LABORATORY SAFETY QUALITY CONTROL
Laboratory Staining: check before issuing to the pathologist
Special stains: accompanied by a control stain
e.g. Giemsa stain for Helicobacter pylori Standard Operating Procedure (SOP): mandated by accrediting/ regulatory agency (ASCP)
MSDS
- Detailed procedure for handling hazardous substance
- Personal hygiene practice (handwashing) - Records of compliance
- Risk assessment
- Cause and prevention of occupational injury/ illness - Health and safety training
- PPE
- Hazardous waste disposal practice HEALTH HAZARDS
1. Biohazards
o Cause diseases in humans
o Infectious agents, contaminated solution, specimen or object
2. Irritants
o Cause reversible inflammatory effects at site of contact with living tissue
3. Corrosive chemicals
o Cause destruction/ irreversible alterations when exposed to living tissue/ destroy inanimate surfaces (generally metals) 4. Sensitizers
o Cause allergic reaction in substantial portion of exposed subjects
o May occur at work (↑exposure levels) 5. Carcinogens
o May induce tumors
o Chloroform, chromic acid, formaldehyde 6. Toxic materials
o Capable of causing death by ingestion, direct contact/ inhalation at certain specific
concentration PHYSICAL HAZARDS 1. Combustible
o Ignite at or above certain temperature (flash point) at which vapors ignite in presence of ignition source
2. Flammables
o Flash points below 1000F/ 37.80C o Requires specially designed cabinets 3. Explosives
o Chemicals that cause sudden instantaneous release of pressure, gas and heat when subjected to sudden shocks, pressure or increase in temperature
4. Oxidizers
o Harmless alone but irritates combustion in other materials
o Can cause fire (through release of oxygen or other gas)
PEL: Permissible exposure limit TLV: Threshold limit value
OEL: Occupational exposure limit
LABELLING
- Chemical name, mixture ingredients
- Manufacturer’s name, address/ name of person who made it
- Date purchased/ made - Expiration date
- Hazardous warning and safety procedure STORAGE OF HAZARDOUS CHEMICALS - Conventional cabinet
- Dangerous liquids must be stored below countertop for minimum risk of bodily exposure
- Reagents: stored in plastic containers/ plastic coated glass bottle
- Do not store flammable liquids at ref temperature HANDLING SPILLS
- Gloves
- Disposable plastic aprons (for possible chemical spill)
- Disposable gowns (for biohazard) - Dustpan and brush (for powders) - Sponge, towel, mop (for liquid)
- Absorbent material (commercial absorbent) - Bleach (NaOCl for biohazard)
- Baking soda (for acids) - Vinegar (for alkali)
- Commercial formalin neutralizing product - Sealable plastic bucket
**For small amount of spill: wipe, dispose sponge **Evacuate room if dangerous
**For large spill: call emergency response team FIRST AID
1. Skin contact
o Wash 15-30 mins o Emergency shower
o Soap with water (if not water soluble) o Remove contaminated clothing 2. Splashing on eyes
o Eyewash solution o Water temp: 15-350C o Rinsing: 15-30 mins
HANDLING POTENTIAL INFECTIOUS SPECIMEN - Fresh tissue/ body fluids
- Fixed specimen (↓risk, formalin treated) - Complete penetration by alcohol destroys
infectious agents except prions
**Prions cause spongiform encelopathies (CJD, scrapie, mad cow disease)
CJD: Creutzfeld Jakob Disease
Treat with formalin/phenol (48hrs) or formic acid (1hr)
Normal Steam Sterilization: inactivates prions Sodium Hypochlorite & Phenol: effective but cause artifacts
Cutting areas: treat w/ chlorine bleach or with suitable commercial disinfectant
HAZARDS AND HANDLING COMMON HISTOLOGICAL CHEMICALS
- During process of dilution, conc. acids should be added to water (never water to acid) to prevent splashing & should be done under a fume hood Acetic Acid
- Direct contact with conc. acid irritates skin, eyes & repiratory system
- 1-10% dilute solution: safe to use
- Conc. glacial acetic acid should not be mixed with chromic acid, nitric acid or Na/KOH
Ammonium Hydroxide
- Should be stored away from acids
- Should not be mixed with formaldehyde, generates heat that is irritating to respiratory system
Aniline
- Toxic when absorbed by skin - Cause severe irritation of eyes - Potential carcinogen
- Excessive exposure may cause drowsiness, headache, nausea & cyanosis
- Avoid routine use Chloroform
- Toxic when inhaled/ingested - Carcinogenic
- Affects liver, reproductive organ, CNS, blood & gastrointestinal tract
- Excessive exposure may cause disorientation, loss of consciousness & death
- Avoid use
Chromic Acid (Potassium Dichromate) - Toxic to kidneys
- Corrosive to skin, mucous membranes - Carcinogenic
- Enviromental toxin
- Do not subject to drain disposal Ethanol
- Skin & eye irritant
- Not likely to cause significant toxicity if used under standard conditions
Ether
- Causes mild to moderate irritation of skin & eyes - Flammable, extremely volatile
- Do not store in refrigerator/ freezer
- Excessive exposure may cause disorientation, loss of consciousness & death
Ethylene Glycol
- Toxic by inhalation/ingestion
- Toxic to reproductive, urinary & blood systems & with additional exposure toxic to skin
- Use propylene-based glycol ethers as substitute - Handle under fume hood with butyl gloves Formaldehyde & Paraformaldehyde - Toxic by inhalation/ ingestion - Severe skin & eye irritant - Carcinogenic
- Corrosive to most metals
- Workers exposed to formaldehyde must be periodically monitored for exposure levels - Formadehyde waste can be recycled by
distillation/drain disposal
- Can be detoxified by commercial product/ disposed by licensed waste hauler
Formic Acid
- Irritates skin & eyes - Corrodes metal
- Handle under fume hood Glutaraldehyde
- Severe irritation of eyes, skin - Toxic by ingestion
Hydrochloric Acid
- Causes severe irritation of skin, eyes, RT - Corrosive to metals
- Conc. acid is dangerous because of its fumes - Handle under a fume hood, use goggles, apron &
gloves
Hydrogen Peroxide - Harmless if used in <5% Hydroxide (Na/K)
- Corrosive to eyes & skin Isopentane
- Extremely flammable - Highly volatile
- Store only in refrigerator/freezer - Chilled isopentane can cause frostbite
- Excessive exposure causes irritation of RT, cough, irregular breathing
- Accidental ingestion can cause vomiting, headache, depression & abdominal swelling Isopropanol
- Cause mild to moderate irritation of skin & eyes - Toxic by ingestion
- Cause severe irritation of eyes & skin - Corrosive to metal (contains mercury)
- Solutions will be contaminated if specimen is fixed with B-5, Helly’s/ Zenker’s fixative
- Reagents used to “de-zenkerize” sections will release mercury
- Must not go through drain disposal
- May be replaced with zinc formalin/glyoxal solutions
Methanol
- Moderate skin & eye irritant - Toxic by ingestion & inhalation
- May cause blindness/ death if taken excessively Nitric Acid
- Corrosive to skin, mucous membranes & metals - Toxic by inhalation
Nitrogen (Liquid)
- Causes frost bite/ thermal burns
- Excessive inhalation may cause dizziness, loss of consciousness, death from aphyxia
Osmium Tetroxide
- Corrosive to eyes & mucous membranes
- Vapors are extremely toxic to reproductive, sensory & RT
- Vials must be scored, broken & opened under hood Oxalic Acid
- Safe when used in dilutions prescribed for histology - Conc. acid is corrosive & causes severe burns of
the eyes, skin & mucous membrane
- Repeated skin contact causes dermatitis & slow healing ulcers
Periodic Acid
- Safe when used in quantities prescribed for histology
Phenol
- Readily absorbed through skin
- May cause increased heart rate, convulsions/death - May burn eyes & skin
- Combustible & should be used with extreme caution under a hood
Picric Acid
- Explosive when dry/ combined with metal & metallic salts
- Should not be disposed by pouring down the drain - Toxic when absorbed through skin
Potassium Ferricyanide & Potassium Ferrocyanide
- Safe when handled in conc. prescribed for histological use
Potassium Permanganate
- Cause irritation of skin & eyes
- Accidental ingestion causes severe gastrointestinal symptoms
- Strong oxidant, do not mix with acetic acid, ammonium hydroxide, ethanol, ethylene glycol, formaldehyde, glycerol, HCl, hydrogen peroxide, sulfurix acid
Propylene Glycol
- Less toxic substitute for ethylene-based ethers Silver Salts
- Safe when used in fresh solution - Explosive when old
- Iriitates skin & eyes
- Causes severe gastrointestinal discomfort if ingested
- Serious environmental hazard - Do not subject to drain disposal Sodium Azide
- Toxic
- Fatal when swallowed/ absorbed through skin/ mixed with acids
- Can explode when in contact with metals - Do not subject to drain disposal
Sodium Bisulfate
- Safe when used in dilute solutions - Keep away from oxidants
Sodium Hypoclorite - Liquid chlorine bleach - Strong oxidant - Eye irritant
- Corrosive to most metals
- Do not mix with formaldehyde/ DAB Sodium Iodate
- Used to replace mercuric oxide when reconstituting Harris hematoxylin
- Little risk when used in laboratory quantities Sodium Thiosulfate
- Minimal health risk when used in histology under normal conditions
Sulfuric Acid
- Strong skin, eyes & RT irritant - Corrosive to most metals - Dilute solutions are safe
- Concentrated solutions produces fumes that are dangerous, requires use of fume hood, apron, goggles, gloves
Toluene
- Skin & eye irritant
- Repeated exposure can cause impaired memory, poor coordination, mood swings & permanent nerve damage
- Should be restricted/ avoided
- Diluent in mounting media for removing coverslips Xylene
- Same risk as toluene Zinc Chloride
- Corrosive to most metal including stainless steel - Do not use in tissue processors
- Skin & eye irritant
- Causes severe gastrointestinal problems when ingested
TYPES OF MICROSCOPE 1. Bright Field Microscope
o Most common type of light microscope o Can view stained/unstained specimen
o Best for stained/naturally pigmented specimen for tissue section/ living photosynthetic
organism
2. Phase Contrast Microscope o For colorless/transparent
o Difficult to distinguish from their surrounding Cell parts of protozoan
Bacteria Sperm trails
Other types of unstainable cells 3. Polarizing Microscope
o For studying rocks and minerals under polarized light
Crystals in urine Biochemistry Biomedical research 4. Fluorescence Microscope
o Use fluorochrome to stain microorganism o Thru excitation of fluorescent dye by light o Light: Xenon arc lamp/ Mercury-vapor lamp
Properties of organic/inorganic substance 5. Dark Field Microscope
o Objects are illuminated at very low angle from the side
o Background is dark
Unstained microorganism suspended in liquid
Living microorganism that are invisible in ordinary light
6. Dissecting Microscope
Surface of solid specimen
Carry out close work such as sorting, dissection, microsurgery, watch making, small circuit manufacturer/inspection 7. Electron Microscope
o Uses electrons
o Can view as small as 0.2 micron (highest magnification: 100x) Virus Fungi Microsporidian parasite MICROTOME 1. Rotary Microtome o Highest performance
o For manual/motorized sectioning of biological specimen
Parts
o Coarse advance hand wheel
Turning the wheel in opposite direction turns the object away from the knife edge For rapid and simple horizontal movement
of specimen arm
Autotrim feature: control specimen arm advance
o Sliding clutch or coarse advance
Prevents continued horizontal movement of specimen arm
o Fine advance wheel
When turned 3600 it provides vertical movement of the specimen arm past the knife area
Compensates for the distance of the retraction plus thickness on the fine advance
o Hand wheel locking device Safety lock
o Fine advance micrometer dial/ thickness scale Sets desired thickness of tissue ribbon o LD information display
Read out display
Shows the number of revolution of the hand wheel
o Object clamps and adapters Specimen holder o Object orientation adapter
Part A: attaches to the specimen arm Part B: attaches to the back of object clamp o Knife holder
o Knife holder base unit o Clearance angle adjustment o Handpad
Power on/off switch Emergency stop button
Speed control (0-150 cutting stroke/min) 2. Freezing Microtome
o For cutting thin to semi-thin fresh frozen tissue o Freeze using liquid CO2 or ↓ temperature
recirculating coolant 3. Rocking Microtome
o 2-24 microns in steps of 2 microns each 4. Sledge Microtome
o For cutting large locks of paraffin and resin embedded
o For large and hard blocks 5. Sliding Microtome
o Optimal stability
o For hard and large blocks (>80 x 60 mm) 6. Cryostat
o Cold microtome
o Within ref chamber with glass window o -200C
7. Ultrathin Microtome
o Can cut up to 0.5 micron o For electron microscopy
o Knife: fragments of broken plate glass MICROTOME KNIVES
- For trimming and section cutting - Knives bevel angle: 27-320 1. Plane concave knife
o 25 mm
o One side is flat, the other is concave 2. Biconcave knife
o For paraffin embedded sections on rotary microtome
o Both side are biconcave 3. Plane wedge knife
o Both sides are straight
o For frozen sections/ cutting hard and tough specimen embedded in paraffin
o For base sledge and sliding microtome o Cutting angle/ clearance angle: 150
Lesser compression on block HONING AND STROPPING
- Sharpening of badly nicked knives to ensure optimum sectioning of tissue blocks
A. Honing
- Removal of gross nicks on the knife edge (coarse honing)
- Cutting the edge of the knife on a stone (honing proper)
Types of Hone 1. Belgium yellow
o For manual sharpening when cutting edge has been rendered blunt/nicked
2. Arkansas
o More polishing effect than Belgium yellow 3. Fine carborundum
o Much more coarser than the two o Used only for badly nicked knives
**Hone is wiped clean with xylene then coarsed with thin film of: (10-20 strokes)
Mineral and clove oil Xylene
Liquid paraffin/ soapy water (for lubrication)
Procedure
1. Fit knife to corresponding knife back (maintain angle, hold knife)
2. One end of the hone, knife heel first
3. Draw obliquely/ diagonally towards operator until toe is reached
4. Turn over, repeat 1-3 **Edge first, Heel to toe
**Plane concave: only concave side should be rubbed on the hone
Precautions
Hone: 8” x 3” to accommodate length of knife edge
Hone should be lubricated
Pressure should be gentle and steady Hone should be clean
After honing, wipe off the oil or soap from the knife with xylene
B. Stropping
- “burr” formed during honing is removed & cutting edge of knife is polished
- Knife is stropped before every object is sectioned - Paddle strop made up of horse leather (attached to
slid back)
- Toe to heel direction - 40-120 strokes Precautions
Oil/grease to prevent rusting Use light pressure
Speed should be avoided
Leather strops are dry, requires oiling before use (vegetable or castor oil)
Should be used for at least 24-48 hours after oiling
Do not use mineral oil or wax Disposable blades
- Common
- Cheaper conventional steel knives - Can cut up to 2-4 micron thick sections Glass knives
- For trimming and semi thin sectioning - For tissue blocks for electron microscopy - Can cut up to 40 x 2.5 cm, plate glass strip - Cracked to form 25 x 25 mm square
- Broken down to two triangular shape knives Diamond knives
- For cutting resin block for electron microscopy - Brittle and expensive
OTHER EQUIPMENTS USED IN HISTOPATHOLOGY 1. Paraffin oven
o “wax oven”
o Maintain temperature of 2-50C above melting point of wax (routinely 560C)
o Store paraffin in its liquid form o Allow section to dry
2. Hot plate
o For delicate tissues such as brain, lower drying point is used to avoid splitting and cracking of the section
o Prone to over heating. Tissue is exposed o Paraffin oven is preffered for drying 3. Floatation waterbath
o 5-100C below melting point of the paraffin wax (45-500C)
o Fish out in less than 30 seconds to prevent tissue morphology distortion
o Add 20% ethanol/ detergent for easy fishing out
o Can hold 2L water 4. Slides
o 76 x 25 mm, 1-1.2 mm thick o Frosted edge slide
o Label with diamond pencil 5. Coplin Jar
o Wide mouthed glass jars o Vertically grooved interior walls
o Used for storage/ staining of slides containing blood smears or tissue section
6. Tissue cassettes & embedding molds a. Leukhart’s embedding mold
2 L shaped strips of heavy brass/metal arranged on a flat metal surface b. Compound embedding unit
Made up of series of interlocking plates resting on flat metal base
c. Plastic embedding rings and base mold
Consist of special stainless steel base mold fitted with plastic embedding ring
d. Disposable embedding mold
Peel away disposable thin plastic embedding mold (perfect even without trimming)
Plastic ice trays (for busy routine lab) Paper boat (for colloid blocks)
Other Embedding Methods: 1. Celloidin/ Nitrocellulose
o Use to be recommended for embedding hard tissues such as bones, teeth and for large sections
2. Double embedding method
o Process in which tissues are first infiltrated with celloidin and then embedded in paraffin mass 3. Resin embedding
o For electron microscopy
o For undecalcified bone and for high resolution light microscopy of tissue sections thinner than usual 2-6um such as renal biopsy
4. Plastics: Epoxy, polyester, acrylic a. Epoxy
Epoxy plastic, catalysts + accelerators Hydrophobic
Reduce antigenicity with VCD (vinylcyclohexane dioxide) Carcinogenic
Bisphenol A (Arab elite) Glycerol (Epon)
Cyclohexane dioxide (spurr) b. Polyester plastic
For EM
c. Acrylic plastic
Esters of acrylic or methacrylic acid Used for LM
GMA (Polyglycol methacrylate) MMA (Methyl methacrylate)
TISSUE EMBEDDING CENTER - Leica EG1160
- Compact bench top TEC which enables the user to produce paraffin embedded tissues that can later be successfully sectioned with ease
- Features digital program interface for individual temperature setting for the paraffin reservoir, cassette bath, mold warmer and work surface Essential Parts
1. Refrigerating system 2. Paraffin melting chamber
3. Microscreen (filters particle/sediments) 4. Hot and cold orientation platforms 5. Waste drawer
6. Hot well (for preheating forceps) Components and Features
1. Paraffin reservoir o Holds 3L paraffin
o 45-700C (paraffin liquid temp) 2. Paraffin dispenser with illumination
o Dispenser is separately heated and always has the same temp as paraffin reservoir
o Dispenser handle is used for manually operating the paraffin flow with a dispenser handle and extension clamp
3. Mold warmer o 33-700C 4. Cassette bath
o 45-700C
o Can hold more than 100 cassettes 5. Cold plate
o -50C
o Optimal consistency of the blocks minimizes the risk of brittleness as a result of rapid cooling and high level of productivity 6. Refrigeration spot
o Integrated in the cold plate ensuring consistent low temp
o Mold containing the sample filled with liquid paraffin (1/3) are placed in refrigeration spot to allow partial solidifying
7. Paraffin collecting tray
o Located under the heated work area to collect excess paraffin derived from surface
8. Work area o 45-700C
o Embedding area, forceps holder, recessed area for cassettes and space to reove the lids o Forceps holder is separately heated
Examination may be done on fresh or preserved tissues depending upon necessity.
Methods of Fresh tissue examination
1. Teasing or Dissociation
- Is a process whereby a selected tissue specimen is immersed in a watch glass containing isotonic salt solution, carefully dissected or separated
2. Squash Preparation (Crushing)
- Process whereby small pieces of tissue not more than 1mm in diameter are places in a microscopic slide and forcibly compressed with another slide.
3. Smear preparation
- Is the process of examining sections or sediments whereby cellular materials are spread lightly over slide by means of a wire loop or applicator or by making a apposition smear with another slide.
o Streaking – w/ an applicator stick or a platinum loop, the material is rapidly and gently applied in a direct or zigzag line throughout the slide
o Spreading – a selected portion of the material is transferred to a clean slide and gently spread into a moderately thick film by teasing the mucous apart with an applicator stick
o Pull Apart – done by placing a drop of secretion or sediment upon slide and facing it to another clean slide o Touch Preparation (impression
smear) – whereby the surface of a freshly cut piece of tissue is brought into contact and perused on to the surface of a clean slide
4. Frozen Section
- This method is normally utilized when a rapid diagnosis of the tissue in question is required and is especially recommended when lipids and nervous tissue elements are to be demonstrated.
o Temperature: -10 to -20 C
o Applications in histotechnology: Rapid pathologic diagnosis
during surgery
Diagnostic and research enzyme histochemistry Demonstration of soluble
substances such as lipids and carbohydrates
Immunofluorescent and immunohistochemical staining Some specialized sliver stains ,
particularly in neuropathology o More commonly used methods for
freezing:
Liquid nitrogen – most rapid freezing agent
Formation of vapor phase (uneven pulling of tissue
Not done in muscle biopsy
Isopentane - cooled by liquid nitrogen – for muscle biopsies Carbon dioxide gas –
freezing microtome, cylinder Aerosol sprays – widely used PROCESSING OF TISSUES 1. Fixation 2. Dehydration 3. Clearing 4. Infiltration (Impregnation) 5. Embedding 6. Trimming 7. Section-Cutting 8. Staining 9. Mounting 10. Labeling
FIXATION AND FIXATIVES Fixation – the most and critical step in
histotechnology involves fixing or preserving fresh tissue for examination.
- Aim:
o To preserve the morphologic and chemical integrity of the cell in as life like a manner as possible.
o To harden and protect the tissue from the trauma of further handling. - Fixatives have the property of forming cross
links between proteins.
- Soluble proteins are fixed to structural proteins and thus rendered insoluble.
Fixation Prevents: - Degeneration - Decomposition - Putrefaction
- Distortion of tissues
Two Basic Mechanisms in Fixation: 1. Additive Fixation
- Chemical constituent of fixative becomes part of the tissues by forming cross-links (ex.formalin, mercury, and osmium tetroxide)
2. Non Additive Fixation
- Not incorporated but alters the composition of tissues by removing the bound water attached to H bonds (ex. Alcoholic fixative)
Main factors involved in fixation: 1. Hydrogen ion concentration
o pH: 6 to 8 2. Temperature
o room temperature
o electron microscopy and histochemistry: 0-40oC o Rapid Fixation: 60oC
o Tissue with tuberculosis: 100oC 3. Thickness of section
o 1 to 2 mm2 – electron microscope o 2cm2 – light microscope
4. Osmolality
o Hypertonic solutions: cell shrinkage o Isotonic solutions: cell swelling
o 400-40 mOsm (recommended)
o Sucrose is commonly added to osmium tetroxide fixatives in electron
microscope 5. Concentration
o Formaldehyde: 10% o Glutaraldehyde :3%
o Glutaraldehyde: (0.25) ideal for immune-electron microscopy 6. Duration of fixation
o Primary fixation in buffered formalin is usually carried out for 2-6 hours but can remain in fixative over the weekend
o Electron microscopy: 3 hours then placed in a holding buffer
Practical consideration of Fixation: 1. Speed – specimen should be fixed
immediately
2. Penetration – formalin diffuses into the tissues at the rate of 1mm per hour 3. Volume – 10-25 times the volume
4. Duration of fixation – fibrous organs take longer fixation
*fixation time can be cut down by using heat, vacuum, agitation or microwave
Types of fixative ACCORDING TO COMPOSITION 1. Simple Fixatives – are made up of only one
component substance a. Aldehydes b. Metallic Fixatives
i. Mercuric chloride ii. Chromate fixatives iii. Lead fixatives iv. Heat
- Formalin – A gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37-40% weight in volume
o Commonly used as a 4% solution, giving 10% formalin for tissue fixation o Buffered to pH 7 with phosphate buffer. - 10% Formol Saline – saturated formaldehyde
(40% by weight volume diluted to 10% sodium chloride)
o For central nervous tissues and general post mortem tissues for histochemical staining
- 10% Neutral Buffered Formalin or Phosphate buffered formalin (pH7)
o Prevents precipitation of acid formalin pigments
o Recommended for preservation and storage of surgical, post mortem and research specimens.
Preparation:
Sodium Dihydrogen phosphate (anhydrous) 3.5gm
Disodium hydrogen phosphate (anhydrous) 6.5gm
Formaldehyde 40% 100ml
Distilled h2o 900ml
- Formal corrosive (formal sublimate) o Formol mercuric chloride solution for
routine post mortem - Gendre’s Fixative
o Alcoholic formalin
- Glutaraldehyde 2 formaldehyde residues linked by 3 carbon chain
o 2-5% for small tissue fragments and needle biopsies fixed in 2-4hrs at room tempt.
o 4% for large tissues less than 4mm thick in 6-8 hrs up to 24hrs
o Especially used for central nervous tissues
2. Compound fixatives- are those that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.
Type of fixative ACCORDING TO ACTION
1. Microanatomical fixatives – are those that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.
2. Cytological fixatives – are those that preserve specific parts and particular microscopic elements of the cell itself.
DECALCIFICATION
- Is it a process whereby calcium or lime salts are removed from tissues (most especially bone and teeth: tuberculous organs and arteriosclerotic vessels).
- It should be done after fixation
- Calcium may be removed by the following: o Acid
o Chelating agents o Ion exchange resins
o Electrical ionization (electrophoresis) ACID DECALCIFYING AGENTS - They are the most widely used agents for
routine decalcification of large amounts of bony tissues because they are stable, easily available and relatively inexpensive.
1. NITRIC ACID
This is the most common and the fastest decalcifying agent used so far
Recommended concentration: 5-10%
Disadvantage: inhibiting nuclear stains and destroying tissues especially in
concentrated solutions. A. Aqueous Nitric Add solution
10%
Decalcification time: 12 – 24 hours
B. Formol-Nitric Acid
C. Perenyi’s fluid
Decalcification time: 2 – 7 days D. Phloroglucin-Nitric Acid
Decalcification time: 12 – 24 hours
2. HYDROCHLORIC ACID
Is inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue.
It will produce good nuclear staining and if used in 1% solution with 70% alcohol may be recommended for surface decalcification of the tissue blocks.
3. FORMIC ACID
Is a moderate acting decalcifying agent which produces better nuclear staining
It is recommended for routine decalcification of postmortem research tissues
Formula: Formic acid (SG: 1.20) – 10ml / Formal saline 10% - 90ml
Decalcification time: 2 – 7 days A. Formic Acid-Sodium Citrate
Solution
Formula:
Aqueous sodium citrate 20% - 50 ml Formic Acid 45% - 50 ml Decalcification time: 3 – 14 days 4. TRICHLOROACETIC ACID Formula: Trichloroacetic acid – 5 g Formol saline 10 % - 95 ml
Decalcification time: 4 – 8 days CHELATING AGENTS
- Substances which combine with calcium ions and other salts
- The most common chelating agents: EDTA - Tissue is placed in EDTA from 1 – 3 weeks for
small specimens, but it may take 6 – 8 weeks or longer to totally decalcify dense cortical bone.
- pH : 7 – 7.4 - Formula:
o EDTA disodium salt – 5.5 gm o Distilled water – 90 ml o Formaldehyde – 10 ml
DEHYDRATION
- Process or removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation.
- Dehydrating agents are alcohols of various types that are generally used in increasing strengths to remove aqueous tissue fluids with little disruption to the tissue
- Commonly used dehydrating agents: o Alcohol o Acetone o Dioxane 4- cellosolve o Triethyl phosphate o Tetrahydrofuran A. Alcohol
o Ethyl alcohol is the alcohol
recommended for routine dehydration of tissues.
o It is a clear, colorless and flammable fluid
o Fast acting
o Methyl Alcohol is a toxic dehydrating agent, primarily employed for blood and tissue films and for smear preparations
o Butyl alcohol is utilized in plant and animal micro-techniques, Slow dehydrating agent
o Temperature: 37C will hasten dehydration
B. Acetone
o It is a cheap, rapid acting dehydrating agent utilized for most urgent biopsies which it dehydrates in ½ to 2 hours. o Limited only to small pieces of tissues
due to its extreme volatility and inflammability
C. Dioxane
o Is an excellent dehydrating and clearing agent readily miscible in water, melted paraffin, alcohol and xylol.
o Ribbon poorly D. Cellosolve
o It dehydrates rapidly
o Ethylene glycol ethers are combustible ar 110 – 120F
o Toxic in inhalation, skin contact, and ingestion
E. Triethyl phosphate
o It is soluble in alcohol, water, ether, benzene, chloroform, acetone and xylene
F. Tetrahydrofuran
o It both dehydrates and clears tissue since it is miscible in both water and paraffin.
o May cause conjunctival irritation *when 4% phenol is added to each 95% ethanol baths part of dehydration process, it acts as a softener for hard tissues such as tendon, nail, dense fibrous tissue.
CLEARING
- Is the process whereby alcohol or a
and replaced with a substance that will dissolve the wax.
- It is should be miscible also with paraffin in order to facilitate the penetration of this embedding medium.
A. Xylene
o Is colorless clearing agent that is commonly used in histology lab. o Clearing time is usually ½ to 1 hour. B. Toluene
o Used as substitute to xylene or benzene
o Clearing time: 1-2 hours C. Benzene
o Is preferred by some as clearing agent in embedding process of tissues because it penetrates and clears tissues rapidly.
o It is rapid acting (15 -30 minutes) D. Chloroform
o Causes less brittleness
o Thicker tissue blocks, even those up to 1cm can be processed.
o Tissues placed in chloroform do not become translucent
E. Cedarwood oil
o Used to clear both paraffin and celloidin sections during embedding process.
o It is especially recommended for central nervous system and cytological studies.
o Clearing time: 2 – 3 days IMPREGNATION AND EMBEDDING - Impregnation (Infiltration): is the process
whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill the tissue cavities
- Embedding (Casting or Blocking): is the process by whichh the impregnated tissue is places into a precisely arranged position in a mold containing a medium which is then allowed to solidify.
4 general types of tissue impregnation and embedding medium:
1. Paraffin wax - rotary
2. Celloidin – sledge microtome 3. Gelatin
4. Plastic
PARAFFIN WAX IMPREGNATION - Paraffin: is the simplest, most common and
best embedding medium used for routine tissue processing.
- Disadvantages:
o Prolonged impregnation can cause excessive tissue shrinkage
o Paraffin must be free from dust, water droplets
- 56C – is the normally used for routine work - Three ways by which paraffin wax
impregnation and embedding of tissues may be performed:
o By manual Processing
o By automatic processing o By vacuum embedding - MANUAL PROCESSING
o At least four changes of wax are required at 15 minutes intervals in order to insure complete removal of the clearing agent from the tissue.
o Immersed the specimen for another 3 hours in the paraffin wax to insure complete embedding or casting of tissue. o FIXATION 10% buffered formalin – 24hrs o DEHYDRATION 70% Alcohol- 6 hours 95% alcohol – 12 hours 100% alcohol- 2 hours 100% alcohol- 1 hour 100% alcohol- 1 hour o CLEARING
Xylene/ Toluene- 1 hour Xylene/Toluene- 1 hour o IMPREGNATION
Paraffin Wax- 15 minutes Paraffin Wax- 15 minutes Paraffin Wax- 15 minutes Paraffin Wax- 15 minutes o EMBEDDING
Paraffin Wax- 3 hours - AUTOMATIC PROCESSING
o Only 2 or 3 changes of paraffin wax are required to remove the clearing agent and properly impregnate the specimen - VACUUM EMBEDDING
o Involves the wax impregnation under negative atmospheric pressure inside an embedding oven to hasten removal of air bubbles and clearing agent from the tissue block thereby promoting a more rapid wax penetration of tissue. o Prevent: brittleness, shrinkage and
hardening of tissues. EMBEDDING
- Orientation : is the 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. Several types of Blocking out Molds:
1. Leuckhart’s Embedding Mold- consists of L shaped strips of heavy brass or metal arranged on flat metal plate and which can be moved to adjust the size of the mold to the size of specimen.
2. Compound Embedding unit – is made up of a series of interlocking plates resting on a flat metal base
3. Plastic Embedding Rings and Base Mold – consists of a special stainless steel base mold fitted with a plastic embedding ring.
4. Disposable Embedding Mold
a. Peel Away- disposable thin plastic embedding molds available in 3 different sizes. Giving perfect block even without trimming
c. Paper boats – are normally utilized for embedding celloidin blocks but are equally for paraffin wax blocks.
Other embedding methods:
1. Celloidin or Nitrocellulose Method o Used to be recommended for
embedding hard tissues such as bones, teeth and for large sections of whole organs.
2. Double Embedding Method
o Is the process in which tissues are first infiltrated with celloidin and
subsequently embedded in paraffin mass.
3. Plastic or Resin Embedding o Used in hard tissues such as
undecalcified bone and for high resolution light microscopy of tissue sections thinner than usual 4- 6 um. o Plastic are classified as: epoxy,
polyester and acrylic
o EPOXY: are made up of a carefully balanced mixture of epoxy plastic, catalysts and accelerators.
3 types: Bisphenol A (araldite) Glycerol (Epon) Cyclohexene dioxide (spurr) Disadvantages: Hydrophobic Reduce antigenicity Compromise the result
of
immunohistochemistry staining
Vinylcyclohexane dioxide – carcinogenic
o POLYESTER: were originally introduced for electron microscopy.
o ACRYLIC PLASTICS: are made up of esters of acrylic of methacrylic acid.
Used extensively for light microscopy
Polyglycol methacrylate (GMA) *hydrophilic* and Methyl Methacrylate – widely used because of its hardness as the ideal embedding medium for undecalcified bone
