tissue processing
TRANSCRIPT
![Page 1: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/1.jpg)
TISSUE PROCESSING
![Page 2: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/2.jpg)
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
![Page 3: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/3.jpg)
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
![Page 4: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/4.jpg)
![Page 5: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/5.jpg)
![Page 6: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/6.jpg)
![Page 7: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/7.jpg)
TISSUE PROCESSING
1. Fixation
2. Dehydration
3. Clearing
4. Impregnation
5. Embedding and blocking
6. Section cutting
7. Routine staining
![Page 8: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/8.jpg)
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!
![Page 9: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/9.jpg)
Tissue get fixed in complete physical and partial chemical state
Principle : denaturation / precipitation of cell proteins , soluble component is made insoluble
![Page 10: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/10.jpg)
Fixatives produce the following effect…
![Page 11: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/11.jpg)
IDEAL FIXATIVE
![Page 12: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/12.jpg)
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
![Page 13: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/13.jpg)
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
![Page 14: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/14.jpg)
![Page 15: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/15.jpg)
![Page 16: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/16.jpg)
![Page 17: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/17.jpg)
![Page 18: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/18.jpg)
![Page 19: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/19.jpg)
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
![Page 20: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/20.jpg)
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
![Page 21: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/21.jpg)
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
![Page 22: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/22.jpg)
TISSUE PROCESSOR
Dehydration + clearing + impregnation
Automated tissue processorOpen (hydraulic )Closed (vaccum)
![Page 23: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/23.jpg)
OPEN / HYDRAULIC PROCESSOR
12 stations 1 jar – formalin 6 jars – grades of alcohol 3 jars – xylene 2 jars – molten paraffin wax
![Page 24: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/24.jpg)
![Page 25: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/25.jpg)
CLOSED / VACCUM PROCESSOR
Different processing fluids are moved in and out of a single station sequentially
![Page 26: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/26.jpg)
EMBEDDING & BLOCKING
Embedding – with molten waxWax blocks –
Metallic L (Leuckahart’s) blocks Plastic moulds
![Page 27: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/27.jpg)
![Page 28: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/28.jpg)
Embedding centre
Wax reservoirHeated area for steel
mouldsWax dispenserSeparate hot and cold
plates
![Page 29: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/29.jpg)
![Page 30: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/30.jpg)
![Page 31: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/31.jpg)
![Page 32: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/32.jpg)
SECTION CUTIING
Microtome – equipmentMicrotomy – technique5 types of microtomes :
1. Rotatory – MC used
2. Sliding
3. Freezing
4. Rocking
5. Base - sledge
![Page 33: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/33.jpg)
![Page 34: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/34.jpg)
![Page 35: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/35.jpg)
![Page 36: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/36.jpg)
ROUTINE STAINING (H&E)
Haematoxylin – nuclear stainEosin – cytoplasmic stainMounted in DPX/Canada balsm End result :-
![Page 37: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/37.jpg)
![Page 38: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/38.jpg)
![Page 39: Tissue processing](https://reader035.vdocument.in/reader035/viewer/2022062316/587303771a28ab99088b4d0d/html5/thumbnails/39.jpg)
THANK YOU