HISTOPATHOLOGIC TECHNIQUES BOARD REVIEW

By: Rene Jesus Alfredo R. Dinglasan, RMT

I.) Quality Assurance and Documentation
A. Histopath Reports 1. Surgical pathology 2. Cytopathology report 3. Autopsy report
‡

Number of copies prepared per report: Three copies = Doctor Patient File

SAMPLE BOARD QUESTIONS 

March 1992 Board Exams: Forensic and anatomic pathology report should be kept in the laboratory for a period of: a. one year b. two years c. six years d. permanently

SAMPLE BOARD QUESTIONS 

March 1998 Board Exams In most private hospitals, the histopathologic report is typed in: a. Four copies b. Triplicate c. Duplicate d. One copy

B. Signatories 1. Request Forms = patient¶s doctor 2. Result Forms = Pathologist

C. Specimen Handling 1. FIX FIRST! 2. Label

D. Routine Turn-over of Results Turn1. Surgical pathology and cytology = 24 hours 2. Frozen section = 5-15 minutes 53. Autopsy report = 1 week

SAMPLE BOARD QUESTIONS 

March 1997 Board Exams An autopsy, to be most informative and helpful, should be done within: a. 36 hours b. 72 hours c. 1 week d. 24 hours

E. Storage of Specimen, Tissue blocks, Slides Specimen = 1 month to 1 year Tissue Blocks = 3 years to 10 years Slides = Indefinite

II. Fresh Tissue Exam 

Methods: 1. Teasing/Dissociation selected tissue spx

watch glass (w/ isotonic salt sol¶n)

Microscopes used: Phase contrast/Bright field 

Methods: 2. Crushing/ Squash preparation

Tissues less than 1 mm in diameter slides or slip

sandwiched between two a slide and a cover

a vital stain may be used 

Methods: 3. Smear prep = process of examining sections or sediments = cellular materials are spread lightly over a slide by means of a wire loop/ applicator stick/ another slide.

Different smear prep techniques include: 

Streaking Spreading PullPull-apart Touch prep/Impression    

Methods: 4.Frozen Section =normally used when a rapid diagnosis of a tissue is required. = APPLICATIONS: 1. Rapid pathologic diagnosis during surgery 2. Enzyme histochemistry 3. Demonstration of soluble substances such as lipids and carbohydrates 4. Immunofluorescent and immunocytochemical staining 5. Some specialized silver stains, particularly in neuropathology.

2 Methods of Preparing Frozen Sections: 

Cold knife procedure optimum condition for sectioning: KNIFE = -40 to -60 C TISSUE = 5 to -10 C ENVIRONMENT = 0 to -10 C

Reminders:
If tissues are frozen too hard = chip into fragments when cut. Remedy: Surface of the block maybe be softened by warming slightly with the finger.  If tissues have not been sufficiently frozen = thick and crumble = block may come away from the stage. Remedy: More bursts of carbon dioxide gas should then be given to refreeze the block. 

2 Methods of Preparing Frozen Sections: 

Cryostat procedure (Cold Microtome) optimum working temp. = -18 to -20 C CRYOSTAT ± a refrigerated cabinet in which a modified microtome is housed. All the controls to the microtome are operated from outside the cabinet. Presently, the rotary microtome is the type of choice.

Commonly Used Methods of Freezing 

Liquid Nitrogen  Isopentane cooled by liquid nitrogen  Carbon dioxide gas  Aerosol sprays = adequate for freezing small pieces of tissue EXCEPT muscle.

PRESERVED TISSUE EXAMINATION

FIXATION
Preserving fresh tissue for examination  First and most critical step in histotechnology  Primary aim: to preserve the aim: morphologic and chemical integrity of the cell in as life-like manner as lifepossible. 

FIXATION 

Secondary aim: to harden and protect the tissue from the trauma of further handling.

FIXATION 

Practical considerations of Fixation: 1. Speed ± the specimen should be placed in fixative as soon as it is removed from the body. 2. Penetration ± formalin diffuses into the tissue at approximately 1 mm/Hr. 3.Volume 3.Volume ± 20 times the tissue volume 4.Duration of fixation

FIXATION 

Two mechanisms involved in Fixation: 1. Additive fixation ± whereby the chemical constituent of the fixative is taken in and becomes part of the tissue. Examples: Formalin Mercuric fixatives Osmium tetroxide

FIXATION 

Two mechanisms involved in Fixation: 2. Non-additive fixation ± whereby the Nonfixing agent is NOT taken in, but changes the tissue composition and stabilizes the tissue by removing the bound water attached to hydrogen bonds of certain groups within the protein molecule. Example: Alcoholic fixatives

FIXATION 

Main factors involved in fixation: 1. Hydrogen ion concentration (pH) satisfactory fixation = pH 6-8 62. Temperature surgical spx ± Rm. Temp. Electron Microscopy and some histochem ± 0-4 C

*Formalin heated at 60 C = sometimes used for rapid fixation of very urgent biopsy specimens *Formalin heated at 100 C = can be used to fix tissues with TB

FIXATION
3. Thickness of section 4. Osmolality 5. Concentration 6. Duration of Fixation

FIXATION
I. Aldehyde fixatives 1. Formaldehyde 2. 10% Formol-Saline = CNS tissues Formol3. 10% BNF (Buffered Neutral Formalin) = best fixative for tissues containing iron pigments = BEST GENERAL TISSUE FIXATIVE

FIXATION
4. Formol-Corrosive (Formol-Sublimate) Formol(Formol5. Glutaraldehyde = preserves plasma protein better. 6. Formol-calcium = for the preservation Formolof lipids

FIXATION
II. Metallic fixatives 1. Mercuric Chloride = may produce black granular deposits on tissues a. Zenker¶s Fluid (with glacial acetic acid) b. Zenker-Formol (Helly¶s Sol¶n) Zenker-

FIXATION
II. Metallic fixatives 1. Mercuric Chloride c. Heidenhain¶s SuSa ±for tumor biopsies of the skin d. Schaudinn¶s fluid e. Ohlmacher¶s fluid f. Carnoy-Lebrun fluid Carnoyg. B-5 fixative B-

FIXATION
II. Metallic fixatives 2. Chromate a. chromic acid b. potassium dichromate c. Regaud¶s (Moller¶s) d. Orth¶s fluid ± for Rickettsia and other bacteria - for study of early degenerative process

FIXATION
II. Metallic fixatives 3. Lead fixatives ±are generally for ACID MUCOPOLYSACCHARIDES (for example: Umbilical Cord/ Wharton¶s jelly)

FIXATION
III. Picrate fixatives -highly explosive when dry - will produce excessive yellow staining of tissues -picrates are formed upon protein; precipitates are soluble in water; hence tissues must be first rendered insoluble by direct immersion in 70% ETOH

FIXATION
III. Picrate fixatives -picrate fixatives MUST NEVER be washed in water before dehydration. TISSUES 70% ETOH 5% Sod. Thiosulfate

wash in running water

FIXATION
III. Picrate fixatives A. Bouin¶s Solution ± for fixation of embryos B. Brasil¶s Alcoholic Picroformol ± less messy than Bouin¶s

FIXATION
IV. Glacial Acetic Acid = fixes nucleoprotein V. Alcoholic Fixatives general disadvantage: POLARIZATION 

Polarization ± glycogen granules move towards the poles or ends of cells. 1. Methanol ± blood smears and BM tissues 2. Ethanol 3. Carnoy¶s fluid ± MOST RAPID FIXATIVE ( Fixation time: 1 to 3 hours ) 4. Alcoholic Formalin ( Gendre¶s Fixative) -useful in preserving sputum 5. Newcomer¶s fluid

FIXATION
VI. Osmium Tetroxide Fixatives - should be kept in a dark-colored, darkchemically clean bottle to prevent evaporation and reduction by sunlight or organic matter. - inhibits hematoxylin and makes counterstaining difficult.

VI. Osmium Tetroxide Fixatives 

produces black precipitate (Osmic oxide)  Prevention: add saturated aqueous mercuric chloride  Remedy: Black osmic oxide crystals may be dissolved in cold water. water.  Precaution: may cause conjunctivitis or blindness.

VI. Osmium Tetroxide Fixatives 

FLEMMING¶S SOL¶N FLEMMING¶S W/O ACETIC ACID 

removal of this serves to improve cytoplasmic details

FIXATION
VII. Tricholoroacetic acid VIII. Acetone ± used for the Dx of Rabies IX. Heat Fixation ± direct flaming fixation
-

microwave fixation (optimum temp. 45-55 C) 45*underheating ± poor sectioning *overheating (above 65 C) -vacuolation -overstained cytoplasm -pyknotic nuclei

FIXATION
1. 2. 3. 4. 5. 6. FIXATIVES FOR E.M. Glutaraldehyde Platinic chloride (PtCl3) Platinic Chloride-formalin Chloride(Zamboni¶s fixative) Gold chloride (AuCl) Osmium tetroxide 10% BNF = acceptable but not recommended

DECALCIFICATION 

A procedure whereby calcium or lime salts are removed from tissue FOLLOWING FIXATION Should be done after fixation and before impregnation 

DECALCIFICATION
More concentrated acid solutions decalcify bone more rapidly but are more harmful to the tissue.  High concentrations and greater amount of fluid will increase the speed of the process.  The recommended ratio of fluid to tissue volume for decalcification is 20 to 1. 

Heat will serve to hasten decalcification BUT it also increases the damaging effects on tissues.  At 37 C = impaired nuclear staining of Van Gieson¶s stain for collagen fibers.  At 55 C = tissue will undergo complete digestion within 24-48 hours. 24 Optimum temperature = RM TEMP (18(1830 C) 

The ideal time required for decalcifying tissue is 24-48 hours. 24 Dense bone tissues usually require up to 14 days or longer in order to complete the process. 

Decalcifying agents 

Nitric acid ± MOST COMMON examples: Perenyi¶s fluid ± acts as BOTH tissue softener and decalcifying agent. PhloroglucinPhloroglucin-Nitric Acid -MOST RAPID DECALCIFYING AGENT!

Decalcifying agents
Formic acid ± BOTH fixative and decalcifying agent  5% formic acid is considered to be the BEST GENERAL DECALCIFYING AGENT  Formic acid is recommended for small pieces of bones and teeth. 

Decalcifying agents 

Hydrochloric acid > Von Ebner¶s fluid ± recommended for teeth and small pieces of bones and teeth.

PostPost-Decalcification 

After decalcification is complete, acid can be removed from tissues or neutralized chemically by immersing the decalcified bone in either: 1. saturated lithium carbonate sol¶n. 2. 5-10% aqueous sodium 5bicarbonate solution for several hours. 

Adequate water rinsing can usually be accomplished in 30 minutes for small samples and 1-4 hrs. for larger 1specimens.

Tissue Softeners
For unduly hard tissues that may damage the microtome knives.  4% aq. phenol.  Molliflex  2% HCl  1% HCl in 70% alcohol Note: Tissues immersed in Molliflex may appear swollen and soapy. (Does not affect normal processing) 

DEHYDRATION
Aim: to remove fixative and water from the tissue and replacing them with dehydrating fluid in preparation for impregnation.  Dehydrating fluids are generally used in increasing strengths.  Increasing strengths = all the aqueous tissue fluids are removed but with little disruption to the tissue due to diffusion currents. 

DEHYDRATION 

For delicate tissues, particularly embryonic and animal tissues, it tissues, is recommended to start processing with 30% ethyl alcohol. alcohol.

DEHYDRATION
COMMONLY USED DEHYDRATING AGENTS: 1.) Alcohol ± MOST COMMON a. Ethanol = for routine dehydration of tissues. = BEST DEHYDRATING AGENT. b. Methyl alcohol = employed for blood and tissue films

DEHYDRATION
c. Butyl alcohol =utilized in plant and animal microtechniques. d. Industrial methylated spirit(denatured alcohol) = ethanol + small amt. of methanol used in the same way as ETOH

DEHYDRATION
e. Isopropyl alcohol = many of the processing methods for use in a microwave oven recommend this agent. 2. Acetone ± BOTH fixative and dehydrating agent. 3. Dioxane (Diethylene dioxide) ± BOTH dehydrating and clearing agent

DEHYDRATION
4. Cellosolve (Ethylene glycol monoethyl ether) ± BOTH dehydrating and clearing agent 5. THF (Tetrahydrofuran) ± BOTH dehydrating agent and clearing agent 6. Triethyl phosphate

Additives to dehydrating agents:
1.) 4% phenol + each 95% ETOH baths acts as a tissue softener for hard tissues such as tendons, nails, or dense fibrous tissues.

Additives to dehydrating agents
2.) Anhydrous copper sulfate = can act as BOTH dehydrating agent and an indicator of water content of the last bath (100% ETOH). *Water(present) = anhydrous copper sulfate = turns to blue (100% ETOH should be changed.)

Remember! 

WHATEVER dehydrating agent is used, the amount in each stage should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution.

CLEARING 

Also known as DEALCOHOLIZATION Process of replacing the dehydrating fluid with a fluid that is miscible with BOTH the dehydrating fluid and the impregnating/embedding medium. medium. 

CLEARING
Clearing agents suitable for routine use: 1. xylene/xylol 2. Toluene 3. Chloroform 4. Methyl benzoate and methyl salicylate 5. Cedarwood oil and clove oil

CLEARING
6. Citrus fruits oils 7. Trichlorethane and petrol 8. Benzene 9. Aniline oil 10. Carbon tetrachloride

CLEARING 

Exemption to the rule: GLYCERIN AND GUM SYRUP NO DEALCOHOLIZATION These clearing agents merely make the tissue clearer!

IMPREGNATION 

Also known as INFILTRATION Process of replacing the clearing agent with the infiltrating medium. The medium used to infiltrate the tissue is usually the same medium used for embedding.  

IMPREGNATION
Four types of tissue impregnation and embedding media: 1.) Paraffin wax 2.) Celloidin (Collodion) 3.) Gelatin 4.) Plastic

IMPREGNATION 

Paraffin ± simplest, most common and the BEST infiltrating/embedding medium. - is NOT recommended for fatty tissues ( the dehydrants and clearing agents used in the process dissolve and remove fat from the tissues).

Paraffin«. 

After clearing, tissue is submerged in 2 or more changes of melted paraffin wax. 

Temperature of paraffin oven = 5555-60 C (Paraffin oven must be maintained at a temperature 2-5 C above the MP of the paraffin wax to be used.)

Paraffin«. 

Wax with melting point = 56 C is normally used for routine work. If the lab temperature = 20-24 C 20- 

paraffin wax MP to use: (54-58 C) (54-

Paraffin«. 

If the lab temp. = 15-18 C 15-

paraffin wax MP to use: 50-54 C 50-

Paraffin«.
When wax has been reused, some water is mixed with it.  If excessive water accumulates, this may impair the impregnating capacity of the medium.  To remove excess water = heat the wax to 100-105 C 100 Paraffin wax may be used twice only! 

SUBSTITUTES FOR PARAFFIN WAX 1. Paraplast = MP: 56-57 C 56= mixture of highly purified paraffin and synthetic plastic polymers = more elastic and resilient than paraffin = for large dense tissue blocks such as bones and brain

SUBSTITUTES FOR PARAFFIN WAX 2. Embeddol = MP: 56-58 C 56=less brittle and less compressible than paraplast. 3. Bioloid = recommended for embedding eyes. 4. Tissue Mat = a product of paraffin, containing rubber, with the same property as paraplast.

SUBSTITUTES FOR PARAFFIN WAX 5. Ester Wax = MP: 46-48 C 46= harder than paraffin =not soluble in water =soluble in 95% ETOH and other clearing agents. =can be used for impregnation without prior clearing of the tissue. =sectioning of ester waxwaximpregnated tissues should be done on a sliding or sledge type microtome due to the relative hardness of the wax.

SUBSTITUTES FOR PARAFFIN WAX 6. Water-soluble waxes = MP: 38-42 C Water38or 45-56 C 45= mostly polyethylene glycols *Most commonly used: CARBOWAX 

Carbowax ± soluble and miscible with water (hence does not require dehydration and clearing of the tissue). - tissues are fixed, washed out and transferred directly into melted carbowax. - suitable for many enzyme histochemical studies.

IMPREGNATION 

Celloidin (Collodion) ± purified form of nitrocellulose =suitable for specimens with large hollow cavities, hard and dense tissues (bones and teeth), large tissue sections of the whole embryo.

Celloidin«.
Two methods for celloidin impregnation: 1. Wet Celloidin ± recommended for bones, teeth, large brain sections and whole organs. 2. Dry Celloidin ± preferred for processing of whole eye sections.

Celloidin«.
L.V.N. (Low Viscosity Nitrocellulose) is another form of celloidin  It is soluble in equal concentration of ether and alcohol, with a lower viscosity, allowing it to be used in higher concentrations and still penetrate tissues rapidly. 

IMPREGNATION 

Gelatin ± rarely used except when dehydration is to be avoided. - used when tissues are for histochem and enzyme studies. - embedding medium for delicate specimens and frozen sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut.

Gelatin«. 

It is water-soluble waterDoes not require dehydration and clearing 

IMPREGNATION 

Plastic/Resin ±classified into: epoxy polyester acrylic

EMBEDDING 

CASTING OR BLOCKING Process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify. ORIENTATION ±process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining.  

EMBEDDING
Temperature of melted paraffin used for embedding = 5-10 C above its MP.  To solidify embedded tissue = cooled rapidly in a ref (-5 C) or immersed in (cold water.  The surface of the section to be cut should be placed parallel to the bottom of the mold in which it is oriented. 

TRIMMING
Process of removing excess wax after embedding.  Excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting.  Knife/blade may be used 

SECTIONING
CUTTING OR MICROTOMY  The process by which a processed tissue is cut into uniformly thin slices (sections) to facilitate studies under the microscope.  4-6 u in thickness for routine histologic procedure.  10-15 u for frozen section. 10section.  0.5 u for electron microscopy. 

SECTIONING
KINDS OF MICROTOMES: 1. Rocking Microtome (Cambridge Rocking Microtome) *inventor: Paldwell Trefall in 1881 *simplest among the microtomes *disadvantage: difficulty in reorienting the block.

SECTIONING
2. Rotary/Minot Microtome 1885*inventor: Minot in 1885-1886 *inventor: *MOST COMMON type used today especially for paraffin-embedded paraffintissues.

SECTIONING
3. Sliding Microtome = MOST DANGEROUS TYPE DUE TO MOVABLE EXPOSED KNIFE! *inventor/developer: Adams in 1789 *inventor/developer: *There are 2 types: a. Base-Sledge Base> for all forms of media >block holder: moving >knife: stationary

SECTIONING
3. Sliding Microtome b. Standard Sliding Microtome >block: stationary >knife: moving

SECTIONING
4. Rotary Rocking Microtome 5. Vibrotome ± used for unfixed, unfrozen specimen sectioning for enzyme demonstrations. - disadvantage: sections are liable to disintegrate.

SECTIONING
6. Ultrathin Microtome ± for cutting sections for Electron Microscopy >uses DIAMOND KNIVES

SAMPLE BOARD EXAM QUESTION
March 1993: What is the optimum temperature of the water bath that is used to float tissue cut from the microtome? a. 30 C b. 37 C c. 45-50 C 45d. 50-56 C 50-

Answer: ³The sections are then floated out on a water bath set at 45-50 C, 45approximately 6-10 C lower than the MP of the wax used for embedding the tissue.´ Page 107 of the New Gregorios book

Thing you must not forget! 
  

Clearance angle = 0-15 degrees 0Bevel angle = 27-32 degrees 27The temperature of the hot plate or drying oven used to dry paraffin sections onto slides should be at the MP of the paraffin. For routine work, 76 x 25 mm. slides that are 1.0-1.2 mm. thick are usually 1.0preferred because they do not break easily.

STAINING
Definition of terms: 1. Chromophores = (Gr. ³color³colorbearers´) 2. Auxochromes = (Gr. ³increasers´) >when attached to the dye molecule, they serve to intensify the color of the dye. They do this by acting as electron donors to the chromophore. 3. Lake = the resultant complex of stain-mordantstain-mordant-tissue.

STAINING
H and E staining: Hematoxylin ± a natural dye derived from extraction from the heartwood of the Mexican tree known as ³Hematoxylin Campechianum´

STAINING
Ripening/Oxidation >may be done by exposing the substance to air and sunlight (SLOW) >may be done by adding oxidizing agents such as: hydrogen peroxide mercuric oxide Potassium permanganate sodium perborate sodium iodate

STAINING
I. Alum Hematoxylins  Used in routine H and E  Produce good nuclear stain (RED)  Examples: 
Ehrlich¶s ±slowly ripened Delafield¶s ±slowly ripened Mayer¶s ± sod. Iodate Gill¶s ± sod. Iodate Harris ± mercuric oxide

STAINING
II. Iron Hematoxylins > iron salts are used as oxidizing agents and mordant > examples: 1. Weigert¶s ± Ferric chloride -for mucles/connective tissue fibers 2. Heidenhain¶s- Ferric ammonium Heidenhain¶ssulfate -for mitochondria, muscle striations, chromatin, and myelin

STAINING
III. Tungsten Hematoxylin > Mallory¶s PTAH (Phophotungtic Acid Hematoxylin) -for staining muscle striations

STAINING
H and E staining Steps: XYLOL ( 2 CHANGES) DESCENDING GRADE OF ALCOHOL WATER *Removal of pigments is done after rehydration and right before primary staining

*Removal of mercuric pigments: -place in Weigert¶s iodine -wash in dist. Water -remove iodine with 5% sod. Thiosulfate -wash in running water -proceed with stain

Stain with Harris/ Ehrlich¶s/Delafield¶s Rinse slides in tap water

Acid alcohol (Differentiator) Ammonia water (Ammonium hydroxide, lithium carbonate, Scott¶s tap water)

Wash well in running tap water Stain with Eosin Y Ascending grade of alcohol Xylol/xylene Mount then label

Results:
Nuclei ± blue  Cartilage and calcium deposits ± dark blue  Cytoplasm and other tissue constituents ± varying shades of red  Blood ± bright red 

PAP Smear Staining 

Uses 3 stains: Hematoxylin, OG-6, OGEA Steps: Fix with 95% ETOH Stain with Harris Hematoxylin Acid Alcohol

PAP Smear Staining
Blueing step Stain with OG-6 = stains the cytoplasm of mature OG(superficial cells) 7070-95% ETOH = for washing

Stain with EA 36/50/65 = Stains the cytoplasm of immature cells (intermediate, parabasal)

PAP Smear Staining
Dehydrate

Xylol

Mount and label

PAP Smear Staining
Eosin azure = Eosin Bismarck brown Lithium carbonate Phosphotungstic acid Light green SF (36,50,65)

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