Histopathology-Review-Booklet 221104 105446

Hanzjethro Villanueva Santos,RMT (+63917)8974844
This review booklet belongs to: ___________________________

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Histopathologic Techniques
Biopsy: obtained from LIVING source Autopsy: obtained from DEAD source
Histotechnique: Art and Science performed by Medical Laboratory Scientists/ Medical Technologist to produced Good Quality
tissue sections that will enable the Pathologist/s to diagnose presence or absence of malignancy.
STEPS:
1. FIXATION: considered the MOST CRITICAL, must be done adequately. If not, other steps following fixation will
be affected. Fixation should be done first before LABELLING.
2. DECALCIFICATION and DEHYDRATION
A. DECALCIFICATION: procedure whereby calcium ions or lime salts are removed from the tissue.
B. DEHYDRATION: process of removing intracellular and extracellular waste, water from the tissue.
*Following fixation and prior to wax impregnation.
3. CLEARING: process of removing the alcohol used as a dehydrating fluid from the tissue and replaced by a substance.*
make the tissue transparent
4. IMPREGNATION: process whereby the clearing reagent used is completely removed from the tissue and
replaced by a medium that will fill all the cavities of the tissue.
5. EMBEDDING: process by which the impregnated tissue is placed into a precisely arranged position in a mold.
6. MICROTOMY/SECTIONING: procedure whereby the processed tissue is trimmed and cut uniformly thin
slices or sections to facilitate studies under the microscope.
7. STAINING: process of applying dyes on the sections to see and study the architectural pattern of the tissue and
cells that make up the whole tissue.
8. MOUNTING: procedure whereby the processed tissue is placed in a slide containing adhesives, mounting medium
and cover slip to protect the tissue section.
9. LABELLING: done to facilitate proper and correct specimen identification.
1. FIXATION:
Primary Goal: to preserve the morphology and chemical integrity of the tissue as close to the original as possible.
Secondary Goal: 1.) to harden the tissue 2.) to protect the tissue from trauma of further handling
Fixation time: Minimum of 6 hours and Maximum of 48 hours.
For Electron Microscopy: 3 Hours
2 types of Fixative:
A. ADDITIVE: The chemical component of the fixative becomes part of the tissue.
Ex: Mercuric fixatives, Formalin
B. NON-ADDITIVE: The chemical component of the fixative does not become part of the tissue but
alters the tissue component. Ex: Alcohol fixatives
*Hydrogen Ion Concentration: pH should be 6-8 (Slightly Hypertonic)

*Amount of fixative: should be 20x the volume of the specimen (15-20:1 ratio)
*Electron Microscopy: 20x the volume of the specimen
*Osmium tetroxide: 5-10x the volume of the specimen
*Museum preparations: not less than 50x the volume of the specimen
*Factors that will retard/prolong fixation time *Factors that will accelerate fixation time
1. Size and Thickness: the larger, the longer time. 1. Size and Thickness: the smaller, the shorter
time
2. Cold temperature 2. Agitation: Continuous mixing
3. Presence of Mucus and blood: wash with Normal Saline 3. Heat and Pressure: 37-56degC
*If the specimen is Human Brain, wash with Ringer’s Lactate
*Factors to consider in choosing the appropriate fixative:

1. The need for immediate examination 3. The tissue structure to be studied
2. The type of tissue to be processed 4. The type of stain to be used
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*Effects of fixative in general:

1. Harden the tissue 3. Reduce the risk of infection
2. Prevent bacterial growth 4. Increase optical differentiation of cells and tissue
Simple Fixative: contains only one substance Compound Fixative: contains more than one
subs.
*Heat Fixation: Thermal coagulation of proteins, usually use for bacterial smears and frozen sections.
*Acetone: Acts as both Fixative and Dehydrating agent. Should be in Ice cold temperature ( -5 to 4 degC)
Used for the preservation of Lipase and Phosphatase, Enzyme studies.
Used small pieces of tissues and for Brain specimen for the diagnosis of RABIES.
Dissolves Fats, Evaporates easily and FLAMMABLE.
*Trichloroacetic Acid: Acts as both Fixative and Decalcifying agent.
*Poor penetrating fluid, often incorporated in compound fixative.
*Glacial Acetic Acid: for Nucleoproteins and Nuclear chromatin and Cytoplasmic differentiation.
Often used in conjunction with other fixatives. SOLIDIFIES at 17degC.
*Alcohol fixatives: causes Glycogen polarization, rapidly denatures and precipitates proteins.
Acts as both Fixative and Dehydrating agent. Ideal for Small tissue Fragments.
A. Methyl Alcohol: for fixing Wet and Dry preparations, smears (blood smears and BM tissues)
B. Ethyl Alcohol: for Blood and Tissue films
C. Isopropyl Alcohol: for Touch Preparations
D. Carnoy’s: Most rapid alcohol fixative, for urgent biopsies (Chromosomes, Lymph glands)
Also used for the diagnosis of Rabies.
E. Newcomer’s: Compatible with FUELGEN. Used for Mucopolysaccharide and Nuclear
proteins.
*Aldehyde fixatives:
A. Formaldehyde
*10% Formaldehyde: commonly used for mailing specimen, with 1mm/hr penetration
rate. Fumes are irritating to the eyes and Nasal mucosa. may cause dermatitis. Precipitation of
white paraformaldehyde is possible.
*37-40% Formaldehyde: diluted form and is also knows as 100% formalin.
B. Gendre’s Fluid: fixes Sputum specimen, used for Microincineration Technique.
C. 10% Formol Saline: used for Central Nervous System, Fats and Enzymes
Diluted with Sodium Chloride, Used generally for Post-mortem tissues.
D. Formol Corrosive: contains 100% formalin and Mercuric chloride.
recommended for Lipids, Phospholipids and Neutral fats.
F. Glutaraldehyde: recommended for Electron Microscopy and Enzyme Histochemistry
*2.5% solution recommended for small tissue fragments
*4% solution recommended for larger tissues with thickness less than 4mm
G. Karnovsky’s paraformaldehyde-glutaraldehyde and Aclrolein:
* Used for Electron Microscopy and Immunohistochemistry
*Picric Acid Fixatives: Imparts Yellow color (treat with Acid Lithium Carbonate). Can cause cell shrinkage.
* Can act as both Fixative and Stain for small tissues.
A. Bouin’s fluid: recommended for fixation of Pituitary biopsies, Embryo and Endometrial curetting
NOT SUITABLE FOR KIDNEY SPECIMEN. Abolishes Fuelgen reaction.
B. Brasil’s Alcoholic Picroformol: Excellent fixative for Glycogen
*Metallic Fixatives:
A. Lead fixatives: fixatives used for Acid Mucopolysaccharide and Connective tissue mucin
B. Chromate fixatives: Highly corrosive to skin and mucus membrane.
B.1 Chromic acid: fixative used for Carbohydrates
B.2 3% Potassium Dichromate: preserves lipids and mitochondria
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B.3 Regaud’s/Moller’s: used for Chromatin, Mitochondria, Golgi bodies, RBC cont. Colloid
B.4 Orth’s fluid: used for early degenerative processes and tissue necrosis.
used for fixation of Rickettsia and other microorganisms.
C. Mercuric Fixatives: most common metallic fixative. Highly Toxic fluid.
excellent for Tissue photography and Trichrome staining.
C.1 Zenker’s fluid: contains Glacial Acetic Acid. For Liver, Spleen, CT fibers and Nuclei.
C.2 Zenker’s formol/ Helley’s: for pituitary glands, bone marrow and blood containing organs
C.3 Heidenhain’s Susa: for Tumor biopsies of the Skin.
C.4 B5 fixative: contains anhydrous sodium acetate, for Bone marrow specimen
*Osmium Tetroxide/Osmic acid: used for Electron Microscopy, Myelin and Peripheral nerves specimen.
A. Flemming’s: most common chrome osmium acetic acid fixative. Excellent for NUCLEAR structures
B. Flemming’s without acetic acid: for CYTOPLASMIC structures.
*Microwave Technique: Accelerates FIXATION, DECALCIFICATION and STAINING.
*Physical ageant with similar mechanism to oven, vacuum and agitation.
*Maybe used for neurochemical substances in brain like ACETYLCHOLINE.
SECONDARY FIXATION: placing an already POST-CHROMATIZATION: form of secondary

Fixed Tissue in a second fixative in order to: fixation using any CHROMATE FIXATIVE.
1. Facilitate and improve demonstration of
particular substances WASHING OUT: removing of excess fixatives.
2. Make special staining techniques possible 1. Tap water: removes excess Osmic acid,
chromates
3. Ensure further and complete hardening and (Kelly’s, Zenker’s and Flemming’s soln.,
Formalin
preservation of tissues. 2. 50-70% alcohol: wash out excess picric acid
3. Alcoholic Iodine: removes excess mercuric fix.
2. DEHYDRATION
*The amount of the dehydrating fluid should be NOT LESS THAN 10X THE VOLUME of the specimen.
*Starts by placing the fixed tissue in 70% alcohol to ASCENDING GRADES of alcohol.
*ASCENDING GRADES: DEHYDRATION *DESCENDING GRADES: HYDRATION
*ALCOHOL: used for ROUTINE tissue processing, used in increasing concentration to avoid distortion.
- for smaller and delicate tissues, low concentration of alcohol can MACERATE the tissue.
- Longer storage in 70-80% may affect the process of staining, Hastens at 37degC
- ANHYDROUS COPPER SULFATE: used as an indicator that will accelerate the process.
Removes the water from the agent.
Turns to BLUE when it its fully/completely dehydrated.
A. Ethyl Alcohol: used for routine dehydration of urgent biopsies, NON-TOXIC but,
FLAMMABLE.
BEST DEHYDRATING agent, FAST ACTING suitable for FATTY TISSUES
B. Methyl Alcohol: Toxic dehydrating agent, used for Blood and Tissue smear preparations.
C. Butyl Alcohol: Dehydrating agent suitable for Plants and Animals.
D. Isopropanol: Dehydrating agent substitute for Ethanol and Xylene. Used in Microwave
technique.
*ACETONE: Evaporates easily, can cause TISSUE SHRINKAGE,
RAPID ACTING, suitable for small pieces of tissue
*DIOXANE: Excellent Dehydrating and Clearing agent. The tissue can be left for long period of time.
- Toxic in man, TISSUE MAY TEND TO FORM RIBBON POORLY.
*2 methods:
1. GRAUPNER’S: involves 3 changes of pure Dioxane soln. and 3 changes of Paraffin wax.
2. WEISEBERGER’S: The tissue is wrapped in a gauze bag. Calcium Oxide absorbs the water.
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*ETHYLYNE GLYCOL/CELLOSOLVE: dehydrates rapidly but is COMBUSTIBLE at 110-120degF.

- Removes ANILINE DYES, toxic by inhalation, skin contact and ingestion.
- Prolong exposure can be toxic to fetal and reproductive system.
*TETRAHYDROFURAN: used for ATHEROMATOUS ARTERIES and HARD COLLAGENOUS specimen.

*Acts as both Dehydrating and Clearing fluid. May cause Dizziness and Nausea.
*TRIETHYL PHOSPHATE: removes water readily and produces minimum shrinkage.
*Used to dehydrate sections and smears following certain stains.
*Produce little distortion and hardening of tissue.
*CARBOXYL: acts as both Dehydrating and Clearing agent.
3. CLEARING
* REMOVES ALCOHOL and MAKE THE TISSUE TRANSPARENT prior to infiltration
*Clearing agent must be miscible with: 1. DEHYDRATING FLUID 2. INFILTRATING MEDIUM 3. MOUNTING
MEDIUM
*Frozen Sections: Glycerin and Gum syrup *Factors affecting clearing:
is used. No dealcoholisation is involved. 1. Viscosity: affects PENETRATION
2. Boiling point: low boiling pt. replaced by wax
*XYLENE: most rapid clearing agent for urgent biopsies. Miscible with absolute alcohol and Paraffin.
- Can be used for celloidin sections. However, the fluid evaporates quickly in paraffin oven.
- Not suitable for NERVOUS TISSUE and LYMPH NODES.
- Disadvantage is it is CARCINOGENIC.
*TOLUENE/TOLUOL: substitute for XYLENE or BENZENE. Tends to acidify in partially filled vessel.
*BENZENE: Best reagent for EMBEDDING process, Penetrates and clears rapidly for urgent biopsies.
- Does not make the tissue hard and brittle but causes minimum shrinkage.
- May damage the Bone Marrow of man in prolong exposure. May cause APLASTIC ANEMIA.
- Highly Inflammable
*CHLOROFORM: Slower in action compared with Xylene. DOES NOT MAKE THE TISSUE TRANSPARENT
- Used for Thicker tissue block and large tissue specimen.
- Recommended for TOUGH TISSUES (SKIN, FIBROID and DECALCIFIED Tissues)
- Tissues may tend to float (wrap the tissue in a gauze), may also attack the rubber seal.
- Not inflammable but toxic to LIVER.
*CEDARWOOD OIL: used to clear both PARAFFIN and CELLOIDIN sections during embedding.
- recommended for cytological studies. (Smooth muscles of skin and Central Nervous System)
*CLOVE OIL: tissues tend to become ADULTERATED.
- when used, the wax impregnation is slow and difficult
- tissue becomes brittle, Removes Aniline dye, dissolves Celloidin
*ANILINE OIL: not normally utilizes as a clearing agent.
- recommended for EMBRYOS, INSECTS and other DELICATE specimens.
*CARBON TETRACHLORIDE: properties and disadvantages are similar with chloroform.
- Dangerous to inhale due to its toxicity.
*METHYL BENZOATE: slow acting, used when DOUBLE EMBEDDING TECHNIQUE is required.
*Other Clearing Agents: *Clearing agent miscible with absolute alcohol

1. Oil of Bergamot: for Skin and Smooth Muscles 1. Xylene (miscible with paraffin)
2. Oil of Origanum: for Skin 2. Toluene (miscible with paraffin)
3. Oil of Wintergren: for Delicate tissue 3. Benzene
4. Carbon disulfide: for Smooth Muscle 4.Chloroform
5. Carbon Xylene: for Friable tissue 5. Carbon tetrachloride
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6. Terpineol: for Eyes

7. Phenol: for Smooth Muscle
8. Limonil: Substitute for Xylene
9. High Test Aviation Lead Free Gasoline
- Excellent clearing agent
- Produces LESS SHRINKAGE.
10. Creosole: for Celloidin section
11. Carboxylol: Dehydrating and Clearing agent.
DECALCIFICATION:
* Procedure whereby calcium ions or lime salts are removed from the tissue
*Done to ensure and facilitate the normal cutting of sections, prevent obscuring the microanatomical
detail
of such sections and other cellular debris. *Calcified bones/tissues are usually cut into small pieces.
*Both decalcification and processing depend on bone thickness. Ideal thickness: 1-3mm
METHODS OF DECALCIFICATION:
1. Use of Acid
A. NITRIC ACID: Most commonly used decalcifying agent. The FASTEST used so far.
A.1 10% Aqueous Nitric Acid: used for urgent biopsies, Fast acting.
- Recommended for HEAVILY CORTICAL BONES.
- Can damage tissue stainability, Imparts YELLOW color with Nitrous Acid.
A.2 Formol Nitric Acid: Rapid Acting for urgent biopsies.
- Yellow imparted by nitric acid formation impair staining reaction of the cell
- Neutralizes the tissue with 5% Sodium Sulfate and wash in running water.
- Addition of 0.1% Urea to pure concentration nitric acid can also disappear the
the tissue discoloration.
A.3 Perenyi’s Fluid: composed of NITRIC ACID + CHROMIC ACID + ETHYL ALCOHOL
- Recommended for routine purposes, DECALCIFY and SOFTEN the tissue.
- When used, Nuclear and Cytoplasmic staining is good.
- Slow decalcifying agent for DENSE BONES.
A.4 Phloroglucin Nitric Acid: MOST RAPID so far. Recommended for urgent biopsies.
- may cause EXTREME TISSUE DISTORTION when prolong decalcification.
- Nuclear staining is poor.
B. HYDROCHLORIC ACID: used for Surface decalcification of blocks and small pieces of bones.
B.1 Von Ebner’s Fluid: permits relatively GOOD CYTOLOGIC STAINING.
- DOES NOT REQUIRE WASHING OUT before dehydration.
- Recommended for TEETH and SMALL PIECES OF BONES.
C. FORMIC ACID: produces BETTER NUCLEAR STAINING and less tissue distortion.
- Safer to handle for routine decalcification of POST-MORTEM research tissues
- recommended for Research and Autopsy, Cartilage and Bone Marrow
- Addition of CITRATE accelerates decalcification time by chelating calcium.
D. TRICHLOROACETIC ACID: Permits GOOD NUCLEAR STAINING, WEAK AGENT.

E. SULFUROUS ACID: Very weak decalcifying agent. Used only for MINUTE PIECES OF BONES
F. CHROMIC ACID: may be used as both FIXATIVE and DECALCIFYING agent

- recommended for MINUTE BONE SPICULES
- considered as an ENVIRONMENTAL TOXIN, Carcinogenic.
- COROSSIVE TO SKIN and MUCUS MEMBRANE.
G. CITRIC ACID CITRATE BUFFER: Weak decalcifying agent.
- Uses CHLOROFORM as preservative
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2. Use of Chelating Agent

- Substances which combines with calcium ions and other salts like iron and magnesium deposits.
A. EDTA/VERSENE: recommended for detailed microscopic studies.
- Potent anticoagulant but WEAK AGENT. Excellent for Electron Microscopy
- Binds with calcium to form weakly dissociated complex.
- Inactivates ALKALINE PHOSPHATASE (Add Magnesium Chloride)
*For small specimens: 1-3 weeks *For dense tissues: 6-8 weeks
3. Ion Exchange Resin

- hastens decalcification by removing calcium ions from FORMIC ACID containing decal. agent.
- the amount of volume should be 20-30x the volume of the specimen. 1-14days duration
- uses Ammonium form Polystyrene resin. X-ray measures the process.
4. Electrolytic Method/Electrophoresis: (MOST RAPID METHOD)

- process whereby positively charged ions are attracted to a negative electrode and subsequently
Remove from the decalcifying agent.
- Satisfactory for SMALL BONE FRAGMENTS
- Principle is similar to chelating agents, utilizes electricity and dependent upon a supply of direct
current remove calcium deposits.
*MEASURING EXTENT OF DECALCIFICATION:

1. Physical Method/Mechanical: Inaccurate, Damages the tissue
2. X-ray or Radiologic Method: Most Sensitive and Reliable
- can detect even smallest focus of calcium.
- if calcium is still present, it will appear OPAQUE.
3. Chemical Method: involves using of Ammonium Oxalate

- Turbidity indicates incomplete decalcification
*Post Decalcification: procedure that involves washing the tissue with LITHIUM CARBONATE.
*To remove acids: 30mins. Washing with Running tap water (small) 1-4hours (larger specimen)
*FACTORS INFLUENCING RATE OF DECALCIFICATION:

1. CONCETRATION and VOLUME 3. STRUCTURE, TEMPERATURE
More concentrated : Rapid but harmful Fluid to tissue ratio: 20:1
2. HEAT 4. MECHANICAL AGITATION

Hastens decalcification nut increases Influence fluid exchange, Accelerates
damaging effects. The rate of diffusion and speeds up the process.
*TISSUE SOFTENERS:
1. Molliflex: 1%HCL in 70%Alcohol
2. Perenyi’s Fluid: 2%HCL in 70% Alcohol
3. Lendrums method: 4% Aqueous phenol
IMPREGNATION/INFILTRATION:
*Process whereby the CLEARING AGENT used from the tissue is removed and fill all cavities and spaces.
*Provides a FIRM CONSISTENCY to the specimen to allow easier HANDLING and CUTTING of
suitably
thin section without any damage or distortion.
*Clearing agent EASILY REMOVED: BENZENE and XYLENE
*Clearing agent DIFFICULT TO REMOVE: CHLOROFORM and CEDARWOOD OIL
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1. PARAFFIN WAX IMPREGNATION: Most commonly used, RAPID ACTING.

- Allows cutting SERIAL SECTIONS and compatible with many staining procedure
- NOT RECOMMENDED FOR FATTY TISSUES
- Prolonged process may lead to EXCESSIVE HARDENING of the tissue and tissue shrinkage
- Inadequate process may case retention of the clearing agent used
Melting Point for ROUTINE WORK 56 degree Celsius
If the laboratory temperature is between 20-24degC 54-58 degree Celsius
If the laboratory temperature is between 15-18degC 50-54 degree Celsius
*METHODS OF PARAFFIN WAX IMPREGNATION:

A. MANUAL: makes use of a PARAFFIN OVEN (2-5degC higher than the Melting Point) or
55-60degC temperature. Involves 4 CHANGES OF PARAFFIN WAX
B. AUTOMATIC: makes us of AUTOTECHNICON
- Usually, 2-3 CHANGES OF WAX for more rapid diagnosis and less technicalities.
C. VACCUM: Infiltration under NEGATIVE ATMOSPHERIC PRESSURE
- Recommended for urgent biopsies and delicate tissues: Lung, Connective Tissue, Brain
Decalcified bones, Eyes, Spleen, Central Nervous System
---------------------------------*SUBSTITUTE FOR PARRAFIN WAX*---------------------------------
1. PARAPLAST (MP: 56-57degC) 3. ESTER WAX (MP: 46-48degC)
- Mixture of HIGHLY PURIFIED PARAFFIN - Harder than paraffin. Makes use of HEAVY
and SYNTHETIC PLASTIC POLYMER DUTY MICROTOME. Eliminates Process of
- For BONES and BRAIN SPECIMEN clearing.
- With better RIBBONING of SECTIONS
4. BIOLOID: semi-synthetic wax
recommended
2. EMBEDDOL (MP: 56-58degC) for embedding EYES
- Similar to paraplast but LESS BRITTLE
and LESS COMPRESSIBLE 5. TISSUE MAT: product of paraffin with
RUBBER
*WATER SOLUBLE WAX: CARBOWAX (MP: 38-42degC or 45-56degC)

- for THICKER SECTIONS, Recommended for NEUROLOGICAL TISSUES
- Eliminates process of DEHYDRATION and CLEARING, recommended for ENZYMES and
HISTOCHEM STUDIES. Cytologic pictures are preserved but carbowax is very easily to be dissolved in
water. “VERY HYGROSCOPIC “or Water Soluble.
2. CELLOIDIN IMPREGNATION:
- A purified form of NITROCELLULOSE. For tissues with LARGE HOLLOW CAVITIES
that tends to collapse (TEETH, BONES, BRAIN, EYES), for Hard and Dense Tissues
- 2% (for thin), 4% (thicker), and 8% (more thick) medium solutions dissolved in equal parts of
ether and alcohol. Very Slow Acting
- Serial section: Difficult to Cut; Photomicrograph: difficult to obtain
*METHODS:
2.1 WET CELLOIDIN: recommended for BONES and BRAIN specimen. Uses 70% alcohol (storage)
* 2-4% for 5-7days transferred to medium celloidin of 4-6% for another 5-7days
*Drained off and poured with thick celloidin of 8-12% usually for 3-5 days.
2.2 DRY CELLOIDIN: Same as wet but does not involve 70% alcohol before cutting
- recommended for EYE specimen.
*GILSON’S MIXTURE: combination of Chloroform and Cedarwood Oil
3. GELATIN IMPREGNATION: rarely used for FROZEN TISSUES.
- Recommended for histochemical and enzyme studies, WATER SOLUBLE
- Tissue for processing should be 2-3mm; Add 1% Phenol to prevent MOLDS
4. USE OF PLASTIC RESIN: provides superior results for LIGHT MICROSCOPY\
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- For Hard tissues like UNCALCIFIED BONES

- For high resolution of light microscopy of tissue sections thinner than 4-6u
- For RENAL BIOPSIES, BONE MARROW BIOPSIES
4.1 EPOXY: components are TOXIC
a. Bisphenol A (Araldite): Slow acting for it is a LARGE MOLECULE
b. Glycerol based (Epon): Have lower viscosity
c. Cyclohexine dioxide (Spurr): Have very low viscosity but can infiltrate faster
4.2 POLYESTER: Originally introduced for Electron Microscopy in 1950’s
4.3 ACRYLIC: Made up of Esthers of Acrylic or Methacyclic acid. Used for Light Microscopy
a. Polyglycol methacrylate: Hydrophillic, Allows many staining method
b. Methyl methacrylate: Ideal medium for Uncalcified bones and other hard tissues.
EMBEDDING:
*Process of placing infiltrated tissue in a mold containing the embedding medium then is allowed to solidify.
*Placing the tissue in a précised position in the mold.
1. Leuckhart’s: consist of 2 L-shaped strips of Temperature of melted paraffin: 5-10degC
Heavy brass or Metal. higher than the melting point.
2. Compound Embedding: Series of interlocking
places or spaces on where to place the tissue. To allow solidification of block: immerse in
water
3. Plastic Embedding rings and Base molds: and refrigerate at -5degC
Can hold the tissue during cutting
4. Tissue-tek: equipped with warm and cold plate *DOUBLE EMBEDDING:” Infiltrate” using
5. Disposable molds: Paper boats, Peel away, Ice trays Celloidin, “Embedd” using Paraffin
MICROTOMY:
*Processed tissue is trimmed and cut into uniformly thin slices or sections to facilitate studied under the
microscope.
-----------------------*ESSENTIAL PARTS OF MICROTOME*----------------------------
1. BLOCK HOLDER CHUCK: Where the tissue 3. PAWL, RACHET FEED, ADJUSTMENT
is hold in position. CREWS: To line up the tissue block, improper
2. KNIFE CARRIER and KNIFE: for Actual position with the knife, adjusting the proper
Cutting of the tissue sections. Thickness of the tissue
---------------------*TYPES OF MICROTOME*--------------------------
1.ROCKING MICROTOME SIMPLEST MICROTOME 10-12u
- Invented by PALDWELL For serial sections of large paraffin embedded tissue.
2.ROTARY MICROTOME MOST COMMON TYPE 4-6u

- Invented by MINOT For routine paraffin embedded tissues and
RESEARCH LABORATORY
3.SLIDING MICROTOME MOST DANGEROUS TYPE 7-9u

- developed by ADAMS Recommended for celloidin, ester wax blocks
a. Standard Sliding Movable part is the KNIFE for extremely hard and
rough tissues
b. Base Sledge Movable part is the BLOCK HOLDER
4.FREEZING MICROTOME Uses INTERMITTENT BURST OF CO2 that will 10-15u

- Invented by QUECKETT freeze the block.
For cutting undehydrated tissues, neurological
tissues, demonstration of fats.
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5.Cryostat or Cold Microtome For FRESH TISSUE MICROTOMY 4u

Tissues are refrigerated: For FLOURESCENT ANTIBODY STAINING or
(-5 to -30 degC) HISTOCHEMICAL ENZYME STUDIES
average -20degC
6.ULTRATHIN MICROTOME For ELECTRON MICROCOPY 0.5-1u
Uses DIAMOND KNIFE
-------------------*MICROTOME KNIVES*-------------------
PLANE CONCAVE One side is FLAT = One side is CONCAVE 25mm long
BICONCAVE BOTH sides are CONCAVE 120mm long
PLANE WEDGE BOTH sides are FLAT Usually 100mm long
------------------------*ANGLES*-------------------------
BEVEL ANGLE Formed between CUTTING EDGES 27-32
CLEARANCE ANGLE Formed between SURFACE of the BLOCK and 0-15
CUTTING EDGE of KNIFE
WEDGE ANGLE Formed by the SIDES of WEDGE KNIFE 14-15
*HONING: Grinding the cutting edge to acquire *STROPPING: Polishing and Sharpening the
even size edge cutting edge
Purpose: REMOVAL OF GROSS NICKS Purpose: REMOVAL OF BURRS
*HEEL TO TOE MOVEMENT *TOE TO HEEL MOVEMENT
Uses 10-20 or 20-30 strokes Uses 40-120 DOUBLE STROKES
Uses Hones/ Oil stones: Uses paddle strop made of HORSE LEATHER
a. Belgium Yellow: gives BEST RESULT that are usually treated with VEGETABLE OIL
b. Arkansas: With more polishing Effects or CASTOR OIL at the back and not the
surface
c. Fine Carborundum: for badly nicked knives
Uses Lubricants: *NEVER USE MINERAL OIL, it will blister and
a. Soapy water c. Clove oil destroy the leather
b. Mineral Oil d. Xylene or Liquid Paraffin
*FISHING OUT: Removal of TISSUE RIBBONS from the float out bath.
*FLOTATION WATER BATH: Temperature: 5-10degC LOWER than the Melting Point or 45-50degC
---------------------------*Drying Technique for Slides*------------------------
1. Leave the slide in 37degC INCUBATOR overnight
2. Place the slide in OVEN for 2hours in 56-60degC
3. Use a HOT PLATE with 45-55degC for 30-45minutes
*ADHESIVES:
1. Mayer’s Egg Albumin: Most common 8. APES or 3-aminopropylthriethoxysilane:
(Equal amount of EGG WHITE and GLYCERIN) recommended for CYTOLOGY
2. Dried Albumin: Dried albumin + Sodium chloride

Addition of THYMOL to prevent growth of molds.
3. 1% Gelatin: composed of Gelatin, Distilled water, *HOPE is the

Glycerol and Phenol crystals
only thing
4. Gelatin-Formaldehyde Mixture: 1%Gelatin +
2% Formaldehyde STRONGER
5. Starch Paste: Powdered Starch, Distilled water than FEARS.
and HCL. Addition of THYMOL CRYSTALS to
prevent growth of molds.
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6. Plasma/ Pooled serum: Readily available from

outdated blood stored in blood banks
7. Poly-L-Lysine: Effective as an adhesive, slowly *Read pages 109-113 of the book “Histopathologic
Decreases in time techniques 2nd edition by Bruce-Gregorios
“Faults occurring during tissue processing.”
STAINING:
*Process of applying dyes on the section to see and study the ARCHITECTURAL PATTERN of the tissue
and PHYSICAL CHARACTERISTICS of the cells.
*GROUPS OF TISSUE STAINING:

1. Histological: tissues are stained after they are made to react with the dye.
- Tissue constituents are demonstrated in section by direct interaction with a dye.
- Produce coloration of the ACTIVE TISSUE COMPONENT
2. Histochemical/ Histochemistry: Tissue is demonstrated thru CHEMICAL REACTIONS

2.1 Perl’s Prussian Blue: for HEMOGLOBIN
2.2 Periodic Acid Schiff: for CARBOHYDRATES
*Enzyme histochemistry: active agent of the stain is used as SUBSTRATE upon which enzyme acts
3. Immunohistochemical: combination of Immunologic and Histochemical Staining

- detecting of PHENOTYPIC MARKERS that are detected by ANTIBODIES
*METHODS OF STAINING:
1. DIRECT STAINING: process of giving color to the sections by using AQUEOUS or ALCOHOLIC dye
Ex.: Methylene Blue, Malachite Green
2. INDIRECT STAINING: the action of the dye is intensified by addition of another agent
*MORDANT: substance that serves as a LINK or BRIDGE between the tissue and the dye.
ERLICH’S HEMATOXYLIN Potassium Allum
WEIGERT’S HEMATOXYLIN Ferric Ammonium Chloride
HEIDENHAIN’S HEMATOXYLIN Ferric Ammonium Sulfate
*ACCENTUATOR: ACCELERATES or HASTENS the staining process.

LEOFFLER’S METHYLENE BLUE KOH
CARBOL FUCHSIN PHENOL
CARBOL THIONINE
3. PROGRESSIVE STAINING: No differentiation or decolorization/ differentiation is involved.

- Gradual application of dye to the tissue, may OBSCURE the details of the tissue
4. REGRESSIVE STAINING: Tissue is OVERSTAINED first until the desired color is achieved.
- Excess stain is removed by the process of DECOLORIZATION
5. METACHROMIC STAINING: staining tissue with a color that is DIFFERENT from the stain itself
- particularly employed for the staining of CONNECTIVE TX, EPITHELIAL MUCINS,
CARTILAGE, MAST CELLS GRANULES and AMYLOID
*Methyl Violet or Crystal Violet *Bismarck Brown *Thionine
*Cresyl Blue for Retics *Basic Fuchsin *Toluidine Blue
*Safranin *Methylene Blue *Azure A, B, C
6. COUNTERSTAINING: involves application of different color to produce contrast and background
7. METALLIC IMPREGNATION: tissue elements are demonstrated NOT BY STAINS but by colorless
solutions of metallic salts.
- agent may tend to produce BLACK DEPOSITS on surface. Ex.: Gold Chloride, Silver Nitrate
11
8. VITAL STAINING: selective staining of LIVING CELLS CONSTITUENTS

*Nucleus is RESISTANT and therefore not demonstrated
8.1 INTRAVITAL STAIN: Stain is INJECTED to any part of living body.
*(Intravenous, Intraperitoneal, Subcutaneous) Ex: LITHIUM, CARMINE and INDIA INK
8.2SUPRAVITAL STAIN: the stain is APPLIED IMMEDIATELY after removal of cells
from the living body.
*Neutral Red: Best Vital Stain *Nile Blue
*Janus Green: for Mitochondria *Thionine
*Trypan Blue *Toluidine Blue
*ROUTINE H and E STAINING: most common method for microanatomical studies of tissues.
1. Initial Xylene Bath For further DEPARAFINIZATION
2. Descending Grades of Alcohol For HYDRATION
3. Application of Hematoxylin NUCLEAR STAIN (Primary dye)
4. Application of Alcohol For DIFFERENTIATION/ DECOLORIZATION
5. Application of Ammonia Water Bluing agent, To INTENSIFY the color of the NUCLEUS
6. Application of Eosin CYTOPLASMIC STAIN (Counterstain)
7. Ascending Grades of Alcohol For DEHYDRATION
8. Last Xylene Bath Clearing prior to MOUNTING
Result:
Nuclei: Blue to Blue black Calcium and Decalcified bones: Purplish blue
Karyosome: Dark Blue RBCs, Eosinophilic granules, Keratin: Bright Orange
Cytoplasm: Pale pink Decalcified Bone matrix, Collagen and Osteoid: Pink
Muscle Fibers: deep Pink
*Staining methods for Frozen Sections: H and E, Polychromic Methylene Blue, Thionin
Stains:
1. NATURAL DYES: stains that are derived from PLANTS and ANIMALS
Ex: Hematoxylin, Cochineal dyes, Orcein Saffron
1.1 HEMATOXYLIN: formed from the oxidation of Hematoxylin either by RIPENING or by

adding oxidizing agents.
*Hydrogen peroxide *Sodium perborate
*Mercuric oxide *Potassium permanganate
*Sodium iodate
*HEMATEIN: ACTIVE coloring agent of Hematoxylin
- used in combination with Alum, Iron Chromium and Copper salts.
A. ALUM HEMATOXYLIN: uses Potassium Allum as mordant
Ripening Agent
Ehrlich’s Hematoxylin Sodium Iodate
Harris Hematoxylin Mercuric Oxide
Cole’s Hematoxylin Alcoholic Iodine
Mayer’s Hematoxylin Sodium Iodate
B. IRON HEMATOXYLIN: generally used for PHOTOMICROGRAPHY

Mordant
Weigert’s Hematoxylin For Muscles and CT fibers Ferric Ammonium Chloride
Heidenhain’s Hematoxylin For Nuclei and Cytoplasmic Ferric Ammonium Sulfate
inclusions
C. COPPER HEMATOXYLIN: recommended for SPERMATOGENESIS study.

D. TUNGSTEN HEMATOXYLIN/PTAH: Uses 1% Phosphotungstic acid as Mordant
1.2 COCHINEAL DYES: dyes that are extracted from BUG treated with ALUM to produce carmine
*PICROCARMINE: contains Picric acid. Recommended for NEUROPATH STUDIES
*BEST CARMINE: contains Aluminum Chloride. Recommended for GLYCOGEN
12
1.3 ORCEIN: Vegetable dyes extracted from LICHENS, used for staining ELASTIC FIBERS
2. SYNTHETIC DYES: also known as “COAL TAR DYES” derived from Hydrocarbon
Benzene
CHROMOPHORE: Responsible for the COLORING PROPERTY
AUXOCHROME: Responsible for DYEING PROPERTY
*EOSIN: RED acid dye (most commonly used COUNTERSTAIN)

1. EOSIN Y
2. BLUISH
3. ETHYL EOSIN
3. AMPHOTERIC DYES: Dyes with a NUETRAL pH

4. LYSOCHROME DYES: regarded as OIL SOLUBLE DYES
4.1 SUDAN BLACK: most sensitive, has much greater affinity to PHOSPHOLIPIDS, Neutral
Fats
4.2 SUDAN III: First sudan dye to be introduced into IMMUNOHISTOCHEMISTRY
- Good Fats stain for CENTRAL NERVOUS SYSTEM tissues
4.3 SUDAN IV: Recommended for staining TRIGLYCERIDES
-Benzoic Acid: intensifies fat and prevents rapid deterioration of the solution
MOUNTING:
*Process whereby a syrupy fluid is applied between the section and the coverslip after staining.
Goals:
1. Protects the stained smear from getting scratched and from bleaching or deterioration due to
oxidation
2. Preserve the slides for permanent keeping to facilitate easy handling and storage
3. Promote better refraction for better microscopic visualization
*Factors to consider in choosing appropriate Mounting Medium:

1. Should have Refractive Index close to that of slide = 1.518
2. Should set hard permanent mounting of tissue section
3. Should not dry quickly
4. Should not cause bleaching
5. Should not change the color of the tissue
6. Should be miscible with Xylene
1. AQUEOUS MOUNTING MEDIA: usually made up of GELATIN, GLYCERIN JELLY an

GLYCEROL
1.1 WATER: has low refractive index, moderately transparent and evaporates easily
- Good only for TEMPORARY MOUNTING, does not allow tissue to be examined under
OIO
1.2 GLYCERIN: may also be used as a preservative, provides greater visibility, Semi-Permanent
1.3 GLYCERIN JELLY/ KAISSER’S: stains may tend to fade, requires RINGING
1.4 FARRANT’S MEDIUM: does not require heat before using, takes longer time to harden
1.5 APATHY’S MEDIUM: recommended for Methylene blue stained nerve preparation
- addition of 50g Potassium acetate or 10g Sodium chloride prevents bleeding of
metachromatic stains for amyloid.
1.6 BRUN’S FLUID: recommended for mounting FROZEN SECTIONS and sections from water
13
REFRACTIVE INDEX
GLYCERIN 1.46
GLYCERIN JELLY/ KAISSER’S 1.47
FARRANT’S MEDIUM 1.43
APATHY’S MEDIUM 1.52
2. RESINOUS MOUNTING MEDIA: used for preparations that have been dehydrated and cleared
in XYLENE or TOULUENE. Recommended for majority of staining method.
2.1 CANADA BALSAM: Natural resin extracted from a Canadian tree “ABUS BALSAMEA”
- usually dissolved in xylene in an incubator @ 37degC or paraffin oven @ 58degC
- Recommended for whole mounts and thick sections, does not promote much shrinkage
- Adheres consistently to the glass and sets hard consistency without granulation
2.2 DPX: recommended for small tissue sections, not for whole mounts, dries rapidly
2.3 XAM: synthetic resin mixture in Xylene, Preserves stain well
2.4 CLARITE: Synthetic resin soluble in xylene, generally preferred than DPX
REFRACTIVE INDEX
CANADA BALSAM 1.524
DPX 1.532
XAM 1.52
CLARITE 1.544
*Other recommended Mountant: *RINGING: process of sealing the margins of the coverslip to
1. Permount prevent the escape of fluid or semi-fluid mounts.
2. Harleco Synthetic Resin *Ringing Medium: DUROFIX: cellulose adhesive
3. Clearmount KRONIG CEMENT: 2parts par. wax + 4-9parts colopohonium
resin
*DEPARAFFINIZATION: Removal of paraffin wax once fixed on the slide

1. Immersion of slide in XYLENE
2. Pass the slide over a flame using alcohol lamp
3. Place slide inside an oven at 55-60degC
14
--------------*CYTOLOGY*-------------
DIAGNOSTIC CYTOLOGY: microscopic examination of cells from different body sites for DIAGNOSTIC PURPOSES
*2 Divisions of CYTOLOGY:
1. Exfoliative Cytology: microscopic examination of the shed, desquamated cells from the body surfaces or
cells harvested by rubbing or brushing a lesional tissue (epithelial) surfaces.
2. Fine-Needle Aspiration: procedure whereby a thin needle is inserted into an area of abnormal-appearing
tissue or body fluid. As with other types of biopsies, the sample collected
during fine needle aspiration can help make a diagnosis or rule out conditions
such as cancer.
I. EXFOLIATIVE CYTOLOGY:
DESQUAMATED CELLS: Cells that SHED or REMOVED from the EPITHELIUM.
Goals of Exfoliative Cytology:

1. To detect malignancy or cancerous conditions 3. To determine genetic sex
2. To detect asymptomatic cancer 4. To detect possible infections related to
3. To evaluate female hormonal activity reproductive system
*IMPRINT/ ABRADED CYTOLOGY:

- Study of cells taken from surfaces of excised/incised specimens by touching them with a clean glass slides
*TECHNIQUES IN CYTOLOGY:
1. SMEAR TEACHNIQUE:
Reminders:
A. Smears are usually made from fresh materials
B. Smears must be prepared and immerse immediately in a fixative while still moist and before drying
C. Fluid specimen must be centrifuged at 2000rpm for 2minute
D. Extra sediment is used for cell block
*If smears CANNOT BE MADE IMMEDIATELY:

1. Place in 50% alcohol 9Ethanol)
2. SACCOMANO preservative: 50% Alcohol + 2% Carbowax
*FIXATIVES for CYTOLOGY
1. Equal parts of 95% ethyl alcohol and ether 3. Carnoy’s fluid for Bloody specimen
2. 95% ethyl alcohol 4. Spraycyte or Cytospray
*SMEAR PREPARATIONS:
1. STREAKING: direct ZIGZAG LINE throughout the slide. (Sputum and bronchial aspirates)
2. SPREADING: Specimen is applied and spread by TEASING using applicator
3. PULL-APART: Specimen is spread evenly on the surface of two slide (for thick secretions)
4. TOUCH PREPARATIONS: The slide is made to touch the cut surface of an organ or tissue
*ADHESIVES:
1. POOLED HUMAN SERUM *MAYERS EGG ALBUMIN is not
2. LEUCONOSTOC CULTURE recommended because it can be stained by
the counterstain. (OG-6 or EA50)
3. combination of CELLOIDIN and ETHER
2. CELL BLOCK TECHNIQUE: also considered as MICROBIOPSY
Uses of cell block:
1. Architectural Evaluation 2. Special stain and Histochemistry
2. Categorization of tumors 3. Archival material for future studies
3. MEMBRANE FILTER METHOD: technique in collecting cells using filter paper with a specified pore size
*The FILTER is made up of POLYCARBONATE and CELLULOSE ESTERS
4. LIQUID BASED CYTOLOGY: technique that allows the cells to be spread in a monolayer
15
GYNECOLOGICAL SPECIMENS:
1. Cervicovaginal Smear or PAP smear:
2. Preparations:
a. The patient should not have been douched or undergone vaginal exam for at-least 1-2days
b. The gloves to be used by the examiner should have no lubricants or powder
c. Smear should be prepared thinly in rotating motion instead of pull apart
*Sites of Collection:
1. Upper Lateral 3rd of Vaginal Wall: for Evaluation of HORMONAL ACTIVITY
2. Ectocervix and Endocervix
3. T-zone: where pre-cancerous lesions can be detected
*Specimen Collection:
1. Vaginal Scrape: recommended for patient with HYSTERECTOMY
2. Vulvar Scrape: recommended for detecting HERPECTIC LESIONS or CARCINOMA
3. 4 Quadrant Vaginal Scrape: recommended for the LOCALIZATION of VAGINAL ADENOSIS
*PAPANICOLAU’S/PAP’S STAIN
1. Harris Hematoxylin Nuclear stain (Primary stain)
2. Orange Green-6 (OG 6) Stains the Cytoplasm of SUPERFICIAL CELLS (Counterstain)
3. Eosin Azure-50 (EA-50) Stains the Cytoplasm of PARABASAL CELLS and
INTERMEDIATE CELLS. (Counterstain)
CELLS FOUND in CERVICOVAGINAL SMEARS:

1. SUPERFICIAL CELLS: most mature, Polygonal cells with DARK PYKNOTIC NUCLEI
- Cells that exhibit “TRUE ACIDOPHILIA” characteristics of vaginal cell under the
influence of ESTROGEN
ESTROGEN Influence desquamation of SUPERFICIAL CELLS
PROGESTERONE Influence desquamation of INTERMEDIATE CELLS
E1 ESTRONE Most predominant during menopause
E2 ESTRADIOL Most predominant during reproductive years
E3 ESTRIOL Plenty but least potent
2. INTERMEDIATE CELLS: medium cells, Polyhedral with BASOPHILIC and VACUOLATED cytoplasm
2.1 NAVICULAR CELLS: found in the latter half of MENSTRUAL CYCLE, MENOPAUSE and
during pregnancy. Presence suggest Progesterone-Estrogen effect
- BOAT-SHAPED CELLS with a tendency to fold or curl on egdes
2.2 PARABASAL CELLS: thick ROUND OVAL CELLS, Nucleus is Large (FRIED EGG app.)
*Normally found in: ABORTION, AFTER MENOPAUSE, AFTER CHILD BIRTH
And 2 weeks of AGE to PUBERTY
2.3 PREGNANCY CELLS: Nucleus pushed at periphery with double walled boundary appearance
- Round oval BOAT SHAPED CELLS with translucent basophilic cytoplasm due to
glycogen accumulation.
3. Other Cells found in Cervico Vaginal Smears:
a. ENDOMETRIAL CELLS: Occurring in groups of three or more, Found during first week of
menstruation. Similar to parabasal but less basophilic.
b. ENDOCERVICAL/GLANDULAR CELLS: Occurs in large groups
- with HONEYCOMB APPEARACE
c. DODERLAIN BACILLUS/ LACTOBACILLUS ACIDOPHILUS: normal vaginal flora
- Last Trimester of Pregnancy
- Infection
- Estrogen deficiency
- Diabetes Mellitus
d. CANDIDA ALBICANS: yeast cell that is commonly seen in patients with Diabetes, Taking
contraceptives and patient with Immunocompromised states.
16
e. TRICHOMONAS VAGINALIS: Stains blue to blue gray

f. GARDNERELLA VAGINALIS: may appear as cells surrounded by bacilli “CLUE CELLS”
g. KOILOCYTES: cells that shows CYTOPATHIC EFFECT of Human Papilloma Virus.
- Cells with atypical nucleus surrounded by perinuclear halo.
- Nucleus may appear like WRINKLED PRUNES.
*FERNING: phenomenon in which cervical mucus exhibit a “PALM LEAF” pattern on drying
- possible to happen in patients with HIGH ESTROGEN LEVEL
- can be used for detection of EARLY PREGNANCY
*Quantitative Evaluation for Vaginal Cytology:

MATURATION INDEX ACIDOPHILIC/EOSINOPHILIC PYKNOTIC INDEX
INDEX
Asses the percentage of cells Asses the percentage of cells appearing Asses percentage of cells with
coming from the main layers of PINK, ORANGE, RED following Pap’s SHRUNKEN, DARK,
the epithelium smear SMALL, STRUTURELESS
nucleus
*REPORTING CANCER CYTOLOGY:

CLASS
Class I ABSENCE OF ATYPICAL CYTOLOGIC PICTURE
Class II Atypical cytologic picture present but NO EVIDENCE OF MALIGNANCY
Class III Cytologic picture suggestive but NOT CONCLUSIVE OF MALIGNANCY
Class IV Cytologic picture STRONGLY SUGGESTIVE OF MALIGNANCY
Class V Cytologic picture CONLUSIVE OF MALIGNANCY
NON-GYNECOLOGICAL SPECIMENS:
1. RESPIRATORY SPECIMEN:
a. SPUTUM: note for presence of ALVEOLAR MACROPHAGE
b. BRONCHIAL BRUSHINGS
c. BRONCHOALVEOLAR LAVAGE: for detection og Pneumocystis jirovecci in AIDS patients
d. BRONCHIAL ASPIRATES
2. PLEURAL, PERITONEAL and PERICARDIAL FLUIDS
- Accumulation of fluid is always indicative of process.
- To prevent jelly clots, add 300units of Heparin/ 100cc or ml aspirate
3. BREAST SECRETIONS: nipple discharge, low diagnostic value
- Post lactation is considered abnormal
4. URINARY TRACT SPECIMENS: first morning urine is discarded
- Detects urothelial malignancy
- 50-100mL is the minimum required amount
*Voided urine (Male) Catheterized specimen (Female)
5. BODY CAVITY EFFUSIONS: 1CC is the minimum required amount of fluid
- Must be collected in a clean and dry container
- Must be submitted fresh to the laboratory
- Refrigerate if there will be a delay
6. GIT SPECIMENS (Gastric lavage, gastric brush)
- Fasting for at-least 8hours is required, specimen must be processed immediately
delay may cause digestion of cells (after 30 minutes of delay)
- 5-10mL is the minimum amount required
II. FINE NEEDLE ASPIRATION: process whereby thin hallow needle is inserted into the mass for
sampling of the cells.
- Only few cells may be obtained, Problematic cells might be missed
17
- For superficial masses, safe and simple technique for cancer detection
*Palpable Masses: BREAST is the most common site.
- Thyroid, Soft Tissues and Lymph Nodes
- 22-23 gauge of needle is recommended to use
*Non-Palpable Masses: Process with the help of FLUOROSCOPY, CT SCAN or any appropriate
radiologic technique
*Record and Specimen Retention:
RECORDS:
Clinical Pathology and Laboratory Reports 10 years
Autopsy Forensic Reports Indefinitely
Surgical Pathology and Bone Marrow Reports 10 years
Cytogenetics report 10 years
SPECIMENS:
Pathology/ Bone Marrow Slides 10 years
Pathology blocks 10 years
Cytogenetic Slides 3 years
Cytogenetic Diagnostic Images 20 years
*Turn-Over/ Releasing of Results:

Surgical Pathology and Cytology 24 hours
Frozen Sections 5-15 minutes
Autopsy Report 1 week
--------------*PATHOLOGY*-------------
*CYTOPATHOLOGIC CHANGES IN DISEASE:
*INFLAMMATION: sum total of changes in the living tissues, in response to an injured agent,
including the local reaction and the repair of an injury.
- Composed of series og physiologic and morphologic alterations in the blood vessels, blood
components and surrounding connective tissues to protect the body against injury.
*CARDINAL SIGNS OF INFLAMMATION
RUBOR Redness Arteriolar and capillary dilatation with increase rate of
blood flow towards the site of injury
TUMOR Swelling Increase capillary permeability, allowing the extravasations
of blood fluid associated with increase hydrostatic pressure
CALOR Heat Transfer of internal heat to the site or surface of the injury
DOLOR Pain Pressure upon the sensory nerve by the exudates or tumor
FUNCTIO LAESA Diminished Function Pain interference with nerve supply and to destruction of the
functioning units of the tissue
*TYPES OF INFLAMMATION:
1. ACUTE INFLAMMATION: inflammatory reaction in which the dominant anatomic changes are
vascular and exudative.
2. CHRONIC INFLAMMATION: involves persistence of the injurious agent for weeks or years,
characterized by proliferation.
3. SUBCHRONIC INFLAMMATION: represent intergrade between acute and chronic inflammation.
*TYPES OF INFLAMMATION ACCORDING TO CHARACTER:
1. SEROUS INFLAMMATION: characterized by 2. HEMORRHAGIC INFLAMMATION:

extensive outpouring of a watery, low-protein fluid characterized by the admixture of blood and
derived from either the blood serum or secretions other elements of the exudates
from serosal mesothelial cells.
18
3. FIBRINOUS INFLAMMATION: characterized 4. SUPPURATIVE INFLAMMATION:

by exudation of large amout of fibrinogen and the characterized by production of large amount
precipitation of fibrin of pus or purulent exudate.
5. CATARRHAL INFLAMMATION: characterized

by hypersecretion of the mucosa with degenerative
changes in the epithelium, characteristic of
involvement of respiratory, gastrointestinal and other
mucus secreting glands.
*ABNORMALITIES IN CELL GROWTH

A. RETROGRESSIVE CHANGES
1. Developmental Defects
1.1 APLASIA: incomplete or defective development of tissue or organ
1.2 AGENESIA: complete non-appearance of organ
1.3 HYPOPLASIA: failure of an organ to reach its mature or adult size
1.4 ATRESIA: failure of an organ to form an opening
2. ATROPHY: reduction in size of cells, tissues or organs resulting from the reduction in the cell
size or reduction in the number cells
2.1 Physiologic Atrophy: occurs naturally
2.2 Pathologic Atrophy: occurs as a consequence of a disease
2.3 Endocrine Atrophy: lack of tropic hormone
2.4 Vascular Atrophy: diminished blood supply
2.5 Starvation Atrophy: lack of nutritional supply
2.6 Atrophy of Disuse: reduced functional demand
2.7 Exhaustion Atrophy: too much work load
2.8 Pressure Atrophy: caused by pressure
B. PROGRESSIVE CHANGES:
1. HYPERTROPHY: Increase in the tissue/organ size due to increase in the cell size
*Physiologic: due to natural process *Pathologic: removal of paired organ
2. HYPERPLASIA: Increase in the tissue/organ size due to increase in cell numbers
C. DEGENERATIVE CHANGES:
1. METAPLASIA: transformation of one adult cell type to another adult cell type REVERSIBLE
2. DYSPLASIA: change in structural components of one mature cell type
3. ANAPLASIA: when one mature cell transforms to more primitive cell type
4. NEOPLASIA: continuous abnormal proliferation of cells without control.
- TUMOR FORMATION, represent a pathologic condition or overgrowth of tissue.
PARENCHYMA Active elements of the tumor
STROMA Connective tissue framework with lymphatic and vascular channels
*Differentiation of Tumors:
A. Depending upon CAPACITY TO PRODUCE DEATH:
1. BENIGN TUMORS: those that do not produce death
2. MALIGNANT TUMORS: usually causes death no matter how small
and no matter where they are located.
B. Depending on HISTOLOGIC CHARACTERISTICS:

1. MEDULLARY: where there are more cells than supporting tissue, SOFT AND MALIGNANT
2. SCIRRHOUS: where there is more connective tissue than cells
*GRADING OF TUMORS:
1. DIFFERENTIATED CELLS: resembling normal cells
2. UNDIFFERENTIATED CELLS: younger forms
19
*BROADER’S CLASSIFICATION:
- Used as a guid for treatment, as a Prognostic guide
DIFFERENTIATED CELLS UNDIFFERENTIATED CELLS
GRADE I 100%-75% 0%-25%
GRADE II 75%-50% 25%-50%
GRADE III 50%-25% 50%-75%
GRADE IV 25%-0% 75%-100%
*STAGING OF TUMORS: determination the spread of cancer within the individual

T Primary Tumor
• T1, T2, T3: with increasing size of primary lesion
N Regional Lymph nodes involvement
• N0, N2, N3: indicates progressively advancing nodal disease
M Metastasis: whether there is a distant metastasis
*CELL DEATH
1. APOPTOSIS: programmed cell death
2. PYKNOSIS: reduction in the size and condensation of the nucleus
3. KARYORRHEXIS: fragmentation of the nucleus
4. KARYOLYSIS: dissolution of the nucleus
5. NECROSIS: rapid process that brings death of a group of cells
TYPES of NECROSIS:
1. COAGULATIVE: encountered when the arterial supply is cut off producing ANEMIC and
ISCHEMIC INFARCTION.
2. LIQUEFACTIVE: rapid enzyme dissolution of the cells leading in producing of pus.
3. CASEOUS: cell death produced by tubercle bacilli; gross state may appear friable cheese
4. GANGRENOUS: due to interruption of blood supply to lower extremities
5. FAT: seen in pancreatic degeneration, release of lipase that will degrade surrounding fats
6. FIBRINOID: deposition of fibrin like material in arterial wall, seen in small blood vessels
*SOMATIC DEATH:
Refers to the DEATH or complete cessation of metabolic and functional activities of the body.
*PRIMARY SIGNS OF DEATH
1. CIRCULATORY FAILURE: occur when cardiac function ceases, absence of pulse and heart beat
2. RESPIRATORY FAILURE: absence of OXYGEN and accumulation of Carbon Dioxide with
loss of oxidative process needed for life
3. NERVOUS FAILURE: loss of coordination of various body functions, chiefly loss of reflexes.
*SECONDARY CHANGES AFTER DEATH:
1. ALGOR MORTIS: first to be demonstrable change observed. Cooling of the body 7degF/hour
2. LIVOR MORTIS: purplish discoloration of the skin or lividity of the skin.
3. RIGOR MORTIS: rigidity or stiffening of the muscles, occurring 6-12hours after death.
Persist for 3-4 days
3. POST-MORTEM CLOTTING: settling and separation of red cells from the fluid phase
of the blood
4. DESSICATION: drying and wrinkling of the cornea and anterior chamber of the eye
5. PUTREFACTION: production of foul-smelling gases due to invasion of the tissue with saprophytic
organisms.
6. AUTOLYSIS: self-digestion of cells
20
*AUTOPSY : Process of taking pieces of tissues from dead person for the purpose of examination
or investigation in order to determine the cause of death or extent of injury leading to the death of the person
*Types of AUTOPSY:
1. According to its purpose:
A. Routine/Hospital Autopsy: done in private hospitals for the purpose of ascertaining the cause of
death of the person
B. Medico-Legal Autopsy: done in NBI or other government institutions for prosecution puposes
2. According to the completeness of the procedure or technique:

A. COMPLETE: request for the examination of the WHOLE BODY from HEAD TO FOOT
B. PARTIAL: request for the examination of REGION or part from the dead body.
3. According to the manner of incision or opening the cadaver:

A. Y-shaped incision: the cadaver is both open from both shoulder regions down to the xiphoid area
then incised down to the pubis. Commonly done in Female Cadaver
B. Straight Cut incision: the cadaver is open from the midline of the body from suprasternal notch
down to the pubis. Commonly done among children.
* TECHNIQUES OF AUTOPSY:
RUDOLPH VIRCHOW Organs are removed ONE BY ONE
CARL ROKITANSKY Characterized by “IN-SITU” dissection
ANTON GHON Thoracic and cervical organs, abdominal organs, and urogenital system are
removed as blocks
“EN’ BLOC” removal technique
M. LETULLE Thoracic cervical abdominal and pelvic organs are removed
“EN MASSES” and subsequently dissected into organ blocks.
*Record and Specimen retention:

RECORDS:
Clinical Pathology and Laboratory Reports 10 years
Autopsy Forensic Reports Indefinitely
Surgical Pathology and Bone Marrow Reports 10 years
Cytogenetics report 10 years
SPECIMENS:
Pathology/ Bone Marrow Slides 10 years
Pathology blocks 10 years
Cytogenetic Slides 3 years
Cytogenetic Diagnostic Images 20 years
NOTE: This Review Booklet for Histopathology was prepared with love, time, and effort.
Should you want to share this with your friends, please do not bypass the professional and
ethical process of asking permission from the compiler, Mister Hanzjethro Villanueva Santos.
Checked and Approved for Distribution:
_______________________________________
Hanzjethro Villanueva Santos, RMT
Founder, Chief Executive Officer
21

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Hanzjethro Villanueva Santos,RMT (+63917)8974844

This review booklet belongs to: ___________________________

*Hydrogen Ion Concentration: pH should be 6-8 (Slightly Hypertonic)

*Factors to consider in choosing the appropriate fixative:

*Effects of fixative in general:

SECONDARY FIXATION: placing an already POST-CHROMATIZATION: form of secondary

*ETHYLYNE GLYCOL/CELLOSOLVE: dehydrates rapidly but is COMBUSTIBLE at 110-120degF.

*TETRAHYDROFURAN: used for ATHEROMATOUS ARTERIES and HARD COLLAGENOUS specimen.

*Other Clearing Agents: *Clearing agent miscible with absolute alcohol

6. Terpineol: for Eyes

D. TRICHLOROACETIC ACID: Permits GOOD NUCLEAR STAINING, WEAK AGENT.

F. CHROMIC ACID: may be used as both FIXATIVE and DECALCIFYING agent

2. Use of Chelating Agent

3. Ion Exchange Resin

4. Electrolytic Method/Electrophoresis: (MOST RAPID METHOD)

*MEASURING EXTENT OF DECALCIFICATION:

- if calcium is still present, it will appear OPAQUE.

3. Chemical Method: involves using of Ammonium Oxalate

*FACTORS INFLUENCING RATE OF DECALCIFICATION:

2. HEAT 4. MECHANICAL AGITATION

1. PARAFFIN WAX IMPREGNATION: Most commonly used, RAPID ACTING.

*METHODS OF PARAFFIN WAX IMPREGNATION:

*WATER SOLUBLE WAX: CARBOWAX (MP: 38-42degC or 45-56degC)

- For Hard tissues like UNCALCIFIED BONES

2.ROTARY MICROTOME MOST COMMON TYPE 4-6u

3.SLIDING MICROTOME MOST DANGEROUS TYPE 7-9u

b. Base Sledge Movable part is the BLOCK HOLDER

4.FREEZING MICROTOME Uses INTERMITTENT BURST OF CO2 that will 10-15u

5.Cryostat or Cold Microtome For FRESH TISSUE MICROTOMY 4u

2. Dried Albumin: Dried albumin + Sodium chloride

3. 1% Gelatin: composed of Gelatin, Distilled water, *HOPE is the

6. Plasma/ Pooled serum: Readily available from

*GROUPS OF TISSUE STAINING:

2. Histochemical/ Histochemistry: Tissue is demonstrated thru CHEMICAL REACTIONS

3. Immunohistochemical: combination of Immunologic and Histochemical Staining

*ACCENTUATOR: ACCELERATES or HASTENS the staining process.

3. PROGRESSIVE STAINING: No differentiation or decolorization/ differentiation is involved.

6. COUNTERSTAINING: involves application of different color to produce contrast and background

8. VITAL STAINING: selective staining of LIVING CELLS CONSTITUENTS

1.1 HEMATOXYLIN: formed from the oxidation of Hematoxylin either by RIPENING or by

B. IRON HEMATOXYLIN: generally used for PHOTOMICROGRAPHY

C. COPPER HEMATOXYLIN: recommended for SPERMATOGENESIS study.

*EOSIN: RED acid dye (most commonly used COUNTERSTAIN)

3. AMPHOTERIC DYES: Dyes with a NUETRAL pH

*Factors to consider in choosing appropriate Mounting Medium:

1. AQUEOUS MOUNTING MEDIA: usually made up of GELATIN, GLYCERIN JELLY an

*DEPARAFFINIZATION: Removal of paraffin wax once fixed on the slide

Goals of Exfoliative Cytology:

*IMPRINT/ ABRADED CYTOLOGY:

*If smears CANNOT BE MADE IMMEDIATELY:

CELLS FOUND in CERVICOVAGINAL SMEARS:

e. TRICHOMONAS VAGINALIS: Stains blue to blue gray

*Quantitative Evaluation for Vaginal Cytology:

*REPORTING CANCER CYTOLOGY:

*Turn-Over/ Releasing of Results:

*TYPES OF INFLAMMATION ACCORDING TO CHARACTER:

1. SEROUS INFLAMMATION: characterized by 2. HEMORRHAGIC INFLAMMATION:

3. FIBRINOUS INFLAMMATION: characterized 4. SUPPURATIVE INFLAMMATION:

5. CATARRHAL INFLAMMATION: characterized

*ABNORMALITIES IN CELL GROWTH

B. Depending on HISTOLOGIC CHARACTERISTICS:

*STAGING OF TUMORS: determination the spread of cancer within the individual

2. According to the completeness of the procedure or technique:

Other Clearing Agents: Clearing agent miscible with absolute alcohol