This presentation is about introduction to 'PATHOLOGY' and the very Basic process of 'TISSUE PROCESSING' along with Introduction to the Body Cells, Epithelium and Tissues.... It will be helpful to 2nd year MBBS students for better understanding of the subject.. Thank You..!!

1. STUDY OF CELLS
& TISSUES and
TISSUE
PROCESSING
DR. SHARAYU SURYAWANSHI
Junior Resident
Dept. of Pathology
Govt. Medical College, Latur

3. PATHOLOGYincludes :
 HISTOPATHOLOGY
 AUTOPSY
 CYTOPATHOLOGY
 BLOOD BANKING

12. SIMPLE
CUBOIDAL
EPITHELIUM
SITES-
 Thyroid
 Surface of
ovaries
 Lining of Nephron
 Walls of Renal
tubules PCT,
DCT
 Parts of eye

13. SIMPLE
COLUMNAR
EPITHELIUM
SITES-
CILIATED TYPE-
 Lining of uterus
 Fallopian tubes
 Small bronchi
NON CILIATED TYPE-
 Stomach to Anal canal
 Gallbladder
 Excretory ducts of
glands

14. SITES-
 ENDOTHELIUM-
Lining of Blood
vessels
 ENDOCARDIUM-
Lining of Heart
 Alveoli of Lungs
 MESOTHELIUM of
Body cavities-
 Pericardium
 Pleura
 Peritoneum

15. SITES-
KERATINISED
Type-
 EPIDERMIS of
skin (Dry
Membrane)
NON
KERATINISED
Type-
Moist linings of-
 Mouth
 Vagina
 Urethra
 Anus
STRATIFIED SQUAMOUS
EPITHELIUM

18. White Blood Cells

19. Red Blood
Cells
PLATELETS

20. Histopathology:
Microscopic examination of stained tissue sections
for diagnosis of disease
Steps involved-
i. Specimen receiving
ii. Tissue Fixation
iii. Grossing- Sections cutting
iv. Tissue Processing
v. Staining
vi. Reporting

22. Basic Steps in Receiving
Specimen:-
 Proper identification and orientation of the specimen
 Unlabelled specimen should never be processed.
 A properly completed histopathology requisition form
containing patient's name, age, sex, relevant clinical data
submitted
 Careful search and examination of all the tissue submitted
for-
 Surgical findings,
 Nature of operation and
 Name of tissue in order

23. Tissues Collection & Fixation:-
 Surgery /Biopsy /Autopsy specimen
 The tissue is removed from the animal body.
 Placed in a fixative which stabilizes the tissues to
prevent decay.
 Most common fixative used is 10% Formalin
 10% Formalin is prepared by:
Addition of nine parts of water to the one part of
40%(aqueous) Formaldehyde gas
 (9:1) proportion of
Water : 40% aqueous Formaldehyde

24. Importance of Tissue Fixation:
 To prevent cell autolysis and bacterial attack.
 To fix the tissues so they will not change their volume and
shape during processing.
 To prepare tissue and leave it in a condition which allow clear
staining of sections.
 To leave tissue as close as their living state as possible, and
no small molecules should be lost.

26. Gross Examination & Sections
Cutting:-
 Describing the specimen’s colour, texture, consistency, areas of
congestion or paleness seen externally.
 Taking its appropriate sections of 2-3mm thickness after
cutting open.
 Placing all parts of it into a small plastic cassette which holds
the tissue while is being processed to a paraffin block.
 Initially, the tissue cassettes are placed into a fixative overnight.

28. TISSUE
HOLDING
CASSETTES:-

29. TISSUE PROCESSING

31. Dehydration:
Water is completely removed from fixed tissue
Procedure:
 Tissue blocks are placed in Cassettes
 These tissues are passed through a series of
increasing concentration of alcohol-
2 baths in 80 % alcohol for 1 hour each
1 bath in 90 % alcohol for 1 hour
3 baths in 100% alcohol for 1 hour each

32. Clearing:
 During dehydration water in tissue has been replaced by
alcohol
 Alcohol should be replaced by paraffin wax.
 As paraffin wax is not alcohol soluble, we replace alcohol with
a substance in which wax is soluble
 Xylene is the substance, miscible in both water and paraffin
wax
 So, Most commonly used clearing agent is Xylene
 Other Reagents:
Chloroform
Benzene
Carbon tetrachloride

33. Impregnation:
Placing tissue in molten wax (50-56’C) for 2 hours
Wax diffuses in tissues by replacing Xylene
Infiltration:
Pouring molten wax in the tissue placed properly in
L-Mould (Leukhart Mould)
Allow it to set in blocks for 20-30 min at room
temperature
Embedding:
Embed the tissue in a solid medium (wax) firm
enough to support the tissue and give sufficient rigidity.
It enables thin sections to be cut and yet soft enough
not to damage the knife or tissue.

35. PARAFFIN TISSUE BLOCKS

36. AUTOMATED TISSSUE PROCESSOR

37.  Now, Paraffin tissue blocks are cut in the special
instrument –Microtome into thin slices of thickness 2-
50 microns
 These slices or ribbons of tissues are kept in ‘Hot water
bath’ (temp 40-46’C) for 1-2 min
 These tissue slices are then carefully placed over
albumin coated slides with the help of needle, to
remove any air Bubble or artefact
 These slides are then dried by ‘Heat Fixation’ over the
burner flame for 3-5 min
 The slides then further processed

38. MICROTOME

39. CLEARING n DEPARAFFINISING:-
Place the dried slides in Xylene for 3 to 4 times.
Hydration:-
Hydrate the section by passing it in the increasing concentrations
alcohol bath and water.
 Bath in 100 % alcohol for 30 to 60 seconds
 Bath in 90 % alcohol for 30 to 60 seconds
 Bath in 80 % alcohol for 30 to 60 seconds
 Bath in 70 % alcohol for 30 to 60 seconds
Next step is Staining with H&E Stains-
H- Haematoxylin- Basic dye (Blue colour) to Basic structures
E- Eosin- Acidic Dye (Pink colour) to Acidic structures

40. Staining:-
 Stain the section with hematoxylin solution for 3 to 5
minutes
 Wash in running tap water
 Quickly dip the slide in 0.5% HCL.
 Quickly rinse the slide in tap water 30 t0 60 second.
 Dip the slide several time in dilute ammonia water
 Wash in tap water and then rinse 95% alcohol
 Agitate the slide in Eosin solution for 10 to 60 second drain the
staining solution

41. Dehydration:-
 Bath in 70 % alcohol for 30 to 60 seconds
 Bath in 95 % alcohol for 30 to 60 seconds
 Bath in 100 % alcohol for 30 to 60 seconds
Clearing:-
Place the slide twice in Xylene for 30-60 sec
Mounting:-
Drain the excess Xylene and mount DPX or Canada Balsam
with a cover slip.
(DPX- Distyrene, Plasticizer, Xylene)

44. Thank You..!