1. TISSUE PROCESSING
DR. UMME KULSUM MUNMUN
ASSISTANT PROFESSOR
DEPARTMENT OF PATHOLOGY
CHANDPUR MEDICAL COLLEGE

2. TISSUE PROCESSING
• Tissue processing is defined as the process of
preparing the tissue by - embedding it in a solid
medium that is firm enough to enable thin
cutting
• And soft enough to enable the knife to cut the
sections with little damage to the knife or the
tissue
• Tissue for study can be obtained from 1) Biopsies
and 2) Autopsies

3. TYPES OF BIOPSY
• Excisional biopsy
• Core / true cut biopsy
• Punch biopsy etc

4. STAGES OF TISSUE PROCESSING
• To fill up the requisition form properly
• Fixation
• Dehydration
• Clearing
• Impregnation
• Embedding
• Section cutting
• Staining
• Mounting

5. • Specimen is received with a requisition form with
adequate information regarding –
- Patient information
- Relevant clinical data
- Surgical findings, name of operation, name of
tissue
• Must be received in 10% formalin solution
• The specimens are given a number for
identification

6. GROSS EXAMINATION
• Describing the specimen and placing all or parts
of it into a small plastic cassette
• The cassettes are placed into a fixative

8. FIXATION
• Specimen received in pathology laboratory is first
adequately fixed
• Fixation is the process by which specimen is
treated with an appropriate fixative for a
particular period of time in order to preserve
tissue as in life like state as possible
• The most commonly used fixative in
histopathology laboratory is 10% formalin
• Some other fixatives: gluteraldehyde, acetic
acid, Carnoy’s fixative, Clarke’s fixative etc.

9. PREPARATION OF 10% FORMALIN
• Formalin is prepared by dissolving 40%
formaldehyde gas in 100 ml of distilled water
• The resultant mixture is 100% formalin
• Routinely 10% formalin is used
• 10% formalin is prepared by mixing 10 ml of
100% formalin in 90 ml of distilled water
• It forms cross links between amino acids of
protein thereby make them insoluble

10. DECALCIFICATION
• Is the process of removing calcium salts from the
specimen
• Done for bony specimen
• Decalcifying agents:
- Nitric acid
- hydrochloric acid
- formic acid etc

11. DEHYDRATION
• It is the process by which the water content in
the tissue is reduced by passing the specimen
through increasing concentrations of dehydrating
agents
• The most commonly used dehydrating agent is
ethanol

12. DEHYDRATION
• 70% alcohol – 1 hour
• 80% alcohol – 1 hour
• 95 % alcohol – 1 hour
• Absolute alcohol – 1 hour
• Absolute alcohol – 1 hour
• Absolute alcohol – 1 hour

13. CLEARING
• It is the process by which alcohol in tissue is
removed which will facilitate the paraffin wax to
be impregnated in tissue
• Commonly used clearing agent: Xylene

14. IMPREGNATION
• Permeating the tissue with a supporting medium
is called impregnation
• Liquid paraffin is usually used for impregnation
• Paraffin impregnation is usually carried out in a
hot air oven at 52-56 ° C

15. EMBEDDING
• Placing the tissue which has been cleared of
alcohol to a metallic mould filled with molten
wax
• After that it is allowed to cool and solidify
• After solidification a wax block is prepared which
is ready for cutting sections

18. SECTIONING
• Paraffin blocks are fixed in the microtome
machine to be sectioned and thin strips of
varying thickness are prepared
• Commonly used microtome machine: Rotary
microtome

21. STAINING
• Routinely used stain in histopathology laboratory
:
Hematoxillin & eosin stain
Hematoxillin (basic stain) : stains acidic structure
(nucleus): deep blue or purple
Eosin (acidic stain) : stains basic structures
(cytoplasm) : pink

23. SPECIAL STAIN
• Special stains are used to identify certain normal
and abnormal substances present in the cells and
tissue which cannot be identified on routine H&E
stain

24. PERIODIC ACID SCHIFF (PAS
STAIN)
• To demonsrate glycogen (glycogen storage
disease) and neutral mucoprotein
• To differentiate poorly differentiated
adenocarcinoma
• To demonstrate basement membrane
• To demonstrate fungi

25. PAS

26. PAS

27. • Mucicarmine, Alcian blue (stains mucin)
• Reticulin fibres : reticulin fibres
• Masson trichrome stain: fibrosis
• Congo red, crystal violet: amyloid – brick red in Congo-red
or Apple green birefringence on polarized microscope
• Oil red O, sudan black B: lipid
• Masson Fontana: melanin
• Von kossa: calcium
• Perl’s Prussian blue: iron

28. MASSON TRICHROME

29. STAINS FOR MICROORGANISM
• Ziehl Neelsen stain : Mycobacteria bacilli - red
• Fite Faraco / Modified Ziehl Neelsen stain : Lepra
bacilli
• Gomori methenamime silver stain : fungus
• Giemsa stain : H. Pylori
• Gram staining

30. ZN STAIN

31. FITE FARACO

32. GIEMSA H. PYLORI

33. FROZEN SECTION
• Pathologic procedure to perform rapid
microscopic analysis of specimen
• Fresh specimen is received
• Cryo-section procedure
• Produces sections without dehydrating, clearing
agents and embedding media
• Section is made in cryostat machine
• Rapid intra-operative diagnosis : malignancy,
margin clearance etc

34. AUTOPSY
• Medical procedure to determine the cause and
manner of death and to evaluate any disease or
injury in the dead body
• Usually performed by a pathologist

35. AUTOPSY
• Forensic – medico-legal purpose
• Clinical / academic

36. CYTOPATHOLOGY
• Abrasive - Pap’s smear , bronchial brushing
• Exfoliative – naturally shedded body fluid , ex.
Sputum, urine, plural fluid, ascitic fluid
• Imprint cytology – during frozen section , bone
marrow biopsy, core biopsy procedure, an
imprint of the specimen is done
• FNAC: routine: superficially palpable lump:
breast, thyroid, lymph node
• Guided: USG guided FNAC, CT guided FNAC

41. • Common fixative in cytopathology: 95% Ehanol
• Stain used in cytopathology, Papanicolaou stain,
H&E stain
• Routine stain in preparation of PBF: leishman
stain

42. THANK YOU