tissue processing
TRANSCRIPT
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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