HISTOTECHNIQUE

Histopathology

It is a branch of pathology which deals with

the study of disease in a tissue section.

The tissue undergoes a series of steps before it

reaches the examiners desk.

PROTOCOLS FOLLOWED IN

HISTOTECHNIQUES :

Receipt and identification

Labelling of the sample with a UNIQUE lab identification number

Fixation

Dehydration

Clearing

Impregnation

Embedding

Section cutting

Staining

Mounting

Type of material obtained in laboratory

The human tissue comes from the surgery and the

autopsy room.

From surgery two types of tissue are obtained.

1. A small piece of lesions or tumor which is sent for

diagnosis before final removal of the lesion or the

tumor (Incisional biopsy).

2. If the whole of the tumor or lesion is sent for

examination and diagnosis by the pathologist, it is

called excisional biopsy.

Tissues from the autopsy are sent for the study of

disease and its course.

Specimen

As soon as the specimen is received in the

laboratory

Check label (name, age, Hospital Registration No. and

the nature of tissue to be examined, clinical diagnosis

and the requisition form is also duly filled.)

Check if the specimen is in proper fixative.

Check if the financial matters have been taken

care off.

Make the entries in biopsy register and give the

specimen a pathology number which is also

known as an accession number.

FIXATION

It is a complex series of chemical events which

brings about changes in the various chemical

constituents of cell like hardening, however the cell

morphology and structural detail is preserved.

Unless a tissue is fixed soon after the removal from

the body it will undergo degenerative changes due

to autolysis and putrefaction hence, tissue

architecture will be lost.

Principle of fixation- The fixative brings about

crosslinking of proteins which produces

denaturation or coagulation of proteins; so that

it maintains everything in their in vivo relation.

Aims & effects of fixation

The preservation of cells & tissue constituents in

as life-like manner as possible.

To check against bacterial contamination.

Hardening : the hardening effect of fixatives allows

easy manipulation of soft tissue like brain,

intestines etc.

Solidification: solidify colloid material.

Properties of fixatives

Coagulation and precipitation (as described above).

Penetrate rapidly.

Solubility of fixatives - All fixatives should be

soluble in a suitable solvent, preferably in water so

that adequate concentrations can be prepared.

Concentration - It is important that the

concentration of fixative is isotonic.(Osmosis)

Reaction - Most fixatives are acidic. It may help in

fixation but can affect staining so has to be

neutralized.

NO fixative will penetrate a piece of tissue thicker

than 1 cm hence while dealing with thicker specimen

following methods are recommended.

SOLID ORGAN :- cut slices as necessary ,but not

thicker than 5 mm.

HOLLOW ORGAN :- either open or pack lightly

with wool soaked in fixative

LARGE ORGAN :- It requires dissection, inject

fixative along the vessels or bronchi as in case of

lung so that it reaches all parts of the organ.

Common fixative used for fixation of tissue

10% neutral formalin

Cheap, easily available, penetrates rapidly

Does not over harden the tissue when kept indefinitely.

Time required for fixation 24-48 hrs.

Disadvantage : formalin pigment is formed in areas of

hemorrhage.

The amount of formalin required for fixation of tissues

10 –20 times the volume of the specimen.

Mechanism of action :

Act by forming cross link between amino acids of the

proteins thereby making them insoluble.

Factors affecting fixation:

Size & thickness of tissue.

Organs containing large amount of blood fix slowly.

Fatty & lipomatous tissues fix slowly.

Time required for fixation.

At room temperature - 12 hours, For small biopsies - 4-6

hours. At 65°C fixation occurs in - 2 hours

Other fixatives

Glutaraldehyde

Mercuric Chloride (HgCl2)

Alcohol (Ethyl Alcohol)

Acetone

Potassium dichromate

Osmium tetraoxide

Acetic acid

Bouin’s fluid

GROSSING

Decalcification

Bone is a hard tissue so it is difficult to cut by microtome it hence needs to be softened before sectioning. The process of softening is done by removing the calcium from the bone. This is called Decalcification.

Decalcifying Agents : Two major types are :

1) Acids

Strong acids :Nitric acid (5-10 % -commonest), HCL

Weak acids : Formic acid, acetic acid, picric acid

2) Chelating Agents : EDTA

Histokinette

Histokinette

• 1st Jar Formalin 10% 1 Fixative

• 2nd Isopropanol 50% 1 Dehydration

• 3rd Isopropanol 70% 1.5

• 4Th Isopropanol 80% 1.5

• 5th Isopropanol 80% 1.5

• 6th Isopropanol 90% 1.5

• 7th Isopropanol 100% 1.5

• 8th Isopropanol 100% 1.5

• 9th Xylene 1 1.5 Clearing

• 10th Xylene 2 1.5

• 11th Paraffin 1 1.5 Impregnation

• 12th Paraffin 2 1.5

THE DIFFERENT STEPS IN TISSUE

PROCESSING

Fixation

Dehydration : stagnant water is a source of bacterial

contamination & tissue destruction. Water in cells is

removed with dehydrating agent . In ascending

grades of alcohol.

Clearing : removing alcohol on one side & replacing it

with paraffin on the other side. Xylene is used due to its

fairly rapid action. Other egs. Chloroform, Benzene, Carbon

tetrachloride, Cedar-wood oil.

Xylene: clearing

Xylene

Impregnation : The empty spaces in the tissue ,after

removal of water by dehydration is filled with

paraffin wax. This is known as impregnation. Done

by immersing the tissue in two changes of molten

wax. Adv: to make the tissue firm in consistency for

cutting.

Done in an oven heated to 52-54 C

Paraplast (purified paraffin + plastic ploymers)

In order to prepare a tissue for embedding, it

must be infiltrated with paraffin. Because water

and paraffin are not miscible, the specimens

must be gradually dehydrated to achieve

replacement of water with alcohol before the

clearing agent is introduced.

Once successfully dehydrated, a clearing agent

that is miscible with alcohol and paraffin (i.e.

xylene ) is infiltrated through the tissue. Finally,

the paraffin is introduced and completes the

tissue for embedding.

Embedding : The impregnated tissue is put in amould & molten wax is added so as to make a waxblock to hold the tissue for cutting.

Wax with melting point of 54-56 C is used.

Once the tissue has been processed it is ready to be orientated into a paraffin block and subsequently sectioned.

Orientation during embedding is crucial for the representation of proper morphology.

(structures in skin, small gastrointestinal biopsies, and vas deferens are among those for which orientation is especially critical.)

L-mould (Leuckhart’s embedding

mould)

L-mould (Leuckhart’s embedding

mould)

Two ‘L’ shaped pieces of heavy brass metal can be

adjusted to make various size moulds.

L-moulds are kept on a glass. Tissue is placed on the

bottom & then the space is filled with molten wax.

The wax is cooled & solidified, L-moulds are

removed.

The brick like square with embedded tissue is

called paraffin block.

Paraffin block

Tissue –Tek-system

Two parts

stainless steel base mould & plastic mould.

Tissue is placed in steel mould & filled with molten wax.

Plastic mould is placed on top of steel mould & again filled with molten wax.

Once the wax is cooled & solidified, steel mould is taken out.

Plastic mould remains & act as a holder for microtome for sectioning.

Microtome

A microtome (from the Greek mikros, meaning

“small”, and temnein, meaning “to cut”) is a tool used

to cut extremely thin slices of material, known as

sections.

TYPES OF MICROTOME

I. Sledge Microtome (Typical cut thickness achievable is

between 10 and 60 micron)

II. Rotary Microtome

III.Auto-cut Microtome

Rotary Microtome

Auto-cut Microtome

WATER BATH

Staining

H & E staining( Hematoxylin & eosin staining)

Mount in DPX.

Cryostat

Microtome is housed within an insulated,

thermostatically controlled refrigerated cabinet.

Few drops of gel is put on chuck . This freezes to

make an ice block, on the tip of which required

tissue is put.

Temp. maintained around -20 degree C.

Frozen section

Rapid diagnosis of tumors

Demonstration of fat by special stains

Quick Demonstration of amyloid

Localization of immunoglobulins

Intraoperative guide to surgeon as to surgical

margins are free of tumor

Mountant

The final stage in the preparation of tissues for

microscopy is mounting.

DPX (DISTRENE, PLASTICISER,

XYLENE)

One of the most commonly used mountants, DPX is a

colourless, neutral medium in which most standard stains

are well preserved. It is prepared by dissolving the

common plastic, polystyrene, in a suitable hydrocarbon

solvent (usually xylene).

Labelling and storage of slides

Slide mounted sections are identified during

preparation by inscribing the slide with the tissue

accession number or suitable code using a diamond

marker or pencil (frosted slides)

slides are usually stored, standing on their short

side, in metal or plastic drawers. Labels need to be

fixed to the opposite end and orientated so the

details can be read with the slide in this upright

position.

REPORTING