion entails destruction of the
secretory cells whose are then discharged by the gland together with
the secretions. Sebaceous.
• In apocrine glands, the apical part of the secretory cells is released
together with the secretory product. Ceruminous & Sudoriferous
gland
CELLULAR ADAPTATIONS
• Hypertrophy is an increase in the size of individual cells
• It usually represents the response the response of a specific organ to an
increased demand for work
• Hypertrophied cells increase their number of intracellular organelles
especially mitochondria.
• Physiologic hypertrophy is the enlargement of muscles of athletes or
weight lifters. It also occurs at puberty with enlargement of sex organs.
• Pathologic hypertrophy caused by an increased functional demand
such as systemic hypertension in which the myocardium must pump
under greater pressure thus increases the size of myocardial muscle cells.
• Hyperplasia is an increase in the number cells. Cells that undergo
hyperplasia are those that are capable of cell division (mitosis).
Physiologic hyperplasia is normal outcome of puberty and pregnancy.
• Renal tumour → erythropoietin → ↑ RBC in circulation
REMOVAL OF FORMALIN PIGMENTS
 ASSESSING EXTENT OF DECALCIFICATION

Chemical Method
• Calcium oxalate test
• simple, reliable, convenient method and is recommended for routine purposes.
• 5ml acid solution + litmus paper turn red due to acidity. Strong ammonia is added drop by
drop
• Litmus will turn blue indicates alkalinity.
• If cloudiness is present it indicates incomplete decalcification.
• 0.5 ml of sat. aq. Solution of ammonium oxalate is added and incubate for 30 minutes
DEHYDRATION
• DEHYDRATION is a process of removing intercellular extracellular
water from tissue following fixation and prior to wax impregnation using a
series of alcohols up to absolute alcohol preferably ethanol
• Ethyl alcohol is recommended for routine dehydration. It is best
dehydrating agent because it is fast acting, not poisonous and inexpensive.
• Methyl alcohol is used for blood smear preparation but toxic.
• Butyl alcohol for plant and animal microtechniques, recommended for
tissue that do not require rapid processing due to its slow acting properties
§ Dioxane
§ Graupner’s method – utilizes pure dioxane and melted paraffin wax
both for 3x
§ Weiseberger’s method – same with Graupner’s except for
utilization of calcium oxide (quicklime)
CLEARING
• DE-ALCOHOLIZATION is the process whereby the dehydrating
agent is removed from tissue and replaced with a substance that will
dissolve the wax with which the tissue is to be impregnated (paraffin)
• Aniline oil not normally utilized. For delicate specimen like embryos
and insects, due to ability to clear 70% alcohol without excessive tissue
shrinkage & hardening.
• Clove oil, its quality is not guaranteed, may be adulterated. Tissue
becomes brittle, aniline dyes are removed, expensive.
• Xylene (Xylol) most commonly used in histology lab due to its rapid
acting nature hence suitable for urgent biopsies. Cheap, does not
dissolve aniline dye, miscible with alcohol and paraffin.
• Benzene similar with xylene. Health hazard (carcinogenic) may damage
BM causing aplastic anemia
• Chloroform is slower than xylene but cause less brittleness. It is
recommended for tough tissues like skin, decalcified tissues, nervous
tissue, embryo and lymph nodes
MOUNTING
• GLASS SLIDE : 1.518
• Canada balsam is a natural resin extracted from North American fir tree
Abies balsamea usually dissolved in xylene. The solution acidifies and
darkens with age and upon exposure to sunlight (LIGHT SENSITIVE)
• Farrants medium is an aq. mounting medium containing gum Arabic
and, glycerol and sodium merthiolate. With RI 1.43. Arsenic trioxide
may be used as a substitute for Na-merthiolate for medium preservation.
Addition of 50gm potassium acetate will produce neutral pH (7.2)
instead of an acid (4.4) and will raise RI to 1.44.
• Cochineal dye is extracted from female cochineal bug Coccus cacti
which is treated with alum to produce dye, carmine, widely used as
powerful chromatin and nuclear stain
• Carmine + picric acid (picrocarmine)
• Carmine + Aluminum chloride (Best’s carmine)