logic techniques

HISTOPATHOLOGIC HISTOPATHOLOGIC TECHNIQUES TECHNIQUES

BOARD REVIEWBOARD REVIEW

By: Rene Jesus Alfredo R. By: Rene Jesus Alfredo R. Dinglasan, RMTDinglasan, RMT

I.) Quality Assurance and I.) Quality Assurance and DocumentationDocumentationA. Histopath ReportsA. Histopath Reports

1. Surgical pathology 1. Surgical pathology 2. Cytopathology report2. Cytopathology report3. Autopsy report3. Autopsy report

• Number of copies prepared per report: Number of copies prepared per report: Three copies = Three copies =

DoctorDoctorPatientPatientFileFile

SAMPLE BOARD QUESTIONSSAMPLE BOARD QUESTIONS

March 1992 Board Exams:March 1992 Board Exams:

Forensic and anatomic pathology Forensic and anatomic pathology report should be kept in the report should be kept in the laboratory for a period of:laboratory for a period of:

a. one yeara. one year

b. two yearsb. two years

c. six years c. six years

d. permanentlyd. permanently


March 1998 Board ExamsMarch 1998 Board Exams

In most private hospitals, the In most private hospitals, the histopathologic report is typed in:histopathologic report is typed in:

a. Four copiesa. Four copies

b. Triplicateb. Triplicate

c. Duplicatec. Duplicate

d. One copyd. One copy

B. SignatoriesB. Signatories

1. Request Forms = patient’s doctor1. Request Forms = patient’s doctor

2. Result Forms = Pathologist2. Result Forms = Pathologist

C. Specimen HandlingC. Specimen Handling

1. FIX FIRST!1. FIX FIRST!

2. Label2. Label

D. Routine Turn-over of ResultsD. Routine Turn-over of Results

1. Surgical pathology and cytology1. Surgical pathology and cytology

= 24 hours= 24 hours

2. Frozen section2. Frozen section

= 5-15 minutes= 5-15 minutes

3. Autopsy report3. Autopsy report

= 1 week= 1 week


March 1997 Board ExamsMarch 1997 Board Exams

An autopsy, to be most informative An autopsy, to be most informative and helpful, should be done within:and helpful, should be done within:

a. 36 hoursa. 36 hours

b. 72 hoursb. 72 hours

c. 1 weekc. 1 week

d. 24 hoursd. 24 hours

E. Storage of Specimen, Tissue blocks, E. Storage of Specimen, Tissue blocks, SlidesSlides

Specimen = 1 month to 1 yearSpecimen = 1 month to 1 year

Tissue Blocks = 3 years to 10 yearsTissue Blocks = 3 years to 10 years

Slides = IndefiniteSlides = Indefinite

II. Fresh Tissue ExamII. Fresh Tissue Exam

Methods:Methods:1. Teasing/Dissociation1. Teasing/Dissociation selected tissue spx watch glassselected tissue spx watch glass

(w/ isotonic(w/ isotonicsalt sol’n)salt sol’n)

Microscopes used: Phase Microscopes used: Phase contrast/Bright contrast/Bright fieldfield

Methods:Methods:

2. Crushing/ Squash preparation2. Crushing/ Squash preparation

Tissues lessTissues less

than 1 mm in diameter sandwiched than 1 mm in diameter sandwiched between two slides between two slides

or or a slide and a cover slip a slide and a cover slip

a vital stain may be useda vital stain may be used

Methods: Methods:

3. Smear prep 3. Smear prep = process of examining = process of examining sections or sedimentssections or sediments

= cellular materials are = cellular materials are spread lightly over a slide by means spread lightly over a slide by means of a wire loop/ applicator stick/ of a wire loop/ applicator stick/ another slide. another slide.

Different smear prep techniques Different smear prep techniques include:include: StreakingStreaking

SpreadingSpreading

Pull-apartPull-apart

Touch prep/ImpressionTouch prep/Impression

Methods:Methods:4.Frozen Section4.Frozen Section=normally used when a rapid diagnosis of =normally used when a rapid diagnosis of a tissue is required.a tissue is required.

= APPLICATIONS:= APPLICATIONS:1. Rapid pathologic diagnosis during 1. Rapid pathologic diagnosis during surgerysurgery2. Enzyme histochemistry2. Enzyme histochemistry3. Demonstration of soluble substances 3. Demonstration of soluble substances such as lipids and carbohydratessuch as lipids and carbohydrates4. Immunofluorescent and 4. Immunofluorescent and immunocytochemical stainingimmunocytochemical staining5. Some specialized silver stains, 5. Some specialized silver stains, particularly in neuropathology. particularly in neuropathology.

2 Methods of Preparing Frozen 2 Methods of Preparing Frozen Sections:Sections: Cold knife procedureCold knife procedure

optimum condition for sectioning:optimum condition for sectioning:KNIFE = -40 to -60 CKNIFE = -40 to -60 CTISSUE = 5 to -10 CTISSUE = 5 to -10 CENVIRONMENT = 0 to -10 CENVIRONMENT = 0 to -10 C

Reminders:Reminders: If tissues are frozen too hard = chip into If tissues are frozen too hard = chip into

fragments when cut.fragments when cut.

Remedy: Surface of the block maybe be Remedy: Surface of the block maybe be softened by warming slightly with the softened by warming slightly with the finger.finger.

If tissues have not been sufficiently frozen If tissues have not been sufficiently frozen = thick and crumble= thick and crumble

= block may come away from = block may come away from the stage.the stage.

Remedy: More bursts of carbon dioxide gas Remedy: More bursts of carbon dioxide gas should then be given to refreeze the block. should then be given to refreeze the block.

2 Methods of Preparing Frozen 2 Methods of Preparing Frozen Sections:Sections:

Cryostat procedure (Cold Microtome)Cryostat procedure (Cold Microtome)

optimum working temp. = -18 to -20 Coptimum working temp. = -18 to -20 C

CRYOSTAT – a refrigerated cabinet in CRYOSTAT – a refrigerated cabinet in which a modified microtome is housed.which a modified microtome is housed.

All the controls to the microtome are All the controls to the microtome are operated from outside the cabinet. operated from outside the cabinet.

Presently, the Presently, the rotary microtomerotary microtome is the is the type of choice.type of choice.

Commonly Used Methods of Commonly Used Methods of FreezingFreezing

Liquid NitrogenLiquid Nitrogen Isopentane cooled by liquid nitrogenIsopentane cooled by liquid nitrogen Carbon dioxide gasCarbon dioxide gas Aerosol sprays = adequate for Aerosol sprays = adequate for

freezing small pieces of tissue freezing small pieces of tissue EXCEPT muscle.EXCEPT muscle.

PRESERVED TISSUE PRESERVED TISSUE

EXAMINATIONEXAMINATION

FIXATIONFIXATION

Preserving fresh tissue for Preserving fresh tissue for examinationexamination

First and most critical step First and most critical step in in histotechnologyhistotechnology

Primary aimPrimary aim: to preserve the : to preserve the morphologic and chemical integrity morphologic and chemical integrity of the cell in as life-like manner as of the cell in as life-like manner as possible.possible.

FIXATIONFIXATION

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

FIXATIONFIXATION

Practical considerations of Fixation:Practical considerations of Fixation:

1. 1. SpeedSpeed – the specimen should be – the specimen should be placed in fixative as soon as it is placed in fixative as soon as it is removed from the body.removed from the body.

2. 2. PenetrationPenetration – formalin diffuses into the – formalin diffuses into the tissue at approximately tissue at approximately 1 mm/Hr.1 mm/Hr.

3.3.VolumeVolume – 20 times the tissue volume – 20 times the tissue volume

4.Duration of fixation4.Duration of fixation

FIXATIONFIXATION Two mechanisms involved in Two mechanisms involved in

Fixation:Fixation:

1. 1. Additive fixationAdditive fixation – whereby the – whereby the chemical constituent of the fixative is chemical constituent of the fixative is taken in and becomes part of the taken in and becomes part of the tissue.tissue.

Examples: FormalinExamples: Formalin

Mercuric fixativesMercuric fixatives

Osmium tetroxideOsmium tetroxide

FIXATIONFIXATION Two mechanisms involved in Two mechanisms involved in

Fixation:Fixation:2. 2. Non-additive fixationNon-additive fixation – whereby the – whereby the

fixing agent is NOT taken in, but fixing agent is NOT taken in, but changes the tissue composition and changes the tissue composition and stabilizes the tissue by removing the stabilizes the tissue by removing the bound water attached to hydrogen bound water attached to hydrogen bonds of certain groups within the bonds of certain groups within the protein molecule.protein molecule.

Example: Alcoholic fixativesExample: Alcoholic fixatives

FIXATIONFIXATION

Main factors involved in fixation:Main factors involved in fixation:1. Hydrogen ion concentration (pH)1. Hydrogen ion concentration (pH)

satisfactory fixation = satisfactory fixation = pH 6-8pH 6-8

2. Temperature2. Temperaturesurgical spx – surgical spx – Rm. Temp.Rm. Temp.Electron Microscopy and some Electron Microscopy and some

histochem histochem – – 0-4 C0-4 C

*Formalin heated at 60 C*Formalin heated at 60 C

= sometimes used for rapid fixation = sometimes used for rapid fixation of very urgent biopsy specimensof very urgent biopsy specimens

*Formalin heated at 100 C*Formalin heated at 100 C

= can be used to fix tissues with TB= can be used to fix tissues with TB

FIXATIONFIXATION

3. Thickness of section3. Thickness of section

4. Osmolality4. Osmolality

5. Concentration5. Concentration

6. Duration of Fixation6. Duration of Fixation

FIXATIONFIXATION

I. Aldehyde fixativesI. Aldehyde fixatives

1. 1. FormaldehydeFormaldehyde

2. 2. 10% Formol-Saline10% Formol-Saline = = CNS tissuesCNS tissues

3. 3. 10% BNF (Buffered Neutral 10% BNF (Buffered Neutral Formalin)Formalin) = = best fixative for best fixative for tissues containing iron pigmentstissues containing iron pigments

= BEST GENERAL = BEST GENERAL TISSUE TISSUE FIXATIVEFIXATIVE

FIXATIONFIXATION

4. Formol-Corrosive (Formol-Sublimate)4. Formol-Corrosive (Formol-Sublimate)

5. Glutaraldehyde = preserves plasma 5. Glutaraldehyde = preserves plasma protein protein

better.better.

6. Formol-calcium = for the preservation 6. Formol-calcium = for the preservation of lipidsof lipids

FIXATIONFIXATIONII. Metallic fixativesII. Metallic fixatives

1. Mercuric Chloride 1. Mercuric Chloride

= may produce black granular = may produce black granular deposits on tissuesdeposits on tissues

a. Zenker’s Fluid (with glacial a. Zenker’s Fluid (with glacial acetic acid)acetic acid)

b. Zenker-Formol (Helly’s Sol’n)b. Zenker-Formol (Helly’s Sol’n)

FIXATIONFIXATIONII. Metallic fixativesII. Metallic fixatives

1. Mercuric Chloride 1. Mercuric Chloride c. Heidenhain’s SuSa –c. Heidenhain’s SuSa –for tumor for tumor

biopsies of the skinbiopsies of the skind. Schaudinn’s fluid d. Schaudinn’s fluid e. Ohlmacher’s fluide. Ohlmacher’s fluidf. Carnoy-Lebrun fluidf. Carnoy-Lebrun fluidg. B-5 fixativeg. B-5 fixative

FIXATIONFIXATION

II. Metallic fixativesII. Metallic fixatives2. Chromate2. Chromate

a. chromic acid a. chromic acid b. potassium dichromateb. potassium dichromatec. Regaud’s (Moller’s)c. Regaud’s (Moller’s)d. Orth’s fluid – for Rickettsia and d. Orth’s fluid – for Rickettsia and

other bacteriaother bacteria- for study of early - for study of early degenerative processdegenerative process

FIXATIONFIXATION

II. Metallic fixativesII. Metallic fixatives

3. Lead fixatives –are generally for 3. Lead fixatives –are generally for ACID MUCOPOLYSACCHARIDESACID MUCOPOLYSACCHARIDES

(for example: Umbilical (for example: Umbilical Cord/ Cord/ Wharton’s Wharton’s jelly)jelly)

FIXATIONFIXATION

III. Picrate fixativesIII. Picrate fixatives-highly explosive when dry-highly explosive when dry- will produce excessive yellow - will produce excessive yellow staining of tissuesstaining of tissues

-picrates are formed upon protein; -picrates are formed upon protein; precipitates are soluble in water; precipitates are soluble in water; hence tissues must be first rendered hence tissues must be first rendered insoluble by direct immersion in 70% insoluble by direct immersion in 70% ETOHETOH

FIXATIONFIXATION

III. Picrate fixativesIII. Picrate fixatives

-picrate fixatives MUST NEVER be -picrate fixatives MUST NEVER be washed in water washed in water before dehydration.before dehydration.

TISSUES 70% ETOH 5% Sod. TISSUES 70% ETOH 5% Sod. ThiosulfateThiosulfate

wash in running waterwash in running water

FIXATIONFIXATION

III. Picrate fixativesIII. Picrate fixatives

A. Bouin’s Solution – A. Bouin’s Solution – for fixation of for fixation of embryosembryos

B. Brasil’s Alcoholic Picroformol – less B. Brasil’s Alcoholic Picroformol – less messy than Bouin’smessy than Bouin’s

FIXATIONFIXATION

IV. Glacial Acetic AcidIV. Glacial Acetic Acid

= fixes nucleoprotein= fixes nucleoprotein

V. Alcoholic FixativesV. Alcoholic Fixatives

general disadvantage: general disadvantage:

POLARIZATIONPOLARIZATION

Polarization – glycogen granules move towards Polarization – glycogen granules move towards the poles or ends of cells.the poles or ends of cells.

1. Methanol – blood smears and BM 1. Methanol – blood smears and BM tissuestissues

2. Ethanol 2. Ethanol

3. Carnoy’s fluid – 3. Carnoy’s fluid – MOST RAPID MOST RAPID FIXATIVEFIXATIVE

(( Fixation time: 1 to 3 hours )Fixation time: 1 to 3 hours )

4. Alcoholic Formalin ( Gendre’s Fixative)4. Alcoholic Formalin ( Gendre’s Fixative)

-useful in preserving -useful in preserving sputumsputum

5. Newcomer’s fluid 5. Newcomer’s fluid

FIXATIONFIXATION

VI. Osmium Tetroxide FixativesVI. Osmium Tetroxide Fixatives

- should be kept in a dark-- should be kept in a dark-colored, chemically clean bottle to colored, chemically clean bottle to prevent evaporation and reduction prevent evaporation and reduction by sunlight or organic matter. by sunlight or organic matter.

- inhibits hematoxylin and makes - inhibits hematoxylin and makes counterstaining difficult.counterstaining difficult.


produces produces black precipitate (Osmic black precipitate (Osmic oxide)oxide)

Prevention: add saturated aqueous Prevention: add saturated aqueous mercuric chloridemercuric chloride

Remedy: Black osmic oxide crystals Remedy: Black osmic oxide crystals may be dissolved in may be dissolved in cold watercold water..

Precaution: Precaution: may cause conjunctivitis may cause conjunctivitis or blindness.or blindness.


FLEMMING’S SOL’N FLEMMING’S SOL’N

FLEMMING’S W/O ACETIC ACIDFLEMMING’S W/O ACETIC ACID

removal of this serves to removal of this serves to improve cytoplasmic detailsimprove cytoplasmic details

FIXATIONFIXATIONVII. Tricholoroacetic acidVII. Tricholoroacetic acid

VIII. Acetone – used for the Dx of VIII. Acetone – used for the Dx of RabiesRabies

IX. Heat Fixation IX. Heat Fixation – – direct flaming fixationdirect flaming fixation

- microwave fixation (optimum temp. 45-55 C)microwave fixation (optimum temp. 45-55 C)

**underheatingunderheating – poor sectioning – poor sectioning

**overheatingoverheating (above 65 C) (above 65 C) -vacuolation-vacuolation -overstained cytoplasm-overstained cytoplasm -pyknotic nuclei-pyknotic nuclei

FIXATIONFIXATION

FIXATIVES FOR E.M.FIXATIVES FOR E.M.1. Glutaraldehyde1. Glutaraldehyde

2. Platinic chloride (PtCl2. Platinic chloride (PtCl33))3. Platinic Chloride-formalin 3. Platinic Chloride-formalin

(Zamboni’s fixative)(Zamboni’s fixative)4. Gold chloride (AuCl)4. Gold chloride (AuCl)5. Osmium tetroxide5. Osmium tetroxide6. 10% BNF = acceptable but not 6. 10% BNF = acceptable but not

recommendedrecommended

DECALCIFICATIONDECALCIFICATION

A procedure whereby calcium or lime A procedure whereby calcium or lime salts are removed from tissue salts are removed from tissue FOLLOWING FIXATIONFOLLOWING FIXATION

Should be done after fixation and Should be done after fixation and before impregnationbefore impregnation

DECALCIFICATIONDECALCIFICATION

More concentrated acid solutions More concentrated acid solutions decalcify bone more rapidly but are decalcify bone more rapidly but are more harmful to the tissue.more harmful to the tissue.

High concentrationsHigh concentrations and greater and greater amount of fluid will increase the amount of fluid will increase the speed of the process.speed of the process.

The recommended ratio of fluid to The recommended ratio of fluid to tissue volume for decalcification is 20 tissue volume for decalcification is 20 to 1.to 1.

Heat will serve to hasten decalcification Heat will serve to hasten decalcification BUT it also increases the damaging BUT it also increases the damaging effects on tissues.effects on tissues.

At 37 C = impaired nuclear staining of At 37 C = impaired nuclear staining of Van Gieson’s stain for collagen fibers.Van Gieson’s stain for collagen fibers.

At 55 C = tissue will undergo complete At 55 C = tissue will undergo complete digestion within 24-48 hours. digestion within 24-48 hours.

Optimum temperature = RM TEMP (18-Optimum temperature = RM TEMP (18-30 C)30 C)

The ideal time required for The ideal time required for decalcifying tissue is 24-48 hours.decalcifying tissue is 24-48 hours.

Dense bone tissues usually require Dense bone tissues usually require up to 14 days or longer in order to up to 14 days or longer in order to complete the process. complete the process.

Decalcifying agentsDecalcifying agents

Nitric acid – MOST COMMONNitric acid – MOST COMMON

examples: examples: Perenyi’s fluid – Perenyi’s fluid – acts as acts as BOTH BOTH tissue softener and tissue softener and decalcifying agent.decalcifying agent.

Phloroglucin-Nitric Acid Phloroglucin-Nitric Acid

--MOST RAPID MOST RAPID DECALCIFYING AGENT!DECALCIFYING AGENT!


Formic acid – BOTH fixative and Formic acid – BOTH fixative and decalcifying agentdecalcifying agent

5% formic acid is considered to be 5% formic acid is considered to be the BEST GENERAL DECALCIFYING the BEST GENERAL DECALCIFYING AGENTAGENT

Formic acid is recommended for Formic acid is recommended for small pieces of bones and teeth.small pieces of bones and teeth.


Hydrochloric acidHydrochloric acid

> Von Ebner’s fluid – recommended > Von Ebner’s fluid – recommended for teeth and small pieces of bones for teeth and small pieces of bones and teeth.and teeth.

Post-DecalcificationPost-Decalcification

After decalcification is complete, acid After decalcification is complete, acid can be removed from tissues or can be removed from tissues or neutralized chemically by immersing neutralized chemically by immersing the decalcified bone in either:the decalcified bone in either:1. saturated lithium carbonate sol’n.1. saturated lithium carbonate sol’n.

2. 5-10% aqueous sodium 2. 5-10% aqueous sodium bicarbonate solution for several bicarbonate solution for several hours. hours.

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

Tissue SoftenersTissue Softeners

For unduly hard tissues that may For unduly hard tissues that may damage the microtome knives.damage the microtome knives.

4% aq. phenol.4% aq. phenol. Molliflex Molliflex 2% HCl2% HCl 1% HCl in 70% alcohol1% HCl in 70% alcoholNote: Tissues immersed in Molliflex Note: Tissues immersed in Molliflex

may appear swollen and soapy. may appear swollen and soapy. (Does not affect normal processing)(Does not affect normal processing)

DEHYDRATIONDEHYDRATION

Aim: to remove fixative and water Aim: to remove fixative and water from the tissue and replacing them from the tissue and replacing them with dehydrating fluid in preparation with dehydrating fluid in preparation for impregnation. for impregnation.

Dehydrating fluids are generally used Dehydrating fluids are generally used in increasing strengths. in increasing strengths.

Increasing strengths = all the Increasing strengths = all the aqueous tissue fluids are removed aqueous tissue fluids are removed but with little disruption to the tissue but with little disruption to the tissue due to diffusion currents.due to diffusion currents.


For delicate tissues, particularly For delicate tissues, particularly embryonic and animal tissuesembryonic and animal tissues, it , it is recommended to start processing is recommended to start processing with with 30% ethyl alcohol30% ethyl alcohol. .


COMMONLY USED DEHYDRATING AGENTS:COMMONLY USED DEHYDRATING AGENTS:

1.) Alcohol1.) Alcohol – MOST COMMON – MOST COMMON

a. Ethanola. Ethanol = for routine dehydration of = for routine dehydration of tissues.tissues.

= BEST DEHYDRATING = BEST DEHYDRATING AGENT.AGENT.

b. Methyl alcohol b. Methyl alcohol = employed for blood = employed for blood and tissue filmsand tissue films


c. Butyl alcohol c. Butyl alcohol =utilized in plant and =utilized in plant and animal microtechniques.animal microtechniques.

d. Industrial methylated d. Industrial methylated spirit(denatured alcohol) spirit(denatured alcohol) = =

ethanol + small amt. of ethanol + small amt. of methanolmethanol

used in the same way as ETOHused in the same way as ETOH


e. Isopropyl alcohol e. Isopropyl alcohol = many of the = many of the processing methods for use in a processing methods for use in a microwave oven recommend this microwave oven recommend this agent. agent.

2. Acetone 2. Acetone – BOTH fixative and – BOTH fixative and dehydrating agent.dehydrating agent.

3. 3. DioxaneDioxane (Diethylene dioxide) (Diethylene dioxide) – BOTH – BOTH dehydrating and clearing agentdehydrating and clearing agent


4. 4. Cellosolve (Ethylene glycol Cellosolve (Ethylene glycol monoethyl ether)monoethyl ether) – BOTH dehydrating – BOTH dehydrating and clearing agentand clearing agent

5. 5. THF (Tetrahydrofuran)THF (Tetrahydrofuran) – BOTH – BOTH dehydrating agent and clearing agentdehydrating agent and clearing agent

6. Triethyl phosphate6. Triethyl phosphate

Additives to dehydrating agents:Additives to dehydrating agents:

1.) 4% phenol + each 95% ETOH baths1.) 4% phenol + each 95% ETOH baths

acts as a tissue softener for acts as a tissue softener for hard tissues such as tendons, nails, hard tissues such as tendons, nails, or dense fibrous tissues.or dense fibrous tissues.

Additives to dehydrating agentsAdditives to dehydrating agents

2.) Anhydrous copper sulfate2.) Anhydrous copper sulfate

= can act as BOTH dehydrating = can act as BOTH dehydrating agent and an indicator of water content agent and an indicator of water content of the last bath (100% ETOH).of the last bath (100% ETOH).

*Water(present) = *Water(present) =

anhydrous copper sulfate = anhydrous copper sulfate = turns to turns to blue blue

(100% ETOH should be changed.)(100% ETOH should be changed.)

Remember!Remember!

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

CLEARINGCLEARING

Also known as Also known as DEALCOHOLIZATIONDEALCOHOLIZATION

Process of replacing the dehydrating Process of replacing the dehydrating fluid with a fluid that is miscible with fluid with a fluid that is miscible with BOTH the BOTH the dehydrating fluiddehydrating fluid and the and the impregnating/embedding mediumimpregnating/embedding medium..

CLEARINGCLEARINGClearing agents suitable for routine Clearing agents suitable for routine

use:use:

1.1. xylene/xylolxylene/xylol

2.2. TolueneToluene

3.3. ChloroformChloroform

4.4. Methyl benzoate and methyl Methyl benzoate and methyl salicylatesalicylate

5.5. Cedarwood oil and clove oilCedarwood oil and clove oil

CLEARINGCLEARING

6. Citrus fruits oils6. Citrus fruits oils

7. Trichlorethane and petrol7. Trichlorethane and petrol

8. Benzene8. Benzene

9. Aniline oil9. Aniline oil

10. Carbon tetrachloride10. Carbon tetrachloride

CLEARINGCLEARING

Exemption to the rule:Exemption to the rule:

GLYCERIN AND GUM SYRUPGLYCERIN AND GUM SYRUP

NO DEALCOHOLIZATIONNO DEALCOHOLIZATION

These clearing agents merely make These clearing agents merely make the tissue clearer!the tissue clearer!

IMPREGNATION IMPREGNATION

Also known as Also known as INFILTRATIONINFILTRATION

Process of replacing the clearing Process of replacing the clearing agent with the infiltrating medium.agent with the infiltrating medium.

The medium used to infiltrate the The medium used to infiltrate the tissue is usually the same medium tissue is usually the same medium used for embedding.used for embedding.

IMPREGNATIONIMPREGNATIONFour types of tissue impregnation and Four types of tissue impregnation and

embedding media:embedding media:1.) Paraffin wax1.) Paraffin wax

2.) Celloidin (Collodion)2.) Celloidin (Collodion)

3.) Gelatin3.) Gelatin

4.) Plastic4.) Plastic

IMPREGNATIONIMPREGNATION

ParaffinParaffin – simplest, most common – simplest, most common and the BEST infiltrating/embedding and the BEST infiltrating/embedding medium.medium.

- is NOT recommended for - is NOT recommended for fatty tissues ( the dehydrants and fatty tissues ( the dehydrants and clearing agents used in the process clearing agents used in the process dissolve and remove fat from the dissolve and remove fat from the tissues).tissues).

Paraffin….Paraffin….

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

Temperature of paraffin ovenTemperature of paraffin oven = = 55-60 C55-60 C

(Paraffin oven must be maintained at a (Paraffin oven must be maintained at a temperature temperature 2-5 C2-5 C above the MP of above the MP of the paraffin wax to be used.)the paraffin wax to be used.)


Wax with melting point = Wax with melting point = 56 C56 C is is normally used for routine work. normally used for routine work.

If the lab temperature = 20-24 CIf the lab temperature = 20-24 C

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


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

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


When wax has been reused, some When wax has been reused, some water is mixed with it. water is mixed with it.

If excessive water accumulates, this If excessive water accumulates, this may impair the impregnating may impair the impregnating capacity of the medium. capacity of the medium.

To remove excess water = heat the To remove excess water = heat the wax to 100-105 Cwax to 100-105 C

Paraffin wax may be used Paraffin wax may be used twice only!twice only!

SUBSTITUTES FOR PARAFFIN WAXSUBSTITUTES FOR PARAFFIN WAX

1. Paraplast = MP: 56-57 C1. Paraplast = MP: 56-57 C

= mixture of highly = mixture of highly purified paraffin and synthetic plastic purified paraffin and synthetic plastic polymerspolymers

= more elastic and = more elastic and resilient than paraffinresilient than paraffin

= for large dense tissue = for large dense tissue blocks such as bones and brain blocks such as bones and brain

SUBSTITUTES FOR PARAFFIN WAXSUBSTITUTES FOR PARAFFIN WAX2. Embeddol = MP: 56-58 C2. Embeddol = MP: 56-58 C

=less brittle and less =less brittle and less compressible than paraplast. compressible than paraplast.

3. Bioloid = 3. Bioloid = recommended for recommended for embedding eyes.embedding eyes.

4. Tissue Mat = a product of paraffin, 4. Tissue Mat = a product of paraffin, containing rubber, with the same containing rubber, with the same property as paraplast. property as paraplast.

SUBSTITUTES FOR PARAFFIN WAXSUBSTITUTES FOR PARAFFIN WAX5. Ester Wax = MP: 46-48 C5. Ester Wax = MP: 46-48 C

= harder than paraffin= harder than paraffin=not soluble in water=not soluble in water=soluble in 95% ETOH =soluble in 95% ETOH

and other clearing agents.and other clearing agents.=can be used for =can be used for

impregnation without prior clearing of the impregnation without prior clearing of the tissue. tissue.

=sectioning of ester wax-=sectioning of ester wax-impregnated tissues should be done on a impregnated tissues should be done on a sliding or sledge type microtomesliding or sledge type microtome due to due to the relative hardness of the wax. the relative hardness of the wax.

SUBSTITUTES FOR PARAFFIN WAXSUBSTITUTES FOR PARAFFIN WAX

6. Water-soluble waxes = MP: 38-42 C6. Water-soluble waxes = MP: 38-42 C

or 45-56 Cor 45-56 C

= mostly polyethylene = mostly polyethylene glycolsglycols

*Most commonly used: *Most commonly used: CARBOWAXCARBOWAX

Carbowax – soluble and miscible with Carbowax – soluble and miscible with water (hence does not require water (hence does not require dehydration and clearing of the dehydration and clearing of the tissue).tissue).

- tissues are fixed, - tissues are fixed, washed out and transferred directly washed out and transferred directly into melted carbowax. into melted carbowax.

- suitable for many - suitable for many enzyme histochemical studies.enzyme histochemical studies.


Celloidin (Collodion) Celloidin (Collodion) – purified form of – purified form of nitrocellulosenitrocellulose

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

Celloidin….Celloidin….

Two methods for celloidin Two methods for celloidin impregnation:impregnation:

1. Wet Celloidin – recommended for 1. Wet Celloidin – recommended for bones, teeth, large brain sections bones, teeth, large brain sections and whole organs.and whole organs.

2. Dry Celloidin – preferred for 2. Dry Celloidin – preferred for processing of whole eye sections. processing of whole eye sections.

Celloidin….Celloidin….

L.V.N. (Low Viscosity Nitrocellulose) L.V.N. (Low Viscosity Nitrocellulose) is another form of celloidinis another form of celloidin

It is soluble in equal concentration of It is soluble in equal concentration of ether and alcohol, with a lower ether and alcohol, with a lower viscosity, allowing it to be used in viscosity, allowing it to be used in higher concentrations and still higher concentrations and still penetrate tissues rapidly.penetrate tissues rapidly.


Gelatin – Gelatin – rarely used except when rarely used except when dehydration is to be avoided.dehydration is to be avoided.

- used when tissues are for - used when tissues are for histochem and enzyme studies. histochem and enzyme studies.

- embedding medium for - embedding medium for delicate specimens and delicate specimens and frozen frozen sections sections because it prevents because it prevents fragmentation of tough and friable fragmentation of tough and friable tissues when frozen sections are cut. tissues when frozen sections are cut.

Gelatin….Gelatin….

It is water-solubleIt is water-soluble

Does not require dehydration and Does not require dehydration and clearingclearing


Plastic/Resin Plastic/Resin –classified into: –classified into:

epoxyepoxy

polyesterpolyester

acrylicacrylic

EMBEDDING EMBEDDING

CASTING OR BLOCKINGCASTING OR BLOCKING

Process by which the impregnated tissue Process by which the impregnated tissue is placed into a precisely arranged position is placed into a precisely arranged position in a mold containing a medium which is in a mold containing a medium which is then allowed to solidify.then allowed to solidify.

ORIENTATION –ORIENTATION –process by which a tissue is process by which a tissue is arranged in precise positions in the mold arranged in precise positions in the mold during embedding, on the microtome during embedding, on the microtome before cutting, and on the slide before before cutting, and on the slide before staining.staining.

EMBEDDINGEMBEDDING

Temperature of melted paraffin used Temperature of melted paraffin used for embedding = for embedding = 5-10 C 5-10 C aboveabove its its MP.MP.

To solidify embedded tissue = cooled To solidify embedded tissue = cooled rapidly in a ref (-5 C) or immersed in rapidly in a ref (-5 C) or immersed in cold water. cold water.

The surface of the section to be cut The surface of the section to be cut should be placed parallel to the should be placed parallel to the bottom of the mold in which it is bottom of the mold in which it is oriented.oriented.

TRIMMINGTRIMMING

Process of removing excess wax Process of removing excess wax after embedding.after embedding.

Excess wax is cut off from the block Excess wax is cut off from the block to expose the tissue surface in to expose the tissue surface in preparation for actual cutting.preparation for actual cutting.

Knife/blade may be usedKnife/blade may be used

SECTIONINGSECTIONING

CUTTING OR MICROTOMYCUTTING OR MICROTOMY The process by which a processed The process by which a processed

tissue is cut into uniformly thin slices tissue is cut into uniformly thin slices (sections) to facilitate studies under (sections) to facilitate studies under the microscope. the microscope.

4-6 u4-6 u in thickness for in thickness for routine routine histologic procedure.histologic procedure.

10-15 u10-15 u for for frozen sectionfrozen section.. 0.5 u0.5 u for for electron microscopy.electron microscopy.


KINDS OF MICROTOMES:KINDS OF MICROTOMES:

1. Rocking Microtome (Cambridge 1. Rocking Microtome (Cambridge Rocking Microtome)Rocking Microtome)

**inventor:inventor: Paldwell Trefall in 1881 Paldwell Trefall in 1881

*simplest among the microtomes*simplest among the microtomes

*disadvantage: difficulty in *disadvantage: difficulty in reorienting the block.reorienting the block.


2. Rotary/Minot Microtome2. Rotary/Minot Microtome

*inventor*inventor: Minot in 1885-1886: Minot in 1885-1886

*MOST COMMON type used today *MOST COMMON type used today especially for paraffin-embedded especially for paraffin-embedded tissues.tissues.

SECTIONING SECTIONING

3. Sliding Microtome = MOST 3. Sliding Microtome = MOST DANGEROUS TYPE DUE TO MOVABLE DANGEROUS TYPE DUE TO MOVABLE EXPOSED KNIFE!EXPOSED KNIFE!

*inventor/developer*inventor/developer: Adams in 1789: Adams in 1789

*There are 2 types:*There are 2 types:

a. Base-Sledgea. Base-Sledge

> for all forms of media> for all forms of media

>block holder: >block holder: movingmoving

>knife: >knife: stationarystationary


3. Sliding Microtome3. Sliding Microtome

b. Standard Sliding Microtomeb. Standard Sliding Microtome

>block: >block: stationarystationary

>knife: >knife: movingmoving


4. Rotary Rocking Microtome4. Rotary Rocking Microtome

5. Vibrotome – used for unfixed, 5. Vibrotome – used for unfixed, unfrozen specimen sectioning for unfrozen specimen sectioning for enzyme demonstrations.enzyme demonstrations.

- disadvantage: - disadvantage: sections are liable to disintegrate. sections are liable to disintegrate.


6. Ultrathin Microtome – for cutting 6. Ultrathin Microtome – for cutting sections for Electron Microscopysections for Electron Microscopy

>uses DIAMOND KNIVES>uses DIAMOND KNIVES

SAMPLE BOARD EXAM SAMPLE BOARD EXAM QUESTIONQUESTION

March 1993:March 1993:

What is the optimum temperature of What is the optimum temperature of the water bath that is used to float the water bath that is used to float tissue cut from the microtome?tissue cut from the microtome?

a. 30 Ca. 30 C

b. 37 Cb. 37 C

c. 45-50 Cc. 45-50 C

d. 50-56 Cd. 50-56 C

Answer:Answer:

““The sections are then floated out on a The sections are then floated out on a water bath set at 45-50 C, water bath set at 45-50 C, approximately approximately 6-10 C 6-10 C lowerlower than the than the MP of the wax used for embedding MP of the wax used for embedding the tissue.”the tissue.”

Page 107 of the New Gregorios bookPage 107 of the New Gregorios book

Thing you must not forget!Thing you must not forget!

Clearance angle = 0-15 degreesClearance angle = 0-15 degrees Bevel angle = 27-32 degreesBevel angle = 27-32 degrees The temperature of the hot plate or drying The temperature of the hot plate or drying

oven used to dry paraffin sections onto oven used to dry paraffin sections onto slides should be slides should be at the MP of the paraffin. at the MP of the paraffin.

For routine work, 76 x 25 mm. slides that For routine work, 76 x 25 mm. slides that are 1.0-1.2 mm. thick are usually preferred are 1.0-1.2 mm. thick are usually preferred because they do not break easily. because they do not break easily.

STAININGSTAININGDefinition of terms:Definition of terms:1. Chromophores = (Gr. “color-1. Chromophores = (Gr. “color-

bearers”)bearers”)2. Auxochromes = (Gr. “increasers”)2. Auxochromes = (Gr. “increasers”)

>when attached to the dye >when attached to the dye molecule, they serve to intensify the molecule, they serve to intensify the color of the dye. They do this by color of the dye. They do this by acting acting as electron donorsas electron donors to the to the chromophore.chromophore.

3. Lake = the resultant complex of 3. Lake = the resultant complex of stain-mordant-tissue.stain-mordant-tissue.

STAININGSTAINING

H and E staining:H and E staining:

Hematoxylin – a natural dye derived Hematoxylin – a natural dye derived from extraction from the heartwood from extraction from the heartwood of the Mexican tree known as of the Mexican tree known as “Hematoxylin Campechianum”“Hematoxylin Campechianum”

STAININGSTAINING

Ripening/OxidationRipening/Oxidation>may be done by exposing the substance to air and >may be done by exposing the substance to air and

sunlight (SLOW)sunlight (SLOW)

>may be done by adding oxidizing agents such as:>may be done by adding oxidizing agents such as:hydrogen peroxidehydrogen peroxidemercuric oxidemercuric oxide

Potassium permanganatePotassium permanganatesodium perboratesodium perboratesodium iodatesodium iodate

STAININGSTAINING

I. Alum HematoxylinsI. Alum Hematoxylins Used in routine H and EUsed in routine H and E Produce good nuclear stain (RED)Produce good nuclear stain (RED) Examples:Examples:

Ehrlich’s –slowly ripenedEhrlich’s –slowly ripenedDelafield’s –slowly ripenedDelafield’s –slowly ripenedMayer’s – sod. IodateMayer’s – sod. IodateGill’s – sod. IodateGill’s – sod. IodateHarris – mercuric oxideHarris – mercuric oxide

STAININGSTAINING

II. Iron HematoxylinsII. Iron Hematoxylins> iron salts are used as oxidizing agents > iron salts are used as oxidizing agents and mordantand mordant> examples:> examples:

1. Weigert’s – Ferric chloride1. Weigert’s – Ferric chloride-for mucles/connective tissue -for mucles/connective tissue

fibersfibers2. Heidenhain’s- Ferric ammonium 2. Heidenhain’s- Ferric ammonium

sulfatesulfate-for mitochondria, muscle -for mitochondria, muscle

striations, chromatin, and myelinstriations, chromatin, and myelin

STAININGSTAINING

III. Tungsten HematoxylinIII. Tungsten Hematoxylin

> Mallory’s PTAH (Phophotungtic > Mallory’s PTAH (Phophotungtic Acid Hematoxylin)Acid Hematoxylin)

-for staining muscle -for staining muscle striationsstriations

STAININGSTAINING

H and E staining Steps:H and E staining Steps:XYLOL ( 2 CHANGES)XYLOL ( 2 CHANGES)

DESCENDING GRADE OF ALCOHOLDESCENDING GRADE OF ALCOHOL

WATERWATER

**Removal of pigments is done after Removal of pigments is done after rehydration and right before primary rehydration and right before primary stainingstaining

**Removal of mercuric pigments:Removal of mercuric pigments:

-place in Weigert’s iodine-place in Weigert’s iodine

-wash in dist. Water-wash in dist. Water

-remove iodine with 5% sod. -remove iodine with 5% sod. ThiosulfateThiosulfate

-wash in running water-wash in running water

-proceed with stain-proceed with stain

Stain with Harris/ Ehrlich’s/Delafield’sStain with Harris/ Ehrlich’s/Delafield’s

Rinse slides in tap waterRinse slides in tap water

Acid alcohol (Differentiator)Acid alcohol (Differentiator)

Ammonia water (Ammonium hydroxide, Ammonia water (Ammonium hydroxide, lithium carbonate, Scott’s tap water)lithium carbonate, Scott’s tap water)

Wash well in running tap waterWash well in running tap water

Stain with Eosin YStain with Eosin Y

Ascending grade of alcoholAscending grade of alcohol

Xylol/xyleneXylol/xylene

Mount then labelMount then label

Results:Results:

Nuclei – blueNuclei – blue Cartilage and calcium deposits – dark Cartilage and calcium deposits – dark

blueblue Cytoplasm and other tissue Cytoplasm and other tissue

constituents – varying shades of redconstituents – varying shades of red Blood – bright redBlood – bright red

PAP Smear StainingPAP Smear Staining

Uses 3 stains: Hematoxylin, OG-6, Uses 3 stains: Hematoxylin, OG-6, EAEA

Steps:Steps:

Fix with 95% ETOHFix with 95% ETOH

Stain with Harris HematoxylinStain with Harris Hematoxylin

Acid AlcoholAcid Alcohol


Blueing stepBlueing step

Stain with OG-6 = stains the cytoplasm of mature Stain with OG-6 = stains the cytoplasm of mature (superficial (superficial

cells)cells)

70-95% ETOH = for washing70-95% ETOH = for washing

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


DehydrateDehydrate

XylolXylol

Mount and labelMount and label


Eosin azure = Eosin azure =

EosinEosinBismarck brownBismarck brownLithium carbonateLithium carbonatePhosphotungstic acidPhosphotungstic acidLight green SF Light green SF

(36,50,65)(36,50,65)

logic techniques

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