1|Page Rachelle Mae F.

Ferranco, RMT

DEFINITION OF HISTOTECHNOLOGY / De: Dental appliances and teeth with no attached soft tissue TAKE NOTE: Terminologies and
HISTOPATHOLOGIC TECHNIQUES S: Saphenous vein segments harvested for coronary artery bypass. sentences marked with are
P: Placentas that do not meet institutionally specified criteria for examination.
R: Rib segments or other tissues if and only if the patient has no malignancy unexpected questions from Dr.
HISTOPATHOLOGIC TECNIQUE: I: Intrauterine contraceptive devices without attached soft tissue Liwanag’s quiz / exam. Hope it could
It is an art of science performed by medical technologists to produces good quality N: Normal toenails and fingernails that are incidentally removed. help you CEU peeps.
tissue sections that will enable the pathologists to come up with accurate diagnosis. T: Therapeutic radioactive sources.
S: Skin or other normal tissue removed during cosmetic or reconstructive procedure if and only
if the patient has no contagious lesion or history of malignancy.
REMEMBER:
PATHOLOGISTS: PRE-ANALYTIC FACTORS: WARM ISCHEMIA AND COLD ISCHEMIA
They are responsible for cutting the specimen and for microscopic exam. Types of specimen in HP:
PRE-ANALYTICAL FACTORS: - Biopsy / Surgical specimen
TAKE NOTE: These are factors affecting the tissues that already have produced artefact rendering the - Autopsy specimen
1. Medical technologist is responsible for the preparation of the specimen. An tissues suboptimal for histologic preparation before arriving in laboratory.
MT does not cut the specimen. Specimens excluded for mandatory
2. Surgeon is responsible for preparation for the removal of organ from the 1. WARM ISCHEMIA submission to the laboratory are
patient. Follow Follow Follow MMo BeBe Co!
 Initial anoxic insult a tissue suffers due to lack of blood supply.
De-SPRINTS.
TYPES OF SPECIMENS SUBMITTED FOR HISTOPATHOLOGIC  Duration depends on the surgery, speed, and skill of the surgeon, and other
factors affecting the conduct of surgery.
EXAMINATION Pre-analytical factors:
 It is beyond the control of the histopathology laboratory. - Warm ischemia
TWO TYPES OF SPECIMEN SUBMITTED IN HP LAB: - Cold ischemia
2. COLD ISCHEMIA
1. BIOPSY (SURGICAL SPECIMEN)
-to determine malignant conditions  Refers to the lack of oxygen once the tissue sample is removed from the DISSECTION:
-comes from the living source, regardless of the size patient’s body and before all metabolic processes are stopped by fixation. - It is done by the
2. AUTOPSY SPECIMEN Pathologists.
-to determine the cause of death PURPOSE OF TISSUE PROCESSING - It is also known as Bread
-comes from non-living or dead source Loafing for the pathologists.
Tissue processing is done to prepare for microscopic examination.
SPECIMENS EXCLUDED FOR MANDATORY SUBMISSION TO THE
LABORATORY GROSS EXAMINATION OF SPECIMENS AND DISSECTION
(Follow Follow Follow MMo BeBe Co! De-SPRINTS)
GROSS EXAMINATION:
Follow: Fats removed by liposuction It is the first step and is done by the pathologists.
Follow: Foreskin removed from circumcision
Follow: Foreign bodies (bullets or other medico-legal evidence given directly to law
THREE LISTS FOR GROSS EXAMINATION:
enforcement personnel)
M: Medical devices (such as catheters, gastronomy tubes, stents, and sutures) 1. Dimension
M: Middle ear ossicles 2. Weight: It is the most important physical characteristics in gross
O: Orthopaedic hardware and other radiopaque medical examination. It is always rounded off to the nearest 0.1 gram.
Be: Bone donated to the bone bank 3. Color and consistency.
Be: Bone segments removed as part of corrective or reconstructive orthopaedic procedures

Co: Cataracts removed by phacoemulsification

2|Page Rachelle Mae F. Ferranco, RMT

SPECIMENS FOR GROSS DESCRIPTION ONLY OBJECTIVES OF FIXATION REMEMBER:

1. Accessory digits 1. To preserve the tissue.
2. Bunions and hammer toes 2. To prevent breakdown of cellular elements.  Fixation is the first and most
3. Extraocular muscle from corrective surgical procedures 3. To coagulate or precipitate protoplasmic substances. critical step of histotechnology.
4. Inguinal hernia sacs in adults
5. Nasal bone and cartilage from rhinoplasty  Additive fixation includes: FMO
TWO BASIC MECHANISMS INVOLVED IN FIXATION
6. Prosthetic breast implants
7. Prosthetic cardiac valve without attached tissue  Non-additive fixation includes:
1. Additive fixation
Alcoholic fixatives
8. Tonsils and adenoids from children  The chemical constituent of the fixative is taken in and becomes part
9. Torn meniscus of the tissue by forming cross-links or molecular complexes and
10. Umbilical hernia sacs in children  Tissue + Hypotonic solution =
giving stability to the protein. SWELL
11. Varicose veins  Examples: Formalin, Mercury, Osmium tetroxide
 Tissue + Hypertonic solution =
FIXATION 2. Non-additive fixation SHRINK
It is the first and most critical step in histotechnology that involves fixing  The fixative does not become part of the tissue.
and preserving tissue for examination.  Examples: Alcoholic fixatives
It preserves that morphology and chemical integrity of the tissue as close BENEFITS OF FIXATION
as the original as possible. It prevents autolysis and putrefaction.
1. Allows thin sectioning of the tissue by hardening the tissue.
The quality of the section on the slide is only as good as the quality of 2. Prevents autolysis and inactivates infectious agents.
fixed tissue specimen. 3. Improved cell avidity for special stains.
If fixation is not adequate, the other processes that follow, such as
dehydration, cleaning, infiltration embedding, microtomy and staining, will
also be inadequate.

Fixatives inactivates lysosomal enzymes to prevent autolysis.

PRIMARY GOAL OF FIXATION

To preserve the tissue.

SECONDARY GOALS OF FIXATION

1. To harden the tissue.
2. To protect the tissue from trauma of further handling.
3|Page Rachelle Mae F. Ferranco, RMT

E. Thickness of sections REMEMBER:

MAIN FACTORS INVOLVED IN FIXATION INSTRUMENTATION / THICKNESS OF SECTIONS
SPECIMEN  Common buffers include:
A. Hydrogen ion concentration Electron Microscopy 1 to 2 mm2 - Phosphate
 Best carried out close to neutral pH: 6-8 Light Microscopy 2 cm2 - Bicarbonate
 Hypoxia of the tissue lowers the pH - Cacodylate
Large solid tissues Should be opened or sliced thinly. - Veronal
 Acidic pH = formation of formalin-heme pigment (black) (Example: Uterus)
 To maintain an adequate fixation time of 4 to 6 hours, the recommended size of  Sucrose is commonly added to
B. Osmolality the tissue is 2 cm3 and no more than 4mm thick. osmium tetroxide fixatives for
 Hypertonic fixatives cause cell shrinkage.
electron microscopy.
 Isotonic and hypotonic fixatives cause cell swelling. F. Concentration
 Cell swelling and/or shrinkage = poor fixation  Formaldehyde = 10% solution  Whole brain is suspended to
 Recommended osmolality: Slightly hypertonic solutions (400 to 450 mOsm)  Glutaraldehyde = 3% solution 10% neutral buffered formalin for
 Isotonic solutions are 340 mOsm  Low concentrations of glutaraldehyde (0.25%) = Immuno-Electron Microscopy 2-3 weeks to ensure excellent
fixation.
C. Temperature G. Duration of fixation
SPECIMEN / FIXATION TEMPERATURE  Penetration of formalin to tissue:
FIXATIVE DURATION OF FIXATION
INSTRUMENTATION / 1 mm per hour and slows down
FIXATIVES Formalin 24 hours as it go deeper into the tissue.
Surgical specimens Room Temperature Neutral Buffered Formalin 2 to 6 hours
and (can remain for a week without  Fixation time can be cut down
Mast cells using electron much adverse effect) using:
microscopy - Heat
Electron Microscopy 3 hours
Tissue processors 40 degrees Celsius - Vacuum
(Then place in a holding buffer) - Agitation
Electron microscopy and 0 to 4 degrees Celsius - Microwave
some histochemistry
 Most tissues can be cut and
 Formalin at 60 degrees Celsius = for rapid fixation of very urgent biopsy trimmed without prior fixation,
specimen. EXCEPT for brain. The brain should
 Formalin at 100 degrees Celsius = to fix tissues with tuberculosis. be fixed before grossing or
sectioning.
D. Volume
 Gregorios’: 10-20x the volume of the specimen  Refrigeration:
 Lo’s: 15-20:1 - It is used to slow down the
decomposition if the tissue
needs to be photographed
and cannot be fixed
immediately.
4|Page Rachelle Mae F. Ferranco, RMT

EFFECTS OF FIXATIVES IN GENERAL: FACTORS THAT RETARD FIXATION REMEMBER:

1. Size / Thickness of section  Presence of blood and mucus

1. They harden soft and friable tissues and make the handling and cutting of sections 2. Presence of blood: such as blood vessels and spleen can be resolved by washing with
easier. This is usually accelerated by the action of alcohol during dehydration 3. Presence of mucus NSS.
process. 4. Fatty tissues: Remedy is to cut fatty tissue thinly.
2. They make cells resistant to damage and distortion caused by hypotonic and 5. Cold temperature: inactivates enzymes  Brain tissue is a bloody
hypertonic solutions used during tissue processing. specimen that must undergo
3. They inhibit bacterial composition. FACTORS THAT ACCELERATE FIXATION intravascular perfusion.
4. They increase the optical differentiation of cells and tissue components thereby
rendering them more readily visible during examination. 1. Size / Thickness of section
5. They act as mordants or accentuators to promote and hasten staining, or they 2. Agitation: constant mixing using autotechnicon.
3. Heat application  Intravascular perfusion: it is a
may inhibit certain dyes in favor of another ( e.g. formaldehyde intensifies while
o Ideal: 37 – 56 degrees Celsius process of washing out the blood
osmium tetroxide inhibits hematoxylin staining).
o Higher than 56 degrees Celsius can damage the tissue. using Ringer’s lactate.
6. They reduce the risk of infections during handling and actual processing of
tissues.
FACTORS TO BE CONSIDERED WHEN CHOOSING THE RIGHT FIXATIVE
(USTTT)
CHARACTERISTICS OF A GOOD FIXATIVE: 1. U: Urgency of case or need for immediate exam.
2. S: Staining technique to be applied.
1. It must be cheap. o Fixative must be compatible with stain to be used.
2. It must be stable o Right pair: Newcomer’s & Feulgen
3. It must be safe to handle. o Wrong pair: Bouin’s & Feulgen
4. It must kill the cell quickly thereby producing minimum distortion of cell 3. T: Type of tissue to be processed.
constituents. o Liver: Zenker’s fluid
5. It must inhibit bacterial decomposition and autolysis. o Pituitary biopsies: Bouin’s
6. It must produce minimum shrinkage of tissue. o Kidney: contraindicated (not used) for Bouin’s
7. It must permit rapid and even penetration of tissues. 4. T: Tissue structure to be studied.
8. It must harden tissues thereby making the cutting of sections easier. o Rickettsiae and Bacteria = Orth’s fluid
9. It must be isotonic causing minimal physical and chemical alteration of cells and o Glycogen = Brasil’s
their constituents. This is not, however, a strict rule since there are some 5. T: Type of section to be made whether serial or individual.
hypotonic solutions (I.e. glacial acetic acid) producing tissue swelling, which are
being used in conjunction with hypertonic solutions (e.g. picric acid) causing
shrinkage of cells, to produce a compound which would give an optimal effect
on the tissue structure.
10. It must make cellular components insoluble to hypotonic solutions and
render them insensitive to subsequent processing.
11. Rapid action it must penetrate the tissue.
5|Page Rachelle Mae F. Ferranco, RMT

METHODS OF FIXATION
2. Cytoplasmic Fixatives: without glacial acetic acid and have a pH of more
1. PHYSICAL METHODS than 4.6.
A. Heat Fixation o Flemming’s fluid without acetic acid
o Principle: Thermal coagulation of proteins o Helly’s fluid
o Seldom used in HP laboratory. o Formalin with post chroming
o In HP lab it is used for frozen sections. o Regaud’s fluid (Moller’s fluid)
o Often used to fix bacterial smears. o Orth’s fluid
B. Microwave Technique 3. Histochemical fixatives
o Can produce toxic vapors. o 10% Formal saline
o Can accelerate both decalcification and staining. o Absolute ethyl alcohol
o To preserve acetylcholine in brain. o Acetone
o Can penetrate tissues up to 10-15 mm. o Newcomer’s
C. Freeze-drying

2. CHEMICAL METHODS: use of either additive or non-additive fixation.

A. Coagulant fixatives: Alcohol, Acetone, TCA, Picric acid FIXATIVES FOR ENZYME HISTOCHEMISTRY
B. Cross-linking fixatives: Formaldehyde, Glutaraldehyde, Aldehydes
C. Compound fixatives: Alcoholic formalin (for fatty tissue like breast) - 4% Formaldehyde or Formol Saline

TYPES OF FIXATIVE ACCORDING TO ACTION FIXATIVES FOR ELECTRON MICROSCOPY

MICRO-ANATOMICAL FIXATIVES: - Osmium tetroxide

- Glutaraldehyde
1. Bouin’s solution - Paraformaldehyde
2. Brasil’s solution
3. 10% formol saline FIXATIVES FOR ELECTRON HISTOCHEMISTRY & ELECTRON
4. 10% neutral buffered formalin IMMUNOCYTOCHEMISTRY
5. Formol sublimate (Formol corrosive) - Karnovsky’s paraformaldehyde-glutaraldehyde
6. Heidenhain’s Susa - Acrolein
7. Zenker’s solution
8. Zenker-formol (Helly’s solution)

CYTOLOGICAL FIXATIVES:

1. Nuclear Fixatives: with glacial acetic acid & pH of 4.6 or less

o Flemming’s fluid
o Carnoy’s fluiGd
o Bouin’s fluid
o Newcomer’s fluid
o Heidenhain’s Susa
1|Page Rachelle Mae F. Ferranco, RMT

SUMMARIZED IMPORTANT INFORMATION ABOUT FIXATIVES  Principle: denaturation and precipitation of

ALCOHOL FIXATIVES proteins.
 Common disadvantage: Glycogen
 Basic component polarization
 Both fixative and decalcifying agent  Glycogen polarization: accumulation of
TRICHLOROACETIC ACID  Often used in combination with other fixatives glycogen granules at both ends of the cell.
to produce compound.  It should not be used in low concentration if
 Often used in combination with other fixatives the purpose is for fixation to prevent lysis
to produce compound. of the cell.
GLACIAL ACETIC ACID  Unique characteristic: solidifies at 17 degrees  Required concentration: 70% to 100%
Celsius  80% alcohol is used to preserve the color
 Contraindicated for cytoplasmic fixation. of the specimen for photographic work.
 Raw material in making shabu.  Glycerin is also used in combination with
 Both fixative and dehydrating agent. alcohol to preserve the color of specimen.
 Must be used at ice cold temperature (-5 to 4  For wet and dry smears, blood smears and
degrees Celsius) (1) METHANOL (100%)
bone marrow tissues.
ACETONE  For lipases and phosphatases  For blood and tissue films.
 For diagnosis of rabies  May be used as simple fixative.
 Evaporates easily (Volatile) (2) ETHANOL (70-100%)  More frequently incorporated into
 Dissolves fats compound fixatives for better results.
 Both fixative and stain.  Strong reducing agent
 In CC, used in Jaffe reaction.  For touch preparations.
PICRIC ACID  Imparts yellow color. (3) ISOPROPYL  It is used for certain special staining
 Remedy: procedures such as Wright-Giemsa.
1. Treat the tissue with another acid dye.  It is the most rapid fixative.
2. Use lithium carbonate.  CULDOG:
3. Place in 20% ethyl alcohol followed by 5% - Chromosomes
sodium thiosulfate and wash with water. (4) CARNOY’S - Urgent biopsy specimens for paraffin
 Types of Picric acid: processing within 5 hours
1. Bouin’s Solution - Lymph glands
-embryos, pituitary biopsies and - Dog: rabies diagnosis
endometrial curetings  It is used for fixing mucopolysaccharides
-never for kidneys and nuclear proteins.
-abolishes feulgen’s (5) NEWCOMER’S  It is compatible with Feulgen’s.
2. Brasil’s
-glycogen demonstration
-less messy than Bouin’s
3. Hollandes
-GIT biopsies and endocrine tissues
2|Page Rachelle Mae F. Ferranco, RMT

(6) CLARKE’S SOLUTION  Produces good results with H & E. caps. Also avoid contact with personal
 Preserves nucleic acid. jewelries.

 Causes less hardening and shrinkage than  For small pieces of liver, spleen, and
(7) METHACARN Carnoy’s. connective tissue fibers and nuclei.
 Glacial acetic acid is added to prevent
 Good for CT mucins and umbilical cord. (a) ZENKER’S turbidity and formation of dark precipitate.
(8) ROSSMAN’S SOLUTION  De-zenkerization: removing mercury
deposits by immersing the tissues in
alcoholic iodine prior to staining.
 For cytology of bone marrow biopsies.
METALLIC FIXATIVES (b) B5
 Excellent microanatomic fixative for
pituitary gland, bone marrow, blood
(1) LEAD  For acid mucopolysaccaharides and (c) HELLY’S / ZENKER-FORMOL containing organs such as spleen and liver.
connective tissue mucins.  For tumor skin biopsies.
 It is used in aqueous solution of basic lead (d) HEIDENHAIN’S SUSA
acetate.
(2) CHROMATE FIXATIVE  Basic component: Potassium dichromate (e) SCHAUDINN’S OHLMACHER’S
(a) Chromic acid  To preserve carbohydrates. (f) CARNOY-LEBRUN SOLUTION
(b) Potassium dichromate  To preserve lipids and mitochondria.  It is proposed as replacement for
 To demonstrate chromatin, mitochondria, ZINC SULFATE mercuric chloride because mercuric
(c) Regaud’s mitotic figures, golgi bodies, RBC and chloride causes irritation to skin, eye and
colloid containing tissues. respiratory.
 For rickettsia and other bacteria.  For Electron Microscopy.
(d) Orth’s fluid  For early degenerative processes and  Required volume: 5 to 10x of the volume
tissue necrosis. of the specimen.
 Main disadvantage: toxic to man  For preserving neurological structures
OSMIUM TETROXIDE / OSMIC ACID like myelin and peripheral nerves.
 2 Important advantages:
- Can be used for preserving tissues for  Principle: Gels and precipitates proteins.
tissue photography.  Should be kept in a dark-colored,
(3) MERCURIC CHLORIDE - Compatible with trichrome stain. chemically clean bottle to prevent
 It can produce black mercury deposits. evaporation and reduction by sunlight or
Remedy for black mercury deposits: wash organic matter.
the tissue using alcoholic iodine. (a) FLEMMING’S WITH ACETIC ACID  Preserves the nucleus.
 Avoid using metallic forceps and metal (b) FLEMMING’S WITHOUT ACETIC ACID  Preserves cytoplasm esp. mitochondria.
3|Page Rachelle Mae F. Ferranco, RMT

- Forms white precipitate

paraformaldehyde that may cause
 These are satisfactory for routine paraffin turbidity. Remedy: Add 10% methanol
ALDEHYDE FIXATIVES sections, for Electron Microscopy, and or filter.
when histochemical and enzyme studies - Can produce brown formalin pigments.
are indicated.  Methods of removing formalin pigments:
(1) GLUTARALDEHYDE  Three concentrations: - Kardasewitch: 28% ammonia water
- 4% for large tissues less than 4mm and 70% ethanol
thick (6-8 hours up to 24 hours) - Lillie’s method: acetone, hydrogen
- 2.5 % for small tissue fragments and peroxide and ammonia water
needle biopsies (2-4 hours at room - Picric acid method: saturated picric
temperature) acid.
- 0.25% Immuno-Electron Microscopy (4) 10% FORMOL SALINE  Saturated formaldehyde diluted to 10%
(2) GLYOXAL (C2H2O2)  Smallest aldehyde. sodium chloride.
 Rapid fixing agent (45 minutes for small  Recommended fixative for CNS tissues
tissues and 4-6 hours for large tissues) and general post-mortem tissue for
 Commercially supplied as 40& aqueous histochemical exam.
solution. (5) 10% NEUTRAL BUFFERED FORMALIN  Recommended for preservation and
(3) FORMALDEHYDE (34-45%)  AKA 100% Formalin OR PHOSPHATE BUFFERED storage of surgical, post-mortem and
 Produced by oxidation of methanol. FORMALIN research specimen.
 Dilution: 1:10 or 1:20  For elastic fibers and tissues with ion
 Solution: 10% or 5% pigments.
(6) FORMOL – CORROSIVE (FORMOL-  Formaldehyde + mercuric chloride
10% FORMALIN SUBLIMATE)  Recommended for routine post-mortem
 Usual fixation time: 24 hours tissues.
 Penetration time: 1mm/hr (7) ALCOHOLIC FORMALIN (GENDRE’S  For preserving sputum.
 Buffer: Phosphate buffer FIXATIVE)  For microincineration (to burn tissues into
 Advantages: ashes to identify mineral)
- Cheap, easy to prepare, stable, readily
available.
- Tolerant fixative.
- For mailing specimens.
 Disadvantages:
- Fumes irritating.
- Cause dermatitis on prolong contact.