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
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

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
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

 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.
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

 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.
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

 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
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

 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
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

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.
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

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.
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

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.
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

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.
 Title: Histopathology

Doc. Number:
KAVGDD_016_003 Date Issued:
KAVG Rev No.01 05/06/09
STANDARD OPERATING
PROCEDURE

 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