HISTOCYTOPATHOLOGY NOTES COMPLETE

HISTOPATHOLOGY TECHNIQUES

                    CLASSIFICATION OF FIXATIVES

 

Based on the parts of tissue or cells to be demonstrated

NATURE OF FIXATIVES	NAME OF FIXATIVES
Microanatomical fixatives	•   10% formalin •   Neutral buffered formalin •   Formal saline •   Buffered Gluteraldehyde •   Zenker Fluid, Zenkers formol •   Formal Calcium
Cytological Fixatives
Cytoplasmic Fixative      (pH ≥ 4.6)	•  Zenkers fluid •  Flemming Fluid without acetic acid •  Schaudinn fluid •  Regaud's fluid •  Formal saline and formal calcium
Nuclear Fixative      (pH ≤ 4.6)	•  Carnoy's fluid •  Clarke fluid •  Flemming Fluid •  Newcomer's fluid
Histochemical Fixatives	•  Formal saline •  Cold acetone •  Absolute alcohol
Based on fixative's ability to fix by coagulating protein
NATURE OF FIXATIVES	NAME OF FIXATIVES
Coagulant Fixatives	Mercuric chloride, Chromium, Picric Acid Ethanol
Non Coagulant fixatives	Formaldehyde, osmium tetroxide, Pot. Dichromate, Acetic acid
Based on number of compounds mixed in a fixative
NATURE OF FIXATIVES	NAME OF FIXATIVES
Simple Fixatives
Aldehydes	•  Formaldehyde •  Gluteral dehyde
Oxidizing Agents	•  Osmium tetroxide •  Potassium dichromate •  Potassium permanganate
Protein Denaturing Agents	•  Acetic acid •  Ethyl alcohol •  Methyl alcohol
Unknown Mechanism	•  Picric acid •  Mercuric chloride
Compound Fixatives -    Neutral buffered formalin -    10% Formol saline -    Zenker's fluid, Zenker's formol
Metallic Fixative
NATURE OF FIXATIVES	NAME OF FIXATIVES
Mercuric fixatives	•    Zenker's •    Helly's •    Heidenhain's Susa •    Schaudinn fluid •    B5 fixative
Chromate fixatives	•    Zenker's  fluid •    Orth's fluid •    Regaud's
Lead fixatives	• Lillies fluid • Lead sub acetate
OTHER FIXATIVES
NATURE OF FIXATIVES	NAME OF FIXATIVES
Picric acid containing fixatives	•    Bouin's fixative •    Gendre's fixative •    Brasil's alcoholic picroformol
Alcoholic Fixatives	Methyl alcohol Ethyl alcohol Carnoy's fluid Clarke's fluid

Fixatives for different target substances

S.N.	Target Substance	Fixative
	Proteins	Neutral Buffered formalin
	Lipids	Osmium tetroxide Gluteraldehyde
	Glycogen	Carnoy's fluid Bouin's fluid
	Carbohydrates	Heidenhain's Susa Buffered Gluteraldehyde Formal calcium
	Biogenic amines	Bouin's fluid Neutral buffered formalin
	Myelin sheath	Flemming's fluid

 

TIME OF FIXATION REQUIRED FOR VARIOUS TISSUES

 

Tissue	Fixative of choice	Time for fixative
Routine.	Formalin	10-12 hours
GIT biopsies	buffered formaldehyde	4-6 hours
Testicular biopsy and embryos	Bouin's fixative	4-6 Hours.
Liver Biopsy		4-12 hours
Bone marrow and pituitary tissue	Helly's fluid (Zenker's formol)
Spleen and liver	Buffered formaldehyde, Zenker's fluid	1-6 hours
Lymph node	B5	12-18 hours
Mictocondria, phosphatides and Nissil substance	Carnoy's fluid	1-2 hours
Chromosome / cell culture	Clarke's fluid	1-2 hours
Smears for H&E Staining	Schaudinn's fluid	2-5 min
Fibrous tissues	Heidenhain's Susa	12-24 hr
Whole Organs	Neutral Buffered Formalin

 

Vapour fixatives

1.  Formaldehyde- Vapour is obtained by heating paraformaldehyde at temperature between 50°  and 80°C. Blocks of tissue require 3-5 hours whereas section require ½- 1 hours.

2. Acetaldehyde- Vapour at 80°C for 1-4 hours.

3. Glutaraldehyde- 50% aqueous solution at 80°C for 2 min to 4 hours. 4. Acrolein /chromyl chloride- used at 37°C for 1-2 hours

Secondary fixation:- Mercuric chloride for 4 hours

Post chromation:- 3% potassium dichromate following normal fixation for myelin and phospholipids demonstration ( for pre and post mordanting).

 

REMOVAL OF PIGMENT:

 A.     Formalin pigment (Three methods)

Method1-Saturated alcoholic picric acid followed by 30 minutes running tap water (15 minutes).

Method2-One per cent sodium hydroxide in 70% ethanol followed by five minutes running tap water (10 minutes).

Method3-Ten volumes hydrogen peroxidase followed by five minutes running tap water (five hours)

 

B.      Mercuric chloride pigment

Gram’s or lugol’s iodine for 5 minute  followed by treatment with 5 % sodium thiosulphate for 5 min

C.     Osmium tetroxide deposit

Hydrogen peroxide in 70% alcohol treatment followed by sunlight bleaching followed by 0.5% potassium permanganate followed by sulfurous acid treatment

 

  

DECALCIFICATION

 

1.       Removal of calcium by acids:- (a) Strong acid, (b) weak acid.

a. Strong acid

Nitric acid- 5-10% aqueous solution used(Most commonly used)- 1-3 days

Formalin Nitric acid- 1-3 days

Hydrochloric acid (Von Ebner's Fluid): 5-10% aqueous solution : 3-5 Days

b.   Weak acid:- formic, acetic and picric acid (5-10% aqueous solution or with additives like formalin)

2.       Gooding and Stelwart's fluid:- formic acid,Formalin and D/W- 2-7 Days

3.       Evans and Krajian fluid:- trisodium citrate,formic acid 2-7 Days

4.       Ion exchange resins

5.       Chelating agents- EDTA- 3 weeks

6.       Electrolytic method:- The electrolyte is HCL and Formic acid in D/W

 

Determination of end point of decalcification

-          X-ray method

-          Chemical Method :-  uses 5% ammonium oxalate and 5% ammonium hydroxide   solution

 

Softening of Hard tissues:-

-       Perenyi's fluid (10% HNO3+Absolute alcohol+0.5% Chromic acid)

-       4% aqueous solution of phenol for 1-3 days (lendrum’s technique)

 

 

 TISSUE PROCESSING

 

Steps:- Fixation, Dehydration, Clearing , Embedding and mounting

1.       Fixation

2.       Dehydrating agents:

-       Ethanol

-       Methanol

-       Acetone- missible with epoxy

-       Isopropyl alcohol

-       Dioxane- Both dehydrating and clearing agent

 

3.       Clearing agents

-       Xylene:- Causes brittleness to tissue on prolonged use

-       Toluene or  Benzene : Does not make the tissue brittle

-       Chloroform:

-       Carbon tetrachloride

-       Cedar wood oil (Histological): best clearing agent, does not harden the tissue but expensive

 

 

4.       Impregnation

a)      Waxes (Temperature 56-60⁰c)

-       Paraffin wax

-       Bee wax

-       Paraplast

-       Bioloid

-       Ester wax

-       Water soluble waxes

b)      Celloidin - for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo

c)       Nitrocellulose Method:-

d)      Dry celloidin method is preferred for processing of whole eye sections

e)       Gelatin Impregnation - when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies

5.       Embedding

-       Leuckhart’s Embedding Mold

-       Compound Embedding Unit 

-       Plastic Embedding Rings and Base mold

Double Embedding Method:- tissues are first infiltrated with celloidin and subsequently embedded in paraffin wax. Eg cutting of large blocks of dense firm tissues like the brain.

 

SECTION CUTTING

 

Types of knieves

Plane wedge : for paraffin and frozen sections.

Planoconcave : celloidin section

Biconcave : paraffin section cutting on rocking and sledge type of microtome.

Tool edge : used with a heavy microtome for cutting very hard tissues like undecalcified bone

 

Honing

Grinding of knife on a hone to restore straight cutting edge and correct bevel.

Hones

-       Belgian black vein or Belgian yellow

-       Arkansas – Not very fast

-       Aloxide

-       Carborundum

-       Plate glass

Stroping

It is the process of polishing an already fairly sharp edge. It removes burrs formed during honing.

-       Horse neck leather

-       Heart woods

 

 

TYPES OF MICROTOME

1. Cambridge Rocking microtome:- knife is fixed, block moves

2. Sliding microtome:-  Moving knife and fixed block

3. Rotary microtome:-

-   Knife is fixed and block moves.

-   Most commonly used

-   Best for serial sections

4. Freezing microtome(cryo microtome):-

-   For frozen sections

-   Useful when lipids, enzymes, and neurological structures are to be demonstrated.

5. Base sledge microtome

-   Used for cutting whole organ or large tissue blocks

 

 TISSUE THICKNESS REQUIRED FOR VARIOUS SAMPLES

Tissue Types	Micron Requirement
Brain and central nervous system (CNS) tissue	8-10 µ Requires much thicker sections to show neurons
Tissue for amyloid diagnosis employing Congo red stain	8-10 µ
Renal (kidney) biopsies	2 µ
Lymph nodes	No thicker than 3 µ

 

Faults in section cutting, causes and their remedy

	Fault	Cause	Remedy
1	Tear or scratch across the section or splitting of ribbon Sharpen the knife	- Jagged knife edge - Dirt or hair on knife edge	Clean the knife
2	Tear or scratch across part of section,	- Calcium, Carbon, or Suture etc., in the tissue or wax	Examine block under magnifying glass. If calcium is present, decalcify block. Remove suture from the tissue with scalpel point. If dust is in wax - Re-embed
3	Holes in the section.	- Air bubbles in the tissue or wax - A piece of hard material in tissue - A soft piece of tissue in block	Re-embedd Reprocess specimen Remove hard material if possible
4	Cracks across the section parallel to knife	- A blunt knife - Knife tilt too small. - Block too hard for thickness of specimen	Sharpen knife Adjust tilt Warm block slightly or re-embed in soft wax.
5	Section shows thin and thick horizontal lines (chatters)	- A loose knife - A loose block A blunt knife - Extremely hard tissue	Tighten knife and/or block Sharpen the knife Soften the tissue if possible or embed in harden wax
6	section cut thick and thin alternative	- Knife tilt is too great and is compressing the block.	Adjust tilt
7	Section compress at one end	- Blunt spot on the knife - A soft spot in the wax, due to presence of clearing agent	Move block along the knife or sharp knife. Re infiltrate tissue and re-embed
8	Section curves to one end.	- Edge of block is not parallel to knife. - A dull spot on knife.	Trim edges   Move block along knife or sharpen knife.
9	Section curl as the they are cut	- Blunt knife - Sections too thick - Too much tilt to knife	Sharpen knife   Adjust microtome Correct the tilt
10	The tissue is shrunken away from wax	- Insufficient dehydration	Reprocess
11	The tissue is too soft when block is trimmed	- Insufficient fixation	Reprocess
12	Specimen crumbles and drops out of the wax leaving a rim of wax as a section	- Insufficient infiltration - Overheated paraffin bath causing tissue to become hard and brittle	Re infiltrate and re-embed   Service the paraffin bath
13	Tissue is dried out or mummified	- Mechanical failure of tissue processing  machine  or  a basket was out of balance and hung up.	Place the specimen in the following rehydration solution for 18-24 hrs. Sodium Carbonate  -   1.0 gm Dist. Water                                                                                         - 70.0 ml Absolute ethyl alcohol - 30.0 ml Re hydrate the reprocess
14	Sections crumble on cutting	- Knife is blunt - Wax is too soft; has crystallized due to slow cooling or contamination with water or clearing agent. - Defective processing e.g. incomplete fixation, dehydration, clearing or embedding.	Sharpen knife.   Re-embed and block with fresh wax Reprocess
15	Failure of block to ribbon	- Block not parallel to ribbon   - Paraffin too hard. Knife tilted too much Sections too thick	Correct the alignment   Re-embed Correct the tilt Adjust the section thickness

 

 

Staining

 

Some common terms used in Histopathology staining

 

S.N.	Term	Meaning	Example
1.	Premordanting	The mordant is applied first, followed by the dye.	Heidenhain’s iron hematoxylin
3.	Metachrome	Mordant and dye are mixed together then applied.	Alum hematoxylin solutions
4.	Progressive staining	Decolorization is not done.	Mayer’s, Delafield’s, Gill’s, Harris’, Carrazzis'
5.	Regressive staining	The slide is over stained and then decolourised.	Delafield’s, Harris’,Ehrlich’s
6.	Birefringence	The colour (different from original stain) shown by slides when viewed under polarized light	Amloyds: Pink to red colour under plain light and Apple-Green colour under polarized light
7.	Argentaffin cells	the cells having strong reducing groups reduce the  ionic silver to metallic silver by itself	E.g. Melanin: Massons Fontana stain
8.	Argyrophilic cells	Cells absorb the silver ions but require the addition of a separate reducing agent in order to converted silver into elemental silver	Warthin-Starry stain for spirochetes

 

Hematoxylin

Extraction: extracted by boiling the heart wood of Hematoxylon Campechianum in hot water.

 

Classification of Hematoxylin

A.      Based on type of oxidation

1.       Natural oxidation:

§  Ehrlich's Hematoxylin

§  Delafield's Hematoxylin

2.        Chemical oxidation:

§  Sodium or Potassium Iodate

§  Mercuric chloride

B.      Based on the mordant used

1.  Alum haematoxylins: Mordant used are aluminum salts, either aluminum

potassium sulphate (potashalum) or aluminum ammonium...

 

TYPES OF ALUM HEMATOXYLIN

a)       Ehrilch’s haematoxylin.

b)      Mayer’s haematoxylin.

c)       Harris’s haematoxylin.

d)      Gill’s haematoxylin.

e)      Cole’s

f)        Delafield's

g)       Carazzi's

 

2.  Iron haematoxylins

3.  Tungsten haematoxylins

4.  Molybdenum haematoxylins

5.  Lead hematoxylins

6. Hematoxylin without mordants

 

Progressive Hematoxylins: Mayer’s, Delafield’s, Gill’s, Harris’, Carrazzis'

Regressive Hematoxylins: Delafield’s, Harris’,Ehrlich’s

 

Type of Hematoxylin	Mordant Used	Oxidant	Uses	Staining time	Remarks
A.       Alum Hematoxylin
Ehrilch’s	Potassium alum	Natural	Nuclear stain used with eosin. Fading of colour is much slower	20-45 min
Mayer’s	Potassium or ammonium alum	Sodium iodate	Celestine Blue Hemalum method of Nuclear Staining	Progressive: 5-10 min Regressive:10-20 min
Harris’s	Potassium alum	Sodium iodate	Routine histopathology; Exfoliative Cytology and for staining of sex chromosome	4-6 minutes
Gill’s	Aluminum sulfate	Sodium iodate	Nuclear stain used with eosin	Progressive:5-15 min
Cole’s	Potassium or ammonium alum	Alcoholic iodine	Nuclear stain used with eosin. used in sequence with Celestine Blue	20-45 min Used with Celestin Blue stain
Delafield's	Ammonium alum	Natural	Blood films for spirochaetes, Protozoa
Carazzi's	Potassium alum	Sodium iodate	For Frozen section	Progressive:
B.       Iron haematoxylins :- The iron acts both as mordant and oxidizer. (Resist decolourization , used especially for acid stains e.g. Trichrome stains, PTAH, Picrosirius Red Stain)
Weigert’s	Ferric chloride	Natural	Masson’s Trichrome Stain, van Geison's stain	Regressive: 20-30 min	Differentiator:
Heidenhain’s	Ferric ammonium sulphate	Natural	For Intestinal parasites, for fungal mycelia in cytology; for mitochondria, muscle striations, chromatin, myelin	Regressive: 1 hour	Differentiator: 5% iron alum
Verhoeff’s			Elastic fibres		2% aquous ferric chloride
Loyez			myelin
C.       TUNGSTUN HEMATOXYLIN
Mallory's Phosphotungstic acid hematoxylin (PTAH)	Phosphotungstic acid	Potassium permanganate or  Natural	Nervous tissues:	Regressive: Over night	Chemically ripened hematoxylin can be used only for 24 hr
D.      Molybdenum Hematoxylin
Thomas Phosphomolybdic  acid hematoxylin	Phosphomolybdic  acid		Used for argentafin cells		Not Widely used
E.       Lead Hematoxylin
Solcia Hematoxylin	Lead salts	No oxidation	For secretory granules in endocrine cells.
F.       Hematoxylin without mordant
Unripened Hematoxylin	…..	……..	For demonstration of minerals: Lead, Iron and Copper

 

S.N.	STAIN Method	Used For	Result
1.	Hematoxylin & Eosin Stain	Most routine stain	Nuclei: blue (hematoxylin) Endoplasmic reticulum: blue (hematoxylin) Elastic fibers: pink (eosin) Collagen fibers: pink (eosin) Reticular fibers: pink (eosin) Red blood cells: orange/red
2.	Cresyl Fast Violet	NISSL
3	Congo Red	Amyloid (Mostly used in patients with Alzheimer’s disease)	pale orange-red (apple green birefringence under polarized light)
4	Luxol Fast Blue	Nerve fibers	Myelin fibres: blue to blue/green Neurons: violet (when counterstained) Red blood cells: blue
5	Masson's Trichrome
6	Mallory’s Muscle Fiber
7	Grocott-Gomori's (or Gömöri) Methenamine Silver	Fungal elements Including Pneumo	black (with sharp margins and cleared center) Background: light green
8	Verhoeff-van Gieson Stain	Elastic fibre	Elastic fibers and cell nuclei: black (Verhoeff component: Iron Hematoxylin) Collagen and muscle: Red (van Gieson component: Picric acid and acid fuschin) Cell cytoplasm and other components: yellow
9	Warthin-Starry Silver impregnation	Spirochaetes and small bacilli	Microorganism: dark brown to black Background: golden brown (due to lower concentrations of silver deposits)
10	PTAH	Intracellular filaments
11	Nissl Staining	Brain & Spinal Cord
12	Thionin	Nissl
13	Toluidine Blue	Mast Cells
14	4′, 6-diamidino-2-phenylindole (DAPI)	stain DNA	Nuclei: blue
15	Alkaline phosphatase	Pluripotent cells:	stem cell membrane: red/purple
16	Bielschowsky Stain	visualize nerve fibers.	Axons, plaque neurites, and tangles: black Plaque and vascular amyloid: generally brown to dark brown
17	Oil Red O	Lipids stain	Neutral triglycerides and lipids (frozen sections), lipoproteins (paraffin sections), and lipofuscin: Bright red
18	Sudan Black B	Lipids stain	Neutral triglycerides and lipids (frozen sections); some lipoproteins (paraffin-embedded sections): blue-black Nuclei: red stains myeloblasts, but not lymphoblasts.
19	Periodic-acid Schiff	Glycogen	Glycogen and other carbohydrates: magenta Nuclei: blue Collagen fibers: pink
20	Periodic-acid Schiff with Diastage	To differentiate mucin from glycogen	Glycogen is digested but mucin is not and stains Magenta
21	Alcian Blue -     pH-2.5 -     pH 5.8	-    Acid Mucin -    Neutral Mucin	-          Blue
22	Perl’s Prussian Blue	hemosiderin (type of iron-storage complex found inside cells) deposits, for instance in some liver diseases or hemolytic anemia.	Iron deposits: Blue or purple Other tissue components: red (when counterstained with neutral red)
23	Toluidine Blue	Mast cells	Mast cell granules and polysaccharides: violet Nuclei: blue Cytoplasm: blue Red blood cells: blue Collagen fibres: blue

 

 

STAINING METHODS BY NATURE OF SUBSTANCES

Stain	Substance	Interpretation
Amyloid
Congo red under polarizing microscope	Amyloid	Green-birefringence
Carbohydrates
Periodic acid-Schiff (PAS)	Glycogen, mucin, mucoprotein, glycoprotein, fungi, basement membranes of glomeruli and tubules	Magenta color
Mucicarmine/Best’s carmine	Epithelial mucin	Red color
Alcian blue	Acid mucin	Blue
Lipids
Sudan III	Lipid	Orange
Oil Red O		Red
Osmium tetroxide		Brown black
Connective tissue
Van Gieson	Extracellular collagen	Red
Masson’s trichrome	Collagen, smooth muscle	Collagen-blue, smooth muscle-red
Phosphotungstic acid hematoxylin (PTAH)	Cross striation of skeletal muscles, glial filaments, fibrin	Dark blue
Verhoeff’s elastic	Elastic fibers	Black
Microorganisms
Gram’s stain	Bacteria	Gram+ve = blue Gram-ve = red
Ziehl-Neelsen’s (Acid-fast) stain	Tubercle bacilli and other acid-fast organisms	Red
Fite-Faraco	Lepra bacilli	Red
Silver methanamine	Fungi	Black
Pigments and minerals
Prussian blue stain (Perl’s stain)	Hemosiderin	Blue
Masson Fontana	Melanin	Black
Von Kossa	Calcium	Orange red
Alzarine Red S		Black
Rubeanic acid	Copper	Greenish-black