HISTOPATHOLOGY

 HISTOPATHOLOGY

Decalcification

This is a process of removing calcium from bone or other mineralized hard tissue to make it soft enough for sectioning.

Method of Decalcification

         i.            Acid method

       ii.            Ion exchange method

     iii.            Chelation

     iv.            Eletrical ionization.

Acid method

This is the common method done in most laboratory.

Procedure

Calcification hard tissue is cut in to small pieces (2-6mm in size) with a thin blade, or sharp knife. The cut pieces are fixed in buffered formalin or natural formalin for 8-10 minutes.

Tissue is washed thoroughly in water to remove fixative.

Tissue is kept in decalcified solution which may be one of the following type

a.       5% HNO3

b.       5% Formic acid

c.       A mixture of formic acid & HCL (5% formic acid 10ml+HCL 8ml

d.       D/W 82ml)

The tissue is suspended in decalcified solution by mean of gauze bag tied by string which has been dipped in matted paraffin.

The bag is suspended in large quantity of decalcified solution (more then 20 times the volume of the tissue) string of the bag is tied with a support in the cover of the beaker so that tissue can be regularly. The decalcification takes few days usually 2-4 day, But it can be done rapidly if the beaker is kept in an incubator at 56 ̊c Decalcify solution is change every day until the process decalcification is completed.

 

Test for complete Decalcification

Roughly it can be done by mechanically bending the tissue if the tissue can be bent like a soft tissue. It is suggestive of complete decalcification. It can also be done by the tissue with fine needle if the needle the tissue easily it means there is no calcium in it. But final test for decalcification is done by chemical method.

 

Chemical is done ib following ways.

Take 5ml of decalcified fluid which was in contain, with tissue in a clean test tube. The fluid is take in a test tube A piece of litmus paper turns blue color. If the fluid turns turvid during this process it means calcium is present in the solution.

If the fluid does not turn turvid it means there is no calcium in it.

The tissue is again suspended in fresh decalcified solution for few hours (2-4 hours). The decalcified solution of this is also tested for presence of calcium it means decalcification is completed.

The decalcified tissue is removed from the bag & is washed running tap water for 4 hours to remove decalcify solution.

To neutralized the acid in decalcified tissue. The tissue is treated with magnesium carbonate to neutralize the intracellular acid in the decalcified cells. The tissue is now ready for dehydration.

 

 

 

 

Dehydration

This is a process through which water from cells & tissue is removed so that space is subsequently taken up by wax. The dehydration is done by passing the tissue through ascending grades of alcohol 50%. Alcohol, 70% alcohol, 80% alcohol, 90% alcohol, absolute alcohol  1hrs in each. Ideal in ethyl alcohol, if this not available isopropyl alcohol of methyl alcohol may be used.

 

 

Clearing

It is process of making the dehydration tissue transparent to necked eye by removing alcohol. So that tissue can be made for paraffin embedding. Usual chemical used for this process is xylene for a tissue of 5 mm thickness it takes one hour. Prolonged stain xylene makes the tissue brittle. Other chemical which may be used as clearing agents are.

         i.            Cedar wood oil :- It is expensive viscus and needs removal by xylene.

       ii.            Benzene :- It causes less shrinkage by xylene but it is carcinogenic.

     iii.            Chloroform :- It does not change refractive of the tissue and hence and point of tissue can not be made up.

     iv.            Toluene :- It is poisonous and hence should not be used.

 

 

 

Paraffin infiltration

Infiltration is process of removal of xylene from the tissue by diffusion in the surrounding melted way. This is done by placing the clear tissue in to paraffin bath. Counting molten wax. After xylene has be removed the molten wax replaces the clearing agent. This process is known as impregnation.

Paraffin wax is maintain is condition by temperature of 55-60 ̊c.  This temperature is maintain it temperature is known as paraffin bath. The tissue kept in paraffin bath for 2-3 hours if temperature of paraffin bath becomes too hot (75 ̊c) the tissue will get cooked which becomes unfit for histopathological Examination.

 

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.

 

STUDY OF SEMEN

SEMEN ANALYSIS

Introduction

Problem with male semen a counts for 40% of all infertility another 40% released to female reproducts and harmon problem. The remaining 20% the pather semen Analysis is the cornerstone of testing for male in fertility this test provides important information about the quality and quantity of the sperm.

Semen is a composite solution consisting basically of spermatozoa suspended in the seminal plasma.

Semen is viscid neutral by slightly Alkaline and pale yellow colour due to its flavien content.

 

Collection of specime3n (Semen)

The usually recommended specimen one collected following a three day period of continence.

The most satisfactory – The specimen collected by masturbation in the clinical pathological Laboratory. This aloss a complete examination of the sperm particularly the liquification.

The specimen should be delivered with in 30min in laboratory.

(Note: - Wide mouth clean and dry bottle (50ml).

Precaution

The specimen should not be collected in condoms ,  Since the  power or lubricate applied in condoms may be spermici.The container in which the specimen is collected should be free from detergent.

Storage

The specimen should be examin immediately after collection. If it is necessary to dtore then it should be kept at R.T (25 ± 5^0C )

Sperm are easly damaged by either excessive heat or excessive cold.

 

Morphology of human spermatozoa

Each normal human spermatozoa is made up of two main parts.

1.       A Head :- It is flat and measure measure approximetly 0.5 micrometer in length 3 micrometer in wide and 1.5 micrometer in thick.

 

2.       A Tail  :- The tail measure about 50µm. It consist of 3 parts. The mid piece, The principal piece thr terminal piece.

 

3.       The tail means by which the sperms move.

 

 

 

Precaution

Name of the patient, Date & Time of Sample (semen) collection , Length of absentise and the time of interval between the collection analysis should be reconered in the semen analysis report.

 

Composition

The prostrating fluid in semen is acidic contain acid  phosphate and proteolytic enzymes which acts on the coagulum of the semen vesicles, thus resulting liquefication on the semen problem may their for inferty lesation.

Spermatozoa are cells which fertilized the egg, they are the main focus when performing a semen analysis.

Source of secration                                                                                         % ejaculate

         i.            Testis                                                                                                    5%

       ii.            Seminal                                                                                                46 – 80 %

     iii.            Prostate                                                                                               13 – 33 %

     iv.            Bulbo urethral and urethral gland                                                       2 – 5 %

 

Routine examination of semen

Throughly mix the sample before Examination.

Physical examination :-  Which is performed after a maximum of 60 min by which the time of liquiofication is complete.

1.       Colour and Appearance :- Normal semen is grey white, Vicid and Opque.

Case of Altered semen colour

                                            i.            Yellow Colour – Pyo spermmia (inflammatory cause) billrubin presence.

                                           ii.            Rust Colour (blooding) – Minnar bleeding in the seminal vesical.

2.       Volume :- Normal volume is 2-5 ml per ejaculate.

       Abnormal Volume

                                i.            Hypo spermia - <1.5 ml is called Hypo spermia.

                               ii.            Hyper spermia – Increase in semen volum >5.5 ml (A very rare phenomena )

                             iii.            A Spermia – Means the total Absess ejaculate.

                             iv.            AZoo spermia – Means the Abscess of Spermatozoa in semen.

                               v.            Oligo Spermia – Oligo spermia known as hyper spermia.

 

3.       Viscosity :- Take the specimen in the pasture piyette and expel it. The specimen whith normal viscosity can be poured drop by drop.

Ø  Inccreased viscosity result in poor invasion of cervical mucus as demonstrate by post coital studies.

4.       Liquefication time :- Normally a sample of semen fealiure liquefy in 60 minute indicates in adequate prostratic secresion.

 

 

Chemical Examinattion

Determination of PH :- By Ph paper strip method, normal 7.2 – 7.6 . Cause of ulteration in the ph.

Ø  7< - Obstruction to ejaculatory duct and conjenitial absence of seminal vesical. Such specimen contain mainly prostatic secretions and hence and acitic Ph.

Ø  Lesser then >7 – acute infection of the prostatic (Accute prostaties). Seminal vesicle and epididymis.

Quantitative determination of semen.

This is based on the principal that the fructose react with resorcinol. In the strong acidic medium to give a red colour complete which is compare with the known fructose slandered at 490nm.

Normal range :- 150 – 300 mg/dL

Ø  High fruetose lavel; (> 300 mg/dL) is Associated with low counts.

Ø  Low fruetose lavel (<150 mg/dL) it associated with decreased testrone and respond well to testestrone tharepy.

Study of motility of sperm

a.       Place a small drop of liquefied semen on a glass slide and cover it with cover slide.

b.       Examine the cover slip preparation under the high power objective reduced elimination.

c.       Count the number of active motile sperm and out  of the total count of the sperm.

d.       Culculate the sperm showing the actual progressing motion.

e.       Examine the slide after 2 hrs, 3 hrs and 6 hrs, To present the drying effect of cover slip preparation should be placed under a petridish with moist filter paper.

f.        Observe for pus cells, epithelial; cell , red blood cell and for other finding.

 

Determination of sperm count

a.       After liquification gently mix the specimen.

b.       Draw the semen up to the 0.5 mark of a WBC Pipette.

c.       Draw in the semen diluting fluid to the 11 mark and mix well.

d.       Load the improved neubour chamber and allow the sperm to the settle for about 5 minute.

e.       Count sperm in the four corners sequare.

 

Calculating Sperm Count

The formula for calculating the sperm count, when 5 small squares within the large center square are counted is:

Number of sperm counted x dilution factor/volume x 1000 = sperm/ml.

Example: 50 sperm are counted in the five small squares. The dilution is 1:20.

Sperm/ml = 50 x 20/0.02 mm³ x 1000 mm³/ml = 50,000,000 sperm/ml.

If the dilution is 1:20, and the usual 5 small squares are counted, then the formula can be simplified as follow:

Sperm/ml = Sperm counted x 1,000,000.

 

Composition of semen diluting fluid

 

Sodium bicarbonate                                                                        5 gms

Formalin                                                                                              1 gm

D/W                                                                                                       99 ml

It should be stored at R.T

Normal Range                                                                                   40 – 300 millions/ml

 

Cause of Low count

a.       Infection :- mumps or chetis prostanitis

b.       Obstruction :- Occlusion or absence of efferent duct.

Endo crinopathies :- Hipo pitutarism, hypo gonodism, hypo and hyper throdisim &estrogen secration tumors.

 

Determination of morphology of sperms

a.       After liquification make a thin smear of the semen on a glass (similar to blood smear)

b.       Let it dry in the air and then heat very gently to fix. If necessary remove the mucous by diping the smear in the semen diluting fluid & then in D/W.

c.       Stain the smear by using Leishman stain or stain 0.2% (weight/volum). Aqueous bnasic fuchsin for 5 min.

Normal Observatiom                                                                     Colour

Spermetozoa heat caps                                                                 light blue

Nuclear posterior                                                                             Dark blue

Bodies & Tail                                                                                  Red or Pink

Spermetazoa size                                                                            50 – 70 m

Head size                                                                                         3-6µ x 2-3µ

 

Observation for other abnormalities

         i.            Abnormally shaped head.

       ii.            Abnormally size head (Giant or minut head).

     iii.            Double heads

     iv.            Vacuoles in the chromatin.

       v.            Middle section – Absent, swollen.

     vi.            Tail – May be rudimentary, double or absent.

                                                                     RRANDHIR KUMAR

                                                    RDK PARAMEDICAL STUDENT STUDY