The document provides information about routine histopathology techniques and staining. It discusses fixation of histology samples, ideal fixatives, changes after fixation, types of fixatives, and the mechanisms of fixation. It also covers tissue processing techniques including dehydration, clearing, infiltration, embedding, and sectioning of tissue blocks using a microtome. Key steps in processing like fixation, dehydration, clearing and infiltration are described in detail. Common fixatives, dehydrating agents, and clearing agents used are also listed.

1. ROUTINE HISTOPATHOLOGY
TECHNIQUES AND STAINING
PRESENTER- DR. CHANDRESH KUMAR
MODERATOR- DR. MANJULA LADER MADAM
DATE- 18.04.2023

2. FIXATION OF HISTOLOGY SAMPLES
• APPROPRIATE FIXATION IS CENTRAL TO ALL HISTOLOGY TESTS
AIMS OF FIXATION-
1. TO PRESERVE THE TISSUE NEAREST TO ITS LIVING STATE
2. TO PREVENT ANY CHANGE IN SHAPE AND SIZE OF THE TISSUE AT THE TIME OF
PROCESSING
3. TO PREVENT ANY AUTOLYSIS
4. TO MAKE THE TISSUE FIRM TO HARD
5. TO PREVENT ANY BACTERIAL GROWTH IN THE TISSUE
6. TO MAKE IT POSSIBLE TO HAVE CLEAR STAIN
7. TO HAVE BETTER OPTICAL QUALITY OF THE CELLS

3. IDEAL FIXATIVE -
AN IDEAL FIXATIVE SHOULD HAVE THE FOLLOWING QUALITIES-
1. PREVENTION OF AUTOLYSIS OF THE CELLS OR TISSUE
2. PREVENTION OF DECOMPOSITION OF THE TISSUE BY BACTERIA
3. MAINTAINING THE VOLUME AND SHAPE OF THE CELL AS FAR AS
POSSIBLE
4. CONSISTENTLY HIGH-QUALITY STAINING PARTICULARLY ROUTINE STAIN
5. RAPID ACTION
6. CHEAP
7. NON-TOXIC

4. CHANGE IN TISSUE AFTER FIXATION –
• VOLUME CHANGES – SHRINKAGE OF THE VOLUME BY FORMALIN (33%).
• HARDENING OF TISSUE – MILD DEGREE HARDENING MAY OCCUR.
• INTERFERENCE OF STAINING – INHIBITS ROUTINE STAIN: OSMIUM TETROXIDE
INHIBITS HAEMATOXYLIN AND EOSIN STAINING.
• CHANGES OF OPTICAL DENSITY BY FIXATION – NUCLEI MAY LOOK LIKE
HYPERCHROMATIC.

5. TYPES OF FIXATIVES

6. ESSENTIAL PRECAUTIONS FOR FIXATION –
• THE TISSUE SHOULD BE FREE FROM EXCESSIVE BLOOD BEFORE PUTTING IT INTO
FIXATIVE.
• TISSUE SHOULD BE THINLY CUT IN 3–5 MM THICKNESS.
• THE AMOUNT OF FIXATIVE FLUID SHOULD BE 20 TIMES MORE THAN THE
VOLUME OF THE
TISSUE.
• THE TISSUE WITH FIXATIVE SHOULD BE IN A TIGHTLY SCREW-CAPPED BOTTLE.

7. MECHANISM OF FIXATION
• DEHYDRATION AND COAGULATION OF PROTEIN –
• ALCOHOLS REMOVE WATER FROM THE TISSUE, DESTABILIZE THE HYDROGEN
BONDS & DISRUPT THE TERTIARY STRUCTURE OF PROTEIN.
• THE SECONDARY STRUCTURE OF THE PROTEIN IS MAINTAINED.
• ETHANOL IS RELATIVELY STRONGER DEHYDRATING AGENT THAN METHANOL.
• THE ETHANOL AND METHANOL START WORK FROM 60–80% CONCENTRATION,
RESPECTIVELY.
• THE DEHYDRATING FIXATIVE HAS TWO DISADVANTAGES: –
- SHRINKAGE OF THE CELLS
- REMOVAL OF THE SOLUBLE SUBSTANCES FROM THE TISSUE

8. • CROSS-LINKING FIXATIVES:
FORMALDEHYDE-
• IN AQUEOUS SOLUTION IT COMBINES WITH WATER TO FORM METHYLENE
HYDRATE, A METHYLENE GLYCOL
• ON LONG-STANDING, THIS METHYLENE GLYCOL FURTHER REACT WITH WATER &
FORM A POLYMER KNOWN AS POLYOXYMETHYLENE GLYCOL.
• THIS AGAIN DEPOLYMERIZED IN METHYLENE GLYCOL IN A NEUTRAL BUFFER
SYSTEM.
• FORMALDEHYDE REACTS WITH VARIOUS SIDE CHAIN OF THE PROTEIN AND
FORMS HYDROXYMETHYL SIDE GROUP.
• THESE COMPOUNDS ARE HIGHLY REACTIVE AND SUBSEQUENTLY CROSS-LINKING
OCCURS BY FORMING A METHYLENE BRIDGE (PRIMARY REACTION)

9. CONT….
• SUBSEQUENT INTERMOLECULAR AND INTRAMOLECULAR CROSS-LINKING OF THE
MOLECULES OCCURS AS A SLOW-GROWING PROCESS.
• THIS ULTIMATELY PRODUCES AN INSOLUBLE PRODUCT.
• THE FORMALIN CAN BE REMOVED FROM TISSUE BY
PROLONGED WASHING.
• ONCE METHYLENE BRIDGE IS FORMED IN THE TISSUE,
THE REACTION IS STABLE, AND IT IS DIFFICULT TO
REMOVE FORMALIN FROM THE TISSUE.
• FORMALDEHYDE ALSO REACTS WITH THE NUCLEIC ACID
BY REACTING WITH THE AMINO GROUP OF NUCLEOTIDES

10. GLUTARALDEHYDE-
• THE ALDEHYDE GROUP OF GLUTARALDEHYDE REACTS WITH AMINO GROUP OF THE
PROTEIN PREDOMINANTLY LYSINE.
• GLUTARALDEHYDE RAPIDLY AND IRREVERSIBLY CROSS-LINKS THE PROTEIN.
• THE PENETRATION OF GLUTARALDEHYDE IS SLOWER THAN FORMALDEHYDE.
OSMIUM TETROXIDE
• IT CAUSES OXIDATION OF UNSATURATED BONDS IN LIPID.
• IT CONVERTS THE UNSATURATED FATTY ACID INTO A STABLE PRODUCT KNOWN AS
GLYCOL OSMATE.
• THE TETRAVALENT OS BECOMES HEXAVALENT IN THIS REACTION. OSMIC ACID
MONOESTER FORMED IN THIS REACTION IS EASILY HYDROLYSED TO A DIOL AND
OSMIC ACID.
• OSMIUM TETROXIDE MAY REACT WITH TWO UNSATURATED CARBON ATOM OF THE
LIPIDS AND MAY CROSS-LINK

11. FIXATION MECHANISM OF DIFFERENT FIXATIVES

16. .

17. .

18. .

19. PROCESSING OF TISSUE
• AIMS OF TISSUE PROCESSING: TO PROVIDE SUFFICIENT RIGIDITY TO THE TISSUE
SO THAT IT CAN BE CUT INTO THIN SECTION FOR MICROSCOPIC EXAMINATION.
• PRINCIPLE OF PROCESSING: WATER WITHIN THE TISSUE IS REMOVED, AND
ANOTHER MEDIUM (USUALLY PARAFFIN WAX) IS IMPREGNATED IN THE TISSUE
THAT PROVIDES THE ADEQUATE SUPPORT TO THE TISSUE.
• THE ESSENTIAL STEPS IN TISSUE PROCESSING:

20. INFLUENCING FACTORS OF TISSUE PROCESSING
• SIZE OF THE TISSUE: – THE SMALLER THE SIZE, THE BETTER THE PROCESSING.
• AGITATION: – AGITATION FACILITATES THE CONTACT OF TISSUE WITH FRESH
SOLUTION.
• HEAT: – INCREASES THE BETTER PENETRATION OF FLUID.
• VISCOSITY: – THE HIGHER THE VISCOSITY OF THE MEDIUM, LOWER THE
PENETRATION.
• NEGATIVE PRESSURE: – NEGATIVE PRESSURE REMOVES TRAPPED AIR IN THE
TISSUE.
– REMOVAL OF CLEARING AGENT BY INCREASING
VOLATILITY.

21. DEHYDRATION
• REMOVES FREE OR UNBOUND WATER MOLECULE OF THE TISSUE AS THE
SUPPORTING
MEDIUM (PARAFFIN) IS NOT MISCIBLE WITH WATER.
• SHARP DIFFERENCE OF CONCENTRATION GRADIENT OF THE DEHYDRATING
FLUID MAY
DAMAGE THE DELICATE TISSUE.
• GRADUAL DEHYDRATION IS NECESSARY.
• TOO MUCH TIME IN THE DEHYDRATING FLUID: THE TISSUE BECOMES HARD AND
BRITTLE.
• ROUTINE LABORATORY: 70, 90 AND 100% ALCOHOL FOR 2 H EACH.
• COMMON DEHYDRATING AGENTS: – ETHYL ALCOHOL, METHYLATED SPIRIT,
METHANOL,
BUTYL ALCOHOL, ISOPROPYL ALCOHOL

22. COMPARISON OF DIFFERENT DEHYDRATING AGENTS
Dehydrating
agents
Advantages Disadvantage
Ethyl alcohol • Rapid and efficient
dehydrating agent
• Needs licence from the government
• Inflammable
• Hard and brittle tissue if kept for
long time
Methanol Equally effective as ethanol • Volatile • High cost
Isopropyl
alcohol
• Relatively rapid action
• Non-toxic
• Minimal tissue shrinkage
Not possible to use in celloidin
technique
Dioxane • Rapid action
• No shrinkage of tissue
Highly toxic gas is generated
Ethylene glycol • Rapid
• No graded solution is needed
• Tissue can be kept in it for
long time
• Very expensive
• Clearing agent is needed
Acetone • Rapid action
• Cheaper than ethanol
• Good for fatty tissue
• Quickly evaporates
• Inflammable
• Prolonged use may cause shrinkage

23. CLEARING

24. COMPARISON OF CLEARING AGENTS

25. INFILTRATION AND EMBEDDING
• AIMS: TO PROVIDE SUPPORT TO THE TISSUE.
• PRINCIPLE: CLEARING AGENT IS REMOVED BY THE PROCESS OF DIFFUSION, AND THE
TISSUE
SPACE IS NOW INFILTRATED WITH THE EMBEDDING MEDIA.
• IDEAL IMPREGNATING MEDIUM:
• MISCIBLE WITH CLEARING AGENT
• LIQUID IN HIGHER TEMPERATURE AND SOLID IN ROOM TEMPERATURE
• HOMOGENOUS AND STABLE • NON-TOXIC AND CHEAP
• TRANSPARENT
• FIT FOR SECTIONING
• THE TISSUE TIME DURATION AND THE NUMBER OF CHANGES OF EMBEDDING
MEDIUM:
• SIZE OF TISSUE: LARGE VERSUS SMALL.
• TYPE OF TISSUE: HARD VERSUS SOFT.
• THE TYPE OF CLEARING AGENT: CEDARWOOD OIL TAKES LONGER TIME.
• TYPE OF PROCESSING: VACUUM PROCESSING ACCELERATES.

26. • PARAFFIN WAX- HYDROCARBON, BY-PRODUCT OF CRUDE PETROLEUM.
• MOST POPULAR EMBEDDING MEDIUM FOR TISSUE PROCESSING.
• THE MELTING POINT VARIES FROM 39 °C TO 70 °C.
• IN INDIAN SUBCONTINENT, THE PARAFFIN WAX WITH MELTING POINT AROUND
60 °C IS THE MOST SUITABLE FOR LABORATORY USE.
• TOTAL 3–4 HR. TIME IN PARAFFIN WAX IS SUFFICIENT FOR IMPREGNATION OF
TISSUE BY WAX.
• ADVANTAGES OF PARAFFIN WAX:
• TISSUE BLOCK CAN BE STORED FOR LONG DURATION.
• NON-TOXIC • CHEAP
• SAFE
• DISADVANTAGES OF PARAFFIN WAX:
• MAY CAUSE TISSUE SHRINKAGE AND HARDENING IN CASE OF PROLONGED
IMPREGNATION.
• PARAFFIN WAX TAKES LONG DURATION FOR THE IMPREGNATION OF THE BONE

27. TISSUE PROCESSING METHODS
• MANUALLY OR BY AUTOMATED PROCESSOR.
• AUTOMATED TISSUE PROCESSOR: THE BASIC PRINCIPLE OF IS TO TRANSFER THE
TISSUE IN DIFFERENT FLUID FOR A SPECIFIED TIME IN A DESIRED ENVIRONMENT.
• TWO TYPES OF PROCESSOR:
1. TISSUE TRANSFER PROCESSOR 2. FLUID TRANSFER
PROCESSOR

28. Overall Precautions of Tissue Processing-
1. The bulk of the tissue should be
optimum for adequate penetration of
fluid.
2. The amount of fluid should be adequate,
fluid level should be always higher than
the tissue level.
3. The tissue basket and cassettes should
be clean and any spillage of wax should
be cleaned.
4. The temperature of the infiltrating
medium should be optimum, and it is
preferable to keep the temperature 3–
4 °C above the melting point.
5. There should be a proper record of the
change of fluid, number of tissues

29. TROUBLESHOOTING IN PROCESSING

30. EMBEDDING OF TISSUE
• THE TISSUE IS SURROUNDED IN A MOLTEN MEDIUM BY USING A MOULD.
• SUBSEQUENTLY THIS MEDIUM IS SOLIDIFIED TO MAKE A BLOCK FOR CUTTING THIN SECTION
OF TISSUE.
• AIMS OF EMBEDDING:
1. TO GIVE SUPPORT OF THE TISSUE
2. TO PREVENT DISTORTION OF THE TISSUE DURING CUTTING
3. TO PRESERVE THE TISSUE FOR ARCHIVAL USE
• THE CHOICE OF THE EMBEDDING MEDIUM:
• PARAFFIN WAX, EPOXY RESIN, METHACRYLATE, CARBOWAX, ETC. ARE USED.
• PARAFFIN WAX IS THE MOST COMMONLY USED EMBEDDING MEDIUM.
• THE CHOICE OF THE EMBEDDING MEDIUM DEPENDS ON :
1. TYPE OF TISSUE: THE DENSITY OF THE TISSUE AND THE EMBEDDING MEDIUM SHOULD BE
CLOSE OTHERWISE TISSUE MAY NOT BE SECTIONED PROPERLY, AND TISSUE WILL BE
DEFORMED.
2. TYPE OF MICROTOME
3. TYPE OF MICROSCOPE

31. DIFFERENT TYPES OF MOULD USED FOR BLOCK
• LEUCKHARD EMBEDDING MOULDS
• STAINLESS STILL MOULD
• PLASTIC MOULD

32. TISSUE EMBEDDING PROCESS

33. TISSUE ORIENTATION DURING EMBEDDING

34. TISSUE MARKING
NEEDED FOR-
1. TO IDENTIFY THE RESECTION PLANE OR OUTER MARGIN OF THE TISSUE
2. TO HELP IN EMBEDDING THE TISSUE
3. ANY AREA OF INTEREST TO IDENTIFY SUCH AS THE AREA OF TRANSITIONAL
ZONE IN CONE BIOPSY OF CERVIX
THE TISSUE MARKERS SHOULD HAVE THE FOLLOWING CHARACTERISTICS
FEATURES:
• THE MARKER SUBSTANCE SHOULD NOT BE DISSOLVED IN FIXATIVE AND TISSUE
PROCESSING
AGENTS.
• THE MARKER SHOULD NOT PENETRATE THE DEEPER TISSUE.
• IT SHOULD BE RECOGNIZABLE IN THE STAINED SECTION BOTH
MICROSCOPICALLY AND
MACROSCOPICALLY.

35. TROUBLESHOOTING IN TISSUE EMBEDDING

36. DECALCIFICATION
AIM: REMOVAL OF CALCIUM SALT FROM TISSUE WITHOUT DAMAGING IT’S MORPHOLOGY
CALCIUM-CONTAINING TISSUE: (1) BONE (2) TOOTH (3) PATHOLOGICAL CALCIFICATION
REQUISITES FOR SUCCESSFUL DECALCIFICATION:
• SMALL TISSUE
• ADEQUATE FIXATION
• CONSISTENCY
• ADEQUATE VOLUME OF DECALCIFYING AGENT
• SUITABLE CHOICE OF THE DECALCIFYING AGENT
METHODS OF DECALCIFICATION
• ACID DECALCIFICATION
• ION-EXCHANGE RESIN
• ELECTRICAL IONIZATION • CHELATING SOLUTION • SURFACE
DECALCIFICATION

37. ACID DECALCIFYING AGENTS

38. TISSUE MICROTOMY
• MICROTOMES- INSTRUMENT BY WHICH WE CUT THE EMBEDDED TISSUE IN THE
PARAFFIN BLOCK AS THIN SECTION.
• THE DIFFERENT TYPES OF MICROTOMES-
• ROTARY MICROTOME
• ROCKING MICROTOME
• BASE SLEDGE MICROTOME
• SLIDING MICROTOME
• CRYOMICROTOME
• ULTRAMICROTOME
• LASER MICROTOME

39. • ROTARY MICROTOME- MOST COMMONLY USED
• THE CUTTING BLADE IS KEPT IN HORIZONTAL POSITION, AND THE BLOCK CONTAINING TISSUE
MOVES UP AND DOWN WITH THE HELP OF ROTATORY HANDLE ATTACHED WITH THE
MICROTOME.
• IN EACH 360° ROTATION OF THE WHEEL HANDLE, THE BLOCK MOVES DOWN FOLLOWED BY UP,
AND THE TISSUE IS CUT AS THIN RIBBON.
• THIS MICROTOME HAS THE OPTION TO BE SEMIAUTOMATED OR AUTOMATED
• ADVANTAGES:
1. GOOD-QUALITY 2–3-ΜM-THIN SECTION IS POSSIBLE.
2. HEAVY AND STABLE AUTOMATED ROTARY MICROTOME REDUCES HEALTH HAZARD AND
GIVES THE BEST-QUALITY SECTION.
3. GOOD TISSUE RIBBON PRODUCTION.
4. EASY-TO-CUT VARIOUS TYPES OF TISSUE: FIRM, FRAGILE, SMALL BIOPSY, ETC.
• DISADVANTAGES:
1. EXPENSIVE.
2. UNSUITABLE TO CUT LARGE BLOCK.
3. KNIFE FACES UP AND SO MAY BE DANGEROUS TO THE TECHNICAL STAFF.

40. .

41. • SECTIONING THE PARAFFIN BLOCK
• THE FOLLOWING INSTRUMENTS ARE ESSENTIAL –
1. MICROTOME WITH BLADE
2. WATER BATH
3. PARAFFIN BLOCK WITH EMBEDDED TISSUE TO CUT
4. ICE TRAY
5. A BLUNT FORCEPS OR CAMEL BRUSH
6. SLIDE RACK WITH SLIDES WATER BATH (FLOATATION CHAMBER)
7. ADHESIVE- USED FOR BRAIN SECTIONS, DECALCIFIED TISSUE, USING STRONG ALKALI AT THE
TIME OF STAINING
THE MOST COMMONLY USED ADHESIVES INCLUDE:
• MAYER’S EGG ALBUMIN AND GLYCEROL
• POLY-L-LYSINE
• 3-AMINOPROPYLTRIETHOXYSILANE (ACEP)

43. TROUBLESHOOTING IN TISSUE SECTIONING

44. .

45. .

46. FROZEN SECTION
• INDICATIONS-
• RAPID DIAGNOSIS OF THE LESION FOR INTRAOPERATIVE MANAGEMENT
• TO KNOW THE EXTENT OF THE LESION
• TO DO ENZYME IMMUNOCYTOCHEMISTRY
• TO DO IMMUNOFLUORESCENCE STUDY
• TO STAIN LIPID AND CERTAIN CARBOHYDRATE IN THE TISSUE
• PRINCIPLE-
• RAPID FREEZING OF THE TISSUE SAMPLE CONVERTS THE WATER INTO ICE. THE
FIRM ICE WITHIN THE TISSUE ACTS AS EMBEDDING MEDIA TO CUT THE TISSUE.
• THE CRYOSTAT IS THE INSTRUMENT THAT HAS THE ARRANGEMENT TO FREEZE
THE TISSUE AND ALSO TO CUT THE FROZEN TISSUE FOR MICROSCOPIC SECTION.

47. CRYOSTAT

49. TROUBLESHOOTING IN FROZEN SECTION

50. • STAINING- HAEMATOXYLIN AND EOSIN (H&E) STAINING-
• RINSE THE SLIDE IN TAP WATER.
• PUT IN HAEMATOXYLIN FOR 1 MIN.
• RINSE IN TAP WATER FOR 5 S.
• RINSE IN SCOTT’S TAP WATER FOR 5 S FOR BLUING.
• DIP IN EOSIN FOR 20 S.
• RAPIDLY RINSE IN TAP WATER.
• 95% ETHANOL FOR 10 S.
• 100% ETHANOL FOR 10 S.
• 100% ETHANOL FOR 10 S.
• DIP IN XYLENE FOR 20 S.
• MOUNT BY DPX.

51. STAINING

52. .

53. ESSENTIAL TYPES OF HAEMATOXYLIN

54. DYE-MORDANT COMPLE –
• HAEMATEIN IS A WEAK ANION AND CANNOT COMBINE WITH NUCLEIC ACID IN THE NUCLEUS.
• WHEN A METALLIC SALT (MORDANT) IS COMBINED WITH HAEMATEIN, THEN A CATIONIC
DYEMETAL COMPLEX IS FORMED THAT BEHAVES AS A STRONG BASIC DYE AND COMBINES
WITH NUCLEIC ACID.
• THE TYPE OF MORDANT DETERMINES THE TYPE OF TISSUE AFFINITY OF THE DYE AND THE
COLOUR OF THE STAIN.
• COMMONLY ALUMINIUM (AL3+), IRON (FE3+), MOLYBDENUM, TUNGSTEN AND LEAD SALTS
ARE USED AS MORDANT.
• TYPES OF HAEMATOXYLIN ON THE BASIS OF MORDANT:
1. IRON HAEMATOXYLIN 2. ALUM HAEMATOXYLIN
3. TUNGSTEN HAEMATOXYLIN 4. LEAD HAEMATOXYLIN
5. MOLYBDENUM HAEMATOXYLIN
6. ONLY HAEMATOXYLIN (NO MORDANT ATTACHED)

55. • BLUING
• THE MOST OF THE REGRESSIVE STAINING OF HAEMATOXYLIN NEEDS BLUING.
• THE REMOVAL OF EXCESS HYDROGEN ION FROM THE STAIN IS KNOWN AS
BLUING.
• HERE THE HAEMALUM WHICH IS SOLUBLE IS CONVERTED TO INSOLUBLE FORM.
• BLUING GIVES CRISP BLUE COLOUR OF THE NUCLEI.
• IN THE PROCESS OF BLUING, THE PH OF THE SOLUTION IS RAISED TO 8.5
(ALKALINE SIDE).
• THE TISSUE SECTION IS TREATED WITH ALKALINE REAGENT, AND THE ACIDIC
REAGENTS ARE NEUTRALIZED IN BLUING PROCESS.
• METHODS:
• RUNNING TAP WATER FOR SEVERAL MINUTES
• TREATING THE SECTION BY SCOTT’S TAP WATER (PH IS 8): 2–3 MIN
• AMMONIUM HYDROXIDE (5%): 2–3 MIN
• AMMONIA VAPOUR: FEW SECONDS

56. BASIC STEPS IN ROUTINE H&E STAINING

57. TROUBLESHOOTING IN STAINING

58. .

60. COMPARISON OF DIFFERENT MOUNTING MEDIA

61. REFERENCES-
1. BASIC AND ADVANCED LABORATORY TECHNIQUES IN HISTOPATHOLOGY AND
CYTOLOGY
– PRANAV DEY
2. BANCROFT’S THEORY AND PRACTICE OF HISTOLOGICAL TECHNIQUES- 8TH
ED.-
S. KIM SUVARNA, CHRISTOPHER LAYTON, JOHN D. BANCROFT

62. THANK YOU
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