Histopathologic and Cytologic Techniques

Fixatives | Lecture | Lesson 2 & 3

FORMALDEHYDE • Preservation and storage of the surgical post

mortem
• One of the most widely used fixatives for tissue
fixation, usually buffered to a pH of 7 with the FORMOL-CORROSIVE (FORMOL SUBLIMATE)
addition of Phosphate buffer.
• Recommended for a routine post mortem tissue
• Advantages:
o Cheap, readily available easy to prepare GLUTARALDEHYDE
and stable
o Compatible with many stains • Made of two formaldehyde residue
o Doesn’t overhardened tissue • More stable effect on tissue especially central
o Penetrates tissue well nervous system as a preservative, could preserve
o Preserves fats and mucin plasma proteins better than the other
o Preserves glycogen preservative
o Preserves but doesn’t precipitate protein • Can preserve immunoglobulin, albumin, and
o Allows natural tissue colors to be globulin
retained after fixation • Produces less tissue shrinkage and also preserve
• Disadvantages: cell tissue better
o Fumes are irritating to the nose and eyes • Has less irritation in the nose and less irritation if
o Irritating to skin expose to skin, it doesn’t cause dermatitis
o May produce considerable shrinkage of
Remember:
tissue
o Soft fixative and doesn’t harden some • If the specimen has microbiology test, it should
cytoplasmic structures be submitted first in the microbiology section,
afterward is to histopath lab
10% FORMOL SALINE
• This is done to lessen the contamination and to
• For central nervous tissue identify specific microbial bacteria
• Advantages: • This is also done if there’s only one specimen
o Penetrates and fixes tissues evenly and need to undergo multiple tests
o Preserves microanatomic and cytologic
METALLIC FIXATIVE
details with minimum shrinkage and
distortion • Utilizes heavy metals in fixing a preserving tissue
o Preserves enzymes and nucleoprotein
o Demonstrate fats and mucin MERCURIC CHLORIDE
o Doesn’t overhardened tissue • Zenker’s Fluid
o Ideal for most staining techniques, o fixing a small pieces of liver tissue,
including silver impregnation spleen, connective tissue and nuclei
o Allows natural tissue color to be restored o a good general fixative for adequate
• Disadvantages: preservation of all kind of tissue
o Slow fixative o gives excellent staining
o Tissues tend to shrink during alcohol o recommended for trichrome staining
dehydration • Zenker’s Formol (Helly’s Solution)
10% NEUTRAL BUFFERED FORMALIN/PHOSPHATE o Excellent microanatomic fixative for
BUFFERED FORMALIN (pH 7) pituitary gland, bone marrow and blood
containing tissue
• Heidenhain’s Susa Solution
 o Consider as excellent cytologic fixative • Could precipitate chromosomes or chromatin
o Is recommended for tumor biopsy reactions
especially skin biopsy
ALCOHOL FIXATIVE
• B-5 Fixative
o Bone marrow biopsy and use in some • Concentration used ranges from 70-100%
lymph nodes or when there’s a
suspected lymphoma Types of Alcohol Fixative:

CHROMATE FIXATIVE • Methyl Alcohol 100%

o This is excellent for fixing dry and wet
• Chromic Acid smears, blood smear and bone marrow
o can precipitate all proteins and can tissue
adequately fixed carbohydrate such as o Poisonous type of alcohol it can irritate
glucose and glycogen eyes and skin. It can also cause
• Potassium Dichromate dermatitis.
o to preserved lipids (fats) and • Ethyl Alcohol
mitochondria o Used in concentration of 70-100%
• Regaud’s (Muller’s) Fluid o Using a lower concentration of ethyl
o Mitochondria, mitotic figure, golgi alcohol can cause hemolyze or destroy
apparatus/body, RBC, tissues containing RBC and inadequately preserve WBC
a colloidal materials • Isopropyl Alcohol 95%
o Chromatin material, chromosomes o Used for preserving touch
• Orth’s Fluid preparation/fixing touch preparation
o Recommended for the study of early • Carnoy’s Fluid
degenerative process and necrosis o Preserving chromosomes, lymph gland
o Is also used for demonstrating bacteria and urgent biopsy
such as rickettsia or other bacteria; o Also used for fixing and preserving
rocky mounted fever is caused by central nervous system for rabies
rickettsia o Yung aso na nakakagat, ung brain nila
ung may rabies, if they bite someone
LEAD FIXATIVE
hahanapin ung dog na yon at gagamitin
• Used in 4% aqueous solution of basic lead ung brain tissue nila for diagnosing rabies
structure • Newcomer’s Fluid
• Recommended for acid mucopolysaccharides o Used for fixing
• Fixing connective tissue especially the mucin mucopolysaccharides and nuclear
structure proteins
o Can also act as both nuclear and
PICRIC ACID FIXATIVE
histochemical fixative
• Preserves glycogen well but causes OSMIUM TETROXIDE (OSMIC ACID)
considerable tissue shrinkage
• Allows brilliant staining with the Trichrome • It preserves cytoplasmic structure well
method • Also used extensively for neurological
Example: tissues
• Bouin’s Solution – recommended for the
Has two types:
fixation of embryos and pituitary biopsy
• Brasil’s Alcoholic Picroformol Fixative – • Fleming’s Solution
excellent fixative for glycogen o Is the most commonly used chrome
osmium acetic acid fixative
GLACIAL ACETIC ACID
 • Fleming’s Solution without Acetic Acid Remember:
o Recommended for cytoplasmic
• The fixative volume ratio is 10:1 and 20:1 / 1:10
structure
and 1:20
ACETONE • Volume ng fixative 10-20x vs. volume ng tissue
• The tissue should be submerged (nakalubog)
• Used in fixing brain tissues for diagnosing brain
• The tissue that is not needed is sinusunog na or
rabies
nililibing
HEAT FIXATION
COMPUTATION OF FORMALIN SOLUTIONS
• Thermal coagulation of tissue protein
N1V1 = N2V2
• For the preparation of bacteriological smear
preparation N1: concentration of concentrated formalin (constant
100%)
Terminologies
N2: concentration of formalin solution to be prepared
• Secondary Fixation V1: volume of concentrated formalin
o A process wherein an already fixed V2: total volume of formalin solution to be prepared
tissue is placed in a second / another Sample Problem:
fixative To make 10% formalin solution, how many ml of distilled
• Post Chromatization water should be added to 190ml of 37% formaldehyde
o a form of secondary fixation whereby a stock solution?
primarily fixed tissue is placed in Answer:
aqueous solution of 2.5-3% potassium N1: 100%
dichromate for 24 hours V1: 190ml
o has 1 day fixation period to enhance and N2: 10% formalin
optimized the target cell tissue V2: ?
o after ma fixed sa unang fixation ibabad
sa potassium dichromate (N1)(V1)_= (100%)(190ml) = 1,900 x 37% = 703ml
• Washing-Out (N2) (10%)
o A process of removing excess fixative
from the tissue after fixation to improve REMINDERS WHEN PERFORMING MACROSCOPIC
staining and remove artefacts (GROSS EXAM)
o Example of Washing Reagent:
o Tap water • Tissue thickness:
o 50-70% of Alcohol o Should not be thicker than .5cm /
o Alcoholic Iodine 5mm (para hindi rin malaglag ung
specimen sa tissue cassette)
FACTORS AFFECTING FIXATION:
• Paper tags:
• Retarded by: o Use pencil for labeling para ma retain
o Size and thickness ung nakasulat kapag binabad; dot
o Presence of mucus matrix printer (a type of impact printer
o Presence of fats that prints using a fixed number of
o Presence of blood pins or wires)
o Cold Temperature • Dissect fat away from lymph nodes before
• Enhanced by: loading
o Size and thickness – should not be • Labeling of sections and blocks
thicker to .5cm / 5mm o Indicate number of sections and
o Agitation blocks(cassettes) taken
 o Document samples taken B. CHELATING AGENTS
systematically
• Substances which combines with calcium ion
o document color code of inked margins
and other salts to form weakly dissociated
(if >1 color)
complexes and facilitate removal of calcium
• Minute and multiple fragments
salts
o wrap tissues in filter paper or similar
• EDTA (Ethylenediaminetetraacectic acid) is the
material to prevent loss in processing
most commonly used chelating agent and
DECALCIFICATION recommended for microscopic study

• A procedure whereby calcium and lime salts are C. ION EXCHANGE RESIN
removed from the tissues following fixation
• Hastens decalcification by removing calcium ion
• Decalcification is performed after fixation and
from formic acid containing agents
before impregnation
• The degree calcification is measured by physical
GOALS: or pricked method

• To ensure and calcificate D. ELECTROPHORESIS (ELECTRICAL IONIZATION)

• To prevent obsecuring of the microanotomic
• Process whereby positively charged calcium ions
detail of sections by bone dusts and cell debris
are attracted to a negative electrode and
DECALCIFYING AGENTS: subsequently removed from decalcifying agent.
• Decalcification time is shortened due to
• Acids
electrolyte present and heat
• Chelating Agents
• Ion exchange resin
• Electrophoresis MEASURING THE EXTENT OF DECALCIFICATION

• I – PHYSICAL/MECHANICAL TEST
• A. ACID DECALCIFYING AGENTS
o Decalcified tissues are softer to touch
o Most widely used agents for routine
o Checking through pricking or probe of
decalcification
needle
o 5-10% concentration for routine
• II – XRAY/RADIOLOGIC METHOD
o Acidic agent can inhibit nuclear stains
o Very expensive but the most ideal and
and also to destroys tissue
reliable method of determining the
o Stable, readily available and relatively
extent of calcification
inexpensive
• III – CHEMICAL METHOD
Rate of Decalcification Depends Upon: o Simple, reliable and convenient method
for routine purposes
• Structure o Involves detection of calcium in acid
• Volume of the solution solution by precipitation of insoluble
• Temperature hydroxide calcium oxilate
MINERAL ACIDS USED AS DECALCIFYING AGENTS: LESSON 3
• Nitric Acid DEHYDRATION AND CLEARING
• Hydrochloric acid • Kapag nag clear sa xray yung na decalcified na
• Formic acid tissue, it means complete na yung process ng
• Trichloroacetic acid decalcification
• Sulphurous acid
DEHYDRATION
• Chromic acid
• Citric acid
 • Process of removing intracellular and IV – CELLOSOLVE
extracellular water from the tissue following
• Dehydrating rapidly
fixation and prior to impregnation.
• When tissues are placed in cellusolve, it can be
• Gradual procedure
maintained for months without causing
CHARACTERTISTICS OF AN IDEAL DEHYDRATING significant hardening or destruction on tissue
AGENT • Ethylene glycol monoethyl ether

• Must dehydrate rapidly V – TRIETHYL PHOSPHATE

• Should not evaporate very fast
• Used to dehydrate sections and smears
• Be able to dehydrate even fatty tissues
following certain stains and produces minimum
• Should not over harden tissue
shrinkage
• Should not be toxic to the body
• Shout not be fire hazard VI – TETRAHYDROFURAN

COMMONLY USED DEHYDRATING AGENT • Reagent that both dehydrates and clears tissue
• Tissue can be cut easily/sections
I - ALCOHOL (most common)
• Disadvantage: this solution is toxic when inhaled
• Ethyl Alcohol
CLEARING
o Recommended for routine dehydration
o In all types available alcohol, ethyl is the • Also known as “Dealcoholization”
best dehydrating agent • Process whereby alcohol is removed from the
o Fast acting tissue and replace with substance that will
o It mixes with water and many organic dissolve the wax with which the tissue is to be
solvent impregnated or the medium on which the tissue
o It penetrates tissue well is to be impregnated.
o Not poisonous; cheap
CHARACTERISTICS OF A GOOD CLEARING AGENT
• Methyl Alcohol
o Toxic dehydrating agent • Should be miscible with alcohol to promote rapid
o It is intended only to use for dehydrating removal of dehydrating agent
blood smear • Should be miscible with, and easily removed by
• Butyl Alcohol melted paraffin wax and/or by mounting medium
o Slow dehydrating agent to facilitate impregnation and mounting
o Produces less shrinkage and less • Should not produce excessive shrinkage,
hardening of the tissues hardening or damage of the tissue
o Recommended for tissues that does not • Should not dissolve out aniline dyes
require rapid process • Should not evaporate quickly
II – ACETONE • Should make tissue transparent

• Cheap, rapid acting COMMON CLEARING AGENT USED:

• Rapid in action but penetrates tissue properly I – XYLENE
and causes brittleness
• It is not recommended for routine dehydration • Most commonly used clearing agent
• Generally suitable for most routine histologic
III - DIOXANE (Diethylene dioxide) processing
• Excellent dehydrating and clearing agent • Problem: Xylene appears milky if the dehydration
• Produce less tissue shrinkage procedure is not fully done or incomplete
• Not recommended for routine dehydrating agent dehydration
II – TOULENE

• May used as an alternative

III – BENZENE

• Penetrates and clear tissue rapidly

IV – CHLOROFORM

• Slower in action than xylene but causes less

brittleness
• Suitable for large tissue specimen
• Recommended for tough tissues
• Disadvantage: It doesn’t make the tissue
transparent/translucent
• Highly toxic

V – CEDARWOOD OIL

• Used to clear both paraffin and celloidin

sections during embedding process
• Is recommended for central nervous tissues,
cytological studies, smooth muscle and
cutaneous membrane

VI – ANILINE OIL

• Not normally used as routine clearing agent

VII – CLOVE OIL

• Causes minimum shrinkage

VIII – CARBON TETRACHLORIDE

• Cheaper than chloroform

• It overharden tissues
• The fume is dangerous and it can lead to
pulmonary abnormality in chronic exposure
• Kaya dapat gumamit ng makapal na gloves nitrile
and proper PPE during histopath lab.