HISTOPATHOLOGY

         HISTOPATHOLOGY

The study of tissue i.e. called histopathology.

Microscopic study of tissue i.e. called histopathology. It deals with minute structure, composition and function of tissue. Histopathology means study of tissue disease, it needs preparation of microscopic slide of specimen remove through biopsy or surgical dissection.

Specimen has to pass through following staps.

         i.            Fixation

       ii.            Decalcification (Remove the calcium)

     iii.            Dehydration (Wash the tissue)

     iv.            Cleaning

       v.            Paraffin in filtration

     vi.            Embedding

    vii.            Section cutting

  viii.            Staining

      ix.            Mounting (DPX mount)

       x.            Reporting

Fixation

Fixation is a process by which tissue is mentain in the condition in which it as been remove from the body. It serves of following purpose.

         i.            It prevents autolysis and preserve cell and tissue consitutent.

       ii.            It heardness the tissue.

     iii.            It conserves semi solid consitancy of cell.

     iv.            It causes alteration of refractive index of cell which enbels and stain components to be better visualized.

Following reagent are employed as fixed.

a.       Formal dehyde

b.       Mercuric chloride

c.       Potassium dichromate

d.       Picric Acide

e.       Ethyl Alcohol

f.        Glutural dehyde

g.       Osmium teraoxide

Classification of fixative

They are broudly classified in to two (2) groups.

1.       Simple Fixative

2.       Compound Fixative

Simple Fixative :-  They are further classified in to four(4) groups.

1.       Aldehyde

a.       Formal dehyde

b.       Glutural dehyde

2.       Oxidising Agents

a.       Osmium tetraoxide

b.       Potassium permagnet

c.       Potassium dichromate

3.       Protein denaturin Agent

a.       Acetic Acid

b.       Methyl Alcohal

c.       Ethyle Alcohal

4.       Unkown mechanism fixative

a.       Mecuric cxhloride

b.       Picric Acid

 

Compound Fixative :-  That is produce by mixing two or more simple fixative. They are following types.

1.       Micro anatomical fixative

a.       Formaline

b.       Formal  sline

c.       Buffer formaline

d.       Formal calcium

e.       Hieden hains susa

f.        Zenker’s fluid

g.       Zenker’s formal

h.       Bouin’s fluid

i.         Gendre’s fluid

j.         Buffered formal sucrose

2.       Cytological fixative

a.       Nuclear fixative :- such as carnoy’s fluid and clarke’s fluid.

b.       Cytoplasm fixative :- Such as chmpy’s fluid for microchondria and lipid.

c.       Exfoliative cytology fixative:- Equal part of 95% ethyl alcohol.

 

3.       Histo chemical fixative

For demorstrative enzyme.

a.       Cold Acetone at 4 ̊c for demonstrative phosphates.

b.       Absolute Alcohal

c.       Vapours of formal dehyde.

4.       Secondary fixation

This is used after the tissue has been primary fixed to emproved staining.

a.       Zenker’s fluid

b.       Heiden hains susa

 

Alternative to fixation

This is done for immediate reporting during surgical opration. This is done by process known as freez drying. The small piecve of tissue is placed in a contener kept at 160 ̊c with liquid nitrogen intercellular cytoplasm is is flozen in to eyes which heard the Tissue and microsection can the cut with and stoment known as cryostate cut section is immediately and diagnosed.

 

Individual fixative

The following fixative are commonly used.

Formalin :-  Formal dehyde  Dissolve , in water yields formaline.

                It is commest fixative used the following advantage are available.

Advantage

a.       It is in expensive.

b.       Reudiah quilable.

c.       Easy prepare.

d.       Compatiblewith most stains.

e.       Penetrates the tissue rapidly.

f.        Best fixative for neutrological tissue.

Disadvantage of formaline

a.       Cause excessive harding of tissue.

b.       Unpleasent vapour causes irritation eyes.

c.       Its Acidic character may interfare with substain.

d.       May cause allergic reaction to the skin.

Other features of formalin

a.       It is universal fixative.

b.       It is neutrolized by calcium carbonate.

c.       Takes 6-8 hours for fixation on 4mm thick tissue at R.T.

d.       Require 15-20 times volume of specimen.

e.       Common type of preparation are 10% formalin for surgical tissue and post-mortem specimen.

f.        10% formalin sline for brain.

g.       10% buffered formalin, This prevents acid formation by formation buffered is acid sodium phosphate monophydrate and unhydrous di-sodium phosphate.

Ø  Process of removal of formalin pigment.

Ø  Wash briefly the tissue in water, Place tissue in 70% Alcohal, Treat section with picric acid in solution of NAOH – Place the section in Ammonium hydroxide (2% in 70% Alcohal)  Wash the                              tIssue , Wash  the tissue in remove  excess of ammonia this process is known deformalization.

Ø  Gluteral dehyde  The fixative is used when the tissue is going to process is electron microscope.    It is used at 4% solution for 4 hours. There are certain discdvantage.

                                 i.            It is Expensive

                               ii.            Penetration is slow.

Bovin’s fluid

Ø  It is compound fixative.

Ø  Tissue is fixed for 4-15 hours.

Ø  It is used for following purpose.

a.       Renal and testicular needle biopsy.

b.       When glycogen is to be demonstrated in the tissue.

c.       It can also be used for general purpose..

Composition

1.       1.22% Picric acid – 750 ml.

2.       40% Formalin – 250 ml.

3.       Glacial Acidic Acid  --  50 ml.

Disadvantage

They may be yellow colouration of tissue due to picric Acid. It max the tissue heard.

It causes lyses of red blood cell.

Process of removing of yellow pigment

1.       Deparaffinised the tissue.

2.       Wash the tissue in Alcohal.

3.       Wash the section for seviere hour in 70% and 50% alcohol.

Carnoy’s fixative

It is also mixed fixatives.

It is used for following purpose.

a.       When tissue is to be stain for glycogen.

b.       Cytological smear small pieces o0f tissue such as endo metrium.

Composition

a.       Absolute Alcohal – 60ml.

b.       Chloroform   -- 30ml

c.       Glacial Acitic acid  -- 10ml

Advantage

1.       Penetration is good.

2.       Tissue in this fixative does not required already dehydrated during fixation.

3.       Fixation is by act of denaturation of cell protein.

4.       Tissue is used for 12-15 hours in refrigerator or 1-3 hours at R.T.

 

Osmium tetra oxide

It is fixative  for nervous tissue it is also used for electron microscope. It is best for lipid and is used as 2% solution. It in parts black colour in tissue.

Zenker’s fluid

It is fixative for connective tissue

         i.            Mercuric chloride – 50gram

       ii.            Potassium dichromate – 25gram

     iii.            Sodium sulphate  – 10gram

     iv.            D/W made up to – 1leter.

Preparation

Ø  5ml glacial Acidic Acid is called to 95ml of zenker fluid just before use.

Ø  This fluid is used when tissue is to the stained by masson’s Trichomstain for connective tissue.

Ø  Before staining tissue is washed for several hours in running tap water to remove mercuric chloride pigmentation.

Ø  Advantages of this fixative is causes excellent staining for connective tissue.

Ø  Disadvantages is over right washing of fixative tissue is require to remove yellow colouration caused by potassium dichromate.

Ø  Mordanting is required to prevent five granules of mercuric.

Ø  Method of moderating

Deparaffinised tissue with

                Wash with Absalute Alcohal

                Wash in 95% Alcohal

                Keep the slide in Alcoholic iodine salution for 10 minutes.

                Wash in 5% sodium thiosulphate for 5 minute remove iodine.

                Keep in it running water to remove sodium thiosulphate.

                Stain.

 

Hely’s fluid

It is a stain for  reticulam fiber and elastic tissue.

Composition

         i.            Mercuric chloride – 5gram

       ii.            Potassium dichromate – 5gram

     iii.            Sodium sulphate  – 10gram

     iv.            D/W made up to – 1leter.

Mix-well

Preparation of working solution

Ø  95 ml of Hely’s solution is mix with 5 ml of 40% formalin.

Ø  Advantage and Disadvantage is same as Zenker’s fluid.

Ø  Buffered formal sucrose fixative.

Ø  This fixative is used for phospholipid enzymesand electron microscope examination.

Preparation of this fixative  

Formalin is mixed with sucrose and M/15 phosphate. Buffer in equal volumes at PH is Adgested to 7.4.