HISTOPATH UNO

Biopsy

Obtained from LIVING source

Autopsy

obtained from DEAD source

FIXATION

Most Critical step

Primary Goal of Fixation

Preserve Morphology and Chemical Integrity of tissue as close to original

Secondary Goal of Fixation

To harden the tissue

To protect the tissue from trauma of further handliing

Fixation Time

Minimum: 6 hrs

Maximum: 48 hrs

3 hrs

Fixation Time for Electron Microscopy

Additive

component becomes part of the tissue

Non-Additive

Component does not become part but it ALTERS the tissue component

20x the volume of the specimen (15-20:1)

amount of fixative

5-10x

Amount of Fixative for Osmium Tetroxide

20x

Amount of Fixative for Electron Microscopy

NOT LESS THAN 50x

Amount of Fixative for Museum Preparations

Factors that will retard/prolong Fixation

Size and Thickness

Cold Temp.

Mucus and Blood (wash with NSS)

Fat (cut into thin slices)

Factors that will Accelerate Fixation time

Size and Thickness

Agitation

Heat and Pressure (37-56 C)

Factors to consider in choosing the appropriate fixative

Need for immediate examination

Type of Tissue

Tissue Structure to be Studied

Type of stain to be used

Effects of Fixative

Harden tissue

Prevent Bacterial growth

Reduce risk of infection

Increase optical differentiation of cells and tissue

Heat Fixation

Thermal coagulation of Proteins

usually for Bacterial smears and Frozen sections

ACETONE

Preserves Lipase, Phosphatase, Enzyme Studies

ACETONE

should be in ice cold temp. ( -5 -4 C)

ACETONE and CARNOY'S FLUID

for diagnosis of RABIES

TRICHLOROACETIC ACID

Both Fixative and Decalcifying Agent

GLACIAL ACETIC ACID

for nuclear proteins, nuclear chromatin. cytoplasmic differentiation

GLACIAL ACETIC ACID

SOLIDIFIES at 17 degrees celsius

ALCOHOL FIXATIVES

both a Fixative and a Dehydrating Agent

ALCOHOL FIXATIVES

causes Glycogen Polarizing

Methyl Alcohol

for wet and dry prep. (blood smears and BM tissues)

Ethyl Alcohol

for blood and tissue films

Isopropyl Alcohol

for Touch Preparations

Carnoy's Fluid

Most Rapid alcohol fixative

Newcomer's

compatible with Fuelgen

Newcomer's

for mucopolysaccharides and nuclear CHON

10% Formaldehyde

used for mailing specimens

10% Formaldehyde

precipitation of White Paraformaldehyde is possible

10% Formaldehyde

aldehyde fixative which may cause dermatitis

10% Formaldehyde

has a 1mm/hr penetration rate

37 - 40% Formaldehye

"100% formaldehyde"

Gendre's Fluid

aldehyde fixative which fixes sputum

Gendre's Fluid

used for microincineration technique

Formol Corrosive

100% formalin + mercuric chloride

Formol Corrosive

recommended for Neutral fats, Phospholipids, lipids

10% Formol Saline

aldehyde fixative used for CNS, fats and enzymes

10% Formol Saline

aldehyde fixative which is for post-mortem tissue

Glutaraldehyde

aldehyde fixative for electron microscopy and enzyme histochemistry

2.5% Glutaraldehyde

glutaraldehyde for smaller specimens

4% Glutaraldehyde

glutaraldehyde for big specimens

Karnovsky's paraformaldehyde-glutaraldehyde and ACROLEIN

aldehyde fixative for electron microscopy and immunochemistry

PICRIC ACID

imparts yellow discoloration

Acid Lithium Carbonate

treatment to remove yellow color in picric acid fixatives

PICRIC ACID

Both FIXATIVE and STAIN

Bouin's Fluid

Abolishes Fuelgen reaction

Bouin's Fluid

for Pituitary biopsies, Embryo, Endometrial curettings

Bouin's Fluid

not suitable for kidney

Brasil's Alcoholic Picroformol

Excellent fixative for GLYCOGEN

LEAD Fixatives

for ACID MUCOPOLYSACCHARIDES and CT mucin

Chromic acid

fixative for carbohydrates

3% potassium dichromate

preserves lipids and mitochondria

Regaud's/Moller's

for Chromatin, Golgi Bodies, Mitochondria, and RBC containing organs

Orth's fluid

for early degenerative processes and tissue necrosis

Orth's Fluid

fixes Rickettsia org.

MERCURIC Fixatives

excellent for TISSUE PHOTOGRAPHY and TRICHROME staining

Zenker's Fluid

for liver, spleen, CT, fibers, nuclei

Zenker's Formol/HELLEY'S

for PITUITARY gland, BM and other blood containing organs

Heidenhan's Susa

For TUMOR biopsies

B5 Fixative

for Bone Marrow

B5 Fixative

contains Anhydrous Sodium Acetate

Flemming's

osmium tetraoxide fixative that contains acetic acid

Flemming's

excellent for NUCLEAR structure

Flemming's without acetic acid

for CYTOPLASMIC structure

Microwave Technique

accelerates FIXATION, DECALCIFICATION, STAINING

Microwave Technique

for neurochemical substances in the brain like acetylcholine

SECONDARY FIXATION

purposes:

make special staining possible

complete hardening and preservation of tissue

improve demonstration of desired subs

Post-Chromatization

a secondary fixation that uses any chromate fixative

WASHING OUT

removes excess fixatives

tap water

removes excess osmic acid and chromates

50-70% alcohol

removes excess picric acid

Alcoholic Iodine

removes excess mercuric fixative

DEHYDRATION

not less than 10x the volume of the specimen

DEHYDRATION

70% alcohol to ascending grades of alcohol

dehydration

ascending grades of OH

hydration

descending grades of alcohol

Anhydrous Copper Sulfate

indicator that will accelerate the process

Blue color

color when it is fully/completely dehydrated