Seminar on Tissue Processing as (2)

SUBMITTED TO-DR. KEYA SIRCAR (M.D.S.,H.O.D)DR. KENNATH J. ARUL (M.D.S.,READER)DR.SANJEET SINGH (M.D.S.,SENIOR LECTURER)DR.VARUN RASTOGI (M.D.S.,SENIOR LECTURER)DR.ARUN SHARMA (B.D.S.,LECTURER)

.

WHAT IS TISSUE PROCESSING???

The technique of getting fixed tissue into paraffin for histological study is called tissue processing.

TERMS ASSOCIATED WITH TISSUE PROCESSING

HISTOLOGY-It is the microscopic examination or

study of tissues.

HISTOPATHOLOGY-It refers to the microscopic examination of tissue to study the manifestations of the disease.

STEPS BEFORE TISSUE PROCESSING

SPECIMEN ACCESSIONING Tissue specimen received in the

surgical pathology laboratory have a request form that lists the patients information and history along with description of the site of origin. The specimen are accessioned by giving

them a number that will identify each specimen for each patient.

GROSS EXAMINATION Tissues removed from the body for

diagnosis arrive in the pathology department and are examined. Gross examination consist of describing the specimen for:

color, shape, size, number and the time at which the tissue received.

STEPS OF TISSUE PROCESSING

Tissue processing

Hard tissue(Teeth and bone)

Soft tissue

Fixation Fixation

Dehydration

Clearing

Embedding

Sectioning by microtome

Decalcification

STUDY OF HARD TISSUES

Some tissues that that contain calcium salts are extremely hard and will not section properly with paraffin embedding owing to the difference in densities between calcium and paraffin. Such tissues need special technique.

Before regular tissue processing and staining

hard tissues such as teeth and bone undergo-

1. Ground section2. Decalcification

GROUND SECTION

It can demonstrate the inorganic or calcified portion. Calcified tissues may be ground to a thin section.

Ground section is made by using following equipments.

Equipment used for making ground section:

laboratory lathe coarse and fine abrasive lathe wheel with water directed onto wheel wooden block 1x1 inches in size adhesive tape camel hair brush mounting medium microscope slide and coverglass

PROCEDURE FOR GROUND SECTIONThe specimen is cut in mesiodistal or

buccolingual plane into half by means of carborundum wheel if a longitudinal section is to be prepared. For the cross-section the tooth is cut half in horizontal or incisoapical direction.

The wooden block is wrapped with adhesive tape, sticky side directed outwards to which the tooth specimen is attached.

First coarse abrasive wheel of the lathe is used and then a fine abrasive wheel is used.

The tooth is ground upto 1-2mm thichness on the lathe.

Then later it is ground on an abrasive stone (Arkansas) manually.

When the desired thickness is achieved the section is carefully lifted by camel hair brush and mounted on the same slide.

DECALCIFICATIONFor the study of organic component and

structure, it is required that that hard tissue be made soft enough it to be cut by knife into

thin sections and then stain it.This is done by decalcification.

STEPS OF DECALCIFICATIONFor fixation, hard tissue is immersed in neutral

formalin.Decalcification is done by immersing the tooth

in acid of specify concentration until the specimen concentration until the specimen become soft.

The tissues is checked by piercing with needle or X-ray is taken for remains of calcified material.

The tissue is washed in running water for 24 hours to remove the acid compeletely.

The procedure after decalcification is same as for other tissue specimen.

DECALCIFYING AGENTSThe agents are: Nitric acid-5% Formic acid-10% EDTA solutionThe nitric acid decalcifies the tooth faster than

any other reagent,i.e. approximately within a week.The disadvantage of nitric acid is that the tissue integrity may not be preserved intact.

Formic acid and EDTA are slow decalcifiers but give better results.

ROUTINE METHODS FOR HISTOLOGICAL STUDY

FIXATION

Strengthening and prevention of tissue

decomposition.The agent used for

fixation of tissue is called fixative.

AIM OF FIXATIONIt is done to preserve tissues permanently in

a life-like state as much as possible.

PURPOSE OF FIXATION

COMMON TYPES OF FIXATIVES

The fixation commonly used is 10% neutral buffered formalin.

Other fixative solutions are: Zenker’s fluid (mercuric chloride,and potassium

dichromate solution). Lugol’s solution (potassium iodide and iodide

solution).

Boulin’s fluid (picric acid and formalde-hyde solution).

Carnoy solution (absolute alcohol and chloroform).

REMOVAL OF FIXATIVES The fixatives should be removed by over night washing. The tissue cassette is placed in a trough and is kept under running water overnight with a small stream of water into the specimen cassette.

DEHYDRATION It is the procedure to

remove water from the specimen.

WHY IS IT DONE ??? As wet fixed tissues (in aqueous

solutions) can be directly unfilterated with paraffin,therefore, firstly the water is removed from the tissue by dehydration.

METHODS OF DEHYDRATION It is done by using ascending grades of

alcohol starting from 70%, 80%, 90%, 95% and absolute alcohol (two

changes) are used to dehydrate the specimen. The tissues cassette is placed in each of the solution for minimum 1 hour.

CLEARING Removal of the dehydrant with a substance that

is miscible with the embedding medium (i.e. paraffin) is known as clearing.

Clearing agents used are: xylene toulene chloroform

INFILTERATION After clearing the tissue

is immersed in melted paraffin (melting pt. 58-60°C).This is done by an equipment called as paraffin wax bath for two hours with a change of half an hour in each container.

This process is known as Infilteration.

EMBEDDING After the specimen is

completely infilterated is melted paraffin it is embedded in paraffin.

Two L-shaped metal pieces are used to prepare a rectangular block.

The tissue is properly oriented so as to get the desired plane of section required.

SECTIONING WITH MICROTOME

When the tisues have been embedded must be cut into sections that can be placed on a slide.This is done with microtome.

The microtome is an equipment that holds a knife with a mechanism for advancing paraffin block.

The most important necessity for proper sectioning is a very sharp knife.

Microtome has a mechanism for advancing the block across the knife.Usually the distance can be set for most paraffin embedded the tissue at 4µ.

PICKING THE SECTIONS Once sections are cut , they are floated

on a warm water bath that helps to remove wrinkles .

Then they are pricked up on a glass microscope slide which is coated with egg albumin

The glass slides are then placed in a warm oven for about 15 minutes to help the section adhereto the glass slides

The tissue sections are cut and picked up on a glass slide . The sections are then ready for staining .

STEPS BEFORE STAININGThe embedding process must be

reversed in order to get paraffin out of the tissue and allow water soluble dyes to penetrate the sections .

Therefore , before any staining can be done ,the slides are “deparaffinized” by running them through xylenes ( or subtitutes )to alcohols to water

STAININGThe Staining process makes use of a

variety of dyes that have been chosen for their ability to stain various cellular components of tissue

The routine stain used is hematoxylin and eosin ( H and E ).

Other stains are referred to as “ special stains ” because they are employed in specific situations according to the diagnostic need .

Slide rackStained slides

IMPORTANT FACT

Frozen Section Fixation Frozen section is a rapid way to fix and mount histology sections. It is used in

surgical removal of tumors, and allow rapid determination of margin (that the tumor has been completely removed). It is done using a

refrigeration device called a cryostat. The frozen tissue is sliced using a microtome, and the frozen slices are mounted on a glass slide and stained

the same way as other methods.

It is a necessary way to fix tissue for certain stain such as antibody linked immunofluorescence staining. It can also be used to determine if a tumour is malignant when it is found incidentally during surgery on a patient.

APPLICATION OF TISSUE PROCESSING

It is used in microscopic study of diseased tissue(i.e. Histopathology) which is an important tool in anatomical pathology, since accurate diagnosis of cancer and other diseases usually requires histopathological examination of samples. Trained medical doctors, frequently board-certified as pathologists, are the personnel who perform histopathological examination and provide diagnostic information based on their observations.

Stained section of cancerous tissue

Pitfalls Poor sectioning 1. Knife marks (scratches perpendicular to knife

edge) 2. Compression (waves parallel to knife edge)

Mounting sections 1. Folds & tears 2. Excess albumin (stain)

Coverslipping 1. Excess

Permount 2. Two coverslips

THANK YOU

Made by :Amardeep Kaur –roll no. 03Apurva Sareen –roll no.11 Harpreet Kaur-roll no. 26Lucky Singh – roll no. 36Priyanka Gupta –roll no.64Taruna Malhotra –roll no.82