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TISSUE PROCESSING
AT MICROSCOPIC LEVEL-
 HISTOLOGY
 Science of examination of normal tissues
 HISTOPATHOLOGY
 Examination of tissues for...
What happens to the SPECIMEN?
 Specimen received in the lab (10% formalin)
 Grossed (appearance, measurements, noticeabl...
TISSUE PROCESSING
1. Fixation
2. Dehydration
3. Clearing
4. Impregnation
5. Embedding and blocking
6. Section cutting
7. R...
FIXATION
 Any tissue once taken out of the body will decompose
due to:-
 Loss of bloody supply and oxygen
 Accumulation...
 Tissue get fixed in complete physical and partial
chemical state
 Principle : denaturation / precipitation of cell
prot...
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
so...
COMMONLY USED FIXATIVES
 Formalin – MC – routine
 Glutaraldehyde – electron microscopy
 Picric acid(Bouin’s solution) –...
DEHYDRATION
 Water removed from tissue s and cells – this space is
occupied by wax
 Tissue sent through grades of alcoho...
CLEARING
 Alcohol from tissues and cells is removed
(dealcoholisation) and replaced by a fluid in which
wax is soluble – ...
IMPREGNATION
 Empty spaces in tissues and cells , after removal of
clearing agent, are taken by molten wax
 Hardens the ...
TISSUE PROCESSOR
 Dehydration + clearing + impregnation
 Automated tissue processor
Open (hydraulic )
Closed (vaccum)
OPEN / HYDRAULIC PROCESSOR
 12 stations
 1 jar – formalin
 6 jars – grades of alcohol
 3 jars – xylene
 2 jars – molt...
CLOSED / VACCUM PROCESSOR
 Different processing
fluids are moved in
and out of a single
station sequentially
EMBEDDING & BLOCKING
 Embedding – with molten wax
 Wax blocks –
 Metallic L (Leuckahart’s) blocks
 Plastic moulds
Embedding centre
 Wax reservoir
 Heated area for steel
moulds
 Wax dispenser
 Separate hot and cold
plates
SECTION CUTIING
 Microtome – equipment
 Microtomy – technique
 5 types of microtomes :
1. Rotatory – MC used
2. Sliding...
ROUTINE STAINING (H&E)
 Haematoxylin – nuclear stain
 Eosin – cytoplasmic stain
 Mounted in DPX/Canada balsm
 End resu...
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Procedures in Histopathology

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Tissue processing

  1. 1. TISSUE PROCESSING
  2. 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. 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. 4. TISSUE PROCESSING 1. Fixation 2. Dehydration 3. Clearing 4. Impregnation 5. Embedding and blocking 6. Section cutting 7. Routine staining
  5. 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. 6.  Tissue get fixed in complete physical and partial chemical state  Principle : denaturation / precipitation of cell proteins , soluble component is made insoluble
  7. 7. Fixatives produce the following effect…
  8. 8. IDEAL FIXATIVE
  9. 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. 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. 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. 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. 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. 14. TISSUE PROCESSOR  Dehydration + clearing + impregnation  Automated tissue processor Open (hydraulic ) Closed (vaccum)
  15. 15. OPEN / HYDRAULIC PROCESSOR  12 stations  1 jar – formalin  6 jars – grades of alcohol  3 jars – xylene  2 jars – molten paraffin wax
  16. 16. CLOSED / VACCUM PROCESSOR  Different processing fluids are moved in and out of a single station sequentially
  17. 17. EMBEDDING & BLOCKING  Embedding – with molten wax  Wax blocks –  Metallic L (Leuckahart’s) blocks  Plastic moulds
  18. 18. Embedding centre  Wax reservoir  Heated area for steel moulds  Wax dispenser  Separate hot and cold plates
  19. 19. SECTION CUTIING  Microtome – equipment  Microtomy – technique  5 types of microtomes : 1. Rotatory – MC used 2. Sliding 3. Freezing 4. Rocking 5. Base - sledge
  20. 20. ROUTINE STAINING (H&E)  Haematoxylin – nuclear stain  Eosin – cytoplasmic stain  Mounted in DPX/Canada balsm  End result :-
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Procedures in Histopathology

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