HISTOPATHOLO

LABORATORY: RISK MANAGEMENT AND SAFETY IN HISTOPATH LABORATORY

CENTRAL LUZON DOCTORS’ HOSPITAL EDUCATIONAL INSTITUTION – LACAYANGA PMS
CHAPTER 1 Toluene
 Skin and eye irritant
RISK MANAGEMENT AND SAFETY IN  Repeated exposure can cause impaired memory,
HISTOPATH LABORATORY poor coordination, mood swing and permanent
nerve damage.
 Identify the hazards
 SOP - control of hazardous substances, risk Xylene
assessment, and other health safety information.  same as toluene
 properly maintained equipment
 quality reagents Methanol
 moderate skin and eye irritant
HAZARDOUS CHEMICALS  Causes blindness and death in excessive amount

Storage: all chemicals can be safety stored in conventional
cabinet Note: dilution : acid to water not water to acid to prevent
splashing
Dangerous liquids
DEFINITION OF TERMS
Storage: stored below countertop height to minimize the risk
of bodily exposure in case bottle is dropped and broken. TISSUE PROCESSING
- Tissue from the body was taken for diagnosis of
Flammable liquid: disease processes must be processed in the
Storage: do not store refrigerator or freezer unless it is histopathology laboratory to produce microscopic
certified as suitable for an explosive atmosphere . Small slides that are viewed by pathologist.
quantity must be made available and do not store any left
over. HISTOTECHNOLOGY
- Is the art and science performed by a
HAZARDOUS CHEMICAL EXAMPLES histotechnologist to produce a tissue section of
good quality that will enable to diagnose the
Acetic acid presence and absence of diseases.

 Concentrated can cause skin eye and respiratory SPECIMEN ACCESSIONING

infection - Request form
 1-10% concentration is safe - Specimen Number

Ammonium hydroxide GROSS EXAMINATION

 Do not mix with formaldehyde because it will
generate heat and vapors that can irritate the
respiratory system
Aniline
 Toxic to the skin and causes severe irritation of the
eyes
- Tissue for diagnosis is examined by the pathologist,
pathology assistant or pathology resident. Consist
of describing the specimen and placing all parts in
a tissue cassette which holds the tissue while being
processed.

Chloroform
 dangerous when inhaled or ingested
 Excessive exposure to vapor causes disorientation,
lost of consciousness and death

Ethanol
TWO TYPES OF TISSUE EXAMINATION
 skin and eye irritant
1. PRESERVED EXAMINATION
Ether
- Better and more effective means of studying tissues
 causes mild to moderate irritation of eye and skin
whether normal and abnormal.
 Flammable and votile - Examination of sections and smears which have
 Causes disorientation, loss of consciousness and been permanently preserved and stained for
death demonstration of specific structures.
Formaldehyde 2. FRESH TISSUE EXAMINATION
 Cause severe irritation of skin and eye - Done when there is immediate need of evaluation
 Carcinogenic and corrosive.
 - Isotonic and Hypotonic solution: cell swelling and
poor fixation
PRESERVED TISSUE EXAMINATION /
PROCESSING - Best: slightly hypertonic solution (400-450 mOsm)
5. Concentration
Solid structures and tissues must be preserved and carefully - Formaldehyde: 10% solution
processed in the following orders: - Glutaraldehyde: 3% solution (immunoelectron
microscopy: 0.25% solution)
1. Fixation 6. Duration
2. Dehydration - Buffered Formalin: 2 – 6 hours
3. Clearing - Formalin: 24 hours
4. Infiltration / Impregnation - Electron microscopy: 3 hours
5. Embedding
6. Trimming PRACTICAL CONSIDERATIONS OF FIXATION
7. Section cutting
8. Staining 1. Speed
9. Mounting - Asap, specimen should be placed in the fixative as
10. Labeling soon as possible
- To prevent autolysis and putrefaction
FIXATION 2. Penetration
- Formalin diffused in to the tissue at the rate of
- Most critical step in histotechnology. approximately 1mm per hour
- Slows down as it goes deeper.
Primary aim: To preserve the morphologic and chemical 3. Volume
integrity of the cell in as a life-like manner, while preventing - Traditionally fixative is 10-25k the tissue volume
degeneration, decomposition, putrefaction, and distortion of - Maximum effectiveness: 20x the tissue volume.
tissues after removal to the body. 4. Duration
- Some tissue takes longer to fix that others;
Secondary aim: Harden and protect the tissue from the depending on the structure
trauma of further handling for it to be easier to cut during
gross examination. EFFECTS OF FIXATIVES IN GENERAL

2 MECHANISMS INVOLVED IN FIXATION 1. Harden soft and friable tissue.

2. Make cells resistant to damage and distortion.
1.
Additive fixation 3. Inhibit Bacterial decomposition.
- A chemical constituent of fixative is taken 4. Increase the optical differentiation of cells and
in ard becomes part of the tissue by tissue components
forming crosslinks and giving stability to 5. Act as a mordant to promote staining.
protein. 6. Reduce the risk of infections during handling and
2. Non – additive fixation processing
- Fixing agent is not incorporated into the
tissue.
- Stabilize tissue by means of removing
water
- Cross-links are formed and stabilize the
intracellular component, prevent autolysis
and make cells unsuitable for bacterial
decomposition.
FACTORS INVOLVED IN FIXATION CHARACTERISTICS OF GOOD FRATIVE

1. Hydrogen ion concentration 1. Cheap

- Satisfactory fixation occurs between pH 6 to 8.
2. Temperature
- Surgical specimen - room temperature – 37C
- Electron microscopy and histochemistry – 0-4C
- Nucleic acids do not react with fixative at room
temperature.
3. Thickness of section
- Tissue blocks: Should be either small (1-2 mrn2 =>
electron microscopy, 2cm2 => light microscopy) or
thin (not more than 0.4cm => light microscopy or
as prescribed by tissue process or manufacturer
4. Osmolality
- Hypertonic solution: cell shrinkage
2. Stable
3. Safe to handle
4. Kill the cell quickly thereby producing minimum
distortion of cell constituents
5. Inhibit bacterial decomposition and autolysis.
6. Produce minimum shrinkage of the cell.
7. Permit rapid and even penetration of tissue.
8. Harden tissue thereby making the cutting of tissue
easier.
9. Isotonic and causes minimal physical and chemical
alteration of the cell and their constituents.
10. Make cellular component insoluble to hypotonic
solutions
11. Permit the subsequent application of many staining - Preserve cytoplasmic structures of the cell
procedures - Should not contain Glacial Acetic Acid
because it destroy mitochondria and Golgi
bodies of the cytoplasm
TYPES OF FIXATIVE ACCORDING TO - pH more than 4.6
COMPOSITION AND ACTION a. Flemming’s fluid without acetic acid
b.Kelly’s fluid
c. Formalin with “Post – chroming”
ACCORDING TO COMPOSITION
d.Regaud’s Fluid (Muller’s Fluid)
e. Orth’s Fluid
A. Simple Fixative
3. Histochemical Fixatives
1. Aldehydes - Preserve the chemical constituents of cell
a. Formaldehyde a. Formol saline 10%
 10% Formol saline b. Absolute Ethyl Alcohol
 10% Neutral Buffered Formalin c. Acetone
 Alcoholic Formalin d. Newcomer’s Fluid
b. Glutaraldehyde
MIXTURE OF FIXATIVES
2. Metallic fixative
a. Mercuric chloride  Two (2) aldehyde fixative mixtures have been
b. Chromate fixative particularly useful for electron cytochemistry.
 Potassium dichromate  Karsvovsky's Paraformaldehyde- Glutaraldehyde
 Chromic acid
c. Lead Fixatives ALDEHYDES FIXATIVES
 Picric acid
 Acetic acid Satisfactory for routine paraffin sections, for electron
 Acetone microscopy and histochemical and enzymes studies.
 Alcohol
 Osmium Tetroxide (Osmic Acid) FORMALDEHYDE (FORMALIN)
d. Heat
 10% Formalin: Most widely use
 Pure stock is unsatisfactory for fixation.
B. Compound Fixative  Diluted to 1:10 or 1:20 to make 10% or 5%
solution
ACCORDING TO ACTION  Fixation time:
 Recommended for nervous tissue preservation.
A. Microanatomical Fixatives
- permit the general study microscopic study of
tissue structures
ADVANTAGES:
a. 10% Formol Saline
b. 10% Neutral Buffered Formalin 1. Cheap, readily available, easy to prepare, stable if
c. Heidenhain's Susa stored in buffered solutions.
d. Formol Sublimate 2. Compatible with many stains.
e. Zenker's Solution 3. Does not overharden tissues. Even with prolonged
f. Zenker-Formol (Kelly's Solution) periods as long as solution is regularly changed.
g. Bouin's Solution 4. Penetrates tissue well.
h. Brasil's Solution 5. Preserves fat and mucin.
6. Preserves glycogen.
B. Cytologic Fixative 7. Preserves but does not precipitate protein.
- preserve specific parts and particular microscopic 8. Restored natural tissue color.
elements of the cell. 9. Allows frozen tissue section to be prepared easily.
10. Does not required washing out.
1. Nuclear Fixatives 11. Tolerant
- Preserves the nuclear structure of the cell
- Primary Component: Glacial Acetic Acid- has DISADVANTAGES:
affinity to nuclear chromatin
- pH: 4.6 or less 1. Fumes are irritated in the eyes and nose
a. Flemming's Fluid 2. Solution is irritating in the skin.
b. Carnoy's Fluid 3. Produce considerable shrinkage of tissue.
c. Bouin's Fluid 4. Do not harden some cytoplasmic structure
d. Newcomer's Fluid 5. Unbuffered
e. Heldenhan's Susa o Reduces the quality of routine cytologic
staining both basophil and eosinophilic
2. Cytoplasmic fixatives staining of cells.
 o Forms abundant brown pigment granules.
6. Prolonged Fixation may produce:
o Bleaching of specimen.
o Dispersal of fat from the tissue to the fluid.
o Loss of glycogen.
STORAGE:
 Storage at very low temperature induced
precipitation of white formaldehyde deposits and
produces turbidity.
Glacial Acetic Acid 5 mL
A2. ZENKER'S FORMOL (HELLY' S SOLUTION)
 Excellent microanatomical fixative for pituitary
gland, bone marrow, and blood containing organs
like spleen and liver.
 Preserves cytoplasmic granules well.
 Fixation Time: 12-24 hours
A3. HEIDENHAIN'S SUSA SOLUTION

 Recommended for tumor biopsies especially in

8. 10 % FORMOL-SALINE the skin
 Excellent cytologic fixative.
 Simple microanatomical fixative  Fixation Time: 3-12 hours
 Made up of saturated formaldehyde diluted to
10% with sodium chloride.
 Recommended for central nervous system tissues A4. B-5 FIXATIVE
and general post-mortem tissues.  Commonly use for bone marrow biopsies
 Preserves microanatomic and cytologic details  Fixation Time: ½ - 2 hours
with minimum shrinkage and distortion.
 Preserves enzymes and nucleoproteins.
CHROMATE FIXATIVES
Formula:
B1. CHROMIC ACID
40% Formaldehyde 100 mL
Distilled water  Used in 1-2% aqueous solution.
NaCl 9g  Precipitates protein and adequately preserves
carbohydrates.
Fixation Time:  Strong oxidizing agent, add strong reducing agent
(formaldehyde) before use to prevent counter
 24 hours at 35°C effects.
 48 hours at 20-25°C B2. POTASSIUM D CHROMATE

10% NEUTRAL BUFFERED FORMALIN OR  Used in 3% aqueous solution.

PHOSPHATE BUFFERED FORMALIN  Preserves lipid.
 Preserves mitochondria if used in pH: 4.5 -
 pH: 7 5.2, acidified: destroys mitochondria
 Recommended for storage of surgical, post
mortem and research specimen. B3. REGARD'S (MULLER'S FLUID)
 Best fixative for tissues containing iron pigments
 Hardens tissue better and more rapidly than Orth’s
and for elastic fibers.
Fluid
 Time consuming to prepare
 Recommended for demonstration of chromatin,
mitochondria, mitotic figures, golgi bodies, RBC
Formula
and colloid-containing tissues.
10 % General Neutral buffered  Blacken tissue in prolonged fixation
Formalin  Fixation time: 12 – 48 hours

Sodium Dihydrogen Phosphate 3.5g B4. ORTH’S FLUID

 Recommended for study of early degenerative

processes and tissue necrosis
A1. ZENKER'S FLUID  Demonstrate ricketssiae and other bacteria
 Preserves myelin better than buffered formalin
 Made up of the mercuric chloride stock solution  Fixation time: 36 – 72 hours
with glacial acetic acid
 add glacial acetic acid before using to prevent LEAD FIXATIVES
turbidity and formation of dark precipitate.
 Recommended for trichrome staining.  Used in 4% aqueous solution of basic lead acetate.
 Fixation Time: 12-24 hours  Recommended for acid mucopolysaccharides
 Fixes connective tissues mucin.
Formula:
Mercuric Chloride Solution 95 mL PICRIC ACID FIXATIVES
  Excellent fixative for glycogen demonstration.
 Stable and precipitates all proteins.
 Highly explosive when dry.

BOUIN'S SOLUTION

 Recommended for embryos and pituitary biopsies.

 Not suitable for kidney structures
 Fixation Time: 6-24 hours
 Formula:
Saturated solution of 75 mL
Picric Acid
40% strong 25 mL
formaldehyde
Glacial Acetic Acid 5mL

Shrinkage effect of picric acid is balanced by the

swelling effect of glacial acetic acid.
BRASIL'S ALCOHOLIC PICROFORMOL
FIXATIVE
 Better and less messy than Bouin's solution.

GLACIAL ACETIC ACID

 Used in conjunction with other fixatives to form a

compound solution.
 Solidifies at 17 C.
 Causes tissue to swell