DEPARTMENT OF ORAL PATHOLOGY SEMINAR ON TISSUE PROCESSING

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) Fixation

Soft tissue Fixation

Decalcification
Dehydration Clearing Embedding Sectioning by microtome

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 undergo1. Ground section
2. 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.

 laboratory lathe coarse and fine abrasive lathe wheel

Equipment used for making ground section:

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 SECTION

The 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 crosssection 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.

DECALCIFICATION
For 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 DECALCIFICATION
For 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 Xray 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 AGENTS
The agents are: Nitric acid-5% Formic acid-10% EDTA solution The 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 FIXATION
It is done to preserve tissues permanently in a life-like state as much as possible.

PURPOSE OF FIXATION
To

prevent or arrest autolysis and bacterial decomposition and putrefaction. To coagulate the tissue so as to avoid or prevent loss of diffusible substances.
make the tissue strong enough to withstand the tissue processing treatment with reagent and wax embedding.
To

To prepare the tissue for differential staining methods with reagent and dyes.

 The fixation commonly used is 10% neutral buffered

COMMON TYPES OF FIXATIVES

formalin. Other fixative solutions are: Zenkers fluid (mercuric chloride,and potassium dichromate solution). Lugols solution (potassium iodide and iodide solution).

 Boulins 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.

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.

METHODS OF DEHYDRATION

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. 5860C).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.

When the tisues have been embedded

SECTIONING WITH MICROTOME

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 4.

Once sections are cut , they are floated on

PICKING THE SECTIONS

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 STAINING

The 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

The Staining process makes use of a

STAINING

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 rack

Stained slides

IMPORTANT FACT
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.

Frozen Section Fixation

This is accomplished through use of a frozen section. The piece of tissue to be studied are snap frozen in a cold liquid or cold environment(20C to -70C). Freezing makes the tissue solid enough to section with a microtome.

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

Poor sectioning
1. Knife marks (scratches perpendicular to knife edge) 2. Compression (waves parallel to knife edge)

Pitfalls

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

Coverslipping 1. Excess Permount

2. Two coverslips

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