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

7. TISSUE PROCESSING
1. Fixation
2. Dehydration
3. Clearing
4. Impregnation
5. Embedding and blocking
6. Section cutting
7. Routine staining

8. 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!

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

10. Fixatives produce the following effect…

11. IDEAL FIXATIVE

12. 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

13. 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

19. 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

20. 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

21. 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

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

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

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

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

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

32. SECTION CUTIING
 Microtome – equipment
 Microtomy – technique
 5 types of microtomes :
1. Rotatory – MC used
2. Sliding
3. Freezing
4. Rocking
5. Base - sledge

36. ROUTINE STAINING (H&E)
 Haematoxylin – nuclear stain
 Eosin – cytoplasmic stain
 Mounted in DPX/Canada balsm
 End result :-