TISSUE PROCESSING

AT MICROSCOPIC LEVEL-

HISTOLOGY Science of examination of normal tissues

HISTOPATHOLOGY Examination of tissues for presence /

absence of changes in structure due to disease process

What happens to the SPECIMEN?

Specimen received in the lab (10% formalin)Grossed (appearance, measurements, noticeable

pathological changes etc) and kept for formalin fixationBits given from representative areas ( not >4mm thick)Tissue processed…Final outcome : stained slide for microscopic

examination

TISSUE PROCESSING

1. Fixation

2. Dehydration

3. Clearing

4. Impregnation

5. Embedding and blocking

6. Section cutting

7. Routine staining

FIXATION

Any tissue once taken out of the body will decompose due to:- Loss of bloody supply and oxygen Accumulation of products of metabolism Action of autolytic enzymes Putrefaction by bacteria

All the above changes PREVENTED BY FIXATION!

Tissue get fixed in complete physical and partial chemical state

Principle : denaturation / precipitation of cell proteins , soluble component is made insoluble

Fixatives produce the following effect…

IDEAL FIXATIVE

TYPES OF FIXATIVES

[A] Simple (one substance) Eg. Formalin Compound (two or more) Eg. Bouin’s solution,

Zencker’s solution

[B] Microanantomical – preserves anatomy Cytological – cytoplasmic and nuclear features Histochemical – constituents and enzymes

COMMONLY USED FIXATIVES

Formalin – MC – routine Glutaraldehyde – electron microscopyPicric acid(Bouin’s solution) – renal & testicular

tissueAlcohol(Carnoy’s fixative) – cytologic smears,

endometrial samplingOsmium tetraoxide – CNS tissues & electron

microscopy

DEHYDRATION

Water removed from tissue s and cells – this space is occupied by wax

Tissue sent through grades of alcohol : 70%, 80%, 95% and absolute alcohol

Ethyl (MC used), methyl, isopropyl alcohol or acetone can be used

CLEARING

Alcohol from tissues and cells is removed (dealcoholisation) and replaced by a fluid in which wax is soluble – makes tissue transparent

Xylene (MC used) , toluene, benzene, chloroform, cedar wood oil can be used

IMPREGNATION

Empty spaces in tissues and cells , after removal of clearing agent, are taken by molten wax

Hardens the tissue – helps in section cuttingMelting point of wax – 54- 62 degree C

TISSUE PROCESSOR

Dehydration + clearing + impregnation

Automated tissue processorOpen (hydraulic )Closed (vaccum)

OPEN / HYDRAULIC PROCESSOR

12 stations 1 jar – formalin 6 jars – grades of alcohol 3 jars – xylene 2 jars – molten paraffin wax

CLOSED / VACCUM PROCESSOR

Different processing fluids are moved in and out of a single station sequentially

EMBEDDING & BLOCKING

Embedding – with molten waxWax blocks –

Metallic L (Leuckahart’s) blocks Plastic moulds

Embedding centre

Wax reservoirHeated area for steel

mouldsWax dispenserSeparate hot and cold

plates

SECTION CUTIING

Microtome – equipmentMicrotomy – technique5 types of microtomes :

1. Rotatory – MC used

2. Sliding

3. Freezing

4. Rocking

5. Base - sledge

ROUTINE STAINING (H&E)

Haematoxylin – nuclear stainEosin – cytoplasmic stainMounted in DPX/Canada balsm End result :-

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