Title page

STANDARD OPERATING PROCEDURE OF

SUBMITTED BY

S.Kalaiarasi S.I.Geetha

K.Vidhyalakshmi S.Arthi

KAVG

JIVA JANTHU PARIKSHA KENDRA

SASTRA

SUBMITTED TO

Dr.S.PANCHAPAKESAN

JIVA JANTHU PARIKSHA KENDRA

SASTRA

SUBMITTING DATE

June 10, 2009

SOP OF HISTOPATHALOGY

KAVG

SASTRA- JEEVA JHANDHU PARIKSHA KENDRA

THANJAVUR

TAMILNADU

INDIA

AIM:

Histology is the study of tissue. To examine the tissue components, it is necessary

to process the tissue. The steps of processing involve: fixation, sectioning, and

visualization.

SCOPE:

Histopathological examination of tissues starts with surgery, biopsy, or autopsy.

The tissue is removed from the body, and then placed in a fixative which stabilizes

the tissues to prevent decay. The most common fixative is formalin (10%

formaldehyde in water).

SAFETY PROCEDURES:

Blood borne Pathogens (Exposure Control Plan)

Working with Chemicals

Chemical Hygiene Safety

Chemical Inventory

Emergency Procedures

Facilities (engineering controls)

Safety Equipment (engineering controls)

Training Records

Tuberculosis Guidelines

EQUIPMENTS:

Shandon Finesse 325 microtome

Feather Microtome blades

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

Knife box

Knife dispenser

A fine brush

Water bath

Waste tray

Kim wipes

Hot plate for sections to adhere to slides

Glass slides

PROCEDURE:

Trimming

Processing

Embedding

Slicing

Staining

Slide preparing

Microscopic analysis

TRIMMING:

The hand wheel brake is applied before mounting a Specimen.

Push the quick release lever of the cassette clamp backward, insert the

cassette, and release the lever check that the cassette is clamped firmly.

Use the vertical and horizontal tilt controls as appropriate to orientate

the specimen correctly with respect to the knife edge.

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

Lock the orientation head in position when the optimum orientation is

obtained.

Send the block holder to its rear limit by turning the coarse advance

knob (left side of Microtome) anticlockwise. The alarm sounds when the

limit is reached.

Select a cutting thickness using the dial on the right side – 4 µm is a general

thickness

Place a knife into the knife holder, release the clamping lever, located

to the right of the Clamp plate slide a knife from the knife box and slide

the knife beneath the clamp ensuring it is sitting straight – engage the

clamping lever and place the knife guard over the knife .

Bring the block towards the knife by rotating the coarse feed wheel

clockwise

Test if the block is close enough, release the hand wheel brake and

slowly rotate the hand wheel clockwise – the knife should just trim the

block

Slowly turn the hand wheel clockwise. Each time the handle of the

handwheel moves from the 12 o’clock position to the 1 o’clock

position, turn the coarse advance knob

Produced jointly by rmu and the unsw tohss working party Microns.

Continue turning the handwheel and advancing the specimen until

clean sections of the specimen are taken.

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

engage the handwheel brake when trimming is completed and the

specimen presents a clean smooth surface to the knife.

SECTIONING:

Place the block in the clamp and bring the block close to the knife using the

coarse feed Handle.

Set the thickness control to the desired setting.

use a new knife or new part of the existing knife by releasing the

clamping lever and Sliding the knife along – lock the lever when this is

done

If the block is in the correct position, release the brake and begin

cutting by rotating the Handwheel clockwise, collecting sections in a

ribbon

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

once you have the required sections, turn the handwheel to the top of

it’s rotation and Engage the brake

remove sections from the clamp plate using a fine brush underneath

the sections to separate them from the blade, and float sections on the water

bath .

NECROPSY AND TISSUE COLLECTION:

Gross exam with report on abnormalities

Collection and optimal fixation of tissues

KAVG

STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

PROCESSING:

In tissue processing the dissected tissue must be in the formalin for about 24 hours.

Chemical Fixation Tissue Processing:

The samples are transferred to a cassette, a container designed to allow reagents to

freely act on the tissue inside. This cassette is immersed in multiple baths of

progressively more concentrated ethanol, to dehydrate the tissue, followed by

toluene or xylene, and finally extremely hot liquid (usually paraffin). During this

12 to 16 hour process, paraffin will replace the water in the tissue, turning soft,

moist tissues into a sample miscible with paraffin, a type of wax. This process is

known as tissue processing.

The processed tissue is then taken out of the cassette and set in a mold. Through

this process of embedding, additional paraffin is added to create a paraffin block

which is attached to the outside of the cassette.

The process of embedding then allows the sectioning of tissues into very thin (2 - 7

micrometer) sections using a microtome. The microtome slices the tissue ready for

microscopic examination. The slices are thinner than the average cell, and are

layered on a glass slide for staining.

Frozen Section Tissue Processing:

The second method of histology processing is called frozen section histology. In

this method, the tissue is frozen and sliced thinly using a microtome mounted in a

refrigeration device called the cryostat.

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

The thin frozen sections are mounted on a glass slide, dried, and stained using the

same staining techniques as traditional wax embedded sections.

The advantages of this method are rapid processing time, less equipment

requirement, and less need for ventilation in the laboratory.

The disadvantage is the poor quality of the final slide. It is used less in diagnostic

pathology, but more in determining margin of a tumor during surgery.

EMBEDDING:

Correct orientation of samples in paraffin wax

Plastic embedding will be available in the future.

SLICING:

Normally slicing is done in 5 micro meter thickness to view a single cell layer.

A microtome works similarly to a cryostat but does not require cold. Instead, the

tissue is fixed, dehydrated and completely permeated with paraffin. It is then

embedded in a paraffin block. This block is stuck to a chuck, and then placed in a

holder, which is progressed by a rotating wheel.

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

STAINING:

The main staining agents are Hematoxylin and Eosin. This combination of stains

is used very commonly because it is quite useful for the display of general

structural features of the tissue.

Hematoxylin stains nuclear substances, chromosomes, mitochondria and muscle

striations blue to black. Eosin stains the cytoplasm and other structures various

shades of red.

Cresyl Violet is a Nissl stain, used for staining neurons which contain Nissl bodies

(rough endoplasmic reticulum). The cell bodies are stained a violet color.

Silver staining is a technique that has particular significance to the Neuroscience

community. Using it, Ramon y Cajal first demonstrated the shapes and the

interconnections in neural tissue.

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

In the slide that is labeled silver impregnated, neurons appear a yellow/orange

color, neurofibrils are brown to black, and neuroglia will be black. In the silver

stained tissue, only the glial elements remain stained as a black color.

Carmine: Dyes nuclei red.

Gold Chloride/Formic Acid: Muscle fibers red, myelin black; nerve fibers

brown/red.

HPS: Hematoxylin, Phloxine, Safron. Nuclei- blue; cytoplasm, muscle, myelin-

red; connective tissue- yellow.

Mason Stain: chromatin- blue to black; nuclei- red; zymogen granules- purple;

cytoplasmic elements- red to mauve; collagen, mucus or connective tissue- green.

MB&P: Methylene Blue and Phloxine: Methylene blue is also a Nissl stain which

stains the cell body blue. Phloxine stains collagen and other non-nuclear tissue

elements bright rose.

Nuclear Fast Red: Stains nuclei red.

Osmic acid: Myelin is stained black.

Wolke's Myelin Sheath: Myelin sheath, blue; background, clear; glial cells and

nucleoli of neurons, black.

SLIDE PREPARING:

The stained slides are taken out and dried for a required time.

Then they are closed with the slide cover.

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09

First the slide cover is placed on a neat surface and then Canada balsam

is applied as a drop.

Then the slide is placed over that cover & it is left for drying. Then it can

be visualized under microscopes.

MICROSCOPIC ANALYSIS:

When observing a freshly fixed section of tissue through a microscope, very little

contrast is observed between adjacent regions of tissue which may have vastly

different composition and function.

As a result, individual cells or parts of cells are difficult or impossible to visualize.

To solve this problem, scientists over time came up with various dyes, staining and

imaging techniques that allow different cell types, or structural or molecular

components, to be preferentially visualized.

HISTORY:

This SOP is prepared by trainers of group II batch under the

supervision of Dr.P.C.Prabhu with letter number KAVGDD_016_003

dated 05/06/2009 and it is valid till 12/05/2009

KAVG STANDARD OPERATING PROCEDURE

Title: Histopathology Doc. Number: KAVGDD_016_003 Rev No.01

Date Issued: 05/06/09