This document provides an overview of tissue processing procedures in pathology laboratories. It describes the key steps involved, which include: fixation, processing, embedding, sectioning, staining, and examination. Tissue is fixed in formalin to preserve its structure. It then undergoes dehydration, clearing, and impregnation before being embedded in paraffin wax blocks for sectioning. Sections are routinely stained with hematoxylin and eosin (H&E) and special stains may also be used. The document outlines various tissue processing techniques and special procedures like decalcification, frozen sections, and cytopathology specimen preparation.

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