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AUTOMATED TISSUE PROCESSING

Tissue processing
Tissues and cells examined outside the

body with an imagine system,are an artifact of the original. Histology and histopathology are comparative sciences where like is compared with like, like with unlike and even sometime unlike with unlike. It is essentisl to ensure that the artifact produced at any stage during tissue handling and processing is constant.

It is as much for this reason as any other

that the predominant method for producing tissue sections is by cutting paraffin wax-embedded tissue on a microtome. Freezing tissue to obtain a solid block from which thin sections could be cut is now confined to specialist areas such as nueropathology,lipid demonstration and urgent frozen sections.

This method of tissue section production

has recently increased. Paraffin wax is not miscible with water and as most fixatives where water based. There must be treatment of tissues to allow paraffin wax impregnation. This is called tissue processing.

Principles of tissue processing


The aim of tissue processing is to embed

the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut. The most satisfactory embedding material for routine histology is parafffin wax.

Stages involved
Dehydration: to remove fixative and water

from the tissue and replace them with dehydrating fluid. Clearing: replacing the dehydrating fluid with a fluid that is totally miscible with dehydrating fluid and the embedding medium. Impregnation: replacing the clearing agent with embedding medium. embedding.

DEHYDRATION
A. Definition: removal of water

B. Steps 1. wash out fixative 2. graded series of alcohol a. 70%, 95%, 100%, 100% 3. replace water by diffusion 4. not too long, not too short

DEHYDRATION
Procedure 1. automatic tissue processor a. overnight 2. Baths: water, 70,95,100,100 % alcohol 3. Clearing agent: 2 baths of xylene

CLEARING
A. Paraffin solvent B. Xylene, clearing agent C. Makes tissue appear clear

INFILTRATION
A. Replace xylene with paraffin B. Immerse in melted paraffin 55o C MP C. Remove all bubbles,

EMEDDING
D.Procedure 1. Place tissue cassette in melted paraffin 2. Fill mold with paraffin 3. Place tissue in mold 4. Allow to cool

Factors influencing the rate of processing


Agitation Heat Viscosity vaccum

Automated tissue processing


The overwhelming majority of

histopathology department now use machines to process tissue blocks. These machines reduce the time taken to process the routine tissue by manual method by atleast 24 hours.

Processsing machines
Traditional carousel Have 12 separate stages in processing. Minimum time taken is 30 minutes in each stage. Enclosed pump fluid type Programmed electonically processor takes 15 minutes per station.

Important points to take care for tissue processing


Fluid and wax containers must be filled to

the appropriate level and correctly located in the machine. Any spillage shoukld be wiped away. Accumulation of wax on any surface or beaker lids must be removed. Wax bath thermostats are set at least 3 degrees above the melting point of wax.

Paraffin wax bath on the carousel- type machine should be checked to ensure that the electric plux are in contact and the wax is molten.

Automated processing schedules


in most laboratories an overnight schedule is

used. With schedules of approximately 16 to 18 hours duration.

OVERNIGHT PROCESSING SCHEDULES


CONTAINER 1 2 FLUID 10% FORMALIN 70% ALCOHOL TIME IN HOUR 0 0.5

3
4 5

95% ALCOHOL
100% ALCOHOL 100% ALCOHOL

0.5
0.5 1

6
7 8 9 10 11 12

100% ALCOHOL
100% ALCOHOL 100% ALCOHOL/XYLENE XYLENE XYLENE WAX WAX

1
1 0.5 1 2 2.5 4