HISTOPATHOLOGIC TECHNIQUES

Fixation

- preserve the tissue in a lifelike manner

- Harden and protect

- Neutral Buffered Formaldehyde/ Formalin

- crosslink formation between proteins

-leaving the tissue in water swelling (hypotonic)

-leaving the tissue in strong salt- shrinking(hypertonic)

2 Mechanisms involved:

1. Additive Fixation

- fixative becomes part of the tissue

-formalin
-mercury
-osmium tetroxide

2. Non-additive fixation
- not incorporated
-alters tissue composition by removing H2O attached to H bonds
-ROH fixatives

Main Factors involved in fixation

1. Hydrogen Ion Concentration

-pH 6-8
2. Temperature
-Surgical spx: ROOM TEMP
-Tissue Processors: 40oC
-EM & Histochem: 0-4oC
*rapid fixation by FORMALIN: heat to 60oC
*the higher the temperature the greater the risk of distortion
*TUBERCULOSIS 100Oc
3. Thickness of Section
-EM: 1-2 mm2
-LM: 2cm2-4cm2
-BRAIN in 10%BF : 2-3 weeks fixation

4. Osmolality
-hypertonic-shrinks
-hypotonic & isotonic swells
- best results at slightly HYPERTONIC SOLN (400-450mOsm)
ISOTONIC SOLN (340mOsm)
-EM: Osmium tetroxide + Sucrose
5. Concentration
-Formaldehyde: 10%
-Glutaraldehyde: 3%
-Immuno-EM: 0.025% Glutaraldehyde
6. Duration of Fixation
-primary fix: 2-6 hrs
-EM: 3 hrs
TYPES OF FIXATIVES ACCORDING TO:
1. Composition
a. Simple one component
*Aldehydes
1.Formaldehyde- 10%, fixation time:24hrs,buffered to pH 7, dilution (1:10/1:20)
2.10% formol Saline- saturated formaldehyde diluted to 10% NaCl
- CNS, post-mortem, histochem
3.10% NBF surgical, post-mortem, research
4. Formal Corrosive Formol + mercuric chloride
- post-mortem
5. Alcoholic Formalin / Gendres Fixative post fix w/ phenol can enhance I
MMUNOPEROXIDASE studies
6. Glutaraldehyde
*Metallic
1. Mercuric- 5-7%, recommended for: Renal tissues, Fibrin, Con. tissue & Muscle
*mercury deposits are removed by treating with 0.5%iodine solution
in 70% ethanol for 5-10minutes
a.Zenkers fluid- Mercuric Chloride stock soln + GlacHAc
- Liver, spleen, CT fibers & nuclei
-recommended for trichrome staining
-immerse to alcoholic iodine-removal of mercuric pigments
b. Zenker-formol (Hellys soln)- pituitary gland, BM, bld cont. Organs
-produces brown pigments if prolonged staying in fixative but
may be removed by tx. w/ sodium thiosulfate
c. Heidenhains Susa- tumor biopsies esp of Skin
-tissue should not be more than 1cm thick
- must be transferred to high grade alcohol after fixation
d. B5- BM biopsies, rapid fixation
2. Chromate
a. Potassium Dichromate 3% aqueous soln
b. Chromic Acid 1-2% aqueous soln, CHO preserve, ppt. all proteins
- strong oxidizing agent
c. Regauds (Mullers) K2Cr + Formadehyde
- Chromatin, mitochon, mitotic figures, golgi, RBC
d. Orths fluid- early degenerative process, necrosis, demo rickettsiae
3. Lead 4% aqueous soln
- acid mucopolysaccharide
- takes up CO2 = insoluble lead carbonate
*Picric Acid- 1% yellow color may be removed by another acid dye or lithium carbonate
- suitable for Aniline
- ppt. all Proteins
A. Boiuns soln embryos & pituitary biopsy
- sat. Soln of Picric acid + Formaldehyde + GlacHAc
-should not be immersed into water since picrates are soluble in H2O
B. Brasils Alcoholic Picroformol Fixative
- formaldehyde + Picric acid +Ethanol + Trichloroacetic acid
- overnight tissue fixation 2-3 changes of fixative
* Glacial Acetic Acid solididies at 17 oC, destroys mitochondria and cytoplasmic elements
*Alcohol fixatives ppt CHON by destroying hydrogen bonds
- Photographic work 80% ROH
- both fixative and dehydrating agent
- causes glycogen granules polarization
a. Methyl Alcohol 100%
- dry &wet smears, blood smears, BM tx
 b. Isopropyl Alcohol touch prep, Wright Giemsa
c. Ethyl Alcohol 70-100%
d. Carnoys fluid chromosomes, lymph glands, urgent biopsy
-FIX BRAIN TISSUE
-most RAPID fixative, fixes % dehydrates, preserve NISSL GRANULES
e. Newcomers Fluid mucopolysaccharide & nuclear CHON
- better reaction in Feulgen than Carnoy
- both nuclear & histochem fixative
*Osmium tetroxide pale yellow powder dissolves in water, causes complete protein denaturation
a. Flemmings soln- most common chrome-osmium acetic acid fixative
-Nuclear preapration
b. Flemmings soln w/o acetic acid Chromic acid + Osmic acid
-cytoplasmic structures
*Trichloroacetic acid marked swelling effect
- may be used as weak decalcifying agent, ppt. CHON
*Aceetone (-5 C to 4 C), phosphatise &lipases
o o

*Heat fixation thermal coagulation of tissue CHON

- rapid dx
-frozen tx
-bacteirologic smear
c. Acetone
d. Alcohol
e. Osmic Acid
4. Heat
b. Compound- two or more components

2. Action
a. Microanatomical-general microscopy w/o structural alteration
-10% Formol Saline
-10% NBF
-Heidenhains Susa
-Formol Sublimate
- Zenkers Soln
-Zenker-Formol (Kellys soln)
-Bouins soln
-Brasils soln
b. Cytological- preserve specific parts
*Nuclear- contain glac.Hac(great affinity to nuclear chromatin)
-pH:4.6 or <
-Flemmings Fliud
-Carnoys Fluid
-Boiuns
-Newcomers fluid
-Heidenhains Susa
*in situ hybridization NBF, B5, Hollande, Zinc formaldehyde
*Cytoplasmic- must NEVER contain glacHac (destroys mito and golgi)
- pH: >4.6
-Flemmings fluid w/o acetic acid
-Kellys fluid
-Formalin with post chroming
-Regaud fluid (Mullers fluid)
-Orths fluid
c. Histochemical
a. Formol Saline 10%
b. Absolute Ethanol
 c. Acetone
d. Newcomers Fluid

LIPID FIXATION
- Cryostat
- Lipid stain
- Mercuric chloride & Potassium dichromate
- Bakers formol-calcium may preserve phospholipids
- Post fix with imidazole osmium tetroxide for ultrastructure lipid demo
- Cholesterol + Digitonin iltrastructure demo
CARBOHYDRATE FIXATION
- Roh fixatives = glycogen demo
- Alcholic formaldehyde = human skin preservative
PROTEIN FIXATION
- NBF or Formaldehyde Vapor
GLYCOGEN FIXATION
- Rossmans fluid
- Cold absolute ROH
- Better retention with CELLOIDIN coating

Mixture of Fixatives

1. Karnovskys paraformaldehyde-glutaraldehyde
2. Acrolein (aldehyde with gluta or formaldehyde)

SECONDARY FIXATION tissue in 10% NBF + Zenker (mordant)prior to:

Massons trichrome stain (connective tissue)
Mallorys aniline blue (collagen)
Phosphotungstic acid-hematoxylin (striated muscle)

POST CHROMATIZATION- 2O Fixation (tissue in 2.5-3% Potassium dichromate)

FACTORS AFFECTING FIXATION

Retarded by
1. Size & thickness larger *thicker takes longer time to fix
2. Presence of Mucus prevents complete fixative penetration
3. Presence of Fat
4. Presence of blood- flush out w/ saline
5. Cold temp-inactivates enzymes
Enhanced by:
1. Size & thickness
2. Agitation

DECALCIFICATION Calcium & lime salts are removed

-Use of chemical reagents:
a. form soluble Ca salts
b. Chelating agents that bind to Ca+ ions
*microcalcification appears DARK PURPLE GRANULAR MASSES W/ LIGHTER PURPLE HALOS
*grating sensation- block face down on a pad of cotton sat w/ 10% HCl for 1 hr.
ACID DECALCIFYING AGENT
I. Nitric acid most common & FASTEST, 5-10%
a. Aqueous nitric acid soln 10%, urgent small & needle biopsy, imparts yellow color; 12-24hrs
b. Formol Nitric acid yellow color may be removed by 5% NaSo4; 1-3 days
c. Perenyis fluid- routine, decalcifies & softens, but slow acting; 2-7 days
d. Phloroglucin Nitric acidmost rapid decal agent; 12-24 hrs

II. HCl- slower action & greater distortion, 1% soln + 70% ROH = surface decal of tissue blocks
a. Von Ebners fluid- NaCl + conc HCl + distilled water; recommended for teeth & small bones

III. Formic acid- moderate acting, recommended for routine decal; 2-7 days
-addition of CITRATE accelerate decal
-both fixative & decalcifying agent
a. Formic acid-NaCitrate soln recommended for autopsy; 3-14 days

IV. Trichlroacetic acid weak decal agent; 4-8 days

V. Sulfurous acid very weak decal agent

VI. Chromic acid (Flemmings fluid) both fixative & decal
-tends to undergo reduction & ppt formation
-environmental toxin, carcinogenic, extremely corrosive
VII. Citric acid-Citrate Buffer soln pH4.5 no distortion but slow; 6days
-Citric acid +Aammonium citrate +ZnSo4 + Chloroform

CHELATING AGENTS
-EDTA + Ca =insoluble non-ionized complex,
- very slow decal agent
-optimal binding at pH8
-EDTA inactivates ALP activity, restore by adding MgCl
ION EXHANGE RESIN
-removing Ca+ ions
-not recommende for fluids w/ nitric acid or HCl
-decal agent 20-30 times volume of tissue;1-14 days, can be measure by X-RAY
ELECTROPHORESIS
- Ca+ attracted to (-) electrode
- Solutions used: Formic acid + Conc. HCl + Distilled water

DEHYRATION removed fixative &water in tissue

-following fixation prior to wax impregnation
-70% to 95% to 100% ethanol
- for delicate tissues start at 30%
Commonly used dehydrating agents:
1. Alcohol most common
a. Ethanol- recommended for routine, best dehydrating because fast acting
b. Methanol- toxic, blood &tissue films
c. Butanol- slow, utilized in plant & micro animal techniques
*to insure complete dehydration- Anhydrous CuSo4(1/4 in deep covered w/ filter paper at the bottom)

2. Acetone- cheap, rapid acting used in urgent biopsy but not recommended to routine; 1-2 hrs
3. Dioxane (diethylene dioxide) dehydrating &clearing agent
-miscible in: H20, MELTED PARAFFIN, ROH & XYLOL
4. Cellosolve (Ethylene glycol monoethyl ether) rapid dehydration, combustible at 110-120 oF
5. Triethyl PO4- min shrinkage & distortion
6. Tetrahydrofuran dehydrates & clears, improves staining
Tissue softeners: Molliflex, Phenol
CLEARING- ROH and dehydrating agent is removed & replaced w/ substance that will dissolve wax
-translucent appearance
-low boiling points
-prolonged exposure to clearing agents cause tissue to become BRITTLE
A. Xylene- colorless, most commonly used;1/2 to 1hr
-used in clearing embedding & mounting
-most rapid, turns milky when dehydration is incomplete
B. Toluene
-used as a sub for xylene or benzene;1-2hrs
C. Benzene penetrates & clears rapidly;15-60 mins
D. Chloroform slower but less brittleness;6-24 hrs
-tissues tend to float in chloroform
-tough tissues, nervous tissues, lymph nodes & embryos
E. Cedarwood Oil- used to clear both paraffin &celloidin(3-5 days), require 2-3 changes; 2-3 days
-very penetrating
F. Aniline oil- used for delicate specimens
G. Clove oil min shrinkage
H. carbon Tetrachloride tissue hardens, similar to chloroform
I. Methyl benzoate & methyl salicy;ate- slow acting, double embedding techniques

IMPREGNATION /INFILTRATION
-Clearing agent is completely removed

EMBEDDING
-impregnated tissue is arranged into a precise position