Tissue processing

Procedures in Histopathology

1. TISSUE PROCESSING

2. 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

3. What happens to the SPECIMEN? Specimen received in the lab (10% formalin) Grossed (appearance, measurements, noticeable pathological changes etc) and kept for formalin fixation Bits given from representative areas ( not >4mm thick) Tissue processed… Final outcome : stained slide for microscopic examination

4. TISSUE PROCESSING Fixation Dehydration Clearing Impregnation Embedding and blocking Section cutting Routine staining

5. 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!

6. Tissue get fixed in complete physical and partial chemical state Principle : denaturation / precipitation of cell proteins , soluble component is made insoluble

7. Fixatives produce the following effect…

8. IDEAL FIXATIVE

9. 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

10. COMMONLY USED FIXATIVES Formalin – MC – routine Glutaraldehyde – electron microscopy Picric acid( Bouin’s solution) – renal & testicular tissue Alcohol( Carnoy’s fixative) – cytologic smears, endometrial sampling Osmium tetraoxide – CNS tissues & electron microscopy

11. 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

12. 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

13. IMPREGNATION Empty spaces in tissues and cells , after removal of clearing agent, are taken by molten wax Hardens the tissue – helps in section cutting Melting point of wax – 54- 62 degree C

14. TISSUE PROCESSOR Dehydration + clearing + impregnation Automated tissue processor Open (hydraulic ) Closed ( vaccum )

15. OPEN / HYDRAULIC PROCESSOR 12 stations 1 jar – formalin 6 jars – grades of alcohol 3 jars – xylene 2 jars – molten paraffin wax

16. CLOSED / VACCUM PROCESSOR Different processing fluids are moved in and out of a single station sequentially

17. EMBEDDING & BLOCKING Embedding – with molten wax Wax blocks – Metallic L ( Leuckahart’s ) blocks Plastic moulds

18. Embedding centre Wax reservoir Heated area for steel moulds Wax dispenser Separate hot and cold plates

19. SECTION CUTIING M icrotome – equipment Microtomy – technique 5 types of microtomes : Rotatory – MC used Sliding Freezing Rocking Base - sledge

20. ROUTINE STAINING (H&E) Haematoxylin – nuclear stain Eosin – cytoplasmic stain Mounted in DPX/ C anada balsm End result :-

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